PR1 Peptide Vaccine Research Articles

Cathepsin G Is Expressed By B Cell Acute Lymphoblastic Leukemia and Is an Effective Immunotherapeutic Target

The azurophil granule proteases neutrophil elastase (NE) and proteinase 3 (P3) have been established as effective immunotherapeutic targets in hematological malignancies. Expression of PR1, an HLA-A2 restricted nonameric peptide derived from NE and P3, has been demonstrated in acute myeloid leukemia (AML). We have successfully targeted PR1 in AML patients using PR1-peptide vaccine, anti-PR1/HLA-A2 antibody and PR1-specific cytotoxic T-lymphocytes (PR1-CTL). We have also identified the uptake and cross-presentation of NE and P3 by breast cancer, lung cancer and melanoma, which underlines their potential as immunotherapeutic targets in non-myeloid malignancies.Cathepsin G is also an azurophil granule serine protease that is highly expressed by AML blasts and leukemic stem cells. Our previous studies showed a high level of efficacy in targeting AML blasts that express CG1 (FLLPTGAEA) using CG1-specific CTL (CG1-CTL), while sparing normal hematopoietic cells. However, two previous reports (Fujiwara et al., Clinical Cancer Research, 2005 and Gorodkiewicz et al., Analytical Biochemistry, 2012) and our published data (Alatrash G, Leukemia, 2017) have reported CG expression in lymphoid leukemia. We therefore hypothesized that CG is an effective immunotherapeutic target in lymphoid malignancies, specifically acute lymphoid leukemia (ALL).We first analyzed CG expression in ALL patient samples. Using reverse phase protein array (RPPA), we analyzed CG expression in ALL blasts from 345 adult ALL patient samples. We detected CG in 345 patient samples at levels comparable to G-CSF stimulated myeloid progenitors. The differential expression of CG in sorted ALL blasts ranged from -1.8030 to 5.4390, with a median value of 0.3506. In estimating CG levels, we found that more than 15% of our ALL samples expressed Log2 concentrations of CG between 0.3 and 0.4 (Figure 1). This was comparable to what we have previously reported in AML. We then wanted to determine whether the ALL-associated CG was due to uptake or endogenous expression. We performed PCR and western blotting and confirmed CG expression in 4 of 9 and 9 of 9 patient ALL samples, respectively (Figure 2). To assess whether CG is also taken up by ALL, we examined CG uptake by the B-ALL cell lines SB, RS4, Nalm6 and HMY (B-ALL). ALL cell lines were incubated with CG protein at increasing doses and durations. Intracellular staining of CG followed by flow cytometry analysis was used to determine uptake of CG by ALL cell lines. Our data show that all of the ALL cell lines take up CG; uptake correlated directly with increasing doses and time of incubation up to 24 hours.Having characterized the expression of CG by ALL, we sought to determine its potential as an immunotherapeutic target, as we have previously shown for AML. In these experiments, CG1-specific CTLs were generated using standard dendritic cells methodology, interleukin-7 (IL-7), IL-2 and CG1-peptide pulsing. Expanded CG1-CTL were confirmed by staining cells with CG1 tetramer followed by flow cytometry analysis. We employed standard calcein-AM cytotoxicity assays to determine specific lysis. In these experiments, patient ALL sample UPN8 was pulsed with calcein-AM and then co-cultured with CG1-CTL for 4 hours. Our results indicated that UPN8 was effectively lysed by CG1-CTL, reaching a maximum specific lysis of ~30% at the highest effector:target ratio (Figure 3).Overall, our analysis confirms CG endogenous expression and uptake by ALL. Furthermore, we conclude that CG is an effective target for immunotherapy in B-ALL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Characterizing the Mechanism of NE and P3 Cross-Presentation in B Cell-Acute Lymphoblastic Leukemia

IntroductionPR1 is a nonameric HLA-A2 restricted leukemia-associated antigen derived from serine proteases neutrophil elastase (NE) and proteinase 3 (P3). NE and P3 are primarily expressed in cells of myeloid lineage including granulocytes, bone marrow progenitors and myeloid leukemia. However, NE and P3 are also cross-presented by antigen presenting cells (APCs) such as B-cells and some non-hematopoietic solid tumors, a mechanism whereby exogenous antigens are endocytosed and presented on HLA class I molecules. Therefore, identifying non-myeloid tumors capable of PR1 cross-presentation broadens the application of PR1-targeted immunotherapies, which to date include PR1 peptide vaccine, PR1 cellular therapy, and 8F4, a T cell receptor (TCR)-like monoclonal antibody (mAb).One possible source of NE and P3 in the microenvironment is neutrophil extracellular traps (NETs). NETs are composed of deoxyribonucleic acid (DNA)/histones and the antimicrobial proteases, NE and P3, which are extruded from activated polymorphonuclear neutrophils (PMNs) during inflammation. Although a main function of NETs is elimination of pathogens, studies have shown immune-regulatory effects of NETs. Thus there is a great interest in understanding the role of NETs in modulating adaptive immune system in the setting of anti-tumor immunity. We hypothesized that NETs facilitate NE and P3 cross-presentation in malignant B-cell acute lymphoblastic leukemia (ALL) due to their high abundance in the bone marrow, which could lead to susceptibility to PR1-targeted immunotherapies.MethodsB-ALL cell lines SB, Nalm6, RS4 and Nalm6 were co-cultured with soluble NE and P3 (10 ug/mL) and PMNs at a ratio of 3:1 (PMN: ALL) in low serum media and analyzed for NE and P3 cross-presentation by staining for HLA-A2/PR1 complex using 8F4 mAb. T2 cells pulsed with PR1 and HIV peptide were used as a positive and negative control, respectively. To determine susceptibility to killing my PR1-CTLs, SB cell line was co-cultured with 10 μg/mL soluble NE and P3 for 24 hours and incubated with healthy PR1-CTLs for 4 hours following a protocol for a standard calcein-AM cytotoxicity assay. T2 cells were pulsed with PR1 and CG1 peptide as a positive and negative control, respectively.To identify NETs as a source of NE and P3, healthy PMNs were treated with ionomycin to induce NETosis, co-cultured with B-ALL cell lines at a ratio of 3:1 (PMN: ALL) overnight and analyzed for NE and P3 uptake. NETs were also treated with DNAse to determine the effect on uptake.ResultsThe B-ALL cell line, SB, cross-presented PR1 from soluble P3 and NE, as well as supernatants derived from quiescent PMNs. There was a 1.3 fold significant increase in PR1/HLA-A2 surface expression when co-cultured with soluble and PMN-associated protease (Figure 1A). We then investigated whether PR1 cross-presentation by SB cell line rendered it susceptible to killing by PR1-CTLs. Dose-dependent killing of SB cells was observed following PR1 cross-presentation compared to nonpulsed cells (Figure 1B). The percent specific lysis was evident even at a very low ratio of 0.625:1 Effector: Target cells (E:T). As controls, T2 cells pulsed with PR1 showed dose-dependent killing by PR1-CTLs, whereas T2 cells pulsed with CG1 and nonpulsed did not, as expected (Figure 1B).To evaluate whether NET-associated NE and P3 facilitate uptake in B-ALL, healthy PMNs were co-cultured with B-ALL cell lines overnight. The co-culture resulted in both NE (Figure 2A-B) and P3 (Figure 2C-D) uptake in RS4 and Nalm6 cell lines with an average MFI of 350 and 426, respectably, compared to nonpulsed MFI of 145. Measurement of intracellular P3 and NE by flow cytometry showed that although both NET-associated and PMN-associated NE and P3 are taken up, uptake of supernatants from resting PMNs was significantly greater than NET-associated (Figure 2). Similar to uptake of soluble and PMN-associated proteases, uptake between B-ALL cell lines was heterogeneous.ConclusionCollectively, our data demonstrate that B-ALL cell lines are capable of PR1 cross-presentation rendering them susceptible to killing by PR1-CTLs. Further, NET-associated NE and P3 are a source of protease uptake. Because NET-associated antigens are less efficient at uptake, our data suggest that NETs produced during systemic inflammation might be less immunogenic in B-cell malignancies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

PR1 peptide vaccine induces specific immunity with clinical responses in myeloid malignancies.

PR1, an HLA-A2-restricted peptide derived from both proteinase 3 and neutrophil elastase, is recognized on myeloid leukemia cells by cytotoxic T lymphocytes (CTL) that preferentially kill leukemia and contribute to cytogenetic remission. To evaluate safety, immunogenicity and clinical activity of PR1 vaccination, a phase I/II trial was conducted. Sixty-six HLA-A2+ patients with acute myeloid leukemia (AML: 42), chronic myeloid leukemia (CML: 13) or myelodysplastic syndrome (MDS: 11) received three to six PR1 peptide vaccinations, administered subcutaneously every 3 weeks at dose levels of 0.25, 0.5 or 1.0 mg. Patients were randomized to the 3 dose levels after establishing the safety of the highest dose level. Primary endpoints were safety and immune response, assessed by doubling of PR1/HLA-A2 tetramer-specific CTL, and the secondary endpoint was clinical response. Immune responses were noted in 35 of 66 (53%) patients. Of the 53 evaluable patients with active disease, 12 (24%) had objective clinical responses (complete: 8, partial: 1 and hematological improvement: 3). PR1-specific immune response was seen in 9 of 25 clinical responders vs. 3 of 28 clinical non-responders (p=0.03). In conclusion, PR1 peptide vaccine induces specific immunity that correlates with clinical responses, including molecular remission, in AML, CML and MDS patients.

PR1 Is Cross-Presented By Multiple Myeloma Cells in Patients and Renders Multiple Myeloma Susceptible to PR1-CTL and Anti-PR1/HLA-A2 Antibody

PR1 is an HLA-A2-retricted, nonameric peptide that is derived from the azurophil granule proteases neutrophil elastase (NE) and proteinase 3 (P3). PR1 has been targeted successfully in acute (AML) and chronic (CML) myeloid leukemia using anti-PR1/HLA-A2 antibody (8F4), PR1-peptide vaccine and PR1-specific cytotoxic T lymphocytes (PR1-CTL). We have previously reported that NE and P3 are cross-presented by normal B cells and dendritic cells (DC), leading to PR1 expression by HLA-A2. Since multiple myeloma (MM) is a B cell malignancy, we investigated whether MM cells can cross-present PR1 as a possible target for immunotherapy.To study whether PR1 is presented by MM cells, patient bone marrow (BM) was stained with 8F4 antibody and then imaged using confocal microscopy. PR1/HLA-A2 was detected on the surface of CD138+ MM cells in BM samples from 3 of 6 HLA-A2+ MM patients (Fig. 1). We then investigated whether cellular immunity to PR1 is detected in peripheral blood (PB) from patients with MM. PB samples from MM patients who had undergone allogeneic (allo) (n=9) and autologous (auto) (n=2) stem cell transplantation (SCT) were stained with PR1/HLA-A2 dextramer in addition to standard lineage markers. PR1-specific CD8+CTL were detected in PB of 10 of 11 patients (range=0.02%-2.9%).Because P3 and NE expression is limited to myeloid cells, we sought to determine the mechanism of PR1 presentation in MM. We performed RT-PCR and western blotting on seven MM cell lines, including U266, ARK, ARP-1, OPM-2, LP-1, IM-9 and RPMI 8226. Neither NE nor P3 were detected in the MM cell lines studied at either the mRNA or proteins levels. We then investigated whether MM cells took up NE and P3. We cultured MM cells for 30 hours with 10 ug/mL of soluble NE and P3 or irradiated HLA-A2 negative PMN, the latter as a source for cell-associated NE and P3. Cells were then stained intracellularly for NE and P3 at different time points. Flow cytometry analysis showed that all the cell lines analyzed took up NE and P3. Uptake was seen as early as 1 hour after co-culture with soluble NE and P3 and was higher for soluble P3. Additionally, more uptake was seen in the cells that were co-cultured with irradiated PMN in comparison with soluble NE and P3.We then investigated whether PR1 expression in MM was through NE and P3 cross-presentation. We focused our studies on the HLA-A2+ U266 MM cell line. U266 cells were co-cultured with soluble NE, P3 or irradiated PMN, as described in the previous section, and then surface stained with 8F4. We detected PR1/HLA-A2 on the surface of U266 cells by flow cytometry as early as 6 and 24 hours after co-culture with soluble NE and P3, respectively. Cross-presentation was also seen in the cells that were co-cultured with irradiated PMN to a similar extent in comparison with the cells that were cultured with soluble NE and P3, however, cross-presentation occurred at an earlier time point (1 hour) in the cells that were cultured with PMNs. Cross-presentation was abrogated by lactacystin, a proteasome inhibitor, and brefeldin A, an ER/Golgi transport inhibitor, indicating that NE and P3 cross-presentation occurs through conventional cross-presentation mechanisms. Furthermore, because immune modulatory drugs and proteasome inhibitors are part of the standard of care therapy for MM, and since both have been shown to affect cross-presentation by DC, we tested whether lenalidomide and bortezomib altered PR1 cross-presentation by U266 and normal DC. U266 and DC were cultured with irradiated PMN in the presence of increasing doses of bortezomib or lenalidomide and then stained with PR1/HLA-A2. Flow-cytometry analysis showed a significant inhibition of PR1-cross-presentation by U266 after addition of bortezomib, but not lenalidomide. PR1 cross-presentation by DC was not affected by either drug.Finally, we tested whether PR1 cross-presentation caused MM cells to become susceptible to PR1-targeting immunotherapies. U266 cells were cultured with NE or P3 for 24 hours, the time point that showed the maximal cross-presentation, followed by addition of 8F4 antibody or co-culture with PR1-CTL. Using calcein AM cytotoxicity assays, we showed dose dependent killing of U266 by 8F4 and PR1-CTL following PR1 cross-presentation (Fig. 2).Together our data show that PR1 is cross-presented by primary MM cells and cell lines. These findings lay the foundation for the future applications of PR1-targeting immunotherapies in MM. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 646: Bi-specific T-cell engaging antibody activates T-cells to target the tumor associated antigen PR1

Abstract Acute (AML) and chronic (CML) myeloid leukemia together account for approximately 50% of newly diagnosed leukemias. Both AML and advanced CML have dismal survival outcomes but are susceptible to immunotherapy. We discovered PR1 (VLQELNVTV) as an effective human leukocyte antigen (HLA)-A2 restricted peptide and have targeted it in clinical trials using PR1 peptide vaccine. PR1 is derived from the serine proteases proteinase-3 and neutrophil elastase which are highly expressed in myeloid leukemia blasts. PR1 was shown to be immunogenic in myeloid malignancies, and we are currently developing several PR1 targeting therapies. We have completed the development and humanization of a T-cell receptor-like monoclonal antibody (h8F4) that targets PR1/HLA-A2 and eliminates primary human AML xenografts in NSG mice. To improve the efficacy of h8F4, we have developed a bi-specific T-cell engaging (BiTE) antibody that targets leukemia PR1/HLA-A2 and the T-cell receptor CD3. This would enhance the anti-leukemia immune response by bringing the T-cell in close proximity to the leukemia cell, and by further activating the T-cells to eliminate the target leukemia. Here we demonstrate successful construction and expression of the h8F4 BiTE in CHO cells. The antibody was purified from cell culture supernatant via Ni-NTA column chromatography, and identity was confirmed using western blot analysis. Next, flow cytometry was used to test the binding specificities of each targeting region of the BiTE antibody. The h8F4 BiTE antibody showed increased binding to T2 cells pulsed with PR1 peptide compared with T2 pulsed with irrelevant CMV peptide pp65 (MFI=627 and 72.4, respectively). In addition, the h8F4 BiTE demonstrated 6.8 fold higher binding to wild type Jurkat cells, which express CD3, in comparison with the CD3-lacking mutant Jurkat cell line J.RT3-T3.5. These results indicate that both regions of the h8F4 BiTE can efficiently recognize their intended targets. Next, we investigated whether h8F4 BiTE engagement with both PR1/HLA-A2 on T2 cells and CD3 on healthy donor PBMC could activate T-cells. We show with flow cytometry that upon 24h incubation in the presence of h8F4 BiTE and T2 cells pulsed with PR1 peptide, both CD4 and CD8 T-cells increase their surface expression of CD69 (21 fold in CD4; 10.3 fold in CD8), CD25 (35 fold in CD4; 3.5 fold in CD8) and CD71 (26.6 fold in CD4; 640 fold in CD8) compared to co-incubation in the absence of BiTE protein. In conclusion, this study shows that we have successfully developed a h8F4 BiTE which recognizes both the CD3 on T cells and PR1/HLA-A2 complexes on myeloid leukemia. Furthermore, the h8F4 BiTE can activate multiple T-cell subsets from healthy donor PBMC as early as 24h following co-incubation with PR1-pulsed T2 cells. This novel BiTE antibody could provide a safer and more potent treatment option for patients with aggressive myeloid leukemias that will improve upon currently available highly toxic standard therapies. Citation Format: Amanda Herrmann, Jin Im, Sijie Lu, Jeffrey Molldrem. Bi-specific T-cell engaging antibody activates T-cells to target the tumor associated antigen PR1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 646. doi:10.1158/1538-7445.AM2014-646

Multiple Myeloma Cross-Presents Neutrophil Elastase and Proteinase 3 and Becomes Susceptible to PR1-Targeting Immunotherapy

Abstract 4973PR1 (VLQELNVTV) is a human leukocyte antigen (HLA)-A2 restricted nonameric peptide that has been effectively targeted in myeloid leukemia using PR1-peptide vaccine and PR1-specific cytotoxic T cells (CTL). A phase I/II clinical trial has been initiated with PR1 peptide vaccine, which demonstrated complete remission and immunologic responses in patients with acute (AML) and chronic (CML) myeloid leukemia, as well as myelodysplastic syndrome. We recently developed an anti-PR1/HLA-A2 antibody (8F4) and have demonstrated its activity against myeloid leukemia blasts in vitro and in a human leukemia xenograft mouse model.PR1 is derived from the serine proteases proteinase-3 (P3) and neutrophil elastase (NE), which are normally found within neutrophil azurophil granules and are released into the inflammatory milieu. We have shown that through cross-presentation, a process whereby exogenous antigen is taken up, processed and expressed on cell surface in association with human leukocyte antigen class I molecules, PR1 can be expressed on the surface of cells that do not endogenously express NE and P3. Specifically, we showed that P3 and NE are taken up and cross-presented by antigen presenting cells (APC), including B cells, as well as breast cancer and melanoma cells. Furthermore, cross-presentation by these non-myeloid cells led to their killing by PR1-CTL and 8F4 antibody.Since multiple myeloma is derived from B cells, which are deficient in NE and P3 but have intrinsic APC functions including cross-presentation, we hypothesized that P3 and/or NE may be taken up and cross-presented by multiple myeloma cells, hence rendering them targets for PR1-CTL and 8F4 antibody. Using RT-PCR and western blotting, we first show that NE and P3 are absent in myeloma cell lines including ARK, ARP-1, OMP-2, LP-1, U-266, IM9, and RPMI 8226. Intracellular staining for P3 and NE after culturing myeloma cell lines with P3 (10 ug/mL) or NE (10 ug/mL) confirmed uptake of P3 and NE by multiple myeloma cell lines; uptake is time dependent with variable plateauing between the cell lines over a 30-hour time course. Using 8F4, the novel PR1-HLA-A2 monoclonal antibody, we demonstrate that P3 and NE are cross-presented by the HLA-A2 positive U-266 myeloma cell line. Peak P3 and NE cross-presentation occurs at 24 hours and 6 hours, respectively. Next, we studied whether PR1 cross-presentation causes cells to be susceptible to PR1-specific killing by PR1-CTL and 8F4 monoclonal antibody. We show that cross-presentation increases susceptibility of U-266 myeloma cell line to killing by PR1-CTL. Specifically, following incubation with P3 (10 ug/mL) and NE (10 ug/mL), we show 30% and 25% killing, respectively, of U-266 cells by PR1-CTL versus unpulsed U-266 cells (10% killing). Furthermore, we show dose-dependent killing of the U-266 myeloma cell line by 8F4 antibody in a complement dependent cytotoxicity assay, with maximum 30% and 20% killing of U-266 cells after incubation with P3 (10 ug/mL) or NE (10 ug/mL), respectively, versus no killing in the unpulsed group. Finally, using PR1/HLA-A2 dextramer staining, we show PR1-CTL in peripheral blood from patients with multiple myeloma following stem cell transplantation (Median, 0. 06%; range, 0. 03%-0. 1%). Our data therefore show that if the uptake of P3 or NE, present in the inflammatory milieu of multiple myeloma bone marrow and extramedullary tumors, leads to PR1 cross-presentation, then PR1-based immunotherapy may be useful to treat patients with multiple myeloma. These results also support a new paradigm linking inflammation and innate immunity to adaptive immune responses to cancer. Disclosures:No relevant conflicts of interest to declare.

Abstract 788: Uptake and cross-presentation of the leukemia associated antigens neutrophil elastase (NE) and proteinase-3 (P3) increases susceptibility of solid tumors to PR1-targeted immunotherapy

Abstract We have shown that the HLA-A2-restricted nonapeptide PR1 (VLQELNVTV) is a leukemia-associated peptide derived from P3 and NE. Furthermore, immunologic and clinical responses to PR1 peptide vaccination occur in patients with acute (AML), chronic (CML) myeloid leukemia and myelodysplastic syndrome. We have also shown that PR1 expression results from NE and P3 cross-presentation by B-cells and dendritic cells. Because NE and P3 are present in lung and breast cancer tumor tissue, we investigated whether uptake of NE and P3 might result in PR1 expression on solid tumors, which could induce susceptibility of non-hematopoietic tumors to PR1-targeted immunotherapy. We studied melanoma, breast, ovarian and lung cancer cell lines to determine whether NE and P3 are cross-presented. Using flow cytometry, we show that numerous malignant cell lines take up 5 to 10 °g/ml of soluble NE and P3 and was dose- and time-dependent, consistent with a receptor-mediated mechanism. In addition to soluble NE and P3, uptake of granulocyte-associated NE and P3 was also demonstrated; however, uptake of soluble NE and P3 were 3- and 2-fold higher, when compared to granulocyte-associated proteins. Within 4 hours of uptake, confocal microscopy showed that soluble P3 localized to endosomes and lysosomes, suggesting the possibility of lysosomal degradation of P3, a common pathway for MHC-I processing of cross-presented proteins. With the use of 8F4, a monoclonal antibody that is specific for a PR1/HLA-A2 conformational epitope, we show that PR1/HLA-A2 is expressed by 12-hours on P3- and NE-pulsed HLA-A2+ melanoma and breast cancer cell lines. Because 8F4 mediates complement-dependent cytotoxicity (CDC) of AML, we studied whether PR1 expression induced susceptibility of P3- and NE-pulsed tumor cells to killing by 8F4 and PR1-CTL. Twelve hours after pulsing the HLA-A2+ breast cancer cell lines MDA-MB-453 and MDA-MB-231 with P3 or NE, up to 40% and 60% of pulsed breast cancer cells were lysed by PR1-CTL and 8F4, respectively. We conclude that PR1 is cross-presented by solid tumors following receptor-mediated uptake of soluble NE and P3 at concentrations observed in tumors and inflammatory sites. Importantly, this cross-presentation increased susceptibility of embryological distinct tumors to PR1-specific immunotherapy. Together, these data suggest that cross-presentation of extracellular inflammatory proteins that are normally not expressed in the tumor may be a mechanism shared by many tumors, which could be exploited by immunotherapy strategies targeting these proteins. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 788. doi:10.1158/1538-7445.AM2011-788

Repeated PR1 and WT1 peptide vaccination in Montanide-adjuvant fails to induce sustained high-avidity, epitope-specific CD8+ T cells in myeloid malignancies

We previously showed that vaccination with one dose of PR1 and WT1 peptides induces transient anti-leukemia immunity. We hypothesized that maintenance of a sustained anti-leukemia response may require frequent boost injections. Eight patients with myeloid malignancies were enrolled in this phase II study, and 6 completed 6 injections of PR1 and WT1 peptides in Montanide-adjuvant with GM-CSF, every two weeks. Both high- and low-avidity PR1 or WT1-specific CD8(+) T cells were detected in all evaluable patients after the first vaccine dose. Repeated vaccination led to selective deletion of high avidity PR1- and WT1-specific CD8(+) T cells and was not associated with significant reduction in WT1-expression. Additional boosting failed to increase vaccine-induced CD8(+) T-cell frequencies further and in all patients the response was lost before the 6(th) dose. PR1- or WT1-specific CD8(+) T cells were not detected in bone marrow samples, excluding their preferential localization to this site. Following a booster injection three months after the 6(th) vaccine dose, no high-avidity PR1 or WT1-specific CD8(+) T cells could be detected, whereas low-avidity T cells were readily expanded. These data support the immunogenicity of PR1 and WT1 peptide vaccines. However, repeated delivery of peptides with Montanide-adjuvant and GM-CSF leads to rapid loss of high-avidity peptide-specific CD8(+) T cells. These results may offer an explanation for the lack of correlation between immune and clinical responses observed in a number of clinical trials of peptide vaccination. New approaches are needed to induce long-term high-avidity memory responses against leukemia antigens.

Surface Expression of PR1 Peptide on Thymic Dendritic Cells Does Not Eliminate PR1-Specific CD8+ Cells In Umbilical Cord Blood

AbstractAbstract 959Positive and negative selection of developing thymocytes is mediated primarily by cortical and medullary epithelial cells (CEC and MEC), respectively, in the thymus. Tolerance to peripheral tissue antigens (PTA) not normally expressed in the thymus can result when PTA expression is induced by AIRE in MEC or when corticomedullary dendritic cells (DC) present endogenous antigens, both mechanisms contributing to deletion of potentially auto-reactive T cells. However, in some instances, negative selection may be incomplete, leading to the release of autoreactive cells into the periphery. Indeed, T cells specific for PR1, the HLA-A2-restricted self-peptide derived from proteinase 3 (P3) and neutrophil elastase (NE), are present in peripheral blood of healthy adults, albeit at extremely low frequency of fewer than 0.0005% of CD8+ T cells. This suggests there is strong thymic central tolerance to PR1. However, PR1-CTL can increase from 0.1 to 2% of peripheral blood CD8 T cells in CML patients treated with interferon or stem cell transplant, and similar levels can be achieved after PR1 peptide vaccination, which suggests that under some circumstances this central tolerance can be reversed. Because of the increasing use of umbilical cord blood (UCB) as an alternative donor source and because incomplete graft-versus-leukemia (GVL) immunity after UCB transplant contributes to relapse, we sought to determine whether PR1-CTL can be expanded from UCB. We hypothesized that PR1-CTL frequency should be low in UCB due to central tolerance. Surprisingly, we found PR1-CTL at a frequency ranging from 0.007 to 0.345% (mean 0.117%) of CD8+ cells in 57 HLA-A2+ cord blood units, similar to what is observed in immunologically responsive vaccine patients and 100- to 1000-fold higher than in healthy adults. The PR1-CTL were predominantly CCR7+CD45+CD28+ and did not efficiently expand ex vivo following peptide stimulation and low dose IL-2, which was consistent with a naive T cell subset. Therefore, this data suggests that central tolerance to PR1 is incomplete. To study whether PR1 is expressed in human thymus, we used the PR1/HLA-A2-specific antibody 8F4 to study PR1 expression. Thymic CEC and MEC expressed no P3, NE, or PR1 by flow cytometry or immunofluorescence imaging of sectioned fetal thymus, which is consistent with previous reports showing absence of P3 or NE induction by AIRE in MEC. Interestingly, by flow cytometric analysis, we found that PR1 is expressed on the surface of thymic dendritic cells (DC) exclusively. This selective expression of PR1 was further confirmed via immunoflourescent staining of sectioned fetal thymus, and the PR1-expressing DC were localized to the corticomedullary junction and medulla. Thus it appears that PR1 expression by DC in the thymus is insufficient for complete central tolerance to PR1. Furthermore, because we have previously shown that PR1-overexpressing CML cells can induce apoptosis of high affinity PR1-CTL, it is possible that peripheral tolerance mechanisms are most critical for preventing autoreactivity to PR1 in humans. These observations suggest possible strategies to overcome tolerance to PR1 by modifying DC uptake and cross-presentation of soluble P3 and NE, when selective autoimmunity may be desirable for leukemia patients or detrimental for patients with vasculitis such as Wegener's granulomatosis. Disclosures:No relevant conflicts of interest to declare.

Soluble Inflammatory Mediators Proteinase-3 and Neutrophil Elastase Are Targeted by PR1-Specific Immunotherapy After Cellular Uptake and Cross Presentation of PR1 Peptide by Breast Cancer Cells

AbstractAbstract 2089The human leukocyte antigen (HLA)-A2 restricted nonapeptide PR1 (VLQELNVTV) was shown to be immunogenic in leukemia. A phase I/II clinical trial has been initiated with PR1 peptide vaccine and to date has demonstrated clinical efficacy, including complete remission and immunologic responses in patients with acute (AML) and chronic (CML) myeloid leukemia, as well as myelodysplastic syndrome. PR1 is derived from the serine proteases proteinase-3 (P3) and neutrophil elastase (NE), which are normally found within neutrophil azurophil granules and are released into the inflammatory milieu. We have shown that P3 and NE are taken up and cross presented by antigen presenting cells and that their cross presentation elicits PR1 immunity. Because P3 and NE are present in breast cancer biopsies, we hypothesized that P3/NE may be taken up by breast cancer cells and cross presented to PR1-CTL. We recently demonstrated that the breast cancer cell lines MDA-MB-231, MDA-MB-453, MCF-7 and HER18 do not endogenously express NE and that NE is taken up by these cell lines. In this report, using PCR, western blot and flow cytometry, we show that P3 also is NOT endogenously expressed by the breast cancer cell lines MDA-MB-231, MDA-MB-453, MCF-7 or HER18. Using confocal microscopy, we demonstrate that P3 is taken up by these breast cancer cell lines within 10 minutes of pulsing and localizes to LAMP-2 containing lysosomal vesicles by 4 hours, suggesting its processing for presentation by (HLA)-I (i.e. HLA-A2). Using 8F4, the novel PR1-HLA-A2 monoclonal antibody, we show that PR1 is cross presented from P3 by 3 of 4 HLA-A2+ breast cancer cell lines (MDA-MB-231, MDA-MB-453-A2+, MCF-7), and from NE by 1 of 4 breast cancer cell lines (MDA-MB-231). Next, we studied whether PR1 presentation made cells susceptible to PR1-specific killing by PR1-CTL and the 8F4 monoclonal antibody. We show that following 12-hour pulsing of the MDA-MB-231 cell line with NE or P3, PR1 CTLs killed up to 31% and 38% of the NE- or P3-pulsed breast cancer cells respectively, vs. <1% of ovalbumin (ova)-pulsed MDA-MB-231cells. Additionally, in a complement mediated cytotoxicity assay using 8F4 antibody, pulsing of MDA-MB-231 cells with P3 led to 60% cytotoxicity (vs. 40% in ova-pulsed cells). In conclusion, this study shows that 1) PR1 is cross presented by breast cancer cells following uptake of soluble P3 and NE and 2) PR1 expression makes breast cancer a target of PR1-specific immunotherapy. If uptake of P3 or NE, present in the inflammatory milieu of other solid tumors, also leads to PR1 cross presentation, then PR1-based immunotherapy may be useful to treat other non-hematopoietic tumors. These results support a new paradigm linking inflammation and innate immunity to adaptive immune responses to cancer. Disclosures:No relevant conflicts of interest to declare.

Anti-PR1/HLA-A2 Antibody (8F4, A TCR-Mimic) Mediates Specific Lysis of AML Stem Cells but Not Normal Hematopoietic Stem Cells.

Abstract 15PR1 (VLQELNVTV) is an HLA-A2-restricted peptide derived from endogenous myeloid leukemia-associated antigens (LAA) proteinase 3 and neutrophil elastase. PR1/HLA-A2 is targeted by PR1-specific cytotoxic T lymphocytes (PR1-CTL) that contribute to cytogenetic remission in patients with CML after allogeneic stem cell transplantation or interferon therapy, and PR1 peptide vaccination induces specific CD8 immunity in AML, MDS, and CML patients. Because clinical effects after PR1 vaccination are associated with a low amount of leukemia and because the effects on leukemia stem cells (LSCs) are unknown, we sought to develop a PR1/HLA-A2-specific MAB that could be more effective in high disease burden cases and that might also target LSC. We previously reported a mouse IgG2A antibody (8F4) with PR1/HLA-A2 specificity. We now used 8F4 to study PR1 expression by FACS and confocal mircroscopy on leukemia subsets and normal peripheral blood mononuclear cells (PBMC). Circulating blasts from peripheral blood (PB) or leukapheresis products (LP) from AML patients (5 HLA-A2+, 2 control HLA-A2-,) were studied (see Table 1). PR1 expression (MFI±SEM) was higher in AML (23.7±5.18) compared to control (4.0±2.42) and normal PBMC (13.6±0.23; p = 0.046). Confocal microscopy confirmed bright heterogeneous surface expression of PR1 on AML, but was nearly absent on healthy donor PB. Interestingly, PR1 expression was higher on LSC (CD34+CD38-lin-) (76±44) compared to healthy donor HSC (CD34+CD38-lin-) (13.4), which suggests that only differentiated myeloid cells might be more susceptible to 8F4 toxicity. To study whether 8F4 mediated specific lysis of PR1+ cells, we determined cell-mediated cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) of 8F4 compared to isotype control. 8F4 induced 51% specific lysis by CDC and 15% specific lysis by ADCC of PR1-pulsed T2 cells, which was dose-dependent on both 8F4 and PR1, with no lysis of target cells by isotype control. Moreover, 8F4 induced CDC-mediated lysis of blasts from 5/5 HLA-A2+ AML patients, but not blasts from an HLA-A2- AML patient (see Table 1), or mononuclear cells from HLA-A2+ healthy donors. To study the in vivo effects of 8F4 on AML, we used an established xenogeneic model of human AML in NOD/SCID mice. 1 × 106 blasts from 4 HLA-A2+ AML patients (including the HLA-A2+ patients susceptible to 8F4-mediated lysis in vitro) and 1 HLA-A2- patient were mixed for one hour under serum-free conditions with 8F4 or isotype control antibody and adoptively transferred to NOD/SCID mice. Engraftment of AML was compared on day 14 and AML growth was confirmed on day 28 by comparing FACS analysis and histopathology of BM from sacrificed animals. 8F4 treatment of AML cells, but not isotype treatment, abrogated engraftment of 4/4 HLA-A2+ AML patients, but not of 1 HLA-A2- AML patient on d14. By day 28, AML expanded by 150% in one surviving isotype-treated animal. Thus, 8F4-mediated specific lysis of AML and LSC by CDC and ADCC mechanisms in vitro were confirmed by in vivo results of AML engraftment. Taken together, these results show that 8F4, a novel TCR-mimic antibody that targets only PR1/HLA-A2+ cells, mediates lysis of AML blasts and LSC, but not total normal BM cells or HSC. Therefore, 8F4 may be useful as an immunotherapeutic agent in the treatment of HLA-A2+ myeloid leukemia, and as the first antibody therapy that may target and eliminate LSC.Table 1Patients' characteristics and 8F4 effectsUPNDiagnosis+Disease statusHLA-A2SexAgeSample*Blast8F4 FCMIn vitro CDC lysisIn vivo Engraftmentiso8F4AML1AML-M1 secondary MyelofibrosisUntreated+M79LP87%++++−AML2AML-M2Relapsed+M61PB66%++++Low*−AML3AML-M5Refractory+F73PB64%+++++−AML4AML-M1Untreated+F55LP92%+++N/DN/DAML5AML-M2Refractory relapsed+M33PB98%N/DN/D+−AML6AML-M5Untreated+M53LP96%++N/DN/DN/DAML7AML-M1Untreated+M24LP85%++−−AML8AML-M1Untreated−F41PB91%−−LowLow+French-American-British AML subtypeFCM flowcytometry staining intensity (+ indicates staining intensity close to those of normal PBMC, ++ and +++ indicate increased intensity)N/D The experiment was not performed due to low viability of sample (less than 50%) or sample was not availableLow* - indicates engraftment <0.5% of viable mCD45- cells, effect of treatment is inaccessible.*Peripheral blood (PB) or leukopheresis (LP) Disclosures:No relevant conflicts of interest to declare.

Breaking Immune Tolerance to Granule Proteases with Full-Length Antigen Vaccine in Humanized Transgenic Mice Reveals Alternative Antigen Processing and Immunodominance Heirarchy Applicable to Clinical Immunotherapy.

Abstract 2054Poster Board II-31The serine proteinases, human neutrophil elastase (HNE) and proteinase 3 (PR3) are degradative enzymes stored in cytoplasmic azurophilic granules of neutrophils. Both proteins are aberrantly expressed in human myeloid leukemias. Over-expression of PR3 is important in maintaining a leukemia phenotype, while HNE may suppress hematopoietic progenitors. PR3 and HNE contain a conserved nonameric HLA-A*02 restricted T-cell epitope called PR1. The presence of PR1-specific CTL has been correlated with molecular remission in CML. A PR1 nonameric peptide vaccine has been evaluated clinically, with promising results. A limitation of the PR1 peptide vaccine is its restriction to patients with the HLA-A*02 allele. A vaccine comprising a larger portion of the PR3 and/or HNE protein and capable of inducing broader T-cell responses restricted by a wider range of HLA alleles is a potential alternative. We have generated numerous recombinant vaccinia viruses (rVV) expressing forms of PR3 and HNE and have evaluated CTL responses in an HLA-A*02 transgenic mouse model (Tg-A2): (1) rVV-EGFP-PR3, contains a gene encoding EGFP fused to human PR3 lacking the N-terminal signal peptide, while retaining both pro-dipeptide and C-terminal propeptide sequences; (2) EGFP fused to C-terminal truncated PR3 (rVV-EGFP-PR3-T) deleted of PR2 CTL epitope, but maintaining PR1 and; (3) rVV-EGFP-HNE. Tg-A2 were immunized with rVV-EGFP-PR3, and 2 weeks later immune splenocytes were expanded by in vitrostimulation (IVS) with syngeneic naïve mouse splenocytes loaded with an overlapping PR3 peptide library. Intracellular cytokine (ICC) assays using a PR3 peptide library or PR1 9-mer epitope peptide demonstrated a robust PR3-specific CD8+ T-cell response, surprisingly without a detectable response to the PR1 peptide. However, PR2, a decamer, is immunodominant in mice following this immunization. This result was unexpected, since the PR2 epitope was identified by computer prediction as a potential HLA-A*02-restricted epitope within PR3 but, unlike the HLA-A*02-restricted 9-mer epitope PR1, has not been extensively studied as a T-cell epitope in humans. We tested whether the immunodominant PR2-specific response in rVV-EGFP-PR3 immunized mice is also found when a naturally-processed PR3 protein is used as the stimulating antigen, expressed from the cell line K562-A2-PR3, a derivative of K562-A2 endogenously expressing human PR3. Splenocytes from mice immunized with rVV-EGFP-PR3 were expanded by co-culture with irradiated K562-A2-PR3 cells, then tested in ICC assays using PR1 and PR2 CTL epitope peptides. We observed a robust response to PR2 but not PR1. While the PR1 epitope is absolutely conserved between human and murine versions of PR3 and HNE, the PR2 epitope is unique to human PR3, diverging from murine PR3 by 3 amino acids, and by 5 amino acid from human and murine HNE. We speculated that Tg-A2 mice may be tolerant to PR1 due to the presence of this epitope within murine PR3 and HNE, which may account for dominance of PR2-specific responses. To investigate the possibility of inducing PR1-specific CTL in Tg-A2, mice were immunized with rVV-EGFP-HNE, and we detected PR1-specific responses by IVS followed by ICC with the PR1 peptide. These results indicate that tolerance to PR1 can likely be broken by immunization with rVV expressing HNE protein that contains the PR1 epitope. These observations can be explained by either the PR2>PR1 competition hypothesis, or by different amino acid sequences flanking the PR1 epitope in PR3 and HNE proteins affecting processing of the protein to generate the PR1 peptide. To test this question, we used rVV-PR3-T to immunize Tg-A2 mice, then conducted IVS followed by ICC with the PR1 peptide to detect PR1-specific CTL. Results showed that immunization with rVV expressing truncated PR3 reliably induced PR1-specific CTL, unlike immunization with rVV expressing the full-length PR3 antigen. We concluded that PR2 epitope prevented the recognition of PR1. Therefore in Tg-A2 mice, vaccination with poxvirus vaccines expressing PR3 will induce an HLA-A2-restricted CTL response almost entirely focused on PR2, whereas immunization with rVV-HNE will induce PR1-specific CTL. If this were replicated in humans, the majority of PR1-specific CTL detected in CML patients could be derived from HNE rather than PR3 antigen presentation, and that PR2-specific CTL derived from PR3 should be equally frequent as PR1 and likely contribute to clinical GVL and tumor regression. Disclosures:No relevant conflicts of interest to declare.

Randomized Phase II Study of Proteinase 3-Derived PR1 Peptide Vaccine and GM-CSF with or without PEG-Interferon ALFA-2B to Eradicate Minimal Residual Disease in Chronic Myeloid Leukemia

BACKGROUND: Complete molecular eradication of chronic myeloid leukemia (CML) cells occurs only in a minority of patients treated with imatinib (IM). The curative potential of allogeneic transplantation and donor lymphocyte infusions highlights the responsiveness of CML to T-cell-mediated immunity. The proteinase 3-derived PR1 peptide (VLQELNVTV) is a leukemia-associated antigen presented on HLA-A2 to cytotoxic T lymphocytes (CTL) that preferentially kill leukemia over normal hematopoietic progenitors.METHODS: This phase II study was designed to evaluate the anti-leukemic effects of PR1 vaccine (2 mg) with GM-CSF (0.6 mg) and an incomplete Freund's adjuvant (Montanide ISA-51) given subcutaneously to patients with CML on IM in complete cytogenetic response (CCyR) with stable residual molecular disease. Patients were randomized to receive the vaccine alone or in combination with pegylated interferon α (PEG-IFN-α; 0.5 μg/kg) with each vaccination. PR1 vaccination was administered on weeks 0, 3, 6, and 18. Immune responses were assessed by PR1/HLA-A2 tetramer staining and molecular responses by quantitative PCR (qPCR) in peripheral blood before study entry, prior to each vaccination, and every 3 months thereafter.RESULTS: Five of the 20 planned HLA-A2+ patients (3 male) have been accrued. All but patient 4 (b3a2) expressed b2a2 transcripts prior to vaccination. Patients 1 (on IM 600 mg/d for 85 months), 2 (on IM 800 mg/d for 72 months) and 5 (on IM 400 mg/d for 76 months) were randomized to receive vaccine+PEG-IFN-α whereas Patients 3 and 4 (both on IM 400 mg/d, for 43 and 37 months, respectively) received PR1 vaccine alone. BCR-ABL1/ABL1 ratios prior to vaccination were 0.99, 0.79, 0.52, 0.10, and 0.44 respectively. The median follow-up from the first PR1 vaccination is 19 months (range, 4-20). All patients experienced transient mild elevations of BCR-ABL1 transcripts after the first vaccination followed by steady decline in transcript levels in Patient 1 (>1-log), and patients 2, 3, and 5 (<1-log) (Figure 1). Furthermore, patient 1 continues exhibiting an ongoing decrement of transcript levels. BCR-ABL1 transcripts have not decreased in Patient 4. Toxicity was limited to grade 1-2 injection site reactions except for Patient 2, in whom the fourth and last dose of PEG-IFN-α was not administered due to behavioral changes. Peripheral blood lymphocytes were analyzed in all 5 patients with PR1/HLA-A2 tetramer and detailed immunophenotyping (CD8, CCR7, CD45RA). Patients 1, 2 and 3 had immune responses (IR) defined as ≥2-fold increase in PR1/HLA-A2 tetramer+ cells. Patient 4 did not have an IR, but had pre-existing PR1-CTL that decreased transiently 3 weeks after the first vaccination. Patient 5 harbored pre-existing PR1-CTLs as well; follow-up analysis pending. No clear trends regarding memory T cell subsets have been identified. Immunity to control CMV-derived pp65 peptide was evident in all 5 patients but did not change after vaccination. Multiple clones of PR1-CTL could be derived from patients 1, 2 and 3 and studies are underway to analyze their TCR-ab affinity to PR1/HLA-A2.CONCLUSION: PR1 vaccination induces specific immunologic responses and improvement of molecular response in patients with CML in CCyR with stable or rising levels of BCR-ABL1 transcripts receiving imatinib therapy. [Display omitted]

PR1 vaccination in myeloid malignancies

The graft-versus-leukemia (GVL) effect following allogeneic stem cell transplantation is testament to the effectiveness of the immune system in recognizing and eliminating leukemia cells. The successful identification of a range of leukemia-associated antigens (LAAs) that drive the GVL response in recent years has stimulated research in the development of vaccines to treat hematological malignancies. Here, we review the current experience with the PR1 vaccine. PR1 is a nine amino acid, HLA-A*0201-restricted peptide, shared by two myeloid LAAs, proteinase (PR)3 and neutrophil elastase (NE). PR3 and NE are found in the primary (azurophil) granule proteins of normal granulocytes and are overexpressed in myeloid leukemia cells. PR1 induces powerful HLA-A*0201-restricted CD8+ T-cell responses that selectively kill myeloid leukemia cells in vitro. The detection of low frequencies of PR1-specific CD8+ T cells in patients with chronic myeloid leukemia and at higher frequencies in patients entering molecular remission after allogeneic stem cell transplantation supports the concept that there is natural immunity to PR1, which can be boosted further by vaccination to enhance immunity to leukemia. Preliminary reports indicate that PR1 peptide vaccination induces significant increases in PR1-specific CD8+ T cells, with rapid and durable remissions in some patients with myeloid leukemia. These promising early results point the way to optimizing the administration of peptide vaccines to improve the treatment of otherwise refractory myeloid leukemias.

Leukemia-associated antigen-specific T-cell responses following combined PR1 and WT1 peptide vaccination in patients with myeloid malignancies

We describe the safety and immunogenicity of a combined vaccine of 2 leukemia-associated antigenic peptides, PR1 and WT1. Eight patients with myeloid malignancies received one subcutaneous dose each of PR1 and WT1 vaccines in Montanide adjuvant, with granulocyte-macrophage colony-stimulating factor. Patients were reviewed weekly for 4 weeks to monitor toxicity and immunologic responses. Toxicity was limited to grades 1 to 2. Using peptide/HLA-A 0201 tetramers and intracellular interferon-gamma staining, CD8(+) T cells against PR1 or WT1 were detected in 8 of 8 patients after a single vaccination. To monitor the kinetics of vaccine-induced CD8(+) T-cell responses and disease regression after vaccination, absolute PR1 and WT1(+)CD8(+) T-cell numbers and WT1 expression were studied weekly after vaccination. Responses occurred as early as 1 week after vaccination. After vaccination, the emergence of PR1 or WT1(+)CD8(+) T cells was associated with a decrease in WT1 mRNA expression as a marker of minimal residual disease, suggesting a vaccine-driven antileukemia effect. Conversely, loss of response was associated with reappearance of WT1 transcripts (P < .01). This is the first demonstration that a combined PR1 and WT1 vaccine is immunogenic. These results support further studies of combination immunization strategies in leukemia patients.

PR1 peptide vaccine is promising for the treatment of myeloid leukaemia,

PR1 peptide vaccine is promising for the treatment of myeloid leukaemia,

PR1 Vaccine Elicited Immunological Response after Hematopoietic Stem Cell Transplantation Is Associated with Better Clinical Response and Event-Free Survival.

Background: PR1 peptide has been established as a human myeloid leukemia-associated antigen. We studied PR1 peptide vaccine in a phase I/II clinical trial in HLA-A2 + patients with AML, MDS and CML. To address whether prior HSCT or prior use immunosuppressive drugs would prevent PR1-induce cytotoxic T lymphocyte (PR1-CTL) immunity after vaccination with PR1 peptide vaccine, we studied the outcome in 20 patients with a prior HSCT, who were treated on the PR1 vaccine trial.Methods: Twenty patients (13 with AML or MDS, 7 with CML) were vaccinated at a median time of 9.5 months (range: 1–220) after HSCT. Sixteen patients had received a prior allogeneic HSCT (12 had allogeneic related, 3 had allogeneic unrelated, and 1 had syngeneic) and 4 patients had a prior autologous HSCT. At the time of vaccination, 5 patients were in CR, and 15 had measurable disease. Patients could not receive systemic immunosuppressive therapy for at least 4 weeks prior to vaccination, and had to be free of acute or chronic GVHD requiring systemic therapy. The vaccine was given subcutaneously every 3 weeks for a total of 3 injections at one of three dose levels: 0.25, 0.5 and 1.0 mg. GM-CSF at a dose of 75 mg was injected subcutaneously into the same site. Immune response to the vaccine (IRV) was defined as > 2-fold increase in PR1-CTL by PR1/HLA-A2 tetramer assay.Results: After a median follow up of 56.5 months (range: 27–89), toxicity was limited to grade I/II injection site reactions in 7 (35%) patients. IRV were observed in 11/20 (55%) patients. Nine of 11 (82%) IRV+ patients versus 1 of 9 (11%) IRV- patients had clinical responses (p = .005). Median event-free survival (EFS) was 23.8 months in IRV+ patients versus 1.9 months in IRV- patients (p=0.03). Median overall survival (OS) in IRV+ patients has not yet been reached vs. 40 months in IRV- patients (p = 0.08). Only 2 patients with pre-existing, limited chronic GVHD experienced a mild exacerbation within 3 months of vaccination, which was controlled with topical steroids. PR1-CTL were enriched in central memory phenotype (TCM) that persisted up to 4 years in clinical responders. Univariate and multivariate Cox proportional hazards analyses showed a low pre-vaccine bone marrow blast count (<10%) was associated with a lower risk of progression (p=0.001 and 0.001, respectively). Type of HSCT, interval between HSCT and PR1 vaccine, PR1 dose level and disease status at HSCT did not have a significant impact on EFS or OS.Conclusion: PR1 vaccine produced PR1-CTL in 11/20 (55%) patients after HSCT. IRV was associated with significantly better clinical response and longer EFS. [Display omitted]

PR1 Peptide Vaccine-Induced Immune Response Is Associated with Better Event-Free Survival in Patients with Myeloid Leukemia.

Background: PR1 is a nonomeric HLA-A2-restricted peptide derived from the myeloid leukemia-associated antigens, proteinase 3 and neutrophil elastase. PR1-specific cytotoxic T lymphocytes (PR1-CTL) selectively kill MDS, AML and CML and contribute to complete cytogenetic remission. We conducted a phase I/randomized phase II trial to evaluate the safety and efficacy of a PR1 peptide vaccine to elicit PR1-CTL in leukemia patients.Methods: Sixty-six HLA-A2+ patients with acute myeloid leukemia (42), chronic myeloid leukemia (13) or myelodysplastic syndrome (11) were treated with PR1 peptide vaccine. The first 54 patient received three vaccinations, and the last 12 patients received 6 vaccinations. PR1 was injected with incomplete Freud's adjuvant (Montanide ISA) subcutaneously, at 3 week intervals at one of three dose levels: 0.25, 0.5 or 1.0 mg per vaccination. GM-CSF at a dose of 75 mg was injected subcutaneously into the same site. Immune response to the vaccine was defined as a ≥ 2-fold increase in peripheral blood PR1-CTL. Fifty-three patients had measurable disease and 13 were in complete remission at enrollment.Results: The vaccine was well tolerated, with toxicity limited to grade I and II injection site reactions. PR1-specific immune response was observed in 25/53 (47%) patients with measurable disease. Clinical responses were observed in 9/25 (36%) immune responders versus 3/28 (10%) immune non-responders (p=0.03). The PR1 vaccine-induced immune response was associated with a longer event-free survival, 8.7 months vs. 2.4 months (p = 0.03), and a trend towards longer overall survival. Among the 13 patients treated in complete remission, 4 have remained in remission for a median of 30.5 months (range 11–59 months). This includes continued complete remissions in 3/10 who were IRV+, and 1/3 who were IRV-. PR1-specific immune response lasted for up to 4 years in some patients. In a multivariate model, low bone marrow blast count (<10%) emerged as a significant predictor of an immune response and a longer event-free survival (p=0.001).Conclusion: PR1 peptide vaccine-induced immune response is associated with a clinical response and better event-free survival in patients with myeloid leukemia, who had measurable disease at vaccination. Patients with ≤10% bone marrow blasts are likely to obtain the maximum benefit. [Display omitted]

Leukemia-Associated Antigen Specific T-Cell Responses Following Combined PR1 and WT1 Peptide Vaccination in Patients with Myeloid Malignancies.

The graft-versus-leukemia (GVL) effect following allogeneic stem cell transplantation (SCT) is evidence that T lymphocytes can eradicate leukemia. The successful identification of a range of leukemia-associated antigens such as proteinase 3 (PR3) and Wilms tumour-1 (WT1) has stimulated efforts to induce leukemia-specific T-cell responses to these antigens using peptide vaccines. Here we describe the safety and immunogenicity of a combined vaccine of two leukemia-associated antigenic peptides, PR1 and WT1. Eight HLA-A*0201 positive patients with myeloid malignancies (2 myelodysplasia, 5 acute myeloid leukemia and 1 chronic myeloid leukemia) received one subcutaneous dose each of PR1 and WT1 vaccines in Montanide adjuvant, with granulocyte-macrophage colony-stimulating factor (GM-CSF). All patients completed 4 weeks follow-up to monitor toxicity and immunological responses. Toxicity was limited to grade 1–2. All remain alive at a median of 252 days (range 105–523). We analyzed the immunological response to vaccination using PR1/HLA-A*0201 and WT1/HLA-A*0201 tetrameric complexes and flow cytometry for intracellular interferon-gamma (IFN-γ) in samples obtained pre- and weekly post-vaccination. A significant CD8+ T-cell response to the vaccine was defined as the emergence of PR1 or WT1-specific CD8+ T-cells when the pre-study analysis was negative or a twofold increase in frequencies when responses were present pre-vaccination. Following vaccination, a significant CD8+ T-cell response to PR1 was seen in 7/8 patients (median 0.34%, range 0.04–0.48%), to WT1 in 5/8 patients (median 0.29%, range 0–0.42%) and to one or both antigens in 8/8 patients. Vaccine-induced CD8+ T-cells were seen as early as 1 week post-vaccination, produced IFN-γ and were preferentially expanded in the effector compartment (CD45RO+/-CD27−). Post-vaccination, there was a strong correlation between the emergence of PR1 or WT1+CD8+ T-cells and a reduction in WT1 mRNA expression, a marker of minimal residual disease, suggesting a vaccine-driven anti-leukemia effect. Loss of response was associated with reappearance of WT1 transcripts (P<0.01). Two patients with detectable CD8+ T-cell responses to PR1 who failed to have a reduction in MRD relapsed 3–6 months following completion of vaccination. This is the first demonstration that a combined PR1 and WT1 vaccine is immunogenic. Based on these results we have initiated a phase 2 study of repeated vaccination with PR1 and WT1 peptides in patients with myeloid malignancies.

PR1 peptide vaccination for patients with myeloid leukemias

7017 Background: PR1 is a nanomeric HLA-A2-restricted peptide derived from the myeloid leukemia-associated antigens proteinase 3 and neutrophil elastase. Methods: Sixty-six HLA-A2+ patients with acute myeloid leukemia (42), chronic myeloid leukemia (13) or myelodysplastic syndrome (11) were treated with PR1 peptide vaccine. The first 54 patient received three vaccinations, and the last 12 patients were given six vaccinations. The vaccine was injected subcutaneously, at 3 week intervals at one of three dose levels: 0.25, 0.5 or 1.0 mg per vaccination. Immune response to the vaccine was defined as a ≥ 2-fold increase in PR1-specific cytotoxic T lymphocytes. Results: Fifty-three patients had measurable disease (MD) and 13 were in complete remission. The vaccine was well tolerated, with toxicity limited to grade I and II injection site reactions. Forty-four of the 53 patients with MD were evaluable for both immune and clinical responses. PR1-specific immune response was observed in 35/44 (57%) of the evaluable patients. Clinical responses were observed in 10 of the 25 immune responders versus 2 of the 19 immune non-responders (p=0.04). PR1 vaccine-induced immune response was associated with a longer event-free survival, 8.7 months vs. 4.1 months in immune non-responders (p = 0.17). Older age and high blast count were associated with short event-free survival (p=0.01 and &lt;0.001). Conclusion: PR1 peptide vaccine-induced immune response is associated with a superior overall clinical response and a trend towards longer event-free survival in patients with persistent myeloid leukemia. [Table: see text]