IL-15/IL-15Ra/CD80-expressing AML cell vaccines eradicate minimal residual disease in leukemic mice

Yimin Shi, Lillia Dincheva-Vogel, Charles E. Ayemoba, Jeffrey P. Fung, Cristina Bergamaschi, George N. Pavlakis, Farzin Farzaneh, and Karin M. L. Gaensler Department of Medicine, University of California San Francisco School of Medicine, San Francisco, CA; Human Retrovirus Section, Vaccine Branch, National Cancer Institute, National Institutes of Health, Frederick, MD; and Department of Haematological Medicine, Rayne Institute, King’s College London, London, United Kingdom


Introduction
Older individuals with acute myelogenous leukemia (AML) have poor outcomes because of more frequent high-risk features and comorbidities. 1 The improved survival achieved with allogeneic hematopoietic stem cell transplantation because of curative graft-versus-leukemia responses conferred by donor T cells provides evidence for the potential efficacy of immunotherapies (reviewed by Dombret and Gardin 1 ). Because many older individuals are ineligible for transplants, there is an unmet need for novel therapeutic approaches.
Although immunotherapies for AML have been explored, to date none have reliably reduced relapse rates. [2][3][4][5] In this context, autologous cell vaccines may have advantages for stimulating antileukemic immunity because responses are directed to multiple leukemiaassociated antigens, some of which are patient specific. Immune responses generated against an autologous AML vaccine would obviate the problem of lack of a priori knowledge of the dominant antigens present in each patient's leukemia. Previous trials with autologous cell vaccines have induced antileukemic immunity, but responses were variable. 6 This is in part because AML blasts are ineffective in T-cell stimulation, because of their downregulation or absent expression of specific costimulators such as CD80, 7,8 and because of their immune evasive effects including upregulation of checkpoint molecules and stimulation of inhibitory immune effectors (reviewed by Teague and Kline 9 ). In older patients, immunotherapeutic efficacy may be further limited by a decline in T-cell responsiveness. [10][11][12][13] Because engineering patient AML cells to express the missing costimulatory protein CD80 has shown promise, 7,8,14,15 we engineered AML vaccines to express a novel combination of CD80 and the heterodimeric complex interleukin-15 (IL-15) and IL-15 receptor a (IL-15Ra) to improve the induction of antileukemic cytolytic responses. The IL-15/IL-15Ra heterodimer is the naturally occurring form of the cytokine and is a member of the gc cytokine family that engages a heterodimeric receptor comprising the IL-2Rb/ IL-15Rb subunit (shared with the IL-2 receptor) and gc. 16 Efficient IL-15 production requires coexpression of IL-15 and IL-15Ra in the same cell, [17][18][19][20][21][22] which substantially increases IL-15 half-life and activity through the IL-2/IL-15R gc receptors. [23][24][25] IL-15 has major advantages as an immune-stimulatory cytokine because in contrast to the effects of IL-2, previously used in immunotherapy, IL-15 reverses CD8 1 T-cell unresponsiveness to tumor-associated antigens, renders T effector cells resistant to suppressive regulatory T cells (Treg's), and participates in antiapoptotic signaling to effector T cells (reviewed by Waldmann 23 ). IL-15 also stimulates more effective induction of antigen-specific cytotoxic lymphocytes and more durable immunity through actions on memory T cells, and it has important roles in natural killer (NK) and NK T-cell activation, proliferation, and survival. 23 Although systemic IL-15 administration has less toxicity than does high-dose IL-2 infusion, it does cause neutropenia, fever, and other side effects. 26 Thus, local expression of IL-15 by genetically modified AML vaccines has the potential advantages of IL-15 immune stimulation, with reduced risk of systemic toxicities. Finally, local expression of membrane-bound and secreted heterodimeric IL-15/IL-15Ra together with costimulation by CD80 may mimic the interactions of professional antigen-presenting cells with lymphocytes, required for triggering effective cell-mediated immune responses.
In these studies, the leukemia-specific cytolytic activity stimulated by coexpression of IL-15/IL-15Ra and CD80 in lentivirally transduced irradiated AML cell vaccines was evaluated in 32Dp210 myeloid leukemia-bearing mice. [27][28][29][30] We also developed a novel model of postremission minimal residual disease (MRD) in leukemic mice that in part recapitulates the clinical setting in which AML cell vaccines would be administered. Our studies show that in mice with prior high leukemic burdens, postremission therapy with 32Dp210-IL-15/IL-15Ra/CD80 vaccines can produce long-term survival and eradication of leukemia.

Methods
Cell lines 32Dp210 leukemia cells transformed by the p210 bcr abl transcript 31 were provided by R. Arlinghaus (University of Texas MD Anderson Cancer Center). For longitudinal in vivo bioluminescence studies, 32Dp210 leukemia cells were transfected with a luciferase vector (Cat. VSL-0074; Capital Bioscience, Gaithersburg, MD). Other 32Dp210 cells were transduced with enhanced green fluorescent protein for flow cytometric analysis, or with the herpes simplex thymidine kinase (HSV-TK) suicide gene, to confer sensitivity to ganciclovir (GCV) and tumor-specific lethality after drug administration. For each experiment, 32Dp210 aliquots were thawed; expanded in RPMI, 10% fetal bovine serum (Gibco, Life Technology), and 1% glutamine; and then washed in phosphate-buffered saline and injected IV via lateral tail vein. 32Dp210-Luc 1 , 32Dp210-GFP 1 , and 32Dp210-Luc-HSV-TK 1 cells all showed comparable growth in vivo.

Lentivirus construction and transduction
Lentiviral vectors were constructed by homologous recombination in the pMLV-mIL-2-Furin-CD80 backbone, containing the murine leukemia virus promoter, murine IL-2, and murine CD80. 32 The murine IL-2-Furin-CD80 cassette in this vector was replaced either with mouse CD80 alone or with codon-optimized mouse IL-15 with the leader sequence from granulocyte-macrophage colony-stimulating factor to increase expression, and a RNA/codon-optimized, membrane associated, complete IL-15Ra. 17,33 The IL-15 cassette was linked by a P2A sequence, to generate a self-cleaving peptide 34 producing mouse IL-15-P2A-IL-15Ra. A tri-cistronic lentiviral vector was constructed by linking the murine IL-15-P2A-IL-15Ra to murine CD80 with an F2A sequence (coding for a second self-cleaving peptide) 35 39 to the IL-15/IL-15Ra cassette generating IL-15-P2A-IL-15Ra-F2A-CD80 (supplemental Figure 1). Lentivirus production and transduction was performed as described. 36 Generation of 32Dp210-derived vaccines 32Dp210 cells were transduced with 8 mg/mL polybrene in 6-well plates. To achieve comparable levels of expression of mouse IL-15, IL-15Ra, and CD80 in 32Dp210 vaccines, transduced cells were first purified based on high-level and comparable CD80 expression. Then, purified 32Dp210-IL-15/IL-15Ra 1 and 32Dp210-IL-15/ IL-15Ra/CD80 cells with comparable expression of IL-15Ra were isolated and expanded.

Mice
Female C3H mice (8-10 weeks; Charles River, San Diego, CA) were housed under pathogen-free conditions at University of California, San Francisco. Experiments were conducted with supervision of University of California, San Francisco Institutional Animal Care and Use Committee according to approved protocol (AN108913-02A).

Vaccine protocols
Lentivirally transduced or untransduced 32Dp210-luc parental cells were irradiated with 40 Gy prior to subcutaneous administration. At lower doses of irradiation, occasional viable tumors were observed. Tumor progression and responses to vaccinations were analyzed in a Xenogen in vivo imaging system (IVIS), after intraperitoneal injection with 150 mg/kg luciferin and anesthesia with 3% isofluorane. Images were analyzed using Living Image software (Xenogen, Alameda, CA).

MRD model
Ten days after IV injection of 1 3 10 5 32Dp210-luc, all mice had detectable leukemia by IVIS and began GCV treatment (50 mg/kg/day 3 14 days) (APP Pharmaceuticals LLC, Schaumburg, IL). To confirm that GCV had no effects on peripheral blood counts, counts were assessed after administration of 100 mg/kg per day 3 14 days (supplemental Table 1).

In vivo cell depletion studies
In vivo cell depletion by intraperitoneal injection of antibodies was carried out on day 0 after inoculation of 1 3 10 4 32Dp210 cells. Antibodies (100 mL diluted rabbit anti-mouse anti-asialo-GM1, or 500 mg rat-anti-mouse CD4, or 500 mg rat anti-mouse CD8 [BioXCell, West Lebanon, NH]), produced .90% depletion of target cells 24 hours after injection, verified by staining for CD3, CD4, CD8, and NKp46 in blood and spleen.

Measurement of IL-15
Secreted mouse IL-15 levels were measured by enzyme-linked immunosorbent assay (ELISA) using recombinant mouse IL-15 as a standard and polyclonal rabbit anti-mouse IL-15 antibody (H114; Santa Cruz Biotechnology Inc., Santa Cruz, CA).

Statistical analyses
Experiments were repeated at least 2 times unless indicated otherwise. Statistical analyses were performed using Prism 6 (GraphPad Software Inc., La Jolla, CA). Results are reported as the mean 6 standard error of the mean (SEM) in independent experiments. The significance of differences was determined using Student paired t test.

Murine 32Dp210 leukemia recapitulates features of clinical AML
Dose-finding studies with 32Dp210 leukemia demonstrated that the minimal cell dose that produced reliable leukemic engraftment, and adequate survival to allow induction of immunity (;30-40 days [38][39][40], was 10 4 cells administered IV ( Figure 1A, upper panel). Because leukemia-associated stimulation of inhibitory immune responses poses challenges in the clinical setting, 9 we assessed whether 32Dp210 leukemia has immunosuppressive effects comparable to those observed in patients. Fourteen days after leukemia inoculation, T cells increased the expression of the negative regulatory receptor PD-1, and 32Dp210 cells upregulated expression of the PD-1 ligand, PD-L1, on engrafted leukemia cells ( Figure 1A lower panels). The numbers of CD3 1 CD4 1 T cells, CD3 1 CD8 1 T cells, and the frequency of CD3 1 CD8 1 CD44 hi T cells were all decreased in leukemic mice, whereas the frequency of inhibitory CD4 1 FoxP3 1 Treg's and CD11b 1 Ly6G 1 myeloid derived suppressor cells increased, demonstrating 32Dp210mediated immunosuppression (supplemental Figures 2 and 3). 32Dp210 leukemia also reproduced early homing of blasts to the bone marrow compartment seen clinically (0.2% GFP 1 cells at day 3) ( Figure 1B).

Lentiviral transduction produces high-level IL-15, IL-15Ra, and CD80 expression
Because coexpression of IL-15 and IL-15Ra markedly increases the secretion, half-life, and bioactivity of IL-15, codon-optimized murine IL-15 and murine IL-15Ra constructs were both incorporated in vectors, with or without CD80 (supplemental Figure 1). 17,33 As expected, IL-15 was barely detected in transduced 293T cell supernatants in the absence of IL-15Ra expression; however, coexpression of IL-15 and IL-15Ra resulted in high levels of IL-15 secretion (

32Dp210-derived vaccines stimulate ex vivo T-cell proliferation in splenocytes from naïve mice
Primary stimulation of splenocytes from naïve mice with irradiated 32Dp210-IL-15/IL-15Ra or 32Dp210-IL-15/IL-15Ra/CD80 vaccines produced a fivefold increase in CD3 1 CD8 1 T-cell proliferation compared with coculture with untransduced 32Dp210 cells ( Figure 3A). Lower levels of proliferation were observed after stimulation with 32Dp210-CD80. In the case of CD3 1 CD4 1 T cells, proliferation levels stimulated by coculture with either parent 32Dp210 cell vaccine, 32Dp210-CD80, or 32Dp210-IL-15/ IL-15Ra were similar; however, the greatest responses were seen after 32Dp210-IL-15/IL-15Ra/CD80 vaccination ( Figure 3B). To assess whether the combination of IL-15 and CD80 expression is synergistic in stimulating T cells in the absence of antigen presenting cells, and other coexisting populations in spleen, CD3 1 CD4 1 and CD3 1 CD8 1 T cells were purified and then stimulated with irradiated 32Dp210-derived cells. Consistent with our hypothesis, coexpression of IL-15/IL-15Ra and CD80 showed striking synergy  Figure 3C) and CD3 1 CD4 1 T cells ( Figure 3D).
In ex vivo assays, leukemia-specific cytolytic activity stimulated by 32Dp210-derived vaccines against 32Dp210 targets was greatest  in splenic T cells from 32Dp210-IL-15/IL-15Ra/CD80 vaccinated mice at all effector to 32Dp210 target cell ratios ( Figure 4A). Vaccination with 32Dp210-IL-15/IL-15Ra also stimulated significant responses. ELISpot assays demonstrated increased frequencies of IFN-g-expressing cells after secondary stimulation of splenocytes, consistent with activation and expansion of cytotoxic T cells ( Figure 4B). In these assays, it was necessary to restrict the number of peripheral blood mononuclear cell to 3 3 10 5 , as higher numbers of peripheral blood mononuclear cells produced too many spots for accurate quantification, because of the high magnitude of immune stimulation. Robust T-cell stimulation by vaccines was further supported by demonstration of increased intracellular IFN-g expression in CD3 1 CD8 1 and CD3 1 CD4 1 T cells after 20 hours coculture with unmodified 32Dp210 cells. Again, highest levels of immune stimulation were induced by 32Dp210-IL-15/IL-15Ra/ CD80 vaccination ( Figure 4C-D).
To determine whether expression of the human BCR-ABL oncogene expressed by 32Dp210 is the dominant antigen driving antileukemic responses, intracellular IFN-g expression in T cells from vaccinated animals was assayed after stimulation with either (1) 32Dp210 or (2) naïve C3H splenocytes loaded with a BCR-ABL fusion peptide GFKQSSKAL shown to be targeted in other studies, 41 or an irrelevant peptide. Intracellular IFN-g expression in splenocytes from vaccinated mice after in vitro stimulation with BCR-ABL peptide-loaded cells was comparable to background levels seen after coculture with media alone or control peptide ( Figure 4C-D). The absence of a dominant response to the BCR-ABL peptide was confirmed by ELISpot assays where BCR-ABL-loaded cells provided no significant stimulation ( Figure 4E). The lack of a major response to BCR-ABL expressed by 32Dp210 supports our hypothesis that the potent antileukemic responses observed are induced by multiple leukemia-associated epitopes on 32Dp210 vaccines. Clinically, the magnitude of the protective response, elicited by a single peptide, is likely to be substantially lower than the aggregate response to a larger panel of patient specific and leukemia associated antigens, which collectively provide greater protective immunity. To further examine the role of BCR-ABL expression in vaccine responses, the ex vivo cytolytic activity of splenocytes against either unmodified 32Dp210, BCR-ABL-loaded, or control peptide-loaded C3H targets was compared. As previously, splenocytes from 32Dp210-IL-15/IL-15Ra/CD80 treated animals showed highest levels of cytolytic activity to 32Dp210 targets, whereas stimulation with either BCR-ABL or control HYLSTQSALSK peptide did not exceed background levels ( Figure 4F).

32Dp210-IL-15/IL-15Ra/CD80 vaccination can eradicate disease in leukemic mice
The therapeutic efficacy of 32Dp210-derived vaccines was then tested in mice with established leukemia. Mice received 3 weekly vaccinations, 3 days after injection of 1 3 10 4 32Dp210 cells, when leukemia has engrafted ( Figure 1B). All unvaccinated leukemic controls died by day 70 ( Figure 5A). Vaccination with parent 32Dp210 cells slightly prolonged survival, consistent with the previously described low-level immunogenicity of irradiated tumor cells. 42,43 Treatment with any of the lenti-engineered vaccines produced greater survival after tumor challenge than did treatment with unmodified 32Dp210 cells ( Figure 5A), but highest survival rates (80%) were observed after vaccination with 32Dp210-IL-15/ IL-15Ra/CD80. These mice also rejected leukemia on rechallenge. Thus, coexpression of CD80, and IL-15/IL-15Ra, induces highly effective antileukemic activity, even in the context of leukemiaassociated immunosuppression.
In vivo depletion of CD8 1 populations abrogates 32Dp210-IL-15/IL-15Ra/CD80 vaccine efficacy To define immune effectors mediating leukemia-specific responses, in vivo antibody-mediated depletion studies were performed with either anti-CD4 or anti-CD8 or anti-asialo-GM1 (AS-GM1) antibodies. The AS-GM1 antibody, which binds to NK cells and also to some activated T cells 44,45 and to basophils, 46 was used instead of anti-NK1.1 antibody, as the NK1.1 antigen (CD161b/CD161c) is not expressed in C3H mice. 47,48 While depletion with anti-CD4 1 or C3H splenocytes were loaded with either BCR-ABL peptide or control peptide and cocultured with splenocytes from vaccinated animals as described in panel A, depicted on the x-axis. The mean number of spots per well per containing 3 3 10 5 cells in triplicate samples is depicted on the y-axis (6 SEM). (F) Splenocytes from non-tumor-bearing mice treated with the 32Dp210-derived vaccines show high levels of lytic activity to 32Dp210 targets, but not to human BCR-ABL peptide loaded syngeneic C3H cells.
Splenocytes from vaccinated non-tumor-bearing C3H mice, as described in panel A were stimulated ex vivo for 5 days with irradiated 32Dp210 cells and used as effectors.
Unmodified 32Dp210 cells, BCR-ABL, or control peptide-loaded splenocytes from naïve C3H mice were used as targets. Ex vivo lytic activity was assayed by intracellular staining of active caspase-3 after coculture of effectors and targets at an effector-to-target ratio 5 10:1 for 24 hours. X-axis depicts cells from different experimental vaccine groups as in panel A. The mean percentage of apoptotic cells, defined by detection of activated caspase-3 by antibody staining, is depicted on the y-axis (6 SEM). (G) Treatment with transduced 32Dp210-derived vaccines confers greater survival after leukemia challenge than does administration of untransduced irradiated 32Dp210 cell vaccines. Mice were treated weekly 3 times with 32Dp210-derived vaccines as described previously. Thereafter, they were inoculated IV with 1 3 10 4 32Dp210-luc cells and longitudinally monitored by in vivo bioluminescence imaging for tumor progression and survival. Percent survival is depicted on the y-axis, and duration of survival on the x-axis.
Animals surviving the initial leukemia challenge were rechallenged with a second IV inoculation of 32Dp210 leukemia (indicated by downward arrow), 150 days after the initial tumor challenge.

The 32Dp210-IL-15/IL-15Ra/CD80 vaccine has efficacy in eradicating postremission MRD
The clinical application of an autologous AML vaccine strategy would be as postremission immunotherapy. To recapitulate this clinical setting, we developed a murine model of MRD for testing our vaccine. Several chemotherapeutic regimens previously shown to induce transient responses in 32Dp210 leukemia were tested. 49 However, advanced 32Dp210 leukemia proved to be highly chemorefractory as dose-finding studies with cytosine arabinoside (AraC), and/or high dose dasatinib to target the BCR-ABL tyrosine kinase, were either ineffective or resulted in toxicity (data not shown). We therefore adopted an alternative strategy for achieving postremission MRD in mice with high leukemia burdens by engineering 32Dp210 cells to express the HSV-TK suicide gene. 36 Treatment with GCV could induce remission without the confounding immune and cytopenic effects of conventional chemotherapy. 50 Because effects of GCV are primarily restricted to suppression of marrow proliferation, but not inhibition of immune function, treatment should not alter leukemia-associated immune deviation, except as associated with reduction of tumor burden. 51 Blood samples confirmed that there was no significant effect on counts after 2 weeks of GCV administration (supplemental Table 1).
To validate this model, mice were treated daily with GCV once a significant leukemia burden was evident by IVIS. Although expression of HSV-TK has sometimes been reported to be immunogenic, 52 HSV-TK expression did not affect leukemic engraftment/progression ( Figure 6). Remission was arbitrarily defined as a level of bioluminescence comparable to background bone marrow), but persistence of MRD (,0.5% GFP 1 cells in BM, n 5 7/7) ( Figure 6C).
The efficacy of postremission vaccination with 32Dp210-IL-15/IL-15Ra/CD80 was then tested in mice with GCV-induced MRD. A 10-fold larger dose of 32Dp210-luc-HSV-TK 1 cells (1 3 10 5 ) was administered to ensure that all mice had evidence of leukemia on day 10 by IVIS, when GCV treatment started ( Figure 7A, upper panel). By day 5 of GCV administration (day 15), most leukemic mice responded ( Figure 7A, lower panel). All animals that received no further therapy after achieving remission with 14-day GCV, relapsed; however, postremission treatment with 32Dp210-IL-15/ IL-15Ra/CD80 was curative, eliminating MRD in 50% of mice, despite the persistence of leukemia-mediated immune suppression ( Figure 7B).

Discussion
Prior AML vaccines have not reliably increased relapse-free survival, because of insufficient induction of antileukemic cytolytic activity; however, the promise of IL-15 in cancer immunotherapy provided a compelling rationale for combining IL-15 and IL-15Ra, with CD80-mediated costimulation to improve vaccine efficacy.
In ex vivo assays, treatment with the 32Dp210-IL-15/IL-15Ra/ CD80 vaccine stimulated the highest levels of cytolytic activity when compared with effects of vaccination with 32Dp210-IL-15/ IL-15Ra, 32Dp210-CD80, or unmodified 32Dp210 cells. The effects of CD80 costimulation and IL-15/IL-15Ra were additive in this context and correlated with induction of cytotoxic lymphocytes with increased IFN-g expression. Major responses most likely reflect responses to multiple leukemia-associated antigens and were not targeted to the human BCR-ABL antigen as demonstrated by the lack of responses after secondary stimulation with the BCR-ABL peptide GFKQSSKAL. 41 The 32Dp210 leukemia recapitulates many features of human AML including early homing to bone marrow and negative effects on host immunity. Although treatment with all of the engineered vaccines increased survival in leukemic mice, 32Dp210-IL-15/   IL-15Ra/CD80 vaccination produced the highest survival rates, consistent with the effects of combined IL-15/IL-15Ra and CD80 expression in stimulating cytolytic activity, also shown ex vivo. Tumor-specific cytotoxic treatment with GCV in HSV-TKexpressing 32Dp210 leukemia induced remission in the majority of mice, as defined pathologically, and by IVIS. However, similar to the clinical setting, MRD persisted after remission induction as evidenced by (1) the presence of MRD (,5% leukemia in bone marrow) in all mice meeting remission criteria by IVIS and (2) the universal relapse in mice that achieved remission with GCV treatment but no further treatment. Postremission 32Dp210-IL-15/IL-15Ra/CD80 vaccination was curative in 50% of GCVtreated subjects, while depletion of CD8 1 cells, but not CD4 1 cells or asialo-GM1 1 NK cells, abrogated vaccine efficacy. Although it is possible that differential levels of in vivo depletion could account for the modest effects of anti-CD4 or anti-AS-GM1 antibodies, dependence on CD8 1 T cells for immunotherapeutic efficacy is consistent with recent studies with the IL-15/IL-15Ra/Fc superagonist ALT-803 in a myeloma model, 53 and with effects of IL-15 in a transgenic adenocarcinoma of the mouse prostate (TRAMP-C2) cancer model. 54 Other studies have also demonstrated the superiority of the IL-15/IL-15Ra heterodimer in stimulating cell-mediated antitumor responses. 53,55-57 Thus, a 10-fold decrease in breast cancer metastases was observed in tumor-bearing mice treated with IL-15/IL-15Ra, compared with outcomes in untreated mice. 58 Addition of IL-15 and IL-15Ra during ex vivo T-cell expansion for adoptive immunotherapy stimulated T cells responding to lower peptide concentrations that lysed targets at lower effector-to-target ratios, than did cells without IL-15 treatment. 59 Although IL-15 has been shown to play an important role in NK cell proliferation and homeostasis, 23 the role of NK cells in some studies of IL-15-mediated antitumor immunity has been less clear. For example, treatment with IL-15-and IL-12-expressing tumor cell vaccines resulted in similar effects on tumor rejection in normal vs NK-depleted mice. 60,61 In contrast, the efficacy of immunotherapy in murine lymphoma 62 and neuroblastoma 63 with IL-15 and IL-12 required the presence of both CD8 1 T cells and NK cells. In our studies, NK depletion by in vivo administration of anti-As-GM antibodies during 32Dp210-derived vaccination did not have a major effect. This could in part be because of the persistence of some tissue-resident NK cells, recently shown to be depleted by administration of anti-NK1.1 antibodies in C57Bl/6 mice, but not by anti-asialo-GM antibodies. 48 As stated previously, NK1.1 is not expressed in the C3H strain and therefore could not be tested in our model.
The feasibility of using IL-15 to enhance stimulation of autologous antileukemic immunity has been demonstrated with T cells from leukemia patients, previously shown to have an exhausted phenotype. 64 Patient-derived T cells, collected at presentation, were expanded and then stimulated in vitro with IFN-g-treated autologous AML cells weekly for 3 weeks, in combination with IL-15 and agonistic anti-CD28 antibodies. AML-reactive, leukemiaspecific T cells were generated in 5/8 patients. 64 The availability of clinical grade IL-15/IL-15Ra, and of an Fc fusion to a mutated form of the heterodimer (ALT803), has allowed the ongoing clinical evaluation of the protein in cancer patients (www.clinicaltrials.gov; #NCT02452268, #NCT01670994). ALT 803 produced clinical responses with CD8 1 and NK cell activation and expansion, without stimulatory effects on Treg's, in patients relapsing after allo-transplant (#NCT01885897). 65 Although significant toxicity was observed with systemic administration of ALT803, these effects were mitigated by subcutaneous administration.
Our studies indicate that AML cell vaccines may enable induction of leukemia-specific immunity by amplification of responses to subdominant leukemia antigens, even when those against the immune dominant antigens are lost. There is now evidence from preclinical studies and recently published phase 1 trials with engineered autologous AML vaccines showing that they (1) are readily generated as cells can be reliably collected, cryopreserved, and engineered 7,66 ; (2) can overcome tumorrelated immunosuppression; and (3) stimulate leukemia-specific cytolytic immune responses when administered either after remission/consolidation chemotherapy or in immunosuppressed patients early after hematopoietic stem cell transplantation. 66,67 The potent immune-stimulatory combination of heterodimeric IL-15/IL-15Ra and CD80 expression in autologous leukemia cells provides a promising and universally applicable approach to generation of personalized leukemia vaccine therapy that could improve progression-free survival.