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Small Molecule Therapeutics

Lenalidomide Augments the Antitumor Activities of Eps8 Peptide-Specific Cytotoxic T Lymphocytes against Multiple Myeloma

Xiaoling Xie, Yiran Chen, Yuxing Hu, Yanjie He, Honghao Zhang and Yuhua Li
DOI: 10.1158/1535-7163.MCT-19-0424 Published December 2019

Abstract

Cancer immunotherapy is a promising new approach to cancer treatment. It has been demonstrated that a high number of tumor-specific cytotoxic T cells (CTL) is associated with increased survival in patients with multiple myeloma. Here, we focused on EGFR pathway substrate 8 (Eps8) as a candidate tumor-associated antigen (TAA) in multiple myeloma. Previous work has shown that Eps8-based immunotherapy in HLA-A2+ cancer patients may result in efficient antitumor immune responses against diverse tumor types. To improve immunotherapy for patients with multiple myeloma, we constructed a cocktail vaccine by combining several HLA-A2–restricted epitopes derived from Eps8 (Eps8cocktail), including Eps8101-2L (WLQDMILQV), Eps8276-1Y9V (YLDDIEFFV), and Eps8455-1Y (YLAESVANV). The CTLs induced by Eps8cocktail (Eps8cocktail-CTLs) showed highly effective anti–multiple myeloma activity, including Th1 cytokines production, cell proliferation, and cytotoxicity against HLA-A2+ multiple myeloma cells. This study highlights the importance of using a cocktail vaccine instead of a single-peptide vaccine to induce a robust response. Importantly, we revealed that lenalidomide effectively stimulated the antitumor activity of the Eps8cocktail-CTLs, with increasing expression trends for T-cell markers (CD28, CD40L, 41BB, and OX40). Compared with unstimulated CTLs and Eps8cocktail-CTLs, lenalidomide-treated Eps8cocktail-CTLs showed superior anti–multiple myeloma activity in humanized multiple myeloma models, including delaying tumor burden increases due to enhanced immune function. These results provide the framework for an Eps8 cocktail vaccination therapy to induce effective Eps8-specific CTLs in HLA-A2+ patients with multiple myeloma. Moreover, these studies further demonstrate that lenalidomide augments the immune response, providing a possibility for its use in combination with peptide vaccines to improve patient outcomes.

Introduction

Multiple myeloma is the second most frequent hematologic malignancy characterized by the expansion of bone marrow plasma cells in association with bone lesions, hematopoietic insufficiency, and immunosuppression (1, 2). The current therapies include conventional chemotherapy, autologous stem cell transplantation, proteasome inhibitors (bortezomib), and immunomodulatory drugs (IMiD), such as thalidomide and lenalidomide. However, multiple myeloma remains difficult to cure, and novel therapies are urgently needed (3, 4). Although immunotherapy has demonstrated good potential in cancer, this approach is suboptimal in multiple myeloma and requires further evaluation (5–7). Among several potential immunotherapeutic approaches for the treatment of multiple myeloma, peptide-based vaccines can effectively enhance the CD8+ T-cell response and improve survival in patients with multiple myeloma. Synthetic peptides also offer several advantages over whole-protein vaccines with regard to safety, production costs, and monitoring for specific immune responses in patients (8). Moreover, peptides have the ability to induce “epitope spreading,” whereby lysed target cancer cells release new antigenic epitopes that are then taken up, processed, and presented by antigen-presenting cells (APC) to a new repertoire of CTLs, thereby furthering tumor lysis. Various tumor-associated antigens (TAA) expressed in cancer cells have been identified and utilized as targets for tumor vaccines (9, 10). These TAAs are antigens that are highly expressed by tumor cells but are not detectable in normal cells (11).

In this study, the validated target we selected was EGFR pathway substrate 8 (Eps8). Eps8 has been documented to play roles in the pathogenesis of solid tumors and hematologic malignancies (12–14). Here, we performed qRT-PCR and Western blotting to confirm significantly higher Eps8 expression in patients with multiple myeloma than in healthy donors. We identified that Eps8 associated with multiple myeloma pathogenesis in vivo and that targeting Eps8 may be a promising therapeutic strategy in patients with multiple myeloma. In our previous study, we reported the identification of three immunogenic peptides including Eps8101-2L (WLQDMILQV), Eps8276-1Y9V (YLDDIEFFV), and Eps8455-1Y (YLAESVANV; ref. 15), which elicited strong antitumor activity. We propose these three peptides as immunogenic epitopes for multiple myeloma, as Eps8 is a potential therapeutic target in multiple myeloma.

Numerous clinical trials have been designed using multiple peptide vaccines, and significant clinical benefits have been observed. The use of these vaccines was safe and feasible, eliciting stronger therapeutic antitumor immunity than a single-peptide vaccine (16). We conceived a cocktail vaccination strategy for superior efficiency and efficacy. On the basis of previous studies, we evaluated the immunogenicity of a cocktail of three HLA-A2–restricted peptides derived from the Eps8 antigen (Eps8cocktail) because HLA-A2 is the most prevalent MHC class I molecule, which would allow the use of the cocktail in large patient populations.

The IMiD immunomodulatory compounds are agents with antineoplastic activity among patients with multiple myeloma. Among these compounds, lenalidomide, a derivative of thalidomide, is an FDA-approved drug for the treatment of multiple myeloma (17–19). A recent study suggested that the immunomodulatory activity of lenalidomide provides a potential opportunity to use this antimyeloma drug as a vaccine adjuvant. On the basis of previous studies of the immunologic effects of lenalidomide, we combined lenalidomide with the Eps8 cocktail vaccine to synergistically improve anticancer therapy (20, 21). Taken together, our results hereby provide a simple and robust vaccination strategy for peptide vaccines. We particularly focus on the role of lenalidomide in stimulating and enhancing the immune function of the CTLs induced by Eps8cocktail (Eps8cocktail-CTLs), which holds tremendous promise for immunotherapy against multiple myeloma.

Materials and Methods

Cell lines

Multiple myeloma cell lines (RPMI 8226, MM1.S, H929, IM-9, ARH-77, and U266) and chronic myeloid leukemia (CML) cell lines (K562) were purchased from Guangzhou Jenniobio Biotechnology Co., Ltd. The human transporter-associated protein (TAP)-deficient cell line T2 purchased from ATCC was used as APCs. All cell lines were maintained in RPMI1640 medium (Invitrogen) supplemented with 10% FBS in a humidified atmosphere incubator with 5% CO2 at 37°C. To ensure authentication and consistency throughout the study, only low passage cells (<passage 5–8) were used in the experiments. All cell lines were authenticated via STR analysis and Mycoplasma testing was performed every 2 months by PCR on in vitro propagated cultures.

Isolation of bone marrow mononuclear cells from patients with multiple myeloma

BMMCs were isolated from bone marrow aspirates from patients with multiple myeloma through Ficoll–Paque density gradient centrifugation according to standard protocols. This study was approved by the Ethics Committee of the Zhujiang Hospital, Southern Medical University (Guangzhou, China) in accordance with the Declaration of Helsinki and all patients signed informed consent documents.

qRT-PCR

Total RNA isolation from cells was conducted according to the manufacturer's instructions for TRIzol reagent (TAKARA). RNA was reverse transcribed into cDNA using a PrimeScript RT reagent kit (TaKaRa Bio Inc.). qRT-PCR analysis was performed for Eps8 using a SYBR Green Dye detection system (Applied Biosystems). The primers for qRT-PCR were purchased from Invitrogen, the following sequences were used. Eps8 (XM.006719057.1): forward primer: 5′-CTG CTC CAT CAC CTC CTC CAA-3′; reverse primer: 5′-CGA TAC TGC CAC CAC TGT CAC T-3′. The expression of GAPDH (NM.001289746.1) was set as the endogenous control: forward primer: 5′-GGA GCG AGA TCC CTC CAA AAT-3′; reverse primer: 5′-GGC TGT TGT CAT ACT TCT CAT GG-3′. All samples were assessed in duplicate.

Mouse xenograft multiple myeloma model

All animal experiments were performed in accordance with the Guide for the Care and Use Committees of Southern Medical University. In brief, NOD-Prkdcem26il2rgem26/Nju (NCG) mice were irradiated on day 0 and injected with U266/NC or U266/shEps8 (no Eps8 expression) cells one day later. For survival curve experiments, death was recorded. The mouse spleen was removed and imaged.

Synthetic peptides

Eps8101-2L (WLQDMILQV), Eps8276-1Y9V (YLDDIEFFV), and Eps8455-1Y (YLAESVANV) peptides were synthesized by standard Fmoc chemistry (Chinese Peptide). The purity of each peptide was validated by mass spectrometry.

Peptide binding assay

A cocktail of 3 HLA-A2–restricted Eps8-derived peptides including Eps8101-2L (WLQDMILQV), Eps8276-1Y9V (YLDDIEFFV), and Eps8455-1Y (YLAESVANV) was assessed for in vitro binding affinity with the T2 cell line. In brief, after incubating the T2 cells in culture medium at room temperature overnight, the cells were pulsed with the peptide cocktail at increasing concentrations (0, 6.25, 12.5, 25, and 50 μg/mL), the positive-control peptide IVMP58-66 or the single peptide Eps8101-2L plus 3 μg/mL human β2-microglobulin (Sigma-Aldrich). After an overnight incubation, HLA-A*0201 expression was measured by flow cytometry (BD Pharmingen) using an anti-human HLA-A2-FITC mAb.

Peptide stability assay

Briefly, T2 cells (2 × 106/mL) were cultured in serum-free RPMI1640 medium supplemented with 3 μg/mL human β2-microglobulin and 25 μg/mL peptide cocktail. The cells were then stained with an anti-human HLA-A2-FITC mAb, and peptide/HLA-A2 complex stability was analyzed at 0, 2, 4, 6, and 14 hours after incubating with 10 μg/mL brefeldin A (Sigma).

Animal studies

C57BL/6-Transgenic (HLA-A2.1)1Enge/J mice (HLA-A*0201 transgenic mice) were purchased from The Jackson Laboratory. The transgenic mice were 6 to 8 weeks old and were immunized subcutaneously twice on days 0 and 7 with a mixture of 3 synthesized peptides. Seven days after the last immunization, splenocytes were harvested for in vitro proliferation, cytokine response, and mouse IFNγ ELISPOT assays (Dakewe Biotech Co., Ltd.).

Isolation of CD3+ T cells and induction of Eps8cocktail-CTLs

Human CD3+ T cells were collected from the peripheral blood mononuclear cells (PBMC) of HLA-A2+ multiple myeloma patients and healthy donors using a pan-T Cell Isolation Kit (Miltenyi Biotec) and purified to >99%. Eps8cocktail-CTLs were generated by repeated stimulation of the CD3+ T lymphocytes with the Eps8 peptide cocktail. Cultures were restimulated every 7 days with T2 cells pulsed with the peptide cocktail for a total of 4 cycles in the presence of human IL2.

Determination of cytokine levels produced by CTLs against multiple myeloma cells

ELISAs were performed to determine the secretion of IL2, TNFα, IFNγ, and granzyme B according to the manufacturer's instructions (Dakewe). In brief, CTLs were coincubated with different multiple myeloma cells for 24 hours at 37°C in a 5% CO2 incubator. The supernatants were harvested after incubation using previously described protocols (22).

Phenotypic analysis of Eps8cocktail-CTLs

Seven days after the last stimulation, Eps8cocktail-CTLs and control T cells were collected for further analysis of different T-cell phenotypes. Total CD3+CD8+ T cells and T-cell surface markers (CD28, CD38, CD40L, and 41BB) were analyzed by flow cytometry.

CD107a assay and intracellular IFNγ expression

Intracellular IFNγ expression and CD107a degranulation were analyzed by flow cytometry to evaluate the functional activity of CTLs stimulated with multiple myeloma cell lines. Briefly, Eps8cocktail-CTLs or Eps8101-2L-CTLs were stimulated with multiple myeloma cell lines in the presence of an anti-CD107a mAb. After 2 hours of incubation at 37°C, brefeldin A (10 μg/mL) was added for another 6 hours. After that incubation, the cells were washed with PBS, and anti-CD3 and anti-CD8 mAbs were added for 30 minutes. After washing with PBS, the cells were fixed with Cytofix/Cytoperm solution and permeated with Fix/Perm working solution (BD Biosciences). Then, the cells were stained with an anti-IFNγ-FITC mAb. Finally, the cells were analyzed by flow cytometry.

Cell proliferation assessed by carboxyfluorescein succinimidyl ester tracking

The cell proliferation of Eps8cocktail-CTLs was measured by flow cytometry using the CellTrace CFSE Cell Proliferation Kit (Invitrogen). A CellTrace working solution was prepared and incubated with the Eps8cocktail-CTLs for 20 minutes at room temperature in the dark. Five times the original staining volume of culture medium was added to quench the reaction. Carboxyfluorescein succinimidyl ester (CFSE)–labeled Eps8cocktail-CTLs (1 × 106 cells/mL) were coincubated with target multiple myeloma cells (2 × 105 cells/mL) at 37°C and 5% CO2 in humidified air. CFSE-labeled Eps8cocktail-CTLs in medium alone were used as control T cells. After 5 days of incubation, the cells were harvested, stained with human anti-CD3 and anti-CD8 mAbs, and examined by flow cytometry.

In vitro cytotoxicity assays

Eps8cocktail-CTLs were mixed with target cells at various effector:target (E/T) ratios as indicated, and a standard lactate dehydrogenase (LDH) release assay (Sigma) was carried out. Briefly, a constant number of target cells (5 × 103 cells/well) was plated into 96-well plates and cocultured with Eps8cocktail-CTLs for 4 hours at 37°C in a CO2 incubator. After incubation, 50 μL of supernatant was collected from each well and transferred to new 96-well plates. The fluorescence of each supernatant was measured by a multilabel counter.

ELISPOT assay

A human IFNγ ELISPOT Kit was used according to the manufacturer's instructions. T2 cells suspended in RPMI1640 medium were stimulated with the Eps8 peptide cocktail for 4 hours, followed by a coincubation with Eps8cocktail-CTLs. After a 20-hour incubation at 37°C, the plates were incubated with a biotinylated detection antibody at 37°C for 1 hour and an HRP-conjugated streptavidin working solution for another 1 hour. AEC substrate solution was added to each well for 30 minutes in the dark at room temperature. Spots were counted by an automated ELISPOT reader.

Analysis of Eps8cocktail-CTLs post-lenalidomide treatment

One week after the fourth stimulation, Eps8cocktail-CTLs were harvested and treated with lenalidomide (0, 2.5, 5, or 10 μmol/L, Selleck) for 4 days. The treated cells were then evaluated for phenotypic changes in the expression of costimulatory markers. To measure cytotoxicity, untreated control Eps8cocktail-CTLs or lenalidomide-treated Eps8cocktail-CTLs (Eps8cocktail@len-CTLs) were stimulated with HLA-A2+Eps8+ tumor cell lines at the indicated E/T ratio as previously described. In addition, Eps8cocktail-CTLs were evaluated for cytotoxicity by assessing CD107a expression upregulation and IFNγ production upon stimulation with multiple myeloma cells, as described above.

Immune reconstitution and multiple myeloma cell transplantation in NCG mice

Female NCG mice (aged 4–6 weeks; Nanjing Biomedical Research Institute of Nanjing University, Nanjing, China) were housed at the Animal Center of Southern Medical University. All procedures followed institutional and national guidelines for the care and use of laboratory animals. The immune system of the NCG mice was reconstructed with a human PBMC (hPBMC) injection, and the mice were subcutaneously transplanted with human multiple myeloma cell lines labeled with luciferase (U266-luc cells). After tumor cell transplantation, tumor growth and immune responses in the mice were observed at regular intervals. Treatment was administered twice in a 7-day interval when the tumor volume reached 100 to 120 mm3. Within 2 weeks after the third immunization, the tumor volumes in each group were examined, and tumor inhibition rates were calculated.

Institutional review

This study was performed after approval by the Zhujiang Hospital institutional review board and all the investigators obtained informed written consent from the subject.

Statistical analysis

All the data were presented as mean ± SD. Unpaired Student t test (two-tailed) was used for comparison between two groups. Statistical significance was set at *, P < 0.05, **, P < 0.01, and ***, P < 0.001.

Results

Eps8 is highly expressed in multiple myeloma cell lines and multiple myeloma patients

The Eps8 protein was detected in all six multiple myeloma cell lines (ARH-77, IM-9, U266, H929, MM1.S, and RPMI 8226), and the acute myelomonocytic leukemia cell line U937 was used as a positive control (12). As shown in Fig. 1A, U266 and ARH-77 cells displayed higher levels of Eps8 protein expression than IM-9, MM1.S, and RPMI 8226 cells. In addition, the Eps8 protein was also highly expressed in primary cells from 8 patients with multiple myeloma, whereas in healthy donors (HD#1), the abundance of Eps8 was low (Fig. 1B). Similarly, the mRNA level of Eps8 in patients with multiple myeloma (n = 12) was markedly higher than that in healthy donors (P < 0.01, Fig. 1C). HLA-A2 expression was detected in different multiple myeloma cell lines by flow cytometry, as described above. Among the multiple myeloma cell lines, ARH-77 and U266 showed HLA-A2+/Eps8high expression, and MM1.S and RPMI 8226 showed HLA-A2−/Eps8medium expression (Supplementary Fig. S1). To confirm the involvement of Eps8 in the progression of multiple myeloma, we established an Eps8-silenced model by introducing an Eps8-specific shRNA vector into U266 cells. We assessed tumorigenicity by intravenously injecting Eps8-specific shRNA-expressing cells (U266/shEps8) into NCG mice, and NC shRNA cells were used as the control. The spleens of the mice receiving cells were removed and imaged (Fig. 1D). The spleens from the mice in the U266/shEps8 group displayed normal morphology, whereas the spleens from the mice in the U266/NC group were enlarged. The quantitative results showed that the spleens from the mice in the U266/shEps8 group were 1/2–3/4 of the weight of those from the mice in the U266/NC group. Compared with the recipients in the U266/NC group, the recipients in the U266/shEps8 group showed significantly prolonged survival (Fig. 1E). Taken together, the expression profile and the role of Eps8 in multiple myeloma progression make Eps8 an attractive target for immunotherapeutic purposes.

Figure 1.
Figure 1.

Eps8 expression in primary multiple myeloma (MM) cells and multiple myeloma cell lines. A, Immunoblots of Eps8 expression in 6 multiple myeloma cell lines. U937 served as a positive control. B, Immunoblots of Eps8 expression in 8 primary multiple myeloma patient samples, 1 normal donor sample, and 1 multiple myeloma cell line. GAPDH served as a loading control. C, Difference in Eps8 mRNA expression between primary multiple myeloma cells (n = 12) and normal cells (n = 9). D, NCG mice injected intravenously with 5 × 106 U266/NC or U266/ShEps8 cells. Morphologies and weights of spleens from mice receiving different cells are shown. E, Survival curves of mice intravenously injected with U266/NC or U266/shEps8 cells. Data are from one representative experiment out of three performed and are presented as the mean ± SD. Significance is indicated as **, P < 0.01.

A peptide cocktail of Eps8 peptides demonstrates high HLA-A2 binding affinity and strong stability

We previously identified that Eps8101-2L (WLQDMILQV), Eps8276-1Y9V (YLDDIEFFV) and Eps8455-1Y (YLAESVANV) are promising epitopes for targeted antitumor immunotherapy. Here, we evaluated these 3 immunogenic peptides as a peptide cocktail, and the binding of the peptide cocktail (Eps8cocktail) to HLA-A2 molecules as well as the stability of the binding were further confirmed by cell-surface HLA class I affinity and stabilization assays, respectively. The affinity of Eps8 peptides for HLA-A2 molecules is expressed as the fluorescence index (FI), which was calculated as [HLA-A2 MFI of T2 cells pulsed with the peptide plus β2-microglobulin/HLA-A2 MFI of T2 cells alone plus β2-microglobulin]. An FI value >1 indicated the upregulation of HLA-A2 molecule expression due to the specific binding of a peptide to the molecules on T2 cells (23). As illustrated in Fig. 2A, compared with the positive control (IVMP58-66) and single peptide (Eps8101-2L), Eps8cocktail showed strong and substantial binding to HLA-A2 molecules. Each of the peptides was tested at concentrations of 0, 6.25, 12.5, 25, 50, and 100 μmol/L. The results showed that Eps8cocktail significantly increased the levels of HLA-A*0201 molecules on the surface of T2 cells in a dose-dependent manner. In the HLA-A2–binding stability assay, T2 cells were incubated overnight with a total multipeptide concentration of 25 μg/mL per peptide. Among the three groups we evaluated, the peptide cocktail group showed highly stable binding with HLA-A*0201 molecules, which was almost as strong as that of the positive control peptide IVMP58-66 group. In contrast, the level of HLA-A*0201-Eps8101-2L complexes decreased rapidly. Altogether, these results suggest that the peptide cocktail vaccine binds with high affinity to HLA-A*0201 molecules and significantly increases the levels of HLA-A*0201 molecules on the surface of T2 cells, suggesting that the Eps8 peptide cocktail could be selected for further analysis.

Figure 2.
Figure 2.

HLA-A2–specific binding affinity and stability of Eps8 cocktail peptides and the induction of functional T cells in HLA-A2.1/Kb transgenic mice. A, HLA-A2–binding capacity and stability of Eps8 cocktail peptides. Various concentrations of peptides were analyzed for binding to T2 cells, as described in the Materials and Methods. Influenza virus matrix protein 58-66 (IVMP58-66; GILGFVFTL) was used as an HLA-A2–specific positive control peptide. B, Representative ELISPOT assays for IFNγ performed with splenic lymphocytes obtained from Eps8 peptide cocktail-immunized HLA-A2.1/Kb transgenic mice. Eps8327-335 (EFLDCFQKF) was used as an HLA-A24–specific negative control peptide. The number of IFNγ spots obtained is shown for each animal (n = 3). C, IL2, TNFα, IFNγ, and granzyme B secretion by Eps8cocktail-CTLs measured by ELISA in culture supernatants collected 1 day following stimulation with different multiple myeloma cell lines. The data shown are representative of three experiments. Data are shown as the mean ± SD. Significance is indicated as *, P < 0.05, **, P < 0.01, and ***, P < 0.001.

In vivo induction of functional Eps8cocktail-CTLs in HLA-A2.1/Kb transgenic mice

The Eps8 peptide cocktail was used to induce CTLs in HLA-A2.1/Kb transgenic mice, and the activity of the induced Eps8cocktail-CTLs was tested by measuring mouse IFNγ. Splenocytes were harvested and restimulated in vitro with peptides. Then, a mouse ELISPOT assay was employed to test the ability of the peptides to induce a CTL response in vitro. The data showed that compared with that from the mice immunized with PBS, the negative control vaccine Eps8327-335 (EFLDCFQKF) or the single Eps8101-2L vaccine, the number of IFNγ-secreting T cells from the mice immunized with the Eps8cocktail vaccine was significantly increased (Fig. 2B).

The primed Eps8cocktail-CTLs were evaluated for their ability to release IL2, TNFα, IFNγ, and granzyme B upon stimulation with different multiple myeloma cells via ELISA. We confirmed that the Eps8cocktail-CTLs produced high levels of IL2, TNFα, IFNγ, and granzyme B in response to HLA-A2+ multiple myeloma cells (U266 and ARH-77 cells), revealing a Th1-polarized immune response (Fig. 2C). Thus, these results suggest that the peptide cocktail vaccine exhibited strong immunogenic potential in generating multiple myeloma-specific CTLs.

Induced Eps8cocktail-CTLs from multiple myeloma patients demonstrate HLA-A2–restricted cell proliferation and specific cytotoxicity against multiple myeloma cell lines

Next, we determined the ability of the Eps8 peptide cocktail to induce antigen-specific CTLs from the blood of patients with multiple myeloma. As in previous experiments, potent Eps8cocktail-CTLs were established from the PBMCs of HLA-A*0201–positive donors by in vitro stimulation with the peptide cocktail. The peptide cocktail was able to induce Eps8cocktail-CTLs from four donors (donors 1–4), and compared with control CTLs that did not receive peptide stimulation, the Eps8cocktail-CTLs showed potent IFNγ production when exposed to T2 cells preloaded with the peptide cocktail (Fig. 3A and B). CTLs were also analyzed for their antitumor functional activities against various tumor cell lines. In LDH cytotoxicity assays, the Eps8101-2L-CTLs generated from the T lymphocytes of four multiple myeloma patients displayed direct cytotoxic activity against HLA-A2+ multiple myeloma cells, including U266 and ARH-77 cells, but not against HLA-A2− cells at E/T ratios of 6.25:1, 12.5:1, 25:1, and 50:1 (Fig. 3C). Next, we examined the capacity of Eps8cocktail-CTLs to kill HLA-A2+ multiple myeloma cells. T cells primed with Eps8cocktail demonstrated higher lytic abilities against U266 and ARH-77 cells at different E/T ratios than single Eps8101-2L-CTLs (Fig. 3D). In contrast, the Eps8cocktail-CTLs did not target and kill the MM1.S or RPMI 8226 (HLA-A2−) multiple myeloma cell line or the K562 (HLA-A2−) CML cell line, indicating that the Eps8cocktail-CTLs showed potent cytotoxicity against HLA-A2+ multiple myeloma cell lines.

Figure 3.
Figure 3.

Induction of Eps8cocktail-CTLs in blood from patients with multiple myeloma and the cytotoxic activity of Eps8cocktail-CTLs against HLA-A2+ multiple myeloma cells compared with that of Eps8101-2L-CTLs. A, IFNγ ELISPOT assays were performed with peptide-primed PBMCs from four different donors. Experiments were performed in triplicate. B, After the in vitro stimulation of PBMCs with a cocktail of Eps8 peptides for 1 week, the frequency of Eps8cocktail-CTLs was detected by an IFNγ ELISPOT assay. C, CTLs induced by Eps8101-2L were incubated with U266, MM1.S, RPMI 8226, ARH-77, and K562 at the indicated effector/target ratios (50:1, 25:1, 12.5:1, and 6.25:1) in a standard LDH release assay. D, The specific cytotoxic activity of Eps8cocktail-CTLs was analyzed using an LDH release assay. E, The HLA-A2–restricted cell proliferation of Eps8101-2L-CTLs and Eps8cocktail-CTLs upon stimulation with multiple myeloma cells was evaluated by CFSE analysis. Eps8101-2L-CTL and Eps8cocktail-CTL proliferation is shown as the percent decrease in CFSE expression on day 5 of culture. Data are shown as the mean ± SD (n = 3). Significance is indicated as *, P < 0.05 and **, P < 0.01.

The antitumor activities of Eps8cocktail-CTLs were further analyzed using a CFSE assay. An increase in the proportion of CD3+CD8+ T cells was detected in both the Eps8101-2L- and Eps8cocktail-CTLs. Flow cytometric analyses showed a significantly higher level of Eps8cocktail-CTL proliferation (shown as % CFSE-low) than Eps8101-2L-CTL proliferation in response to U266 (proliferating cells: 71.4% vs. 42.2%) and ARH-77 (proliferating cells: 77.2% vs. 40.5%) HLA-A2+ multiple myeloma cells (Fig. 3E). In contrast, the Eps8cocktail-CTLs did not proliferate in response to HLA-A2− cells, including MM1.S (proliferating cells: 15.9%), K562 (proliferating cells: 29.0%), and RPMI 8226 (proliferating cells: 28.8%) cells. Therefore, these results demonstrate the functional anti–multiple myeloma activities of CTLs induced by peptide vaccination.

Eps8cocktail-CTLs display functional activities in response to stimulation with HLA-A2+ multiple myeloma cell lines

To further characterize the antitumor response, we evaluated the abilities of Eps8cocktail-CTLs to produce IFNγ and CD107a following stimulation with various multiple myeloma cell lines or K562 cells. We demonstrated a sharp rise in CD107a degranulation in the Eps8101-2L-CTLs stimulated with HLA-A2+ cells (U266 and ARH-77) compared with the Eps8101-2L-CTLs stimulated with HLA-A2− cells (Fig. 4A). Interestingly, we detected higher levels of antitumor activities against an HLA-A2+ multiple myeloma cell line by CTLs induced with Eps8cocktail compared with CTLs induced with Eps8101-2L. The level of CD107a degranulation was increased upon stimulation with either U266 cells (Donor 1: Eps8101-2L vs. Eps8cocktail-CTL: 6.29% vs. 7.85%; Donor 2: 5.9% vs. 12.9%; Donor 3: 9.89% vs. 18.8%; Donor 4: 3.68% vs. 7.68%) or ARH77 cells (Donor 1: Eps8101-2L vs. Eps8cocktail-CTL: 7.20% vs. 16.40%; Donor 2: 6.43% vs. 7.89%; Donor 3: 7.89% vs. 11.70%; Donor 4: 5.12% vs. 7.12%). These observations were also confirmed in further analyses of IFNγ production. Our data showed that the Eps8cocktail-CTLs had increased IFNγ production in response to U266 cells (Donor 1: 17.80%; Donor 2: 7.89%; Donor 3: 6.78%; Donor 4: 11.89%) and ARH-77 (Donor 1: 17.20%; Donor 2: 7.71%; Donor 3: 7.89%; Donor 4: 8.76%; Fig. 4B). These functional immune responses were higher than those against HLA-A2− cells, offering evidence that the immunogenicity of the Eps8 peptide cocktail can induce polyfunctional CTLs with HLA-A2-restricted anti–multiple myeloma activities.

Figure 4.
Figure 4.

Comparison of peptide-specific degranulation and IFNγ production in response to multiple myeloma cells between Eps8cocktail-CTLs and Eps8101-2L-CTLs. A, The degranulation of Eps8cocktail-CTLs and Eps8101-2L-CTLs was evaluated by measuring CD107a expression upregulation on CD8+ CTLs after stimulation with various multiple myeloma cells using flow cytometry. The Eps8cocktail-CTLs stimulated with HLA-A2+ multiple myeloma cells showed a higher level of CD107a+/CD8+ cells than the Eps8101-2L-CTLs. B, Eps8cocktail-CTLs and Eps8101-2L-CTLs were analyzed for IFNγ production upon stimulation with various multiple myeloma cell lines. Representative flow cytometric analysis confirmed the HLA-A2–restricted IFNγ production in response to multiple myeloma cells.

Lenalidomide increases the expression of T-cell markers on multiple myeloma vaccine-induced CTLs and augments their anti–multiple myeloma activities

Lenalidomide has been shown to enhance T-cell immunity, providing a potential opportunity to use this anti–multiple myeloma drug as a vaccine adjuvant. We found that compared with the Eps8101-2L vaccine, the Eps8cocktail vaccine alone did not alter T-cell marker expression (Supplementary Fig. S2). In these studies, we evaluated whether lenalidomide could regulate the expression of key molecules involved in T-cell costimulation (CD28, 41BB, CD40L, and OX40) or enhance the antitumor activities of Eps8cocktail-CTLs against multiple myeloma cells. We observed that short-term treatment of Eps8cocktail-CTLs with lenalidomide (4 days) did not change the total CD3+CD8+ T-cell percentage of the CTLs with increasing concentrations of lenalidomide (0, 2.5, 5, and 10 μmol/L; Supplementary Fig. S3). However, lenalidomide treatment of the Eps8cocktail-CTLs increased the expression of T-cell markers (Fig. 5A). Indeed, lenalidomide induced a dose-dependent (0, 2.5, 5, and 10 μmol/L) increase in the frequency of CD28+ T cells within the Eps8cocktail-CTL population. In addition to increasing the expression of CD28, lenalidomide increased the expression of the secondary costimulatory markers CD40L, 41BB, and OX40 in a dose-dependent manner (0, 2.5, 5, and 10 μmol/L). These results therefore provide additional support for the activity of lenalidomide in enhancing multiple costimulatory pathways, thereby augmenting antigen-specific CD8+ CTL activity. To determine the potential of lenalidomide to augment antigen-specific T-cell immunity, cytotoxicity was measured at baseline and after each lenalidomide treatment. We observed a higher level of specific lysis of the ARH-77 and U266 cell lines by Eps8cocktail@len-CTLs than by Eps8cocktail-CTLs without lenalidomide treatment (Fig. 5B). Importantly, compared with the baseline values of the Eps8cocktail-CTLs without lenalidomide treatment, the anti–multiple myeloma activities against ARH-77 or U266 cells of the Eps8cocktail-CTLs increased following lenalidomide treatment, as shown by the increase in IFNγ production (Fig. 5C). Furthermore, the Eps8cocktail@len-CTLs showed a higher proportion of CD8+ cells expressing the CD107a degranulation marker upon recognition of HLA-A2+ multiple myeloma cells than the Eps8cocktail-CTLs (Fig. 5D). Therefore, these studies demonstrate that lenalidomide treatment affects critical T-cell markers and augments the antitumor response of Eps8cocktail-CTLs, suggesting a critical role for lenalidomide in enhancing vaccine efficacy.

Figure 5.
Figure 5.

Modulation of the T-cell marker expression and cytotoxicity of Eps8cocktail-CTLs by treatment with lenalidomide. A, Eps8cocktail-CTLs treated with various doses of lenalidomide (4 days, 0, 2.5, 5, 10 μmol/L) were analyzed for CD28, CD40L, OX40, and 41BB expression. B, The cytotoxicity of Eps8cocktail-CTLs and lenalidomide-treated Eps8cocktail-CTLs (Eps8cocktail@len-CTLs) against U266 and ARH-77 cells (HLA-A2.1+, Eps8+) was assessed at a 50:1 E/T ratio. C, Eps8cocktail@len-CTL degranulation (CD107a) in response to multiple myeloma cells was analyzed by flow cytometry. Eps8cocktail@len-CTLs showed a higher proportion of CD8+ cells expressing the CD107a degranulation marker upon recognition of U266 and ARH-77 cells than Eps8cocktail-CTLs. D, Intracellular IFNγ production was measured by flow cytometry after stimulation of Eps8cocktail-CTLs and Eps8cocktail@len-CTLs with the HLA-A2+ U266 and ARH-77 myeloma cell lines. A higher level of IFNγ production was detected in the Eps8cocktail@len-CTLs than in the Eps8cocktail-CTLs. Data are shown as the mean ± SD (n = 3). Significance is indicated as *, P < 0.05, **, P < 0.01, and ***, P < 0.001.

The peptide cocktail vaccine increases survival in a humanized multiple myeloma model

To optimize xenograft models to validate the in vivo therapeutic effects of the Eps8cocktail-CTLs and Eps8cocktail@len-CTLs, we first established a humanized mouse model of multiple myeloma. Experiments performed in multiple myeloma model mice specifically use hPBMCs as sources of T cells. Therefore, we first established humanized mice by intravenously injecting hPBMCs into NCG mice to reconstitute the mice with human T cells. At 3 weeks post-hPBMC implantation, the mice were checked for engraftment of the human cells. The hPBMC-injected mice demonstrated engraftment of human leukocytes (26.7 ± 3.3% hCD45+ cells, n = 18). In the hPBMC-injected mice, the majority of the hCD45+ cells were T cells (90.1 ± 3% hCD3+hCD19− cells; Supplementary Fig. S4A). The NCG mice injected with hPBMCs demonstrated no human B-cell (hCD3−hCD19+) engraftment. At the time of sacrifice, histologic analysis of the liver and spleen indicated that the mice injected with hPBMCs displayed greater leukocyte infiltration than uninjected mice (Supplementary Fig. S4B). Overall, the transplantation of hPBMCs into NCG mice led to human T-cell reconstitution.

A total of 5 × 106 U266 cells stably expressing luciferase (U266-luc cells) were subcutaneously transplanted into immune-reconstituted NCG mice. Compared with the control group (CTLs without peptide and lenalidomide stimulation), both the Eps8cocktail-CTL and Eps8cocktail@len-CTL groups showed a reduction in tumor growth after intravenous injection. However, a remarkable inhibition of tumor volume was observed in the group treated with Eps8cocktail@len-CTLs beginning 14 days after tumor implantation, suggesting that Eps8cocktail vaccine treatment was able to delay primary tumor growth. In addition, the addition of lenalidomide to immune-based anti–multiple myeloma strategies could augment their efficacy. Tumor growth was monitored over time by bioluminescence analysis (Fig. 6A). The mice treated with Eps8cocktail@len-CTLs showed improved control of tumor growth (Fig. 6B and C) without mouse death or obvious body weight loss (Fig. 6D). Furthermore, the percentages of CD28+ T cells and IFNγ-producing cells within the CD8+ T-cell population were significantly increased, and the PD-1+ T-cell frequency was decreased in the tumors after treatment with Eps8cocktail and lenalidomide compared with the other two treatments (Fig. 6E; Supplementary Fig. S5). The antitumor effect of Eps8cocktail@len was further supported by Ki67 and TUNEL staining of tumor sections. As shown in Fig. 6F, the Ki67 expression decreased, whereas the TUNEL signal increased in the Eps8cocktail@len-treated tumors compared with those in other treatment groups. Semiquantitative analysis of the ratios (positive cells to all cells) showed that the numbers of proliferative cells in the Eps8cocktail@len group was significantly less than that in the control and Eps8cocktail groups, suggesting that the Eps8cocktail@len-CTL group exhibited significant apoptosis and proliferation inhibition in the tumor cells.

Figure 6.
Figure 6.

A novel Eps8 peptide cocktail vaccine for the inhibition of U266 multiple myeloma growth in vivo. A, In vivo bioluminescence imaging of U266-luc tumors in control and treated groups. Three representative mice per treatment group are shown. B, Tumor tissues obtained from each group of euthanized mice 35 days after administration of control CTLs, Eps8cocktail-CTLs, or Eps8cocktail@len-CTLs. C, Tumor volume of U266-luc tumor xenografts in mice from each group after the different administrations. D, Body weight of the mice from each group without any significant differences. E, Representative flow cytometric analysis of CD28+ and IFNγ+ T-cell infiltration within the tumor gated on CD3+ cells (left) and the quantified results (right) for the different groups. F, Ki67 and TUNEL staining images of tumor tissue samples from the three groups after treatment (left). Percentages of positive cells to all cells in islet (right). Data are presented as the mean ± SD (n = 6). Significance is indicated as *, P < 0.05, **, P < 0.01, and ***, P < 0.001.

Discussion

Cancer immunotherapy is attracting more attention as a next-generation cancer therapeutic strategy (24, 25). Among several types of cancer immunotherapies, peptide vaccines are one of the most successful public health interventions, preventing and controlling tumor progression in an efficient and cost-effective manner. It is essential that more suitable and immunogenic TAAs should be chosen for the induction of more robust antitumor immune responses using multiple-peptide cocktail vaccines (26). Here, we focused on the HLA-A*0201 allele as it is the most common HLA class I subtype in the Caucasian population (27). In previous studies, we identified highly immunogenic HLA-A2–specific Eps8-derived peptides, Eps8101-2L (WLQDMILQV), Eps8276-1Y9V (YLDDIEFFV) and Eps8455-1Y (YLAESVANV). In this study, we further demonstrated Eps8 protein expression by primary multiple myeloma patient cells and multiple myeloma cell lines via Western blot and qRT-PCR analyses. We predicted that Eps8 plays an important role in multiple myeloma. Our data suggested that Eps8 is involved in the growth of myeloma cells and might be responsible for tumor progression in vivo. The involvement of Eps8 in multiple myeloma suggests a key role for Eps8 in multiple myeloma cell growth and survival, making Eps8 a desirable target for multiple myeloma immunotherapy. Combining CTL epitopes will contribute to a synergistic effect of immunotherapy on multiple myeloma. Therefore, we evaluated a cocktail of 3 immunogenic HLA-A2–specific peptides, Eps8101-2L (WLQDMILQV), Eps8276-1Y9V (YLDDIEFFV) and Eps8455-1Y (YLAESVANV), for its ability to evoke anti–multiple myeloma immunity in T lymphocytes obtained from HLA-A2+ patients with multiple myeloma. The results showed that the CTLs induced by Eps8cocktail had polyfunctional antitumor activities, IFNγ production, CD107 degranulation, and cell proliferation triggered by the peptide cocktail.

Given the inherent weak immunogenicity of cancer vaccines, there is a need for clinical-grade immune adjuvants that can potentiate cancer-specific immune responses. Combination therapies involving adjuvants may prove critical for the long-term success of cancer vaccines (28, 29). Currently, clinically approved vaccine adjuvants are limited. Several vaccine adjuvants have shown promise but are still in development. Among the potential candidates, lenalidomide has been shown to enhance the antitumor immune responses of tumor antigen-specific CTLs and may prove beneficial when used in conjunction with cancer vaccines (30).

To optimize vaccination strategies, combinations with lenalidomide may provide an approach that enhances the therapeutic potency of cancer vaccines (31, 32). Our studies emphasized the impact of lenalidomide on antigen-specific CTLs and showed that short-term treatment of Eps8cocktail-CTLs with lenalidomide enhanced the immune function of Eps8cocktail-CTLs by increasing T-cell marker expression, IFNγ production, and specific cytotoxicity. Accordingly, T-cell production is improved upon lenalidomide treatment. These data, along with data from other studies, provide evidence for the use of lenalidomide as an efficient combination therapy to enhance vaccine efficacy in patients with multiple myeloma.

In summary, we evaluated a cocktail of three immunogenic HLA-A2–specific peptides, Eps8101-2L (WLQDMILQV), Eps8276-1Y9V (YLDDIEFFV) and Eps8455-1Y (YLAESVANV), for its ability to elicit anti–multiple myeloma activity in CD3+ T lymphocytes collected from HLA-A2+ patients with multiple myeloma. In this study, we demonstrated the efficacy of an Eps8 peptide cocktail vaccine for multiple myeloma immunotherapy. The peptide cocktail vaccine we proposed herein might offer a new strategy for multiple myeloma immunotherapy. In future studies, we will attempt to identify other HLA-A*0201 epitopes and, by connecting multiple epitopes, overcome antigen loss and immune escape.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Authors' Contributions

Conception and design: X. Xie, Y. Li

Development of methodology: X. Xie, Y. Hu, H. Zhang, Y. Li

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): X. Xie, Y. Chen, Y. Hu, Y. He, H. Zhang

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): X. Xie, Y. Chen, Y. Li

Writing, review, and/or revision of the manuscript: X. Xie, Y. Chen, Y. Li

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): Y. Li

Study supervision: Y. Li

Acknowledgments

This work was supported by the Science and Technology Program of Guangzhou, China (grant no. 201704020216, to Y. Li); the Natural Science Foundation of Guangdong Province, China (grant no. 2018B030311042 to Y. Li); and the Guangzhou Regenerative Medicine and Health Guangdong Laboratory, China (grant no. 2018GZR110105014 to Y. Li).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Footnotes

  • Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).

  • Mol Cancer Ther 2019;18:2258–69

  • Received April 20, 2019.
  • Revision received June 19, 2019.
  • Accepted August 7, 2019.
  • Published first August 14, 2019.

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Lenalidomide Augments the Antitumor Activities of Eps8 Peptide-Specific Cytotoxic T Lymphocytes against Multiple Myeloma
Xiaoling Xie, Yiran Chen, Yuxing Hu, Yanjie He, Honghao Zhang and Yuhua Li
Mol Cancer Ther December 1 2019 (18) (12) 2258-2269; DOI: 10.1158/1535-7163.MCT-19-0424
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