Jooeun Bae

Immunomodulatory drug lenalidomide (CC-5013, IMiD3) augments anti-CD40 SGN-40-induced cytotoxicity in human multiple myeloma: Clinical implications

SGN-40, a humanized immoglobulin G1 (IgG1) anti-CD40 monoclonal antibody, mediates cytotoxicity against human multiple myeloma (MM) cells via suppression of interleukin (IL)-6-induced proliferative and antiapoptotic effects as well as antibody-dependent cell-mediated cytotoxicity (ADCC). Here, we studied the clinical significance of an immunomodulatory drug lenalidomide on SGN-40-induced cytotoxicity against CD138(+)CD40(+) MM lines and patient MM cells. Pretreatment with lenalidomide sensitized MM cells to SGN-40-induced cell death. Combined lenalidomide and SGN-40 significantly induced MM apoptosis, evidenced by enhanced cleavage of caspase-3/8/poly(ADP-ribose)polymerase and increased sub-G(0) cells, compared with either single agent at the same doses. Pretreatment of effector cells with lenalidomide augmented SGN-40-induced MM cell lysis, associated with an increased number of CD56(+)CD3(-) natural killer (NK) cells expressing CD16 and LFA-1. Importantly, pretreatment with lenalidomide or lenalidomide and SGN-40 markedly enhanced NK-cell-mediated lysis of autologous patient MM cells triggered by SGN-40. Lenalidomide also up-regulated CD40L on CD56(+)CD3(-) NK cells, facilitating IL-2-mediated activation of NK cells. In addition, lenalidomide induced the CD56(dim) NK subset, which are more potent mediators of ADCC against target MM cells than the CD56(bright) NK subset. Finally, pretreatment of both effector and target MM cells with lenalidomide markedly enhanced SGN-40-mediated ADCC against CD40-expressing MM cells. These studies, therefore, show that the addition of lenalidomide to SGN-40 enhances cytotoxicity against MM cells, providing the framework for combined lenalidomide and SGN-40 in a new treatment paradigm to both target MM cells directly and induce immune effectors against MM.

Myeloma-specific multiple peptides able to generate cytotoxic T lymphocytes: A potential therapeutic application in multiple myeloma and other plasma cell disorders

Purpose: The efficacy of peptide vaccines may be enhanced by stimulating immune cells with multiple peptides derived from distinct tumor-associated antigens. We have evaluated the heteroclitic XBP1-US184–192 (YISPWILAV), heteroclitic XBP1-SP367–375 (YLFPQLISV), native CD138260–268 (GLVGLIFAV), and native CS1239–247 (SLFVLGLFL) peptides, which have strong HLA-A2 affinity and immunogenicity in combination, for their ability to elicit multiple myeloma antigen–specific responses. Experimental Design: Multipeptide-specific cytotoxic T lymphocytes (MP-CTL) were generated by the stimulation of CD3+ T lymphocytes from HLA-A2+ individuals with either autologous mature dendritic cells or T2 cells pulsed with a cocktail of these four peptides. Results: The peptide cocktail did not compromise tumor antigen–specific activity of CTLs. MP-CTLs displayed increased total, effector memory (CCR7−CD45RO+), and activated (CD69+) CD3+CD8+ T lymphocytes. In addition, MP-CTL showed IFN-γ production, cell proliferation, and cytotoxicity against HLA-A2+ multiple myeloma cells, including cells of HLA-A2+ patients with multiple myeloma. Importantly, MP-CTLs showed specific responses in functional assays to each relevant peptide but not to an irrelevant HLA-A2–specific CMV pp65 (NLVPMVATV) peptide. Conclusions: These results highlight the potential therapeutic application of vaccination with a cocktail of HLA-A2–specific peptides to induce CTLs with a broad spectrum of immune responses against multiple myeloma antigens. Clin Cancer Res; 18(17); 4850–60. ©2012 AACR.

Identification of novel myeloma-specific XBP1 peptides able to generate cytotoxic T lymphocytes: a potential therapeutic application in multiple myeloma.

The purpose of these studies was to identify human leukocyte antigen (HLA)-A2+ immunogenic peptides derived from XBP1 antigens to induce a multiple myeloma (MM)-specific immune response. Six native peptides from non-spliced XBP1 antigen and three native peptides from spliced XBP1 antigen were selected and evaluated for their HLA-A2 specificity. Among them, XBP1184−192, XBP1 SP196−204 and XBP1 SP367−375 peptides showed the highest level of binding affinity, but not stability to HLA-A2 molecules. Novel heteroclitic XBP1 peptides, YISPWILAV or YLFPQLISV, demonstrated a significant improvement in HLA-A2 stability from their native XBP1184−192 or XBP1 SP367−375 peptide, respectively. Cytotoxic T lymphocytes generated by repeated stimulation of CD3+ T cells with each HLA-A2-specific heteroclitic peptide showed an increased percentage of CD8+ (cytotoxic) and CD69+/CD45RO+ (activated memory) T cells and a lower percentage of CD4+ (helper) and CD45RA+/CCR7+ (naïve) T cells, which were distinct from the control T cells. Functionally, the cytotoxic T lymphocytes (CTL) demonstrated MM-specific and HLA-A2-restricted proliferation, interferon-γ secretion and cytotoxic activity in response to MM cell lines and importantly, cytotoxicity against primary MM cells. These data demonstrate the distinct immunogenic characteristics of unique heteroclitic XBP1 peptides, which induce MM-specific CTLs and highlights their potential application for immunotherapy to treat the patients with MM or its pre-malignant condition.

Phenotypic and functional effects of heat shock protein 90 inhibition on dendritic cell

The 90-kDa heat shock protein (Hsp90) plays an important role in conformational regulation of cellular proteins and thereby cellular signaling and function. As Hsp90 is considered a key component of immune function and its inhibition has become an important target for cancer therapy, we here evaluated the role of Hsp90 in human dendritic cell (DC) phenotype and function. Hsp90 inhibition significantly decreased cell surface expression of costimulatory (CD40, CD80, CD86), maturation (CD83), and MHC (HLA-A, B, C and HLA-DP, DQ, DR) markers in immature DC and mature DC and was associated with down-regulation of both RNA and intracellular protein expression. Importantly, Hsp90 inhibition significantly inhibited DC function. It decreased Ag uptake, processing, and presentation by immature DC, leading to reduced T cell proliferation in response to tetanus toxoid as a recall Ag. It also decreased the ability of mature DC to present Ag to T cells and secrete IL-12 as well as induce IFN-γ secretion by allogeneic T cells. These data therefore demonstrate that Hsp90-mediated protein folding is required for DC function and, conversely, Hsp90 inhibition disrupts the DC function of significant relevance in the setting of clinical trials evaluating novel Hsp90 inhibitor therapy in cancer.

Activated platelets rapidly up-regulate CD40L expression and can effectively mature and activate autologous ex vivo differentiated DC

Background DC are a promising immunotherapeutic for treatment of infectious/malignant disease. For clinical trials, immature DC generated from precursor cells such as monocytes, using serum-free media containing GM-CSF and IL-4, can be matured with specific cytokines/factors. CD40 ligand (CD40L) plays an important role in DC activation/maturation but is not available for clinical applications. These studies evaluated the feasibility of using activated platelets with elevated CD40L surface expression to stimulate autologous DC maturation. Methods Pilot and clinical scale studies were executed using magnetic/centrifugal separation. Monocyte precursors were differentiated to immature DC with GM-CSF and IL-4 and the ability of activated autologous platelets to mature these cells was evaluated on the basis of phenotype and function. Results In small-scale studies, DC cultures stimulated with activated autologous platelets (CD40L-AP), tumor necrosis factor-alpha (TNF-alpha) or soluble CD40L (sCD40L) up-regulated expression of phenotype markers indicative of activation and maturation. CD86 expression was significantly enhanced (P<0.05) by stimulation with either CD40L-AP (75.5+/-14.5%) or sCD40L (80.5%+/-5.3%) compared with immature DC (55.2+/-14.8%), as were CD80 and CD83. Similarly, in large-scale studies using Isolex 300I to enrich for monocytes and platelets for DC generation/maturation on a clinical scale, stimulation with CD40L-AP increased CD86 and CD80 expression as well as the ability to stimulate allogeneic lymphocytes compared with control cultures. Discussion These results demonstrate that thrombin-activated platelets express CD40L and are effective at inducing matured DC with potent immunogenic activity. Furthermore, these studies demonstrate the feasibility of this approach for clinical immunotherapeutic interventions.

Heat Shock Protein 90 Is Critical for Regulation of Phenotype and Functional Activity of Human T Lymphocytes and NK Cells

The 90-kDa heat shock protein (Hsp90) has become an important therapeutic target with ongoing evaluation in a number of malignancies. Although Hsp90 inhibitors have a high therapeutic index with limited effects on normal cells, they have been described to inhibit dendritic cell function. However, its effect on human immune effector cells may have significant clinical implications, but remains unexplored. In this study, we have evaluated the effects of Hsp90 inhibition on human T lymphocyte and NK cells, including their Ag expression, activation, proliferation, and functional activities. These studies demonstrate that Hsp90 inhibition irreversibly downregulates cell surface expression of critical Ags (CD3, CD4, CD8), the costimulatory molecule (CD28, CD40L), and αβ receptors on T lymphocytes, as well as activating receptors (CD2, CD11a, CD94, NKp30, NKp44, NKp46, KARp50.3) on NK cells. Hsp90 inhibition significantly reduced CD4 protein expression on T lymphocytes at both the cell surface and intracellular level, which was shown to be associated with aberrant regulation of Src-kinase p56Lck. Downregulation of the Ags triggered by Hsp90 inhibition on CD3+ T lymphocytes, both in CD4+ and CD8+ T cell subsets, was associated with a disruption in their cellular activation, proliferation, and/or IFN-γ production, when the inhibition occurred either in activated or inactivated cells. In addition, downregulation of key activating receptors on NK cells following Hsp90 inhibition resulted in decreased cytotoxicity against tumor cells. Therefore, these observations demonstrate the need to closely monitor immune function in patients being treated with a Hsp90 inhibitor and may provide a potential therapeutic application in autoimmune diseases.

A multiepitope of XBP1, CD138 and CS1 peptides induces myeloma-specific cytotoxic T lymphocytes in T cells of smoldering myeloma patients.

We evaluated a cocktail of HLA-A2-specific peptides including heteroclitic XBP1 US184–192 (YISPWILAV), heteroclitic XBP1 SP367–375 (YLFPQLISV), native CD138260–268 (GLVGLIFAV) and native CS1239–247 (SLFVLGLFL), for their ability to elicit multipeptide-specific cytotoxic T lymphocytes (MP-CTLs) using T cells from smoldering multiple myeloma (SMM) patients. Our results demonstrate that MP-CTLs generated from SMM patients’ T cells show effective anti-MM responses including CD137 (4-1BB) upregulation, CTL proliferation, interferon-γ production and degranulation (CD107a) in an HLA-A2-restricted and peptide-specific manner. Phenotypically, we observed increased total CD3+CD8+ T cells (>80%) and cellular activation (CD69+) within the memory SMM MP-CTL (CD45RO+/CD3+CD8+) subset after repeated multipeptide stimulation. Importantly, SMM patients could be categorized into distinct groups by their level of MP-CTL expansion and antitumor activity. In high responders, the effector memory (CCR7−CD45RO+/CD3+CD8+) T-cell subset was enriched, whereas the remaining responders’ CTL contained a higher frequency of the terminal effector (CCR7−CD45RO−/CD3+CD8+) subset. These results suggest that this multipeptide cocktail has the potential to induce effective and durable memory MP-CTL in SMM patients. Therefore, our findings provide the rationale for clinical evaluation of a therapeutic vaccine to prevent or delay progression of SMM to active disease.

A novel immunogenic CS1-specific peptide inducing antigen-specific cytotoxic T lymphocytes targeting multiple myeloma

The CS1 antigen provides a unique target for the development of an immunotherapeutic strategy to treat patients with multiple myeloma (MM). This study aimed to identify HLA‐A2+ immunogenic peptides from the CS1 antigen, which induce peptide‐specific cytotoxic T lymphocytes (CTL) against HLA‐A2+ MM cells. We identified a novel immunogenic HLA‐A2‐specific CS1239‐247 (SLFVLGLFL) peptide, which induced CS1‐specific CTL (CS1‐CTL) to MM cells. The CS1‐CTL showed a distinct phenotype, with an increased percentage of effector memory and activated CTL and a decreased percentage of naïve CTL. CS1239‐247 peptide‐specific CD8+ T cells were detected by DimerX analyses and demonstrated functional activities specific to the peptide. The CTL displayed HLA‐A2‐restricted and antigen‐specific cytotoxicity, proliferation, degranulation and γ‐interferon (IFN‐γ) production against both primary MM cells and MM cell lines. In addition, the effector memory cells subset (CD45RO+CCR7−/CD3+CD8+) within CS1‐CTL showed a higher level of CD107a degranulation and IFN‐γ production as compared to effector cells (CD45RO−CCR7−/CD3+CD8+) against HLA‐A2+ primary MM cells or MM cell lines. In conclusion, this study introduced a novel immunogenic HLA‐A2‐specific CS1239‐247 peptide capable of inducing antigen‐specific CTL against MM cells that will provide a framework for its application as a novel MM immunotherapy.

Identification of CD19 and CD20 Peptides for Induction of Antigen-Specific CTLs against B-Cell Malignancies

The purpose of these studies was to develop immunogenic peptides derived from the CD19 and CD20 self-antigens for the induction of antigen-specific CTLs against B-cell malignancies. A total of seven peptides were designed and examined for their HLA-A2.1 affinity and immunogenicity. Of these peptides, we identified two highly immunogenic HLA-A2.1-specific peptides, CD19150-158 (KLMSPKLYV) and CD20188-196 (SLFLGILSV), which were capable of inducing peptide-specific CTLs. The CTLs displayed HLA-A2.1-restricted and antigen-specific cytotoxicity against Burkitts lymphoma, chronic B cell leukemia, and multiple myeloma cell lines. The CD19 or CD20 peptide–specific CTL cytotoxicity was confirmed using HLA-A2.1+ T2 cells presenting the appropriate peptide. No cytotoxic activity was observed against T2 cells presenting the irrelevant MAGE-3 peptide or T2 cells alone. In addition, the CTLs displayed a significant (P < 0.05) increase in cell proliferation and IFN-γ secretion (>830 ng/mL) following restimulation with HLA-A2.1+/CD19+/CD20+ tumor cells. The CTLs also displayed a distinct phenotype consisting of a high percentage of CD69+/CD45RO+ and a low percentage of CD45RA+/CCR7+ CD4+ or CD8+ T cells characteristic of effector memory cell population. Cyclic guanosine 3′,5′-monophosphate culture conditions using serum-free AIM-V medium containing human AB serum, recombinant human interleukin 2 (Proleukin) and CD3/CD28 Dynabeads were developed resulting in a 35-fold expansion of CD20 peptide–specific CTLs. The expanded CD20-CTLs retained their cytotoxic activity (28-49%) against the Burkitts lymphoma cell line. In conclusion, we report here on the identification of novel immunogenic CD19150-158 (KLMSPKLYV) and CD20188-196 (SLFLGILSV) peptides that have immunotherapeutic potentials as peptide vaccines or targeted T-cell therapies for treating B-cell malignancies.

Novel epitope evoking CD138 antigen-specific cytotoxic T lymphocytes targeting multiple myeloma and other plasma cell disorders.

The development of an immunotherapeutic strategy targeting CD138 antigen could potentially represent a new treatment option for multiple myeloma (MM). This study evaluated the immune function of CD138 peptide‐specific cytotoxic T lymphocytes (CTL), generated ex vivo using an HLA‐A2‐specific CD138 epitope against MM cells. A novel immunogenic HLA‐A2‐specific CD138260–268 (GLVGLIFAV) peptide was identified from the full‐length protein sequence of the CD138 antigen, which induced CTL specific to primary CD138+ MM cells. The peptide‐induced CD138‐CTL contained a high percentage of CD8+ activated/memory T cells with a low percentage of CD4+ T cell and naive CD8+ T cell subsets. The CTL displayed HLA‐A2‐restricted and CD138 antigen‐specific cytotoxicity against MM cell lines. In addition, CD138‐CTL demonstrated increased degranulation, proliferation and γ‐interferon secretion to HLA‐A2+/CD138+ myeloma cells, but not HLA‐A2−/CD138+ or HLA‐A2+/CD138− cells. The immune functional properties of the CD138‐CTL were also demonstrated using primary HLA‐A2+/CD138+ cells isolated from myeloma patients. In conclusion, a novel immunogenic CD138260–268 (GLVGLIFAV) peptide can induce antigen‐specific CTL, which might be useful for the treatment of MM patients with peptide‐based vaccine or cellular immunotherapy strategies.