Vitamin D insufficiency is associated with an increased risk of early clinical failure in follicular lymphoma.

We evaluated whether vitamin D insufficiency (VDI; 25(OH)D <20 ng/ml) was associated with adverse outcomes among follicular lymphoma (FL) patients using an observational prospective cohort study of 642 FL patients enrolled from 2002-2012. The median age at diagnosis was 60 years. At a median follow-up of 59 months, 297 patients (46%) had an event (progression, treatment failure), 78 had died and 42 (6.5%) had a lymphoma-related death. VDI was associated with inferior event-free survival (EFS) at 12 months (EFS12, odds ratio (OR)=2.05; 95% confidence interval (CI) 1.18-3.54), overall survival (OS, hazards ratio (HR)=2.35; 95%CI 1.37-4.02), and lymphoma-specific survival (LSS, HR=2.97; 95% CI 1.52-5.80) for the full cohort. Among patients treated with immunochemotherapy (IC), VDI was associated with inferior EFS12 (OR=3.00; 95% CI 1.26-7.13), OS (HR=2.86; 95% CI 1.39-5.85), and LSS (HR=2.96; 95% CI 1.29-6.79). For observed patients, VDI was associated with inferior OS (HR=2.85; 95% CI 1.20-6.76). For other therapies, VDI was associated with inferior OS (HR=3.06; 95% CI 1.01-9.24). Our work is the first to reveal an association of VDI with early clinical failure, and to demonstrate an association of VDI with adverse outcomes among patients who are observed or treated with therapies other than IC. Our findings suggest a potentially modifiable prognostic factor to address in patients with FL.

Good prognosis cytogenetics in B-cell chronic lymphocytic leukemia is associated in vitro with low susceptibility to apoptosis and enhanced immunogenicity.

Chromosomal abnormalities in B-cell chronic lymphocytic leukemia (B-CLL) have been shown to correlate with prognosis. Little is known about the relationship between chromosomal abnormalities and biological behavior of B-CLL cells in vitro. The present study was designed to explore the impact of chromosomal abnormalities determined by interphase fluorescence in situ hybridization (FISH) on the in vitro survival and immunogenicity of B-CLL. Considerable heterogeneity was noted in the in vitro survival and expression of costimulatory, adhesion, and antigen-presenting molecules by B-CLL cells. Spontaneous apoptosis of B-CLL cells in vitro was significantly lower in samples with good prognosis cytogenetics when compared to samples with poor prognosis cytogenetics. In contrast, B-CLL cells from samples with good prognosis cytogenetics exhibited higher basal expression of molecules involved in costimulation, cellular adhesion, and antigen presentation, and induced significantly more T-cell proliferation in mixed lymphocyte cultures. We conclude that chromosomal aberrations of B-CLL cells correlate with the in vitro biological behavior of B-CLL. Our data indicate that good prognosis cytogenetics correlates with less spontaneous apoptosis but greater in vitro immunogenicity. These findings could have significant implications on the design of future therapeutic approaches in patients with CLL, and the likelihood of response based on cytogenetics.

Mitochondria control of cell death induced by anti-HLA-DR antibodies.

We have previously reported that crosslinking HLA-DR directly induces programmed cell death of malignant B cells. The present study further characterizes the biochemical mechanism for HLA-DR-mediated programmed cell death of tumor cells. Phosphatidylserine exposure on the plasma membrane and propidium iodide incorporation occur with very rapid kinetics and are observed as early as 10 min after the induction of cell death with anti-HLA-DR. In striking contrast to anti-CD95, we observe no activation of caspase-3, -8, or -9 upon anti-HLA-DR addition. Furthermore, the irreversible caspase inhibitor Z-VAD.fmk also failed to inhibit anti-HLA-DR-mediated cell death, further supporting the conclusion that HLA-DR induces cell death via a caspase-independent mechanism. We demonstrate that anti-HLA-DR-induced cell death is instead associated with a rapid disruption of the inner mitochondrial transmembrane potential, DeltaPsi(m), a process that is significantly inhibited by Bcl-2 overexpression. Furthermore, we find that DeltaPsi(m) disruption results in the selective release of apoptosis-inducing factor (AIF) from the mitochondria. We propose that AIF is acting to initiate the morphological and biochemical changes observed in HLA-DR-mediated cell death.

Divergent therapeutic and immunologic effects of oligodeoxynucleotides with distinct CpG motifs.

Immune stimulatory oligodeoxynucleotides (ODN) with unmethylated CpG motifs are potent inducers of both innate and adaptive immunity. It initially appeared that a single type of optimal CpG motif would work in all applications. We now report that specific motifs of CpG ODN can vary dramatically in their ability to induce individual immune effects and that these differences impact on their antitumor activity in different tumor models. In particular, a distinct type of CpG motif, which has a chimeric backbone in combination with poly(G) tails, is a potent inducer of NK lytic activity but has little effect on cytokine secretion or B cell proliferation. One such NK-optimized CpG ODN (1585) can induce regression of established melanomas in mice. Surprisingly, no such therapeutic effects were seen with CpG ODN optimized for activation of B cells and Th1-like cytokine expression (ODN 1826). The therapeutic effects of CpG 1585 in melanoma required the presence of NK but not T or B cells and were not associated with the induction of a tumor-specific memory response. In contrast, CpG 1826, but not CpG 1585, was effective at inducing regression of the EL4 murine lymphoma; this rejection was associated with the induction of a memory response and although NK cells were necessary, they were not sufficient. These results demonstrate that selection of optimal CpG ODN for cancer immunotherapy depends upon a careful analysis of the cellular specificities of various CpG motifs and an understanding of the cellular mechanisms responsible for the antitumor activity in a particular tumor.

Humanization and characterization of the anti-HLA-DR antibody 1D10.

1D10 is a previously described antibody that binds to cells from a majority of B-cell malignancies. The current studies were designed to further evaluate the antigen specificity of 1D10 and its potential as an immunotherapeutic agent. Studies with transfectants and immunoprecipitation demonstrated that 1D10 recognizes some, but not all, of the human HLA-DR beta chains. Both normal and malignant B cells can express the 1D10 antigen. A humanized version of 1D10 was produced using CDR grafting. The resulting antibody has an affinity that is similar to that of the parental murine antibody. In addition, the humanized antibody is capable of inducing complement-mediated cytotoxicity, antibody-dependent cell cytotoxicity, and direct apoptosis of 1D10-expressing B cells. Based on these in vitro anti-tumor activities, we conclude humanized 1D10 deserves further evaluation as an immunotherapeutic agent.

CpG DNA increases primary malignant B cell expression of costimulatory molecules and target antigens.

Multiple factors, including expression of costimulatory molecules, antigen-presenting molecules, and target antigens, likely impact the efficacy of antibody therapy and other approaches to the immunotherapy of B cell malignancy. Unmethylated CpG-dinucleotides in select base contexts ("CpG motifs") that resemble sequences found in bacterial DNA are potent immunostimulatory agents capable of inducing a complex immune response, including a strong B cell stimulus. We examined the effect of a potent human CpG oligonucleotide (CpG ODN 2006) on different types of primary human malignant B cells and reactive follicular hyperplasia. CpG oligodeoxynucleotide (CpG ODN), but not control (non-CpG ODN), increased the expression of costimulatory molecules (CD40, CD80, CD86, CD54) on malignant B cells without altering the phenotype of B cells obtained from reactive follicular hyperplasia. CpG ODN also enhanced expression of class I and class II MHC in most samples. CD20 expression was increased in response to CpG ODN, most notably in B-CLL and marginal zone lymphoma. An inverse correlation was found between baseline expression of CD20 and CD40 and their expression after exposure to CpG ODN, thus the most significant increase in expression of these molecules was found in those samples that had the lowest baseline levels. In conclusion, CpG ODN can lead to increasing expression of molecules involved in costimulation, antigen presentation, and as targets for antibody-based therapy and deserve further evaluation as potential immunotherapeutic agents for B cell malignancy.

APC stimulated by CpG oligodeoxynucleotide enhance activation of MHC class I-restricted T cells.

Oligonucleotides containing unmethylated CpG motifs (cytosine-phosphorothioate-guanine oligodeoxynucleotide (CpG ODN)) are potent immunostimulatory agents capable of enhancing the Ag-specific Th1 response when used as immune adjuvants. We evaluated the cellular mechanisms responsible for this effect. Development of a CTL response was enhanced when mice were immunized with peptide-pulsed dendritic cells (DCs) treated with CpG ODN. However, in vitro, CpG ODN had no direct effect on highly purified T cells. In vitro, CpG ODN treatment of peptide- or protein-pulsed DCs enhanced the ability of the DCs to activate class I-restricted T cells. The presence of helper T cells enhanced this effect, indicating that treatment with CpG ODN does not obviate the role of T cell help. The enhanced ability of CpG ODN-treated DCs to activate T cells was present but blunted when DCs derived from IL-12 knockout mice were used. Fixation of Ag-pulsed, CpG ODN-treated DCs limited their ability to activate T cells. In contrast, fixation had little effect on DC activation of T cells when DCs were not exposed to CpG ODN. This indicates that production of soluble factors by DCs stimulated with CpG ODN plays a particularly important role in their ability to activate class I-restricted T cells. We conclude that CpG ODN enhances the development of a cellular immune response by stimulating APCs such as DCs, to produce IL-12 and other soluble factors.

The immunobiology and clinical potential of immunostimulatory CpG oligodeoxynucleotides.

Over 100 years ago, Coley first explored the use of bacterial products as immunostimulatory therapy for nonbacterial disease. It is now clear that bacterial DNA, and synthetic oligodeoxynucleotides containing specific motifs centered on a CpG dinucleotide (CpG ODN), are potent immunostimulatory agents. The molecular mechanisms responsible for the immunostimulatory effects of CpG ODN have yet to be elucidated fully, although it is clear that CpG ODN act rapidly on a variety of cell types. This includes activation of B cells, natural killer cells, and antigen-presenting cells including monocytes, macrophages, and dendritic cells. These effects have led to evaluation of CpG ODN as immune adjuvants in immunization where they have been shown in animal models to enhance the development of a TH1-type immune response. Preliminary results from clinical trials using CpG ODN as an immune adjuvant are promising. Preclinical studies suggest CpG ODN can also enhance innate immunity against a variety of infections, synergize with monoclonal antibody to enhance antibody-dependent cellular cytotoxicity, and alter the Th1/Th2 balance as a possible treatment for allergic diseases and asthma. Clinical evaluation has recently begun to determine whether promising preclinical results with CpG ODN can be translated into effective and tolerable clinical treatment approaches.

Combination immunotherapy of relapsed or refractory low-grade or follicular non-Hodgkin's lymphoma with rituximab and interferon-alpha-2a.

Rituximab and IFN have each demonstrated single-agent activity in patients with low-grade non-Hodgkin's lymphoma (NHL). A single-arm, multicenter, Phase II trial was conducted to assess the safety and efficacy of combination therapy with rituximab and IFN-alpha-2a in 38 patients with relapsed or refractory, low-grade or follicular, B-cell NHL. IFN-alpha-2a [2.5 or 5 million units (MIU)] was administered s.c., three times weekly for 12 weeks. Starting on the fifth week of treatment, rituximab was administered by i.v. infusion (375 mg/m2) weekly for 4 doses. All 38 patients received four complete infusions of rituximab and were evaluable for efficacy, although 11 patients (29%) did not-receive all 36 injections of IFN. The mean number of IFN-alpha-2a injections was 31 doses; the mean total units received were 141 MIU (maximum, 180 MIU). The study treatment was reasonably well tolerated with no unexpected toxicities stemming from the combination therapy. No grade 4 events were reported. Frequent adverse events during the treatment period included asthenia (35 of 38 patients), chills (31 of 38), fever (30 of 38), headache (28 of 38), nausea (23 of 38), and myalgia (22 of 38). The overall response rate was 45% (17 of 38 patients); 11% had a complete response, and 34% had a partial response. The Kaplan-Meier estimates for the median response duration and the median time to progression in responders are 22.3 and 25.2 months, respectively. Further follow-up is needed to determine whether this treatment combination leads to a significantly longer time to progression than single-agent treatment with rituximab.

Uses of granulocyte-macrophage colony-stimulating factor in vaccine development.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a potent cytokine capable of inducing differentiation, proliferation, and activation of a variety of immunologically active cell populations. In addition to its effects on stimulating granulocytic hematopoiesis, it also facilitates development of both humoral and cellular mediated immunity. Accordingly, strategies involving the use of GM-CSF as a vaccine adjuvant have attracted considerable attention. These strategies include the systemic administration of soluble GM-CSF with an immunogen, and also its use as part of gene therapy approaches to immunization. Because of the potency of this cytokine as an immune adjuvant, particular interest has focused on its use to overcome poorly immunogenic antigens such as those associated with intracellular infections and cancer. This review focuses on recent advances in the use of GM-CSF as a vaccine adjuvant.

CpG oligodeoxynucleotides enhance monoclonal antibody therapy of a murine lymphoma.

Bacterial DNA and synthetic oligodeoxynucleotides containing unmethylated cytosine-guanine dinucleotides known as cytosine phosphorothioate guanine oligodeoxynucleotides (CpG ODN) can activate various immune-cell subsets, including cells that participate in antibody-dependent cell-mediated cytotoxicity (ADCC). Studies have shown that CpG ODN enhance the efficacy of antitumor monoclonal antibody (MoAb) therapy in the 38C13 murine B-cell lymphoma. We performed a series of in vivo experiments using this tumor model to better characterize combination therapy with MoAb and CpG ODN. CpG ODN enhanced the efficacy of MoAb therapy of lymphoma in a dose-dependent manner. This effect was seen whether the CpG ODN was given before or after the MoAb therapy, but was decreased when CpG ODN was given more than 2 days after MoAb therapy. Three doses of CpG ODN and MoAb were more effective than single doses. There was no obvious toxicity with multiple dosing. These studies confirm that immunostimulatory CpG ODN enhance the efficacy of MoAb therapy, and that multiple courses of combination therapy with CpG ODN can serve as an effective therapy for lymphoma. Further exploration of this potentially potent combination of treatments, including clinical evaluation, is indicated.

Bispecific antibodies in cancer therapy.

Based upon in vitro and animal studies, a number of Phase I and II clinical trials have been initiated to test whether bispecific antibodies could redirect immune effectors against tumor cells in cancer patients. Recently, results from those trials showed beneficial effects in some patients but it is clear many problems remain to be solved. In addition, molecular engineering approaches are providing new and improved sources of clinically relevant bispecific antibodies.

CpG DNA: a potent signal for growth, activation, and maturation of human dendritic cells.

DNA molecules containing unmethylated CpG-dinucleotides in particular base contexts ("CpG motifs") are excellent adjuvants in rodents, but their effects on human cells have been less clear. Dendritic cells (DCs) form the link between the innate and the acquired immune system and may influence the balance between T helper 1 (Th1) and Th2 immune responses. We evaluated the effects of CpG oligodeoxynucleotides alone or in combination with granulocyte-macrophage colony-stimulating factor (GMCSF) on different classes of purified human DCs. For primary dendritic precursor cells isolated from human blood, CpG oligonucleotides alone were superior to GMCSF in promoting survival and maturation (CD83 expression) as well as expression of class II MHC and the costimulatory molecules CD40, CD54, and CD86 of DCs. Both CD4-positive and CD4-negative peripheral blood dendritic precursor cells responded to CpG DNA which synergized with GMCSF but these DCs showed little response to lipopolysaccharide (LPS). In contrast, monocyte-derived DCs did not respond to CpG, but they were highly sensitive to LPS, suggesting an inverse correlation between CpG and LPS sensitivity in different subsets of DCs. Compared with GMCSF, CpG-treated peripheral blood DCs showed enhanced functional activity in the mixed lymphocyte reaction and induced T cells to secrete increased levels of Th1 cytokines. These findings demonstrate the ability of specific CpG motifs to strongly activate certain subsets of human DCs to promote Th1-like immune responses, and support the use of CpG DNA-based trials for immunotherapy against cancer, allergy, and infectious diseases.

5E10: a prostate-specific surface-reactive monoclonal antibody.

We created mAb that reacted solely with prostate epithelial cells. The strategy involved immunizing mice with a mixture of six different prostatic carcinoma cell lines and selecting by flow cytometry only those antibodies that bind whole cells. The primary screening was performed using a mixture of all six prostate cell lines used in immunization together with six non-prostate cell lines in the same tube. Antibodies that gave a bimodal pattern of surface staining were selected for further evaluation. The most attractive clone, designated 5E10, produced IgG1 mAb that recognized four of the six prostatic cell lines and did not react with non-prostate tumor cell lines, peripheral blood and bone marrow mononuclear cells and endothelial and bone marrow stromal cells. 5E10 mAb reacted with both benign and malignant prostate in eight of eight histological samples and no reactivity was noted with non-prostate normal tissues. The 5E10 antigen is a transmembrane glycoprotein with a molecular weight of 110 kDa and the epitope recognized by 5E10 is extracellular. Ongoing studies are exploring the nature of this antigen in more depth.

Monoclonal antibodies in the treatment of human B-cell malignancies.

Monoclonal antibody technology emerged in the 1970's and was greeted by a wave of optimism. Many believed this new form of therapy would be effective in the treatment of human cancers. Early clinical trials in B-cell lymphomas demonstrated both the potential and limitations of unlabeled murine monoclonal antibody therapy, and taught us valuable lessons regarding the importance of the antibody structure, and nature of the targeted antigen. Since that time modifications in antibody structure and careful selection of target antigen have improved the clinical efficacy of these agents. Clinical trials using humanized antibodies have demonstrated that human/mouse chimeric antibodies and humanized antibodies have enhanced anti-tumor activity, decreased immunogenicity, and a very favorable toxicity profile. Radiolabeled monoclonal antibodies can induce durable remissions in lymphoma with toxicity limited largely to bone marrow suppression. Clinical trials with immunotoxins have demonstrated anti-tumor activity but also have been associated with significant toxicity. Standard treatment options for B-cell lymphoma will soon include antibody-based therapies. Further basic and clinical research is needed so we can understand more thoroughly the mechanisms responsible for the observed anti-tumor effects, and explore more extensively the best approach to their clinical use.

Immunostimulatory CpG oligodeoxynucleotides enhance the immune response to vaccine strategies involving granulocyte-macrophage colony-stimulating factor.

Immunostimulatory oligodeoxynucleotides containing the CpG motif (CpG ODN) can activate various immune cell subsets and induce production of a number of cytokines. Prior studies have demonstrated that both CpG ODN and granulocyte-macrophage colony-stimulating factor (GM-CSF) can serve as potent vaccine adjuvants. We used the 38C13 murine lymphoma system to evaluate the immune response to a combination of these two adjuvants. Immunization using antigen, CpG ODN, and soluble GM-CSF enhanced production of antigen-specific antibody and shifted production towards the IgG2a isotype, suggesting an enhanced TH1 response. This effect was most pronounced after repeat immunizations with CpG ODN and antigen/GM-CSF fusion protein. A single immunization with CpG ODN and antigen/GM-CSF fusion protein 3 days before tumor inoculation prevented tumor growth. CpG ODN enhanced the production of interleukin-12 by bone marrow-derived dendritic cells and increased expression of major histocompatibility complex class I and class II molecules, particularly when cells were pulsed with antigen/GM-CSF fusion protein. We conclude that the use of CpG ODN in combination with strategies involving GM-CSF enhances the immune response to antigen and shifts the response towards a TH1 response and that this approach deserves further evaluation in tumor immunization approaches and other conditions in which an antigen-specific TH1 response is desirable.

Anti-CD3-based bispecific antibody designed for therapy of human B-cell malignancy can induce T-cell activation by antigen-dependent and antigen-independent mechanisms.

Anti-CD3 x anti-B-cell antigen bispecific monoclonal antibodies (bsAbs) can redirect T-cell-mediated lysis toward malignant B cells. Clinical trials with CD3-based bsAbs have shown toxicity in patients which is likely related to nonspecific T-cell activation and targeting. Our current studies were designed to explore the mechanisms responsible for the observed in vivo toxicity by evaluating the immunologic effects of 2 different bsAb preparations in vitro. 1D10 was used as the tumor specific arm of the bsAbs. This antibody reacts with a variant of HLA-DR found on a majority of pre-B- and B-cell malignancies, and normal B cells in some individuals. Anti-CD3 served as the T-cell specific arm. A 1D10 x anti-CD3 bispecific IgG (bsIgG) produced using the hybrid-hybridoma method was compared to a 1D10 x anti-CD3 bispecific F(ab')2 [bsF(ab')2] produced using the leucine zipper technique. In cytotoxicity assays, both bsIgG and bsF(ab')2 induced lysis by pre-activated T cells of 1D10 (+) malignant B cells. bsIgG at high concentrations also induced lysis of 1D10 (-) tumor cells, while bsF(ab')2 did not. Proliferation of T cells induced by bsIgG and bsF(ab')2 was also evaluated. Both forms of bsAbs induced T-cell proliferation in the presence of antigen (+) Raji cells, while only bsIgG did so in the presence of antigen (-) malignant B cells. bsF(ab')2 induced T-cell activation in the absence of any tumor cells when testing was performed on samples where the 1D10 target antigen was present on normal peripheral blood B cells. We conclude that non-specific T-cell activation from bsAbs can occur in an antigen-independent manner due to the Fc/Fc receptor (FcR) interaction, or in an antigen-dependent manner when antigen is expressed on normal or tumor cells. Both mechanisms may have been responsible for the toxicity observed in prior clinical studies.