Showing papers in "Journal of Immunotherapy in 2006"
TL;DR: This report is the first to describe the administration of recombinant interleukin-7 to humans and demonstrates the ability of this cytokine to mediate selective increases in CD4+ and CD8+ lymphocytes along with a decrease in the percentage ofCD4+ T-regulatory cells.
Abstract: Lymphopenia is a serious consequence of HIV infection and the administration of cancer chemotherapeutic agents. Although growth factors can be administered to patients to increase circulating neutrophils, there is no effective method to stimulate CD8+ lymphocyte production in humans, in vivo. This report is the first to describe the administration of recombinant interleukin-7 to humans and demonstrates the ability of this cytokine to mediate selective increases in CD4+ and CD8+ lymphocytes along with a decrease in the percentage of CD4+ T-regulatory cells. These studies suggest an important role for interleukin-7 in the treatment of patients with lymphopenia.
407 citations
TL;DR: Clinical studies to estimate the frequency of specific immune evasion mechanisms in individual patients, to correlate specific events with clinical outcome, and to develop strategies to counter resistance mechanisms should receive a high priority.
Abstract: The identification of tumor-expressed antigens that can be recognized by specific T lymphocytes has made it possible both to study the properties of T cells participating in anti-tumor immune responses in patients and also to develop antigen-specific immunotherapies as a treatment modality. Interestingly, moves toward intervention have proceeded at a faster pace than have investigations toward understanding. In melanoma in particular, many clinical trials of active immunization have been performed, and many of these have shown increases in tumor antigen-specific T cells circulating in the blood. However, clinical responses have been infrequent, arguing that mechanisms of resistance downstream from initial T cell priming may be dominant in many cases. In fact, may patients show spontaneous generation of immune effector cells and/or antibodies, implying that the priming phase has occurred already in such individuals even without vaccination. Recent attention has turned toward mechanisms of immune evasion at the effector phase of the anti-tumor immune response, predominantly within the tumor microenvironment. Evidence is accumulating that T cell-intrinsic hyporesponsiveness or anergy, extrinsic suppression by regulatory cell populations, inhibitory ligands such as PD-L1, soluble factors such as TGF-beta, and the activity of nutrient-catabolizing enzymes such as indoleamine 2,3-dioxygenase (IDO), may contribute to immune escape in different settings. Murine preclinical models have shown that interfering with each of these processes can translate into T cell-mediated tumor control. Clinical studies to estimate the frequency of specific immune evasion mechanisms in individual patients, to correlate specific events with clinical outcome, and to develop strategies to counter resistance mechanisms should receive a high priority.
276 citations
TL;DR: Higher serum levels and prolonged administration of anti-CTLA-4 antibody resulted in a trend toward a greater incidence of grade III/IV autoimmune toxicity than previously reported, but did not seem to increase objective response rates.
Abstract: We previously reported our experience in treating 56 patients with metastatic melanoma using a human anti-cytotoxic T-lymphocyte antigen 4 (CTLA-4) antibody. Durable tumor regressions were seen that correlated with the induction of autoimmune toxicities. In this study, we treated 46 additional patients using an intrapatient dose escalation schema to test whether higher doses of anti-CTLA-4 antibody would induce increased autoimmunity and concomitant tumor regression. Twenty-three patients started anti-CTLA-4 antibody administration at 3 mg/kg and 23 patients started treatment at 5 mg/kg, receiving doses every 3 weeks. Patients were dose-escalated every other dose to a maximum of 9 mg/kg or until objective clinical responses or grade III/IV autoimmune toxicity were seen. Escalating doses of antibody resulted in proportionally higher plasma concentrations. Sixteen patients (35%) experienced a grade III/IV autoimmune toxicity. Five patients (11%) achieved an objective clinical response. Two of the responses are ongoing at 13 and 16 months, respectively. Flow cytometric analysis of peripheral blood revealed significant increases in both T-cell surface markers of activation and memory phenotype. Thus, higher serum levels and prolonged administration of anti-CTLA-4 antibody resulted in a trend toward a greater incidence of grade III/IV autoimmune toxicity than previously reported, but did not seem to increase objective response rates.
258 citations
TL;DR: The present results justify the design of larger follow-up studies to assess the clinical response to DC vaccines loaded with killed allogeneic tumor cells in patients with metastatic melanoma and suggest that cross-priming/presentation of melanoma antigens by DC vaccine had occurred.
Abstract: Dendritic cells (DCs) loaded with killed allogeneic tumors can cross-prime tumor-specific naive CD8 T cells in vitro, thereby providing an option to overcome human leukocyte antigen restriction inherent to loading DC vaccines with peptides. We have vaccinated 20 patients with stage IV melanoma with autologous monocyte-derived DCs loaded with killed allogeneic Colo829 melanoma cell line. DCs were generated by culturing monocytes with granulocyte macrophage-colony stimulating factor (granulocyte macrophage-colony stimulating factor) and interleukin (IL-4) and activated by additional culture with tumor necrosis factor and CD40 ligand. A total of 8 vaccines were administered at monthly intervals. The first patient was accrued December 2002 and the last November 2003. Fourteen patients were alive at 12 months, 9 patients were alive at 24 months, and 8 patients are alive as of January 2006. The estimated median overall survival is 22.5 months with a range of 2 to 35.5 months. Vaccinations were safe and tolerable. They induced, in 2 patients who failed previous therapy, durable objective clinical responses, 1 complete regression (CR) and 1 partial regression (PR) lasting 18 and 23 months, respectively. Three out of 13 analyzed patients showed T-cell immunity to melanoma antigen recognized by autologous T cells (MART-1) tissue differentiation antigen. Two of 3 patients showed improved immune function after vaccinations demonstrated by improved secretion of interferon (IFN)-gamma or T-cell proliferation in response to MART-1 derived peptides. In one of these patients, vaccination led to elicitation of CD8 T-cell immunity specific to a novel peptide-derived from MART-1 antigen, suggesting that cross-priming/presentation of melanoma antigens by DC vaccine had occurred. Thus, the present results justify the design of larger follow-up studies to assess the clinical response to DC vaccines loaded with killed allogeneic tumor cells in patients with metastatic melanoma.
235 citations
TL;DR: The reduced immunogenicity of this new generation of mAbs is expected to enhance efficacy, safety, and ease of use.
Abstract: Monoclonal antibody (mAb) therapy has been facilitated by a number of technologic advances over the past 30 years. Whereas hybridoma development of murine mAbs was requisite for the development of mAbs as drugs, the inherent immunogenicity of rodent sequences in humans has presented obstacles to the clinical application of mAbs. Sensitization to mAb therapeutics poses significant risk to the patient and may blunt the efficacy of these therapies. The advent of chimeric antibodies lessened but did not eliminate the rodent content of mAbs; thus, immunogenicity remained a concern. Further elimination of rodent sequences enabled the production of humanized mAbs, followed by current technology using phage display and, finally, transgenic mice technology, which allows for the generation of fully human therapeutic mAbs. The reduced immunogenicity of this new generation of mAbs is expected to enhance efficacy, safety, and ease of use. In addition to providing replacements for existing mAb drugs, new technologies have greatly facilitated the optimization and modification of mAbs, opening numerous therapeutic avenues.
180 citations
TL;DR: CpG 7909 can be safely given as a 2-hour IV infusion to patients with previously treated non-Hodgkin lymphoma at doses that have immunomodulatory effects, and two subjects demonstrated late response.
Abstract: Oligodeoxynucleotides containing CpG motifs (CpG ODN) can alter various immune cell subsets important in antibody therapy of malignancy. We undertook a phase I trial of CPG 7909 (also known as PF-3512676) in patients with previously treated lymphoma with the primary objective of evaluating safety across a range of doses, and secondary objectives of evaluating immunomodulatory effects and clinical effects. Twenty-three patients with previously treated non-Hodgkin lymphoma received up to 3 weekly 2-hour intravenous (IV) infusions of CPG ODN 7909 at dose levels 0.01 to 0.64 mg/kg. Evaluation of immunologic parameters and clinical endpoints occurred for 6 weeks. Infusion-related toxicity included grade 1 nausea, hypotension, and IV catheter discomfort. Serious adverse hematologic events observed more than once included anemia (2=Gr3, 2=Gr4), thrombocytopenia (4=Gr3), and neutropenia (2=Gr3), and were largely judged owing to progressive disease. Immunologic observations included: (1) The mean ratio of NK-cell concentrations compared with pretreatment at day 2 was 1.44 (95% CI=0.94-1.94) and at day 42 was 1.53 (95% CI=1.14-1.91); (2) NK activity generally increased in subjects; and (3) Antibody-dependent cellular cytotoxicity activity increased in select cohorts. No clinical responses were documented radiographically at day 42. Two subjects demonstrated late response. We conclude CpG 7909 can be safely given as a 2-hour IV infusion to patients with previously treated non-Hodgkin lymphoma at doses that have immunomodulatory effects.
148 citations
TL;DR: Macrophage recruitment by Ab-opsonized tumor cells did not result in enhanced cytokine secretion, suggesting that the cytokine shower observed in rituximab-treated patients is not caused by macrophage activation, and that cytokines have no role in CLL killing.
Abstract: Antibody-dependent cellular cytotoxicity (ADCC) is one of the mechanisms of tumor killing during antibody (Ab) immunotherapy, and a role for myeloid cells as effectors has been observed in several models. We are developing immunotherapy approaches based on administration of large numbers of ex vivo interferon-gamma-activated macrophages to cancer patients. With a quantitative assay measuring killing of nonproliferating tumor cells, we evaluated whether, in physiologic conditions, these macrophages synergize with the anti-CD20 Ab rituximab for killing primary B-cell chronic lymphocytic leukemia (B-CLL) cells. ADCC reached levels of 70% to 80% at effector to target ratios as low as 1:1. Macrophage recruitment by Ab-opsonized tumor cells did not result in enhanced cytokine secretion, suggesting that the cytokine shower observed in rituximab-treated patients is not caused by macrophage activation, and that cytokines have no role in CLL killing. We observed that uptake of tumor material by macrophages was not directly correlated to tumor killing. Nonetheless, experiments in the presence of cytochalasin D showed that ADCC occurred mainly by phagocytosis. Tumor killing was largely mediated by Fc gammaRI and inhibited by increasing concentration of serum. Importantly, complement deposition on B-CLL cells did not seem to enhance macrophage ADCC in this model, as complement-depleted and complement-repleted human plasmas exerted comparable inhibition.
116 citations
TL;DR: It is concluded that Pan02 tumor promotes the prevalence of Treg, in part through the secretion of TGF-β, which may result in immune evasion.
Abstract: We reported earlier that patients with breast or pancreas cancer have an increased prevalence of regulatory T cells (Treg) in the blood and tumor draining lymph nodes (TDLNs) compared with healthy individuals. In the current study, we tested the hypothesis that tumor cells promote the prevalence of Treg. The transforming growth factor-beta (TGF-beta) secreting murine pancreas adenocarcinoma, Pan02 cell line was injected into syngeneic C57BL/6 mice and the prevalence of Treg in the TDLNs and tumor spleen was measured weekly. Compared with control mice, the prevalence of CD25+ CD4+ cells in TDLNs and in tumor spleen increased with tumor growth. Analysis of these CD25+ CD4+ T cells in vitro confirmed expression of the Treg marker, Foxp3. In addition, their functional activity resembled that of Treg, as evidenced by a poor proliferative capacity; suppression of proliferation of CD25- CD4 or CD8T cells and inhibition of interferon-gamma release by CD25- CD4+ T cells. Reconstitution of Pan02-bearing Rag-/- mice with naive syngeneic CD25- CD4+ T cells induced CD25+ CD4+ Foxp3+ T cells in TDLNs, but not in the spleen. In contrast, Foxp3 was not detected in unreconstituted Pan02-bearing Rag-/- mice, or reconstituted mice bearing a TGF-beta-negative esophageal tumor. Furthermore, administration of neutralizing anti-TGF-beta antibody blocked the induction of Foxp3 in reconstituted Pan02-bearing Rag-/- mice. These results mimic earlier in vitro studies showing induction of Foxp3 through CD3 plus CD28 stimulation in the presence of TGF-beta. We conclude that Pan02 tumor promotes the prevalence of Treg, in part through the secretion of TGF-beta, which may result in immune evasion.
110 citations
TL;DR: It is shown that TNF/DC are not terminally differentiated but can still respond to the microbial stimulus lipopolysaccharide, which is important for selecting the appropriate injection route of human DC for tumor immunotherapy.
Abstract: Dendritic cell (DC) maturation can occur by different types of stimuli Previously, we described that murine DC matured with tumor necrosis factor (TNF) up-regulate surface MHC and costimulatory molecules but lack cytokine release, and therefore termed them semi-mature DC These TNF/DC-induced tolerance after intravenous (iv) injection in a model of experimental autoimmune encephalomyelitis (EAE) Here, we show that TNF/DC are not terminally differentiated but can still respond to the microbial stimulus lipopolysaccharide Subcutaneously injected TNF/DC induce an unpolarized T(H)1/T(H)2 response and are not protective in the experimental autoimmune encephalomyelitis model Although TNF/DC home to the draining lymph node, they remain negative for intracellular cytokine stainings However, the nonmigrating, endogenous DC started to produce interleukin (IL)-12p40, TNF and little IL-6, IL-10, and MCP-1 in a bystander fashion Together, DC matured with the inflammatory stimulus TNF remains responsive to further signals in vitro and in vivo These signals can be provided by pathogens or the subcutaneous injection route, which can convert them from tolerogenic to immunogenic DC These findings are important for selecting the appropriate injection route of human DC for tumor immunotherapy
91 citations
TL;DR: These studies demonstrate the immune-stimulatory properties and safety of anti-OX40 in primates and provide a strong scientific rationale to pursue clinical trials in humans.
Abstract: The immune-stimulatory properties of anti-CD134 (OX40) antibodies have been well documented in rodents, including their ability to enhance antitumor immunity. In this study, an anti-OX40 antibody (Ab) known to costimulate monkey T cells in vitro, was infused into rhesus macaque monkeys during immunization with the simian immunodeficiency virus protein, gp130. The draining lymph nodes from immunized monkeys treated with anti-OX40 were enlarged compared with immunized monkeys injected with mouse Ig. Anti-OX40-treated monkeys had increased gp130-specific Ab titers, and increased long-lived T-cell responses, compared with controls. There were no overt signs of toxicity in the anti-OX40-treated monkeys. The encouraging immune-stimulatory effects led to the good manufacturing practice production of an anti-OX40 Ab for clinical trials in cancer patients. A detailed toxicology study was performed with anti-OX40 in nonhuman primates. Three groups of 8 monkeys received anti-OX40 at 1 of 3 dose levels (0.4, 2.0, and 10 mg/kg) and a control group received saline. No clinical toxicity was observed, but acute splenomegaly and enlarged gut-associated lymph nodes were observed in the anti-OX40-treated animals; splenomegaly and lymphadenopathy resolved by day 28. These studies demonstrate the immune-stimulatory properties and safety of anti-OX40 in primates and provide a strong scientific rationale to pursue clinical trials in humans.
89 citations
TL;DR: A novel overnight method using Flt3 ligand-mobilized DCs loaded with antigenic peptides and pulsed with peptides to produce DCs for cancer immunotherapy is developed, which primes immune responses to cancer antigens.
Abstract: Flt3 ligand mobilizes dendritic cells (DCs) into blood, allowing generation in vivo of large numbers of DCs for immunotherapy. These immature DCs can be rapidly activated by soluble CD40 ligand (CD40L). We developed a novel overnight method using these cytokines to produce DCs for cancer immunotherapy. Flt3 ligand-mobilized DCs (FLDCs) were isolated, activated with CD40L, loaded with antigenic peptides from influenza matrix protein, hepatitis B core antigen, NY-ESO-1, MAGE-A4, and MAGE-A10, and injected into patients with resected melanoma. Three injections were given at 4-week intervals. Study end points included antigen-specific immune responses (skin reactions to peptides alone or peptide-pulsed FLDCs; circulating T-cell responses), safety, and toxicity. No patient had a measurable tumor. Six patients were entered. FLDCs were obtained, enriched, and cultured under Good Manufacturing Practice grade conditions. Overnight culture with soluble CD40L caused marked up-regulation of activation markers (CD83 and HLA-DR). These FLDCs were functional and able to stimulate antigen-specific T cells in vitro. No significant adverse events were attributable to FLDCs. Peptide-pulsed FLDCs caused strong local skin reactions up to 60 mm diameter with intense perivascular infiltration of T cells, exceeding those seen in our previous peptide-based protocols. Antigen-specific blood T-cell responses were induced, including responses to an antigen for which the patients were naive (hepatitis B core antigen) and MAGE-A10. MAGE-A10-specific T cells with a skewed T-cell receptor repertoire were detected in 1 patient in blood ex vivo and from tumor biopsies. Vaccination with FLDCs pulsed with peptides is safe and primes immune responses to cancer antigens.
TL;DR: In vitro incubation of human PBMCs with LMB-2 reduced the levels of CD4+CD25+ and Foxp3-expressing cells without impairing the function of the remaining lymphocytes, making it an attractive candidate for reducing human Treg cells in vivo before the administration of cancer vaccine or cell transfer immunotherapy approaches.
Abstract: CD4(+)CD25(+) T-regulatory cells (T(reg)) can inhibit the proliferation and cytokine secretion of CD4(+)CD25(-) helper T cells in mice and humans. In murine tumor models, the presence of these T(reg) cells can inhibit the antitumor effectiveness of T-cell transfer and active immunization approaches. We have thus initiated efforts to eliminate T(reg) cells selectively from human peripheral blood mononuclear cells (PBMCs) to potentially bolster antitumor responses. LMB-2 is a recombinant immunotoxin that is a fusion of a single-chain Fv fragment of the anti-Tac anti-CD25 monoclonal antibody to a truncated form of the bacterial Pseudomonas exotoxin A. In vitro incubation of human PBMCs with LMB-2 reduced the levels of CD4(+)CD25(+) and Foxp3-expressing cells without impairing the function of the remaining lymphocytes. The short in vivo half-life of LMB-2 makes it an attractive candidate for reducing human T(reg) cells in vivo before the administration of cancer vaccine or cell transfer immunotherapy approaches.
TL;DR: In this article, the efficacy of a GM-CSF-producing tumor vaccine given before and after docetaxel in mice bearing established lung tumors was evaluated, and significant tumor regression and prolonged survival were observed compared with chemotherapy alone.
Abstract: In this report, we evaluated the efficacy of a GM-CSF-producing tumor vaccine given before and after docetaxel in mice bearing established lung tumors. Mice bearing established 3LL tumors were treated with docetaxel and tumor vaccines transduced with either control or GM-CSF adenoviral vectors. Docetaxel (5-20 mg/kg) treatment alone had only a minimal effect on growth of established 3LL tumors in vivo, although docetaxel was cytotoxic to 3LL cells in vitro. When mice bearing established 3LL tumors were pretreated with docetaxel followed by vaccination with irradiated GM-CSF- transduced 3LL tumor cells, significant tumor regression and prolonged survival were observed compared with chemotherapy alone. Delaying docetaxel treatment until after tumor vaccination abrogated the vaccine's anti-tumor effects. Mice that survived treatment were able to resist a lethal rechallenge of 3LL tumor cells. Memory CTL specific for an epitope (MUT-1) derived from 3LL were detected in surviving mice. Docetaxel induced a mild lymphodepletion in mice, both CD4 and CD8 subsets were reduced in LN and spleens. Interestingly, docetaxel also diminished the number of memory CD8+ T cells (CD122+) and possible CD4+ CD25+ Foxp3+ natural Treg cells. Docetaxel treatment did not affect antigen-driven proliferation of naive T cells but significantly promoted survival of activated T cells. Thus, augmentation of vaccine induced antitumor immunity in docetaxel-treated mice primarily due to the enhanced survival of antigen-experienced T cells.
TL;DR: In in vivo experiments, the anticancer effect of OK-432 was significantly inhibited by suppression of phagocytosis activity by cytochalasin B, and in vitro experiments elucidated the mechanism mediated by phagocytes and TLR4 signaling in the immune effect ofOK-432.
Abstract: It has previously been reported by our group that Toll-like receptor (TLR) 4 is involved in anticancer immunity induced by OK-432, a Streptococcus-derived immunotherapeutic agent. However the detailed mechanism of the OK-432-induced immune response via TLR4 remained uncertain, because it may not be possible for OK-432, which consists of whole bacterial bodies, to bind directly to TLR4. In the current study, we conducted in vitro and in vivo experiments to investigate the hypothesis that OK-432 may first be captured and dissolved by phagocytes and that the active components released by the cells may then induce host responses via TLR4. TS-2 monoclonal antibody, which recognizes an active component of OK-432 designated OK-PSA was used in the current study. First, it was observed that OK-432-induced cytokine production by dendritic cells (DCs) and macrophages was significantly inhibited in vitro by cytochalasin B, a phagocytosis inhibitor. Immunofluorescence staining using TS-2 demonstrated that OK-432 was captured and dissolved by phagocytes. OK-PSA was detected in the supernatants derived from OK-432-treated DC culture by enzyme-linked immunosorbent assay using TS-2. Supernatants from OK-432-treated DC culture increased nuclear factor (NF)-kappaB activity in TLR4-expressing cells, and the increased activity was inhibited by TS-2 antibody. OK-432 itself did not activate NF-kappaB in these cells. In in vivo experiments, the anticancer effect of OK-432 was significantly inhibited by suppression of phagocytosis activity by cytochalasin B. In this case, the amount of OK-PSA, an active component of OK-432, in the sera was also reduced by cytochalasin B. These findings elucidated the mechanism mediated by phagocytosis and TLR4 signaling in the immune effect of OK-432.
TL;DR: Vaccination with 1E10 was immunogenic and relatively well tolerated, and the 0.5-mg dose level was selected for future trials because similar results were observed with the 3 tested dose levels.
Abstract: Summary: In this study, the immunogenicity and toxicity profile of 1E10, an anti-idiotypic vaccine mimicking the N-glycolylGM3 ganglioside, was investigated with an extended vaccination protocol. The year-long vaccination scheme consisted of 6 biweekly intradermal injections (induction phase), followed by 10 monthly boosters (maintenance). Nineteen patients with high-risk (stage III) or metastatic breast cancer were vaccinated with different dose levels of 1E10 (0.5, 1, and 2 mg). The humoral and cellular responses to 1E10 and the targeted ganglioside were assessed at baseline and throughout the treatment. Local skin reactions represented the most common adverse event (National Cancer Institute Toxicity Criteria (NCIC) grades I and II), followed by mild flu-like symptoms lasting for 1 to 2 days. Two patients were removed from the study because of vaccine-related hypersensitivity reactions. A third patient was removed from the study after a transient loss of consciousness with uncertain relation to the vaccine. All patients showed a strong antibody response to the targeted ganglioside. In addition, ganglioside-specific T-cell responses were recorded in 5 of 13 evaluable patients. Vaccination with 1E10 was immunogenic and relatively well tolerated. Because similar results were observed with the 3 tested dose levels, the 0.5-mg dose level was selected for future trials.
TL;DR: The findings clearly show that the concomitant activation of both CD4+ and CD8+ (memory) T-cells using mRNA-electroporated CD40-B cells is feasible in CMV and HIV-1–seropositive persons, which indicates the potential value of this approach for application in cellular immunotherapy of infectious diseases.
Abstract: Recently, it has become obvious that not only CD8 + T-cells, but also CD4 + T-helper cells are required for the induction of an effective, long-lasting cellular immune response. In view of the clinical importance of cytomegalovirus (CMV) and human immunodeficiency virus (HIV) infection, we developed 2 strategies to simultaneously reactivate viral antigen-specific memory CD4 + and CD8 + T-cells of CMV-seropositive and HIV-seropositive subjects using mRNA-electroporated autologous CD40-activated B cells. In the setting of HIV, we provide evidence that CD40-activated B cells can be cultured from HAART-naive HIV-1 seropositive patients. These cells not only express and secrete the HIV p24 antigen after electroporation with codon-optimized HIV-1 gag mRNA, but can also be used to in vitro reactivate Gag antigen-specific interferon-γ-producing CD4 + and CD8 + autologous T-cells. For the CMV-specific approach, we applied mRNA coding for the pp65 protein coupled to the lysosomal-associated membrane protein-1 to transfect CD40-activated B cells to induce CMV antigen-specific CD4 + and CD8 + T-cells. More detailed analysis of the activated interferon-γ-producing CMV pp65 tetramer positive CD8 + T-cells revealed an effector memory phenotype with the capacity to produce interleukin-2. Our findings clearly show that the concomitant activation of both CD4 + and CD8 + (memory) T-cells using mRNA-electroporated CD40-B cells is feasible in CMV and HIV-1-seropositive persons, which indicates the potential value of this approach for application in cellular immunotherapy of infectious diseases.
TL;DR: Vaccination with liposome-encapsulated peptide antigen and CpG ODN allows for the in vivo loading and activation of DC, thereby generating reactive CTL populations even against poorly immunogenic self-peptide presenting tumors resulting in a potent anti-tumor immune response.
Abstract: The induction of a potent and specific T cell response is a major challenge in the development of efficacious cancer vaccine strategies. We applied a novel liposomal formulation (AVE3) for efficient delivery of antigenic peptides into APCs of the skin. These liposomes resulted in a long-lasting deposition of encapsulated compounds at the injection site and the draining lymph nodes. Using a peptide from the melanocyte differentiation antigen tyrosinase-related protein (TRP2) 2 we could show that vaccination with liposome-encapsulated peptide in combination with oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODNs) as adjuvant leads to the induction of tumor cell-specific cytotoxic T cells. The most potent immune response was observed when both, TRP2 peptide and CpG ODNs, were encapsulated into AVE3. Importantly, in contrast to vaccination with free TRP2 liposomal TRP2 peptide generated T cells which respond to 1000-fold lower antigen concentration. Using the poorly immunogenic B16 melanoma model we could demonstrate that vaccination with liposomal TRP2 peptide plus CpG ODNs but not vaccination with free peptide or adjuvant alone resulted in tumor protection in subcutaneous and metastatic tumor models. In summary, vaccination with liposome-encapsulated peptide antigen and CpG ODN allows for the in vivo loading and activation of DC, thereby generating reactive CTL populations even against poorly immunogenic self-peptide presenting tumors resulting in a potent anti-tumor immune response.
TL;DR: An IgG liposome with physicochemical properties suitable for delivering antigens to DCs is demonstrated and paves the way to the application of IgGliposomes for tumor immunotherapy using DCs.
Abstract: Liposomes represent a promising vehicle to deliver exogenous antigens to dendritic cells (DCs) for tumor immunotherapy. Targeting exogenous antigens to Fcgamma receptors on DCs has been shown to result in efficient presentation of antigen-derived peptides on major histocompatibility complex (MHC) class I and class II molecules. In this study, it was investigated whether DCs that endocytosed physicochemically optimized antigen-containing liposomes conjugated with IgG efficiently present antigens on MHC class I and class II molecules, and consequently induce strong antitumor immune responses. IgG-conjugated liposomes that were 200 nm in diameter without attaching polyethylene glycol were most efficiently endocytosed by DCs. Human monocyte-derived DCs that endocytosed tetanus toxoid (TT)-containing IgG liposomes via CD32 stimulated CD4(+) T cells more strongly than DCs pulsed with TT-containing bare liposomes or with soluble TT. Immunization of mice with DCs that endocytosed ovalbumin (OVA)-containing IgG liposomes but not OVA-containing bare liposomes or soluble OVA completely prevented the growth of OVA-expressing lymphoma cells. Importantly, administration of DCs that endocytosed OVA-containing IgG liposomes to the mice with established OVA-expressing tumors strongly suppressed tumor growth. This study demonstrates an IgG liposome with physicochemical properties suitable for delivering antigens to DCs and paves the way to the application of IgG liposomes for tumor immunotherapy using DCs.
TL;DR: ReGel is an aqueous, filter sterilizable ABA tri-block polymer consisting of poly-(lactide-co-glycolide) and polyethylene glycol as mentioned in this paper.
Abstract: ReGel is an aqueous, filter sterilizable ABA tri-block polymer consisting of poly-(lactide-co-glycolide) and polyethylene glycol We tested the suitability of this polymer to provide sustained interleukin-2 (IL-2) delivery for cancer immunotherapy ReGel/IL-2 is liquid at or below room temperature, and is easily injectable through narrow gauge needles, but undergoes a reversible thermal transition into a bioerodible depot at body temperature We demonstrated that ReGel/IL-2 releases IL-2 over 72 to 96 hours in vitro, without loss of bioactivity Pharmacokinetic studies after peritumoral injection of 01 mL ReGel/IL-2 in mice demonstrated an early burst of IL-2 release, followed by more sustained release kinetics over 96 hours (T(1/2)beta 48 h) Less than 15% of the injected dose was detectable in blood or kidneys during the first 48 hours A single peritumoral dose of ReGel/IL-2 [1 to 4 million international units (MIU) ReGel/IL-2, split into 4 quadrant injections] was administered to mice bearing subcutaneous RD-995 spindle cell carcinoma Only the highest dose of ReGel/IL-2 tested (40 MIU) resulted in significant hypotension on day 3 after injection Weekly treatment of Meth A fibrosarcoma and RENCA renal carcinoma with ReGel/IL-2 (2 MIU/dose) induced a significant reduction in tumor growth and improved survival Reduction in tumor growth at implants remote from treated lesions was also observed, suggesting systemic activation of antitumor immunity These findings establish that peritumoral injection of ReGel/IL-2 is an effective delivery system for cancer immunotherapy, while decreasing IL-2 toxicity This polymer delivery system is likely to be broadly applicable for sustained delivery of other cytokines and peptides
TL;DR: An in vitro expansion protocol for human TREG is developed and it is demonstrated that FoxP3 is not a reliable marker forhuman TREG as it is transiently inducible in CD4+CD25− cells upon activation with cytokines or via their T cell receptor.
Abstract: CD4+CD25+ regulatory T cells (TREG) are engaged in the regulation of murine and human immune responses as well as graft-versus-host disease (GvHD) after allogeneic stem-cell transplantation. Despite their suppression of GvHD they do not impair graft-versus-tumor activity in the mouse, which makes TR
TL;DR: In vitro generation of melanoma-reactive T lymphocytes was compared using 3 common γ-chain cytokines, interleukin (IL)-2, IL-7, and IL-15, alone or in combination, and the proliferation, function, and phenotype were evaluated.
Abstract: The adoptive transfer of human tumor-reactive T lymphocytes into autologous patients can mediate the regression of metastatic melanoma. Here, the in vitro generation of melanoma-reactive T lymphocytes was compared using 3 common gamma-chain cytokines, interleukin (IL)-2, IL-7, and IL-15, alone or in combination. The proliferation, function, and phenotype were evaluated for tumor-reactive T cells derived from peripheral blood mononuclear cells (PBMCs) from patients previously immunized with the melanoma-associated peptide gp100:209-217(210M) and PBMCs transduced with a retrovirus encoding the alpha and beta chains of a gp100-reactive T-cell receptor (TCR). IL-7 alone did not induce significant proliferation of any tumor-reactive T-cell population, whereas IL-2 and IL-15 induced significant proliferation of tumor-reactive T lymphocytes from both sources. Cells cultured in the presence of IL-2 or IL-15 secreted comparable amounts of interferon-gamma and IL-2 in response to melanoma cells in vitro and were phenotypically similar in terms of costimulatory molecules (CD27 and CD28), cytokine receptors (CD25, CD122, and CD127), and a lymphoid homing molecule (CD62L). In addition, the proliferation, function, and phenotype of T cells cultured with combinations of IL-2, IL-7, and IL-15 were similar to those grown with IL-2 alone. The effects of these cytokines on TCR stimulation of CD45RA+ naive cells derived from adult patients and from human umbilical cord blood were also compared. Similar to the data with activated tumor-reactive T lymphocytes, IL-7 alone did not support significant proliferation of naive T cells after TCR stimulation with anti-CD3, although IL-2 and IL-15 induced comparable proliferation of T lymphocytes with similar phenotypic attributes.
TL;DR: In this paper, the authors showed that transduction of cytotoxic T lymphocytes (CTLs) with a retroviral vector expressing the dominant-negative TGFbeta type II receptor (DNR) overcomes this tumor evasion in a model of Epstein-Barr virus (EBV)-positive Hodgkin disease.
Abstract: Transforming growth factor (TGF)-beta, a pleiotropic cytokine that regulates cell growth, is secreted by many human tumors and markedly inhibits tumor-specific cellular immunity. It has previously been shown by our group that transduction of cytotoxic T lymphocytes (CTLs) with a retroviral vector expressing the dominant-negative TGFbeta type II receptor (DNR) overcomes this tumor evasion in a model of Epstein-Barr virus (EBV)-positive Hodgkin disease. TGFbeta is an important physiologic regulator of T-cell growth and survival, however, abrogation of this regulatory signal in genetically modified cells is potentially problematic. To ensure that unresponsiveness to TGFbeta did not lead to the unregulated growth of genetically modified CTLs, the characteristics of DNR-transduced CTLs in vivo were studied. Donor C57BL6 mice were vaccinated with human papillomavirus-E7 plasmid DNA to induce production of E7-specific CTLs. The E7-specific CTLs were genetically modified to express enhanced green fluorescent protein (GFP) or DNR and administered to syngeneic mice. All mice received monthly boosts with E7 DNA for 9 months, and during this time, transduced CTLs were detected in the peripheral blood of most of the mice using a quantitative real-time polymerase chain reaction. By 12 months, 3 months after cessation of vaccination, no DNR-transduced CTLs or GFP-transduced CTLs were detected in the peripheral blood. There were 4 cases of lymphoma (2 DNR-transduced mice and 2 control mice): all tumors were CD3-/CD8- and were also negative for the DNR transgene. Hence, mature antigen-specific cytotoxic T cells can be genetically modified to resist the antiproliferative effects of TGFbeta without undergoing spontaneous lymphoproliferation in vivo. They may be of value for treating human cancers, which use TGFbeta as a powerful immune evasion mechanism.
TL;DR: Its low toxicity and the induction of a predominantly cellular immune response suggest that the addition of 300 to 400 μg GM-CSF to VACCIMEL is useful in increasing the immune response.
Abstract: We investigated whether recombinant human granulocyte-monocyte-colony-stimulating factor (rhGM-CSF) increased the immunogenicity of VACCIMEL, a vaccine consisting of 3 irradiated allogeneic melanoma cell lines. A phase I clinical trial was performed on 20 melanoma patients in stages IIB (n=2), III (n=10), and IV (n=8), who were disease free after surgery (n=16) or had minimal disease (n=4). Cohorts of 4 patients were vaccinated 4 times with VACCIMEL and bacillus Calmette Guerin (BCG) as adjuvant. Besides, the patients received placebo (group 1) or GM-CSF: 150 microg (group 2), 300 microg (group 3), 400 microg (group 4), and 600 microg (group 5) per vaccine. The combination of VACCIMEL and GM-CSF had low toxicity. Only in group 5, grade 2 thoracic pain (3/4 patients) and abdominal cramps (2/4 patients) were observed. Delayed-type hypersensitivity increased after vaccination and it was highest in group 4. Phytohemagglutinin stimulation of peripheral blood lymphocytes was analyzed in 9 patients: 4/9 had normal stimulation; 3/9 had low basal stimulation, which recovered after vaccination; and 2/9 were not stimulated. Antimelanoma antibodies preexisted in 9/19 patients; in 3/19 patients, antibodies anti-33 kd, 90 kd, and 100 kd antigens were induced by vaccination. IgG2 but not IgG1 antibodies were detected. Anti-BCG antibodies, mostly IgG2, reached the highest post/prevaccination ratio in group 4. Median serum interleukin-12 was lower in progressing patients (61.6 pg/mL) than in those without evident disease (89 pg/mL). Thus, its low toxicity and the induction of a predominantly cellular immune response suggest that the addition of 300 to 400 microg GM-CSF to VACCIMEL is useful in increasing the immune response.
TL;DR: Repeated immunization of mice enhanced short-term tumor suppression, resulting in the complete regression of primary tumors in up to 40% of the mice, but did not improve long-term survival owing to recurrence.
Abstract: A single intratumoral injection of interleukin-12 and granulocyte-macrophage colony-stimulating factor-encapsulated microspheres induced the regression of advanced spontaneous mammary tumors, suppressed additional tumor development, and enhanced survival in her-2/neu transgenic mice. Posttherapy tumor eradication was dependent on both CD4 + and CD8 + T cells and correlated with the tumor infiltration kinetics of a transient effector T-cell response. Upon long-term monitoring, tumor regression was found to be temporary, and disease-free survival was not achieved despite the development of systemic anti-tumor cytotoxic T-cell memory and antibody responses. Repeated immunization of mice enhanced short-term tumor suppression, resulting in the complete regression of primary tumors in up to 40% of the mice, but did not improve long-term survival owing to recurrence. The failure of chronic therapy to achieve complete cure was associated with an inability to maintain the intensity of the posttherapy effector T-cell response in this model.
TL;DR: The data demonstrate the influence of the N-glycosylation pattern on the ADCC activity of chimeric CD19 antibodies and point to the importance of suitable expression systems for the production of highly active therapeutic antibodies.
Abstract: To investigate the influence of N-linked oligosaccharides at asparagines-297 on the cytolytic potential of chimeric CD19 antibodies, three distinct variants were generated by production in different expression systems. The same chimeric CD19 antibody was produced in Sf21 insect cells, human 293 T cells, and 293 T cells expressing a co-transfected beta1,4-N-acetylglucosaminyltransferase III (GnTIII). The N-glycan structures and the cytolytic potential of the antibodies produced in these three systems were directly compared. After expression in insect cells, the antibody carried paucimannosidic N-linked oligosaccharides, distinct from the complex biantennary carbohydrate moieties attached to the product from human cells. After co-expression with GnTIII in human cells, the antibody carried an eightfold greater percentage of oligosaccharides with a bisecting N-acetylglucosamine (78.7% versus 9.6%) and a 30-fold increased proportion of bisecting, defucosylated oligosaccharides (15.9% versus 0.5%). The insect cell product triggered stronger antibody-dependent cellular cytotoxicity (ADCC) of a human leukemia-derived cell line than the product from non-re-engineered 293 T cells and was equally effective at 50- to 100-fold lower concentrations. The antibody from glyco-engineered 293 T cells had comparable lytic activity as the insect cell product. Both mediated significant ADCC at lower effector-to-target cell ratios than the antibody from non-re-engineered 293 T cells, and both were highly effective against primary blasts from pediatric leukemia patients. The data demonstrate the influence of the N-glycosylation pattern on the ADCC activity of chimeric CD19 antibodies and point to the importance of suitable expression systems for the production of highly active therapeutic antibodies.
TL;DR: The findings suggest that patients with recurrent or advanced tumor disease and metastatic spread could benefit from this modern therapeutic regime, especially after insufficient radioiodine therapy.
Abstract: Epithelial cell adhesion molecule (EpCAM) is expressed by a broad variety of carcinoma cells. It is recognized by the monoclonal antibody 17-1A, which has already been applied for immunotherapy of several carcinoma types in preclinical and small clinical studies. In the present study the immunohistochemical properties of 17-1A were evaluated in 121 cases of thyroid carcinomas of follicular cell origin, comprising of 75 differentiated (DTC; 35 papillary and 40 follicular carcinomas), 24 poorly differentiated (PDTC) and 22 anaplastic thyroid carcinomas. Overexpression of EpCAM, as recently defined, was found with a distinct membranous staining pattern in 81.3% of DTCs and in 66.6% of PDTCs. In contrast, all anaplastic thyroid carcinomas (0%) completely lacked EpCAM expression. Normal thyroid tissue presented with weak and heterogeneous EpCAM staining. This study demonstrates EpCAM overexpression as a common finding in DTCs and PDTCs, and thus these tumors as possible novel targets for EpCAM-directed immunotherapy. Our findings suggest that patients with recurrent or advanced tumor disease and metastatic spread could benefit from this modern therapeutic regime, especially after insufficient radioiodine therapy.
TL;DR: It is indicated that mixing peptides in the same emulsion can alter reactivity compared with peptides injected separately by mechanisms that may include the induction of localized nonspecific inflammation or competitive binding of peptides to major histocompatibility complex molecules.
Abstract: Efforts to develop effective cancer vaccines often use combinations of immunogenic peptides to increase the applicability and effectiveness of the immunizations. The immunologic consequences of combining more than 1 self/tumor antigen in a single vaccine emulsion remain unclear, however. We performed 2 sequential clinical trials in patients at high risk for melanoma recurrence. Patients were given the highly immunogenic gp100:209–217(210M) peptide and the less immunogenic tyrosinase:368–376(370D) peptide once every 3 weeks for 4 weeks. This vaccination course was 12 weeks long, and patients were vaccinated for up to 4 courses (16 total vaccinations). In the first trial in 31 patients, the peptides were emulsified separately in incomplete Freund adjuvant and injected at 2 different sites. In the second trial in 33 patients, the peptides were emulsified together and injected at the same site. Cryopreserved lymphocytes were obtained by apheresis after each course and were evaluated for antipeptide activity using tetramer, enzyme-linked immunospot, and in vitro sensitization boost assays. When the peptides were injected at separate sites, robust specific reactivity to the native gp100:209–217 peptide was measured by each of the assays, whereas immunization with the tyrosinase:368–376(370D) peptide was far less effective. When the peptides were emulsified and injected together at the same site, immunization to the gp100:209–217(210M) epitope dropped precipitously, whereas reactivity to the tyrosinase: 368–376(370D) peptide was enhanced. These cautionary data indicate that mixing peptides in the same emulsion can alter reactivity compared with peptides injected separately by mechanisms that may include the induction of localized nonspecific inflammation or competitive binding of peptides to major histocompatibility complex molecules.
TL;DR: Informatics approaches can be used to identify novel targets that are more specific for tumors, thereby enhancing safety and providing a means of directing toxic agents to the tumor, identify novel pathways essential for disease progression, and identify Fc mutants that have enhanced immune effector function.
Abstract: INTRODUCTION The antibodies currently approved for the treatment of diseases, including cancer, have been developed predominantly based on the understanding and identification of key targets involved in disease pathology. Thus, for oncology, currently marketed antibodies to epidermal growth factor receptors (EGFR/HER1 and HER2) and vascular endothelial growth factor (VEGF) treat cancer by blocking the function of these targets that are crucial for tumor progression. Other targets for launched products are those that are highly up-regulated on neoplastic cells, including CD20, CD52, and CD33. Antibodies are generally highly specific for their molecular targets and can be used to affect disease-specific targets, thereby sparing normal cells and causing less toxicity than traditional cytotoxic chemotherapies. Effective antibodies act through one or more of a variety of mechanisms, including (a) blocking essential cellular growth factors or receptors, (b) directly inducing apoptosis, (c) binding to target cells and recruiting ‘‘effector functions’’ such as antibodydependent cellular cytotoxicity (ADCC), or complementdependent cytotoxicity (CDC), and (d) delivering cytotoxic payloads such as chemotherapies, radioisotopes and toxins. The use of informatics will be essential as we develop new waves of products that provide additional efficacy, specificity, or safety over currently marketed products. Informatics approaches can be used to (a) identify novel targets either upstream or downstream of already validated targets to enhance or complement efficacy, (b) identify targets that are more specific for tumors, thereby enhancing safety and providing a means of directing toxic agents to the tumor, (c) identify novel pathways essential for disease progression, and (d) identify Fc mutants that have enhanced immune effector function. ANTIBODY TECHNOLOGY The first monoclonal antibodies from mice were generated in 1975. In humans, mouse-derived antibodies are highly immunogenic, and therefore ‘‘chimeric’’ antibodies were created by replacing mouse constant domains (non-antigen binding domains) with human constant domains. This improvement considerably reduced the immune response to therapeutic antibodies. Additional modifications of framework regions within the antigenbinding variable regions further reduce immunogenicity and result in what are termed ‘‘humanized’’ antibodies. Fully human antibodies can be derived from human cells or from genetically engineered mice transgenic for human antibody genes. Human antibodies can also be generated from antibody-expressing phage libraries as single chain Fv or Fab fragments that can subsequently be converted to full-length antibodies.
TL;DR: Assessment of antigen presentation by human DCs genetically modified with plasmid cDNAs, RNAs, adenoviruses, or retroviruses, encoding the melanoma antigen gp100 or the tumor-testis antigen NY-ESO-1 suggests that DCs transduced with viral vectors may be more efficient thanDCs transfected with c DNAs or RNAs for the induction of tumor reactive CD8+ and CD4+ T cells in vitro and in
Abstract: Genetic modification of dendritic cells (DCs) with recombinant vectors encoding tumor antigens may aid in developing new immunotherapeutic treatments for patients with cancer. Here, we characterized antigen presentation by human DCs genetically modified with plasmid cDNAs, RNAs, adenoviruses, or retroviruses, encoding the melanoma antigen gp100 or the tumor-testis antigen NY-ESO-1. Monocyte-derived DCs were electroporated with cDNAs or RNAs, or transduced with adenoviruses. CD34+ hematopoietic stem cell-derived DCs were used for retroviral transduction. Genetically modified DCs were coincubated with CD8+ and CD4+ T cells that recognized major histocompatibility complex class I- and class II-restricted epitopes from gp100 and NY-ESO-1, and specific recognition was evaluated by interferongamma secretion. Cytokine release by both CD8+ and CD4+ T cells was consistently higher in response to DCs modified with adenoviruses than cDNAs or RNAs, and maturation of DCs after genetic modification did not consistently alter patterns of recognition. Also, retrovirally transduced DCs encoding gp100 were well recognized by both CD8+ and CD4+ T cells. These data suggest that DCs transduced with viral vectors may be more efficient than DCs transfected with cDNAs or RNAs for the induction of tumor reactive CD8+ and CD4+ T cells in vitro and in human vaccination trials.
TL;DR: The fact that the efficacy of coramsine is potentiated by CpG ODNs suggests thatCoramsine-induced cell death is an immunologic null event.
Abstract: Coramsine is a novel chemotherapeutic agent isolated from Solanum linnaeanum (devil's apple) Topical treatment provides clinical benefit for skin tumors To evaluate the potential broader applicability of the drug, its in vivo anticancer efficacy in a murine model of malignant mesothelioma and its mode of action were investigated Systemic administration of coramsine slowed tumor growth and prolonged survival time Importantly, the antitumor efficacy of coramsine was enhanced when treatment was combined with stimulation of innate immunity using unmethylated CpG-containing oligodeoxynucleotides (ODNs) Combination treatment further slowed tumor growth and provided a survival benefit Coramsine seems to kill tumor cells by direct cell lysis Using 2 different assays to detect apoptosis (caspase activation and DNA fragmentation), we found no evidence that coramsine induces any form of programmed cell death The fact that the efficacy of coramsine is potentiated by CpG ODNs suggests that coramsine-induced cell death is an immunologic null event