Concomitant blockade of A2AR and CTLA-4 by siRNA-loaded polyethylene glycol-chitosan-alginate nanoparticles synergistically enhances antitumor T-cell responses

Inhibitory immune checkpoint (ICP) molecules are important immunosuppressive factors in a tumor microenvironment (TME). They can robustly suppress T-cell-mediated antitumor immune responses leading to cancer progression. Among the checkpoint molecules, cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) is one of the critical inhibitors of anticancer T-cell responses. Besides, the expression of adenosine receptor (A2AR) on tumor-infiltrating T cells potently reduces their function. We hypothesized that concomitant silencing of these molecules in T cells might lead to enhanced antitumor responses. To examine this assumption, we purified T cells from the tumor, spleen, and local lymph nodes of CT26 colon cancer-bearing mice and suppressed the expression of A2AR and CTLA-4 using the small interfering RNA (siRNA)-loaded polyethylene glycol-chitosan-alginate (PCA) nanoparticles. The appropriate physicochemical properties of the produced nanoparticles (NPs; size of 72 nm, polydispersive index [PDI] < 0.2, and zeta potential of 11 mV) resulted in their high efficiency in transfection and suppression of target gene expression. Following the silencing of checkpoint molecules, various T-cell functions, including proliferation, apoptosis, cytokine secretion, differentiation, and cytotoxicity were analyzed, ex vivo. The results showed that the generated nanoparticles had optimal physicochemical characteristics and significantly suppressed the expression of target molecules in T cells. Moreover, a concomitant blockade of A2AR and CTLA-4 in T cells could synergistically enhance antitumor responses through the downregulation of PKA, SHP2, and PP2Aα signaling pathways. Therefore, this combination therapy can be considered as a novel promising anticancer therapeutic strategy, which should be further investigated in subsequent studies.     

CTLA-4 blockade enhances the CTL responses to the p53 self-tumor antigen

p53 is an attractive target for cancer immunotherapy because it is overexpressed in a high proportion of many different types of tumors. However, it is also expressed in normal tissues and acts as a toleragen in vivo. Previously, detailed examination of the repertoire specific for the murine p53(261-269) epitope in conventional and p53-deficient mice demonstrated that because of expression of p53, the CD8(+) T cells that respond to this epitope express low-affinity TCRs. It has been reported that tolerance to tumor Ags can be broken by in vivo administration of anti-CTLA-4 mAb. With the goal of overriding tolerance and achieving optimal activation of p53-specific CTL, the current study has assessed the effect of anti-CTLA-4 mAb on the p53-specific repertoire. It was found that blockade of CTLA-4 engagement at the time of antigenic stimulation induced a vigorous amplification of the CTL responses to p53 as well as proportionate expansion of the memory T cell pool. This effect was dependent on the presence of CD4(+) T cell help and correlated with an enhancement of helper function. However, anti-CTLA-4 treatment did not enhance the avidity of the resultant p53-specific CTL populations and, therefore, could not reverse this important consequence of tolerance.     

CTLA-4 blockade and the renaissance of cancer immunotherapy

Cytotoxic T-lymphocyte associated antigen 4 (CTLA-4) plays a key role in restraining the adaptive immune response of T-cells towards a variety of antigens including tumor associated antigens (TAAs). The blockade of this immune checkpoint elicits an effective anticancer immune response in a range of preclinical models, suggesting that naturally occurring (or therapeutically induced) TAA specific lymphocytes need to be “unleashed” in order to properly fight against malignant cells. Therefore, investigators have tested this therapeutic hypothesis also in humans: the favorable results obtained with this strategy in patients with advanced cutaneous melanoma are revolutionizing the management of this highly aggressive disease and are fueling new enthusiasm on cancer immunotherapy in general.     

Activity and safety of CTLA-4 blockade combined with vaccines in cynomolgus macaques

The immune modulatory molecule CTLA-4 (CD152), through interactions with the B7 costimulatory molecules, has been shown to be a negative regulator of T cell activation in various murine model systems. Abs that block CTLA-4 function can enhance immune responses that mediate potent antitumor activity. However, CTLA-4 blockade can also exacerbate autoimmune disease. The safety and activity of anti-CTLA-4 Abs in primates has not been addressed. To that end, we generated human Abs against CTLA-4 using transgenic mice expressing human Ig genes. A high affinity Ab (10D1) that blocked the binding of CTLA-4 to the B7-1 and B7-2 ligands and had cross-reactivity with macaque CTLA-4 was chosen for further development. Administration of 10D1 to cynomolgus macaques significantly enhanced Ab responses to hepatitis surface Ag and a human melanoma cell vaccine. Anti-self Ab responses as measured by immunoassays using lysate from melanocyte-rich tissues were elicited in those animals receiving the melanoma cell vaccine and anti-CTLA-4 Ab. Remarkably, chronic administration of 10D1 did not result in measurable polyclonal T cell activation, significant alteration of the lymphocyte subsets, or induce clinically observable autoimmunity. Repeated dosing of the 10D1 did not elicit monkey anti-human Ab responses in the monkeys. These observations support the development of CTLA-4 blockade for human immunotherapy.     

Myeloid-derived suppressor cells and their role in CTLA-4 blockade therapy

Immune checkpoints are a series of inhibitory pathways that are crucial for modulating the intensity and duration of immune response. Among these checkpoints, cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) has been shown to be a key regulator of the early activation of naïve and memory T cells. Immune checkpoint blockade is emerging as one of the most promising therapeutic approaches directed toward the activation of the immune response against tumors. The first of these therapies that has been FDA approved is ipilimumab, a fully human monoclonal antibody that blocks CTLA-4. The in cis effects that CTLA-4 blockade has on T cells have been properly described, but there are still questions to be answered regarding the indirect or in trans effects. One of the alternative cellular populations that may play a role in the outcome of CTLA-4 blockade therapy is myeloid-derived suppressor cells (MDSCs), which have recently been associated with clinical outcome in advanced melanoma. In addition to this, MDSCs have been shown to be decreased in number and functional potential after treatment with ipilimumab. A better clarification of what effects CTLA-4 blockade may have on these cellular populations is likely to provide insights on possible predictive biomarkers for CTLA-4 blockade therapy.     

CTLA-4 blockade increases antigen-specific CD8+ T cells in prevaccinated patients with melanoma: three cases

Background

Anti-cytotoxic T-lymphocyte antigen-4 (CTLA-4) antibodies, such as ipilimumab, have generated measurable immune responses to Melan-A, NY-ESO-1, and gp100 antigens in metastatic melanoma. Vaccination against such targets has potential for immunogenicity and may produce an effector-memory T-cell response.

Methods

To determine the effect of CTLA-4 blockade on antigen-specific responses following vaccination, in-depth immune monitoring was performed on three ipilimumab-treated patients prevaccinated with gp100 DNA (IMF-24), gp100^209?C217 and tyrosinase peptides plus GM-CSF DNA (IMF-32), or NY-ESO-1 protein plus imiquimod (IMF-11); peripheral blood mononuclear cells were analyzed by tetramer and/or intracellular cytokine staining following 10-day culture with HLA-A*0201-restricted gp100^209?C217 (ITDQVPFSV), tyrosinase^369?C377 (YMDGTMSQV), or 20-mer NY-ESO-1 overlapping peptides, respectively. Tumors from IMF-32 were analyzed by immunohistochemistry to help elucidate mechanism(s) underlying tumor escape.

Results

Following vaccination, patients generated weak to no CD4⁺ or CD8⁺ T-cell response specific to the vaccine antigen but demonstrated increases in effector-memory (CCR7^loCD45RA^lo) tetramer⁺CD8⁺ T cells. After ipilimumab induction, patients experienced a robust, although sometimes transient, antigen-specific response for gp100 (IMF-32 and IMF-24) or NY-ESO-1 (IMF-11) and produced polyfunctional intracellular cytokines. Primary and metastatic tumors expressed tyrosinase but not gp100 or class I/II MHC molecules.

Conclusion

Vaccination induced a measurable antigen-specific T-cell response that increased following CTLA-4 blockade, potentially ??boosting?? the vaccine-primed response. Tumor escape may be related to antigen loss or lack of MHC expression necessary for immune activity. These results in a limited number of patients support the need for further research into combining vaccination with ipilimumab and provide insight into mechanisms underlying tumor escape.     

Synergy between chemotherapeutic agents and CTLA-4 blockade in preclinical tumor models

Ipilimumab, a cytotoxic T-lymphocyte antigen-4 (CTLA-4) binding agent, has proven to be an effective monotherapy for metastatic melanoma and has shown antitumor activity in trials when administered with other therapeutic agents. We hypothesized that the combination of ipilimumab with chemotherapeutic agents, such as ixabepilone, paclitaxel, etoposide, and gemcitabine, may produce therapeutic synergy based on distinct but complementary mechanisms of action for each drug and unique cellular targets. This concept was investigated using a mouse homolog of ipilimumab in preclinical murine tumor models, including SA1N fibrosarcoma, EMT-6 mammary carcinoma, M109 lung carcinoma, and CT-26 colon carcinoma. Results of CTLA-4 blockade in combination with one of various chemotherapeutic agents demonstrate that synergy occurs in settings where either agent alone was not effective in inducing tumor regression. Furthermore, when combined with CTLA-4 blockade, ixabepilone, etoposide, and gemcitabine elicited prolonged antitumor effects in some murine models with induction of a memory immune response. Future investigations are warranted to determine which specific chemo-immunotherapy combinations, if any, will produce synergistic antitumor effects in the clinical setting.     

CTLA-4.FasL inhibits allogeneic responses in vivo

CTLA-4.Fas ligand (CTLA-4.FasL), a paradigmatic 'trans signal converter protein (TSCP)', can attach to APC (via CTLA-4 binding to B7) and direct intercellular inhibitory signals to responding T cells (via FasL binding to Fas receptor), converting an activating APC-to-T cell signal into an inhibitory one. Our previous studies established that CTLA-4.FasL inhibits human primary mixed lymphocyte reactions (MLR) and induces alloantigen-specific hyporesponsiveness ex vivo. The present study extends this to an in vivo context. Using splenocytes from MHC-mismatched C57BL/6 and Balb/c mice, we demonstrated that his(6)CTLA-4.FasL, effectively inhibits murine MLR. Moving in vivo, we demonstrated that subcutaneously administered his(6)CTLA-4.FasL modulates the in vivo response of infused allogeneic splenocytes. his(6)CTLA-4.FasL reduces the number of cells in each cell division, and increases the percentage of dead cells in each division. These findings are consistent with an antigen-induced cell death of the alloreactive cells, and bolsters recombinant TCSP promise as a therapeutic for transplantation diseases.     

TGF-beta mediates CTLA-4 suppression of cellular immunity in murine kalaazar

Recent studies indicate important roles for CTLA-4 engagement in T cells, and for TGF-beta production in the immunopathogenesis of murine kalaazar or visceral leishmaniasis, but a functional link between these two pathways in helping intracellular parasite growth is unknown. Here we report that Ag or anti-CD3 activation of splenic CD4+ T cells from visceral leishmaniasis leads to intense CTLA-4-mediated TGF-beta1 production, as assessed either by CTLA-4 blockade or by direct CTLA-4 cross-linkage. Production of TGF-beta1 accounted for the reciprocal regulation of IFN-gamma production by CTLA-4 engagement. Following CD4+ T cell activation, intracellular growth of Leishmania chagasi in cocultured splenic macrophages required both CTLA-4 function and TGF-beta1 secretion. Cross-linkage of CTLA-4 markedly increased L. chagasi replication in cocultures of infected macrophages and activated CD4+ T cells, and parasite growth could be completely blocked with neutralizing anti-TGF-beta1 Ab. Exogenous addition of rTGF-beta1 restored parasite growth in cultures protected from parasitism by CTLA-4 blockade. These results indicate that the negative costimulatory receptor CTLA-4 is critically involved in TGF-beta production and in intracellular parasite replication seen in murine kalaazar.     

CTLA-4 blockade differentially influences the outcome of non-lethal and lethal Plasmodium yoelii infections

An immune response against malaria has to be tightly controlled. The production of pro-inflammatory cytokines is required to control parasites but the same cytokines are also involved in severe malaria. We have shown that CTLA-4 expression during Plasmodium berghei malaria dampens the immune response. This strain provokes a pro-inflammatory immune response that is associated with the pathology of cerebral malaria. Accordingly a blockade of CTLA-4 during the blood-stage of P. berghei malaria leads to an exacerbation of disease. To analyze the effects of a CTLA-4 blockade in a malaria model which is not prone to immune pathology we employed P. yoelii infection. Blood-stage infection led to a rapid induction of CTLA-4 on T cells. Using the non-lethal P. yoelii strain Py17NL we found that a blockade of CTLA-4 resulted in an increased T cell activation and IFN-gamma production, which was accompanied by a lower peak parasitemia and earlier parasite clearance. In contrast, blockade of CTLA-4 during infection with a P. yoelii strain exhibiting a higher parasitemia induced markedly increased serum-levels of TNF-alpha, which was associated with severe inflammation and reduced survival.     

CTLA-4 overexpression inhibits T cell responses through a CD28-B7-dependent mechanism

CTLA-4 has been shown to be an important negative regulator of T cell activation. To better understand its inhibitory action, we constructed CTLA-4 transgenic mice that display constitutive cell surface expression of CTLA-4 on CD4 and CD8 T cells. In both in vivo and in vitro T cell responses, CTLA-4 overexpression inhibits T cell activation. This inhibition is dependent on B7 and CD28, suggesting that overexpressed CTLA-4 inhibits responses by competing with CD28 for B7 binding or by interfering with CD28 signaling. In addition, expression of the transgene decreases the number of CD25+Foxp3+ T cells in these mice, but does not affect their suppressive ability. Our data confirm the activity of CTLA-4 as a negative regulator of T cell activation and that its action may be by multiple mechanisms.     

CTLA-4 engagement and regulatory CD4+CD25+ T cells independently control CD8+-mediated responses under costimulation blockade

Blockade of costimulatory signals is a promising therapeutic target to prevent allograft rejection. In this study, we sought to characterize to what extent CTLA-4 engagement contributes to the development of transplantation tolerance under the cover of CD40/CD40L and CD28/CD86 blockade. In vitro, we found that inhibition of the primary alloresponse and induction of alloantigen hyporesponsiveness by costimulation blockade was abrogated by anti-CTLA-4 mAb. In addition, regulatory CD4(+)CD25(+) T cells (T(REG)) were confirmed to play a critical role in the induction of hyporesponsiveness by anti-CD40L and anti-CD86 mAb. Our data indicated that CTLA-4 engagement is not required for activation or suppressor function of T(REG). Instead, in the absence of either CTLA-4 signaling or T(REG), CD8(+) T cell division was enhanced, whereas the inhibition of CD4(+) T cell division by costimulation blockade remained largely unaffected. In vivo, the administration of additional anti-CTLA-4 mAb abrogated anti-CD40L- and anti-CD86 mAb-induced cardiac allograft survival. Correspondingly, rejection was accompanied by enhanced allograft infiltration of CD8(+) cells. We conclude that CTLA-4 signaling and T(REG) independently cooperate in the inhibition of CD8(+) T cell expansion under costimulation blockade.     

Regulatory T-cell depletion synergizes with gp96-mediated cellular responses and antitumor activity

Despite its potent immunostimulatory properties, vaccination with autologous tumor-derived gp96 has relatively modest antitumor effect in a range of clinical trials. Based on our previous study showing a gp96-mediated immune balance between CTL and Tregs, here we investigated possible synergy between gp96 vaccine and systemic Treg depletion on induction of antitumor T-cell immunity and the mechanisms accounting for synergistic efficacy. In gp96-peptide complex immunized BALB/c mice, anti-CD25 mAb treatment significantly increased IFN-γ-producing CD8(+) and CD4(+) T cells by about 1-2-fold in spleen and 40-50% in lymph node. A significantly higher number of peptide-specific CTL were observed under anti-CD25 mAb treatment compared with no treatment. Moreover, Treg depletion synergistically improved the anticancer activity of tumor-derived gp96 vaccine in the poorly immunogenic and highly tumorigenic B16 melanoma model in C57BL/6?J mice. While gp96 immunization alone led to the modest enhancement of CTL activities in spleen, the combination with Treg depletion dramatically increased tumor-specific CTL responses. In addition, the combination resulted in a significant increase of CD8(+) T-cell infiltration in tumor, which correlated with an enhanced inhibition of tumor growth. Our results provide evidence that targeting Tregs may provide a more efficient strategy to potentiate gp96-mediated T-cell responses and enhance the antitumor efficiency of gp96-based therapeutic vaccine.     

The role of CTLA-4 in the regulation of T cell immune responses

Over the past few years a great deal of research has examined how T cell-dependent immune responses are initiated and subsequently regulated. Ligation of the TCR with an antigenic peptide bound to an MHC protein on a professional APC provides the crucial antigen-specific stimulus required for T cell activation. Interaction of CD28 with CD80 or CD86 molecules on APC initiates a costimulatory or second signal within the T cell which augments and sustains T cell activation initiated through the TCR. However, recently it has become clear that T cell immune responses are a result of a balance between stimulatory and inhibitory signals. Cytotoxic T lymphocyte-associated molecule-4 (CTLA-4) is a cell surface molecule that is expressed nearly exclusively on CD4+ and CD8+ T cells. Investigation into the role of CTLA-4 in the regulation of T cell immune responses has revealed that CTLA-4 is a very important molecule involved in the maintenance of T cell homeostasis. In the present review, evidence for the proposed inhibitory role of CTLA-4 is examined and a model suggesting a role for CTLA-4 in both early and late stages of T cell activation is presented.     

Polymorphism analysis of the CTLA-4 gene in paracoccidioidomycosis patients

The CTLA-4 protein is expressed in activated T cells and plays an essential role in the immune response through its regulatory effect on T cell activation. Polymorphisms of the CTLA-4 gene have been correlated with autoimmune, neoplastic and infectious illnesses. This work aimed to verify possible associations between single nucleotide polymorphisms (SNPs) in CTLA-4, -318C/T in the promoter and +49A/G in exon 1 and paracoccidioidomycosis (PCM) caused by Paracoccidioides brasiliensis. For this purpose, 66 chronic form PCM patients and 76 healthy controls had their allele, genotype and haplotype frequencies determined. The genetic admixture structure of the patients and controls was evaluated to eliminate ancestral bias. The comparison of frequencies indicated no significant differences between patients and controls that could link the SNPs to PCM. Groups were admixture matched with no difference observed in population ancestry inference, indicating that the absence of association between CTLA-4 polymorphisms and PCM could not be attributed to ancestral bias. This study showed that there was no association between the CTLA-4 SNPs -318 and +49 and the resistance or susceptibility to PCM.     

CTLA-4 blockade augments human T lymphocyte-mediated suppression of lung tumor xenografts in SCID mice

Previous studies by others using transplantable murine tumor models have demonstrated that the administration of antibodies that block CTLA-4 interaction with B7 can provoke the elimination of established tumors, and that the tumor suppression is mediated by T-cells and/or cells expressing NK1.1. Studies from our lab have established in a human/severe combined immunodeficient (SCID) mouse chimeric model that autologous peripheral blood leukocytes (PBL) can suppress the growth of tumor xenografts in a PBL dose-dependent fashion, and that this suppression is dependent upon the patients T and NK cells. Using this human/mouse chimeric model, we sought to determine whether an antibody blockade of CTLA-4 would enhance the anti-tumor response of a patients PBL. It was first important to determine whether the tumor suppression observed in the SCID model was dependent upon CD28/B7 co-stimulation. Blockade of B7 with a human CTLA-4-Ig fusion protein completely abrogated the lymphocyte-mediated tumor suppression, confirming in this model that tumor suppression is dependent upon a CD28/B7 co-stimulation. Using two different CTLA-4 specific monoclonal antibodies, we observed that CTLA-4 blockade significantly enhanced the human lymphocyte-mediated tumor suppression in mice co-engrafted with PBL and tumor cells. This enhancement was observed in both an allogeneic setting (in which the PBL were allogeneic with respect to the tumor) and an autologous setting (in which the PBL and tumor were from the same patient). These results sustain the notion that human anti-tumor immune response can be augmented (in vivo) by blocking the interaction between CTLA-4 and B7.     

CTLA-4 blockade by a human MAb enhances the capacity of AML-derived DC to induce T-cell responses against AML cells in an autologous culture system

BACKGROUND: Cells from AML patients can differentiate into the phenotype of DC when cultured with GM-CSF and IL-4. Such cytokine-treated AML-derived DC (AML-DC) can stimulate autologous T cells. In this study we examined whether an anti-CTLA-4 MAb (MDX-010) could enhance the generation of autologous anti-AML T cells. METHODS: MAb MDX-010 was added to AML PBMC cultures in the presence of GM-CSF and IL-4, a previously reported AML-DC culture method of generating anti-AML T cells. T-cell activation and proliferation were examined thereafter. RESULTS: Addition of MDX-010 to GM-CSF/IL-4-conditioned AML-DC cultures induced a mean seven-fold increase in the numbers of autologous T cells compared with cultures without MDX-010 (P < 0.007). T cells stimulated by AML-DC with CTLA-4 blockade were significantly more cytotoxic towards autologous AML cells than those without MDX-010 (42 +/- 23% vs. 26 +/- 15%, E:T ratio of 20). T cells stimulated by AML-DC with CTLA-4 blockade had significantly greater proportions of T cells producing IFN-gamma in response to autologous AML cells than those cultured with AML-DC alone (10.7 +/- 4.7% vs. 4.5 +/- 2.4% for CD4+ IFN-gamma+ CD69+ and 9.8 +/- 4.1% vs. 4 +/- 2.1% for CD8+ IFN-gamma+ CD69+ with or without MDX-010; n = 7; P < 0.007, P < 0.003, respectively). DISCUSSION: CTLA-4 blockade enhances the activity and numbers of AML-reactive T cells that can be stimulated by autologous AML-DC and may enhance the efficacy of adoptive immunotherapy of AML.     

Paradoxical inhibition of T-cell function in response to CTLA-4 blockade; heterogeneity within the human T-cell population

T-cell co-stimulation delivered by the molecules B7-1 or B7-2 through CD28 has a positive effect on T-cell activation, whereas engagement of cytotoxic T-lymphocyte antigen 4 (CTLA-4) by these molecules inhibits activation. In vivo administration to mice of blocking monoclonal antibodies or Fab fragments against CTLA-4 can augment antigen-specific T-cell responses and, thus, therapy with monoclonal antibody against CTLA-4 has potential applications for tumor therapy and enhancement of vaccine immunization. The effects of B7-1 and B7-2 co-stimulation through CD28 depend on the strength of the signal delivered through the T-cell receptor (TCR) and the activation state of T cells during activation. Thus, we sought to determine whether these factors similarly influence the effect of B7-mediated signals delivered through CTLA-4 during T-cell activation. Using freshly isolated human T cells and Fab fragments of a monoclonal antibody against CTLA-4, we demonstrate here that CTLA-4 blockade can enhance or inhibit the clonal expansion of different T cells that respond to the same antigen, depending on both the T-cell activation state and the strength of the T-cell receptor signal delivered during T-cell stimulation. Thus, for whole T-cell populations, blocking a negative signal may paradoxically inhibit immune responses. These results provide a theoretical framework for clinical trials in which co-stimulatory signals are manipulated in an attempt to modulate the immune response in human disease.