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Immunization of amyloid precursor protein transgenic mice with fibrillar β-amyloid (Aβ) prevents Alzheimer’s disease (AD)-like neuropathology. The first immunotherapy clinical trial used fibrillar Aβ, containing the B and T cell self epitopes of Aβ, as the immunogen formulated with QS21 as the adjuvant in the vaccine. Unfortunately, the clinical trial was halted during the phase II stage when 6% of the participants developed meningoencephalitis. The cause of the meningoencephalitis in the patients that received the vaccine has not been definitively determined; however, analysis of two case reports from the AN-1792 vaccine trial suggest that the meningoencephalitis may have been caused by a T cell-mediated autoimmune response, whereas production of anti-Aβ Abs may have been therapeutic to the AD patients. Therefore, to reduce the risk of an adverse T cell-mediated immune response to Aβ immunotherapy we have designed a prototype epitope vaccine that contains the immunodominant B cell epitope of Aβ in tandem with the synthetic universal Th cell pan HLA DR epitope, pan HLA DR-binding peptide (PADRE). Importantly, the PADRE-Aβ1–15 sequence lacks the T cell epitope of Aβ. Immunization of BALB/c mice with the PADRE-Aβ1–15 epitope vaccine produced high titers of anti-Aβ Abs. Splenocytes from immunized mice showed robust T cell stimulation in response to peptides containing PADRE. However, splenocytes from immunized mice were not reactivated by the Aβ peptide. New preclinical trials in amyloid precursor protein transgenic mouse models may help to develop novel immunogen-adjuvant configurations with the potential to avoid the adverse events that occurred in the first clinical trial.

BackgroundThe development of a safe and effective AD vaccine requires a delicate balance between providing an adequate anti-Aβ antibody response sufficient to provide therapeutic benefit, while eliminating an adverse T cell-mediated proinflammatory autoimmune response. To achieve this goal we have designed a prototype chemokine-based DNA epitope vaccine expressing a fusion protein that consists of 3 copies of the self-B cell epitope of Aβ42 (Aβ1–11) , a non-self T helper cell epitope (PADRE), and macrophage-derived chemokine (MDC/CCL22) as a molecular adjuvant to promote a strong anti-inflammatory Th2 phenotype.Methods and FindingsWe generated pMDC-3Aβ1–11-PADRE construct and immunized 3xTg-AD mouse model starting at age of 3–4 months old. We demonstrated that prophylactic immunizations with the DNA epitope vaccine generated a robust Th2 immune response that induced high titers of anti-Aβ antibody, which in turn inhibited accumulation of Aβ pathology in the brains of older mice. Importantly, vaccination reduced glial activation and prevented the development of behavioral deficits in aged animals without increasing the incidence of microhemorrhages.ConclusionsData from this transitional pre-clinical study suggest that our DNA epitope vaccine could be used as a safe and effective strategy for AD therapy. Future safety and immunology studies in large animals with the goal to achieve effective humoral immunity without adverse effects should help to translate this study to human clinical trials.

Despite the strides that immunotherapy has made in mediating tumor regression, the clinical effects are often transient, and therefore more durable responses still are needed. The temporary nature of the therapy-induced immune response can be attributed to tumor immune evasion mechanisms, mainly the effect of suppressive immune cells and, in particular, T regulatory cells (Treg). Although the depletion of Treg has been shown to be effective in enhancing immune responses, selective depletion of these suppressive cells without affecting other immune cells has not been very successful, and new agents are sought. We found that PI3K-Akt pathway inhibitors selectively inhibit Treg with minimal effect on conventional T cells (Tconv). Our results clearly show selective in vitro inhibition of activation (as represented by a decrease in downstream signaling) and proliferation of Treg in comparison to Tconv when treated with different Akt and PI3K inhibitors. This effect has been observed in both human and murine CD4 T cells. In vivo treatment with these inhibitors resulted in a significant and selective reduction in Treg both in naïve and tumor-bearing mice. Furthermore, these PI3K-Akt inhibitors led to a significant therapeutic antitumor effect, which was shown to be Treg-dependent. Here, we report the use of PI3K-Akt pathway inhibitors as potent agents for the selective depletion of suppressive Treg. We show that these inhibitors are able to enhance the antitumor immune response and are therefore promising clinical reagents for Treg-depletion.

Programmed death-1 receptor (PD-1) is expressed on T cells following TCR activation. Binding of this receptor to its cognate ligands, programmed death ligand (PDL)-1 and PDL-2, down-regulates signals by the TCR, promoting T-cell anergy and apoptosis, thus leading to immune suppression. Here, we find that using an anti-PD-1 antibody (CT-011) with Treg-cell depletion by low-dose cyclophosphamide (CPM), combined with a tumor vaccine, induces synergistic antigen-specific immune responses and reveals novel activities of each agent in this combination. This strategy led to complete regression of established tumors in a significant percentage of treated animals, with survival prolongation. We show for the first time that combining CT-011 and CPM significantly increases the number of vaccine-induced tumor-infiltrating CD8 1 T cells, with simultaneous decrease in infiltrating Treg cells. Interestingly, we find that CT-011 prolongs Treg-cell inhibition induced by CPM, leading to a sustainable significant synergistic decrease of splenic and tumor-infiltrated Treg cells. Surprisingly, we find that the anti-tumor effect elicited by the combination of CT-011 and CPM is dependent on both CD8 1 and CD4 1 T-cell responses, although the antigen we used is a class I MHCrestricted peptide. Thus, we describe a novel and effective therapeutic approach by combining multiple strategies to target several tumor-mediated immune inhibitory mechanisms.Key words: Cancer immunotherapy . Programmed death-1 receptor . Vaccine IntroductionImmune suppression/evasion is one of the major impediments to the development of effective immune therapy for cancer. Programmed death-1 receptor (PD-1) is a member of the B7 family that is expressed on activated T cells and is found to play an important role in immune evasion. On binding its cognate ligands programmed death ligand (PDL)-1 or PDL-2, PD-1 downregulates signaling by the T-cell receptor (TCR), inducing T-cell anergy and apoptosis and thus leading to immune suppression [1][2][3][4][5][6]. Many human malignancies up-regulate PDL-1, and this upregulation has been directly correlated with immune suppression and poor prognosis in several types of cancer [4,[7][8][9][10][11]. The PD-1/PDL-1 interaction leads to suppression and apoptosis of [27,[29][30][31][32][33].Here, we hypothesize that combining inhibition of Treg cells with strategies that block the PD-1/PDL-1 interaction and vaccine would result in a potent anti-tumor immunotherapeutic strategy. CT-011 is a novel humanized IgG1 k recombinant monoclonal anti-PD-1 antibody that has been shown to promote anti-tumor immunity in animal models in a Phase I clinical trial in hematological malignancies [34].We found that PD-1 blockade with low-dose CPM, given in combination with vaccine, synergistically induces strong antigenspecific immune responses and increases CD8 1 and CD4 1 Foxp3À T-cell infiltration into the tumor, leading to a potent antitumor effect. Interestingly, we demonstrated that the efficacy of the combination relies not only on CD8 1 but...

Different strategies proposed as therapy for Alzheimer disease (AD) have aimed to reduce the level of toxic forms of A␤ peptide in the brain. Here, we directly analyze the therapeutic utility of the polyclonal anti-A␤ 1-11 antibody induced in 3xTg-AD mice vaccinated with the second generation prototype epitope vaccine. Substoichiometric concentrations of purified anti-A␤ 1-11 antibody prevented aggregation of A␤ 42 and induced disaggregation of preformed A␤ 42 fibrils down to nonfilamentous and nontoxic species. Anti-A␤ 1-11 antibody delayed A␤ 42 oligomer formation but ultimately appeared to stabilize nonfibrillar conformations, including oligomer-like assemblies. The reduced oligomer-mediated cytotoxicity observed upon preincubation of A␤ oligomers with the anti-A␤ 1-11 antibody in the absence of oligomer disaggregation suggests a possible oligomer rearrangement in the presence of the antibody. These in vitro observations suggest that preventive vaccination may protect from AD or may delay the onset of the disease, whereas therapeutic vaccination cannot disrupt the toxic oligomers and may only minimally alleviate preexisting AD pathology. AD4 is characterized by deposition of fibrillar forms of A␤ peptide in senile plaques, appearance of A␤ congophilic deposits and neurofibrillary tangles in the cerebrovasculature, and neuronal loss (1-4). A␤ peptide is cleaved from the amyloid precursor protein (APP) by ␤-and ␥-secretases (5-7) and is thought to play a central role in the onset and progression of AD (8 -10). In AD, the normally soluble A␤ molecule (39 -43 aa) undergoes conformational changes and is deposited as insoluble fibrils, oligomers, and protofibrills. Previously, it was demonstrated that A␤ neurotoxicity requires insoluble fibril formation (11) and that these fibrils serve as inducers of neuronal apoptosis (12). Recently, emphasis has shifted to smaller soluble oligomers of A␤ 42 , such as the 12-mers known as A␤-derived diffusible ligands, increased about 70-fold in AD patients' brains over controls (13). More recently, it was shown that extracellular accumulation of 56-kDa soluble A␤ assembly impairs memory in middle-aged APP/Tg 2576 mice in the absence of neuritic plaques (14). A␤ 42 dimers and trimers naturally secreted from a 7PA2 cell line were also suggested to be responsible for the disruption of cognitive functions (15). Importantly, intraventricular injection of such A␤ 42 small oligomers inhibited long term potentiation in rat hippocampus, and an injection of anti-A␤ monoclonal antibody 6E10 prevented this inhibition (16). It has also been demonstrated that passive immunization with monoclonal antibodies (NAB61) that specifically recognize a pathologic conformation present in A␤ oligomers resulted in a rapid improvement in spatial learning and memory (17).The therapeutic potency of polyclonal and monoclonal anti-A␤ antibodies was documented in different mouse models of AD (18 -25). Collectively, these data suggest that antibodies specific to the N-terminal region of A␤ are capable of reduci...

Constitutive activation of the KRAS oncogene in human malignancies is associated with aggressive tumor growth and poor prognosis. Similar to other oncogenes, KRAS acts in a cell-intrinsic manner to affect tumor growth or survival. However, we describe here a different, cell-extrinsic, mechanism through which mutant KRAS contributes to tumor development. Tumor cells carrying mutated KRAS induced highly suppressive T cells, and silencing KRAS reversed this effect. Overexpression of the mutant KRASG12V gene in wild-type KRAS tumor cells led to Treg induction. We also demonstrate that mutant KRAS induces the secretion of interleukin-10 and transforming growth factor-β1 (both required for Treg induction) by tumor cells through the activation of the MEK-ERK-AP1 pathway. Finally, we report that inhibition of KRAS reduces the infiltration of Tregs in KRAS-driven lung tumorigenesis even before tumor formation. This cell-extrinsic mechanism allows tumor cells harboring a mutant KRAS oncogene to escape immune recognition. Thus, an oncogene can promote tumor progression independent of its transforming activity by increasing the number and function of Tregs. This has a significant clinical potential, in which targeting KRAS and its downstream signaling pathways could be used as powerful immune modulators in cancer immunotherapy.

To modulate T-cell function for cancer therapy, one challenge is to selectively attenuate regulatory but not conventional CD4 T-cell subsets [regulatory T cell (Treg) and conventional T cell (Tconv)]. In this study, we show how a functional dichotomy in Class IA PI3K isoforms in these two subsets of CD4 T cells can be exploited to target Treg while leaving Tconv intact. Studies employing isoform-specific PI3K inhibitors and a PI3Kδ-deficient mouse strain revealed that PI3Kα and PI3Kβ were functionally redundant with PI3Kδ in Tconv. Conversely, PI3Kδ was functionally critical in Treg, acting there to control T-cell receptor signaling, cell proliferation, and survival. Notably, in a murine model of lung cancer, coadministration of a PI3Kδ-specific inhibitor with a tumor-specific vaccine decreased numbers of suppressive Treg and increased numbers of vaccine-induced CD8 T cells within the tumor microenvironment, eliciting potent antitumor efficacy. Overall, our results offer a mechanistic rationale to employ PI3Kδ inhibitors to selectively target Treg and improve cancer immunotherapy. .