spliceosomal-peptide-p140 and Lupus-Erythematosus--Systemic

Nowadays, pharmacologic treatments of autoinflammatory diseases are largely palliative rather than curative. Most of them result in non-specific immunosuppression, which can be associated with broad disruption of natural and induced immunity with significant and sometimes serious unwanted injuries. Among the novel strategies that are under development, tools that modulate the immune system to restore normal tolerance mechanisms are central. In these approaches, peptide therapeutics constitute a class of agents that display many physicochemical advantages. Within this class of potent drugs, the phosphopeptide P140 is very promising for treating patients with lupus, and likely also patients with other chronic inflammatory diseases. We discovered that P140 targets autophagy, a finely orchestrated catabolic process, involved in the regulation of inflammation and in the biology of immune cells. In vitro, P140 acts directly on a particular form of autophagy called chaperone-mediated autophagy, which seems to be hyperactivated in certain subsets of lymphocytes in lupus and in other autoinflammatory settings. In lupus, the "correcting" effect of P140 on autophagy results in a weaker signaling of autoreactive T cells, leading to a significant improvement of pathophysiological status of treated mice. These findings also demonstrated ex vivo in human cells, open novel avenues of therapeutic intervention in pathological conditions, in which specific and not general targeting is highly pursued in the context of the new action plans for personalized medicines.

Topics: Animals; Antibodies, Monoclonal; Antigens, CD; Autophagy; B-Cell Activating Factor; Clinical Trials as Topic; Disease Models, Animal; Gene Expression Regulation; Humans; Immune Tolerance; Immunologic Factors; Lupus Erythematosus, Systemic; Lymphocyte Subsets; Mice; Molecular Targeted Therapy; Peptide Fragments; Precision Medicine

Over the last decade there has been a rapid expansion in the use of peptides as drugs. Nowadays, they are being used therapeutically in such diverse areas as endocrinology, neurology, haematology and some types of allergies. In the field of autoimmunity, a few candidates have emerged. Thus, in the pipeline of novel strategies designed to treat patients with systemic lupus erythematosus, the 21-mer peptide P140/Lupuzor raises hopes for the generation of an efficient, specific and safe treatment. This phosphopeptide has successfully completed a phase IIb clinical trial and will enter into a multi-centre, double-blind, placebo-controlled phase III clinical trial. The phase IIb trial showed that after three months of therapy (three subcutaneous injections of 200 µg peptide/patient in addition to standard of care), Lupuzor improved Systemic Lupus Erythematosus Disease Activity Index score of lupus patients under active treatment by 67.6% versus 41.5% in the placebo group (p < 0.025). After three additional months of follow-up, the improvement rate was 84.2% versus 45.8% (p < 0.025). The side-effect profile was unproblematic and the drug was well tolerated as evidenced by a very low drop-out rate. P140 does not behave as an immunosuppressant, it acts primarily as a fine immunomodulator of autoreactive CD4(+) T cells. Its underlying mechanism of action involves autophagy, a cellular process that implicates lysosomal-dependent recycling of intracellular components and controls the pool of major histocompatibility complex class II-displayed peptides that is presented to CD4(+) T cells.

Topics: Animals; Autoimmunity; CD4-Positive T-Lymphocytes; Clinical Trials, Phase II as Topic; Disease Models, Animal; Humans; Immunologic Factors; Lupus Erythematosus, Systemic; Mice; Peptide Fragments

Synthetic peptides can advantageously replace cognate proteins in solid-phase assays designed to help diagnosing autoimmune diseases. They can also represent essential tools for the discovery, pre-clinical and pharmaceutical development of therapeutics designed to attenuate these multifactorial and polymorphic diseases. We comment here on the peptide P140 able to delay the development of lupus in mouse models that spontaneously develop the disease and that has been evaluated in multicenter double-blind phase IIb clinical trials including lupus patients.

Topics: Animals; Autoantigens; Humans; Lupus Erythematosus, Systemic; Peptide Fragments; Peptides

After a long period where the potential of therapeutic peptides was let into oblivion and even dismissed, there is a revival of interest in peptides as potential drug candidates. Novel strategies for limiting metabolism and improve their bioavailability, and alternative routes of administration have emerged. This resulted in a large number of peptide-based drugs that are now being marketed in different indications. Regarding autoimmunity, successful data have been reported in numerous mouse models of autoimmune inflammation, yet relatively few clinical trials based on synthetic peptides are currently underway. This review reports on peptides that show much promises in appropriate mouse models of autoimmunity and describes in more detail clinical trials based on peptides for treating autoimmune patients. A particular emphasis is given to the 21-mer peptide P140/Lupuzor that has completed successfully phase I, phase IIa and phase IIb clinical trials for systemic lupus erythematosus.

Topics: Amino Acid Sequence; Animals; Autoimmune Diseases; Autoimmunity; Biological Availability; Clinical Trials as Topic; Drug Administration Routes; Drug Administration Schedule; Humans; Immunologic Factors; Lupus Erythematosus, Systemic; Mice; Molecular Sequence Data; Peptide Fragments; Survival Rate

Autoimmune diseases are illnesses that occur when the body's tissues are attacked by its own immune system. This review describes the path between the discovery of a synthetic peptide able to delay the development of lupus in mouse models that spontaneously develop lupus-like disease, and the results of multicenter phase IIb clinical trials including lupus patients. If the 2nd phase IIb and phase III results confirm the 1st phase IIb clinical trial, this therapeutic peptide could have a major impact in the treatment of patients with lupus, an autoimmune disease affecting at least five million patients in the world, in majority young women for whom there is no specific treatment today.

Topics: Animals; Clinical Trials, Phase II as Topic; Clinical Trials, Phase III as Topic; Disease Models, Animal; Humans; Lupus Erythematosus, Systemic; Mice; Peptide Fragments

It is well recognised that the historical timeline required for developing a drug, beginning with target identification and validation, is long and often tedious. It requires a large set of competences in various areas of molecular and cellular biology, chemistry, pharmacology, imaging, and model animal experimentation. Once the active molecule appears to be ready for human testing in controlled clinical trials, then the question arises of how to formulate it to render it stable, adequately packaged, according to the chosen route of administration, and bioavailable to reach its target in the affected organs. Historically, excipients have been considered inert and devoid of medicinal effect or influence. In fact, excipients are seldom neutral and some of them have been found to play a significant role, for example by initiating or participating in chemical and physical interactions with the active substance, leading in certain cases to compromise its therapeutic activity. It is difficult today to appreciate the number of potential drugs that have been discarded as a result of limited efficacy due to inappropriate excipients. This matter is presented here, with the peptide P140 (Lupuzor™) as example. Two formulations of P140, differing in the excipients used (mannitol or trehalose), have been evaluated in patients affected by systemic lupus erythematosus in two successive phase IIb clinical trials. P140 was shown to reduce excessive autophagy activity discovered in some lupus immune cell subsets. One of the two excipients, namely trehalose, has been claimed to exert an intrinsic stimulating activity on autophagy process, which was found therefore to counteract the beneficial peptide effects.

Topics: Animals; Autophagy; Disease Models, Animal; Excipients; Humans; Lupus Erythematosus, Systemic; Mice; Peptide Fragments; Trehalose

To evaluate treatment with the peptide-based agent, Lupuzor, in a double-blind, randomised, placebo-controlled study of patients with systemic lupus erythematosus.. Patients who met ≥4 of the American College of Rheumatology criteria, had a score of ≥6 on the Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K) and did not have an A score on the British Isles Lupus Assessment Group (BILAG)-2004 scale were eligible. 149 intention-to-treat (ITT) patients were randomly assigned to receive Lupuzor (200 μg) subcutaneously every 4 weeks (n=49; group 1) or every 2 weeks (n=51; group 2) or placebo (n=49; group 3) in addition to standard of care (SOC). A target population (136 ITT patients) consisting of patients having a clinical SLEDAI score ≥6 at week 0 was considered. The clinical SLEDAI score is the SLEDAI-2K score obtained by omitting low complement and increased DNA binding components.. In the ITT overall population, 53.1% in group 1 (p=0.048), 45.1% in group 2 (p=0.18) and 36.2% in the placebo group achieved an SLE Responder Index (SRI) response at week 12. In the target population, the results were more impressive: 61.9% in group 1 (p=0.016), 48.0% in group 2 (p=0.18) and 38.6% in the placebo group achieved an SRI response at week 12. An interim analysis including 114 patients from the target population demonstrated an even better efficacy (according to SLEDAI score) in group 1 compared with placebo (67.6% vs 41.5% (p<0.025) at week 12 and 84.2% vs 45.8% (p<0.025) at week 24). The most common adverse event was a mild injection-site erythema.. Lupuzor/200 µg given three times at 4-week intervals during 12 weeks in addition to SOC is efficacious and generally well tolerated.

Topics: Adult; Double-Blind Method; Female; Humans; Immunologic Factors; Lupus Erythematosus, Systemic; Male; Middle Aged; Peptide Fragments; Treatment Outcome; Young Adult

To assess the safety, tolerability, and efficacy of spliceosomal peptide P140 (IPP-201101; sequence 131-151 of the U1-70K protein phosphorylated at Ser140), which is recognized by lupus CD4+ T cells, in the treatment of patients with systemic lupus erythematosus (SLE).. An open-label, dose-escalation phase II study was conducted in two centers in Bulgaria. Twenty patients (2 male and 18 female) with moderately active SLE received 3 subcutaneous (SC) administrations of a clinical batch of P140 peptide at 2-week intervals. Clinical evaluation was performed using approved scales. A panel of autoantibodies, including antinuclear antibodies, antibodies to extractable nuclear antigens (U1 RNP, SmD1, Ro/SSA, La/SSB), and antibodies to double-stranded DNA (anti-dsDNA), chromatin, cardiolipin, and peptides of the U1-70K protein, was tested by enzyme-linked immunosorbent assay (ELISA). The plasma levels of C-reactive protein, total Ig, IgG, IgG subclasses, IgM, IgA, and IgE, and of the cytokines interleukin-2 and tumor necrosis factor alpha were measured by ELISA and nephelometry.. IgG anti-dsDNA antibody levels decreased by at least 20% in 7 of 10 patients who received 3 x 200 microg IPP-201101 (group 1), but only in 1 patient in the group receiving 3 x 1,000 microg IPP-201101 (group 2). Physician's global assessment of disease activity scores and scores on the SLE Disease Activity Index were significantly decreased in group 1. The changes occurred progressively in the population of responders, increased in magnitude during the treatment period, and were sustained. No clinical or biologic adverse effects were observed in the individuals, except for some local irritation at the highest concentration.. IPP-201101 was found to be safe and well tolerated by subjects. Three SC doses of IPP-201101 at 200 microg significantly improved the clinical and biologic status of lupus patients.

Topics: Adolescent; Adult; Aged; Antibodies, Antinuclear; C-Reactive Protein; DNA; Female; Humans; Immunotherapy; Lupus Erythematosus, Systemic; Male; Middle Aged; Peptide Fragments; Peptides; Severity of Illness Index; Spliceosomes; Treatment Outcome; Young Adult

In systemic lupus erythematosus, T cells display multiple abnormalities. They are abnormally activated, secrete pro-inflammatory cytokines, help B cells to generate pathogenic autoantibodies, and provoke the accumulation of autoreactive memory T cells. P140, a synthetic peptide evaluated in phase-III clinical trials for lupus, binds HSPA8/HSC70 chaperone protein.

Topics: Animals; Antigen Presentation; B-Lymphocytes; HSC70 Heat-Shock Proteins; Lupus Erythematosus, Systemic; Mice; Peptide Fragments

The phosphopeptide P140/Lupuzor, which improves the course of lupus disease in mice and patients, targets chaperone-mediated autophagy (CMA), a selective form of autophagy that is abnormally upregulated in lupus-prone MRL/lpr mice. Administered intravenously to diseased mice, P140 reduces the expression level of two major protein players of CMA, LAMP2A and HSPA8, and inhibits CMA in vitro in a cell line that stably expresses a CMA reporter. Here, we aimed to demonstrate that P140 also affects CMA in vivo and to unravel the precise cellular mechanism of how P140 interacts with the CMA process. MRL/lpr mice and CBA/J mice used as control received P140 or control peptides intravenously. Lysosome-enriched fractions of spleen or liver were prepared to examine lysosomal function. Highly purified lysosomes were further isolated and left to incubate with the CMA substrate to study at which cellular step P140 interacts with the CMA process. The data show that P140 effectively regulates CMA in vivo in MRL/lpr mice at the step of substrate lysosomal uptake and restores some alterations of defective lysosomes. For the first time, it is demonstrated that by occluding the intralysosome uptake of CMA substrates, a therapeutic molecule can attenuate excessive CMA activity in a pathological pro-inflammatory context and protect against hyperinflammation. This recovery effect of P140 on hyperactivated CMA is not only important for lupus therapy but potentially also for treating other (auto)inflammatory diseases, including neurologic and metabolic disorders, where CMA modulation would be highly beneficial.

Topics: Animals; Autophagy; Chaperone-Mediated Autophagy; Lupus Erythematosus, Systemic; Lysosomal-Associated Membrane Protein 2; Lysosomes; Mice, Inbred CBA; Mice, Inbred MRL lpr; Models, Biological; Peptide Fragments; Phosphopeptides; Spleen

Phosphopeptide P140 (Lupuzor) is an inhibitor of autophagy currently being evaluated in late-stage clinical trials for the treatment of lupus. This study was undertaken to investigate the effect of P140 ex vivo on human T and B cells.. Human B cells, T cells, and dendritic cells were analyzed by flow cytometry and cellular assays. The expression of autophagy markers was evaluated by immunoblotting and flow cytometry. The levels of B cell receptor (BCR) signaling markers and HLA molecules were assessed by flow cytometry. Toll-like receptor ligands were screened using an assay with transfected HEK 293 cells. P140 cell entry and trafficking were measured by immunofluorescence in the presence of various inhibitors of endosomal pathways.. As was previously observed after intravenous injection of the peptide in a mouse model of lupus, P140 entered human B cells by a clathrin coat-dependent endocytosis process and homed into lysosomes. The peptide displayed no direct effect on BCR signaling of memory, naive mature, transitional, and B1 cells. However, it strongly reduced the overexpression of HLA class II molecules on lupus B cells that were acting as antigen-presenting cells, down-regulated the maturation and differentiation of B cells into plasma cells, and decreased IgG secretion.. These findings show that P140 down-regulates HLA class II overexpression in human lupus B cells, and also that P140 hampers the differentiation of B cells into autoantibody-secreting plasma cells, likely due to the resulting lack of T cell signaling and activation. This mechanism appears to switch off the downstream events leading to secretion of pathogenic autoantibodies, thus explaining the highly promising results obtained in clinical trials of P140 (Lupuzor) for the treatment of lupus.

Topics: Autophagy; B-Lymphocytes; Cell Differentiation; HEK293 Cells; Histocompatibility Antigens Class II; Humans; Lupus Erythematosus, Systemic; Lymphocyte Activation; Peptide Fragments; T-Lymphocytes

The P140 peptide, a 21-mer linear peptide (sequence 131-151) generated from the spliceosomal SNRNP70/U1-70K protein, contains a phosphoserine residue at position 140. It significantly ameliorates clinical manifestations in autoimmune patients with systemic lupus erythematosus and enhances survival in MRL/lpr lupus-prone mice. Previous studies showed that after P140 treatment, there is an accumulation of autophagy markers sequestosome 1/p62 and MAP1LC3-II in MRL/lpr B cells, consistent with a downregulation of autophagic flux. We now identify chaperone-mediated autophagy (CMA) as a target of P140 and demonstrate that its inhibitory effect on CMA is likely tied to its ability to alter the composition of HSPA8/HSC70 heterocomplexes. As in the case of HSPA8, expression of the limiting CMA component LAMP2A, which is increased in MRL/lpr B cells, is downregulated after P140 treatment. We also show that P140, but not the unphosphorylated peptide, uses the clathrin-dependent endo-lysosomal pathway to enter into MRL/lpr B lymphocytes and accumulates in the lysosomal lumen where it may directly hamper lysosomal HSPA8 chaperoning functions, and also destabilize LAMP2A in lysosomes as a result of its effect on HSP90AA1. This dual effect may interfere with the endogenous autoantigen processing and loading to major histocompatibility complex class II molecules and as a consequence, lead to lower activation of autoreactive T cells. These results shed light on mechanisms by which P140 can modulate lupus disease and exert its tolerogenic activity in patients. The unique selective inhibitory effect of the P140 peptide on CMA may be harnessed in other pathological conditions in which reduction of CMA activity would be desired.

Topics: Animals; Autoimmunity; Autophagy; B-Lymphocytes; Endocytosis; Endosomes; Histocompatibility Antigens Class II; HSC70 Heat-Shock Proteins; Humans; Lupus Erythematosus, Systemic; Lysosomes; Mice; Mice, Inbred MRL lpr; Molecular Chaperones; NIH 3T3 Cells; Peptide Fragments; Phosphopeptides; Phosphorylation; Ribonucleoprotein, U1 Small Nuclear; Serine; T-Lymphocytes

Peptide therapeutics hold attractive potential. However, the proper stabilization of such therapeutics remains a major challenge. Some peptides are marginally stable and are prone to degradation. Therefore, in addition to chemical modifications that can be introduced in their sequence, a wide variety of excipients are added in the formulation to stabilize them, as is also done routinely for protein therapeutics. These substances are supposed to suppress peptide/protein aggregation and surface adsorption, facilitate their dispersion and additionally to provide physiological osmolality. Particular attention has to be paid to the choice of such excipients. Here we highlight the observation that in certain clinical situations, an excipient that is not totally inert can play a highly damaging role and mask (or even reverse) the beneficial effect of a molecule in clinical evaluation. This is the case, for instance, of trehalose, a normally safe excipient, which notably has proven to act as an activator of autophagy. This excipient, although used efficiently in several therapeutics, adversely impacted a phase IIb clinical trial for human and murine lupus, a systemic autoimmune disease in which it has been recently discovered that at the base line, autophagy is already abnormally enhanced in lymphocytes. Thus, in this particular pathology, while the peptide that was tested was active in lupus patients when formulated in mannitol, it was not efficient when formulated in trehalose. This observation is important, since autophagy is enhanced in a variety of pathological situations, such as obesity, diabetes, certain neurological diseases, and cancer.

Topics: Animals; Clinical Trials as Topic; Drug Interactions; Excipients; Humans; Lupus Erythematosus, Systemic; Peptide Fragments; Trehalose

Dendrons constituted of an adamantane core, a focal point and three arms, were synthetized starting from a multifunctional adamantane derivative. Maleimido groups at the periphery of the scaffold were used to covalently attach the peptide called P140, a therapeutic phosphopeptide controlling disease activity in systemic lupus, both in mice and patients. Biotinylation of the trimers at the focal point was performed using click chemistry and the conjugates were studied in terms of solubility, binding affinity to its receptor, the HSPA8/HSC70 chaperone protein, effect on HSPA8 folding property and in vivo activity. The results showed that the trimerization of P140 peptide does not trigger aggregation or steric hindrances during the interaction with HSPA8 protein. Compared to the monomeric cognate peptide, the trivalent P140 peptide displayed the same capacity, in vitro, to down-regulate HSPA8 activity and, in vivo in MRL/lpr lupus-prone mice, to reduce abnormal blood hypercellularity. The control trimer synthesized with the same scaffold and a scrambled sequence of P140 showed no effect in vivo. This work reveals that adamantane-based scaffolds with a well-defined spatial conformation are promising trivalent systems for molecular recognition and for biomedical applications.

Topics: Adamantane; Animals; Biotin; Dendrimers; Drug Carriers; Female; HSC70 Heat-Shock Proteins; Humans; Lupus Erythematosus, Systemic; Mice, Inbred MRL lpr; Peptide Fragments

The P140 phosphopeptide issued from the spliceosomal U1-70K small nuclear ribonucleoprotein protein displays protective properties in MRL/lpr lupus-prone mice. It binds both major histocompatibility class II (MHCII) and HSC70/Hsp73 molecules. P140 peptide increases MRL/lpr peripheral blood lymphocyte apoptosis and decreases autoepitope recognition by T cells.. To explore further the mode of action of P140 peptide on HSC70+ antigen-presenting cells.. P140 biodistribution was monitored in real time using an imaging system and by fluorescence and electron microscopy. Fluorescence activated cell sorting and Western blotting experiments were used to evaluate the P140 effects on autophagic flux markers.. P140 fluorescence accumulated especially in the lungs and spleen. P140 peptide reduced the number of peripheral and splenic T and B cells without affecting these cells in normal mice. Remaining MRL/lpr B cells responded normally to mitogens. P140 peptide decreased the expression levels of HSC70/Hsp73 chaperone and stable MHCII dimers, which are both increased in MRL/lpr splenic B cells. It impaired refolding properties of chaperone HSC70. In MRL/lpr B cells, it increased the accumulation of the autophagy markers p62/SQSTM1 and LC3-II, consistent with a downregulated lysosomal degradation during autophagic flux.. The study results suggest that after P140 peptide binding to HSC70, the endogenous (auto)antigen processing might be greatly affected in MRL/lpr antigen-presenting B cells, leading to the observed decrease of autoreactive T-cell priming and signalling via a mechanism involving a lysosomal degradation pathway. This unexpected mechanism might explain the beneficial effect of P140 peptide in treated MRL/lpr mice.

Topics: Animals; Antigen Presentation; Autophagy; B-Lymphocytes; Histocompatibility Antigens Class II; HSC70 Heat-Shock Proteins; Lupus Erythematosus, Systemic; Mice; Mice, Inbred MRL lpr; Peptide Fragments; Signal Transduction; Tissue Distribution

The phosphopeptide P140 issued from the spliceosomal U1-70K snRNP protein is recognized by lupus CD4(+) T cells, transiently abolishes T cell reactivity to other spliceosomal peptides in P140-treated MRL/lpr mice, and ameliorates their clinical features. P140 modulates lupus patients' T cell response ex vivo and is currently included in phase IIb clinical trials. Its underlying mechanism of action remains elusive. Here we show that P140 peptide binds a unique cell-surface receptor, the constitutively-expressed chaperone HSC70 protein, known as a presenting-protein. P140 induces apoptosis of activated MRL/lpr CD4(+) T cells. In P140-treated mice, it increases peripheral blood lymphocyte apoptosis and decreases B cell, activated T cell, and CD4(-)CD8(-)B220(+) T cell counts via a specific mechanism strictly depending on gammadelta T cells. Expression of inflammation-linked genes is rapidly regulated in CD4(+) T cells. This work led us to identify a powerful pathway taken by a newly-designed therapeutic peptide to immunomodulate lupus autoimmunity.

Topics: Animals; Apoptosis; B-Lymphocytes; Binding Sites; Down-Regulation; Fluorescent Antibody Technique; HSC70 Heat-Shock Proteins; Lupus Erythematosus, Systemic; Mice; Mice, Inbred MRL lpr; Models, Biological; Peptide Fragments; Receptors, Antigen, T-Cell, gamma-delta; Ribonucleoprotein, U1 Small Nuclear; Surface Plasmon Resonance; T-Lymphocytes; T-Lymphocytes, Regulatory