Peptides and Methods of Use

Abstract

The present invention provides synthetic peptides. The present invention relates to modified 15-amino acid synthetic peptides from the Polar Assortant (PA) peptide, a scrambled peptide derived from a human astrovirus protein. In some embodiments, the present invention relates to peptides that are modified versions of PA, including sarcosine substitutions at certain amino acid positions, that are stapled, and / or have D-enantiomeric substitutions of certain amino acids. The present invention further provides methods for selecting at least one synthetic peptide for treating various conditions. TIFF2023548344000031.tif38142

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 108,732, filed November 2, 2020, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on October 28, 2021, is named 251110_000158_SL.txt, and is 25,380 bytes in size.

[0003] 1. Field of the Invention Aspects of the present invention relate generally to synthetic peptides and their uses for therapy and diagnosis, and more particularly to stapled synthetic peptides, either alone or in combination with D-enantiomers of specific amino acids of the synthetic peptides. [Background technology]

[0004] 2. Background complement system The complement system, an essential component of the innate immune system, plays a crucial role as a defense mechanism against invading pathogens, stimulating the adaptive immune response and helping to remove immune complexes and apoptotic cells. Three distinct pathways comprise the complement system: the classical, lectin, and alternative pathways. C1q and mannose-binding lectin (MBL) are structurally related recognition molecules in the classical and lectin pathways, respectively. IgM or clustered IgG serve as the primary ligands for C1q, while MBL recognizes polysaccharides such as mannan. Ligand binding by C1q and MBL leads to the sequential activation of C4 and C2 to form the C3 convertases of the classical and lectin pathways, respectively. In contrast, activation of the alternative pathway does not require a recognition molecule but can amplify C3 activation initiated by the classical or lectin pathways. Activation of any of these three pathways results in the formation of inflammatory mediators (C3a and C5a) and the membrane attack complex (MAC), which triggers cell lysis.

[0005] While the complement system plays a key role in many protective immune functions, complement activation is a key mediator of tissue damage in a wide range of autoimmune and inflammatory disease processes (Ricklin and Lambris, "Complement-targeted therapeutics." Nat Biotechnol 2007; 25(11):1265-75).

[0006] There is a need for complement regulators. On the one hand, the complement system is a vital host defense against pathogenic organisms. On the other hand, its unchecked activation can cause devastating host cell damage. Currently, despite the known morbidity and mortality associated with complement dysregulation in many disease processes, including autoimmune diseases such as systemic lupus erythematosus, myasthenia gravis, and multiple sclerosis, only two anticomplement therapies have been recently approved for use in humans: (1) eculizumab (Soliris™) and (2) ultomiris (Ravulizumab™), two humanized, long-acting monoclonal antibodies against C5 used in the treatment of paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS). PNH and aHUS are orphan diseases afflicting only a very small number of individuals. Currently, no complement regulators have been approved for the more common disease processes in which dysregulated complement activation plays a central role. Dysregulated complement activation may play a role in both chronic and acute disease manifestations.

[0007] The development of peptides to inhibit the classical, lectin, and alternative pathways of the complement system is needed because each of these three pathways has been shown to contribute to numerous autoimmune and inflammatory disease processes. Specific blockade of the classical and lectin pathways is particularly needed because both pathways are involved in ischemia-reperfusion-induced injury and other diseases in many animal models. Humans with alternative pathway deficiencies suffer from severe bacterial infections. Therefore, a functional alternative pathway is essential for immune surveillance against invading pathogens.

[0008] Naturally occurring peptides are essential signaling molecules that play important physiological roles in human biology in the form of neurotransmitters, hormones, growth factors, and antimicrobial agents [1]. Given their unique specificity and efficient properties, this class of molecules has received considerable attention as human therapeutic agents for various disease indications. As of March 2018, more than 60 peptides have been approved for therapeutic use in the United States, Europe, and / or Japan, with 155 currently in clinical development [2]. Compared to small molecules (<500 Da), which often suffer from toxicity and off-target effects, the favorable properties of peptides offer considerable advantages. Furthermore, compared to large protein-based molecules such as humanized monoclonal antibodies, peptides typically enjoy low manufacturing costs and can often be chemically synthesized, thus avoiding costly and complex production and purification. Often, naturally occurring peptides cannot be directly translated into therapeutic use due to suboptimal chemical and physical stability and poor pharmacokinetics (half-life). Therefore, several technical approaches are frequently used to rationally design peptides into more druggable molecules suitable for human administration.

[0009] We have identified a novel family of peptides known as PIC1 (also known as EPICC peptides). PIC1 peptides possess multiple anti-inflammatory properties, including inhibition of the classical complement pathway, myeloperoxidase (MPO) inhibition, neutrophil extracellular trap (NET) inhibition, and intrinsic antioxidant and antibacterial activity [3-8]. The precursor to PIC1 peptides was originally based on the discovery that the 787 amino acid capsid protein sequence of human astrovirus type 1, a non-enveloped icosahedral RNA virus [9] that is an endemic pathogen causing gastroenteritis in human infants, can inhibit activation of the classical complement pathway

[10] .

[0010] The PIC1 / EPICC family of molecules comprises a collection of rationally designed peptides with several anti-inflammatory functional properties, including inhibition of the classical complement pathway, myeloperoxidase inhibition, neutrophil extracellular trap inhibition, and antioxidant activity. The original PIC1 peptide is a 15-amino acid peptide sequence derived from a scrambled astrovirus coat protein. TIFF0007804672000001.tif4128. The original PIC1 peptide was modified with a monodisperse 24-mer PEGylated moiety at the C-terminus, TIFF0007804672000002.tif4128, thereby increasing its aqueous solubility. A sarcosine substitution scan of SEQ ID NO:3 revealed that replacing isoleucine at position 8 or cysteine ​​at position 9 with sarcosine improved the performance of the two peptides. TIFF0007804672000003.tif12166 was found to be water soluble without pegylation (as described in U.S. Pat. No. 10,005,818). Additional variants based on the PA-I8Sar and PA-I9Sar molecules were constructed, including stapled forms of the peptide and / or one or more D-enantiomeric substitutions at certain amino acid positions. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] U.S. Patent No. 10,005,818 [Non-patent literature]

[0012] [Non-Patent Document 1] Ricklin and Lambris, “Complement-targeted therapeutics.” Nat Biotechnol 2007; 25(11):1265-75 Summary of the Invention

[0013] Brief Summary of the Invention As noted in the background section, there is a great need in the art to identify technologies for peptide-based inhibitors of different pathways of the complement system and to use this knowledge to develop novel therapeutic peptides. The present invention fulfills this and other needs. Aspects of the present invention relate generally to synthetic peptides, and more specifically, to synthetic peptides that are stapled and / or contain one or more D-enantiomeric forms of amino acids.

[0014] In one aspect, the present invention provides synthetic peptides that regulate the complement system and methods for using these peptides. In particular, in some embodiments, the synthetic peptides can bind to, regulate, and inactivate C1 and MBL, thus efficiently inhibiting the activation of the classical and lectin pathways at their earliest stages while leaving the alternative pathway intact. These peptides have therapeutic value because they selectively regulate and inhibit the activation of C1 and MBL without affecting the alternative pathway, and can be used to treat diseases mediated by dysregulated activation of the classical and lectin pathways. In other embodiments, the peptides regulate the activation of the classical pathway but not the lectin pathway. The peptides are useful for a variety of therapeutic indications.

[0015] In some embodiments, the present invention is based on the identification and modification of a 15-amino acid peptide from the Polar Assortant (PA) peptide (SEQ ID NO:2), modifications of this peptide, and methods for their use. The PA peptide is a scrambled peptide derived from the human astrovirus protein called CP1 (SEQ ID NO:1). The PA peptide is also known as PIC1 (peptide inhibitor of complement C1), AstroFend, AF, or SEQ ID NO:2. The PIC1 peptide was originally so named because it was found to be associated with diseases mediated by the complement system. A pegylated form of the PIC1 peptide, called PA-dPEG24 (SEQ ID NO:3), has 24 PEG moieties at the C-terminus of the peptide and has been shown to have improved efficacy in complement inhibition. A form of the PIC1 peptide with the amino acid derivative sarcosine at position 8, called PA-I8Sar (SEQ ID NO:4), also has improved efficacy in complement inhibition. A form of the PIC1 peptide bearing the amino acid derivative sarcosine at position 9, designated PA-C9Sar (SEQ ID NO:5), also exhibits improved efficacy in complement inhibition. PA-dPEG24, PA-I8Sar, and PA-C9Sar are described, for example, in U.S. Patent No. 10,005,818 and U.S. Patent Application Publication No. US2019 / 0209660. As used herein, the term "PIC1 peptide" includes SEQ ID NOs:6-35 and 54-55, which are stapled forms of SEQ ID NO:4 and / or substitutions of SEQ ID NO:4 with one or more D-enantiomeric forms of amino acids in place of their normal L-enantiomers, and SEQ ID NOs:36-53, which are stapled forms of SEQ ID NO:5 and / or substitutions of SEQ ID NO:5 with one or more D-enantiomeric forms of amino acids in place of their normal L-enantiomers.

[0016] In some aspects, the present invention relates to peptides that are stapled forms of PA-I8Sar and / or contain one or more D-enantiomeric amino acid substitutions in the sequence of PA-I8Sar, which can regulate the activation of the classical pathway and the lectin pathway by binding to C1q and MBL. In some aspects, the present invention relates to peptides that are stapled forms of PA-I9Sar and / or contain one or more D-enantiomeric amino acid substitutions in the sequence of PA-I9Sar, which can regulate the activation of the classical pathway and the lectin pathway by binding to C1q and MBL.

[0017] In some embodiments, the peptide sequence has at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NOs: 6-55. In some embodiments, the peptide sequence has at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NOs: 6-35 and 54-55. In some embodiments, the peptide sequence has at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NOs: 36-53.

[0018] In one aspect, the invention provides synthetic peptides comprising at least about 95% sequence identity to the amino acid sequences of SEQ ID NOs:6-35 and 54-55. In some embodiments, the invention is a synthetic peptide comprising the amino acid sequences of SEQ ID NOs:6-35 and 54-55 and modifications. In one aspect, the invention is a synthetic peptide comprising at least about 95% sequence identity to the amino acid sequences of SEQ ID NOs:36-53. In some embodiments, the invention is a synthetic peptide comprising the amino acid sequences of SEQ ID NOs:36-53 and modifications.

[0019] In another aspect, the present invention provides peptide combinations disclosed herein. In some embodiments, the present invention provides compositions comprising at least one synthetic peptide selected from the group consisting of SEQ ID NOs:6-55 and variants thereof. In another aspect, the present invention provides compositions comprising at least one synthetic peptide selected from the group consisting of SEQ ID NOs:6-35 and 54-55 and variants thereof. In another aspect, the present invention provides compositions comprising at least one synthetic peptide selected from the group consisting of SEQ ID NOs:36-53 and variants thereof. In another aspect, the composition further comprises another D-enantiomeric form and / or stapled peptide form of SEQ ID NO:4 and / or SEQ ID NO:5 and variants thereof. In another aspect, the composition further comprises one or more of SEQ ID NOs:2, 3, 4 and / or 5 and variants thereof.

[0020] In another aspect, the present invention is a pharmaceutical composition comprising a therapeutically effective amount of at least one synthetic peptide selected from the group consisting of SEQ ID NOs:6-55 and variants thereof. In another aspect, the present invention is a pharmaceutical composition comprising a therapeutically effective amount of at least one synthetic peptide selected from the group consisting of SEQ ID NOs:6-35 and 54-55 and variants thereof. In another aspect, the present invention is a pharmaceutical composition comprising a therapeutically effective amount of at least one synthetic peptide selected from the group consisting of SEQ ID NOs:36-53 and variants thereof. In another aspect, the pharmaceutical composition further comprises another D-enantiomeric form and / or stapled peptide form of SEQ ID NO:4 and / or SEQ ID NO:5 and variants thereof. In another aspect, the pharmaceutical composition further comprises one or more of SEQ ID NOs:2, 3, 4 and / or 5 and variants thereof.

[0021] In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of at least one synthetic peptide selected from the group consisting of SEQ ID NOs:6-55 and variants thereof, and at least one pharmaceutically acceptable carrier, diluent, or excipient. In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of at least one synthetic peptide selected from the group consisting of SEQ ID NOs:6-35 and 54-55 and variants thereof, and at least one pharmaceutically acceptable carrier, diluent, or excipient. In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of at least one synthetic peptide selected from the group consisting of SEQ ID NOs:36-53 and variants thereof, and at least one pharmaceutically acceptable carrier, diluent, or excipient. In another aspect, the pharmaceutical composition further comprises another D-enantiomeric form and / or stapled peptide form of SEQ ID NO:4 and / or SEQ ID NO:5. In another aspect, the pharmaceutical composition further comprises one or more of SEQ ID NOs: 2, 3, 4 and / or 5 and variants thereof.

[0022] In one aspect, the present invention provides synthetic peptides comprising at least about 95% sequence identity to an amino acid sequence selected from the group of SEQ ID NOs: 6-55.

[0023] In some embodiments, the present invention provides synthetic peptides comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:6-55. In some embodiments, the present invention provides synthetic peptides comprising at least about 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:9, 19, 22, and 25. In some embodiments, the present invention provides synthetic peptides comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:9, 19, 22, and 25. In some embodiments, the present invention provides synthetic peptides comprising at least about 95% sequence identity to SEQ ID NO:9. In some embodiments, the present invention provides synthetic peptides comprising at least about 95% sequence identity to SEQ ID NO:19. In some embodiments, the present invention provides synthetic peptides comprising at least about 95% sequence identity to SEQ ID NO:22. In some embodiments, the present invention provides synthetic peptides comprising at least about 95% sequence identity to SEQ ID NO:25. In another aspect, the present invention provides a combination of the peptides disclosed herein. In some embodiments, the present invention provides compositions comprising at least one synthetic peptide selected from the group consisting of SEQ ID NOs:9, 19, 22, and 25 and variants thereof. In another aspect, the composition further comprises another D-enantiomeric and / or stapled peptide form of SEQ ID NO:4 and / or SEQ ID NO:5 and variants thereof. In another aspect, the composition further comprises one or more of SEQ ID NOs:2, 3, 4, and / or 5 and variants thereof.

[0024] In another aspect, the invention is a pharmaceutical composition comprising a therapeutically effective amount of at least one synthetic peptide selected from the group consisting of SEQ ID NOs:9, 19, 22, and 25 and variants thereof. In another aspect, the pharmaceutical composition further comprises another D-enantiomeric and / or stapled peptide form of SEQ ID NO:4 and / or SEQ ID NO:5 and variants thereof. In another aspect, the pharmaceutical composition further comprises one or more of SEQ ID NOs:2, 3, 4, and / or 5 and variants thereof.

[0025] In a related aspect, the present invention provides pharmaceutical compositions comprising a therapeutically effective amount of any of the synthetic peptides disclosed herein and at least one pharmaceutically acceptable carrier, diluent, or excipient.

[0026] In a related aspect, the present invention provides a method of modulating the complement system comprising administering to a subject in need thereof a pharmaceutical composition described herein.

[0027] In a related aspect, the present invention provides a method of inhibiting myeloperoxidase activity comprising administering to a subject in need thereof a pharmaceutical composition described herein.

[0028] In a related aspect, the present invention provides a method of inhibiting NETosis comprising administering to a subject in need thereof a pharmaceutical composition described herein.

[0029] In a related aspect, the present invention provides a method of inhibiting oxidant activity comprising administering to a subject in need thereof a pharmaceutical composition described herein.

[0030] In a related aspect, the invention provides a method of inhibiting the binding of PD-1 to PD-L1, comprising administering to a subject in need thereof a pharmaceutical composition described herein.

[0031] In a related aspect, the present invention provides a method of inhibiting T cell depletion comprising administering to a subject in need thereof a pharmaceutical composition described herein.

[0032] In a related aspect, the present invention provides a method of inhibiting angiogenesis comprising administering to a subject in need thereof a pharmaceutical composition described herein.

[0033] These and other objects, features and advantages of the present invention will become more apparent from a reading of the following specification in conjunction with the accompanying description, claims and drawings. [Brief explanation of the drawings]

[0034] The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects described below. [Figure 1A] Figures 1A-1B show the inhibition of complement activation by the PIC1 peptide in an ABO incompatibility assay. Figure 1A shows the modification of PA-I8Sar, and Figure 1B shows the modification of PA-C9Sar. Inhibition of ABO incompatibility hemolysis in a CH50-type assay. Peptides are at a final concentration of 0.5 mM. Values ​​are expressed as a percentage of the positive control consisting of human O serum and AB red blood cells in GVBS++ buffer. Data are the mean ± SEM of n=3 independent experiments. [Figure 1B] See legend to Figure 1A. [Figure 2A] Figures 2A-2B show half-maximal binding values ​​for the binding of PIC1 peptides to C1q. Half-maximal binding concentrations were calculated from the binding curves for (2A) the PA-I8Sar mutant and (2B) the PA-I9Sar mutant. Peptide variant PA-0114 was not analyzed due to a lack of material caused by inefficient synthesis, and PA-0116 could not be analyzed due to insufficient solubility. PA-0130, -0131, -0132, and -0133 could not be analyzed because they were not recognized by the primary polyclonal antibody. [Figure 2B] See legend to Figure 2A. [Figure 3A] Figures 3A-3B show half-maximal values ​​for PIC1 peptide inhibition of MPO activity. Half-maximal values ​​were calculated from the activity curves for (3A) the PA-I8Sar mutant and (3B) the PA-C9Sar mutant. PA-0119 did not titrate down to a half-maximal value, so calculations were not possible. [Figure 3B] See legend to Figure 3A. [Figure 4A] Figures 4A-4B show the PIC1 peptide inhibition of oxidant activity in a total antioxidant capacity (TAC) assay. Antioxidant activity is measured in copper reducing equivalents (CRE). (4A) The PA-I8Sar mutant and (4B) the PA-I9Sar mutant were tested across a range of concentrations, and the maximum amount of antioxidant activity is reported for each peptide. [Figure 4B] See legend to Figure 4A. [Figure 5] Figure 5 shows PIC1 peptide inhibition of free DNA. Free DNA, PicoGreen analysis, after neutrophil stimulation with PMA and hydrogen peroxide (HO) compared to neutrophil-only control. PIC1 peptide was added to serum to a final concentration of 2 mM. The graph shows that all peptides inhibited the release of free DNA by neutrophils, as a marker of NETosis. Data are means ± SEM of n=5 independent experiments. [Figure 6A] Figures 6A-6D show C1q binding curves. Binding of increasing concentrations of PA-I8Sar (6A-6B) and PA-I9Sar (6C-6D) to immobilized C1q in an ELISA-type assay. Peptide variants PA-0130, -0131, -0132, and -0133 could not be analyzed because they were not recognized by the primary polyclonal antibody. PA-0114 was not analyzed due to a lack of material caused by inefficient synthesis, and PA-0116 could not be analyzed due to insufficient solubility. [Figure 6B] See legend to Figure 6A. [Figure 6C] See legend to Figure 6A. [Figure 6D]See legend to Figure 6A. [Figure 7A] Figures 7A-7D show MPO inhibition curves. Inhibition of MPO activity by increasing concentrations of PA-I8Sar modified forms (7A-7B) and PA-I9Sar modified forms (7C-7D) in an ELISA-type assay. PA-0119 did not decrease to half-maximal values, so calculations were not possible. [Figure 7B] See legend to Figure 7A. [Figure 7C] See legend to Figure 7A. [Figure 7D] See legend to Figure 7A. [Figure 8A] Figures 8A-8D show total antioxidant capacity binding curves. Increasing concentrations of PA-I8Sar (8A-8B) and PA-I9Sar (8C-8D) were analyzed for total antioxidant activity. Antioxidant activity is measured as copper reducing equivalents (CRE). [Figure 8B] See legend to Figure 8A. [Figure 8C] See legend to Figure 8A. [Figure 8D] See legend to Figure 8A. [Figure 9] FIG. 9 shows the results of a pharmacokinetic assay for increasing doses of PA-0117 based on C1q target acquisition (left bar, 400 mg / kg; middle bar, 200 mg / kg; right bar, 20 mg / kg). [Figure 10A] 10A-10B show the results of hemolysis and pharmacokinetic assays for increasing doses of PA-0127 (left bar, 400 mg / kg; middle bar, 200 mg / kg; right bar, 20 mg / kg). [Figure 10B] See legend to Figure 10A. [Figure 11A] 11A-11B show the results of hemolysis and pharmacokinetic assays for increasing doses of PA-0130 (left bar, 400 mg / kg; middle bar, 200 mg / kg; right bar, 20 mg / kg). [Figure 11B] See legend to Figure 11A. [Figure 12]FIG. 12 shows the results of the hemolysis assay for increasing doses of PA-0133 (left bar, 400 mg / kg; middle bar, 200 mg / kg; right bar, 20 mg / kg). [Figure 13] Figure 13 shows the inhibition of PD-1 binding to PD-L1 in an ELISA plate-based assay. PIC1 peptide was bound to PD-L1 immobilized on the plate surface. Biotinylated PD-1 was then added, and bound PD-1 was detected with streptavidin-HRP reagent, followed by TMB as the substrate for the colorimetric assay. [Figure 14] Figure 14 shows that the PIC1 peptides RLS-0117, RLS-0118, RLS-0127*, and RLS-0133* were able to inhibit PD-1 / PD-L1-mediated cell signaling. PD-1 effector cells were incubated with PD1-L1 aAPC cells in the absence or presence of increasing concentrations of anti-PD-1 antibody (positive control) and selected PIC1 peptides. Luminescence was detected using a luminometer plate reader. For clarity, PIC1 peptides that did not inhibit signaling are not shown but are outlined in Table 4. [Figure 15] Figure 15 shows the binding of RLS-0117 to CTLA-4, PD-1, and PD-L1 in an ELISA plate-based assay. RLS-0117 was bound to CTLA-4, PD-1, and PD-L1 immobilized on the plate surface. Bound C1q served as a positive control for peptide binding. Increasing amounts of peptide were added to the plate, followed by a rabbit polyclonal antibody recognizing the peptide and then a secondary anti-rabbit antibody conjugated with HRP. The plate was then developed by adding TMB as a substrate for the colorimetric assay. [Figure 16]Figure 16 shows that the PIC1 peptides RLS-0127*, RLS-0130, RLS-0156, RLS-0170, and RLS-0174 were able to inhibit CTLA-4-mediated cell signaling. CTLA-4 effector cells were incubated with aAPC / Raji cells in the absence or presence of increasing concentrations of anti-CTLA-4 antibody (positive control), RLS-0127*, RLS-0130, RLS-0156, RLS-0170, and RLS-0174. Luminescence was detected using a luminometer plate reader. For clarity, other PIC1 peptides that did not inhibit CTLA-4 function are outlined in Table 4. [Figure 17] Figure 17 shows that selected PIC1 peptides were able to inhibit T cell depletion to varying degrees, as measured by the reduction in the levels of the apoptotic cell marker caspase 3 / 7. Purified human pan-T cells were stimulated with Dynabeads every 48 hours over an 8-day period, and PIC1 peptide (2 mg / ml) was also administered to the cells at each stimulation. Cells that did not receive Dynabeads were run in parallel to assess background levels of caspase 3 / 7 signal. On day 8, cells were harvested, and caspase 3 / 7 levels were determined by ELISA. [Figure 18A] Figures 18A-18F show that selected PIC1 peptides were able to rescue T cell exhaustion to various degrees, as measured by increased levels of the cytokines IL-2 (18A-18C) and IFN-gamma (18D-18F). Purified human pan-T cells were stimulated with Dynabeads every 48 hours over an 8-day period, and PIC1 peptide (2 mg / ml) was also administered to the cells at each stimulation. Cell supernatants were collected at each stimulation, and levels of IL-2 and IFN-gamma were assayed by ELISA. [Figure 18B] See legend to Figure 18A. [Figure 18C] See legend to Figure 18A. [Figure 18D] See legend to Figure 18A. [Figure 18E] See legend to Figure 18A. [Figure 18F] See legend to Figure 18A. [Figure 19A] Figures 19A-19C show the binding of the PIC1 peptide to VEGF in an ELISA plate-based assay. The PIC1 peptide was bound to VEGF immobilized on the plate surface. A fixed amount of the PIC1 peptide (1 mg / ml) was added to the plate, followed by a rabbit polyclonal antibody recognizing the peptide and then a secondary anti-rabbit antibody conjugated with HRP. The plate was then developed by adding TMB as a substrate for the colorimetric assay. [Figure 19B] See legend to Figure 19A. [Figure 19C] See legend to Figure 19A. [Figure 20] Figure 20 shows that specific PIC1 peptides were able to inhibit non-VEGF-mediated angiogenesis induced by LPS. HUVEC cells were incubated with the indicated PIC1 peptides, followed by the addition of LPS and plating on extracellular matrix. After overnight incubation, evidence of angiogenesis was determined by fluorescence microscopy. Cells receiving and not receiving LPS served as positive and negative controls for angiogenesis. [Figure 21A]Figures 21A-21C show that RLS-0127* and RLS-0133* both inhibit complement activation and MPO peroxidase activity, and that RLS-0127* also binds to C1q. (21A) RLS-0127* and RLS-0133* were evaluated for complement inhibition in an ABO hemolysis assay using O plasma. Increasing amounts of each peptide were added to the assay, and hemolysis was assessed by absorbance at 450 nm in a plate reader. (21B) RLS-0127* and RLS-0133* were evaluated for MPO peroxidase inhibition in a plate-based assay. Increasing amounts of each peptide were added to wells containing bound, purified human MPO, and reduction of peroxidase activity by the TMB substrate was assessed by absorbance at 450 nm in a plate reader. (21C) RLS-0127* and RLS-0133* were evaluated for C1q binding in a plate-based assay. Increasing amounts of each peptide were added to wells containing bound, purified human C1q, which were then probed with an antibody against the peptide followed by a secondary antibody-HRP conjugate. Color development with the TMB substrate was assessed by absorbance at 450 nm in a plate reader. Because RLS-0133* is not recognized by the rabbit polyclonal antibody, binding to C1q could not be determined. RLS-0088 was used as a positive control for all assays. [Figure 21B] See legend to Figure 21A. [Figure 21C] See legend to Figure 21A. DETAILED DESCRIPTION OF THE INVENTION

[0035] Detailed Description of the Invention As noted in the background section, there is a great need in the art to identify technologies for peptide-based inhibitors of different pathways of the complement system and to use this knowledge to develop novel therapeutic peptides. The present invention fulfills this and other needs. Aspects of the present invention relate generally to synthetic peptides, and more specifically to synthetic peptides that are stapled and / or contain one or more amino acids in D-enantiomeric form.

[0036] To facilitate an understanding of the principles and features of various embodiments of the present invention, various exemplary embodiments are described below. Although exemplary embodiments of the present invention are described in detail, it should be understood that other embodiments are contemplated. Accordingly, it is not intended that the present invention be limited in scope to the details of the construction and arrangement of components set forth in the following description or examples. The present invention is capable of other embodiments and of being practiced or carried out in various ways. Furthermore, in describing exemplary embodiments, specific terminology is used for the sake of clarity.

[0037] It should also be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, a reference to one component is intended to include compositions of multiple components. A reference to a composition containing "a" component is intended to include other components in addition to the one specified. In other words, the terms "a," "an," and "the" do not denote a limitation of quantity, but rather denote the presence of "at least one" of the referenced item.

[0038] As used herein, the term "and / or" can mean "and," can mean "or," can mean "exclusive or," can mean "one," can mean "some but not all," can mean "neither," and / or it can mean "both." The term "or" is intended to mean an inclusive "or."

[0039] Furthermore, in describing exemplary embodiments, terminology is used for the sake of clarity. Each term is intended to be accorded its broadest meaning as understood by one of ordinary skill in the art and to encompass all technical equivalents that operate in a similar manner to accomplish a similar purpose. It is understood that embodiments of the disclosed technology can be practiced without these specific details. In other instances, well-known methods, structures, and techniques have not been shown in detail so as not to obscure an understanding of this description. References to "one embodiment," "an embodiment," "illustrative embodiment," "some embodiments," "certain particular embodiments," "various embodiments," etc., indicate that the embodiments of the disclosed technology so described may include particular features, structures, or characteristics, but not all embodiments necessarily include the particular feature, structure, or characteristic. Furthermore, repeated use of the phrase "in one embodiment" does not necessarily refer to the same embodiment, although it may.

[0040] As used herein, the term "about" should be interpreted to refer to both the numbers specified as the endpoints of any range. Any reference to a range should be considered to support any subset within the range. Ranges can be expressed herein as "about," "approximately," or "substantially" from one particular value and / or to another particular value. When such a range is expressed, other exemplary embodiments include from one particular value and / or to the other particular value. Furthermore, the term "about" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within an acceptable standard deviation, as practiced in the art. Alternatively, "about" can mean within ±20%, preferably ±10%, more preferably ±5%, and even more preferably ±1% of a given value. Alternatively, particularly for biological systems or processes, the term can mean within one order of magnitude, preferably within two-fold, of a value. Where specific values ​​are described in this application and claims, unless otherwise stated, the term "about" is implicit and means within an acceptable error range for the particular value in this context.

[0041] Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within the range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0042] Similarly, as used herein, a characterization such as "substantially free" or "substantially pure" of something can include both "at least substantially free" or "at least substantially pure" of something and "completely free" or "completely pure" of something.

[0043] "Comprising" or "containing" or "including" means that at least the specified compound, element, particle, or method step is present in a composition or article or method, and does not exclude the presence of other compounds, materials, particles, method steps, even if other such compounds, materials, particles, method steps have the same function as the specified one.

[0044] Throughout this description, various components may be identified as having particular values ​​or parameters, but these items are provided as exemplary embodiments. Indeed, the exemplary embodiments do not limit the various aspects and concepts of the present invention, as many comparable parameters, sizes, ranges, and / or values ​​may be implemented. Terms such as "first," "second," "primary," "secondary," etc., do not denote any order, quantity, or importance, but rather are used to distinguish one element from another.

[0045] It should be noted that terms such as "particularly," "preferably," "typically," "generally," and "often" are not used herein to limit the scope of the claimed invention or to imply that a particular feature is critical, essential, or even essential to the structure or function of the claimed invention. Rather, these terms are intended simply to highlight alternative or additional features that may or may not be utilized in particular embodiments of the invention. It should also be noted that terms such as "substantially" and "about" are used herein to indicate the inherent degree of uncertainty that may result from any quantitative comparison, value, measurement, or other representation.

[0046] Dimensions and values ​​disclosed herein should not be understood to be strictly limited to the exact numerical values ​​recited. Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as "50 mm" is intended to mean "about 50 mm."

[0047] It should also be understood that the recitation of one or more method steps does not preclude the presence of additional or intervening method steps between those steps expressly identified. Similarly, it should also be understood that the recitation of one or more components in a composition does not preclude the presence of additional components other than those expressly identified.

[0048] The materials described below as comprising various elements of the present invention are intended to be exemplary and not limiting. Many suitable materials that would perform the same or similar functions as the materials described herein are intended to be encompassed within the scope of the present invention. Such other materials not described herein may include, but are not limited to, materials developed after the development of the present invention. Any dimensions listed in the various figures are for illustrative purposes only and are not intended to be limiting. Other dimensions and proportions are contemplated and are intended to be within the scope of the present invention.

[0049] As used herein, the term "subject" or "patient" refers to a mammal, including, but not limited to, humans and veterinary animals. In a preferred embodiment, the subject is a human.

[0050] As used herein, the term "combination" of a synthetic peptide according to the claimed invention and at least one second pharmaceutically active ingredient means that at least two, but any desired combination of compounds, are delivered simultaneously or sequentially (e.g., within 24 hours). When used to treat various diseases, it is contemplated that the compositions and methods of the present invention can be used with other therapeutic methods / agents suitable for the same or similar diseases. Such other therapeutic methods / agents can be co-administered (simultaneously or sequentially) to produce additive or synergistic effects. Due to additive or synergistic effects, the appropriate therapeutically effective dosage for each agent can be reduced.

[0051] A "disease" is a state of a subject's health in which the subject is unable to maintain homeostasis and in which, if the disease does not improve, the subject's health continues to deteriorate. In contrast, a "disorder" in a subject is a state of health in which the subject is able to maintain homeostasis, but in which the subject's health is less favorable than it would be in the absence of the disorder. If left untreated, a disorder does not necessarily cause a further decline in the subject's health.

[0052] The terms "treating" a situation, disorder, or condition or "treatment" of a situation, disorder, or condition include: (1) preventing or delaying the onset of at least one clinical or subclinical symptom of the situation, disorder, or condition occurring in a subject who may be afflicted with or susceptible to the situation, disorder, or condition, but who has not yet experienced or is exhibiting clinical or subclinical symptoms of the situation, disorder, or condition; or (2) inhibiting the situation, disorder, or condition, i.e., arresting, reducing, or delaying the occurrence of the disease or its recurrence (in the case of maintenance treatment) or at least one clinical or subclinical symptom thereof; or (3) palliating the disease, i.e., causing regression of the situation, disorder, or condition, or at least one clinical or subclinical symptom thereof. The benefit to the subject being treated is either statistically significant or at least perceptible to the patient or physician.

[0053] As used herein, the term "therapeutic" means treatment and / or prophylaxis. A therapeutic effect is achieved by suppressing, diminishing, ameliorating, or eradicating the disease state.

[0054] As used herein, the term "therapeutically effective" as applied to dose or amount refers to the quantity of a compound or pharmaceutical composition that, when administered to a subject to treat (e.g., prevent or ameliorate) a condition, disorder, or state, is sufficient to effect such treatment. A "therapeutically effective amount" will vary depending on the compound or bacterium or analog administered, as well as the disease and its severity and the age, weight, physical condition, and responsiveness of the mammal being treated.

[0055] The phrase "pharmaceutically acceptable," when used in connection with the compositions of the present invention, refers to molecular entities and other components of such compositions that are physiologically tolerable and typically do not produce adverse reactions when administered to a mammal (e.g., a human). Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia or other generally recognized pharmacopoeias for use in mammals, more particularly humans.

[0056] The term "pharmaceutical carrier" or "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a compound is administered. Such pharmaceutical carriers may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water or aqueous saline solutions and aqueous dextrose and glycerol solutions are preferably used as carriers, particularly for injectable solutions. Alternatively, a pharmaceutical carrier may be a solid dosage form carrier, including, but not limited to, one or more of binders (for compressed pills), glidants, encapsulating agents, flavoring agents, and coloring agents. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin.

[0057] The term "analog" or "functional analog" refers to a related modified form of a polypeptide in which at least one amino acid substitution, deletion, or addition has been made such that the analog retains substantially the same biological activity in vivo and / or in vitro as the unmodified form.

[0058] The terms "sequence identity" and "percent identity" are used interchangeably herein. In the present invention, to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (for example, gaps can be introduced in the first amino acid or nucleic acid sequence for optimal alignment with the second amino acid or nucleic acid sequence). The amino acid or nucleotide residues at corresponding amino acid or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid or nucleotide residue as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by these sequences (i.e., identity % = number of identical positions / total number of positions (i.e., overlapping positions) × 100). Preferably, the two sequences are the same length.

[0059] Several different computer programs are available for determining the degree of identity between two sequences. For example, comparison of sequences and determination of percent identity between two sequences can be achieved using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid or nucleic acid sequences is determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. (48): 444-453 (1970)) incorporated into the GAP program of the Accelrys GCG software package (available at www.accelrys.com / products / gcg), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4, and a length weight of 1, 2, 3, 4, 5, or 6. These different parameters will likely produce slightly different results, but the overall percent identity of two sequences will not change significantly when using different algorithms.

[0060] Sequence comparison can be carried out over the entire length of the two sequences being compared, or over a fragment of the two sequences. Typically, comparison will be carried out over the full length of the two sequences being compared. However, sequence identity can also be carried out over a region of, for example, 20, 50, 100 or more consecutive amino acid residues.

[0061] " Sequence identity " as known in the art refers to the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, that is, between a reference sequence and a given sequence that is compared with the reference sequence.Sequence identity is determined by comparing a given sequence with a reference sequence after optimally aligning the sequences so as to produce the highest degree of sequence similarity as determined by the string match between the sequences.When aligning in this way, sequence identity is confirmed position by position, for example, if the nucleotide or amino acid residue at a certain position is the same, the sequence is "identical" at that position.Then, the total number of identical positions is divided by the total number of nucleotides or residues in the reference sequence to obtain sequence identity %. Sequence identity may be determined by any of a number of methods, including, but not limited to, Computational Molecular Biology, Lesk, A.N., ed., Oxford University Press, New York (1988), Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinge, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York (1991); and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073, the teachings of which are incorporated herein by reference. (1988).Preferred methods for determining sequence identity are designed to give the largest match between the sequences tested. Methods for determining sequence identity are codified in publicly available computer programs that determine sequence identity between given sequences. Examples of such programs include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research, 12(1):387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. Molec. Biol., 215:403-410 (1990)). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCVI NLM NIH Bethesda, Md. 20894, Altschul, S. F. et al., J. Molec. Biol., 215:403-410 (1990), the teachings of which are incorporated herein by reference). (1990)). These programs optimally align sequences using default gap weights to produce the highest level of sequence identity between a given sequence and a reference sequence. As one example, by a polynucleotide having a nucleotide sequence having at least, e.g., 95%, e.g., at least 96%, 97%, 98%, 99%, or 100% "sequence identity" to a reference nucleotide sequence, it is intended that the nucleotide sequence of the given polynucleotide is identical to the reference sequence, except that the given polynucleotide sequence may contain up to 5, up to 4, up to 3, up to 2, up to 1, or up to 0 point mutations per 100 nucleotides of the reference nucleotide sequence.In other words, in a polynucleotide having a nucleotide sequence that is at least 95%, for example, at least 96%, 97%, 98%, 99%, or 100% identical to a reference nucleotide sequence, up to 5%, 4%, 3%, 2%, 1%, or 0% of the nucleotides of the reference sequence can be deleted, replaced with other nucleotides, or up to 5%, 4%, 3%, 2%, 1%, or 0% of the total nucleotides of the reference sequence can be inserted into the reference sequence. These mutations of the reference sequence can occur at the 5' or 3' terminal position of the reference nucleotide sequence, or anywhere between these terminal positions, either individually among the nucleotides in the reference sequence or in one or more consecutive groups within the reference sequence. Similarly, by a polypeptide having a given amino acid sequence having at least, e.g., 95%, e.g., at least 96%, 97%, 98%, 99%, or 100% sequence identity to a reference amino acid sequence, it is intended that the given amino acid sequence of the polypeptide is identical to the reference sequence except that the given polypeptide sequence may contain up to 5, up to 4, up to 3, up to 2, up to 1, or up to 0 amino acid changes per every 100 amino acids of the reference amino acid sequence. In other words, to obtain a given polypeptide sequence with at least 95%, for example, at least 96%, 97%, 98%, 99%, or 100% sequence identity with a reference amino acid sequence, up to 5%, 4%, 3%, 2%, 1%, or 0% of the amino acid residues of the reference sequence can be deleted or substituted with other amino acids, or up to 5%, 4%, 3%, 2%, 1%, or 0% of the total number of amino acid residues of the reference sequence can be inserted into the reference sequence. These changes in the reference sequence can occur at the amino or carboxy terminal positions of the reference amino acid sequence, or anywhere between these terminal positions, either individually among the residues in the reference sequence or in one or more consecutive groups within the reference sequence. Preferably, non-identical residue positions differ by conservative amino acid substitutions.However, conservative substitutions are not included as matches when determining sequence identity.

[0062] As used herein, the term "immune response" includes innate immune responses, as well as T cell-mediated immune responses and / or B cell-mediated immune responses. Exemplary immune responses include T cell responses, such as cytokine production and cytotoxicity, and B cell responses, such as antibody production. Furthermore, the term "immune response" includes immune responses that are indirectly affected by T cell activation, such as antibody production (humoral response) and activation of cytokine-responsive cells, such as macrophages. Immune cells involved in the immune response include lymphocytes, such as B cells and T cells (CD4+, CD8+, Th1 and Th2 cells); antigen-presenting cells (e.g., professional antigen-presenting cells, such as dendritic cells, macrophages, B lymphocytes, Langerhans cells, and non-professional antigen-presenting cells, such as keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes); natural killer cells; myeloid cells, such as macrophages, eosinophils, mast cells, basophils, and granulocytes (e.g., neutrophils).

[0063] "Parenteral" administration of the immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intradermal (id) injection or infusion techniques.

[0064] In the medical field, the term "preventing" encompasses any activity that reduces the burden of mortality or morbidity due to a disease. Prevention can occur at the primary, secondary, and tertiary levels. While primary prevention avoids the onset of a disease, secondary and tertiary levels of prevention encompass activities aimed at preventing the progression of the disease and the appearance of symptoms, as well as reducing the negative impact of an already established disease by restoring function and reducing disease-related complications.

[0065] A "variant" of a polypeptide according to the present invention may be (i) one in which one or more amino acid residues are substituted with a conservative or non-conservative amino acid residue (preferably a conservative amino acid residue), and such substituted amino acid residue may or may not be encoded by the genetic code; (ii) one in which one or more modified amino acid residues are present, e.g., a residue modified by the attachment of a substituent group; (iii) a polypeptide that is an alternative splice variant of a polypeptide of the present invention; (iv) a fragment of a polypeptide; and / or (v) a polypeptide fused to another polypeptide, e.g., a leader sequence or secretion sequence, or a sequence used for purification (e.g., a His tag) or detection (e.g., an Sv5 epitope tag). Fragments include polypeptides generated via proteolytic cleavage (including multi-site proteolysis) of the original sequence. Variants may be post-translationally or chemically modified. Such variants are considered to be within the scope of one skilled in the art from the teachings herein. As used herein, the term "variant" includes peptides having at least about 95% identity to the peptides disclosed herein.

[0066] Within the meaning of the present invention, the term "co-administration" is used to refer to the administration of a composition according to the present invention and another therapeutic agent simultaneously in one composition, or simultaneously in different compositions, or sequentially (preferably within 24 hours).

[0067] In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art, such techniques being fully explained in the literature. See, among others, Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (referred to herein as "Sambrook et al., 1989"); DNA Cloning: A Practical Approach, Volumes I and II (D.N. Glover ed. 1985); Oligonucleotide Synthesis (M.J. Gait ed. 1984); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds. (1985); Transcription and Translation (B.D. Hames & S.J. Higgins, eds. (1984); Animal Cell Culture (R.I. Freshney, ed. (1986); Immobilized Cells and Enzymes (I.R.L. Press, (1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); F.M. Ausubel et al. (eds.), Current Protocols in See Molecular Biology, John Wiley & Sons, Inc. (1994).

[0068] Peptide Compositions of the Present Invention Modifications of the amino acid structure of CP1 have led to the discovery of additional peptides capable of modulating complement activation, such as C1q activity. These peptides are also significantly more potent than the parent molecule in in vitro assays of classical complement pathway activation / inhibition, myeloperoxidase (MPO) inhibition, oxidant and NET activity. 8th place compared to TIFF0007804672000004.tif4128 TIFF0007804672000005.tif4128 and 9th place It has previously been shown that the substitution of isoleucine with sarcosine in TIFF0007804672000006.tif5128 results in peptides with increased solubility and enhanced inhibition of biological activity without PEGylation. To determine whether more potent peptides could be identified, amino acid variants based on the PA-I8Sar and PA-C9Sar scaffolds were synthesized. The amino acid variants consisted of stapled peptides or peptides with D-amino acids individually substituted at each position in the PA-I8Sar and PA-C9Sar peptide sequences (Table 1). Four peptides based on the PA-I8Sar scaffold contained a combination of stapled and D-amino acid residues. While not wishing to be bound by theory, stapling techniques can increase peptide stability and enhance biological activity by locking the peptide molecule into a biologically active α-helical secondary structure. While not wishing to be bound by theory, D-amino acid substitutions can confer additional stability to the peptide and extend its in vivo half-life. All but one of these peptides were readily soluble in water and were evaluated for biological activity in a variety of in vitro assays.

[0069] As used herein, the term "peptide" refers to a naturally occurring amino acid sequence or a peptidomimetic, peptide analog, and / or synthetic derivative of about 15 amino acids based on SEQ ID NO:4 or SEQ ID NO:5 (e.g., but not limited to, stapled peptides, sarcosine-substituted, D-amino acid-substituted, and pegylated peptides). Furthermore, a peptide may be less than about 15 amino acid residues, e.g., about 10 to about 15 amino acid residues, e.g., about 5 to about 10 amino acid residues. For example, peptides of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 amino acid residues may also be peptides within the context of the present invention. A peptide may also be more than 15 amino acids, e.g., 16, 17, 18, 19, 20, or more amino acids.

[0070] The disclosed peptides are generally amino acid sequences of about 15 amino acid residues or less, which may be constrained (i.e., have certain elements of structure, such as, for example, the presence of amino acids initiating a β-turn or β-pleated sheet, or be cyclized, for example, by the presence of disulfide-bonded Cys residues) or unconstrained (i.e., linear).

[0071] Substitutes for an amino acid within a peptide sequence can be selected from other members of the class to which the amino acid belongs. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Amino acids containing aromatic ring structures include phenylalanine, tryptophan, and tyrosine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine and lysine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. For example, one or more amino acid residues within a sequence can be substituted with another amino acid of a similar polarity that acts as a functional equivalent, resulting in a silent change.

[0072] Conservative changes generally do not lead to significant changes in the structure and function of the resulting protein. Non-conservative changes are likely to change the structure, activity, or function of the resulting protein. For example, the peptides of the present disclosure comprise one or more of the following conservative amino acid substitutions: aliphatic amino acids, such as alanine, valine, leucine, and isoleucine, are replaced with other aliphatic amino acids; serine is replaced with threonine; threonine is replaced with serine; acidic residues, such as aspartic acid and glutamic acid, are replaced with other acidic residues; residues with amide groups, such as asparagine and glutamine, are replaced with other residues with amide groups; basic residues, such as lysine and arginine, are replaced with other basic residues; and aromatic residues, such as phenylalanine and tyrosine, are replaced with other aromatic residues.

[0073] Particularly preferred amino acid substitutions include: (a) Ala to Glu or vice versa, so that the negative charge can be reduced; (b) Lys to Arg or vice versa, so that a positive charge can be maintained; (c) Ala to Arg or vice versa, so that the positive charge can be reduced; (d) Glu to Asp or vice versa, so that the negative charge can be maintained; (e) Ser to Thr or vice versa, so that a free -OH can be maintained; (f) Gln to Asn or vice versa, so that free NH2 can be maintained; (g) Ile for Leu or Val or vice versa, as roughly equivalent hydrophobic amino acids; (h) Phe for Tyr or vice versa, as roughly equivalent aromatic amino acids; and (i) Ala to Cys or vice versa, such that disulfide bonds are affected.

[0074] Substitutions for amino acids within the peptide sequence can be selected from any amino acid, including, but not limited to, alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, pyrolysine, selenocysteine, serine, threonine, tryptophan, tyrosine, valine, N-formyl-L-methionine, sarcosine, or other N-methylated amino acids. In some embodiments, sarcosine is substituted for an amino acid within the peptide sequence.

[0075] Peptide stapling can be achieved by using, for example, unnatural amino acids with side chains that can be covalently linked at desired positions in the peptide to introduce an alpha helix into the peptide structure (see, e.g., Ali et al., Stapled Peptides Inhibitors: A New Window for Target Drug Discovery, Comput Struct Biotechnol J. 2019; 17: 263-281 and Walensky et al., Hydrocarbon-Stapled Peptides: Principles, Practice, and Progress, J Med Chem. 2014 Aug 14; 57(15): 6275-6288).

[0076] In one aspect, the present invention discloses synthetic peptides derived from human astrovirus coat proteins, the peptides comprising the amino acid sequences and modifications of SEQ ID NOs:6-55. In some aspects, the present invention discloses synthetic peptides derived from human astrovirus coat proteins, the peptides comprising the amino acid sequences and modifications of SEQ ID NOs:6-55, as shown in Table 1 below. Staple amino acids are underlined and D-enantiomeric amino acids are shown in bold.

[0077] Table 1: List of peptides of the present invention TIFF0007804672000007.tif135170TIFF0007804672000008.tif216170TIFF0007804672000009.tif216170TIFF0007804672000010.tif200170

[0078] In some embodiments, the peptide sequence has at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NOs: 6-55. In some embodiments, the peptide sequence has at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NOs: 6-35 and 54-55. In some embodiments, the peptide sequence has at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NOs: 36-53.

[0079] In one aspect, the invention is a synthetic peptide comprising the amino acid sequence and modifications of SEQ ID NOs: 6-55. In some embodiments, the invention is a synthetic peptide comprising the amino acid sequence and modifications of SEQ ID NOs: 6-35 and 54-55. In some embodiments, the invention is a synthetic peptide comprising the amino acid sequence and modifications of SEQ ID NOs: 36-53.

[0080] In another aspect, the present invention provides peptide combinations disclosed herein. In some embodiments, the present invention provides compositions comprising at least one synthetic peptide selected from the group consisting of SEQ ID NOs:6-55 and variants thereof. In another aspect, the present invention provides compositions comprising at least one synthetic peptide selected from the group consisting of SEQ ID NOs:6-35 and 54-55 and variants thereof. In another aspect, the present invention provides compositions comprising at least one synthetic peptide selected from the group consisting of SEQ ID NOs:36-53 and variants thereof. In another aspect, the composition further comprises another D-enantiomeric form and / or stapled peptide form of SEQ ID NO:4 and / or SEQ ID NO:5 and variants thereof. In another aspect, the composition further comprises one or more of SEQ ID NOs:2, 3, 4 and / or 5 and variants thereof.

[0081] In another aspect, the present invention is a pharmaceutical composition comprising a therapeutically effective amount of at least one synthetic peptide selected from the group consisting of SEQ ID NOs:6-55 and variants thereof. In another aspect, the present invention is a pharmaceutical composition comprising a therapeutically effective amount of at least one synthetic peptide selected from the group consisting of SEQ ID NOs:6-35 and 54-55 and variants thereof. In another aspect, the present invention is a pharmaceutical composition comprising a therapeutically effective amount of at least one synthetic peptide selected from the group consisting of SEQ ID NOs:36-53 and variants thereof. In another aspect, the pharmaceutical composition further comprises another D-enantiomeric form and / or stapled peptide form of SEQ ID NO:4 and / or SEQ ID NO:5 and variants thereof. In another aspect, the pharmaceutical composition further comprises one or more of SEQ ID NOs:2, 3, 4 and / or 5 and variants thereof.

[0082] In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of at least one synthetic peptide selected from the group consisting of SEQ ID NOs:6-55 and variants thereof, and at least one pharmaceutically acceptable carrier, diluent, or excipient. In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of at least one synthetic peptide selected from the group consisting of SEQ ID NOs:6-35 and 54-55 and variants thereof, and at least one pharmaceutically acceptable carrier, diluent, or excipient. In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of at least one synthetic peptide selected from the group consisting of SEQ ID NOs:36-53 and variants thereof, and at least one pharmaceutically acceptable carrier, diluent, or excipient. In another aspect, the pharmaceutical composition further comprises another D-enantiomeric form and / or stapled peptide form of SEQ ID NO:4 and / or SEQ ID NO:5. In another aspect, the pharmaceutical composition further comprises one or more of SEQ ID NOs: 2, 3, 4 and / or 5 and variants thereof.

[0083] In one aspect, the present invention provides synthetic peptides comprising at least about 95% sequence identity to an amino acid sequence selected from the group of SEQ ID NOs: 6-55.

[0084] In some embodiments, the present invention provides synthetic peptides comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:6-55. In some embodiments, the present invention provides synthetic peptides comprising at least about 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:9, 19, 22, and 25. In some embodiments, the present invention provides synthetic peptides comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:9, 19, 22, and 25. In some embodiments, the present invention provides synthetic peptides comprising at least about 95% sequence identity to SEQ ID NO:9. In some embodiments, the present invention provides synthetic peptides comprising at least about 95% sequence identity to SEQ ID NO:19. In some embodiments, the present invention provides synthetic peptides comprising at least about 95% sequence identity to SEQ ID NO:22. In some embodiments, the present invention provides synthetic peptides comprising at least about 95% sequence identity to SEQ ID NO:25. In another aspect, the present invention provides a composition comprising at least one synthetic peptide selected from the group consisting of SEQ ID NOs:9, 19, 22, and 25 and variants thereof. In another aspect, the composition further comprises another D-enantiomeric and / or stapled peptide form of SEQ ID NO:4 and / or SEQ ID NO:5 and variants thereof. In another aspect, the composition further comprises one or more of SEQ ID NOs:2, 3, 4, and / or 5 and variants thereof.

[0085] In another aspect, the invention is a pharmaceutical composition comprising a therapeutically effective amount of at least one synthetic peptide selected from the group consisting of SEQ ID NOs:9, 19, 22, and 25 and variants thereof. In another aspect, the pharmaceutical composition further comprises another D-enantiomeric and / or stapled peptide form of SEQ ID NO:4 and / or SEQ ID NO:5 and variants thereof. In another aspect, the pharmaceutical composition further comprises one or more of SEQ ID NOs:2, 3, 4, and / or 5 and variants thereof.

[0086] The disclosed peptides can selectively modulate the activation of C1q and MBL without affecting alternative pathway activity, and are therefore ideal for the prevention and treatment of diseases mediated by dysregulated activation of the classical and lectin pathways, respectively. Specific blockade of the classical and lectin pathways is particularly needed because both pathways are involved in ischemia-reperfusion-induced injury in many animal models [Castellano et al., "Therapeutic targeting of classical and lectin pathways of complement protects from ischemia-reperfusion-induced renal damage." Am J Pathol. 2010; 176(4):1648-59; Lee et al., "Early complement factors in the local tissue immunocomplex generated during intestinal ischemia / reperfusion injury." Mol. Immunol. 2010 February; 47(5):972-81; Tjernberg, et al., "Acute antibody-mediated complement activation mediates lysis of pancreatic islet cells and may cause tissue loss in clinical islet transplantation." Transplantation. 2008 Apr. 27; 85(8):1193-9; Zhang et al. "The role of natural IgM in myocardial ischemia-reperfusion injury.” J Mol Cell Cardiol. 2006 July; 41(1):62-7). The alternative pathway is essential for immune surveillance against invading pathogens, and humans with a deficiency in the alternative pathway suffer from severe bacterial infections.By binding to and inactivating C1q and MBL, the peptide can efficiently modulate activation of the classical and lectin pathways while leaving the alternative pathway intact.

[0087] As used herein, the term "modulate" refers to (i) controlling, reducing, inhibiting, or regulating the biological function of an enzyme, protein, peptide, factor, by-product, or derivative thereof, either individually or in combination; (ii) reducing the amount of a biological protein, peptide, or derivative thereof, either in vivo or in vitro; or (iii) interrupting a biological chain of events, cascade, or pathway known to involve a series of related biological or chemical reactions. Thus, the term "modulate" can be used to describe, for example, reducing the amount of a single component of the complement cascade compared to a control sample, reducing the rate or total amount of formation of a component or component complex, or reducing the overall activity of a complex process or series of biological reactions, resulting in cell lysis, formation of a convertase enzyme, formation of a complement-derived membrane attack complex, inflammation, or inflammatory disease. In in vitro assays, the term "modulate" can refer to a measurable change or reduction in some biological or chemical event, although one of skill in the art will recognize that a measurable change or reduction need not be absolute to be "modulatory."

[0088] In some aspects, the present invention relates to therapeutically active peptides that have the effect of modulating the complement system.

[0089] Modulation of C1q interaction with the C1q receptor The interaction of C1q with the C1q receptor appears to play an important role in homeostatic functions such as the clearance of apoptotic cellular debris and immune complexes, as well as in T cell signaling through antigen-presenting cells (macrophages and dendritic cells). Currently, there are no clinical pharmacological agents that modulate the interaction of C1q with the C1q receptor.

[0090] The disclosed peptides can be used to block the binding of C1q to C1q receptors, including calreticulin / cC1qR. The ability of the disclosed peptides to block the binding of C1q to cellular receptors may have an important role in modulating intracellular signaling processes mediated by the binding of C1q to the C1q receptor.

[0091] Myeloperoxidase (MPO) activity Myeloperoxidase (MPO) is a neutrophil-derived enzyme that produces hypochlorite (bleach) in acute inflammation, damaging invading and host cells alike. This enzyme is known to be destructive to host tissues in many diseases.

[0092] In some embodiments, the peptides disclosed herein block the enzymatic activity of MPO. In some embodiments, the MPO activity present in purified human neutrophil lysates can be directly inhibited by the peptides. In some embodiments, the present invention demonstrates that the peptides have anti-inflammatory activity.

[0093] Hemolysis inhibition Peptides of the present invention, including SEQ ID NOs: 6-56, can block complement-mediated lysis of AB human red blood cells (RBCs) by O serum in vitro, an assay that mimics ABO incompatibility.

[0094] Neutrophil extracellular trap formation NETs are formed during the stimulation of neutrophils in acute inflammation and can damage host tissues. These NETs consist of extracellular DNA coated with neutrophil-derived proteins, such as histones, neutrophil elastase, and MPO, which can be toxic to host cells and tissues. In some embodiments, the peptides disclosed herein block the formation of NETs.

[0095] Oxidant activity Oxidant activity resulting from the formation of reactive oxygen species can be generated in acute inflammation, leading to host cell and tissue damage. In some embodiments, the peptides disclosed herein have antioxidant activity against oxidant-generating molecules such as MPO.

[0096] Pharmaceutical compositions of the present invention The present disclosure provides pharmaceutical compositions capable of regulating the complement system, comprising at least one peptide as described above and at least one pharmaceutically acceptable carrier, diluent, stabilizer, or excipient. Pharmaceutically acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed. They may be solid, semisolid, or liquid. The pharmaceutical compositions of the present invention may be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, or syrups.

[0097] The pharmaceutical compositions of the present invention are prepared by mixing the peptides of appropriate purity with pharmaceutically acceptable carriers, diluents, or excipients. Examples of formulations and methods for preparing such formulations are well known in the art. The pharmaceutical compositions of the present invention are useful as prophylactic and therapeutic agents for various disorders and diseases such as those described above. In one embodiment, the composition contains a therapeutically effective amount of at least one peptide disclosed herein. In another embodiment, the composition contains at least one other active ingredient effective for regulating the complement system. In another embodiment, the composition contains at least one other active ingredient effective for treating at least one disease associated with the complement system. In another embodiment, the composition contains at least one other active ingredient effective for treating at least one disease not associated with the complement system. As used herein, the term "therapeutically effective amount" refers to the total amount of each active ingredient sufficient to show benefit to the subject.

[0098] The therapeutically effective amount of peptide varies depending on several factors, such as the condition to be treated, the severity of the condition, the time of administration, the administration route, the excretion rate of the peptide used, the duration of treatment, the concomitant therapy involved, and the age, sex, weight and condition of the subject.Those skilled in the art can determine the therapeutically effective amount.Therefore, those skilled in the art may need to titrate the dosage and modify the administration route to achieve the maximum therapeutic effect.

[0099] Effective daily doses generally range from about 0.001 to about 200 milligrams per kilogram of body weight (mg / kg), including about 5 to about 160 mg / kg, about 10 to about 160 mg / kg, about 40 mg / kg to about 160 mg / kg, and about 40 mg / kg to about 100 mg / kg. This dose can be achieved with a dosing regimen of 1 to 6 times daily. Alternatively, optimal treatment can be achieved with sustained-release formulations using less frequent dosing regimens.

[0100] In another aspect, the present invention is a pharmaceutical composition comprising a therapeutically effective amount of at least one synthetic peptide selected from the group consisting of SEQ ID NOs:6-55 and variants thereof, and at least one pharmaceutically acceptable carrier, diluent, or excipient. In another aspect, the present invention is a pharmaceutical composition comprising a therapeutically effective amount of at least one synthetic peptide selected from the group consisting of SEQ ID NOs:6-35 and 54-55 and variants thereof, and at least one pharmaceutically acceptable carrier, diluent, or excipient. In another aspect, the present invention is a pharmaceutical composition comprising a therapeutically effective amount of at least one synthetic peptide selected from the group consisting of SEQ ID NOs:36-53 and variants thereof, and at least one pharmaceutically acceptable carrier, diluent, or excipient. In another aspect, the pharmaceutical composition further comprises another D-enantiomeric form and / or stapled peptide form of SEQ ID NO:4 and / or SEQ ID NO:5, and at least one pharmaceutically acceptable carrier, diluent, or excipient. In another aspect, the pharmaceutical composition further comprises one or more of SEQ ID NOs:2, 3, 4 and / or 5 and variants thereof and at least one pharmaceutically acceptable carrier, diluent, or excipient.

[0101] The compositions of the present invention can include carriers and / or excipients. While the peptides of the present invention can be used directly for therapy, it may be preferable to administer them in a pharmaceutical formulation, for example, in a mixture with suitable pharmaceutical excipients and / or carriers selected with respect to the intended route of administration and standard pharmaceutical practice. The excipients and / or carriers must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to their recipient. Acceptable excipients and carriers for therapeutic use are well known in the pharmaceutical field and are described, for example, in Remington: The Science and Practice of Pharmacy. Lippincott Williams & Wilkins (AR Gennaro, ed. 2005). The choice of pharmaceutical excipients and carriers can be selected with respect to the intended route of administration and standard pharmaceutical practice. Oral formulations are easily compatible with additional mixtures, such as milk, yogurt, and infant formula. Oral solid dosage forms can also be used, and examples of oral solid dosage forms include capsules, tablets, caplets, pills, troches, lozenges, powders, and granules. Non-limiting examples of suitable excipients include, for example, diluents, buffering agents (e.g., sodium bicarbonate), preservatives, stabilizers, binders, compression agents, lubricants, dispersion enhancers, disintegrants, antioxidants, flavoring agents, sweeteners, and coloring agents. Those skilled in the art can fully prepare suitable solutions.

[0102] In one embodiment of any of the compositions of the present invention, the composition is formulated for delivery by routes such as oral, topical, rectal, mucosal, sublingual, nasal, naso / oro (naso / oro) gastric gavage, parenteral, intraperitoneal, intradermal, transdermal, intrathecal, intranasal, and intratracheal administration.In one embodiment of any of the compositions of the present invention, the composition is in the form of a liquid, foam, cream, spray, powder, or gel.In one embodiment of any of the compositions of the present invention, the composition comprises a buffering agent (e.g., sodium bicarbonate).

[0103] The administration of compounds and compositions in the method of the present invention can be achieved by any method known in the art.Non-limiting examples of useful routes of delivery include oral, rectal, fecal (by enema), and nasogastric / oral gavage, as well as parenteral, intraperitoneal, intradermal, transdermal, intrathecal, intranasal, and intratracheal administration.The active agent can be systemic after administration, or can be localized by using regional administration, intramural administration, or by using implants that act to retain the active dose at the site of implantation.

[0104] Useful dosages of the compounds and formulations of the present invention can vary widely depending on the nature of the disease, the patient's medical history, the frequency of administration, the mode of administration, the clearance of the agent from the host, and the like. A high initial dose can be administered, followed by a lower maintenance dose. The dose can be administered less frequently, such as weekly or biweekly, to maintain an effective dosage level, or can be divided into smaller doses and administered daily, semiweekly, and the like. It is contemplated that various doses may be effective to achieve a therapeutic effect. While it is possible to use the compounds of the present invention directly for therapy, it may be preferable to administer them in a pharmaceutical formulation, e.g., in admixture with appropriate pharmaceutical excipients, diluents, or carriers selected with regard to the intended route of administration and standard pharmaceutical practice. The excipients, diluents, and / or carriers must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. Acceptable excipients, diluents, and carriers for therapeutic use are well known in the pharmaceutical arts and are described, for example, in Remington: The Science and Practice of Pharmacy. Lippincott Williams & Wilkins (AR Gennaro, ed. 2005). The choice of pharmaceutical excipient, diluent, and carrier can be selected with regard to the intended route of administration and standard pharmaceutical practice.

[0105] Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous suspensions which may contain suspending agents, solubilizing agents, thickening agents, stabilizers, and preservatives.

[0106] The solutions or suspensions can contain any of the following components in any combination: a sterile diluent, such as, but not limited to, water for injection, saline solution, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol and methylparabens; antioxidants, such as ascorbic acid and sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetic acid, citric acid, and phosphate; and agents for the adjustment of tonicity, such as sodium chloride or dextrose.

[0107] When an agent exhibits insufficient solubility, methods for solubilizing the agent can be used. Such methods are known to those skilled in the art and include, but are not limited to, the use of cosolvents such as dimethyl sulfoxide (DMSO), the use of surfactants such as TWEEN® 80, or dissolving in aqueous sodium bicarbonate solution. Pharmaceutically acceptable derivatives of the agent can also be used in formulating effective pharmaceutical compositions.

[0108] The compositions can include, together with the active agent, for example, but not limited to, diluents such as lactose, sucrose, dicalcium phosphate, or carboxymethylcellulose; lubricants such as magnesium stearate, calcium stearate, and talc; and binders such as starch, natural gums such as acacia gum, gelatin, glucose, molasses, polyvinylpyrrolidone, cellulose, and derivatives thereof, povidone, crospovidone, and other such binders known to those skilled in the art. Liquid pharmaceutically administrable compositions can be prepared, for example, by dissolving, dispersing, or mixing the active agent defined above and any pharmaceutical adjuvants in a carrier such as, for example, but not limited to, water, saline, aqueous dextrose, glycerol, glycol, ethanol, etc., thereby forming a solution or suspension. If desired, the administered pharmaceutical composition can also contain small amounts of nontoxic auxiliary substances, such as wetting agents, emulsifying agents, or solubilizing agents, pH buffering agents, and the like, for example, but not limited to, acetic acid, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other such agents. Actual methods for preparing such dosage forms are known or will be apparent to those skilled in the art (e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 15th Edition, 1975). The administered composition or formulation will, in any event, contain a quantity of the active agent sufficient to alleviate the symptoms of the subject being treated.

[0109] The active agents or pharmaceutically acceptable derivatives may be prepared with carriers that protect the agent against rapid elimination from the body, such as time-release formulations or coatings. The compositions may include other active agents to obtain desired combinations of properties.

[0110] Parenteral administration is generally characterized by subcutaneous, intramuscular, or intravenous injection, and is also contemplated herein. Injections can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for liquid solution or suspension before injection, or as emulsions. Suitable excipients include, but are not limited to, water, saline, dextrose, glycerol, or ethanol. In addition, if desired, the administered pharmaceutical composition can also contain small amounts of nontoxic auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, and other such agents, such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, and cyclodextrins.

[0111] The lyophilized powder can be reconstituted for administration into solutions, emulsions, and other mixtures, or can be formulated as a solid or gel. Sterile lyophilized powders are prepared by dissolving the agent provided herein or a pharmaceutically acceptable derivative thereof in a suitable solvent. The solvent can contain excipients to improve stability or other pharmacological components of the powder or the reconstituted solution prepared from the powder. Excipients that can be used include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable agents. The solvent can also contain a buffer, such as citric acid, sodium, or potassium phosphate, or other such buffers known to those skilled in the art, typically at about neutral pH. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those skilled in the art provides the desired formulation. Generally, the resulting solution can be apportioned into vials for lyophilization. Each vial can contain, by way of example and not limitation, a single dose (10-1000 mg, e.g., 100-500 mg) or multiple doses of the agent. The lyophilized powder can be stored under appropriate conditions, e.g., at about 4°C to room temperature. Reconstitution of this lyophilized powder with water for injection provides a formulation for use in parenteral administration.

[0112] How to use Another aspect of the present invention provides a method of regulating the complement system comprising administering to a subject in need thereof a therapeutically effective amount of a peptide and / or pharmaceutical composition of the present invention.

[0113] In another aspect, the present invention provides a method for inhibiting myeloperoxidase activity, comprising administering to a subject in need thereof a therapeutically effective amount of a peptide and / or pharmaceutical composition of the present invention.

[0114] In another aspect, the present invention provides a method of inhibiting NETosis, comprising administering to a subject in need thereof a therapeutically effective amount of a peptide and / or pharmaceutical composition of the present invention.

[0115] In another aspect, the present invention provides a method of inhibiting oxidant activity comprising administering to a subject in need thereof a therapeutically effective amount of a peptide and / or pharmaceutical composition of the present invention.

[0116] In another aspect, the present invention provides methods for inhibiting the binding of PD-1 to PD-L1, comprising administering to a subject in need thereof a therapeutically effective amount of a peptide and / or pharmaceutical composition of the invention.

[0117] In another aspect, the present invention provides a method of inhibiting T cell depletion comprising administering to a subject in need thereof a therapeutically effective amount of a peptide and / or pharmaceutical composition of the present invention.

[0118] In another aspect, the present invention provides a method of inhibiting angiogenesis, comprising administering to a subject in need thereof a therapeutically effective amount of a peptide and / or pharmaceutical composition of the present invention.

[0119] Combination therapy A further aspect of the present invention provides a method for modulating the complement system, comprising administering a pharmaceutical composition of the present invention to a subject. While the pharmaceutical compositions of the present invention can be administered as the sole active pharmaceutical agent, they can also be used in combination with one or more therapeutic or prophylactic agents effective in modulating the complement system. In this aspect, the method of the present invention comprises administering a pharmaceutical composition of the present invention before, simultaneously with, and / or after one or more additional therapeutic or prophylactic agents effective in modulating the complement system.

[0120] The pharmaceutical compositions of the present invention can be administered with additional agents in combination therapy, either together or separately, or by combining the pharmaceutical composition and additional agent in a single composition. Dosages are administered and adjusted to achieve maximum regulation of the complement system. For example, both the pharmaceutical composition and the additional agent are typically present at dosage levels between about 10% and about 150%, more preferably between about 10% and about 80%, of the dosage typically administered in a monotherapy regimen. [Example]

[0121] The present invention is also described and illustrated through the following examples. However, the use of these and other examples anywhere in the specification is merely illustrative and in no way limits the scope and meaning of the invention or any exemplified term. Likewise, the present invention is not limited to any particular preferred embodiment described herein. Indeed, many modifications and variations of the present invention may become apparent to those skilled in the art upon reading this specification, and such variations can be made without departing from the invention in spirit or scope. Therefore, the present invention is to be limited only by the terms of the appended claims, along with the full range of equivalents to which such claims are entitled.

[0122] Example 1: Characterization of SEQ ID NOs: 6-53 The inventors found that a sarcosine amino acid substitution scan of PA-dPEG24 revealed that substitution of this amino acid at different positions in the peptide resulted in peptides that were water-soluble in the absence of PEGylation and also exhibited increased inhibitory activity in in vitro assays of classical complement pathway activation, MPO activation, NET formation, and antioxidant activity.

[11] Two variants in which sarcosine was replaced with isoleucine at position 8 (PA-I8Sar) or cysteine ​​at position 9 (PA-C9Sar) exhibited increased inhibitory activity in the respective assays. Using both the PA-I8Sar and PA-C9Sar peptide scaffolds, the inventors created peptides with D-amino acid substitutions and / or engineered stapling that exhibited increased potency in various functional assays and retained aqueous solubility in the absence of PEGylation.

[0123] Materials and reagents Peptides were synthesized by New England Peptide (Gardner, MA) to >90% purity (Table 1). Stapled peptides were generated by a one-component stapling technique using either S-pentenylalanine (S5) at the i,i+4 positions for one-turn stapling or a combination of R-octenylalanine (R8) and S-pentenylalanine (S5) at the i,i+7 positions. Sequences corresponding to PA-0135, PA-0137, PA-0139, PA-0141, and PA-0146 could not be produced because the cis isomer became excessive over the trans isomer during synthesis, making purification of the normally favored trans isomer difficult (NEP, staff correspondence). Therefore, these sequences were not pursued. Because no enantiomeric forms exist, the D-enantiomeric form of each amino acid was individually substituted at each position in the PA-I8Sar and PA-C9Sar peptide sequences, except for sarcosine at positions 8 (PA-I8Sar) and 9 (PA-C9Sar). All peptides, except PA-0116, were dissolved in water and the pH was adjusted with NaOH. Purified C1q was purchased from Complement Technology (Tyler, TX). Purified MPO was purchased from Lee BioSolutions (Maryland Heights, MO), and tetramethylbenzidine (TMB) was purchased from Thermo Fisher (Waltham, MA). The buffer contained the complement-permissive GVBS ++ Buffer solution (veronal-buffered saline containing 0.1% gelatin, 0.15 mM CaCl2, and 1 mM MgCl2

[12] ) was included.

[0124] method Normal human serum (NHS) Blood type O normal human serum (NHS) was prepared as previously described

[12] . Briefly, blood from at least four healthy human donors was collected in additive-free Vacutainer tubes (red caps). The blood was incubated at room temperature for 30 minutes and on ice for 2 hours to separate the clot and serum. The serum was then pooled, aliquoted, and frozen at -80°C.

[0125] Hemolytic assay of complement activity For hemolytic complement assays, human red blood cells (RBCs) from type AB donors were purified, washed, and diluted to 1.0 × 10 9 Human serum from a blood type O donor at a final concentration of 15% was mixed with 0.5 mM peptide and normalized to cells / ml. ++ and 5.0 × 10 7 The volume was brought up to 0.2 ml with 1 RBC. Samples were incubated at 37°C for 1 hour, then spun at 3,000 rpm for 5 minutes, and the supernatant was collected and read at 412 nm. Values ​​were calculated as GVBS. ++ Expressed as a percentage of the positive control consisting of human O serum and AB red blood cells in buffer.

[0126] C1q binding assay C1q binding assays were performed as previously described

[11] . Briefly, Immunlon-2 HB ELISA plates were coated with 1 μg / ml C1q in bicarbonate buffer overnight at 4°C. Plates were washed with PBS-T (phosphate-buffered saline + 0.1% Tween) and then blocked with 1% gelatin / PBS for 2 hours at room temperature. After washing, plates were incubated with peptides starting at 2.5 mg / ml and then serially diluted in 1% gelatin / PBS for 1 hour at room temperature, followed by washing. Plates were then coated with lead peptides lacking PEGylation. The wells were probed with a rabbit antibody raised against TIFF0007804672000011.tif4128

[11] at 1:1000 in 1% gelatin / PBS for 1 hour at room temperature, followed by goat anti-rabbit HRP (Sigma Aldrich, St. Louis, MO) at 1:1000 in 1% gelatin / PBS for 1 hour at room temperature, with an intermediate wash step. After adding TMB substrate solution to the wells, the reaction was stopped with 1N H2SO4, and the plate was read at 450 nm on a BioTek Synergy HT plate reader.

[0127] MPO activity assay MPO activity assays were performed as previously described

[10] . Briefly, peptides were diluted to 12 mg / ml and serially titrated in 96-well plates in a volume of 0.02 ml. MPO was diluted to 20 μg / ml, and 0.02 ml was added to the titrated peptide. TMB (3,30,5,50-tetramethylbenzidine) (0.1 ml) was added to each well for 2 min, followed by 0.1 ml of 2.5 N H2SO4 for an additional 2 min. The wells were then read at 450 nm using a 96-well plate reader (BioTek).

[0128] Total antioxidant capacity assay The antioxidant capacity of PIC1 mutants was measured using the TAC (total antioxidant capacity) assay (Cell Biolabs, Inc, San Diego, CA) based on the reduction of copper(II) to copper(I), as previously reported [6]. The kit protocol was performed according to the manufacturer's recommendations.

[0129] NETosis assay Free DNA, as a marker of NET formation, was measured by PicoGreen as previously described

[11] . Briefly, 2.0 × 10 cells were cultured in a 96-well plate at 37 °C in a humidified incubator supplemented with 5% CO2. 6 Human neutrophils were stimulated with 12 nM PMA and 0.05% HO in RPMI with or without the indicated peptide variants (2 mM) for 2.5 hours to induce NETosis. Neutrophils in RPMI alone served as a negative control. 50 units of monocytic nuclease (Fisher) were added to each well to digest released extracellular DNA for 10 minutes at 37°C. The preparation was then aliquoted into adjacent wells and mixed 1:1 with prepared PICO green reagent (Fisher). Fluorescence was then quantified using a BioTek microplate reader at excitation 485 nm / emission 528 nm.

[0130] statistical analysis Quantitative data were analyzed to determine the mean, standard error of the mean (SEM), and Student's t-test

[14] using Excel (Microsoft, Redmond, WA).

[0131] result peptide In vitro assays of classical complement pathway, MPO, oxidant and NET activity, parent molecule 8th place compared to TIFF0007804672000012.tif4128 TIFF0007804672000013.tif4128 and 9th place We previously demonstrated that substitution of isoleucine with sarcosine in TIFF0007804672000014.tif5128 resulted in peptides with increased solubility and enhanced inhibition of biological activity without PEGylation

[11] . To determine whether more potent peptides could be identified, we synthesized amino acid variants based on the PA-I8Sar and PA-C9Sar scaffolds. The amino acid variants consisted of stapled peptides or peptides with D-amino acids individually substituted at each position in the PA-I8Sar and PA-C9Sar peptide sequences (Tables 1 and 2, respectively). Three peptides based on the PA-I8Sar scaffold contained a combination of stapled and D-amino acid combinations (PA-0143 to PA-0145). Stapling techniques have been shown to increase peptide stability and enhance biological activity by locking peptide molecules into a bioactive α-helical secondary structure [2], while D-amino acid substitutions can confer additional stability to native peptides and extend their in vivo half-life

[15] . Each of these peptides, with the exception of PA-0116, is readily soluble in water and was evaluated for biological activity in various in vitro assays.

[0132] Complement inhibition and C1q binding To assess the extent to which peptide variants inhibit antibody-induced complement activation, we utilized an ABO-incompatible ex vivo assay, in which purified red blood cells from an "AB+" donor are incubated with serum from an "O" subject containing anti-A and anti-B antibodies

[13] . Peptides were tested at a concentration of 0.8 mg / ml (approximately 0.5 mM). For peptides based on the PA-I8Sar backbone, peptides PA-0114 to PA-0133, consisting of stapled peptides (PA-0114 to PA-0119) and D-amino acid substitutions (PA-0120 to PA-0133), inhibited ABO-incompatible hemolysis to a similar or greater extent than the PA-I8Sar control (Figure 1A). Compared with the PA-I8Sar variant, the stapled peptide PA-0115 dramatically and unexpectedly reduced ABO hemolysis by 30% (P = 0.06), while PA-0117 reduced ABO hemolysis by 25% (P = 0.279). The D-amino acid variants PA-0127 and PA-0133 reduced ABO hemolysis by 20% (P = 0.083) and 25% (P = 0.076), respectively.

[0133] Additional stapled peptides based on the PA-I8Sar backbone were designed (PA-0135 to PA-0141 and PA-0146), but only PA-0136, -0138, and -0140 could be synthesized in sufficient quantities for analysis (Table 2). PA-0136 inhibited complement to the same level as the parent molecule, PA-I8Sar, whereas PA-0134, -0138, and -0140 inhibited complement to a lesser extent (Figure 1A). Given the superior inhibition of hemolytic activity of the stapled peptide PA-0117 and the D-amino acid substitutions PA-0127, -0130, and -0133, peptides combining PA-0117 with each of these D-amino acid variants (PA-0143 to PA-0145) were synthesized and tested for complement inhibition (Figure 1A). These peptides ranged from low complement inhibitory activity to approximately a 10% increase in complement inhibition for PA-0145 compared to PA-I8Sar, suggesting that the combination of stapled peptides and D-amino acid substitutions did not exhibit synergistic complement inhibitory activity (Fig. 1A ).

[0134] Next, we tested stapled and D-amino acid variants of the PA-C9Sar molecule for complement inhibition in hemolysis assays. Most of the peptides inhibited complement activity slightly more than PA-C9Sar, except for variants PA-0162 and -0167, which inhibited complement activity slightly less; PA-0169 had activity similar to the parent molecule (Figure 1B). In contrast, the stapled peptide PA-0155 unexpectedly significantly reduced ABO hemolysis by 64% (P value < 0.001).

[0135] Astrovirus capsid proteins and their derived PIC1 molecules inhibit activation of the classical complement pathway by binding to the pattern recognition molecule C1q [3, 10, 11]. We next tested the binding of peptide mutants to C1q in an ELISA-type assay, in which C1q was used as a capture substrate and bound peptides were detected by PA-dPEG24. Detection was performed with a rabbit polyclonal antibody against the peptide portion of TIFF0007804672000015.tif4128

[11] . Binding curves were obtained for stapled peptides based on the PA-I8Sar and PA-C9Sar backbones and D-amino acid peptides (Figures 6A-6D), from which half-maximal binding concentrations were calculated (Figures 2A and B, respectively). For the stapled peptides based on the PA-I8Sar backbone, these binding curves and half-maximal binding calculations show that PA-0115 and PA-0119 exhibit significantly increased C1q binding compared to PA-I8Sar and PA-0117 and -0118. For the D-amino acid variants, all peptides for which C1q binding was detected exhibited variable, but increased, C1q binding compared to PA-I8Sar (Figure 2A). For peptides based on the PA-C9Sar scaffold, except for PA-0148, C1q bound to the same extent or better than the parent peptides (Fig. 2B). Surprisingly, although some peptides, such as PA-0115, exhibited excellent complement inhibitory and C1q binding activity (compare Figs. 1A and 2A), the strength of C1q binding did not strictly correlate with the inhibition of classical pathway complement activation for other peptide variants (e.g., PA-0162) (compare Figs. 1B and 2B), suggesting that complement inhibitory activity may not be entirely determined by the strength of C1q binding.

[0136] Myeloperoxidase Inhibitor We previously demonstrated that PA-dPEG24 and sarcosine-substituted peptides can bind to and inhibit myeloperoxidase (MPO) activity [4, 11]. To confirm the inhibition of MPO activity by various peptides, various concentrations of stapled peptides and D-amino acid variants were tested (Figures 7A–7D), and half-maximal activity levels were calculated from dose-response curves (Figures 3A and 3B, respectively). For the PA-I8Sar peptide, most variants showed similar or slightly improved levels of MPO inhibition, except for stapled peptide PA-0140, which showed reduced inhibition of MPO activity (Figure 3A). For the PA-C9Sar peptide, most variants maintained similar inhibitory activity to the parent PA-C9Sar peptide, except for stapled peptides PA-0148, -0151, -0153, -0155, and -0163, which showed reduced MPO inhibition (Figure 3B). Thus, for both the PA-I8Sar and PA-C9Sar mutants, although some stapled peptides had varying effects on MPO-binding affinity, D-amino acid substitutions did not dramatically alter MPO binding.

[0137] Antioxidant capacity The antioxidant properties of PIC1 variants were evaluated in a total antioxidant capacity (TAC) assay, as previously reported [6]. For stapled peptides and D-amino acid peptides based on the PA-I8Sar and PA-C9Sar backbones, total antioxidant activity was determined across a range of peptide concentrations (Figures 8A–8D), with activity reported at the highest peptide concentration (1.5 mM) (Figures 4A and B, respectively). For stapled peptides based on the parent PA-I8Sar peptide, PA-0114 and -0015 showed reduced total antioxidant capacity, while PA-0116 had increased activity, and PA-0117 through PA-0119 maintained the same amount of activity as the parent peptide. For the D-amino acid variants, there was variation in the amount of antioxidant activity, with peptides possessing similar, slightly lower, and slightly higher levels of total antioxidant capacity. In contrast, the stapled peptides (PA-0135 to -0140) and peptides combining stapled and D-amino acids showed reduced total antioxidant capacity (Figure 4A). Surprisingly, most of the peptides based on the PA-C9Sar peptide showed reduced total antioxidant capacity, except for the stapled peptide PA-0147, which maintained similar activity (Figure 4B). We found that the parent PA-dPEG24 peptide We have previously shown that both the adjacent cysteine ​​residues at positions 9 and 10 of TIFF0007804672000016.tif4128 are essential for antioxidant activity, and oxidation of both residues inhibits this function.[6] These data suggest that the cysteine ​​at position 10 is sufficient to maintain antioxidant activity.

[0138] Free DNA (NETosis) inhibition PA-I8Sar and PA-C9Sar have previously been shown to inhibit the formation of neutrophil extracellular traps (NETs) by fluorescence microscopy and free DNA as a marker of NETosis [7]. Selected modifications of PA-I8Sar, consisting of a stapled peptide (PA-0117) and three D-amino acid modifications (PA-0127, -0130, and -0133), were screened for the reduction of free DNA by stimulated neutrophils (Figure 5). As expected, the control (PA-I8Sar) reduced the levels of free DNA compared to stimulated neutrophils. PA-0117 and -0127 did not reduce free DNA levels, whereas PA-0130 and -0133 reduced free DNA similar to that of the PA-I8Sar control.

[0139] Consideration We previously demonstrated that sarcosine substitution of the parent 15-residue PEGylated PIC1 molecule (PA-dPEG24) resulted in six peptides that were water-soluble without PEGylation and had enhanced activity in functional assays of complement, MPO, NETosis, and oxidant activity

[11] . PA-I8Sar (in which the isoleucine at position 8 was replaced with sarcosine) was selected for further modification by peptide stapling and D-amino acid substitution to determine whether its functional activity in various assays could be further enhanced. We also performed the same analysis with PA-C9Sar (in which the cysteine ​​at position 9 was replaced with sarcosine). Although PA-C9Sar did not demonstrate significantly enhanced activity in various assays compared to PA-I8Sar

[11] , we were interested in analyzing its function in the context of stapling and D-amino acid substitution to determine whether a single cysteine ​​residue could maintain functional activity. As we have previously shown, both cysteine ​​residues at positions 9 and 10 are important for the functional activity of the PIC1 molecule

[11] . The activities of the PA-I8Sar and PA-C9Sar mutants in various assays are summarized in Tables 2 and 3.

[0140] Table 2. Summary of PA-I8Sar peptides and properties TIFF0007804672000017.tif43170TIFF0007804672000018.tif197170TIFF0007804672000019.tif34170 1 ND: Not determined. Peptide sequences PA-0130, -0131, -0132, -0133, -0136, and -0145 showed minimal binding to C1q. PA-0114 and -0116 were not analyzed due to reduced peptide availability and aqueous solubility, respectively. The MPO half-maximal activity of PA-0119 could not be determined because the peptide did not titrate. 2 NA: Not characterized. The peptide sequences PA-0135, -137, -139, -141, and -146 could not be synthesized efficiently.

[0141] Table 3. Summary of PA-C9Sar peptides and properties TIFF0007804672000020.tif117170TIFF0007804672000021.tif58170 1 ND: Not determined. For C1q binding, peptide sequence PA-0153 did not titrate, and PA-0149, -0155, -0167, and -0169 titrated to the parent peptide sequence PA-0153. It was not recognized by polyclonal antibodies against TIFF0007804672000022.tif5128.

[0142] PA-I8Sar Peptides. Stapling the PA-I8Sar molecule at various positions surprisingly resulted in a subset of peptides (PA-0115, -0116, -0117, and -0118) that exhibited significantly enhanced activity relative to the parent molecule. Unexpectedly, some modifications exhibited differential modulation of functional activity. For example, PA-0115 exhibited enhanced activity compared to PA-I8Sar in hemolysis, C1q binding, and MPO assays, but exhibited much lower antioxidant activity. In contrast, PA-0116 exhibited superior antioxidant activity but similar activity to PA-I8Sar in the hemolysis assay. The other stapled peptides did not appear to enhance functional activity compared to PA-I8Sar.

[0143] Similar to the stapled peptides, D-amino acid substitutions at each position of the PA-I8Sar molecule, except for sarcosine at position 8, resulted in molecules (PA-0121, -0127, -0128, -0130, and -0133) with varying degrees of enhanced activity in various assays. Interestingly, with the exception of -0121 and -0130, the D-amino acid variants did not exhibit enhanced antioxidant activity. Furthermore, combinations of PA-0117 with the superior D-amino acid modifications (PA-0143 to -0145) did not exhibit enhanced activity.

[0144] A subset of PA-I8Sar peptides was also tested for the reduction of free DNA, a biomarker of NETosis, as previously reported

[11] . The stapled peptide PA-0117 and the D-amino acid variant PA-0127 did not show a reduction in free DNA, whereas two other D-amino acid variants (PA-0130 and -0133) demonstrated the ability to inhibit NET formation.

[0145] The PA-C9Sar peptide. The same strategy of stapling and D-amino acid substitution on the PA-C9Sar backbone unexpectedly resulted in peptides with varying levels of functional enhancement in various assays. Regarding the stapled modifications, PA-0155 showed a significant increase in complement inhibitory activity. However, while a few stapled peptides showed enhanced activity in various assays, the majority of these peptides exhibited activity similar to that of the parent molecule. Regarding the D-amino acid variants, PA-0165 and -0166 showed significantly increased activity in various assays.

[0146] The results presented here demonstrate that peptide stapling and the introduction of non-canonical amino acids can significantly improve the functional activity of the PIC1 molecule. Interesting and surprising was the discovery that such modifications can lead to improvements in one or more functional activities of the PIC1 peptide. The ability to isolate peptides with distinct functional activities could potentially be utilized to target specific inflammatory diseases in which dysregulated complement, neutrophil (MPO and NETosis), or oxidant activity plays a central role in pathogenesis.

[0147] Example 2: Pharmacokinetic data for selected peptides In this study, four peptides from Example 1 that showed excellent activity in in vitro assays for complement, NETosis, and MPO inhibition were selected for further study. These peptides were PA-0117, -0127, -0130, and -0133. The four peptides were injected into rats to determine their pharmacokinetic profiles by hemolysis inhibition and binding assays.

[0148] method Male Wistar rats with indwelling jugular catheters were administered PA-0117, -0127, -0130, and -0133; groups of three animals received 20, 200, and 400 mg / kg of the compound as a single bolus IV infusion. Control rats received an IV infusion of saline. At various time points post-infusion (0.5, 2, 5, 20, 60, 120, 240, 480, and 1440 minutes), blood aliquots were drawn, and plasma was isolated and frozen at -70°C until analysis. After terminal blood collection, animals were sacrificed and subjected to gross necropsy.

[0149] result Rats receiving each peptide showed no obvious signs of pathology by gross necropsy across all dose groups, and no abnormalities were noted in blood chemistry or CBC analysis. Peptides PA-0127, -0130, and -0133 all demonstrated inhibition of complement activity in hemolytic assays at the early time point at the highest dose, and as expected, the inhibitory activity disappeared over time (Figures 10A, 11A, and 12). PA-0117 could not be analyzed because functional complement activity was not obtained from plasma samples. For quantitative (ELISA-like) target binding assays to determine peptide levels in samples, different assays had to be used because the various peptides did not efficiently bind C1q as a capture substrate and / or the antibodies used for detection did not efficiently bind the various peptides. PA-0117 was analyzed in a C1q target acquisition assay (C1q binding assay) as described in Example 1 (Figure 9). PA-0117 was detected at the early time point at each dose and decreased over time, as expected. The levels of PA-0127 and -0130 were measured by coating plates with plasma samples and measuring PA-0020 The binding of PA-0133 to the IgG1 gene was determined by direct detection of the peptide with a rabbit antibody raised against TIFF0007804672000023.tif5128 (Figures 10B and 11B). Similar to PA-0117, increased binding was observed at early time points, with a diminishing signal at later time points. PA-0133 could not be detected with the available antibodies.

[0150] Data Overview PA-0117: The hemolytic assay results were unusable (pre-bleeds had almost no activity, and only a few other random samples had any complement activity). PK assays for PA-0117 were performed by sandwich ELISA (Figure 9).

[0151] PA-0127: The results of the hemolytic assay are discussed herein (see also Figure 10A). For the PK assay, samples were coated directly onto plates, and then rabbit anti-PA was used because the peptide did not bind well enough to C1q or chicken anti-I8 for use in the original assay (Figure 10B).

[0152] PA-0130: The results of the hemolytic assay are discussed herein (see also Figure 11A). For the PK assay, samples were coated directly onto plates, and then rabbit anti-PA was used because the peptide did not bind well enough to C1q or chicken anti-I8 for use in the original assay (Figure 11B).

[0153] PA-0133: The results of the hemolysis assay are discussed herein (Figure 12). Because the peptide did not interact with any of the previously generated anti-PA antibodies, it was not possible to complete the PK assay.

[0154] conclusion Four selected peptides were active in vivo as assessed by inhibition of complement activity or C1q binding.

[0155] Example 3: Inhibition of PD-1 binding to PD-L1 The immune checkpoint pathway is an area of ​​considerable interest in cancer research. PD-1 is one of the best-characterized checkpoint proteins. Binding between PD-1 and its ligand PD-L1 inhibits T cell activation, allowing cancer cells to escape the body's immune surveillance. PD-L1 is a 40 kDa type 1 transmembrane protein that suppresses the adaptive arm of the immune system during certain events, such as pregnancy, tissue allografts, autoimmune diseases, and other disease conditions. Some human cancer cells express high levels of PD-L1, and blocking this receptor has been shown to reduce tumor growth in the presence of immune cells, thus allowing tumor cells to evade anti-tumor immunity. Therefore, PD-L1 / PD-1 is a therapeutic target in cancer immunotherapy. We used a commercially available ELISA kit to evaluate whether the PIC1 peptide has the ability to inhibit the binding of PD-1 to its receptor PD-L1. The PIC1 peptide showed various levels of binding inhibition, ranging from 0% to 24% (Figure 13). The data are summarized in Table 4 and demonstrate the ability of these peptides to inhibit the interaction of PD-1 with PD-L1.

[0156] Example 4: Activity of PIC1 peptides in PD-1 blockade bioassays To evaluate whether the PIC1 peptides could block PD-1 inhibitory activity in a cell-based assay, selected peptides were screened in the PD-1 / PD-L1 Blockade Bioassay (Promega). The PD-1 / PD-L1 Blockade Bioassay is a biologically relevant MOA-based assay that can be used to measure the potency and stability of antibodies and other biologics designed to block the PD-1 / PD-L1 interaction. This bioluminescent cell-based assay is used to measure the potency and stability of molecules targeting PD-1 and consists of two genetically engineered cell lines: aAPC / CHO-K1 cells, which contain engineered cell surface proteins designed to activate the cognate TCR in an antigen-independent manner. When the two cell types were cocultured, the PD-1 / PD-L1 interaction inhibited TCR signaling and NFAT-mediated luciferase activity. Addition of inhibitory molecules that block either PD-1 or PD-L1 releases an inhibitory signal, resulting in TCR signaling and NFAT-mediated luciferase activity. Selected PIC1 peptides, as well as anti-PD-1 antibodies (positive controls), were screened in this assay, and RLS-0117, RLS-0118, RLS-0127*, and RLS-0133* showed increased luminescence, indicating inhibition of PD-1 / PD-L1 inhibitory signaling (Figure 14). In contrast, RLS-0115, RLS-0122, RLS-0130, RLS-0142, RLS-0143, RLS-0144, RLS-0150, RLS-0154, RLS-0155, RLS-0156, RLS-0162, RLS-0164, RLS-0168, RLS-0170, RLS-0172, RLS-0173, RLS-0174, and RLS-0175 did not exhibit PD-1 / PD-L1 inhibitory activity in this assay (outlined in Table 4).

[0157] (Table 4) TIFF0007804672000024.tif160170TIFF0007804672000025.tif210170TIFF0007804672000026.tif210170TIFF0007804672000027.tif24170

[0158] Example 5: CTLA-4 binding by PIC1 peptide RLS-0117 We further evaluated whether RLS-0117 could bind to a well-characterized checkpoint inhibitor, namely, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, also known as CD152). Similar to the PD1 / PD-L1 interaction, CTLA-4 is a checkpoint protein that is often upregulated on the surface of cancer cells and binds to its ligands CD80 or CD86 on the surface of T cells, suppressing T cell activation and allowing cancer cells to escape destruction by the immune system. Therefore, pharmaceutical inhibition of CTLA-4 or its ligands is considered a promising strategy by many cancer researchers and is a therapeutic target in cancer immunotherapy. To confirm the ability of RLS-0117 to bind to CTLA-4, we performed a binding assay in which these proteins were coated on microtiter plates and then incubated with increasing amounts of either RLS-0134 or RLS-0150. Plates coated with PD-1, PD-L1, and C1q served as positive controls for peptide binding. RLS-0117 showed dose-dependent binding to PD-1, PD-L1, and C1q, as expected, and also bound to CTLA-4 (Figure 15). The ability of RLS-0117 to bind to CTLA-4 suggests that it may be able to functionally inhibit these interactions in cell-based bioassays.

[0159] Example 6: Activity of PIC1 peptide in CTLA-4 blocking bioassay To assess whether the PIC1 peptide can block CTLA-4 inhibitory activity in a cell-based assay, selected peptides were screened in a CTLA-4 blocking bioassay (Promega). This bioluminescent cell-based assay is used to measure the potency and stability of molecules targeting CTLA-4 and consists of two genetically engineered cell lines: CTLA-4 effector cells, Jurkat T cells expressing human CTLA-4 and a luciferase reporter driven by a native promoter that responds to TCR / CD28 activation, and aAPC / Raji cells, expressing an engineered cell surface protein designed to activate the cognate TCR in an antigen-independent manner and endogenously expressing the CTLA-4 ligands CD80 and CD86. When the two cell types were cocultured, CTLA-4 competed with CD28 for their shared ligands, CD80 and CD86, thereby inhibiting CD28 pathway activation and promoter-mediated luminescence. Addition of molecules that block the interaction of CTLA-4 with its ligands CD80 and CD86 results in promoter-mediated luminescence. A CTLA-4 antibody used as a positive control showed a dose-dependent increase in luminescence, indicating inhibition of CTLA-4 binding to its cognate receptor (Figure 16). PIC1 peptides RLS-0127*, RLS-0130, RLS-0156, RLS-0170, and RLS-0174 all showed inhibitory activity in this blocking bioassay, whereas peptides RLS-0115, RLS-0117, RLS-0118, RLS-0133*, RLS-0156, RLS-0172, RLS-0173, and RLS-0175 did not. These data are summarized in Table 4.

[0160] Example 7: Inhibition of T cell depletion by PIC1 peptide T cell depletion is a form of T cell dysfunction that occurs in cancer. It is defined by insufficient effector function, reduced cytokine release (e.g., IL-2, TNF-alpha, IFN-gamma), and persistent expression of inhibitory receptors (e.g., PD-1, LAG-3, CD244, CD160) accompanied by progressive loss of effector function due to overstimulation. This depletion prevents optimal control of tumor growth. T cell depletion is widespread in the tumor microenvironment (TME) and can lead to T cell apoptosis. T cell depletion is reversible, and pharmaceutical inhibition of T cell depletion is considered a promising strategy by many cancer researchers. We developed a T cell depletion protocol to determine whether the PIC1 peptide could reverse T cell depletion and enhance T cell viability and effector function. To induce T cell depletion, purified human pan-T cells were stimulated with T-Activator CD3 / CD28 Dynabeads, washed, and restimulated every 48 hours. After each Dynabead stimulation, PIC1 peptide was added to the cells. After 3–4 stimulations, cells were harvested for readout, which consisted of assessing T cell apoptosis by measuring caspase 3 / 7 levels and production of the cytokines IL-2 and IFN-gamma, which indicate T cell functionality. T cells stimulated with beads in the absence of peptide showed increased levels of caspase 3 / 7, indicative of apoptosis (untreated, Figure 17). T cells treated with PIC1 peptide showed reduced levels of caspase 3 / 7, and some peptides, such as RLS-0117 and RLS-0130, showed very low to undetectable levels of caspase 3 / 7. In contrast, RLS-0173 and RLS-0174 showed increased levels of caspase 3 / 7. To further assess whether the PIC1 peptide could restore T cell functionality in cells subjected to the depletion protocol, we next assessed the production of the cytokines IL-2 and IFN-gamma. Supernatants from cells were collected after each stimulation, and cytokines were measured by ELISA.As shown in Figures 18A-18C, cells that did not receive peptide exhibited a spike in IL-2 signal at Dynabead stimulation 1, which then became undetectable at stimulation 2. In contrast, cells treated with RLS-0117, RLS-0127*, RLS-0130, RLS-0162, RLS-0173, RLS-0174, and RLS-0175 exhibited an IL-2 signal at stimulation 2, whereas RLS-0115, RLS-0118, and RLS-0133* exhibited no IL-2 signal at stimulation 2. When IFN-gamma levels were measured, the PIC1 peptide exhibited the same pattern of IFN-gamma release as seen with IL-2 (Figures 18D-18F). Lower levels of IFN-gamma signal compared to IL-2 were consistently observed in this assay. These data are summarized in Table 4.

[0161] Example 8: Binding to VEGF and inhibition of VEGF function by PIC1 peptide Angiogenesis, the formation of new blood vessels from established vasculature, is an essential component of tumor growth and metastasis formation. Inhibition of tumor angiogenesis is a major therapeutic strategy in oncology. Vascular endothelial growth factor (VEGF) is a potent and specific angiogenic factor and a key requirement for tumor growth. VEGF inhibitors, such as monoclonal antibodies, are currently utilized to inhibit tumor growth in cancer patients. While these anti-VEGF drug therapies have proven effective in advanced and metastatic cancers, they have been shown to cause side effects such as hypertension, arterial clots, wound healing complications, and, more rarely, gastrointestinal perforation and fistula. Therefore, safe VEGF inhibitors that are not based on monoclonal antibody technology are needed. We tested the ability of these PIC1 peptides to bind to human VEGF and inhibit VEGF function in a cell-based bioassay. To confirm the ability of the PIC1 peptide to bind to VEGF, a binding assay was performed in which VEGF was coated onto a microtiter plate and then incubated with the PIC1 peptide (1.0 mg / ml). As shown in Figures 19A-19C, the PIC1 peptide bound to VEGF at various levels. Next, we used a VEGF bioassay (Promega) to determine whether these PIC1 peptides could functionally inhibit VEGF-mediated cell signaling through its cognate cell surface receptor, VEGFR-2 (KDR). The VEGF bioassay is a bioluminescent cell-based assay that measures VEGF stimulation and inhibition of VEGFR-2 using luciferase as a readout. This assay can be used for the discovery and development of novel biotherapies aimed at either inducing or inhibiting VEGF responses. VEGF-responsive cells were engineered to express an upstream response element (RE) of luc2P and an exogenous VEGF receptor. When VEGF binds to VEGF-responsive cells, the receptor transduces an intracellular signal, resulting in light emission. The bioluminescent signal is detected with a luminometer.A select number of PIC1 peptides were tested in this bioassay (outlined in Table 4), and these peptides did not exhibit inhibitory activity in this assay.

[0162] Example 9: Inhibition of non-VEGF-mediated angiogenesis by PIC1 peptide While VEGF plays a major role in cancer angiogenesis, other non-VEGF factors can induce angiogenesis and promote tumor growth. Currently, there are no drugs on the market that inhibit non-VEGF-mediated angiogenesis. To evaluate whether the PIC1 peptide can inhibit non-VEGF-mediated angiogenesis, we developed an angiogenesis model using human umbilical endothelial vein cells (HUVECs), in which the addition of lipopolysaccharide (LPS) induces angiogenesis. HUVECs were first incubated with Cell Trace Violet dye, followed by the addition of PIC1 peptide (10 mg / ml) at 37°C for 1 hour. They were then treated with 10 μg / ml LPS and plated on an extracellular matrix to promote angiogenesis. The cells were incubated overnight at 37°C in a humidified CO2 incubator. The cells were then visualized by fluorescence microscopy for tube formation, which indicates angiogenesis. Cells that did not receive LPS did not show any aggregation or formation of tube buds, whereas these structures were evident in cells treated with LPS (Figure 20). Varying levels of angiogenesis inhibition were observed in the presence of PIC1 peptides, with some peptides (RLS-0118 and RLS-0175) showing no signs of angiogenesis, similar to control cells not stimulated with LPS. These data are summarized in Table 4.

[0163] Example 10: RLS-0127* and RLS-0133* have complement and myeloperoxidase (MPO) inhibitory activity in vitro RLS-0127* and RLS-0133* were shown to inhibit complement activation in an ex vivo hemolysis assay when administered IV to Wistar rats (Figures 10A and 12), and pharmacokinetic data are also reported for RLS-0127* (Figure 10B). We further characterized the ability of these peptides to inhibit complement activation in vitro using human AB red blood cells sensitized with human O plasma. As shown in Figure 21A, RLS-0127* and RLS-0133* inhibited complement activation in a dose-dependent manner, with RLS-0127* demonstrating a level of inhibition very similar to that of the positive control, RLS-0088. We also evaluated the ability of RLS-0127* and RLS-0133* to inhibit MPO activity, as determined by inhibition of TMB oxidation in a plate-based assay. Similar to the hemolysis assay, RLS-0127* and RLS-0133* inhibited TMB oxidation in a dose-dependent manner, similar to the positive control RLS-0088 (Figure 21B). Furthermore, we evaluated the ability of RLS-0127* and RLS-0133* to bind to C1q in a plate-based assay, in which C1q was coated onto a plate and increasing amounts of peptide were added, followed by a primary antibody against the peptide and a secondary antibody conjugated to HRP. The plate was then incubated with TMB, resulting in a chemiluminescent signal detected at a wavelength of 450 nm in a plate reader. RLS-0127* bound to C1q in a dose-dependent manner, similar to RLS-0088 (positive control) (Figure 21C). Binding of RLS-0133 to C1q could not be detected because the primary antibody was unable to recognize this peptide. These data are summarized in Table 4.

[0164] Example 11: Administration of pharmaceutical compositions To modulate the complement system, a pharmaceutical composition comprising a therapeutically effective amount of any of SEQ ID NOs: 6-55 and variants thereof is administered to a subject in need thereof.

[0165] To inhibit myeloperoxidase activity, a pharmaceutical composition containing a therapeutically effective amount of any of SEQ ID NOs: 6-55 and variants thereof is administered to a subject in need thereof.

[0166] To inhibit NETosis, a pharmaceutical composition comprising a therapeutically effective amount of any of SEQ ID NOs: 6-55 and variants thereof is administered to a subject in need thereof.

[0167] To inhibit oxidant activity, a pharmaceutical composition comprising a therapeutically effective amount of any of SEQ ID NOs: 6-55 and variants thereof is administered to a subject in need thereof.

[0168] To inhibit the binding of PD-1 to PD-L1, a pharmaceutical composition comprising a therapeutically effective amount of any of SEQ ID NOs:6-55 and variants thereof is administered to a subject in need thereof.

[0169] To inhibit T cell depletion, a pharmaceutical composition comprising a therapeutically effective amount of any of SEQ ID NOs: 6-55 and variants thereof is administered to a subject in need thereof.

[0170] To inhibit angiogenesis, a pharmaceutical composition comprising a therapeutically effective amount of any of SEQ ID NOs: 6-55 and variants thereof is administered to a subject in need thereof.

[0171] List of Aspects The following is a non-exhaustive list of aspects provided by the present invention: 1. A synthetic peptide comprising at least about 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 6-55. 2. The synthetic peptide of embodiment 1, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6-55. 3. The synthetic peptide of embodiment 1, comprising at least about 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 19, 22, 25. 4. The synthetic peptide of embodiment 1, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 19, 22, and 25. 5. The synthetic peptide of embodiment 1, consisting of an amino acid sequence comprising at least about 95% sequence identity to SEQ ID NO:9. 6. The synthetic peptide of embodiment 1, consisting of an amino acid sequence comprising at least about 95% sequence identity to SEQ ID NO:19. 7. The synthetic peptide of embodiment 1, consisting of an amino acid sequence comprising at least about 95% sequence identity to SEQ ID NO:22. 8. The synthetic peptide of embodiment 1, consisting of an amino acid sequence comprising at least about 95% sequence identity to SEQ ID NO:25. 9. A composition comprising at least one synthetic peptide of any of embodiments 1 to 8, optionally in further combination with another D-enantiomeric form and / or stapled peptide form of SEQ ID NO:4 and / or SEQ ID NO:5 and variants thereof, and / or one or more of SEQ ID NO:2, 3, 4 and / or 5 and variants thereof. 10. A pharmaceutical composition comprising a therapeutically effective amount of at least one synthetic peptide of any of embodiments 1 to 8 or the composition of embodiment 9, and optionally at least one pharmaceutically acceptable carrier, diluent, or excipient. 11. A method of modulating the complement system, comprising administering the pharmaceutical composition of embodiment 10 to a subject in need thereof. 12. A method of inhibiting myeloperoxidase activity, comprising administering the pharmaceutical composition of embodiment 10 to a subject in need thereof. 13. A method of inhibiting NETosis, comprising administering the pharmaceutical composition of embodiment 10 to a subject in need thereof. 14. A method of inhibiting oxidant activity, comprising administering the pharmaceutical composition of embodiment 10 to a subject in need thereof. 15. A method of inhibiting the binding of PD-1 to PD-L1, comprising administering to a subject in need thereof the pharmaceutical composition of embodiment 10. 16. A method of inhibiting T cell depletion, comprising administering the pharmaceutical composition of embodiment 10 to a subject in need thereof. 17. A method of inhibiting angiogenesis, comprising administering the pharmaceutical composition of embodiment 10 to a subject in need thereof.

[0172] While several possible embodiments are disclosed above, embodiments of the present invention are not so limited. These exemplary embodiments are not intended to be exhaustive or to unnecessarily limit the scope of the invention, but instead were chosen and described in order to explain the principles of the invention so that others skilled in the art may practice the invention. Indeed, various modifications of the invention in addition to those described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[0173] All patents, applications, publications, test methods, literature, and other materials cited herein are incorporated by reference in their entirety as if physically present herein.

[0174] References TIFF0007804672000028.tif207161TIFF0007804672000029.tif93166

Claims

1. A synthetic peptide consisting of the amino acid sequence shown in SEQ ID NO:

7.

2. A synthetic peptide consisting of the amino acid sequence shown in SEQ ID NO:

9.

3. A synthetic peptide consisting of the amino acid sequence shown in SEQ ID NO:

10.

4. A pharmaceutical composition comprising a therapeutically effective amount of the synthetic peptide of any one of claims 1 to 3 and at least one pharmaceutically acceptable carrier, diluent, or excipient.

5. 10. The pharmaceutical composition of claim 4 for use in a method for regulating the complement system.

6. 5. The pharmaceutical composition of claim 4 for use in a method for inhibiting myeloperoxidase activity.

7. 10. The pharmaceutical composition of claim 4 for use in a method of inhibiting oxidant activity.

8. A pharmaceutical composition for use in a method for inhibiting the binding of PD-1 to PD-L1, comprising a therapeutically effective amount of the synthetic peptide of claim 2 or 3, and at least one pharmaceutically acceptable carrier, diluent, or excipient.

9. A pharmaceutical composition for use in a method for inhibiting T cell depletion, comprising a therapeutically effective amount of the synthetic peptide of claim 2 and at least one pharmaceutically acceptable carrier, diluent, or excipient.

10. 10. The pharmaceutical composition of claim 4 for use in a method of inhibiting angiogenesis.

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