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. TIFF2023548343000022.tif40146

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 108,762, 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_000156_SL.txt, and is 5,875 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 crucial 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 molecule family 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. TIFF0007804671000001.tif4128. The original PIC1 peptide was modified with a monodisperse 24-mer PEGylated moiety at the C-terminus. TIFF0007804671000002.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. TIFF0007804671000003.tif11157 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 another aspect, the present invention provides synthetic peptides that inhibit the binding of programmed death-ligand 1 (PD-L1) to the PD-1 receptor. 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 reduces tumor growth in the presence of immune cells, thus allowing tumor cells to evade anti-tumor immunity. PD-L1 acts as a checkpoint protein in myeloid cells and is a therapeutic target in cancer immunotherapy.

[0016] 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. Pat. No. 10,005,818 and U.S. Patent Application Publication No. US2019 / 0209660. As used herein, the term "PIC1 peptide" includes SEQ ID NOs:6-8, 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:9-13, 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.

[0017] 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.

[0018] 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:6-13. 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:6-8. 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:9-13.

[0019] In one aspect, the invention provides synthetic peptides comprising at least about 95% sequence identity to the amino acid sequences of SEQ ID NOs:6-8. In some embodiments, the invention is a synthetic peptide comprising the amino acid sequence of SEQ ID NOs:6-8 and a modification. In one aspect, the invention is a synthetic peptide comprising at least about 95% sequence identity to the amino acid sequence of SEQ ID NOs:9-13. In some embodiments, the invention is a synthetic peptide comprising the amino acid sequence of SEQ ID NOs:9-13 and a modification.

[0020] 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:6-13 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:6-8 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:9-13 and variants thereof, and at least one pharmaceutically acceptable carrier, diluent, or excipient.

[0021] 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: 6-13.

[0022] In some embodiments, the present invention provides synthetic peptides comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6-13.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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]

[0031] 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. (2A) The half-maximal binding concentrations were calculated from the binding curves for the PA-I8Sar mutant and (2B) the PA-I9Sar mutant. The peptide variant PA-0142 did not bind to C1q, whereas the peptide variant PA-0152 did not titrate, so half-maximal binding could not be calculated. The peptide variant PA-0168 was not recognized by the primary polyclonal antibody and therefore could not be analyzed. [Figure 2B] See legend to Figure 2A. [Figure 3A] 3A-3B show half-maximal values ​​for PIC1 peptide inhibition of MPO activity. Half-maximal values ​​were calculated from activity curves for (3A) the PA-I8Sar mutant and (3B) the PA-C9Sar mutant. [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 5A] Figures 5A-5C show C1q binding curves. Binding of increasing concentrations of PA-I8Sar (5A-5B) and PA-I9Sar (5C) to immobilized C1q in an ELISA-type assay. Peptide variant PA-0142 did not bind C1q, whereas peptide variant PA-0152 was not titrated, so half-maximal binding could not be calculated. Peptide variant PA-0168 was not recognized by the primary polyclonal antibody and therefore could not be analyzed. [Figure 5B] See legend to Figure 5A. [Figure 5C] See legend to Figure 5A. [Figure 6A]6A-6C show MPO inhibition curves. Inhibition of MPO activity by increasing concentrations of PA-I8Sar modified (6A-6B) and PA-I9Sar modified (6C) in an ELISA-type assay. [Figure 6B] See legend to Figure 6A. [Figure 6C] See legend to Figure 6A. [Figure 7A] Figures 7A-7C show total antioxidant activity curves. Increasing concentrations of PA-I8Sar (7A-7B) and PA-I9Sar (7C) were analyzed for total antioxidant activity. Antioxidant activity is measured in copper reducing equivalents (CRE). [Figure 7B] See legend to Figure 7A. [Figure 7C] See description of Figure 7A. [Figure 8] Figure 8 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 9A] Figures 9A-9B show the binding of RLS-0134 (9A) and RLS-0150 (9B) to CTLA-4, PD-1, and PD-L1 in an ELISA plate-based assay. RLS-0134 and RLS-0150 were bound to CTLA-4, PD-1, and PD-L1 immobilized on the plate surface. Bound C1q and MAC-1 served as positive and negative controls for peptide binding, respectively. 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 to HRP. The plate was then developed by adding TMB as a substrate for the colorimetric assay. [Figure 9B] See description of Figure 9A. [Figure 10]Figure 10 shows that RLS-0122, RLS-0150, RLS-0154, RLS-0164, and RLS-0168 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 antibodies (positive control), RLS-0122, RLS-0150, RLS-0154, RLS-0164, and RLS-0168, and RLS-0088 (negative control). Luminescence was detected using a luminometer plate reader. The decrease in signal with high concentrations of test peptides is due to cell death as a result of the peptide's buffering effect on the cells. [Figure 11] Figure 11 shows that RLS-0122, RLS-0164, and RLS-0168 were able to inhibit T cell exhaustion, as measured by the reduction 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 not receiving 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. RLS-0150 and RLS-0154 did not exhibit a reduction in caspase levels. [Figure 12A]Figures 12A-12D show that RLS-0122, RLS-0150, RLS-0154, RLS-0164, and RLS-0168 were able to restore T cell exhaustion, as measured by increased levels of the cytokines IL-2 (Figures 12A and 12C) and IFN-gamma (Figures 12B and 12D). RLS-0164 is shown in Figures 12A and 12B, and RLS-0122, RLS-0150, RLS-0154, and RLS-0168 are shown in Figures 12C and 12D. Purified human pan-T cells were stimulated with Dynabeads every 48 hours over an 8-day period, and the cells were also given PIC1 peptide (2 mg / ml) at each stimulation. Cell supernatants were collected at each stimulation, and IL-2 and IFN-gamma levels were assayed by ELISA. [Figure 12B] See description of Figure 12A. [Figure 12C] See description of Figure 12A. [Figure 12D] See description of Figure 12A. [Figure 13] Figure 13 shows the binding of the PIC1 peptide to VEGF in an ELISA plate-based assay. The PIC1 peptide was bound to VEGF immobilized on the surface of the plate. A fixed amount of the PIC1 peptide (1 mg / ml) was added to the plate, followed by a rabbit polyclonal antibody recognizing the peptide, followed by a secondary anti-rabbit antibody conjugated with HRP. The plate was then developed by adding TMB as a substrate for the colorimetric assay. [Figure 14] Figure 14 shows that specific PIC1 peptides were able to inhibit VEGF-mediated cell signaling. VEGF effector cells were incubated with RLS-0122, RLS-0150, RLS-0154, RLS-0164, and RLS-0168, and then VEGF was added. Luminescence was detected using a luminometer plate reader. Cells incubated with VEGF alone served as a positive control for VEGF-mediated cell signaling, as shown by the line indicating a 20,000 relative luminescence unit (RLS) response, indicating VEGF binding to VEGFR-2. [Figure 15] Figure 15 shows that specific PIC1 peptides were able to inhibit non-VEGF-mediated angiogenesis induced by LPS. HUVEC cells were incubated with either RLS-0122, RLS-0150, RLS-0154, RLS-0164, or RLS-0168, 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 16] Figure 16 shows that RLS-0122 can inhibit activation of the classical complement pathway by the human renal carcinoma cell line A498. Supernatants from A-498 cells were added to purified human C1q preincubated with increasing doses of RLS-0122 or RLS-0174 and loaded onto IgG-coated plates. The samples were then incubated at 37°C for 1 hour. After incubation, the samples were washed three times with PBS-T, followed by the addition of purified human C4 (4 μg / ml) and incubation at 37°C for 1.5 hours. Supernatants were collected for analysis by Quidel's MicroVue Complement C4a ELISA. [Figure 17A] Figures 17A-17B show the effects of RLS-0122 on survival (17A) and quality of life (17B) in a murine TC-1 cancer cell model. Animals were injected subcutaneously with 4 x 10^5 TC-1 cells into the flank. Five days later, animals received vehicle treatment with saline (n = 6) or IV drug treatment with RLS-0122 at 160 mg / kg 1x / day (n = 8) for 15 consecutive days. Animals were also evaluated every two days for the duration of the study for behavioral and physical condition scores. If a score of 7 or less was reached, animals were evaluated daily. A score of 5 or less was considered the endpoint for euthanasia. [Figure 17B] See description of Figure 17A. DETAILED DESCRIPTION OF THE INVENTION

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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."

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] "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.

[0041] 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.

[0042] 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.

[0043] 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."

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] " 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.

[0059] 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).

[0060] "Parenteral" administration of the immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intradermal (id) injection or infusion techniques.

[0061] 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.

[0062] 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.

[0063] 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).

[0064] 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).

[0065] 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 TIFF0007804671000004.tif4128 TIFF0007804671000005.tif4128 and 9th place It has previously been shown that the substitution of isoleucine with sarcosine in TIFF0007804671000006.tif4128 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). One peptide based on the PA-I8Sar scaffold contained a combination of stapled and D-amino acids. 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.

[0066] 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.

[0067] 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).

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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).

[0073] 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:6-13. 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:6-13, as shown in Table 1 below. Staple amino acids are underlined and D-enantiomeric amino acids are shown in bold.

[0074] Table 1: List of peptides of the present invention TIFF0007804671000007.tif203161

[0075] 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:6-13. 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:6-8. 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:9-13.

[0076] In one aspect, the invention is a synthetic peptide comprising the amino acid sequence and modifications of SEQ ID NOs:6-13. In some embodiments, the invention is a synthetic peptide comprising the amino acid sequence and modifications of SEQ ID NOs:6-8. In some embodiments, the invention is a synthetic peptide comprising the amino acid sequence and modifications of SEQ ID NOs:9-13.

[0077] 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:6-13 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:6-8 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:9-13 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.

[0078] 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.

[0079] 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."

[0080] In some aspects, the present invention relates to therapeutically active peptides that have the effect of modulating the complement system.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] Hemolysis inhibition Peptides of the present invention, including SEQ ID NOs: 6-13, can block complement-mediated lysis of AB human red blood cells (RBCs) by O serum in vitro, an assay that mimics ABO incompatibility.

[0087] Inhibition of PD-1 / PD-L1 binding The peptides of the present invention, including SEQ ID NOs: 6-13, can block the binding of PD-1 to PD-L1 in vitro. This assay mimics the binding between PD-1 on T cells and its ligand PD-L1 on the surface of cancer cells.

[0088] 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.

[0089] 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 preventive 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. 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.

[0090] 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.

[0091] 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.

[0092] 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:6-13 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:6-8 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:9-13 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.

[0093] 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.

[0094] 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).

[0095] 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.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] How to use Another aspect of the present invention provides a method for modulating the complement system, comprising administering a therapeutically effective amount of a peptide and / or pharmaceutical composition of the present invention to a subject in need thereof. Activation of the classical complement pathway has been shown to contribute to tumor progression for certain cancers, such as renal clear cell carcinoma, and therefore, inhibition of the classical complement pathway may be a therapeutic approach for slowing the progression of such tumors.

[0105] 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.

[0106] 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.

[0107] In another aspect, the present invention provides methods for inhibiting PD-L1 activity, comprising administering to a subject in need thereof a therapeutically effective amount of a peptide and / or pharmaceutical composition of the invention.

[0108] 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.

[0109] 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]

[0110] 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.

[0111] Example 1: Characterization of SEQ ID NOs: 6-13 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.

[0112] 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 single-turn stapling or combining R-octenylalanine (R8) / S-pentenylalanine (S5) at the i,i+7 positions. Because no enantiomeric forms exist, the D-enantiomeric form of each amino acid was individually substituted at various positions in the PA-I8Sar and PA-C9Sar peptide sequences, except for sarcosine at positions 8 (PA-I8Sar) and 9 (PA-C9Sar). All peptides 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). ++Buffer solution (veronal-buffered saline containing 0.1% gelatin, 0.15 mM CaCl2, and 1 mM MgCl2

[12] ) was included.

[0113] 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.

[0114] 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.

[0115] 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 TIFF0007804671000008.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.

[0116] 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).

[0117] 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.

[0118] PD-1:PD-L1 inhibitor screening ELISA assay This inhibitor screening ELISA pair was designed to facilitate the identification and characterization of novel PD-1 pathway inhibitors. The assay utilizes a colorimetric sandwich ELISA platform, with biotinylated human PD-1 bound to immobilized human PD-L1. Plates were coated with human PD-LI and then incubated with various PIC1 peptides. Human PD-1-biotin was then bound to the coated human PD-L1, followed by the addition of streptavidin-HRP. TMB was then added as a colorimetric HRP substrate, and the plate wells were analyzed in a plate reader at absorbance of 450 nm.

[0119] 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).

[0120] result peptide In vitro assays of classical complement pathway, MPO, oxidant and NET activity, parent molecule 8th place compared to TIFF0007804671000009.tif4128 TIFF0007804671000010.tif4128 and 9th place It has previously been shown that the substitution of isoleucine with sarcosine in TIFF0007804671000011.tif4128 results in peptides with increased solubility and enhanced inhibition of biological activity without PEGylation

[11] . To determine whether more potent peptides could be identified, amino acid variants based on the PA-I8Sar and PA-C9Sar scaffolds were synthesized, consisting 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). One peptide based on the PA-I8Sar scaffold contained a combination of stapled and D-amino acid combinations (PA-0142). Stapling techniques have been shown to increase peptide stability and enhance biological activity by locking peptide molecules into a biologically active 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 is readily soluble in water and was evaluated for biological activity in various in vitro assays.

[0121] 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 were 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-0122, PA-0134, and PA-0142, consisting of D-amino acid substitutions (PA-0122), a stapled peptide (PA-0134), and a staple and D-amino acid substitution (PA-0142), inhibited ABO-incompatible hemolysis to a similar extent (PA-0134 and PA-0142) or to a greater extent (PA-0122) compared to the PA-I8Sar control (Figure 1A).

[0122] Next, we tested the stapled and D-amino acid variants of the PA-C9Sar molecule for complement inhibition in a hemolytic assay. In contrast to the PA-I8Sar variant, all peptides based on the PA-C9Sar molecule inhibited complement activity more effectively than PA-C9Sar (Figure 1B).

[0123] 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 TIFF0007804671000012.tif4128

[11] . Binding curves were obtained for stapled and D-amino acid peptides based on the PA-I8Sar and PA-C9Sar backbones (Figures 5A-5C), from which half-maximal binding concentrations were calculated (Figures 2A and 2B, respectively). For peptides based on the PA-I8Sar backbone, these binding curves and calculations of half-maximal binding indicated that the D-amino acid peptide PA-0122 and the stapled peptide PA-0134 exhibited significantly increased C1q binding compared to PA-I8Sar. For peptides based on the PA-C9Sar backbone, C1q bound to a greater extent than the parent peptides (Figure 2B). Surprisingly, although some peptides, such as PA-0150, exhibited excellent complement inhibition and C1q binding activity (compare Figures 1B and 2B), the strength of C1q binding did not strictly correlate with the inhibition of classical pathway complement activation of other peptide variants (e.g., PA-0134) (compare Figures 1A and 2A), suggesting that complement inhibitory activity may not be entirely determined by the strength of C1q binding.

[0124] Myeloperoxidase Inhibitor To confirm the inhibition of MPO activity by various peptides, various concentrations of the stapled peptide and D-amino acid mutants were tested (Figures 3A-3B), and half-maximal activity levels were calculated from dose-response curves (Figures 6A-6C, respectively). For the PA-I8Sar peptide, PA-0122 and PA-0142 exhibited similar levels of MPO inhibition, whereas PA-0134 exhibited reduced inhibition of MPO activity (Figure 3A). For the PA-C9Sar peptide, PA-164 and PA0168 maintained similar inhibitory activity to the parent PA-C9Sar peptide, whereas PA-0150, PA-0152, and PA-0154 exhibited reduced MPO inhibition (Figure 3B). Thus, for both the PA-I8Sar and PA-C9Sar mutants, some peptides exhibited varying effects on MPO binding affinity.

[0125] Antioxidant capacity The antioxidant properties of the PIC1 variants were evaluated in a total antioxidant capacity (TAC) assay, as previously reported [6]. Total antioxidant activity was determined across a range of peptide concentrations for stapled peptides and d-amino acid peptides based on the PA-I8Sar and PA-C9Sar backbones (Figures 4A-4B), with activity reported at the highest peptide concentration (1.5 mM) (Figures 5A-5C). For peptides based on the parent PA-I8Sar peptide, PA-0122 showed a slight increase in total antioxidant capacity compared to the parent peptide, while PA-0134 and PA-0142 showed decreased activity. Surprisingly, most of the peptides based on the PA-C9Sar peptide showed reduced total antioxidant capacity, with the exception of the d-amino acid peptide PA-0164, which showed enhanced activity (Figure 4B). We found that the parent PA-dPEG24 peptide exhibited reduced total antioxidant capacity. We have previously shown that both the adjacent cysteine ​​residues at positions 9 and 10 of TIFF0007804671000013.tif4128 are essential for antioxidant activity, and oxidation of both residues inhibits this function.[6] These data indicate that the cysteine ​​at position 10 is sufficient to maintain antioxidant activity and that the D-enantiomer of this cysteine ​​(PA-0164) can enhance this activity.

[0126] 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. Therefore, pharmaceutical inhibition of PD-1 or its ligand is considered a promising strategy by many cancer researchers. To confirm whether these PD-1 derivatives can inhibit the interaction of PD-1 with PD-L1, we utilized a commercially available ELISA kit. PA-0071 and PA-0088 did not inhibit PD-1 binding to PD-L1. In contrast, PA-0134, PA-0142, PA-0150, PA-0152, and PA-0154 inhibited binding by 29–46% (Figure 8).

[0127] 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.

[0128] Table 2. Summary of PA-I8Sar peptides and properties TIFF0007804671000014.tif71162 1 ND: undecided

[0129] Table 3. Summary of PA-C9Sar peptides and properties TIFF0007804671000015.tif98161 1 ND: Not determined. With regard to C1q binding, the peptide sequence PA-0168 was not recognized by a polyclonal antibody against the parent peptide sequence, IALILEPICCQERAA-dPEG24 (SEQ ID NO: 3).

[0130] 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.

[0131] Example 2. 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. Therefore, pharmaceutical inhibition of PD-1 or its ligand is considered a promising strategy by many cancer researchers. To confirm whether these PD-1 derivatives can inhibit the interaction of PD-1 with PD-L1, we utilized a commercially available ELISA kit. PA-0071 and PA-0088 did not inhibit PD-1 binding to PD-L1. In contrast, PA-0134, PA-0142, PA-0150, PA-0152, and PA-0154 inhibited binding by 29–46% (Figure 8).

[0132] Example 3. Binding of PD-1, PD-L1, and CTLA-4 by PIC1 peptides RLS-0134 and RLS-0150 We further evaluated whether RLS-0134 and RLS-0150 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 upregulated on the surface of cancer cells that 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 could be a therapeutic target in cancer immunotherapy. To confirm the ability of selected PIC1 peptides to bind to CTLA-4, we performed binding assays 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, while MAC-1 served as a negative control for peptide binding. RLS-0134, as expected, showed dose-dependent binding to PD-1, PD-L1, and C1q and also bound to CTLA-4, but showed minimal binding to MAC-1 (Figure 9A). RLS-0150 also showed dose-dependent binding to PD-1, PD-L1, and C1q, and bound more strongly to CTLA-4 (Figure 9B).

[0133] Example 4. Activity of PIC1 peptide in CTLA-4 blocking bioassay To evaluate 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 can be 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 a molecule that blocks the interaction of CTLA-4 with its ligands CD80 and CD86 results in promoter-mediated luminescence. The 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 10). The PIC1 peptides RLS-0122, RLS-0150, RLS-0154, RLS-0164, and RLS-0168 all showed inhibitory activity in this assay, as indicated by increased signal, while RLS-0088 (negative control) showed no inhibition above background in the blocking bioassay. These data are summarized in Table 4 and show that the PIC1 peptide can inhibit the interaction of CTLA-4 with its cognate receptor in a cell-based bioassay and can functionally inhibit CTLA-4-mediated signal transduction.

[0134] Table 4. Summary of PIC1 peptides and properties TIFF0007804671000016.tif79158

[0135] Example 5. Inhibition of T cell depletion by PIC1 peptide T cell depletion is a form of T cell dysfunction that occurs in cancer. It is generally 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. Depletion can prevent 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 a 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. Bead-stimulated T cells without peptide treatment showed increased levels of caspase 3 / 7, indicative of apoptosis, whereas unstimulated cells showed low levels of caspase 3 / 7 signal (untreated, Figure 11). T cells treated with the PIC1 peptides RLS-0122, RLS-0164, and RLS-0168 showed reduced levels of caspase 3 / 7, with some peptides, such as RLS-0122 and RLS-0168, showing very low to undetectable levels of caspase 3 / 7. In contrast, RLS-0150 and RLS-0154 increased caspase 3 / 7 levels. To further assess whether the PIC1 peptides 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 after each stimulation were collected and cytokines were measured by ELISA. As shown in Figures 12A-12B, cells that did not receive peptide exhibited a spike in IL-2 or IFN-gamma signaling at Dynabead stimulation 1, which then became undetectable at stimulation 2. In contrast, cells treated with RLS-0164 exhibited IL-2 and IFN-gamma signaling at stimulation 2. Lower levels of IFN-gamma signaling were consistently observed in this assay. Compared to RLS-0150, which does not inhibit T cell exhaustion, RLS-0122, RLS-0154, and RLS-0168 all exhibited detectable IL-2 at stimulation 2 (Figure 12C). IFN-gamma signaling was detectable at stimulation 2 for RLS-0122 and RLS-0168, but not for RLS-0154 (Figure 12D). These data are summarized in Table 4 and demonstrate that the PIC1 peptide can inhibit human T cell depletion as measured by inhibition of apoptotic markers and restoration of cytokine production.

[0136] Example 6. 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 considered 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 mg / ml). As shown in Figure 13, PIC1 binds to VEGF at various levels, with RLS-0122 and RLS-0164 binding with high affinity. 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 VEGFR-2 inhibition 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.As shown in Figure 14, addition of VEGF incubated with cells without peptide gave a signal of 20,000 relative luminescence units (RLS) response, indicating VEGF binding to VEGFR-2. RLS-0150 and RLS-0154 dose-dependently reduced luminescence, indicating blocking VEGF signaling through VEGFR-2, whereas RLS-0122, RLS-0164, and RLS-0168 did not inhibit signaling. These data are summarized in Table 4.

[0137] Example 7. 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, indicative of 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 15). Varying levels of angiogenesis inhibition were observed in the presence of PIC1 peptides, with some peptides (RLS-0164 and RLS-0168) showing no detectable tube formation, similar to control cells not stimulated with LPS. These data are summarized in Table 4.

[0138] Example 8. Inhibition of complement activation in the human renal carcinoma cell line A498 by RLS-0122 Effective therapy for clear cell renal cell carcinoma (ccRCC) is needed, especially in the metastatic stage when surgery is ineffective. Complement is a key factor in tissue inflammation and aids cancer progression through the production of complement component 5a (C5a) (Roumenina et al., Cancer Immunol Res; 7(7) July 2019). Using data mining techniques, ccRCC was identified as a cancer type that simultaneously expresses high expression of multiple components that are part of the classical complement pathway. A high density of cells producing the classical complement pathway components C1q and C4, as well as the presence of C4 activation fragment deposits in primary tumors, often correlates with poor prognosis. Therefore, inhibition of the classical complement pathway may represent a novel therapeutic strategy for potentially reducing tumor growth in ccRCC, as well as in other cancers where complement activation plays a role in tumor-promoting inflammation, such as lung cancer and head and neck squamous cell carcinoma (HNSCC). We tested the ability of RLS-0122 to block complement activation, as assessed by reduction of C4a levels, in an in vitro human renal carcinoma cell line A498 system. The A-498 cell line, derived from human renal carcinoma, produces the complement component products C1r and C1s. Addition of purified C1q creates a functional C1 complex (Roumenina et al., Cancer Immunol Res; 7(7) July 2019). RLS-0122 was selected for evaluation in this in vitro assay based on its ability to potently block complement activation in a hemolytic assay (Figure 1). Concentrated supernatant (3%) from serum-starved A-498 cells was added to purified human C1q (0.5 μg / ml) preincubated with increasing doses (0-8 mg / ml) of RLS-0122 or RLS-0174 (negative control peptide), and loaded onto IgG-coated plates. Samples were then incubated for 1 hour at 37° C. After incubation, samples were washed three times with PBS-T before adding purified human C4 (4 μg / ml) and incubating for 1.5 hours at 37° C. Supernatants were collected for analysis by Quidel's MicroVue Complement C4a ELISA.RLS-0122 dose-dependently reduced the level of C4a production, whereas a peptide that does not inhibit classical complement activity (RLS-0174) had no significant inhibitory activity (FIG. 16).

[0139] Example 9. RLS-0122 enhances survival and quality of life in a murine TC-1 tumor cell model To evaluate the efficacy of RLS-0122 in an in vivo system, we utilized the TC-1 tumor cell model in C57Bl / 6 mice, as previously reported (Roumenina et al., Cancer Immunol Res; 7(7) July 2019). TC-1 cells are derived from a human papillomavirus-transformed lung epithelial cell line, and complement activation has been shown to contribute to tumor growth. When TC-1 cells were introduced into the flank of mice, highly aggressive tumors formed, resulting in 100% mortality within approximately 26 days. To test the efficacy of RLS-0122 on survival and quality of life in this model, animals were injected subcutaneously with 4 x 10^5 TC-1 cells into the flank. Five days later, animals received either saline vehicle treatment (n = 6) or IV drug treatment with 160 mg / kg 1x / day RLS-0122 (n = 8) for 15 consecutive days. Animals treated with RLS-0122 exhibited a significant median survival time of 8 days longer than those receiving vehicle (p=0.0465) (Figure 17A). Animals were also evaluated every 2 days for the duration of the study for behavioral and physical condition scoring. If a score of 7 or less was reached, animals were evaluated daily. A score of 5 or less was considered the endpoint for euthanasia. Animals receiving RLS-0122 demonstrated an increased quality of life after the end of treatment, with scores reaching statistical significance on days 22-26 (p<0.05) (Figures 17A-17B).

[0140] Example 10: Administration of pharmaceutical compositions To modulate the complement system, a pharmaceutical composition containing a therapeutically effective amount of any of SEQ ID NOs: 6-13 and variants thereof is administered to a subject in need thereof.

[0141] To inhibit myeloperoxidase activity, a pharmaceutical composition containing a therapeutically effective amount of any of SEQ ID NOs: 6-13 and variants thereof is administered to a subject in need thereof.

[0142] To inhibit oxidant activity, a pharmaceutical composition containing a therapeutically effective amount of any of SEQ ID NOs: 6-13 and variants thereof is administered to a subject in need thereof.

[0143] To inhibit the binding of PD-1 to PD-L1, a pharmaceutical composition comprising a therapeutically effective amount of any of SEQ ID NOs:6-13 and their variants is administered to a subject in need thereof.

[0144] To inhibit T cell depletion, a pharmaceutical composition comprising a therapeutically effective amount of any of SEQ ID NOs: 6-13 and variants thereof is administered to a subject in need thereof.

[0145] To inhibit angiogenesis, a pharmaceutical composition comprising a therapeutically effective amount of any of SEQ ID NOs: 6-13 and variants thereof is administered to a subject in need thereof.

[0146] 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: 6-13. 2. 2. The synthetic peptide of embodiment 1, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6-13. 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: 7-11. 4. 2. The synthetic peptide of embodiment 1, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-11. 5. 2. The synthetic peptide of claim 1, comprising an amino acid sequence comprising at least about 95% sequence identity to SEQ ID NO:6. 6. 2. The synthetic peptide of claim 1, comprising an amino acid sequence comprising at least about 95% sequence identity to SEQ ID NO:7. 7. 2. The synthetic peptide of claim 1, comprising an amino acid sequence comprising at least about 95% sequence identity to SEQ ID NO:8. 8. 2. The synthetic peptide of claim 1, comprising an amino acid sequence comprising at least about 95% sequence identity to SEQ ID NO:9. 9. 2. The synthetic peptide of claim 1, comprising an amino acid sequence comprising at least about 95% sequence identity to SEQ ID NO:10. 10. 2. The synthetic peptide of claim 1, comprising an amino acid sequence comprising at least about 95% sequence identity to SEQ ID NO:11. 11. 2. The synthetic peptide of claim 1, comprising an amino acid sequence comprising at least about 95% sequence identity to SEQ ID NO:12. 12. 2. The synthetic peptide of claim 1, comprising an amino acid sequence comprising at least about 95% sequence identity to SEQ ID NO:13. 13. 13. A pharmaceutical composition comprising a therapeutically effective amount of the synthetic peptide of any of claims 1 to 12 and at least one pharmaceutically acceptable carrier, diluent, or excipient. 14. 14. A method of regulating the complement system, comprising administering the pharmaceutical composition of claim 13 to a subject in need thereof. 15. 14. A method of inhibiting myeloperoxidase activity, comprising administering the pharmaceutical composition of claim 13 to a subject in need thereof. 16. 14. A method of inhibiting oxidant activity, comprising administering the pharmaceutical composition of claim 13 to a subject in need thereof. 17. 14. A method of inhibiting the binding of PD-1 to PD-L1, comprising administering the pharmaceutical composition of claim 13 to a subject in need thereof. 18. 14. A method of inhibiting T cell depletion, comprising administering the pharmaceutical composition of claim 13 to a subject in need thereof. 19. 14. A method of inhibiting angiogenesis, comprising administering the pharmaceutical composition of claim 13 to a subject in need thereof.

[0147] Array Table TIFF0007804671000017.tif203161

[0148] 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.

[0149] All patents, applications, publications, test methods, literature, and other materials cited herein are incorporated by reference in their entirety as if physically present herein.

[0150] References TIFF0007804671000018.tif27150TIFF0007804671000019.tif229151TIFF0007804671000020.tif27150

Claims

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

6.

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

7.

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

8.

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 1 and at least one pharmaceutically acceptable carrier, diluent, or excipient.

10. A pharmaceutical composition for use in a method for inhibiting angiogenesis, comprising a therapeutically effective amount of the synthetic peptide of claim 1 and at least one pharmaceutically acceptable carrier, diluent, or excipient.

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