Peptides and methods of use

Abstract

We provide peptide-based inhibitors for different pathways in the complement system. [Solution] The present invention provides synthetic peptides. The present invention relates to the modification of a 15-amino acid synthetic peptide from polar assortant (PA) peptides, which are scrambled peptides derived from human astrovirus protein. In some embodiments, the present invention relates to peptides that are modified PAs, which are stapled and / or have D-enantiomer substitutions of certain amino acids, and which include sarcosine substitutions at certain amino acid positions. The present invention further provides a method for selecting at least one synthetic peptide to treat various conditions.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Application No. 63 / 108,762, filed on November 2, 2020, the disclosure of which is hereby incorporated by reference in its entirety.

[0002] Sequence Listing This application is electronically filed in ASCII format and includes a sequence listing that is hereby incorporated by reference in its entirety. The ASCII copy was created on October 28, 2021, named 251110_000156_SL.txt, and is 5,875 bytes in size.

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

Background Art

[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 adaptive immune responses and helping to eliminate immune complexes and apoptotic cells. The complement system comprises three distinct pathways: the classical pathway, the lectin pathway, and the alternative pathway. C1q and mannose-binding lectin (MBL) are structurally related recognition molecules for the classical and lectin pathways, respectively. While IgM or clustered IgG acts as the primary ligand for C1q, MBL recognizes polysaccharides such as mannan. Ligand binding by C1q and MBL leads to the sequential activation of C4 and C2, forming the C3 convertases of the classical and lectin pathways, respectively. In contrast, activation of the alternative pathway does not require recognition molecules but can amplify the C3 activation induced by the classical or lectin pathway. Activation of any of these three pathways leads to the formation of inflammatory mediators (C3a and C5a) as well as membrane attack complexes (MACs) that cause cell lysis.

[0005] While the complement system plays a crucial role in many protective immune functions, complement activation is a key mediator of tissue damage in the processes of a wide range of autoimmune and inflammatory diseases (Ricklin and Lambris, "Complement-targeted therapeutics." Nat Biotechnol 2007; 25(11):1265-75 (Non-patent Literature 1)).

[0006] Complement regulators are needed. On the one hand, the complement system is a crucial host defense against pathogenic organisms. On the other hand, its unsuppressed activation can cause catastrophic 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 anti-complement therapies have recently been 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 rare diseases that affect only a very small number of people. Currently, there are no approved complement regulators for more common disease processes in which dysregulated complement activation plays a central role. Dysregulation of complement activation can play a role in both chronic and acute disease symptoms.

[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 the processes of numerous autoimmune and inflammatory diseases. Specific blockade of the classical and lectin pathways is particularly necessary because both of these 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, functional alternative pathways are 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 inherent specificity and efficient properties, this class of molecules has attracted considerable attention as human therapeutic agents for a variety of disease symptoms, with more than 60 approved for therapeutic use in the United States, Europe, and / or Japan as of March 2018, and 155 currently in clinical development [2]. Compared to small molecules (<500 Da) that 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-cost production and can often be chemically synthesized, thus avoiding costly and complex generation and purification. Often, naturally occurring peptides cannot be directly translated to therapeutic use due to suboptimal chemical and physical stability, as well as poor pharmacokinetics (half-life). Therefore, several technical approaches are frequently used to rationally design peptides into molecules suitable for human administration and leading to the development of newer drugs.

[0009] The inventors 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 precursors of PIC1 peptides were initially based on the discovery that the 787-amino acid capsid protein sequence of human astrovirus type 1, an endemic non-enveloped icosahedral RNA virus that causes gastroenteritis in human infants [9], can inhibit the activation of the classical complement pathway

[10] .

[0010] The PIC1 molecular family comprises a collection of rationally designed peptides possessing several anti-inflammatory functional properties, including inhibition of the classical complement pathway, myeloperoxidase inhibition, neutrophil extracellular trapping inhibition, and antioxidant activity. The original PIC1 peptide is a 15-amino acid peptide sequence derived from scrambled astrovirus coat protein. The filename is TIFF2026065064000002.tif4128. The original PIC1 peptide is modified with a monodisperse 24-mer pegylated moiety at the C-terminus. TIFF2026065064000003.tif4128 This increases its water solubility. SEQ ID NO:3 sarcosine substitution scan shows that replacing isoleucine at position 8 or cysteine ​​at position 9 with sarcosine results in two peptides. TIFF2026065064000004.tif11157 was found to be water-soluble without pegylation (as described in U.S. Patent No. 10,005,818 (Patent Document 1)). Further variants based on the PA-I8Sar and PA-I9Sar molecules were constructed, including staple forms of the peptide and / or one or more D-enantiomeral 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 [Overview of the Initiative]

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

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

[0015] In another aspect, the present invention provides a synthetic peptide that inhibits 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 polar assortant (PA) peptide (SEQ ID NO:2), the modification of this peptide, and methods of use thereof. PA peptide is a scrambled peptide derived from a human astrovirus protein called CP1 (SEQ ID NO:1). PA peptide is also known as PIC1 (complement C1 peptide inhibitor), AstroFend, AF, or SEQ ID NO:2. PIC1 peptide was initially named as such because it was found to be associated with complement-mediated diseases. A pegylated form of 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 an improved effect against complement inhibition. A form of PIC1 peptide with the amino acid derivative sarcosine at position 8, called PA-I8Sar (SEQ ID NO:4), also shows an improved effect against complement inhibition. A form of the PIC1 peptide having the amino acid derivative sarcosine at position 9, called PA-C9Sar (SEQ ID NO: 5), also exhibits improved efficacy against complement inhibition. PA-dPEG24, PA-I8Sar, and PA-C9Sar are described, for example, in U.S. Patent No. 10,005,818 and U.S. Patent Application Publication No. US2019 / 0209660. As used herein, the term “PIC1 peptide” includes SEQ ID NO: 6-8, which are staple forms of SEQ ID NO: 4 and / or substitutions of SEQ ID NO: 4 having one or more D-enantiomers of amino acids instead of the usual L-enantiomers, and SEQ ID NO: 9-13, which are staple forms of SEQ ID NO: 5 and / or substitutions of SEQ ID NO: 5 having one or more D-enantiomers of amino acids instead of the usual L-enantiomers.

[0017] In some aspects, the invention relates to peptides that are in the stapled form 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 invention relates to peptides that are in the stapled form 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 a synthetic peptide having at least about 95% sequence identity to the amino acid sequence 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 modifications. In one aspect, the invention is a synthetic peptide having 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 modifications.

[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 NO: 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 NO: 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 NO: 9-13 and variants thereof, and at least one pharmaceutically acceptable carrier, diluent, or excipient.

[0021] In one aspect, the present invention provides a synthetic peptide comprising at least about 95% sequence identity to an amino acid sequence selected from the group of SEQ ID NO: 6-13.

[0022] In some embodiments, the present invention provides a synthetic peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 6-13.

[0023] In a related aspect, the present invention provides a pharmaceutical composition 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 the pharmaceutical composition described herein to a subject in need thereof.

[0025] In a related aspect, the present invention provides a method of inhibiting myeloperoxidase activity, comprising administering the pharmaceutical composition described herein to a subject in need thereof.

[0026] In a related aspect, the present invention provides a method for inhibiting oxidative activity, comprising the step of administering a pharmaceutical composition described herein to a subject in need of such inhibition.

[0027] In a related aspect, the present invention provides a method for inhibiting the binding of PD-1 to PD-L1, comprising the step of administering a pharmaceutical composition described herein to a subject in need.

[0028] In a related context, the present invention provides a method for inhibiting T cell depletion, comprising the step of administering a pharmaceutical composition described herein to a subject in need.

[0029] In a related context, the present invention provides a method for inhibiting angiogenesis, comprising the step of administering a pharmaceutical composition described herein to a subject in need of such treatment.

[0030] These and other objects, features and advantages of the present invention will become more apparent upon reading the following specification together with the attached description, claims, and drawings. [Brief explanation of the drawing]

[0031] The accompanying figures incorporated herein and constituting part of this specification illustrate several aspects described below. [Figure 1A] Figures 1A and 1B show the inhibition of complement activation by the PIC1 peptide in the 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 the CH50 type assay. Peptides are at a final concentration of 0.5 mM. Values ​​are expressed as a percentage of positive controls consisting of human O serum and AB erythrocytes in GVBS++ buffer. Data are mean ± SEM of n=3 independent experiments. [Figure 1B] Refer to the description in Figure 1A. [Figure 2A]Figures 2A-2B show the maximum half-binding values ​​for PIC1 peptide binding to C1q. Maximum half-binding concentrations were calculated from the binding curves for (2A)PA-I8Sar mutant and (2B)PA-I9Sar mutant. Peptide mutant PA-0142 did not bind to C1q, while peptide mutant PA-0152 did not titrate, making it impossible to calculate the maximum half-binding value. Peptide mutant PA-0168 was not recognized by the primary polyclonal antibody and therefore could not be analyzed. [Figure 2B] Refer to the description in Figure 2A. [Figure 3A] Figures 3A-3B show the maximum half-values ​​for PIC1 peptide inhibition of MPO activity. For (3A)PA-I8Sar mutant and (3B)PA-C9Sar mutant, the maximum half-values ​​were calculated from the activity curves. [Figure 3B] Refer to the description in Figure 3A. [Figure 4A] Figures 4A-4B show the inhibition of oxidative activity by PIC1 peptides in total antioxidant capacity (TAC) assays. Antioxidant activity is measured by copper reducing equivalents (CRE). (4A)PA-I8Sar mutant and (4B)PA-I9Sar mutant were tested at various concentrations, and the maximum antioxidant activity for each peptide is reported. [Figure 4B] Refer to the description in Figure 4A. [Figure 5A] Figures 5A-5C show the C1q binding curves. These curves illustrate the binding of progressively increasing concentrations of PA-I8Sar modified compounds (5A-5B) and PA-I9Sar modified compounds (5C) to immobilized C1q in an ELISA-type assay. Peptide mutant PA-0142 did not bind to C1q, while peptide mutant PA-0152 did not titrate, making it impossible to calculate the maximum half-binding dose. Peptide mutant PA-0168 was not recognized by the primary polyclonal antibody and therefore could not be analyzed. [Figure 5B] Refer to the description in Figure 5A. [Figure 5C] Refer to the description in Figure 5A. [Figure 6A]Figures 6A-6C show the MPO inhibition curves. Inhibition of MPO activity by gradually increasing concentrations of PA-I8Sar modified (6A-6B) and PA-I9Sar modified (6C) in an ELISA-type assay. [Figure 6B] Refer to the description in Figure 6A. [Figure 6C] Refer to the description in Figure 6A. [Figure 7A] Figures 7A-7C show the total antioxidant activity curves. The total antioxidant activity of PA-I8Sar modified compounds (7A-7B) and PA-I9Sar modified compounds (7C) at gradually increasing concentrations was analyzed. Antioxidant activity was measured using copper reducing equivalents (CRE). [Figure 7B] Refer to the description in Figure 7A. [Figure 7C] Refer to the description in 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 the bound PD-1 was detected with streptavidin-HRP reagent and subsequently with TMB as a substrate for a colorimetric quantitative 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. The bound C1q and MAC-1 served as positive and negative controls for peptide binding, respectively. Gradual doses of peptide were added to the plate, followed by the addition of a rabbit polyclonal antibody that recognizes the peptide, and then a secondary anti-rabbit antibody conjugated with HRP. The plate was then colored by adding TMB as a substrate for a colorimetric quantitative assay. [Figure 9B] Refer to the description in 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 cellular signaling. CTLA-4 effector cells were incubated with aAPC / large cells in the absence or presence of escalating concentrations of anti-CTLA-4 antibody (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 the test peptide is due to cell death resulting from the peptide buffering effect on cells. [Figure 11] Figure 11 shows that RLS-0122, RLS-0164, and RLS-0168 were able to inhibit T cell depletion, as measured by a reduction in the level of caspase 3 / 7, an apoptotic cell marker. Purified human pan-T cells were stimulated with Dynabeads every 48 hours for 8 days, and PIC1 peptide (2 mg / ml) was also administered to the cells at each stimulation. Background levels of caspase 3 / 7 signaling were evaluated in parallel with cells not treated with Dynabeads. On day 8, cells were harvested and caspase 3 / 7 levels were determined by ELISA. RLS-0150 and RLS-0154 did not show 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 depletion, as measured by increased levels of cytokine IL-2 (12A and 12C) and IFN-gamma (12B and 12D). RLS-0164 is shown in Figures 12A and B, 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 PIC1 peptide (2 mg / ml) was also administered to the cells at each stimulation. Cell supernatant was collected at each stimulation, and levels of IL-2 and IFN-gamma were assayed by ELISA. [Figure 12B] Refer to the description in Figure 12A. [Figure 12C] Refer to the description in Figure 12A. [Figure 12D] Refer to the description in 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 PIC1 peptide (1 mg / ml) was added to the plate, followed by the addition of a rabbit polyclonal antibody that recognizes the peptide, and then a secondary anti-rabbit antibody conjugated with HRP. The plate was then colored by adding TMB as a substrate for a colorimetric quantitative 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 were a positive control for VEGF-mediated cell signaling, indicated by a line showing a 20,000 relative luminescence unit (RLS) response indicating the binding of VEGF to VEGFR-2. [Figure 15] Figure 15 shows that a specific PIC1 peptide was able to inhibit LPS-induced non-VEGF-mediated angiogenesis. HUVEC cells were incubated with either RLS-0122, RLS-0150, RLS-0154, RLS-0164, or RLS-0168, and then LPS was added and plated onto the extracellular matrix. After overnight incubation, evidence of angiogenesis was determined by fluorescence microscopy. LPS-treated and untreated cells served as positive and negative controls for angiogenesis. [Figure 16] Figure 16 shows that RLS-0122 can inhibit the activation of the classical complement pathway by the human kidney cancer cell line A498. Supernatant derived from A-498 cells was added to purified human C1q cells pre-incubated with escalating 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, and purified human C4 (4 ug / ml) was added, followed by incubation at 37°C for 1.5 hours. The supernatant was collected for analysis using 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 mouse TC-1 cancer cell model. Animals were subcutaneously injected with 4 × 10^5 TC-1 cells into their flanks. Five days later, animals were given either a saline vehicle (n=6) or IV RLS-0122 at 160 mg / kg 1× / day (n=8) for 15 consecutive days. Behavioral and physical status scores were also assessed every two days throughout the study. If the score reached 7 or below, the animals were assessed daily. A score of 5 or below was considered the endpoint for euthanasia. [Figure 17B] Refer to the description in Figure 17A. [Modes for carrying out the invention]

[0032] Detailed description of the invention As stated in the background section, there is a great need in the art to identify techniques for peptide-based inhibitors of different pathways of the complement system and to use this knowledge to develop novel therapeutic peptides. The present invention satisfies this need and other needs. Aspects of the present invention generally relate to synthetic peptides, more specifically to synthetic peptides comprising stapled and / or one or more D-enantiomerized amino acids.

[0033] To facilitate understanding of the principles and features of various embodiments of the present invention, several exemplary embodiments are described below. While exemplary embodiments of the present invention are described in detail, it should be understood that other embodiments are conceivable. Therefore, the present invention is not intended to be limited to the details of the composition and arrangement of components described below or in the examples. Other embodiments of the present invention are possible and can be implemented or carried out in various ways. Furthermore, specific terminology is used in the description of exemplary embodiments for clarity.

[0034] It should also be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless otherwise specified in the context. 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 specified one. In other words, the terms “a,” “an,” and “the” do not indicate a limitation of quantity, but rather indicate the presence of “at least one” of the items referred to.

[0035] As used herein, the terms "and / or" can mean "and," "or," "exclusive or," "one," "some but not all," "neither," and / or this can mean "both." The term "or" is intended to mean inclusive "or."

[0036] Furthermore, terminology is used for clarity in the description of exemplary embodiments. Each term is intended to have its broadest meaning as understood by those skilled in the art and to include all technical equivalents that operate in a similar manner to achieve similar purposes. It should be understood that embodiments of the disclosed technology can be implemented without these specific details. Where otherwise, well-known methods, structures, and techniques are not described in detail so as not to obscure the understanding of this description. References to “one embodiment,” “a certain embodiment,” “example embodiment,” “several embodiments,” “a particular embodiment,” and “various embodiments” indicate that embodiments of the disclosed technology described in this way may include certain features, structures, or characteristics, but not all embodiments necessarily include those features, structures, or characteristics. 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 as referring to both the number designated as the endpoint of any range. Any reference to a range should be considered as supporting any subset within the range. A range can be expressed herein as “about,” “approximately,” or “substantially” from a particular value and / or “about,” “approximately,” or “substantially” to another particular value. Where such a range is expressed, other exemplary embodiments include from a particular value and / or to another particular value. Furthermore, the term “about” means within an acceptable margin of error for a particular value determined by those skilled in the art, which is considered to depend in part on how the value is measured or determined, i.e., the limitations of the measuring system. For example, “about” may mean within an acceptable standard deviation according to convention in the art. Alternatively, “about” may mean a range of up to ±20%, preferably up to ±10%, more preferably up to ±5%, and more preferably even further up to ±1% of a given value. Or, particularly for biological systems or processes, the term may mean within one order of magnitude of the value, preferably up to twice the value. Where specific values ​​are mentioned in this application and claims, unless otherwise stated, the term “about” is implicit and in this context means that the value is within the acceptable margin of error.

[0038] Throughout this disclosure, various aspects of the invention may be presented in range form. It should be understood that the range form is merely for convenience and brevity and should not be interpreted as an inflexible limitation on the scope of the invention. Therefore, the range description should be considered to specifically disclose all possible subranges and individual numbers within the range. For example, a range description such as 1–6 should be considered to specifically disclose subranges such as 1–3, 1–4, 1–5, 2–4, 2–6, 3–6, 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 width of the range.

[0039] Similarly, as used herein, characterizations such as "substantially absent" or "substantially pure" may include both "at least substantially absent" or "at least substantially pure" and "completely absent" or "completely pure."

[0040] "Comprising," "containing," or "including" means that at least the specified compound, element, particle, or process step is present in the composition, article, or method, and does not exclude the presence of other compounds, materials, particles, or process steps, even if those other compounds, materials, particles, or process steps have the same function as the specified one.

[0041] Throughout this description, various components with specific values ​​or parameters may be identified, but these items are provided in exemplary embodiments. In fact, exemplary embodiments do not limit the various aspects and concepts of the invention, as many comparable parameters, sizes, ranges, and / or values ​​can be implemented. Terms such as “first,” “second,” “primary,” “secondary,” etc., do not indicate 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 certain features are decisive, essential, or even more important to the structure or function of the claimed invention. Rather, these terms are intended to simply highlight alternative or further features that may or may not be available in particular aspects 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 arise from any quantitative comparison, value, measurement, or other expression.

[0043] The dimensions and values ​​disclosed herein should not be understood as being strictly limited to the exact numerical values ​​listed. Instead, unless otherwise specified, each such dimension is intended to mean both the listed value and a functionally equivalent range around that value. For example, a dimension disclosed as "50 mm" is intended to mean "approximately 50 mm."

[0044] It should also be understood that the reference to one or more process steps does not preclude the presence of further or intervening process steps between those explicitly identified steps. Similarly, it should be understood that the reference to one or more components in a composition does not preclude the presence of further components other than those explicitly identified.

[0045] The materials described below as constituting various elements of the present invention are intended to be illustrative and not limiting. Many suitable materials that would perform the same or similar functions as those described herein are intended to be included within the scope of the present invention. Other such materials not described herein include, but are not limited to, materials developed after the development of the present invention. Any dimensions listed in the various drawings are for illustrative purposes only and are not intended to be limiting. Other dimensions and proportions are intended to be contemplated and included within the scope of the present invention.

[0046] As used herein, the terms “subject” or “patient” refer to mammals and, not limited to, humans and veterinary animals. In preferred embodiments, the subject is human.

[0047] As used herein, the term “combination” of the synthetic peptide and at least one second pharmaceutically active ingredient according to the claimed invention means that at least two, but any desired combination of, the compounds are delivered simultaneously or sequentially (e.g., within 24 hours). When used to treat various diseases, the compositions and methods of the present invention are intended to be available in conjunction with other therapeutic methods / agents suitable for the same or similar diseases. Such other therapeutic methods / agents may 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 health condition in which the subject is unable to maintain homeostasis, and if the disease does not improve, the subject's health continues to deteriorate. In contrast, a "disability" is a health condition in which the subject can maintain homeostasis, but the subject's health condition is less desirable than if there were no disability. Even if left untreated, a disability does not necessarily lead to a further decline in the subject's health condition.

[0049] The term "treatment" of a condition, disorder, or state includes: (1) preventing or delaying the onset of at least one clinical or quasi-clinical symptom of a condition, disorder, or state occurring in a subject who is currently suffering from or susceptible to such a condition, disorder, or state, but has not yet experienced or exhibited any clinical or quasi-clinical symptoms of the condition, disorder, or state; or (2) inhibiting the condition, disorder, or state, i.e., stopping, reducing, or delaying the onset or recurrence of the disease (in the case of maintenance treatment) or at least one clinical or quasi-clinical symptom thereof; or (3) reducing the disease, i.e., causing the regression of at least one clinical or quasi-clinical symptom of the condition, disorder, or state. The benefit to the subject being treated is either statistically significant or at least perceived by the patient or physician.

[0050] As used herein, the term “therapeutic” means treatment and / or prevention. Therapeutic effects are obtained by suppression, reduction, remission, or eradication of the disease condition.

[0051] As used herein, the term “therapeutically effective” as applied to dose or quantity means the amount of a compound or pharmaceutical composition that is sufficient to produce, when administered to a subject, to treat (e.g., prevent or improve) a situation, disorder, or condition. “Therapeutically effective amount” is considered to vary depending on the compound or bacterium or analogue administered, as well as the disease and its severity, and the age, weight, physical condition, and responsiveness of the mammal being treated.

[0052] When used in connection with the compositions of the present invention, the phrase “pharmaceutically acceptable” refers to molecular entities and other components of such compositions that are physiologically tolerable and typically do not produce adverse reactions when administered to mammals (e.g., humans). Preferably, as used herein, the term “pharmaceutically acceptable” means that it is approved by federal or state regulatory authorities for use in mammals, more particularly in humans, or that it is listed in the United States Pharmacopeia or other commonly accepted pharmacopoeias.

[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, such as petroleum, animal, vegetable, or synthetic sources, such as peanut oil, soybean oil, mineral oil, or sesame oil. Water or aqueous saline solutions and aqueous dextrose and glycerol solutions are preferably used as carriers, especially for injectable solutions. Alternatively, the pharmaceutical carrier may be a solid dosage form carrier containing, but not limited to, one or more binders (for compressed pills), flow enhancers, 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 so that the analog retains substantially the same biological activity as the unmodified form in vivo and / or in vitro.

[0055] The terms “sequence identity” and “identity percentage” are used interchangeably herein. In this invention, to determine the identity percentage of two amino acid sequences or two nucleic acid sequences, it is defined herein that the sequences are aligned for the purpose of optimal comparison (for example, gaps can be introduced in the sequence of the first amino acid or nucleic acid for optimal alignment with the second amino acid or nucleic acid sequence). The amino acid residues 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, then the molecules are identical at that position. The identity percentage between the two sequences is a function of the number of identical positions common to these sequences (i.e., identity % = number of identical positions / total number of positions (i.e., overlapping positions) × 100). Preferably, the two sequences are of the same length.

[0056] Several different computer programs are available to determine the degree of identity between two sequences. For example, the comparison of sequences and the determination of the identity percentage between two sequences can be achieved using mathematical algorithms. In a preferred embodiment, the identity percentage between two amino acid sequences or nucleic acid sequences is determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. (48): 444-453 (1970)), which is 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, as well as gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6. While these different parameters are expected to yield slightly different results, the overall identity percentage of the two sequences will not change significantly if different algorithms are used.

[0057] Sequence comparisons can be performed over the full length of the two sequences being compared, or over fragments of the two sequences. Typically, the comparison is thought to be performed over the full length of the two sequences being compared. However, sequence identity can be performed over regions of, for example, 20, 50, 100, or more consecutive amino acid residues.

[0058] In the art, "sequence identity" refers to the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, i.e., a reference sequence, and a given sequence that is compared to the reference sequence. Sequence identity is determined by comparing a given sequence to a reference sequence after the sequences have been optimally aligned to yield the highest degree of sequence similarity, which can be determined by matching strings of such sequences. During such alignment, sequence identity is checked position by position; for example, sequences are "identical" at a particular position if they have identical nucleotides or amino acid residues at that position. The sequence identity percentage is then obtained by dividing the total number of such identical positions by the total number of nucleotides or residues in the reference sequence. Sequence identity is incorporated herein by reference, but is not limited to, the teachings of which are included herein by reference: Computational Molecular Biology, Lesk, AN, ed., Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, 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. It can be easily calculated by known methods, including those described in (1988).A preferred method for determining sequence identity is designed to obtain the greatest match between the sequences being tested. Methods for determining sequence identity are systematized 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, SF et al., J. Molec. Biol., 215:403-410 (1990)). The BLASTX program is publicly available from NCBI and other sources (its teachings are incorporated herein by reference: BLAST Manual, Altschul, S. et al., NCVI NLM NIH Bethesda, Md. 20894, Altschul, SF et al., J. Molec. Biol., 215:403-410). (1990)). These programs optimally align sequences using default gap weights to achieve the highest level of sequence identity between a given sequence and a reference sequence. As one example, a given polynucleotide sequence is intended to be identical to the reference sequence, except that the given polynucleotide sequence may contain up to 5, 4, 3, 2, 1, or 0 point mutations per 100 nucleotides of the reference sequence.In other words, in a polynucleotide having a nucleotide sequence with at least 95% sequence identity to a reference nucleotide sequence, for example, at least 96%, 97%, 98%, 99%, or 100%, up to 5%, 4%, 3%, 2%, 1%, or 0% of the nucleotides of the reference sequence can be deleted and 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 in the reference sequence can occur at the 5' or 3' terminal positions of the reference nucleotide sequence, or dispersed individually among nucleotides in the reference sequence or in one or more consecutive groups within the reference sequence, at any location between these terminal positions. Similarly, by a polypeptide having a given amino acid sequence having sequence identity of at least, for example, 95%, for example, at least 96%, 97%, 98%, 99%, or 100% with respect to a reference amino acid sequence, the given amino acid sequence of the polypeptide is intended to be identical to the reference sequence, except that the given polypeptide sequence may contain up to 5, 4, 3, 2, 1, or 0 amino acid changes per 100 amino acids of the reference amino acid sequence. In other words, to obtain a given polypeptide sequence having at least 95% sequence identity with a reference amino acid sequence, for example, at least 96%, 97%, 98%, 99%, or 100%, 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 in the reference sequence can be inserted into the reference sequence. These changes to the reference sequence can occur at the amino or carboxyl terminal positions of the reference amino acid sequence, or dispersed individually between residues in the reference sequence or in one or more consecutive groups within the reference sequence, at any location between these terminal positions. Preferably, the positions of non-identical residues are distinguished by conservative amino acid substitutions.However, when determining sequence identity, conservative substitutions are not included as matches.

[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, e.g., cytokine production and cytotoxicity, and B cell responses, e.g., antibody production. Furthermore, the term “immune response” includes immune responses indirectly affected by T cell activation, e.g., antibody production (humoral response) and cytokine-responsive cell activation, e.g., macrophage activation. 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, and oligodendrocytes); natural killer cells; myeloid cells, such as macrophages, eosinophils, mast cells, basophils, and granulocytes (e.g., neutrophils).

[0060] Examples of "parenteral" administration of immunogenic compositions include subcutaneous (sc), intravenous (iv), intramuscular (im), or intradermal (id) injection or infusion techniques.

[0061] In the field of medicine, the term "prevent" encompasses any activity that reduces the mortality or morbidity burden of a disease. Prevention can occur at primary, secondary, and tertiary levels. Primary prevention avoids the onset of the disease, while secondary and tertiary prevention encompasses activities aimed at reducing the negative effects of an already established disease by preventing disease progression and the appearance of symptoms, as well as by restoring function and reducing disease-related complications.

[0062] A “mutant” of a polypeptide according to the present invention may be (i) one or more amino acid residues substituted with conserved or non-conserved amino acid residues (preferably conserved amino acid residues), where such substituted amino acid residues may or may not be encoded in the genetic code; (ii) one or more modified amino acid residues, e.g., residues modified by substituent attachment; (iii) a polypeptide that is an alternative splicing variant of the polypeptide of the present invention; (iv) a polypeptide fragment; and / or (v) a polypeptide fused with another polypeptide, e.g., a leader sequence or secretion sequence, or a sequence used for purification (e.g., a His tag) or a sequence used for detection (e.g., an Sv5 epitope tag). The fragment contains a polypeptide produced by proteolytic cleavage (including multi-site proteolysis) of the original sequence. The mutant may be post-translationally or chemically modified. Such mutants are considered to be within the scope of the art from the teachings herein. As used herein, the term “mutant” includes peptides having at least about 95% identity to the peptides disclosed herein.

[0063] Within the scope 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, simultaneously in different compositions, or consecutively (preferably within 24 hours).

[0064] According to the present invention, conventional molecular biology, microbiology, and recombinant DNA techniques within the scope of the art can be used. Such techniques are well described in the literature. Among the many available, for example, Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (referred to as "Sambrook et al., 1989" in this specification); DNA Cloning: A Practical Approach, Volumes I and II (DN Glover ed. 1985); Oligonucleotide Synthesis (MJ Gait ed. 1984); Nucleic Acid Hybridization (BD Hames & SJ Higgins eds. (1985); Transcription and Translation (BD Hames & SJ Higgins, eds. (1984); Animal Cell Culture (RI Freshney, ed. (1986); Immobilized Cells and Enzymes (IRL Press, (1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); FM Ausubel et al. (eds.), Current Protocols in See Molecular Biology, John Wiley & Sons, Inc. (1994).

[0065] The peptide composition of the present invention Modification of the amino acid structure of CP1 led to the discovery of further peptides that can regulate complement activation, such as C1q activity. In in vitro assays of classical complement pathway activation / inhibition, myeloperoxidase (MPO) inhibition, oxidized forms, and NET activity, the parent molecule... Compared to TIFF2026065064000005.tif4128, it ranks 8th. TIFF2026065064000006.tif4128 and 9th place Previous studies in TIFF2026065064000007.tif4128 have shown that substitution of isoleucine with sarcosine results in peptides with increased solubility and enhanced inhibitory activity without pegylation. To determine whether more potent peptides could be identified, amino acid variants based on the PA-I8Sar and PA-C9Sar skeletons were synthesized, consisting of staple 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 skeleton included combinations of staple and D-amino acid combinations. While we do not wish to be constrained by theory, stapling techniques can increase peptide stability and enhance bioactivity by immobilizing peptide molecules into bioactive α-helix secondary structures. While we do not wish to be constrained by theory, D-amino acid substitutions can provide further stability to peptides and extend their in vivo half-life. All but one of these peptides are readily soluble in water, and their biological activity was evaluated using various in vitro assays.

[0066] As used herein, the term “peptide” refers to a naturally occurring amino acid sequence, or a peptide mime, peptide analogue, and / or a synthetic derivative of about 15 amino acids based on SEQ ID NO:4 or SEQ ID NO:5 (e.g., staple peptides, sarcosine substitutions, D-amino acid substitutions, and pegylated peptides, etc.). Furthermore, a peptide may be less than about 15 amino acid residues, for example, about 10 to about 15 amino acid residues, for example, a peptide of about 5 to about 10 amino acid residues. For example, peptide residues of amino acids 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 may also be peptides in the context of the present invention. A peptide may also be more than 15 amino acids, for example, 16, 17, 18, 19, and 20, or more amino acids.

[0067] The disclosed peptides are generally amino acid sequences of about 15 amino acid residues or less than about 15 amino acid residues, which are either constrained (i.e., have certain structural elements such as the presence of an amino acid that initiates a β-turn or β-pleat sheet, or are cyclized by the presence of a disulfide-bonded Cys residue, for example) or unconstrained (i.e., linear).

[0068] Substitutions for amino acids in 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 in a sequence can be substituted with other amino acids of similar polarity that act as functional equivalents, 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 more likely to alter the structure, activity, or function of the resulting protein. For example, the peptides of this disclosure include one or more of the following conservative amino acid substitutions: aliphatic amino acids, e.g., replacement of alanine, valine, leucine, and isoleucine with another aliphatic amino acid; serine with threonine; threonine with serine; acidic residues, e.g., replacement of aspartic acid and glutamic acid with another acidic residue; residues with amide groups, e.g., replacement of asparagine and glutamine with another residue with an amide group; basic residues, e.g., replacement of lysine and arginine with another basic residue; and aromatic residues, e.g., replacement of phenylalanine and tyrosine with another aromatic residue.

[0070] Particularly preferred amino acid substitutions include the following: (a) Ala to Glu or vice versa, such that the negative charge can be reduced; (b) Lys relative to Arg, or vice versa, such that a positive charge can be maintained; (c) Ala relative to Arg, or vice versa, such that the positive charge can be reduced; (d) Glu to Asp or vice versa, such that a negative charge can be maintained; (e) Ser relative to Thr, or vice versa, such that the free -OH can be maintained; (f) Gln relative to Asn, or vice versa, such that free NH2 can be maintained; (g) Ile as a roughly equivalent hydrophobic amino acid to Leu or Val, or vice versa; (h) Phe for Tyr or vice versa, as roughly equivalent aromatic amino acids; and (i) Ala to Cys or vice versa, such that the disulfide bond is affected.

[0071] The amino acid substitutions in the peptide sequence are not limited to but can be selected from any amino acid, including alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, pyrrolidine, 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 in the peptide sequence.

[0072] Peptide stapling can be achieved by using unnatural amino acids with side chains that can be linked, for example, by covalent bonds, at desired positions within the peptide to introduce an alpha-helix into the peptide structure. See, for example, 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 embodiment, the present invention discloses a synthetic peptide derived from human astrovirus coat protein, the peptide comprising the amino acid sequence and modifications of SEQ ID NO: 6-13. In several embodiments, the present invention discloses a synthetic peptide derived from human astrovirus coat protein, the peptide comprising the amino acid sequence and modifications of SEQ ID NO: 6-13 as shown in Table 1 below. Staple amino acids are underlined, and D-enantiomer amino acids are shown in bold.

[0074] (Table 1) List of peptides of the present invention TIFF2026065064000008.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 with SEQ ID NO: 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 with SEQ ID NO: 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 with SEQ ID NO: 9-13.

[0076] In one aspect, the present invention is a synthetic peptide comprising amino acid sequences and modifications of SEQ ID NO: 6-13. In several aspects, the present invention is a synthetic peptide comprising amino acid sequences and modifications of SEQ ID NO: 6-8. In several aspects, the present invention is a synthetic peptide comprising amino acid sequences and modifications of SEQ ID NO: 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 NO: 6-13 and their variants, 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 NO: 6-8 and their variants, 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 NO: 9-13 and their variants, and at least one pharmaceutically acceptable carrier, diluent, or excipient. In another aspect, the pharmaceutical composition further comprises another D-enantiomer and / or staple peptide form of SEQ ID NO: 4 and / or SEQ ID NO: 5. In another context, the pharmaceutical composition further comprises SEQ ID NO: 2, 3, 4, and / or 5, as well as one or more of their variants.

[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 classical and lectin pathways is particularly necessary because both of these 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 islets 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). Alternative pathways are essential for immune surveillance against invading pathogens, and individuals lacking these pathways suffer from severe bacterial infections.By binding to and inactivating C1q and MBL, this peptide can efficiently regulate the activation of classical and lectin pathways while leaving alternative pathways intact.

[0079] As used herein, the term “modulate” means (i) controlling, reducing, inhibiting, or regulating the biological function of an enzyme, protein, peptide, factor, by-product, or derivative thereof, either individually or in complex; (ii) reducing the amount of a biological protein, peptide, or derivative thereof, either in vivo or in vitro; or (iii) disrupting a biological chain of events, cascades, or pathways known to involve a series of related biological or chemical reactions. Thus, the term “modulate” can be used, for example, to describe reducing the amount of a single component of a complement cascade compared to a control sample, reducing the rate or total amount of formation of a component or a complex of components, or reducing the overall activity of a complex process or series of biological reactions, which may result in consequences such as cell lysis, conversion of convertase enzymes, formation of complement-derived membrane attack complexes, inflammation, or inflammatory diseases. In in vitro assays, the term “regulate” can refer to a measurable change or reduction of a certain biological or chemical event, but those skilled in the art will recognize that the measurable change or reduction does not need to be absolute for it to be “regulateable.”

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

[0081] Modulation of the interaction between C1q and the C1q receptor. The interaction between C1q and the C1q receptor appears to play a crucial role in homeostatic functions such as the removal of apoptotic cell debris and immune complexes, as well as in T cell signaling mediated by antigen-presenting cells (macrophages and dendritic cells). Currently, there are no clinically proven pharmacological agents that modulate the interaction between C1q and the C1q receptor.

[0082] The disclosed peptides, including calreticulin / cC1qR, can be used to block the binding of C1q to C1q receptors. The ability of the disclosed peptides to block the binding of C1q to cellular receptors may play a crucial role in modulating intracellular signaling processes mediated by the binding of C1q to C1q receptors.

[0083] Myeloperoxidase (MPO) activity Myeloperoxidase (MPO) is a neutrophil-derived enzyme that produces hypochlorite (bleach) in acute inflammation, damaging both invading and host cells. This enzyme is known to be destructive to host tissues in many diseases.

[0084] In some embodiments, the peptides disclosed herein blocked the enzymatic activity of MPO. In some embodiments, the MPO activity present in the solubilized solution of purified human neutrophils can be directly inhibited by the peptide. In some embodiments, the present invention demonstrates that the peptide has anti-inflammatory activity.

[0085] Oxidative activity Oxidative activity resulting from the formation of reactive oxygen species can be generated in acute inflammation leading to damage to host cells and tissues. In some embodiments, the peptides disclosed herein have antioxidant activity against oxidative-producing molecules such as MPO.

[0086] Hemolysis inhibition Including sequence ID numbers 6-13, the peptides of the present invention can block complement-mediated lysis of A / B human erythrocytes (RBCs) by O serum in vitro. This assay mimics ABO incompatibility.

[0087] Inhibition of PD-1 / PD-L1 binding Including sequence ID numbers 6-13, the peptides of the present invention 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 composition of the present invention This disclosure provides a pharmaceutical composition capable of modulating the complement system, comprising at least one peptide as described above and at least one pharmaceutically acceptable carrier, diluent, stabilizer, or excipient. The pharmaceutically acceptable carrier, excipient, or stabilizer is non-toxic to the recipient at the dosage and concentration used. These may be solid, semi-solid, or liquid. The pharmaceutical composition 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 peptide having appropriate purity with a pharmaceutically acceptable carrier, diluent, or excipient. Examples of formulations and methods for preparing such formulations are well known in the art. The pharmaceutical compositions of the present invention are useful as prophylactic and therapeutic agents for various disorders and diseases as described above. In one embodiment, the composition comprises a therapeutically effective amount of at least one peptide. In another embodiment, the composition comprises at least one other active ingredient effective in modulating the complement system. In another embodiment, the composition comprises at least one other active ingredient effective in treating at least one disease associated with the complement system. In yet another embodiment, the composition comprises at least one other active ingredient effective in treating at least one disease not associated with the complement system. As used herein, the term “therapeutably effective amount” means the total amount of each active ingredient sufficient to demonstrate a benefit to the subject.

[0090] The therapeutically effective amount of peptide varies depending on several factors, such as the condition being treated, the severity of the condition, the time of administration, the route of administration, the elimination rate of the peptide used, the duration of treatment, the concomitant therapies 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 route of administration to obtain the maximum therapeutic effect.

[0091] The effective daily dose is generally within the range of approximately 0.001 to 200 milligrams (mg / kg) per kilogram of body weight, including approximately 5 to 160 mg / kg, 10 to 160 mg / kg, 40 mg / kg to 160 mg / kg, and 40 mg / kg to 100 mg / kg. This dose can be achieved with a regimen of 1 to 6 doses per day. Alternatively, optimal treatment can be achieved with a sustained-release formulation using a lower-frequency dosing regimen.

[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 NO: 6-13 and their variants, 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 NO: 6-8 and their variants, 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 NO: 9-13 and their variants, and at least one pharmaceutically acceptable carrier, diluent, or excipient. In another aspect, the pharmaceutical composition further comprises another D-enantiomer and / or staple 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 SEQ ID NO:2, 3, 4 and / or 5 and their variants, and at least one pharmaceutically acceptable carrier, diluent, or excipient.

[0093] The compositions of the present invention may include carriers and / or excipients. While the peptides of the present invention may be used as is for therapeutic purposes, it may be preferable to administer them in a pharmaceutical formulation, for example, in a mixture with appropriate 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 compatible with the other components of the formulation and “acceptable” in the sense that they are not harmful to their recipients. 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 edit. 2005). The choice of pharmaceutical excipients and carriers may be selected with respect to the intended route of administration and standard pharmaceutical practice. Oral formulations readily correspond to further mixtures such as milk, yogurt, and infant formula. Solid dosage forms for oral administration may also be used, such as capsules, tablets, caplets, pills, lozenges, powders, and granules. Suitable excipients, though not limited to specific examples, include diluents, buffers (e.g., sodium bicarbonate), preservatives, stabilizers, binders, compressors, lubricants, dispersion enhancers, disintegrants, antioxidants, flavorings, sweeteners, and colorants. Those skilled in the art can easily prepare suitable solutions.

[0094] In any one embodiment of the compositions of the present invention, the composition is formulated for delivery by routes such as oral, topical, rectal, mucosal, sublingual, nasogastric, naso / oro gastric forced feeding, parenteral, intraperitoneal, intradermal, transdermal, intrathecal, nasogastric, and intratracheal administration. In any one embodiment of the compositions of the present invention, the composition is in the form of a liquid, foam, cream, spray, powder, or gel. In any one embodiment 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 forced feeding, as well as parenteral, intraperitoneal, intradermal, transdermal, intrathecal, nasogastric, and intratracheal administration. The activator may be systemic after administration, or localized by regional administration, intramural administration, or by the use of an implant that acts to retain the active dose at the implantation site.

[0096] The effective dosage 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, and the elimination of the active ingredient from the host. The initial dose can be high, and subsequent maintenance doses can be low. Doses can be administered at low frequencies, such as weekly or bi-weekly, to maintain an effective dosage level, or they can be divided into smaller doses and administered daily, every two weeks, etc. It is conceivable that various doses may be effective in achieving therapeutic effects. While the compounds of the present invention can be used as is for therapeutic purposes, it may be preferable to administer them in a pharmaceutical formulation, for example, in a mixture with appropriate pharmaceutical excipients, diluents, or carriers selected in relation to the intended route of administration and standard pharmaceutical practices. The excipients, diluents, and / or carriers must be compatible with the other components of the formulation and “acceptable” in the sense that they are not harmful to their recipients. Acceptable excipients, diluents, 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 edit. 2005). The choice of pharmaceutical excipients, diluents, and carriers may be selected in relation 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 that may contain antioxidants, buffers, bacteriostatic agents, and solutes that make the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous suspensions that may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives.

[0098] The solution or suspension may contain any combination of the following components: sterile diluents, for example, water for injection, saline solution, non-volatile oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antimicrobial agents, for example, benzyl alcohol and methylparaben; antioxidants, for example, ascorbic acid and sodium bisulfite; chelating agents, for example, ethylenediaminetetraacetic acid (EDTA); buffers, for example, acetic acid, citric acid, and phosphoric acid; and agents for adjusting tonicity, for example, sodium chloride or dextrose.

[0099] If the agent exhibits insufficient solubility, methods for solubilizing the agent can be used. Such methods are known to those skilled in the art and are not limited to, but include the use of a cosolvent, such as dimethyl sulfoxide (DMSO), a surfactant, such as TWEEN® 80, or dissolution in an aqueous sodium bicarbonate solution. In the formulation of effective pharmaceutical compositions, pharmaceutically acceptable derivatives of the agent may also be used.

[0100] The composition may, along with the activator, include, for example, not limited to: diluents, e.g., lactose, sucrose, dicalcium phosphate, or carboxymethylcellulose; lubricants, e.g., magnesium stearate, calcium stearate, and talc; and binders, e.g., starch, natural rubber, e.g., acacia gum gelatin, glucose, molasses, polyvinylpyrrolidone, cellulose, and their derivatives, povidone, crospovidone, and other such binders known to those skilled in the art. Liquid pharmaceutically acceptable compositions may be prepared, for example, by dissolving, dispersing, or mixing the activator and any pharmaceutically acceptable auxiliaries in a carrier, for example, not limited to, water, saline solution, aqueous dextrose, glycerol, glycol, ethanol, etc., thereby forming a solution or suspension. If desired, the administered pharmaceutical composition may also contain small amounts of non-toxic auxiliary substances, such as wetting agents, emulsifiers, or solubilizers, pH buffering agents, for example, not limited to, acetic acid, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, sodium triethanolamine acetate, triethanolamine oleate, and other such agents. Practical 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). In any case, the administered composition or formulation shall contain an amount of the active agent sufficient to alleviate the symptoms of the subject being treated.

[0101] The activator or pharmaceutically acceptable derivative can be prepared using a carrier that protects the agent from rapid elimination from the body, such as a sustained-release formulation or a coating. The composition may contain other activators to obtain a desired combination of properties.

[0102] Parenteral administration generally features, and is intended herein, injection by subcutaneous, intramuscular, or intravenous injection. The injection solution can be prepared in conventional forms, either as a liquid solution or suspension, as a solid suitable for the liquid solution or suspension before injection, or as an emulsion. Suitable excipients include, but are not limited to, water, saline, dextrose, glycerol, or ethanol. Furthermore, if desired, the administered pharmaceutical composition may also contain small amounts of non-toxic 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 cyclodextrin.

[0103] Lyophilized powders can be reconstituted for administration of solutions, emulsions, and other mixtures, or formulated as solids or gels. Sterile lyophilized powders are prepared by dissolving the agents provided herein or pharmaceutically acceptable derivatives thereof in a suitable solvent. The solvent may contain excipients to improve stability, or other pharmacological components of the powder or the reconstituted solution prepared from the powder. Excipients that may be used include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable agents. The solvent may also contain a buffer, e.g., citrate, sodium, or potassium phosphate, or other such buffers known to those skilled in the art, typically at a nearly neutral pH. The desired formulation is then provided by sterile filtration of the solution, followed by lyophilization under standard conditions known to those skilled in the art. Generally, the resulting solution can be distributed into vials for lyophilization. Each vial may, for example, contain, non-limiting, a single dose (10-1000 mg, e.g., 100-500 mg) or multiple doses of the preparation. The lyophilized powder can be stored under appropriate conditions, for example, at approximately 4°C to room temperature. Reconstitution of this lyophilized powder with sterile water for injection provides a formulation for parenteral administration.

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

[0105] In another aspect, the present invention provides a method for inhibiting myeloperoxidase activity, comprising the step of administering a therapeutically effective amount of the peptide and / or pharmaceutically active composition of the present invention to a subject in need.

[0106] In another aspect, the present invention provides a method for inhibiting oxidative activity, comprising the step of administering a therapeutically effective amount of the peptide and / or pharmaceutically active composition of the present invention to a subject in need.

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

[0108] Combination therapy Further aspects of the present invention provide a method for modulating the complement system, comprising the step of administering a pharmaceutical composition of the present invention to a subject. The pharmaceutical compositions of the present invention can be administered as a sole active pharmaceutical agent, but 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 includes the step of administering a pharmaceutical composition of the present invention before, simultaneously with, and / or after, one or more further therapeutic or prophylactic agents effective in modulating the complement system.

[0109] The pharmaceutical compositions of the present invention can be administered in combination therapy with further agents, either together or separately, or by combining the pharmaceutical composition and further agents in a single composition. Dosage is administered and adjusted to achieve maximum regulation of the complement system. For example, both the pharmaceutical composition and further agents 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. [Examples]

[0110] The present invention is also described and illustrated through the following embodiments. However, the use of these and other embodiments anywhere in this specification is illustrative only and does not in any way limit the scope and meaning of the present invention or any exemplified terms. Similarly, the present invention is not limited to any particular preferred embodiment described herein. In fact, many modifications and variations of the present invention may become apparent to those skilled in the art by reading this specification, and such variations can be made without departing in spirit or scope from the present invention. Accordingly, the present invention should be limited only by the terms of the appended claims, together with the entire scope of equivalents to which the appended claims are entitled.

[0111] Example 1: Characterization of SEQ ID NO: 6~13 The inventors found that sarcosine amino acid substitution scanning 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 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 their respective assays. Using both the PA-I8Sar and PA-C9Sar peptide backbones, the inventors created peptides with D-amino acid substitutions and / or manipulated stapling that exhibited increased potency in a variety of functional assays and retained water solubility in the absence of pegylation.

[0112] Materials and Reagents The peptides were synthesized to >90% purity by New England Peptide (Gardner, MA) (Table 1). Staple peptides were produced by a one-component stapling technique using either S-pentenylalanine (S5) at the i,i+4 position for one-turn stapling, or a combination of either R-octenylalanine (R8) / S-pentenylalanine (S5) at the i,i+7 position. Since no enantiomers exist, the D-enantiomer forms of each amino acid were 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). Complement-tolerant GVBS was used as the buffer. ++The solution contained a buffer (veronal buffered saline containing 0.1% gelatin, 0.15 mM CaCl2, and 1 mM MgCl2

[12] ).

[0113] method Normal human serum (NHS) As previously described

[12] , normal human serum (NHS) of blood type O was prepared. 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 blood clots and serum. The serum was then pooled, divided into equal portions and frozen at -80°C.

[0114] Complement activity hemolysis assay For the hemolytic complement assay, human erythrocytes (RBCs) from a type AB donor were purified, washed, and 1.0 × 10⁶ cells were prepared as previously described

[13] . 9 The concentration was standardized to cells / ml. Human serum from a type O donor at a final concentration of 15% was mixed with 0.5 mM peptide and prepared as GVBS. ++ and 5.0×10 7 The volume was increased to 0.2 ml using one RBC. The sample was incubated at 37°C for 1 hour, then spun at 3,000 rpm for 5 minutes, the supernatant was collected, and read at 412 nm. The values ​​were GVBS. ++ Expressed as a percentage of positive controls consisting of human O serum and AB erythrocytes in buffer solution.

[0115] C1q binding assay As previously described

[11] , a C1q binding assay was performed. Briefly, Immunlon-2 HB ELISA plates were coated overnight at 4°C with 1 μg / ml C1q in bicarbonate buffer. The plates were washed with PBS-T (phosphate buffered saline + 0.1% Tween) and then blocked at room temperature for 2 hours with 1% gelatin / PBS. After washing, the plates were incubated at room temperature for 1 hour with peptides starting at 2.5 mg / ml and then serially diluted in 1% gelatin / PBS, followed by washing. The plates were then bound with lead peptides lacking pegylation. Rabbit antibodies produced against TIFF2026065064000009.tif4128

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

[0116] MPO activity assay As previously described

[10] , an MPO activity assay was performed. Briefly, the peptide was diluted to 12 mg / ml and titrated continuously in 0.02 ml volumes in a 96-well plate. 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 minutes, followed by the addition of 0.1 ml of 2.5N H2SO4 for a further 2 minutes, and then read at 450 nm using a 96-well plate reader (BioTek).

[0117] Total antioxidant capacity assay As previously reported [6], the antioxidant capacity of PIC1 mutants was measured based on the reduction of copper(II) to copper(I) using the TAC (Total Antioxidant Capacity) assay (Cell Biolabs, Inc, San Diego, CA). The kit protocol was followed 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. In this assay, biotinylated human PD-1 was conjugated to immobilized human PD-L1, and a colorimetric quantitative sandwich ELISA platform was used. Plates were coated with human PD-L1 and then incubated with various PIC1 peptides. Human PD-1-biotin was then conjugated to the coated human PD-L1, followed by the addition of streptavidin-HRP. TMB was then added as a colorimetric quantitative HRP substrate, and the plate wells were analyzed in a plate reader at 450 nm absorbance.

[0119] statistical analysis Quantitative data were analyzed, and the mean, standard error (SEM), and Student's t-test

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

[0120] result peptide In in vitro assays of classical complement pathway, MPO, oxidized derivatives, and NET activity, parent molecule Compared to TIFF2026065064000010.tif4128, it ranks 8th. TIFF2026065064000011.tif4128 and 9th place It has been previously shown that the substitution of isoleucine with sarcosine in TIFF2026065064000012.tif4128 results in a peptide 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 backbone were synthesized, consisting of staple peptides or peptides having D-amino acids individually substituted at each position of the PA-I8Sar and PA-C9Sar peptide sequences (Tables 1 and 2, respectively). One peptide based on the PA-I8Sar backbone included a combination of staple and D-amino acid combinations (PA-0142). Stapling techniques have been shown to enhance the stability and bioactivity of peptides by immobilizing them into bioactive α-helix secondary structures [2], while D-amino acid substitution can provide further stability to native peptides and extend their in vivo half-life

[15] . Each of these peptides is readily soluble in water, and their bioactivity was evaluated using various in vitro assays.

[0121] Complement inhibition and C1q binding To evaluate the extent to which peptide variants inhibit antibody-induced complement activation, an ABO incompatibility ex vivo assay was used, in which purified erythrocytes from "AB+" donors were incubated with serum from an "O" control containing anti-A and anti-B antibodies

[13] . Peptides were tested at a concentration of 0.8 mg / ml (approximately 0.5 mM). For PA-I8Sar backbone-based peptides, the D-amino acid substitution (PA-0122), the staple peptide (PA-0134), and the staple and D-amino acid substitution (PA-0142) peptides PA-0122, PA-0134, and PA-0142 inhibited ABO incompatibility hemolysis to the same extent (PA-0134 and PA-0142) or greater extent (PA-0122) compared to the PA-I8Sar control (Figure 1A).

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

[0123] Astrovirus capsid protein and the PIC1 molecule derived therefrom inhibit the activation of the classical complement pathway by binding to the pattern recognition molecule C1q [3, 10, 11]. The inventors then tested the binding of peptide variants to C1q using an ELISA-type assay, in which C1q was used as a capture substrate and the bound peptide was captured using PA-dPEG24 Detection was performed using a rabbit polyclonal antibody against the peptide portion of TIFF2026065064000013.tif4128

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

[0124] Myeloperoxidase inhibition To confirm the inhibition of MPO activity by various peptides, staple peptides and D-amino acid variants were tested at various concentrations (Figures 3A-3B), and the maximum half-molecular activity level was calculated from the dose-response curves (Figures 6A-6C, respectively). For the PA-I8Sar peptide, PA-0122 and PA-0142 showed similar levels of MPO inhibition, while PA-0134 showed 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, but PA-0150, PA-0152, and PA-0154 showed decreased MPO inhibition (Figure 3B). Therefore, for both PA-I8Sar and PA-C9Sar variants, some peptides showed various effects on MPO binding affinity.

[0125] Antioxidant capacity As previously reported [6], the antioxidant properties of PIC1 mutants were evaluated in a total antioxidant capacity (TAC) assay. Total antioxidant activity was determined over various peptide concentrations for staple peptides and D-amino acid peptides based on the PA-I8Sar and PA-C9Sar skeletons (Figures 4A-4B), and the activity at the highest peptide concentration (1.5 mM) is reported (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 peptides based on the PA-C9Sar peptide showed a decrease in total antioxidant capacity, with the exception of the d-amino acid peptide PA-0164, which showed enhanced activity (Figure 4B). The inventors also investigated the parent PA-dPEG24 peptide We previously showed that both the adjacent cysteine ​​residues at positions 9 and 10 of TIFF2026065064000014.tif4128 are essential for antioxidant activity, and that oxidation of both residues inhibits this function [6]. These data suggest 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 a field of considerable interest in cancer research. PD-1 is one of the most well-characterized checkpoint proteins. The binding of PD-1 to its ligand, PD-L1, suppresses T cell activation, allowing cancer cells to evade the body's immune surveillance. Therefore, pharmacological inhibition of PD-1 or its ligand is considered a promising strategy by many cancer researchers. A commercially available ELISA kit was used to determine whether these PIC1 derivatives could inhibit the interaction of PD-1 with PD-L1. PA-0071 and PA-0088 did not inhibit the binding of PD-1 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 The inventors previously showed that sarcosine substitution of 15 residues of the parent PIC1 molecule (PA-dPEG24) resulted in six peptides that were water-soluble without pegylation and exhibited enhanced activity in functional assays of complement, MPO, NETosis, and oxidative activity

[11] . PA-I8Sar (isoleucine at position 8 substituted with sarcosine) was selected for further modification by stapling and D-amino acid substitution to determine whether its functional activity could be further enhanced in various assays. The inventors performed the same analysis with PA-C9Sar (cysteine ​​at position 9 substituted with sarcosine). PA-C9Sar did not show significant enhancement of activity in various assays compared to PA-I8Sar

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

[11] . The activity of PA-I8Sar and PA-C9Sar mutants in various assays is summarized in Tables 2 and 3.

[0128] (Table 2) Overview of PA-I8Sar peptide and its properties TIFF2026065064000015.tif71162 1 ND: undecided

[0129] (Table 3) Overview of PA-C9Sar peptide and its properties TIFF2026065064000016.tif98161 1 ND: Undecided. Regarding C1q binding, the peptide sequence PA-0168 was not recognized by the 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-standard amino acids can significantly enhance the functional activity of the PIC1 molecule. Of particular interest and surprise was the finding that such modifications could lead to improvements in one or more functional activities of the PIC1 peptide. The ability to isolate peptides with different functional activities could potentially be utilized to target dysregulated complement, neutrophils (MPO and NETosis), or certain inflammatory diseases in which oxidative activity plays a central role in pathogenesis.

[0131] Example 2. Inhibition of PD-1 binding to PD-L1. The immune checkpoint pathway is a field of considerable interest in cancer research. PD-1 is one of the most well-characterized checkpoint proteins. The binding of PD-1 to its ligand, PD-L1, suppresses T cell activation, allowing cancer cells to evade the body's immune surveillance. Therefore, pharmacological inhibition of PD-1 or its ligand is considered a promising strategy by many cancer researchers. A commercially available ELISA kit was used to determine whether these PIC1 derivatives could inhibit the interaction of PD-1 with PD-L1. PA-0071 and PA-0088 did not inhibit the binding of PD-1 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. The inventors further evaluated whether RLS-0134 and RLS-0150 can bind to a well-characterized checkpoint inhibitor, namely cytotoxic T lymphocyte-associated protein 4 (CTLA-4, also known as CD152). Similar to PD1 / PD-L1 interactions, CTLA-4 is a checkpoint protein upregulated on the surface of cancer cells, binding to ligands CD80 or CD86 on the surface of T cells, thereby suppressing T cell activation and allowing cancer cells to evade destruction by the immune system. Therefore, pharmaceutically inhibiting 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, binding assays were performed, in which these proteins were coated onto microtiter plates and then incubated with escalating 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. RLS-0134 showed dose-dependent binding to PD-1, PD-L1, and C1q as expected, 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 blockade bioassay In cell-based assays, selected peptides were screened using the CTLA-4 blocking bioassay (Promega) to evaluate whether PIC1 peptides could block CTLA-4 inhibitory activity. 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 / large cells: large cells expressing engineered cell surface proteins designed to activate allogeneic TCRs in an antigen-independent manner and endogenously expressing CTLA-4 ligands CD80 and CD86. When the two cell types are co-cultured, CTLA-4 competes with CD28 and their common ligands, CD80 and CD86, thereby inhibiting activation of the CD28 pathway and promoter-mediated luminescence. The addition of molecules that block the interaction between CTLA-4 and its ligands CD80 and CD86 induced 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 alloreceptor (Figure 10). PIC1 peptides RLS-0122, RLS-0150, RLS-0154, RLS-0164, and RLS-0168 all showed inhibitory activity in this assay, as indicated by an increase in signal; however, RLS-0088 (negative control) did not show inhibition beyond the background of the blocking bioassay. These data are summarized in Table 4, demonstrating that PIC1 peptides can inhibit the interaction of CTLA-4 with its alloreceptor and functionally inhibit CTLA-4-mediated signaling in cell-based bioassays.

[0134] (Table 4) Overview of PIC1 peptide and its properties TIFF2026065064000017.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 persistent expression of inhibitory receptors (e.g., PD-1, LAG-3, CD244, CD160) accompanied by insufficient effector function, reduced cytokine release (e.g., IL-2, TNF-alpha, IFN-gamma), and progressive loss of effector function due to hyperstimulation. Depletion can disrupt the optimal control of tumor growth. T-cell depletion is widely observed in the tumor microenvironment (TME) and can lead to T-cell apoptosis. T-cell depletion is reversible, and pharmacological 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 can restore 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 re-stimulated every 48 hours. After each Dynabead stimulation, the PIC1 peptide was added to the cells. After 3–4 stimulations, cells were harvested for readout, which consisted of a step to assess T cell apoptosis by measuring caspase 3 / 7 levels and the production of T cell-functional cytokines IL-2 and IFN-gamma. T cells stimulated with beads but not treated with the peptide showed increased caspase 3 / 7 levels, indicating apoptosis, while unstimulated cells showed low levels of caspase 3 / 7 signaling (untreated, Figure 11). T cells treated with PIC1 peptides RLS-0122, RLS-0164, and RLS-0168 showed a decrease in caspase 3 / 7 levels, with some peptides, such as RLS-0122 and RLS-0168, showing caspase 3 / 7 levels ranging from very low to undetectable. In contrast, RLS-0150 and RLS-0154 increased caspase 3 / 7 levels. To further evaluate whether PIC1 peptides can restore T cell functionality in cells subjected to a depletion protocol, the inventors then evaluated the production of cytokines IL-2 and IFN-gamma.Cell-derived supernatants were collected after each stimulation, and cytokines were measured by ELISA. As shown in Figures 12A-12B, cells not treated with the peptide showed spikes of IL-2 or IFN-gamma signaling with Dynabead stimulation 1, and these spikes were undetectable with stimulation 2. In contrast, cells treated with RLS-0164 showed IL-2 and IFN-gamma signaling with stimulation 2. Lower levels of IFN-gamma signaling were consistently observed in this assay. Compared to RLS-0150, which does not inhibit T cell depletion, RLS-0122, RLS-0154, and RLS-0168 all showed detectable IL-2 with stimulation 2 (Figure 12C). IFN-gamma signaling was detectable with stimulation 2 for RLS-0122 and RLS-0168, but undetectable for RLS-0154 (Figure 12D). These data are summarized in Table 4, and the data demonstrate that the PIC1 peptide can inhibit human T cell depletion, as measured by the inhibition of apoptosis markers and the 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 vascular structures, is an essential element of tumor growth and metastasis formation. Inhibition of tumor angiogenesis is considered a primary treatment strategy in oncology. Vascular endothelial growth factor (VEGF) is a potent and specific angiogenic factor and a crucial requirement for tumor growth. VEGF inhibitors, such as monoclonal antibodies, are currently used 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 blood clots, complications in wound healing, and, more rarely, gastrointestinal perforation and gastrointestinal fistula. Therefore, there is a need for safe VEGF inhibitors that are not based on monoclonal antibody technology. The inventors tested whether these PIC1 peptides possess the ability to bind to human VEGF and inhibit VEGF function in cell-based bioassays. To confirm the ability of PIC1 peptides to bind to VEGF, a binding assay was performed in which VEGF was coated onto a microtiter plate and then incubated with PIC1 peptide (1 mg / ml). As shown in Figure 13, PIC1 bound to VEGF at various levels, with RLS-0122 and RLS-0164 binding with high affinity. Next, the inventors used a VEGF bioassay (Promega) to determine whether these PIC1 peptides could functionally inhibit VEGF-mediated cell signaling via its allogeneic 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 readout information. This assay can be used for the discovery and development of novel biological therapies aimed at either inducing or inhibiting the VEGF response. VEGF-responsive cells are engineered to express the upstream response element (RE) of luc2P and the exogenous VEGF receptor. When VEGF binds to VEGF-responsive cells, the receptor transmits an intracellular signal, resulting in luminescence. The bioluminescent signal is detected by a luminometer.As shown in Figure 14, the addition of VEGF incubated with cells without peptides induced a 20,000 relative luminescence unit (RLS) response signal, indicating VEGF binding to VEGFR-2. RLS-0150 and RLS-0154 dose-dependently reduced luminescence, indicating blockade of VEGFR-2-mediated VEGF signaling, while 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 to 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, then PIC1 peptide (10 mg / ml) was added at 37°C for 1 hour, followed by treatment with 10 ug / ml LPS and placement on the extracellular matrix to promote angiogenesis. The cells were incubated overnight at 37°C in a humidified CO2 incubator. Cells were then visualized for tubular formation indicating angiogenesis by fluorescence microscopy. Cells not treated with LPS showed no aggregation or formation of any tube buds, while these structures were evident in LPS-treated cells (Figure 15). In the presence of PIC1 peptides, varying levels of angiogenesis inhibition were observed, and several peptides (RLS-0164 and RLS-0168) did not show 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 human kidney cancer cell line A498 by RLS-0122. Effective therapy for renal clear cell carcinoma (ccRCC) is particularly needed 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 exhibits high expression of multiple components that are part of the classical complement pathway. High density of cells producing classical complement pathway components C1q and C4, and the presence of C4 activating fragment deposition in the primary tumor, often correlate with poor prognosis. Therefore, inhibition of the classical complement pathway may offer a novel therapeutic strategy to potentially reduce 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). The inventors tested the ability of RLS-0122 to block complement activation in an in vitro human renal cancer cell line A498 system, as assessed by the reduction of C4a levels. The A-498 cell line, derived from human renal cancer, produces complement component products C1r and C1s. Functional C1 complexes are created by the addition of purified C1q (Roumenina et al., Cancer Immunol Res; 7(7) July 2019). RLS-0122 was selected and evaluated in this in vitro assay based on its ability to potently block complement activation in a hemolysis assay (Figure 1). Concentrated supernatant (3%) from serum-deficient A-498 cells was added to purified human C1q (0.5 ug / ml) pre-incubated with escalating doses (0-8 mg / ml) of RLS-0122 or RLS-0174 (negative control peptide) and loaded onto IgG-coated plates. Next, the samples were incubated at 37°C for 1 hour. After incubation, the samples were washed three times with PBS-T, and purified human C4 (4 ug / ml) was added. The samples were incubated at 37°C for 1.5 hours. The supernatant was collected for analysis using Quidel's MicroVue complement C4a ELISA.RLS-0122 dose-dependently reduced the level of C4a production, while peptides that do not inhibit classical complement activity (RLS-0174) showed no significant inhibitory activity (Figure 16).

[0139] Example 9. RLS-0122 enhances survival and quality of life in a mouse TC-1 tumor cell model. To evaluate the efficacy of RLS-0122 in vivo, we utilized a 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 lung epithelial cell line transformed with human papillomavirus, and complement activation has been shown to contribute to tumor growth. When TC-1 cells are introduced into the flanks of mice, highly aggressive tumor formation occurs, with 100% death within approximately 26 days. In this model, to test the efficacy of RLS-0122 on survival and quality of life, animals were subcutaneously injected with 4 × 10^5 TC-1 cells into their flanks. After 5 days, animals were given either a vehicle treatment with saline (n=6) or IV drug treatment with 160 mg / kg 1 × / day of RLS-0122 (n=8) for 15 consecutive days. Animals treated with RLS-0122 showed a significantly longer median survival time of 8 days compared to those treated with a vehicle (p=0.0465) (Figure 17A). Behavioral and physical status scoring was also assessed every two days throughout the study. If the score reached 7 or below, the animal was assessed daily. A score of 5 or below was considered the endpoint for euthanasia. Animals treated with RLS-0122 showed an improved quality of life after the end of treatment, and the score reached statistical significance between days 22 and 26 (p<0.05) (Figures 17A-17B).

[0140] Example 10: Administration of pharmaceutical composition To modulate the complement system, a pharmaceutical composition containing a therapeutically effective amount of any of SEQ ID NO: 6-13 or their variants is administered to the subject in need.

[0141] To inhibit myeloperoxidase activity, a pharmaceutical composition containing a therapeutically effective amount of any of SEQ ID NO: 6-13 or their variants is administered to the subject in need.

[0142] To inhibit oxidative activity, a pharmaceutical composition containing a therapeutically effective amount of any of SEQ ID NO: 6-13 or their variants is administered to the subject in need.

[0143] To inhibit the binding of PD-1 to PD-L1, a pharmaceutical composition containing a therapeutically effective amount of any of SEQ ID NO: 6-13 or their variants is administered to the subject in need.

[0144] To inhibit T cell depletion, a pharmaceutical composition containing a therapeutically effective amount of SEQ ID NO: 6-13 and any of their variants is administered to subjects in need.

[0145] To inhibit angiogenesis, a pharmaceutical composition containing a therapeutically effective amount of any of SEQ ID NO: 6-13 or their variants is administered to the subject in need.

[0146] List of types The following is a non-exhaustive list of embodiments provided by the present invention: 1. A synthetic peptide containing at least approximately 95% sequence identity with an amino acid sequence selected from the group with SEQ ID NO: 6-13. 2. A synthetic peptide according to embodiment 1, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 6 to 13. 3. A synthetic peptide according to embodiment 1, which has at least approximately 95% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 7 to 11. 4. A synthetic peptide according to embodiment 1, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 7 to 11. 5. The synthetic peptide according to claim 1, comprising an amino acid sequence having at least about 95% sequence identity with respect to SEQ ID NO:6. 6. The synthetic peptide according to claim 1, comprising an amino acid sequence having at least about 95% sequence identity with respect to SEQ ID NO:7. 7. The synthetic peptide according to claim 1, comprising an amino acid sequence having at least about 95% sequence identity with respect to SEQ ID NO:8. 8. The synthetic peptide according to claim 1, comprising an amino acid sequence having at least about 95% sequence identity with respect to SEQ ID NO:9. 9. The synthetic peptide according to claim 1, comprising an amino acid sequence having at least about 95% sequence identity with respect to SEQ ID NO:10. 10. The synthetic peptide according to claim 1, comprising an amino acid sequence having at least approximately 95% sequence identity with SEQ ID NO:11. 11. The synthetic peptide according to claim 1, comprising an amino acid sequence having at least about 95% sequence identity with respect to SEQ ID NO:12. 12. The synthetic peptide according to claim 1, comprising an amino acid sequence having at least about 95% sequence identity with SEQ ID NO:13. 13. A pharmaceutical composition comprising a therapeutically effective amount of any synthetic peptide according to claims 1 to 12 and at least one pharmaceutically acceptable carrier, diluent, or excipient. 14. A method for modulating a complement system, comprising the step of administering the pharmaceutical composition of claim 13 to a subject in need thereof. 15. A method for inhibiting myeloperoxidase activity, comprising the step of administering the pharmaceutical composition of claim 13 to a subject in need thereof. 16. A method for inhibiting oxidative activity, comprising the step of administering the pharmaceutical composition of claim 13 to a subject in need thereof. 17. A method for inhibiting the binding of PD-1 to PD-L1, comprising the step of administering the pharmaceutical composition of claim 13 to a subject in need thereof. 18. A method for inhibiting T cell depletion, comprising the step of administering the pharmaceutical composition of claim 13 to a subject in need thereof. 19. A method for inhibiting angiogenesis, comprising the step of administering the pharmaceutical composition of claim 13 to a subject in need thereof.

[0147] Table of arrays TIFF2026065064000018.tif203161

[0148] While several possible embodiments are disclosed above, the embodiments of the present invention are not limited in this way. These exemplary embodiments are not intended to be exhaustive or to unnecessarily limit the scope of the invention, but rather have been selected and described to illustrate the principles of the invention so that others skilled in the art can carry it out. Indeed, it is expected that 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, documents, and other materials cited herein are incorporated herein by reference in their entirety as if they were physically present within this specification.

[0150] References TIFF2026065064000019.tif27150TIFF2026065064000020.tif229151TIFF2026065064000021.tif27150

[0151] Sequence information SEQUENCE LISTING <110> REALTA LIFE SCIENCES, INC. <120> PEPTIDES AND METHODS OF USE <150> US 63 / 108,762 <151> 2020-11-02 <160> 13 <170> PatentIn version 3.5 <210> 1 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 1 Pro Ala Ile Cys Gln Arg Ala Thr Ala Thr Leu Gly Thr Val Gly Ser 1 5 10 15 Asn Thr Ser Gly Thr Thr Glu Ile Glu Ala Cys Ile Leu Leu 20 25 30 <210> 2 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 2 Ile Ala Leu Ile Leu Glu Pro Ile Cys Cys Gln Glu Arg Ala Ala 1 5 10 15 <210> 3 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 3 Ile Ala Leu Ile Leu Glu Pro Ile Cys Cys Gln Glu Arg Ala Ala 1 5 10 15 <210> 4 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (8)..(8) <223> Sarcosine <400> 4 Ile Ala Leu Ile Leu Glu Pro Xaa Cys Cys Gln Glu Arg Ala Ala 1 5 10 15 <210> 5 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (9)..(9) <223> Sarcosine <400> 5 Ile Ala Leu Ile Leu Glu Pro Ile Xaa Cys Gln Glu Arg Ala Ala 1 5 10 15 <210> 6 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (3)..(3) <223> D-amino acid <220> <221> MOD_RES <222> (8)..(8) <223> Sarcosine <400> 6 Ile Ala Leu Ile Leu Glu Pro Xaa Cys Cys Gln Glu Arg Ala Ala 1 5 10 15 <210> 7 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (3)..(3) <223> (R)-2-(7'-octenyl)alanine <220> <221> SITE <222> (3)..(10) <223> Stapled <220> <221> MOD_RES <222> (8)..(8) <223> Sarcosine <220> <221> MOD_RES <222> (10)..(10) <223> (S)-2-(4'-pentenyl)alanine <400> 7 Ile Ala Ala Ile Leu Glu Pro Xaa Cys Ala Gln Glu Arg Ala Ala 1 5 10 15 <210> 8 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (3)..(3) <223> D-amino acid <220> <221> MOD_RES <222> (8)..(8) <223> Sarcosine <220> <221> MOD_RES <222> (10)..(10) <223> (S)-2-(4'-pentenyl)alanine <220> <221> SITE <222> (10)..(14) <223> Stapled <220> <221> MOD_RES <222> (14)..(14) <223> (S)-2-(4'-pentenyl)alanine <400> 8 Ile Ala Leu Ile Leu Glu Pro Xaa Cys Ala Gln Glu Arg Ala Ala 1 5 10 15 <210> 9 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (7)..(7) <223> (R)-2-(7'-octenyl)alanine <220> <221> SITE <222> (7)..(14) <223> Stapled <220> <221> MOD_RES <222> (9)..(9) <223> Sarcosine <220> <221> MOD_RES <222> (14)..(14) <223> (S)-2-(4'-pentenyl)alanine <400> 9 Ile Ala Leu Ile Leu Glu Ala Ile Xaa Cys Gln Glu Arg Ala Ala 1 5 10 15 <210> 10 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (2)..(2) <223> (S)-2-(4'-pentenyl)alanine <220> <221> SITE <222> (2)..(6) <223> Stapled <220> <221> MOD_RES <222> (6)..(6) <223> (S)-2-(4'-pentenyl)alanine <220> <221> MOD_RES <222> (9)..(9) <223> Sarcosine <400> 10 Ile Ala Leu Ile Leu Ala Pro Ile Xaa Cys Gln Glu Arg Ala Ala 1 5 10 15 <210> 11 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (7)..(7) <223> (S)-2-(4'-pentenyl)alanine <220> <221> SITE <222> (7)..(11) <223> Stapled <220> <221> MOD_RES <222> (9)..(9) <223> Sarcosine <220> <221> MOD_RES <222> (11)..(11) <223> (S)-2-(4'-pentenyl)alanine <400> 11 Ile Ala Leu Ile Leu Glu Ala Ile Xaa Cys Ala Glu Arg Ala Ala 1 5 10 15 <210> 12 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (9)..(9) <223> Sarcosine <220> <221> MOD_RES <222> (10)..(10) <223> D-amino acid <400> 12 Ile Ala Leu Ile Leu Glu Pro Ile Xaa Cys Gln Glu Arg Ala Ala 1 5 10 15 <210> 13 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptides <220> <221> MOD_RES <222> (9)..(9) <223> Sarcosine <220> <221> MOD_RES <222> (14)..(14) <223> D-amino acid <400> 13 Ile Ala Leu Ile Leu Glu Pro Ile Xaa Cys Gln Glu Arg Ala Ala 1 5 10 15

Claims

1. A synthetic peptide containing at least approximately 95% sequence identity with an amino acid sequence selected from the group with SEQ ID NO: 6-13.

2. The synthetic peptide according to claim 1, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 6 to 13.

3. The synthetic peptide according to claim 1, which has at least about 95% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 7 to 11.

4. The synthetic peptide according to claim 1, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 7 to 11.

5. The synthetic peptide according to claim 1, comprising an amino acid sequence having at least about 95% sequence identity with respect to SEQ ID NO:

6.

6. The synthetic peptide according to claim 1, comprising an amino acid sequence having at least about 95% sequence identity with SEQ ID NO:

7.

7. The synthetic peptide according to claim 1, comprising an amino acid sequence having at least about 95% sequence identity with respect to SEQ ID NO:

8.

8. The synthetic peptide according to claim 1, comprising an amino acid sequence having at least about 95% sequence identity with respect to SEQ ID NO:

9.

9. The synthetic peptide according to claim 1, comprising an amino acid sequence having at least about 95% sequence identity with respect to SEQ ID NO:

10.

10. The synthetic peptide according to claim 1, comprising an amino acid sequence having at least about 95% sequence identity with SEQ ID NO:

11.

11. The synthetic peptide according to claim 1, comprising an amino acid sequence having at least about 95% sequence identity with SEQ ID NO:

12.

12. The synthetic peptide according to claim 1, comprising an amino acid sequence having at least about 95% sequence identity with SEQ ID NO:

13.

13. A pharmaceutical composition comprising a therapeutically effective amount of the synthetic peptide described in any one of claims 1 to 12 and at least one pharmaceutically acceptable carrier, diluent, or excipient.

14. A method for regulating a complement system, comprising the step of administering the pharmaceutical composition according to claim 13 to a subject in need thereof.

15. A method for inhibiting myeloperoxidase activity, comprising the step of administering the pharmaceutical composition according to claim 13 to a subject in need thereof.

16. A method for inhibiting oxidative activity, comprising the step of administering the pharmaceutical composition according to claim 13 to a subject in need thereof.

17. A method for inhibiting the binding of PD-1 to PD-L1, comprising the step of administering the pharmaceutical composition according to claim 13 to a subject in need thereof.

18. A method for inhibiting T cell depletion, comprising the step of administering the pharmaceutical composition according to claim 13 to a subject in need thereof.

19. A method for inhibiting angiogenesis, comprising the step of administering the pharmaceutical composition according to claim 13 to a subject in need thereof.

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