JP2023145635A - Peptides having immunomodulatory properties - Google Patents

Abstract

To provide novel peptides having immunomodulatory activities in vivo and in vitro.SOLUTION: The peptides can include a particular striapathic region of alternating hydrophilic and hydrophobic modules that can adopt an amphipathic conformation under physiological conditions. This disclosure provides peptides that can specifically bind to key functional regions on one or more signaling proteins, in particular pro-inflammatory cytokines, macrophage inhibition proteins, and histone regulation proteins. This disclosure includes peptides that are sufficiently stable in the circulation to allow for intravenous administration. Pharmaceutical compositions including the subject peptides are also provided. The subject peptides find use in methods of modulating macrophage activity. In some cases, the peptide is a CD206-binding agent. Also provided are methods of treating a subject for a condition associated with chronic inflammation using the peptides and compositions of this disclosure.SELECTED DRAWING: None

Description

Acute inflammation is a tissue's initial response to noxious stimuli. It is a complex process that begins when cells present within the injured tissue, including macrophages, dendritic cells, histiocytes, Kupffer cells, and mast cells, sense damage-associated molecules and become activated. involves a highly controlled process. When activated, these cells release inflammatory mediators such as vasodilators. Vasodilators induce an increase in blood flow and vascular permeability in the vicinity of the injury. This, in turn, results in increased migration of plasma and white blood cells (including neutrophils and macrophages) from the blood to the injured tissue. Since inflammatory mediators are generally rapidly degraded, constant stimulation is required to sustain acute inflammation. As a result, acute inflammation ends when the noxious stimulus is removed.

A variety of agents, including but not limited to bacteria, viruses, physical injury, chemical injury, cancer, chemotherapy, and radiation therapy, may be affected depending on the specific agent and genetic makeup of the animal exposed to it. May cause long-term and excessive inflammation. Such inflammation, known as chronic inflammation, contributes to many widespread and debilitating diseases, including heart disease, cancer, respiratory disease, stroke, neurological diseases such as Alzheimer's disease, diabetes, and kidney disease. This is thought to be a contributing factor. The result of chronic inflammation is the destruction of normal tissue and its replacement with collagen-rich connective tissue. Collagen-rich connective tissue, also known as scar tissue, exhibits reduced tissue function compared to normal tissue. In turn, persistent and long-term formation of scar tissue leads to fibrosis. Fibrosis is one of the common symptoms of diseases that affect the lungs, skin, liver, heart, and bone marrow, including idiopathic pulmonary fibrosis, scleroderma, keloids, cirrhosis, myocardial fibrosis, It is an important factor in diseases such as diabetic kidney disease, myelodysplastic syndromes, and other diseases.

Studies of chronic inflammation and fibrosis have shown that a common network of signaling proteins tends to function together to establish a pro-inflammatory state, regardless of the activator and the tissue affected. This network of signaling proteins includes several different cytokines, cytokine receptors, transcription factors, and microRNAs, including TGFβ, TGFβRII, and miRNA19b. Therefore, therapeutic agents that reduce inflammation without harmful side effects are of great interest.

Novel peptides are provided that have immunomodulatory activity in vitro and in vivo. The peptides may contain specific striapathic regions of alternating hydrophilic and hydrophobic modules that are capable of adopting an amphipathic conformation under physiological conditions. The peptides can specifically bind to important functional regions on one or more signaling proteins, specifically pro-inflammatory cytokines, macrophage inhibitory proteins, and/or histone regulatory proteins. The present disclosure includes peptides that are sufficiently stable in the circulation in vivo after administration to a subject. Pharmaceutical compositions containing the subject peptides are also provided.

The subject peptides find use in methods of modulating macrophage activity. In some cases, the peptide is a CD206 binding agent. Also provided are methods of treating a subject for conditions associated with chronic inflammation using the peptides and compositions of the disclosure.

The features and advantages of the compositions and methods of the invention are set forth in or more fully apparent in the following description and appended claims. For example, suitable immunomodulatory polypeptides can be identified by using the formulas and sequences described herein. Additionally, the features and advantages of the described compositions and methods can be learned by practicing the methods or become apparent from the description.

Figure 3 shows a graph of the results of bleomycin-induced pulmonary fibrosis reduction in a mouse model of pulmonary fibrosis. Fibrosis measurement is Ashcroft score after trichrome staining. Collagen score is a quantitative measurement after hydroxyproline staining. Further details are presented in the Examples below. We demonstrate that the exemplary peptides of interest cooperate with PD-1 checkpoint inhibitors to reduce tumor volume in a mouse tumor inhibition model. Further details are presented in the Examples below. We demonstrate that exemplary peptides RP832C and RP837 reduce macrophage survival in human scleroderma patient samples. Macrophage samples were evaluated after 96 hours of incubation with various concentrations of peptide. Figure 3 shows the characterization of peptides RP832C and RP837 in samples from healthy controls with low arginase: IFNg (interferon-gamma) ratio. Figure 2 shows the characterization of peptides RP832C and RP837 in macrophage samples of scleroderma patients with high arginase: IFNg ratios.

The following description provides specific details to provide a thorough understanding of the invention. However, to avoid obscuring aspects of the described immunomodulatory peptides and related methods of treating a subject, well-known structures, materials, processes, techniques, and operations are not shown or described in detail. is not listed. Additionally, one of skill in the art will understand that the described immunomodulatory peptides and related methods of treating a subject can be implemented and used without these specific details. Indeed, the described immunomodulatory peptides and methods can be practiced by modifying the peptides, compositions, kits, and methods illustrated and with other methods, treatments, devices, and techniques conventionally used. Can be used in combination.

Immunomodulatory Polypeptides As summarized above, the present disclosure provides immunomodulatory peptides, specifically peptides with immunosuppressive properties, and the use of such immunomodulatory peptides in subjects, specifically persistent or chronic and a subject suffering from or at risk of developing a medical condition associated with inflammation. The terms "immunomodulatory" and "immunomodulation" are used interchangeably herein. In some cases, the immunomodulatory peptides described herein may be referred to as anti-inflammatory peptides, and vice versa. In certain cases, an immunomodulatory peptide (eg, as described herein) is an anti-inflammatory peptide, eg, the peptide has at least one anti-inflammatory property.

Certain aspects of subject immunomodulatory polypeptides that can be applied to or adapted for use with the peptides of the present disclosure are described by Jaynes et al. in WO2016/061133, the disclosure of which is incorporated herein by reference. is incorporated herein in its entirety.

The terms "peptide" and "polypeptide" are used interchangeably herein to refer to a polymer constructed from amino acid residues. The term "amino acid residue" as used herein refers to any naturally occurring amino acid, non-naturally occurring amino acid, or amino acid mimetic (such as a peptoid monomer). Amino acid residues can be in the L or D form.

The present disclosure includes immunomodulatory peptides having a striapathic region that makes up at least 25% of the length of the polypeptide and at least one immunomodulatory property. The term "striapathic region" refers to a region or portion of a peptide sequence that consists of a sequence of alternating hydrophobic and hydrophilic modules. A "hydrophobic module" is defined as one to five (e.g., one to three or one to two) hydrophobic amino acid residues, e.g., 1, 2, 3, 4, or 5 hydrophobic amino acid residues. This is the peptide sequence. A "hydrophilic module" is comprised of 1 to 5 (e.g., 1 to 3 or 1 to 2) hydrophilic amino acid residues, e.g., 1, 2, 3, 4, or 5 hydrophilic amino acid residues. This is the peptide sequence.

Thus, a striapathic region can be represented by the formula (X _1-5 J _1-5 ) _n or (J _1-5 X _1-5 ) _n , where each X is a hydrophilic amino acid residue. , each J represents a hydrophobic amino acid residue, and each n is an integer from 1 to 10, such as 2 to 10, 2 to 8, 3 to 8, 4 to 8, or 5 to 10. As described in further detail below, embodiments of the present disclosure include immunomodulatory peptides having striapathic regions with a certain degree of cationic charge. The immunomodulatory peptides of the present disclosure can include a striapathic region with a cationic surface. In certain embodiments, the striapathic region has a cationic charge (ie, a charge greater than 0, eg, +1, +2, +3, +4, +5, +6 or more). In certain embodiments, the immunomodulatory peptide includes a tail region (eg, a hydrophobic tail sequence). In certain embodiments, the immunomodulatory peptide comprises two or more striapathic regions. In such embodiments, the two amphipathic regions of the peptide are in dimeric form, and the two amphipathic regions may have the same or different amino acid sequences. (ie, may be a homodimer or a heterodimer). In certain embodiments, two (or more) striapathic regions are connected via a linker or connecting region. A linker can be a contiguous (or inline) amino acid sequence or a non-amino acid moiety, as desired.

Hydrophobic amino acid residues, for example, are characterized by side group groups that have chemical or physical properties that are primarily nonpolar in the environment, eg, physiological conditions, in which the peptide finds use. Such hydrophobic amino acid residues may be naturally occurring or non-naturally occurring. Hydrophobic amino acid residues can be mimetics of naturally occurring amino acids characterized by side group groups with primarily nonpolar chemical or physical properties. Conversely, hydrophilic amino acid residues, for example, are characterized by side group groups that are predominantly polar (eg, charged or neutral hydrophilic) in the environment, eg, physiological conditions, in which the peptide finds use. Such hydrophilic amino acid residues may be naturally occurring or non-naturally occurring. Hydrophilic amino acid residues can be mimetics of naturally occurring amino acids characterized by side chain groups that are primarily hydrophilic (charged or neutral polar). Examples of hydrophilic and hydrophobic amino acid residues are shown in Table 1 below. Suitable non-naturally occurring amino acid residues and amino acid mimetics are known in the art. For example, Liang et al. (2013), “An Index for Characterization of Natural and
See Non-Natural Amino Acids for Peptidomimetics, "PLoS ONE8(7): e67844.

Although most amino acid residues can be considered either hydrophobic or hydrophilic, some can behave as either hydrophobic or hydrophilic, depending on their context. For example, due to their relatively weak nonpolar character, glycine, proline, serine, and/or cysteine may function as hydrophilic amino acid residues. Conversely, due to their bulky and slightly hydrophobic side chains, histidine and arginine may function as hydrophobic amino acid residues.

The term "anti-inflammatory properties" as used herein refers to reducing or inhibiting, or expected to reduce or inhibiting, pro-inflammatory signals mediated by a protein target and/or Refers to any property of a polypeptide that can be evaluated in silico, in vitro, and/or in vivo that reduces or inhibits inflammation. The term "immunomodulatory property," as used herein, modulates or is capable of modulating the expression or secretion of one or more cytokines involved in autoimmunity and/or immune responses to infectious agents. Refers to any property of a polypeptide that can be predicted and evaluated in silico, in vitro, and/or in vivo or by modulating one or more components of a cytokine signaling pathway.

Selected Immunomodulatory Peptides of Interest The exemplary immunomodulatory peptide sequences described herein are merely examples and are not the only immunomodulatory polypeptides provided herein. Indeed, fragments and variants of the disclosed peptide sequences are also within the scope of this disclosure.

The present disclosure provides immunomodulatory polypeptides, sometimes referred to as "RP peptides," that meet one or more of the structural formulas described below. The present disclosure also provides immunomodulatory polypeptides that share a minimal degree of homology with any of the exemplary RP peptides disclosed herein, or variants thereof, or fragments thereof. Thus, a peptide or polypeptide of the present disclosure satisfies one of the formulas described herein or shares a minimal degree of homology with any of the exemplary RP peptides disclosed herein. It is an immunomodulatory peptide.

A "fragment" of the invention includes at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous amino acid residues (or up to one less than the number of amino acid residues of the subject peptide) and retains at least one immunomodulatory property of the subject peptide. Thus, fragments of the invention may have one, two, three, four, or more amino acids removed from the N-terminus and/or C-terminus compared to the parent immunomodulatory peptide disclosed herein. Contains the missing peptide.

A "variant" of the invention is a polypeptide that is substantially similar to a polypeptide disclosed herein and retains at least one immunomodulatory property of the subject polypeptide. Variants include deletions (i.e., truncation) of one or more amino acid residues at the N-terminus or C-terminus of the subject polypeptides disclosed herein. deletions and/or additions of one or more amino acid residues at one or more internal sites in and/or one or more amino acid residues at one or more positions in the subject polypeptides disclosed herein. Substitutions of (eg, one, two, three, or even more) amino acid residues may be included. For subject polypeptides that are 12 amino acid residues or less in length, the variant polypeptide may contain up to 3 (e.g., 3, 2, 1 (one or none) deleted amino acid residues.

Accordingly, the present invention provides polypeptides that are at least 50% identical (i.e., at least 50% sequence identical) to any one of the immunomodulatory polypeptides disclosed in the tables disclosed herein (e.g., Table 3). (e.g., at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or more) and still retain at least one immunomodulatory property. do. Sequence identity is based on a comparison of two peptide sequences or fragments thereof of the same or similar length.

Thus, in certain embodiments, the present disclosure provides 1 to 10 amino acid differences (e.g., 10 or less, 9 or less, 8 or less) to any one of the polypeptides disclosed herein. 1, 7, 6, 5, 4, 3, 2, or 1 amino acid difference) and still retains at least one immunomodulatory property. provides a polypeptide. "Amino acid difference" as used herein includes amino acid substitutions, amino acid insertions, terminal amino acid additions, amino acid deletions, terminal amino acid truncations, or any combination thereof. Differences between the striapathic region of a homologous immunomodulatory polypeptide and any one of the immunomodulatory polypeptides in Table 3 include the deletions, additions, and/or additions of amino acid residues discussed herein. or may include substitutions. The substituted amino acid residue may be unrelated (e.g., unrelated in terms of hydrophobicity/hydrophilicity, size, charge, polarity, etc.) to the amino acid residue being replaced, or the substituted amino acid residue may be may constitute similar, conservative, or highly conservative amino acid substitutions. As used herein, "similar," "conservative," and "highly conservative" amino acid substitutions are defined as shown in Table 2 below. Determination of whether amino acid residue substitutions are similar, conservative, or highly conservative is important in peptide backbones that can be modified to increase peptide stability, as discussed below. rather than based solely on the side chains of amino acid residues.

Certain immunomodulatory peptides of interest, as well as fragments and variants thereof that find use in the subject pharmaceutical compositions and methods, are described in more detail below. In certain cases, the subject immunomodulatory peptides have macrophage modulating activity.

The "length" of a polypeptide is the number of amino acid residues joined end-to-end that make up the polypeptide, excluding any non-peptide linkers and/or modifications that the polypeptide may contain. In some embodiments, the peptide is 5-30 amino acid residues long (e.g., 5-25, 10-20, or 5-18, 5-12, or 5-10, or 6-30, 6- 25, 6-20, 6-18, 6-12, 6-10, or 7-12, or 7-10 amino acid residues long) and under physiological conditions (e.g., as described herein) It contains striapathic regions of alternating hydrophilic and hydrophobic modules that adopt an amphiphilic conformation. In some embodiments, the peptide is 5 to 12 amino acid residues long (e.g., 6, 7, 8, 9, or 10 amino acid residues long) and has an amphipathic conformation under physiological conditions. It employs striapathic regions of alternating hydrophilic and hydrophobic modules. In certain instances, the striapathic region of the peptide is 5 to 18 amino acid residues long (e.g., 6 to 18, 6 to 14, 6 to 12, 7 to 12, or 5, 6, 7, 8, 9 , 10, 11, or 12 amino acids in length), and the peptide is optionally further modified (eg, as described herein). The striapathic region comprises two or more (e.g., three or more or four or more) hydrophobic modules and one or more (e.g., two or more) hydrophobic modules (e.g., each containing at least one cationic residue). , three or more, or four or more) hydrophilic modules. In some embodiments, a subject immunomodulatory peptide (eg, as described herein) is a CD206-binding peptide. In some cases, the striapathic region of the peptide has a length of 6-12 amino acid residues, such as 7-12 amino acid residues. In some cases, the striapathic region of the peptide has a length of 6-10 amino acid residues.

The hydrophobic module may consist of any convenient residues. In certain cases, the hydrophobic module includes amino acid residues selected from phenylalanine, tryptophan, alanine, valine, and glycine. The striapathic region may contain a total of one, two or more cationic amino acid residues, such as three or more, four or more, five or more, six or more, or even more. The immunomodulatory peptides may contain two, three or more hydrophilic modules of any convenient residues. In some cases, the hydrophilic module includes amino acid residues selected from lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, and glutamine.

In the formulas described herein, J(N) is used to refer to a particular hydrophobic module, where N indicates position in linear form. Similarly, X(N) is used to refer to a particular hydrophilic module, where N indicates the position in linear form.

In the formulas described herein, J _(nx) is used to refer to a particular hydrophobic amino acid residue, where n indicates in which module the residue is located and x is , indicating its position in the module. Similarly, X _(nx) is used to refer to a particular hydrophilic amino acid residue, where n indicates in which module the residue is located and x indicates its position in the module. show.

In the particular case of the present immunomodulatory peptide, the striapathic region includes hydrophobic and hydrophilic modules with the following formula:
[J1]-[X1]-[J2] (Formula 1)

In some embodiments of the present immunomodulatory peptides, the striapathic region includes the following formula of hydrophilic and hydrophobic modules:
[J1]-[X1]-[J2]-[X2] (Formula 2)

In some embodiments of the present immunomodulatory peptides, the striapathic region includes the following formula of hydrophilic and hydrophobic modules:
[X1]-[J1]-[X2]-[J2] (Formula 3)

In some embodiments of the present immunomodulatory peptides, the striapathic region includes the following formula of hydrophobic and hydrophilic modules:
[J1]-[X1]-[J2]-[X2]-[J3] (Formula 4)

In certain embodiments, the striapathic region includes three or more hydrophilic modules and three or more hydrophobic modules, and includes one of the following formulas:
[J1]-[X1]-[J2]-[X2]-[J3]-[X3] (Formula 5)
[J1] - [X1] - [J2] - [X2] - [J3] - [X3] - [J4] (Formula 6)

In certain embodiments, the striapathic region includes three or more hydrophilic modules and three or more hydrophobic modules and includes the formula: ,
[X1]-[J1]-[X2]-[J2]-[X3]-[J3] (Formula 7)

In some cases of Equation 1, the striapathic region has an arrangement defined by one of the following equations:
[J _1a J _1b ]-[X _1a X _1b ]-[J _2a J _2b ] (Formula 1A)
[J _2b J _2a ]-[X _1b X _1a ]-[J _1b J _1a ] (Formula 1B)
During the ceremony,
J _1a , J _1b , J _2a , and J _2b are each independently selected from hydrophobic amino acid residues (e.g., phenylalanine, tryptophan, and valine);
X _1a and X _1b are each independently selected from hydrophilic amino acid residues (eg, lysine or arginine).

In some instances of Formula 1A, the peptide comprises the sequence FWKRFV(RP837N) (SEQ ID NO: 5), or a fragment or variant thereof (eg, a variant containing one substitution).

In some embodiments of Equation 2, the striapathic region has an arrangement defined by the following equation:
[J _1a J _1b ]-[X _1a X _1b ]-[J _2a ]-[X _2a ] (Formula 2A)
During the ceremony,
J _1a , J _1b , and J _2a are each independently selected from hydrophobic amino acid residues (e.g., phenylalanine, tryptophan, or valine);
X _1a , X _1b , and X _2a are each independently selected from hydrophilic amino acid residues (eg, lysine or arginine).

In some instances of Formula 2A, the peptide comprises the sequence FVRKWR (RP837C ¹ ) (SEQ ID NO: 6), or a fragment or variant thereof (eg, a variant containing one substitution).

In some embodiments of Equation 3, the striapathic region has an arrangement defined by the following equation:
[X _1a X _1b ]-[J _1a J _1b J _1c J _1d ]-[X _2a X _2b ]-[J _2a J _2b ] (Formula 3A)
During the ceremony,
J _1a , J _1b , J _1c , J _1d , J _2a , and J _2b are each independently selected from hydrophobic amino acid residues (e.g., leucine, serine, alanine, or phenylalanine);
X _1a , X _1b , X _2a , and X _2b are each independently selected from hydrophilic amino acid residues (eg, glutamic acid, aspartic acid, lysine, asparagine, or arginine).

In some embodiments of Equation 3A, the striapathic region has an arrangement defined by the following equation:
EX _1b LSAFX _2a NJ _2a J _2b (SEQ ID NO: 25)
During the ceremony,
J _2a and J _2b are each independently selected from alanine and phenylalanine;
X _1b and X _2a are each independently selected from lysine and arginine.

In some instances of Formula 3A, the peptide comprises the sequence EKLSAFRNFF (RP843) (SEQ ID NO: 9), or a fragment or variant thereof (eg, a variant containing one or two substitutions).

In the particular case of Equation 4, the striapathic region has an arrangement defined by one of the following equations:
[J _1a J _1b ]-[X _1a X _1b ]-[J _2a J _2b ]-[X _2a X _2b ]-[J _3a J _3b ] (Formula 4A)
[J _3a J _3b ]-[X _2a X _2b ]-[J _2b J _2a ]-[X _1b X _1a ]-[J _1b J _1a ] (Formula 4B)
During the ceremony,
J _1a , J _1b , J _2a , J _2b , J _3a , and J _3b are each independently selected from hydrophobic amino acid residues (e.g., phenylalanine, tyrosine, isoleucine, or leucine);
X _1a , X _1b , X _2a , and X _2b are each independently selected from hydrophilic amino acid residues (eg, lysine or arginine).

In some embodiments of Equations 4A-4B, the striapathic region has an arrangement defined by the following equations.
LJ _1b KKIIKKJ _3a L (SEQ ID NO: 26)
where J _1b and J _3a are independently phenylalanine, tyrosine, or leucine (eg, tyrosine or leucine).

In some instances of Formulas 4A-4B, the peptide comprises the sequence LYKKIIKKLL (RP846) (SEQ ID NO: 12), or a fragment or variant thereof (eg, a variant containing one or two substitutions).

In some embodiments of Equation 4, the striapathic region has an arrangement defined by the following equation:
[J _1a J _1b J _1c ]-[X _1a ]-[J _2a J _2b ]-[X _2a X _2b ]-[J _3a J _3b ] (Formula 4C)
During the ceremony,
J _1a , J _1b , J _1c , J _2a , J _2b , J _3a , and J _3b are each independently selected from hydrophobic amino acid residues (e.g., phenylalanine, tyrosine, or proline);
X _1a , X _2a , and X _2b are each independently selected from hydrophilic amino acid residues (eg, aspartic acid, lysine, or arginine).

In some embodiments of Equation 4C, the striapathic region has an arrangement defined by the following equation:
FYPDJ _2a J _2b X _2a X _2b J _3a J _3b (SEQ ID NO: 27)
During the ceremony,
J _2a , J _2b , J _3a , and J _3b are each independently phenylalanine or tyrosine (e.g., phenylalanine);
X _2a and X _2b are each independently lysine or arginine.

In some instances of Formula 4C, the peptide comprises the sequence FYPDFFKKFF (RP844) (SEQ ID NO: 10), or a fragment or variant thereof (eg, a variant containing one or two substitutions).

In some embodiments of Equation 4, the striapathic region has an arrangement defined by the following equation:
[J _1a J _1b ]-[X _1a X _1b ]-[J _2a ]-[X _2a X _2b X _2c ]-[J _3a J _3b ] (Formula 4D)
During the ceremony,
J _1a , J _1b , J _2a , J _3a , and J _3b are each independently selected from hydrophobic amino acid residues (e.g., phenylalanine, serine, glycine, or isoleucine);
X _1a , X _1b , X _2a , X _2b , and X _2c are each independently selected from hydrophilic amino acid residues (eg, glutamic acid, aspartic acid, lysine, or arginine).

In some embodiments of Equation 4D, the striapathic region has an arrangement defined by the following equation:
J _1a J _1b X _1a X _1b SKEKIG (SEQ ID NO: 28)
During the ceremony,
J _1a and J _1b are each independently phenylalanine or tyrosine (e.g., phenylalanine);
X _1a and X _1b are each independently lysine or arginine.

In some instances of Formula 4D, the peptide comprises the sequence FFRKSKEKIG (RP853) (SEQ ID NO: 18), or a fragment or variant thereof (eg, a variant containing one or two substitutions).

In a particular case, the striapathic region has an arrangement defined by the following equation:
[J _1a J _1b ]-[X _1a X _1b ]-[J _2a J _2b ]-[X _2a X _2b ]-[J _3a ] (Formula 4E)
During the ceremony,
J _1a , J _1b , J _2a , J _2b , and J _3a are each independently selected from phenylalanine, alanine, and isoleucine;
X _1a , X _1b , X _2a , X _2b , and X _2c are each independently selected from ornithine, lysine, and arginine.

In a particular case, the striapathic region has an arrangement defined by the following equation:
[J _1a J _1b ]-[X _1a X _1b ]-[J _2a J _2b ]-[X _2a X _2b ]-[J _3a ]-[X _3a ] (Formula 5A)
During the ceremony,
J _1a , J _1b , J _2a , J _2b , and J _3a are each independently selected from phenylalanine, tryptophan, alanine, and valine;
X _1a , X _1b , X _2a , X _2b , and X _3a are each independently selected from ornithine, lysine, and arginine.

In some embodiments of Equation 5A, the striapathic region has an arrangement defined by the following equation:
J _1a J _1b OOJ _2a J _2b OOJ _3a O (SEQ ID NO: 29)
where J _1a , J _1b , J _2a , J _2b , and J _3a are each independently selected from phenylalanine and alanine (e.g., each J1, J2, and J3 module contains both phenylalanine and alanine). include).

In some embodiments of Equation 5A, the striapathic region has an arrangement defined by the following equation:
FAX _1a X _1b FAX _2a X _2b J _3a FX _3a (SEQ ID NO: 30)
wherein X _1a , X _1b , X _2a , X _2b , and X _3a are each independently selected from ornithine, lysine, and arginine.

In some instances of Formula 5A, the peptide comprises the sequence FAOOFAOOFO (RP850) (SEQ ID NO: 19), or a fragment or variant thereof (eg, a variant containing one or two substitutions).

In some embodiments of Equation 5A, the striapathic region has an arrangement defined by the following equation:
FWKX _1b FVX _2a KWX _3a (SEQ ID NO: 31)
In the formula, X _1b , X _2a , and X _3a are each independently lysine or arginine.

In some instances of Formula 5A, the peptide has the sequence FWKRFVRKWR (RP837) (SEQ ID NO: 4) or FWKKFVKKWK (RP841) (SEQ ID NO: 7), or a fragment or variant thereof (e.g., with one or two substitutions). including variants).

In some cases, the immunomodulatory peptide of Formula 5A is not FFRKFAKRFK (RP183) (SEQ ID NO: 21) or FFKKFFKKFK (RP185) (SEQ ID NO: 22).

In a particular case, the striapathic region has an arrangement defined by the following equation:
[J _1a J _1b ]-[X _1a X _1b ]-[J _2a J _2b ]-[X _2a X _2b ]-[J _3a ]-[X _3a ] (Formula 5A)
During the ceremony,
J _1a , J _1b , J _2a , J _2b , and J _3a are each independently selected from hydrophobic amino acid residues (e.g., phenylalanine, tryptophan, alanine, valine, and glycine);
X _1a , X _1b , X _2a , X _2b , and X _3a are each independently selected from hydrophilic amino acid residues (e.g., lysine, ornithine, arginine, histidine, aspartic acid, glutamic acid, asparagine, or glutamine) Ru.

In some cases of Formula 5A, J _1a , J _1b , J _2a , J _2b , and J _3a are each independently selected from phenylalanine, tryptophan, alanine, and glycine, and X _1a , X _1b , X _2a , X _2b , and _X3a are each independently selected from lysine and arginine. In the particular case of Formula 5A, J _1a , J _1b , J _2a , J _2b , and J _3a are each independently selected from phenylalanine, tryptophan, alanine, and valine, and X _1a , X _1b , X _2a , X _2b and X _3a are each independently selected from ornithine, lysine, and arginine (eg, Lys or Arg). In some instances of Formula 5A, J _1a , J _1b , J _2a , J _2b , and J _3a are each independently selected from phenylalanine and alanine, and X _1a , X _1b , X _2a , X _2b , and Each X _3a is independently selected from lysine and arginine. In the particular case of Formula 5A, J _1a , J _1b , J _2a , J _2b , and J _3a are each phenylalanine, and X _1a , X _1b , X _2a , X _2b , and X _3a are each independently , lysine and arginine. In some cases of Formula 5A, J _1a , J _1b , J _2a , J _2b , and J _3a are each tryptophan, and X _1a , X _1b , X _2a , X _2b , and X _3a are each independently: selected from histidine, lysine, and arginine. In some instances of Formula 5A, J _1a , J _2a , and J _3a are each independently selected from phenylalanine and tryptophan, J _1b is selected from tryptophan and alanine, and J _2b is valine, tryptophan, and alanine, and each of X _1a , X _1b , X _2a , X _2b , and X _3a is independently selected from ornithine, lysine, arginine, or histidine.

In some embodiments of Equation 5A, the striapathic region has an arrangement defined by the following equation:
WWX _1a HWWHX _2b WX _3a (Sequence number 32)
where X _1a , X _2b , and X _3a are each independently histidine, lysine, or arginine.

In some instances of Formula 5B, the peptide has the sequence WWHHWWHHWH (RP847) (SEQ ID NO: 13), WWRHWWHRWR (RP848) (SEQ ID NO: 14), or WWKHWWHKWK (RP849) (SEQ ID NO: 15), or a fragment or variant thereof. (e.g., variants containing one or two substitutions).

In some embodiments of Equation 5, the striapathic region has an arrangement defined by the following equation:
[J _1a J _1b ]-[X _1a X _1b ]-[J _2a J _2b J _2c ]-[X _2b ]-[J _3a ]-[X _3a ] (Formula 5B)
During the ceremony,
J _1a , J _1b , J _2a , J _2b , and J _3a are each independently selected from hydrophobic amino acid residues (e.g., phenylalanine, alanine, threonine, or leucine);
X _1a , X _1b , X _2a , X _2b , and X _3a are each independently selected from hydrophilic amino acid residues (eg, histidine, aspartate, lysine, or arginine).

In some embodiments of Equation 5B, the striapathic region has an arrangement defined by the following equation:
J _1a J _1b X _1a HJ _2a J _2b THLD (SEQ ID NO: 33)
During the ceremony,
J _1a , J _1b , J _2a and J _2b are each independently selected from phenylalanine and alanine;
X _1a is independently selected from lysine and arginine.

In some instances of Formula 5C, the peptide comprises the sequence FFRHFATHLD (RP845) (SEQ ID NO: 11), or a fragment or variant thereof (eg, a variant containing one or two substitutions).

In some embodiments of Equation 5, the striapathic region has an arrangement defined by the following equation:
[J _1a ]-[X _1a ]-[J _2a J _2b J _2c ]-[X _2a ]-[J _3a J _3b ]-[X _3a X _3b ] (Formula 5C)
During the ceremony,
J _1a , J _2a , J _2b , J _2c , J _3a , and J _3b are each independently selected from hydrophobic amino acid residues (e.g., phenylalanine, tyrosine, leucine, glycine, or isoleucine);
X _1a , X _2a , X _3a , and X _3b are each independently selected from hydrophilic amino acid residues (eg, glutamine, lysine, or histidine).

In some embodiments of Equation 5C, the striapathic region has an arrangement defined by the following equation:
J _1a QJ _2a LGX _2a IIHH (SEQ ID NO: 34)
During the ceremony,
J _1a and J _2a are each independently selected from phenylalanine, tyrosine, and leucine;
X _2a is lysine and arginine.

In some instances of Formula 5C, the peptide comprises the sequence FQFLGKIIHH (RP852) (SEQ ID NO: 17), or a fragment or variant thereof (eg, a variant containing one or two substitutions).

In some embodiments of Equation 6, the striapathic region has an arrangement defined by the following equation:
[J _1a ]-[X _1a X _1b ]-[J _2a J _2b ]-[X _2a ]-[J _3a ]-[X _3a X _3b ]-[J _4a J _4b ] (formula 6A)
During the ceremony,
J _1a , J _2a , J _2b , J _3a , J _4a , and J _4b are each independently selected from hydrophobic amino acid residues (e.g., phenylalanine, tryptophan, alanine, isoleucine, valine, and glycine);
X _1a , X _1b , X _2a , X _3a , and X _3b are each independently selected from hydrophilic amino acid residues (eg, lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, or glutamine).

In some embodiments of Equation 6A, the striapathic region has an arrangement defined by the following equation:
GX _1a X _1b GJ _2b X _2a GX _3a X _3b GJ _4b (SEQ ID NO: 35)
During the ceremony,
J _2b and J _4b are each independently selected from phenylalanine, tryptophan, alanine, isoleucine, and valine;
X _1a , X _1b , X _2a , X _3a , and X _3b are each independently selected from lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, and glutamine.

In some embodiments of Equation 6A, the striapathic region has an arrangement defined by the following equation:
GDX _1b GIX _2a GHX _3b GF (SEQ ID NO: 36)
wherein X _1b , X _2a , and X _3b are each independently selected from lysine and arginine.

In some instances of Formula 6A, the peptide comprises the sequence GDRGIKGHRGF (RP842) (SEQ ID NO: 8), or a fragment or variant thereof (eg, a variant containing one or two substitutions).

In some embodiments of Equation 7, the striapathic region has an arrangement defined by the following equation:
[X _1a X _1b ]-[J _1a ]-[X _2a ]-[J _2a ]-[X _3a ]-[J _3a J _3b J _3c ] (Formula 7A)
During the ceremony,
J _1a , J _2a , J _3a , J _3b , and J _3c are each independently selected from hydrophobic amino acid residues (e.g., isoleucine, valine, leucine, serine, or alanine);
X _1a , X _1b , X _2a , and X _3a are each independently selected from hydrophilic amino acid residues (eg, lysine or arginine).

In some embodiments of Equation 7A, the striapathic region has an arrangement defined by the following equation:
X _1a X _1b IX _2a VX _3a LSA (SEQ ID NO: 37)
wherein X _1a , X _1b , X _2a , and X _3a are each independently selected from lysine and arginine.

In some instances of Formula 7A, the peptide comprises the sequence KKIRVRLSA (RP851) (SEQ ID NO: 16), or a fragment or variant thereof (eg, a variant containing one or two substitutions).

Multimeric Peptides The present disclosure includes multimers (eg, dimers) of two or more immunomodulatory peptides (eg, as described herein) connected via branched or liner linkers. Aspects of the present disclosure include dimers of any of the subject immunomodulatory polypeptides. The dimer may be a homodimer or a heterodimer. Any two immunomodulatory polypeptides can be connected via a linker. Any convenient linker can be utilized. Linkers that can be used include covalent bonds, peptide linkers (e.g., glycine-containing linkers or Gly and Ser-containing linkers), C1-C12 linkers with terminal amino and/or carboxylic acid groups, or polymeric linkers (e.g., PEG or modified PEG). The dimer may include a linker that connects the C-terminus of the first polypeptide with the N-terminus of the second polypeptide. In certain cases, two polypeptides may be linked via their C-termini. In certain cases, two polypeptides may be linked via their N-termini.

The disclosure further includes any two immunomodulatory polypeptides linked together. The linkage is Gly-Gly-Gly (GGG), Gly-Gly-Gly-Arg (GGGR, SEQ ID NO: 40), which links the C-terminus of the first immunomodulatory polypeptide to the N-terminus of the second immunomodulatory polypeptide. ), Gly-Pro-Gly (GPG), or Gly-Pro-Gly-Arg (GPGR, SEQ ID NO: 41) sequences. Alternatively, the linkage may be a peptoid linker (e.g., a poly-N substituted version of any of the peptide linkers described above), a g-amino acid containing polymer (e.g., corresponding to any of the peptide linkers described above), or a non- Can be a peptide chemical linker. The linked immunomodulatory polypeptides can be any of the polypeptides disclosed herein (e.g., Table 3), the same polypeptides linked to form a homodimer, or It can include different polypeptides that are linked to form a heterodimer. Techniques for linking peptides via peptide and non-peptide linkers are well known in the art, and the polypeptide combinations of the present invention are intended to encompass all such linkages.

Any two striapathic region-containing peptides (eg, as described herein) can be linked. The two regions of a dimeric peptide may be homodimeric or heterodimeric with respect to each other. Homodimer means that the two peptide regions of the dimeric peptide have the same N to C sequence or vice versa. The subject immunomodulatory polypeptides described herein can be linked in any convenient configuration to generate multimers. In certain cases, a multimer comprises three or more immunomodulatory polypeptides (eg, as described herein), and the polypeptides can be arranged in a linear or branched fashion. Linear multimers of immunomodulatory polypeptides can include a head-to-tail arrangement of linked peptides linked via covalent bonds or any linker (eg, a peptide linker). In some cases, a linear multimer may be referred to as an oligomer, eg, a polypeptide chain that includes a sequence segment of an immunomodulatory polypeptide (eg, as described herein). Alternatively, linear multimeric immunomodulatory polypeptides may have head-to-head (e.g., N-terminus linked) and/or tail-to-tail (e.g., C-terminus linked) configurations. can be connected via In a branched multimer, the immunomodulatory polypeptides can be linked via any convenient branched linker, eg, a group containing three functional groups for attachment to amino acid residues such as lysine amino acids. In some cases, the multimer is a dimer.

In certain cases, the immunomodulatory peptide dimer has the formula:
Z ¹ -T-Z ²
During the ceremony,
T is a linker, e.g. a peptide linker;
Z ¹ is 3-10 (eg, 4-10, 5-10, or 3) consisting of a mixture of hydrophilic and hydrophobic amino acid residues (eg, as described herein). ~6, or 3, 4, 5, or 6) amino acid residues;
Z ² is 3-10 (e.g., 4-10, 5-10, or 3) consisting of a mixture of hydrophilic and hydrophobic amino acid residues (e.g., as described herein). 6, or 3, 4, 5, or 6) amino acid residues.

In the particular case of dimers, the hydrophilic module consists of amino acid residues selected from lysine and arginine, and the hydrophobic module consists of amino acid residues selected from phenylalanine and tryptophan. In certain cases, the first and second polypeptides (Z ¹ and Z ² ) include four amino acid residues. In certain cases, Z ¹ and Z ² each include four amino acid residues, two of which are hydrophilic residues (e.g., as described herein) and the remaining two Amino acid residues are hydrophobic residues (eg, as described herein).

In certain embodiments, the dimer has one of the following formulas:
[X1]-[J1]-T-[J1]-[X1] (Formula 8)
[J1]-[X1]-T-[X1]-[J1] (Formula 9)
[X1]-[J1]-T-[J2]-[X2] (Formula 10)
[J1]-[X1]-T-[X2]-[J2] (Formula 11)
where T is a linker (eg, a peptide linker).

In some cases of formulas 8 and 9, the dimer has a sequence defined by one of the following formulas:
[X _1a X _1b ]-[J _1a J _1b ]-T-[J _1b J _1a ]-[X _1b X _1a ] (Formula 8A)
[J _1a J _1b ]-[X _1a X _1b ]-T-[X _1b X _1a ]-[J _1b J _1a ] (Formula 9A)
During the ceremony,
T is a peptide linker (e.g. a polyglycine linker),
J _1a and J _1b are each independently selected from hydrophobic amino acid residues (e.g. tryptophan or phenylalanine);
X _1a and X _1b are each independently selected from hydrophilic amino acid residues (eg, asparagine or arginine).
In certain instances of Formula 8A and Formula 9A, T is a peptide linker consisting of one, two, or three glycine residues.

In some embodiments of Formula 9A, the dimer has a sequence defined by the following formula:
FW-[X _1a X _1b ]-T-[X _1b X _1a ]-WF (SEQ ID NO: 38)
wherein X _1a , X _1b are each independently selected from lysine and arginine.

In some embodiments of Formula 9A, the dimer has a sequence defined by the following formula:
[J _1a J _1b ]-KR-T-RK-[J _1b J _1a ] (SEQ ID NO: 39)
wherein J _1a and J _1b are each independently selected from tryptophan and phenylalanine.

In some instances of Formula 7A, the peptide comprises the sequence FWKRGGRKWF (RP837A) (SEQ ID NO: 4), or a fragment or variant thereof (eg, a variant containing one or two substitutions).

In some cases of Formulas 10 and 11, the dimer has a sequence defined by one of the following formulas:
[J _1a J _1b ]-[X _1a X _1b ]-T-[X _2a X _2b ]-[J _2a J _2b ] (Formula 10A)
[X _1a X _1b ]-[J _1a J _1b ]-T-[J _2a J _2b ]-[X _2a X _2b ] (Formula 11A)
During the ceremony,
J _1a , J _1b , J _2a , and J _2b are each independently selected from hydrophobic amino acid residues (e.g., tryptophan or phenylalanine);
X _1a , X _1b , X _2a , and X _2b are each independently selected from hydrophilic amino acid residues (eg, asparagine or arginine).
In certain instances of Formula 10A and Formula 11A, T is a peptide linker consisting of one, two, or three glycine residues.

In certain cases, the dimeric first and second polypeptides (Z ¹ and Z ² ) have the following formula of hydrophilic and hydrophobic modules: [X1]-[J1]-[X2]-[ J2] (Formula 3) or [J1]-[X1]-[J2]-[X2] (Formula 2).

In certain embodiments, the dimer has one of the following formulas:
[X1]-[J1]-[X2]-[J2]-T-[J2]-[X2]-[J1]-[X1] (Formula 12)
[J1]-[X1]-[J2]-[X2]-T-[X2]-[J2]-[X1]-[J1] (Formula 13)
[X1]-[J1]-[X2]-[J2]-T-[J3]-[X3]-[J4]-[X4] (Formula 14)
[J1]-[X1]-[J2]-[X2]-T-[X3]-[J3]-[X4]-[J4] (Formula 15)
where T is a peptide linker.

In some instances of Formula 12 and Formula 13, the dimer has one of the following formulas:
[X _1a ]-[J _1a ]-[X _2a ]-[J _2a ]-T-[J _2a ]-[X _2a ]-[J _1a ]-[X _1a ] (Formula 12A)
[J _1a ]-[X _1a ]-[J _2a ]-[X _2a ]-T-[X _2a ]-[J _2a ]-[X _1a ]-[J _1a ] (Formula 13A)
During the ceremony,
T is a peptide linker (e.g. a polyglycine linker),
J _1a and J _2a are each independently selected from phenylalanine and tryptophan;
X _1a and X _2a are each independently selected from lysine and arginine.

In some instances of Formula 12A, the peptide comprises the sequence RWKFGGFKWR (RP832C) (SEQ ID NO: 1), or a fragment or variant thereof (eg, a variant containing one or two substitutions).

In some instances of Formula 13A, the peptide comprises the sequence FKWRGGRWKF (RP837C) (SEQ ID NO: 3), or a fragment or variant thereof (eg, a variant containing one or two substitutions). In certain embodiments, the immunomodulatory peptide includes a tail region.

In some instances of Formula 14 and Formula 15, the dimer has one of the following formulas:
[X _1a ]-[J _1a ]-[X _2a ]-[J _2a ]-T-[J _3a ]-[X _3a ]-[J _4a ]-[X _4a ] (Formula 14A)
[J _1a ]-[X _1a ]-[J _2a ]-[X _2a ]-T-[X _3a ]-[J _3a ]-[X _4a ]-[J _4a ] (Formula 15A)
During the ceremony,
T is a peptide linker (e.g. a polyglycine linker),
J _1a , J _2a , J _3a , and J _4a are each independently selected from phenylalanine and tryptophan;
X _1a , X _2a , X _3a , and X _4a are each independently selected from lysine and arginine.

Immunomodulatory peptides of interest include any one of the polypeptides of Table 3, a fragment thereof (e.g., as described herein), or a variant thereof (e.g., as described herein). Including, but not limited to:

In certain embodiments, the subject immunomodulatory polypeptides are
a) a sequence selected from the peptide sequences in Table 3;
b) a sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, or at least 95% sequence identity) with a sequence defined in a); and c) a). a sequence with one or two amino acid substitutions compared to a sequence defined in Table 2, wherein the one or two amino acid substitutions are amino acid substitutions according to Table 2 (e.g., similar amino acid substitutions, substitutions or highly conservative amino acid substitutions).

In a particular case, the sequence described in a) is RP832C. In a particular case, the sequence described in a) is RP837. In a particular case, the sequence described in a) is RP837C. In a particular case, the sequence described in a) is RP837A. In a particular case, the sequence described in a) is RP837N. In a particular case, the sequence described in a) is RP837C ¹ . In a particular case, the sequence described in a) is RP841. In a particular case, the sequence described in a) is RP842. In a particular case, the sequence described in a) is RP843. In a particular case, the sequence described in a) is RP844. In a particular case, the sequence described in a) is RP845. In a particular case, the sequence described in a) is RP846. In a particular case, the sequence described in a) is RP847. In a particular case, the sequence described in a) is RP848. In a particular case, the sequence described in a) is RP849. In a particular case, the sequence described in a) is RP850. In a particular case, the sequence described in a) is RP851. In a particular case, the sequence described in a) is RP852. In a particular case, the sequence described in a) is RP853.

In certain cases, the sequence described in b) has a sequence that has at least 80% sequence identity with the sequence defined in a). In certain cases, the sequence described in b) has a sequence that has at least 85% sequence identity with the sequence defined in a). In certain cases, the sequence described in b) has a sequence that has at least 90% sequence identity with the sequence defined in a). In certain cases, the sequence described in b) has a sequence that has at least 95% sequence identity with the sequence defined in a).

In a particular embodiment, the sequence shown in c) has one or two amino acid substitutions compared to the sequence defined in a), and the one or two amino acid substitutions are similar to those according to Table 2. This is an amino acid substitution. In a particular embodiment, the sequence shown in c) has one or two amino acid substitutions compared to the sequence defined in a), and the one or two amino acid substitutions are conservative according to Table 2. This is an amino acid substitution. In a particular embodiment, the sequence shown in c) has one or two amino acid substitutions compared to the sequence defined in a), and the one or two amino acid substitutions are highly This is a conservative amino acid substitution. Any of the immunomodulatory peptide variations described herein may be applied to the parent peptides in Table 3.

Excluded Polypeptides Compositions of the present disclosure optionally exclude and include polypeptides described in U.S. Patent Application Nos. 2012/0270770 and 2003/0109452, and U.S. Patent No. 6,559,281. , the disclosures of which are incorporated herein by reference in their entirety. Accordingly, one or more polypeptides and/or uses of such polypeptides described in such publications may be excluded from the scope of the compositions and/or methods of the present disclosure. Additionally, any of the polypeptides disclosed in Tables 3 to 9 of WO2016/061133 by Jaynes et al., the disclosures of which tables are incorporated herein by reference, may be used in the compositions disclosed herein. and/or may be optionally excluded from methods using such compounds. In some cases, any of the polypeptides disclosed in Table 4 below may be optionally excluded from the compositions and/or methods of using such compounds disclosed herein.

In some cases, the immunomodulatory peptide of the formulas described herein is not a polypeptide of Table 4.

Modified Polypeptides Embodiments of the present disclosure include modification of any of the immunomodulatory polypeptides of the present disclosure by chemical or genetic means. Examples of such modifications include construction of partial or full sequence peptides with unnatural and/or natural amino acids in L or D form. For example, any of the peptides disclosed herein and any variants thereof can be produced in the all-D form. Additionally, polypeptides of the present disclosure can be modified to contain carbohydrate or lipid moieties such as sugars or fatty acids covalently attached to the side chains of amino acids or to the N- or C-termini. Additionally, polypeptides of the present disclosure can be modified to enhance solubility and/or half-life upon administration. For example, polyethylene glycol (PEG) and related polymers have been used to enhance the solubility and half-life of protein therapeutics in the blood. Accordingly, polypeptides of the present disclosure can be modified with PEG polymers and the like. Polypeptides of the present disclosure can also be modified to contain sulfur, phosphorous, halogens, metals, and the like. Amino acid mimetics can be used to generate polypeptides of the present disclosure (e.g., having a structure based on a structural algorithm or similar to any of the immunomodulatory polypeptides disclosed herein). . In certain embodiments, polypeptides of the disclosure that include amino acid mimetics have enhanced properties, such as resistance to degradation. For example, a polypeptide of the present disclosure can include one or more (eg, all) peptoid monomers.

An immunomodulatory polypeptide can be linked to another molecule via a biodegradable linkage such as a disulfide bond. Disulfide bonds can be mediated by sulfhydryl groups of cysteine residues found in immunomodulatory polypeptides, as well as sulfhydryl groups in other molecules. Cysteine residues can be located, for example, at either the C-terminus or the N-terminus of the immunomodulatory polypeptide. Using this type of disulfide linkage, polypeptides of the present disclosure can be conveniently linked to a wide variety of useful molecules. For example, the linkage may include another immunomodulatory polypeptide (optionally containing a C-terminal or N-terminal cysteine residue), a fluorescent label (e.g., Dylight 350), a chemotherapeutic agent (e.g., a sulfhydryl at an appropriate position on the taxol ring structure), a fluorescent label (e.g., Dylight 350), a chemotherapeutic agent (e.g., taxol derivatives formed by adding a group and subsequent oxidation with cysteine-containing peptides of the present disclosure.

A linked immunomodulatory polypeptide (e.g., a homodimer or a heterodimer) binds a target molecule (e.g., a pro-inflammatory signaling protein) with a binding energy greater than that of either monomeric polypeptide alone. can bind to target proteins such as Thus, for example, the energy of binding of a linked immunomodulatory polypeptide to an NF-kB class II protein (e.g., RelB) can be at least -700 kcal/mol, and in certain embodiments at least -750, -800, -900, -1000, -1100, -1200, -1250, -1300, -1350, -1400, -1425, -1450, -1475, -1500, -1525, -1550, -1575, -1600kcal /mol or more. Energy of binding can be determined, eg, in silico, in vitro, or in vivo, using methods well known in the art (eg, using the ClusPro™ algorithm).

In some instances where a modified peptide is covalently attached to a molecule of interest, the resulting compound may be referred to as a peptide conjugate. Any convenient molecule of interest may be conjugated to the subject immunomodulatory peptide. The molecules of interest may be peptidic or non-peptidic, and may be naturally occurring or synthetic. Molecules of interest suitable for use in combination with the subject immunomodulatory peptides include protein domains, polypeptides, peptide tags, specific binding moieties (e.g., antibodies or antibody fragments), polyethylene glycols (PEG), carbohydrates. , dextran, or polyacrylates, linkers, half-life extending moieties, labels, and moieties that confer desirable drug-like properties such as solid supports. In some cases, the molecule of interest is characterized by increased aqueous solubility, ease of chemical synthesis, cost, bioconjugation sites, stability, pI, aggregation, reduced non-specific binding, as described herein, for example. Enhanced and/or modified properties and functions may be imparted to the resulting modified peptide, including, but not limited to, specific binding to a second target protein, and/or specific binding to a second target protein.

In some embodiments where any one of the peptide sequences described herein is presented, the peptide sequence includes one or more additional residues at the N-terminus and/or C-terminus of the sequence, e.g. It may be extended to include two or more, three or more, four or more, five or more, six or more, or even more additional residues. Any convenient residues may be included at the N-terminus and/or C-terminus of the peptide to increase solubility via water-soluble groups, linkages, labels or specific binding moieties for dimerization or multimerization. Desirable properties or groups can be provided, such as a linkage to connect to.

In some cases, the subject modified peptides are described by the formula below.
B-LM
where B is an immunomodulatory peptide (e.g., as described herein), L is any linking group, M is the molecule of interest, and L is any convenient position. (eg, via the N-terminus, C-terminus, or a side chain of a residue not involved in binding to the target).

A modified peptide may include one or more molecules of interest. In some cases, the molecule of interest is covalently attached through the alpha-amino group of the N-terminal residue or covalently attached to the alpha-carboxylic acid group of the C-terminal residue.

Molecules of interest may include polypeptide or protein domains. Polypeptides and protein domains of interest include gD tags, c-Myc epitopes, FLAG tags, His tags, fluorescent proteins (e.g., GFP), beta-galactosidase proteins, GSTs, albumins, immunoglobulins, antibodies, Fc domains, or similar antibody-like fragments, polypeptides containing leucine zipper motifs, coiled-coil domains, hydrophobic regions, hydrophilic regions, free thiols that form intermolecular disulfide bonds between two or more multimerization domains, ridge" domain, beta-lactoglobulin, or fragments thereof.

Molecules of interest may include half-life extending moieties. The term "half-life extending moiety" refers to a chemical modification that prevents or reduces the in vivo proteolytic or other activity of a subject compound as compared to an unconjugated form of the subject compound, and that prevents or reduces the half-life or increase other pharmacokinetic properties (e.g., rate of absorption), reduce toxicity, improve solubility, increase biological activity and/or target selectivity of the subject compounds with respect to targets of interest; Refers to a pharmaceutically acceptable moiety, domain, or "vehicle" covalently linked or conjugated to a subject compound that increases the potential and/or reduces the immunogenicity of the subject compound.

In certain embodiments, the half-life extending moiety is a polypeptide that binds to a serum protein, such as an immunoglobulin (eg, IgG) or serum albumin (eg, human serum albumin (HSA)). Polyethylene glycol is an example of a useful half-life extending moiety. Exemplary half-life extending moieties include polyalkylene glycol moieties (e.g., PEG), serum albumin or fragments thereof, transferrin receptor or transferrin-binding portions thereof, and binding sites for polypeptides that enhance in vivo half-life. moieties containing, copolymers of ethylene glycol, copolymers of propylene glycol, carboxymethylcellulose, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymers, polyamino acids such as polylysine ), dextran n-vinylpyrrolidone, polyvinylpyrrolidone, propylene glycol homopolymer, propylene oxide polymer, ethylene oxide polymer, polyoxyethylated polyol, polyvinyl alcohol, linear or branched glycosylated chain, polysialic acid, polyacetal, lipid , long chain fatty acids, long chain hydrophilic aliphatic groups, immunoglobulin Fc domains (see, e.g., U.S. Pat. No. 6,660,843), albumin (e.g., human serum albumin, e.g., U.S. Pat. No. 6,660,843), 926,898 and US 2005/0054051, US Pat. No. 6,887,470), transthyretin (TTR, see e.g. US 2003/0195154, 2003/0191056), or thyroxine-binding globulin ( TBG) is included.

In certain embodiments, the half-life extending moiety is a lipid. In certain embodiments, the half-life extending moiety is a fatty acid. Any convenient lipids and fatty acids can be used in the subject modified compounds. For example, Chae et al. , “The fatty acid conjugated exendin-4 analogs for type2 antidiabetic therapeutics”, J. Control Release. 2010May21;144(1):10-6.

In certain embodiments, immunomodulatory peptides are modified to include specific binding moieties. A specific binding moiety is a moiety that is capable of specifically binding to a second moiety that is complementary to it. In some _cases , the specific binding moiety is at least 10 ⁻ Binds to a complementary second moiety with an affinity of ⁷ M. Complementary binding moiety pairs of specific binding moieties include ligands or activators/promoters and receptors, antibodies and antigens, complementary polynucleotides, complementary protein homo- or heterodimers, aptamers and small molecules. , and polyhistidine tags and nickel. Specific binding pairs can include analogs, derivatives, and fragments of the original specific binding member. For example, antibodies directed against protein antigens may also recognize peptide fragments, chemically synthesized and labeled proteins, derivatized proteins, etc., as long as the epitope is present. Protein domains that may find use as specific binding moieties include Fc domains or similar antibody-like fragments, leucine zipper motifs, coiled-coil domains, hydrophobic regions, hydrophilic regions, and linkages between two or more multimerization domains. These include, but are not limited to, polypeptides containing free thiols that form intermolecular disulfide bonds, or "bulge into the cavity" domains (e.g., WO 94/10308, U.S. Pat. No. 5,731,168, Lovejoy et al. al. (1993), Science 259: 1288-1293, Harbury et.
al. (1993), Science 262: 1401-05, Harbury et al. (1994), Nature 371:80-83, Hakansson et.
al. (1999), Structure 7: 255-64).

In certain embodiments, the peptide is a linked specific binding moiety that specifically binds to a target protein. The linked specific binding moiety can be an antibody, antibody fragment, receptor activator, or aptamer. The linked specific binding moiety can specifically bind to any convenient target protein, such as a target protein that is desired to be targeted in conjunction with the subject therapeutic method. Target proteins of interest include PDGF (e.g., PDGF-B), VEGF-B, VEGF-C, VEGF-D, EGF, EGFR, Her2, PD-1, PD-L1, OX-40, and LAG3. Including, but not limited to: In certain embodiments, the linked specific binding moiety is a receptor activator or ligand, e.g., a protein ligand associated with inflammatory pathways, such as interleukin 13 (IL-13), or a toll-like receptor (TLR). ) family members, such as molecules that activate TLR3. In certain cases, the linked specific binding moiety (eg, protein, antibody, or antibody fragment) can be further linked to an additional active agent (eg, a chemotherapeutic agent as described herein).

An immunomodulatory polypeptide (eg, as described herein) can be conjugated to an additional active agent to provide an immunomodulatory polypeptide conjugate. Once the subject peptides have been produced and/or created and selected in accordance with the teachings herein, they can be linked, fused, (e.g., covalently bonded) with pharmaceutically active or diagnostic moieties or biocompatible modifiers. (bondally or non-covalently) or otherwise associated. The term "peptide conjugate" refers to any biologically active or detectable molecule or drug that is associated with a disclosed immunomodulatory peptide compound, regardless of the method of association. In this regard, such conjugates include, in addition to the disclosed immunomodulatory peptides, peptides, polypeptides, proteins, prodrugs that are metabolized in vivo to active agents, polymers, nucleic acid molecules, small molecules, binding agents, It is understood that mimetics, synthetic drugs, inorganic molecules, organic molecules, and radioisotopes can be included. Additionally, as indicated above, the selected conjugate is covalently or non-covalently associated with or linked to the subject peptide and is used, at least in part, to effect the conjugation. Depending on the method used, various stoichiometric molar ratios may be indicated.

In certain instances, the molecule of interest is a second active agent, such as an active agent or drug that finds use in combination with the target of interest in the subject therapeutic methods. In certain cases, the molecule of interest is a small molecule, chemotherapeutic agent, antibody, antibody fragment, bispecific antibody, aptamer, or L-protein. In some embodiments, peptides are modified to include moieties useful as pharmaceuticals (eg, proteins, nucleic acids, small organic molecules, etc.). Exemplary pharmaceutical proteins include, for example, cytokines, antibodies, chemokines, growth factors, interleukins, cell surface proteins, extracellular domains, cell surface receptors, cytotoxins, and the like. Exemplary small molecule pharmaceuticals include small molecule toxins or therapeutic agents. Any convenient therapeutic or diagnostic agent (eg, as described herein) can be conjugated to the immunomodulatory peptide. A variety of therapeutic agents are listed below in the section entitled "Combination Therapy," including, but not limited to, anti-cancer, anti-proliferative, cytotoxic, and chemotherapeutic agents; Any one can be adapted for use in the subject peptide conjugates.

In certain embodiments, the modified peptide can be conjugated to a bispecific antibody, e.g., an engineered bispecific monoclonal antibody that is capable of simultaneously binding two different types of antigens of interest. can.

In certain embodiments, modified peptides can include cell-penetrating peptides (eg, tat). Cell-penetrating peptides can facilitate cellular uptake of molecules. Any convenient tag polypeptides and their respective antibodies may be used. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; influenza HA tag polypeptide and its antibody 12CA5 [Field et al. , Mol. Cell. Biol. 8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7, and 9E10 antibodies thereto [Evan et al. , Molecular and Cellular Biology, 5:3610-3616 (1985)]; and herpes simplex virus glycoprotein D (gD) tag and its antibodies [Paborsky et al. , Protein Engineering, 3(6):547-553 (1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al. , BioTechnology 6:1204-1210 (1988)], KT3 epitope peptide [Martin et al. , Science 255:192-194 (1992)], tubulin epitope peptide [Skinner et al. , J. Biol. Chem. 266:15163-15166 (1991)], and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al. , Proc. Natl. Acad. Sci. U. S. A. 87:6393-6397 (1990)].

Those skilled in the art will appreciate that several different reactions are available for attachment or association of therapeutic or diagnostic moieties and/or linkers to the subject immunomodulatory peptides. In certain embodiments, this includes the amino terminus, the C-terminal carboxylic acid, the amine group of lysine, the free carboxylic acid groups of glutamic acid and aspartic acid, the sulfhydryl group of cysteine, and various moieties of aromatic amino acids, e.g. This can be achieved by reaction of the amino acid residues of the peptides described herein. One method of covalent attachment is a carbodiimide reaction that links the carboxy (or amino) group of the compound to the amino (or carboxy) group of the subject peptide. Additionally, bifunctional agents such as dialdehydes or imidoesters have been used to link the amino groups of the subject peptides to the amino groups of antibody molecules. Maleimide-thiol conjugation chemistry, such as click chemistry between an azide group and an alkynyl group, can also be used to attach drugs to immunomodulatory peptides. Schiff base reactions can also be used to attach drugs to peptides. This method may involve periodate oxidation of a drug containing glycol or hydroxy groups to form an aldehyde, which is then reacted with a binding agent. Binding occurs through the formation of a Schiff base with the amino group of the binding agent. Isothiocyanates and azlactones can also be used as coupling agents to covalently attach drugs to binding agents.

It is understood that several variations or types of linkers can be used to associate the disclosed immunomodulatory peptides with pharmaceutically active or diagnostic moieties or biocompatibility modifiers. In some embodiments, the linker is cleavable under intracellular conditions such that cleavage of the linker releases drug units from the antibody in the intracellular environment. In certain embodiments, the linker unit is not cleavable. Bivalent linker reagents are known and useful for joining two or more functionally or biologically active moieties such as peptides, nucleic acids, drugs, toxins, antibodies, haptens, and reporter groups. (Hermanson, G.T. (1996) Bioconjugate Techniques; Academic Press: New
York, p234-242).

Compositions Compositions of the present disclosure include immunomodulatory polypeptides that satisfy one of the structural formulas described herein. For example, an immunomodulatory polypeptide can have a striapathic region having a sequence according to any one of the formulas disclosed herein. Typically, the immunomodulatory polypeptides included in the compositions of the present disclosure are synthetic polypeptides (eg, made by chemical synthesis and/or recombinantly produced).

Compositions of the present disclosure can include a single immunomodulatory polypeptide, or a combination thereof. The compositions may be substantially free of proteins and other polypeptides that do not satisfy the structural algorithms disclosed herein. As used herein, the term "substantially free of proteins and other polypeptides" means that less than 5% of the protein content of the composition is protein and other polypeptides that are not immunomodulatory polypeptides of the present disclosure. means composed of other polypeptides. Compositions substantially free of non-immunomodulatory polypeptides of the present disclosure include 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or less, may have proteins or other polypeptides that do not satisfy the structural algorithms disclosed herein. Thus, the composition may be substantially free of blood proteins such as serum albumin, globulin, fibrinogen, and clotting factors. Alternatively, the composition may be substantially free of globulin, fibrinogen, and clotting factors, but may include purified or recombinantly produced serum albumin.

Compositions of the present disclosure, in certain embodiments, contain immunomodulatory polypeptides that are not naturally found in humans or other mammals or animals. However, the compositions of the present disclosure may include an immunomodulatory polypeptide found naturally in humans or other mammals or animals, provided that the composition is associated with the immunomodulatory polypeptide in vivo or provided that it is substantially free of biological molecules (such as non-immunomodulatory polypeptides, nucleic acids, lipids, carbohydrates, and metabolites) that co-purify with the immunomodulatory polypeptide. As used herein, the term "substantially free of biological molecules" means that less than 5% of the dry weight of a composition is comprised of biological molecules that are not immunomodulatory polypeptides. It means that. Compositions that are substantially free of such biological molecules include 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or It may have biological molecules that are less than immunomodulatory polypeptides. Thus, for example, the composition may be substantially free of biological molecules abundant in blood, such as the proteins, fatty acids, cholesterol, non-protein clotting factors, metabolites discussed above. Additionally, the composition may be substantially free of cells and cell fragments, including red blood cells, white blood cells, and platelets.

Compositions of the present disclosure may contain at least 1 mg (e.g., at least 5, 10, 20, 30, 40, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000 mg , or more) immunomodulatory polypeptides. Thus, for example, the composition may be about 1 mg to about 1000 mg (eg, about 5 mg to about 900 mg, about 5 mg to about 800 mg, about 5 mg to about 700 mg, about 5 mg to about 600 mg, about 10 mg to about 500 mg, about 10 mg to about 400mg, about 10mg to about 300mg, about 10mg to about 250mg, about 10mg to about 200mg, about 10mg to about 150mg, about 10mg to about 100mg, about 50mg to about 500mg, about 50mg to about 400mg, about 50mg to about 300mg, about 50 mg to about 250 mg, about 50 mg to about 200 mg, about 50 mg to about 150 mg, about 50 mg to about 100 mg, about 75 mg to about 500 mg, about 75 mg to about 400 mg, about 75 mg to about 300 mg, about 75 mg to about 250 mg, about 75 mg ~ about 200 mg, about 75 mg to about 150 mg, about 75 mg to about 100 mg, about 100 mg to about 500 mg, about 100 mg to about 400 mg, about 100 mg to about 300 mg, about 100 mg to about 250 mg, about 100 mg to about 200 mg, or the terminus above any other range containing two of the immunomodulatory polypeptides).

Compositions of the present disclosure may contain at least 1 mg/ml (e.g., at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 , 95, 100 mg/ml or more) of an immunomodulatory polypeptide. Thus, for example, the composition may be about 1 mg/ml to about 1000 mg/ml (eg, about 5 mg/ml to about 900 mg/ml, about 5 mg/ml to about 800 mg/ml, about 5 mg/ml to about 700 mg/ml, about 5 mg/ml to about 600 mg/ml, about 5 mg/ml to about 500 mg/ml, about 10 mg/ml to about 500 mg/ml, about 10 mg/ml to about 400 mg/ml, about 10 mg/ml to about 300 mg/ml, about 10 mg/mL to about 250 mg/mL, about 10 mg/mL to about 200 mg/mL, about 10 mg/mL to about 150 mg/mL, about 10 mg/mL to about 100 mg/mL, about 50 mg/mL to about 500 mg/mL, about 50 mg/mL to about 400 mg/mL, about 50 mg/mL to about 300 mg/mL, about 50 mg/mL to about 250 mg/mL, about 50 mg/mL to about 200 mg/mL, about 50 mg/mL to about 150 mg/mL, about 50 mg/ml to about 100 mg/ml, about 75 mg/ml to about 500 mg/ml, about 75 mg/ml to about 400 mg/ml, about 75 mg/ml to about 300 mg/ml, about 75 mg/ml to about 250 mg/ml, about 75 mg/ml to about 200 mg/ml, about 75 mg/ml to about 150 mg/ml, about 75 mg/ml to about 100 mg/ml, about 100 mg/ml to about 500 mg/ml, about 100 mg/ml to about 400 mg/ml, about 100 mg/ml to about 300 mg/ml, about 100 mg/ml to about 250 mg/ml, about 100 mg/ml to about 200 mg/ml, about 10 mg/ml to about 150 mg/ml, or two of the above termini. or any other range of immunomodulatory polypeptide concentrations.

Compositions of the present disclosure include pharmaceutical compositions. Such pharmaceutical compositions can include one or more immunomodulatory polypeptides and a pharmaceutically acceptable carrier. Pharmaceutical compositions can further include proteins other than the immunomodulatory polypeptides of the present disclosure and/or chemotherapeutic agents. Other proteins may be therapeutic agents, such as therapeutic antibodies. The therapeutic protein or antibody may have immunomodulatory properties or other properties that the immunomodulatory polypeptides of the present disclosure enhance or are enhanced by. Alternatively, the other protein may be a carrier protein such as serum albumin (eg, HSA). Serum albumin (eg, HAS, BSA, etc.) can be purified or recombinantly produced. By mixing the immunomodulatory polypeptide(s) in a pharmaceutical composition with serum albumin, the immunomodulatory polypeptide is effectively "loaded" onto the serum albumin, resulting in a greater amount of the immunomodulatory polypeptide. It can enable successful delivery to the site of inflammation. The chemotherapeutic agent can be, for example, an anti-cancer chemotherapeutic agent. Such chemotherapeutic agents include gemcitabine, docetaxel, bleomycin, erlotinib, gefitinib, lapatinib, imatinib, dasatinib, nilotinib, bosutinib, crizotinib, ceritinib, trametinib, bevacizumab, sunitinib, sorafenib, trastuzumab, ad-trastuzumab emtansine, rituxi mabu , ipilimumab, rapamycin, temsirolimus, everolimus, methotrexate, doxorubicin, Abraxane, forfilinox, cisplatin, carboplatin, 5-fluorouracil, Teysumo, paclitaxel, prednisone, levothyroxine, and pemetrexed. .

In some instances of the subject pharmaceutical compositions, the composition comprises an immunomodulatory polypeptide that is a CD206 binding peptide (eg, as described herein) and a chemotherapeutic agent. In some embodiments, the immunomodulatory polypeptides that find use in combination compositions are the peptides of Table 3. In certain cases, immunomodulatory peptides (eg, the peptides of Table 3) are combined with chemotherapeutic agents. In certain cases of pharmaceutical compositions, the chemotherapeutic agent is gemcitabine. In some cases of the pharmaceutical composition, the chemotherapeutic agent is docetaxel. In some cases of the pharmaceutical composition, the chemotherapeutic agent is Abraxane.

In some instances of the subject pharmaceutical compositions, the composition is a CD206-binding peptide (e.g., as described herein) and a second additional agent (e.g., as described herein). conjugated immunomodulatory polypeptides. In some cases, the additional agent is a chemotherapeutic agent. In some embodiments, the immunomodulatory polypeptides that find use in the subject peptide conjugates are the peptides of Table 3. In certain cases, immunomodulatory peptides (eg, the peptides of Table 3) are conjugated to chemotherapeutic agents. In the particular case of the subject peptide conjugates, the chemotherapeutic agent is gemcitabine. In some cases of the subject peptide conjugates, the chemotherapeutic agent is docetaxel. In some cases of the subject peptide conjugates, the chemotherapeutic agent is Abraxane. In some cases of the subject peptide conjugates, the chemotherapeutic agent is paclitaxel.

In some cases, the subject pharmaceutical compositions that find use in the treatment of cancer, such as ovarian cancer, include immunomodulatory polypeptides, such as dendritic cells (DCs) that promote Th1/Th17 immunity, in combination with vaccination therapy. ) including vaccination agents. In some cases of the pharmaceutical composition, the immunomodulatory polypeptide is an adjuvant in combination with the Th17-inducing vaccination agent.

The pharmaceutical compositions of the present invention can be formulated for oral, parenteral, inhaled, topical, mucosal, and the like. In some embodiments, administration is via a route selected from oral, intravenous, intraperitoneal, inhalation, intranasal, intraprostatic, and intratumoral. The invention is not limited by route of administration. Compositions formulated for oral delivery can, for example, include an enteric coating to ensure that the peptides contained therein reach the intestinal tract and beyond. Enteric-coated formulations such as gastroresistant capsules for oral administration, suppositories for rectal or vaginal administration also form part of this disclosure. Compositions formulated for topical delivery, for example, suspended in a gel or cream, coated onto microneedles, or injected into a bandage or topical patch, contain the peptides contained therein. can extend the duration of action. Any inhalable formulation capable of providing an aerosolized form containing the subject peptides for delivery to a patient via an intrapulmonary route can be used in conjunction with the present disclosure. In some cases, the subject compositions are administered by, for example, injectable skin, subcutaneous, and/or intratumoral injection into nodular tumors.

In some embodiments, the compositions are prepared using standard techniques (e.g., as described in Remington: The Science and Practice of Pharmacy, Mack Publishing Company, Easton, Pa., 19th edition, 1999). 5 (for example, for mucosal delivery techniques including intranasal, pulmonary, vaginal, and rectal techniques), and European Publication No. 517,565 and Illum et al., J. Controlled Rel., 1994, 29:133-141 (e.g., intranasal For techniques of administration, see section 1.). In some cases, the compositions of the invention can be administered cutaneously or transdermally using standard techniques. The method of intranasal vaccination involves administering the subject composition in droplet or spray form to the nasopharynx of the subject being treated. In some embodiments, a nebulized or aerosolized composition is provided.

Liposomal pharmaceutical compositions comprising the subject immunomodulatory peptides are also provided. Any convenient nanocarriers and liposomes are described in “Liposomes in drug delivery: a patent review” by Arias. Expert Opinion on Therapeutic Patents, 23, 2013, issue 11, p. 1399-1414, and “Multifunctional nanocarriers” by Torchilin, Advanced Drug Delivery Reviews, Volume 58, Issue 14, 1 December 2006, Pages 1 Suitable for use in the preparation of liposomal formulations of the subject peptides, such as the nanocarriers and liposomes described in US Pat. can be done.

Also provided are nanoparticle formulations or compositions comprising the subject immunomodulatory peptides. Nanoparticle formulations or compositions can increase the aqueous solubility of peptides of interest and achieve protection, sustained, and targeted delivery of peptides in therapeutic applications (e.g., as described herein). be able to. In some cases, the formulation is a polymer-based nanoparticle formulation. Nanoparticle formulations of interest include albumin nanoparticles, such as human serum albumin-containing nanoparticle formulations. In some cases, desolvation techniques can be used to prepare albumin nanoparticles. Particle size, peptide drug release, encapsulation efficiency, and peptide drug polymer interactions can be determined and selected using any convenient in vitro method. For example, in vivo pharmacokinetic cell culture studies in rats can be used for biological characterization of desired formulations.

In some cases, nanoparticle formulation compositions comprising the subject immunomodulatory peptides consist of iron oxide nanoparticles (IONPs). IONPs find use in a variety of biomedical applications. In some cases, IONP formulations may exhibit high uptake within macrophages and/or target cancer cells. IONPs with the desired cytotoxicity, in vivo distribution, and/or clearance can be selected for use in combination with the subject immunomodulatory peptides. A variety of well-characterized IONPs with different sizes and coatings can be utilized in the subject compositions and formulations. In some cases, polyethyleneamine (PEI) coated IONPs or PEGylated IONPs are utilized. IONPs can enhance the cytotoxicity of the subject formulations through multiple mechanisms such as ROS generation and apoptosis.

Kits also include an immunomodulatory polypeptide that is a CD206-binding peptide (e.g., as described herein) and an additional agent (e.g., a chemotherapeutic or immunotherapeutic agent) for use in the treatment of cancer. Also provided. The kit can include a dose of an immunomodulatory peptide in an amount effective to inhibit the growth of cancer cells in a subject. The kit can also include a dose of an additional agent, such as a chemotherapeutic or immunotherapeutic agent (eg, as described herein), in an amount effective to inhibit the growth of cancer cells in the subject. Optionally, the kit includes an insert with instructions for administration of the immunomodulatory peptide and/or additional agent (eg, chemotherapeutic or immunotherapeutic agent). In some cases, the set of instructions for combination therapy includes (i) a lower dose of an immunomodulatory peptide when used in combination with a chemotherapeutic agent; (ii) a lower dose of an immunomodulatory peptide when used in combination with a chemotherapeutic agent; (iii) recommend a lower dose of an additional drug (e.g., a chemotherapeutic or immunotherapeutic agent); and/or (iii) a regimen for one or both drugs that differs from the normally recommended regimen. There is.

Methods The present disclosure provides methods of modulating macrophage activity using immunomodulatory peptides (eg, as described herein). In some of the methods, the macrophage activity modulated is macrophage polarization. The method can include contacting a macrophage with a CD206 binding agent that is a peptide of the present disclosure to modulate the activity of the macrophage. In some cases, modulating acidity means inhibiting macrophage activity. The methods of the invention can provide for a reduction in macrophage survival, which can be determined using any convenient method.

In certain embodiments, the immunomodulatory polypeptides of the present disclosure have at least -650 kcal/mol, and in certain embodiments at least -700, -750, -800, -850, -900, -925, -950, It can bind to human CD206 with an affinity of -975, -1000, -1025, -1050 kcal/mol or higher. The required binding affinity may correspond to a binding affinity that can be detected in vitro or in vivo. Alternatively, the required binding affinity may correspond to a binding affinity that can be detected in silico, for example using the ClusPro™ algorithm.

Macrophages targeted using the subject methods can be M2 macrophages or tumor-associated macrophages (TAM). The targeted macrophages can be in vitro or in vivo.

In certain embodiments, the peptides of the present disclosure bind to more than one target (eg, a pro-inflammatory target). In some embodiments, the variant polypeptide binds three, four, five, or more pro-inflammatory targets. For example, variant polypeptides are capable of binding any combination of targets disclosed herein (e.g., NF-kB class II protein and human serum albumin (HSA)), as discussed below. can. Such binding can be based on in silico, in vitro, or in vivo data.

Exemplary RP peptides of interest include various signaling molecules associated with inflammation, including the NF-kB class II subunits RelB, TGFβ, Notch1, Wnt8R, TRAIL, IL6R, IL10R, EGFR, and CDK6; It can also interact with other membrane-associated signaling molecules including CD206, CD47, and SIRP-α, the translational modification protein transglutaminase 2 (TGM2), and the histone modification enzyme histone methyltransferase (HMT). In certain cases, the subject peptide is a CD206 binding peptide. Folding of these protein targets into their normal three-dimensional conformation often generates amphipathic clefts that have high affinity for the immunomodulatory peptides described herein.

Further details of the target signaling molecules to which the subject immunomodulatory peptides specifically bind are described in WO2016/061133 by Jaynes et al., the disclosure of which is incorporated herein in its entirety.

An immunomodulatory polypeptide that finds use in the subject methods can be based on its ability to bind to the mannose binding site on CD206 and/or to interfere with or block the binding of SIRP-mannose to CD206. For example, the immunomodulatory polypeptide comprises at least one amino acid residue of CD206 selected from the group consisting of Glu-725, Tyr-729, Glu-733, Asn-747, and Asp-748, or CD206 of another species. Can bind to equivalent amino acid residue(s) in a protein. Alternatively, the immunomodulatory polypeptide is Phe-708, Thr-709, Trp-710, Pro-714, Glu-719, Asn-720, Trp-721, Ala-722, Glu-725, Tyr-729, Glu- at least one amino acid residue of human CD206 selected from the group consisting of 733, Asn-747, Asp-748, Ser-1691, Cys-1693, Phe-1694, and Phe-1703, or a CD206 protein of another species. can be attached to equivalent amino acid residue(s) within. In certain embodiments, the immunomodulatory polypeptide comprises at least one amino acid residue of CD206 selected from the group consisting of Phe-708, Trp-710, Trp-721, Glu-725, Tyr-729, Glu-733. , or to the equivalent amino acid residue(s) in the CD206 protein of another species.

In certain instances, the immunomodulatory polypeptide binds to the fibronectin (FBN) domain of CD206 and/or interferes with or blocks collagen binding to CD206. In some cases, the immunomodulatory polypeptide can specifically bind to the fibronectin (FBN) domain of CD206. In some cases, the subject immunomodulatory polypeptides bind to the C-type carbohydrate recognition domain (CRD) of CD206 and modulate (eg, activate) the activity of CD206. In some cases, the subject immunomodulatory polypeptides bind to the C-type carbohydrate recognition domain (CRD) of CD206 to modulate (eg, block, block, or inhibit) the activity of CD206. In certain cases, the CRD domain to which the subject immunomodulatory polypeptides specifically bind to modulate the activity of CD206 is the CRD4 or 5 domain.

In certain embodiments, an immunomodulatory polypeptide binds more than one target (eg, a pro-inflammatory target). In some embodiments, the immunomodulatory polypeptide binds three, four, five, or more pro-inflammatory targets. For example, an immunomodulatory polypeptide can bind to any combination of targets disclosed herein. Such binding can be based on in silico, in vitro, or in vivo data. Thus, an immunomodulatory polypeptide comprises two or more NF-kB class II subunits (e.g., RelB and at least one other NF-kB class II subunit, such as RelA, cRel, NF-kB1, or NF-kB2). unit). Alternatively (or in addition), the immunomodulatory polypeptide comprises an NF-kB class II subunit (e.g., RelB) as well as at least one other signaling molecule (e.g., TGFβ, Notch1, Wnt8R, TRAIL, IL6R, IL10R, at least one signaling molecule selected from the group consisting of EGFR, CDK6, CD206, CD47, SIRP-α, HMT, and TGM2). For example, the immunomodulatory polypeptide comprises an NF-kB class II subunit (e.g., RelB) and at least one signaling member selected from the group consisting of TGFβ, Notch1, Wnt8R, TRAIL, IL6R, IL10R, EGFR, and CDK6. Can bind to molecules. Alternatively, the immunomodulatory polypeptide is capable of binding an NF-kB class II subunit (e.g., RelB) and at least one signaling molecule selected from the group consisting of CD206, CD47, SIRP-α, and TGM2. can. In other alternatives, the immunomodulatory polypeptide can bind to the NF-kB class II subunit (eg, RelB) and HMT. In other alternatives, the immunomodulatory polypeptide comprises at least one signaling molecule selected from the group consisting of TGFβ, Notch1, Wnt8R, TRAIL, IL6R, IL10R, EGFR, and CDK6, as well as CD206, CD47, SIRP-α , and TGM2. In another alternative, the immunomodulatory polypeptide binds to at least one signaling molecule selected from the group consisting of TGFβ, Notch1, Wnt8R, TRAIL, IL6R, IL10R, EGFR, and CDK6, and also binds to HMT. be able to. In yet other embodiments, the immunomodulatory polypeptide is at least selected from the group consisting of NF-KB class II subunit (e.g., RelB), TGFβ, Notch1, Wnt8R, TRAIL, IL6R, IL10R, EGFR, and CDK6. It can also bind to at least one signaling molecule selected from the group consisting of CD206, CD47, SIRP-α, and TGM2, and HMT. In certain embodiments, the immunomodulatory polypeptide also binds two or more pro-inflammatory targets and serum albumin (eg, human serum albumin).

The immunomodulatory polypeptides of the present disclosure provide a powerful tool for reducing inflammation and/or treating conditions associated with excessive inflammation (whether acute or chronic). As used herein, the terms "treat," "treating," and similar words mean stabilizing, reducing symptoms of, preventing the occurrence of, or curing a medical condition. It shall be.

Accordingly, the present disclosure reduces the expression level and/or activity of at least one (e.g., 2, 3, 4, 5, or more) proinflammatory cytokine(s) at a site of inflammation in a subject. provide a method. The method includes administering to a subject an immunomodulatory polypeptide of the present disclosure (or, eg, a pharmaceutical composition comprising an immunomodulatory polypeptide). Pro-inflammatory cytokines consist of NF-kB, TNFα, IL-1, IL-6, IL-8, IL-12, IL-17, IL-23, MCP-1, MMP-1, and MMP-9 can be selected from the group. The reduction can be at least a 10% (eg, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more) reduction in cytokine expression or activity.

The present disclosure also provides that the expression level and/or activity of at least one (e.g., 2, 3, 4, 5, or more) proinflammatory cytokine(s) at a site of potential inflammation in a subject is A method of inhibiting the increase is also provided. The method includes administering to a subject an immunomodulatory polypeptide of the present disclosure (or, eg, a pharmaceutical composition comprising an immunomodulatory polypeptide). Pro-inflammatory cytokines consist of NF-kB, TNFα, IL-1, IL-6, IL-8, IL-12, IL-17, IL-23, MCP-1, MMP-1, and MMP-9 can be selected from the group. The method increases cytokine expression and/or activity by 20% or less (e.g., 15%, 12.5%, 10%, 7.5%, 5%, 4%, 3%, 2%, 1%, or Such an increase can be inhibited by limiting the amount (below).

Modulation of the levels and/or activity of pro-inflammatory cytokine(s) at a site of inflammation in a subject may optionally involve the use of targeted immune cells (e.g., effector T cells, regulatory T cells (Tregs), natural killer cells). It will be appreciated that downstream regulation of the activity of cells (NK cells, B cells, etc.) and control of targeted immune or inflammatory responses can be provided.

The present disclosure also provides methods of treating or preventing conditions associated with chronic inflammation. Conditions associated with chronic inflammation include irritable bowel disease, ulcerative colitis, colitis, Crohn's disease, idiopathic pulmonary fibrosis, asthma, keratitis, arthritis, osteoarthritis, rheumatoid arthritis, autoimmune diseases, and cats. or human immunodeficiency virus (FIV or HIV) infection, cancer, age-related inflammation and/or stem cell dysfunction (e.g., age-related increase in Nlrp3 expression, age-related increase in SOCS3 in muscle stem cells, etc.), Graft-versus-host disease (GVHD), keloids, scleroderma, obesity, diabetes, diabetic wounds, other chronic wounds, atherosclerosis, multiple sclerosis, Parkinson's disease, Alzheimer's disease, macular degeneration, gout , gastric ulcers, gastritis, mucositis, toxoplasmosis, and chronic viral or microbial infections, such as chronic bacterial or protozoal infections. The method comprises administering an immunomodulatory polypeptide of the present disclosure (or, e.g., a pharmaceutical composition comprising an immunomodulatory polypeptide) to a subject suffering from or likely to develop a disease condition. include.

The disclosure also provides methods of treating or preventing fibrosis. Fibrosis can be, for example, pulmonary fibrosis, skin fibrosis, liver fibrosis, renal fibrosis, or fibrosis caused by ionizing radiation. The method comprises administering an immunomodulatory polypeptide of the present disclosure (or, e.g., a pharmaceutical composition comprising an immunomodulatory polypeptide) to a subject suffering from or likely to develop fibrosis. including.

The disclosure also provides methods of treating cancer. Cancers include colon cancer, breast cancer, leukemia, lymphoma, ovarian cancer, prostate cancer, liver cancer, lung cancer, testicular cancer, cervical cancer, bladder cancer, endometrial cancer, kidney cancer, melanoma, thyroid cancer, or brain cancer. Or it could be eye cancer. The method includes administering an immunomodulatory polypeptide of the present disclosure (or, eg, a pharmaceutical composition comprising an immunomodulatory polypeptide) to a subject suffering from cancer. The subject matter of the present disclosure also provides methods for treating solid tumor cancer in a subject. In some embodiments, the method includes administering to the subject a therapeutically effective amount of a compound disclosed herein.

For any of the aforementioned methods, the subject may be a domestic animal (e.g., horse, cow, pig, goat, sheep, rabbit, chicken, turkey, duck, etc.), a pet (e.g., dog, cat, rabbit, hamster, etc.) gerbils, birds, fish, etc.), laboratory animals (e.g. mice, rats, monkeys, chimpanzees, owls, fish, etc.), zoo animals (e.g. gorillas, orangutans, chimpanzees, monkeys, elephants, camels, zebras, wild boars, lions, The animal may be an animal such as a tiger, giraffe, bear, bird, etc.), a wild animal (eg, deer, big wolf, mountain lion, bird, etc.), or a human.

In combination with any of the aforementioned methods, the immunomodulatory polypeptide(s) can be administered at a dose and frequency that depends on the species of animal, the size of the animal, and the condition being treated. Typically, the immunomodulatory polypeptide will be about 1 mg to about 1000 mg (eg, about 5 mg to about 900 mg, about 5 mg to about 800 mg, about 5 mg to about 700 mg, about 5 mg to about 600 mg, about 10 mg to about 500 mg, about 10 mg to about 400 mg, about 10 mg to about 300 mg, about 10 mg to about 250 mg, about 10 mg to about 200 mg, about 10 mg to about 150 mg, about 10 mg to about 100 mg, about 50 mg to about 500 mg, about 50 mg to about 400 mg, about 50 mg to about about 300 mg, about 50 mg to about 250 mg, about 50 mg to about 200 mg, about 50 mg to about 150 mg, about 50 mg to about 100 mg, about 75 mg to about 500 mg, about 75 mg to about 400 mg, about 75 mg to about 300 mg, about 75 mg to about 250 mg , about 75 mg to about 200 mg, about 75 mg to about 150 mg, about 75 mg to about 100 mg, about 100 mg to about 500 mg, about 100 mg to about 400 mg, about 100 mg to about 300 mg, about 100 mg to about 250 mg, about 100 mg to about 200 mg, or (or any other range containing two of the above-mentioned endpoints) once daily (or every other day or once a week). The daily dose may be administered once during the day or may be divided into smaller doses taken at multiple times during the day. In humans (and other similarly sized mammals), a dose of 5 mg/kg can be administered every other day. Immunomodulatory polypeptides may be administered over a period of time (e.g., 2-3 weeks), at intervals (e.g., administer polypeptide for 2-3 weeks, wait 2-3 weeks, then repeat the cycle), or during inflammation. It can be administered until such time that promotive cytokine levels are reduced or stabilized, chronic inflammatory conditions or fibrosis are reversed, or the cancer is in remission.

Administration of an immunomodulatory polypeptide (or a pharmaceutical composition comprising such a polypeptide) in combination with any of the aforementioned methods may be via intravenous, intraperitoneal, parenteral, orthotopic, subcutaneous, topical, inhalation. , by nasal, oral, sublingual, intraocular, implantable depot means, nanoparticle-based delivery systems, microneedle patches, microparticles, beads, osmotic or mechanical pumps, and/or other mechanical means. It can be executed using

In combination with any of the foregoing methods, an immunomodulatory polypeptide (e.g., as described herein) (or a pharmaceutical composition comprising such a polypeptide) reduces or prevents inflammation or chronic It can be administered in combination with another drug designed to treat or prevent inflammation or fibrosis, or to treat cancer. In either case, the immunomodulatory polypeptide can be administered before, simultaneously with, or after administration of the other drug. For the treatment of cancer, immunomodulatory polypeptide(s) may include taxanes, nucleoside analogs, steroids, anthracyclines, thyroid hormone replacement drugs, thymidylate targeting drugs, chimeric antigen receptor/T cell therapy, chimeric antigen receptors, etc. body/NK cell therapy, apoptosis regulator inhibitors (e.g., B-cell CLL/lymphoma 2 (BCL-2) BCL-2-like 1 (BCL-XL) inhibitors), CARP-1/CCAR1 (cell division cycle and apoptosis Regulator 1) selected from the group consisting of inhibitors, colony stimulating factor-1 receptor (CSF1R) inhibitors, CD47 inhibitors, cancer vaccines (e.g. Th17-induced dendritic cell vaccines), as well as other cell therapies It can be administered in combination with additional therapeutic agents (eg, chemotherapeutic agents or immunomodulatory agents). Certain chemotherapeutic agents include, for example, gemcitabine, docetaxel, bleomycin, erlotinib, gefitinib, lapatinib, imatinib, dasatinib, nilotinib, bosutinib, crizotinib, ceritinib, trametinib, bevacizumab, sunitinib, sorafenib, trastuzumab, ad-trastuzumab emtansine, They include rituximab, ipilimumab, rapamycin, temsirolimus, everolimus, methotrexate, doxorubicin, Abraxane, forfilinox, cisplatin, carboplatin, 5-fluorouracil, Teismo, paclitaxel, prednisone, levothyroxine, pemetrexed, navitoclax, and ABT-199.

In some embodiments, the immunomodulatory polypeptides that find use in combination therapy are peptides that have macrophage modulating activity (eg, as described herein). In certain cases, the immunomodulatory polypeptide is a CD206 binding peptide (eg, as described herein). In some embodiments, the immunomodulatory polypeptides that find use in combination therapy are the peptides of Table 3. In certain cases, immunomodulatory peptides (eg, the peptides of Table 3) can be administered in combination with chemotherapeutic agents to treat cancer. In certain cases, the chemotherapeutic agent is gemcitabine. In some cases, the chemotherapeutic agent is docetaxel. In some cases, the chemotherapeutic agent is Abraxane.

For the treatment of cancer (eg, melanoma, non-small cell lung cancer, or lymphoma such as Hodgkin's lymphoma), the immunomodulatory polypeptide(s) can be administered in combination with an immunotherapeutic agent. An immunotherapeutic agent is any convenient agent that finds use in the treatment of disease by inducing, enhancing, or suppressing an immune response. In some cases, the immunotherapeutic agent is an immune checkpoint inhibitor. Any convenient checkpoint inhibitor may be used, including but not limited to cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) inhibitors, programmed death 1 (PD-1) inhibitors, and PD-L1 inhibitors. , may be utilized in combination with the subject peptides. Exemplary checkpoint inhibitors of interest include, but are not limited to, ipilimumab, pembrolizumab, and nivolumab. In certain embodiments, for the treatment of cancer and/or inflammatory diseases, the immunomodulatory polypeptide(s) can be administered in combination with a colony stimulating factor-1 receptor (CSF1R) inhibitor. CSF1R inhibitors of interest include, but are not limited to, emactuzumab.

Any convenient cancer vaccine therapy and agent can be used in combination with the subject immunomodulatory polypeptide compositions and methods. For the treatment of cancer, e.g., ovarian cancer, the immunomodulatory polypeptide(s) can be administered in combination with vaccine therapy, e.g., a dendritic cell (DC) vaccination agent that promotes Th1/Th17 immunity. . Th17 cell infiltration is correlated with significantly prolonged overall survival in ovarian cancer patients. In some cases, immunomodulatory polypeptides find use as adjuvant treatments in combination with Th17-inducing vaccination.

Rishi et al. , Journal of Biomedical Nanotechnology, Volume 11, Number 9, September 2015, pp. 1608-1627 (20), CARP-1/CCAR1 (cell division cycle and apoptosis regulator 1) inhibitors, and anti-CD47 antibody agents such as Hu5F9-G4. Also of interest are agents that are CD47 inhibitors, including but not limited to.

In certain instances, the combination provides an enhanced effect compared to either component alone, and in some cases the combination provides an enhanced effect compared to the combined or additive effects of the components. Provide additive or synergistic effects. Various combinations of subject polypeptides and chemotherapeutic agents can be used, either sequentially or simultaneously. For multiple doses, the two agents may be used in direct alternation, or two or more doses of one agent may be alternated with, for example, a single dose of the other agent. Simultaneous administration of both drugs may also be repeated alternately or the dosages of the individual drugs may be distributed differently. In some cases, the time between dosing is from about 1 to 6 hours, up to about 6 to 12 hours, up to about 12 to 24 hours, up to about 1 to 2 days, up to about 1 to 2 weeks after the start of treatment. or even longer.

In some embodiments, a method of reducing cancer cell proliferation comprises contacting a cell with an effective amount of a subject immunomodulatory polypeptide (e.g., as described herein). It's a method. The methods can be practiced in combination with chemotherapeutic agents (eg, as described herein). Cancer cells can be in vitro or in vivo. In certain cases, the method includes contacting the cell with an immunomodulatory peptide (eg, a peptide of Table 3) and contacting the cell with a chemotherapeutic agent. Any convenient cancer cell can be targeted. In certain cases, the chemotherapeutic agent is gemcitabine. In some cases, the chemotherapeutic agent is docetaxel. In some cases, the chemotherapeutic agent is Abraxane.

Alternatively, for methods of treating cancer, the immunomodulatory polypeptide(s) (or pharmaceutical compositions comprising such polypeptides) can be administered in combination with radiation therapy. Again, the immunomodulatory polypeptide(s) may be administered before or after the administration of radiation therapy.

Any of the foregoing methods of this disclosure further include the step of evaluating the effectiveness of the therapeutic treatment. The immunomodulatory polypeptides of the present disclosure have demonstrable ability to reduce tissue inflammation and suppress overproduction of inflammatory mediators such as IL-1, IL-6, IL-12, and TNFα, both in tissue and serum. (data not shown), the effectiveness of therapeutic treatment can be determined by measuring the levels of such cytokines (e.g., in serum) to determine whether the levels responded appropriately to treatment. It can be evaluated by Depending on cytokine levels, the dosage of the immunomodulatory polypeptide(s) can be adjusted up or down as needed.

Definitions It is to be understood that this invention is not limited to the particular embodiments described herein, which as such may vary, of course. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the invention is limited only by the claims appended hereto. It should also be understood that this is not the case.

When a range of values is provided, unless the context clearly dictates otherwise, each intervening value up to one-tenth of a unit between the upper and lower limits of the range, and within that stated range. It is to be understood that any other mentioned or intervening values of are encompassed by the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed by the invention, subject to any specifically excluded limit within the stated range. . Where the stated range includes one or both of the limits, ranges excluding one or both of those included limits are also included in the invention.

Certain ranges are presented herein in which numerical values are preceded by the term "about." The term "about" is used herein to provide literal support for the exact number to which it precedes, as well as to a number that is near or approximates the number to which it precedes. In determining whether a number is close to or approximates a specifically enumerated number, the unenumerated number that is close to or approximates the specifically enumerated number in the context in which it is presented. may be a number that provides a substantial equivalent of the number given.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative exemplary methods and materials are described herein. written in the book.

All publications and patents cited herein are herein incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. , is incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. Citation of any publication is for its disclosure prior to the filing date and is not to be construed as an admission that the invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may differ from the actual publication dates, which may need to be independently confirmed.

It is noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. sea bream. It is further noted that the claims may be drafted to exclude any optional element. This statement therefore serves as an antecedent to the use of exclusive terms such as "merely," "only," or "negative" limitations in connection with the recitation of claim elements. It is intended that

As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has individual components and features that are within the scope or spirit of the invention. can be easily separated from or combined with features of any of the other embodiments without departing from the invention. Any recited method can be performed in the order in which the events are recited or in any other order that is logically possible.

Subject methods and materials that find use in the preparation and evaluation of the subject immunomodulatory peptides include those disclosed in the experimental section of WO2016/061133 by Jaynes et al., the disclosure of which is incorporated herein in its entirety. Incorporated into the specification.

The following examples are presented to provide those skilled in the art with a complete disclosure and description of how to make and use the invention, and are not intended to limit the scope of what the inventors consider to be their invention. , nor is it intended to represent that the following experiments are all or the only experiments performed.

Example 1: Inhibition of Tumor Growth Polypeptides of the present disclosure are also tested for their effect on tumor growth in a mouse model of non-metastatic breast cancer. MCF-7 human non-metastatic breast cancer cells are cultured in normal growth medium at 37°C, 5% _CO2 . Cells are harvested at 80%-90% confluence. Immune-compromised athymic nude mice (J:NU) are divided into two groups (9 mice per group). All mice are injected with approximately 4.5×10 ⁶ MCF-7 cells stained with VIVO Tracker 680 and suspended in 200 μl of PBS/Matrigel mixture. Cells are injected subcutaneously onto the dorsal surface of the treated animal using a 22 gauge needle fitted with a 500 μl syringe.

Animals are treated with the indicated vehicle and peptide. Animals treated with peptides are treated with the subject polypeptides. Freshly prepared peptides were dissolved in sterile saline at a concentration of 100 μM and used to treat animals in the peptide group. Animals treated with vehicle are injected with saline buffer alone. All treatments are injected into the tumor mass twice a week for 5 weeks using a 27.5 gauge needle fitted with a 1 ml syringe. Animal body weight and tumor volume are measured three times per week and fluorescent labeling is followed by VIVO Tracker 680 and IVIS imaging.

Figure 2 shows that peptides RP832C and RP185 reduce tumor volume in a mouse tumor inhibition model. The data demonstrate that polypeptides of the present disclosure inhibit tumor growth in vivo.

Example 2: Administration of peptides in combination with chemotherapy Considering the significant role of inflammation in tumor development and metastasis, and the known association between M2 macrophage activity and tumor development, peptides of the present disclosure (e.g., Table 3 It was anticipated that the administration of selected peptides) could have a positive impact on the outcome of cancer treatment.

To test this theory, approximately 5×10 ⁶ human triple-negative breast cancer cells (MDA-MB-231) are injected under the left upper nipple of a cohort of immunocompromised (“nude”) mice. Following this administration, one cohort received vehicle only and two of the cohorts received the chemotherapeutic agent gemcitabine at a once-every-four-day dose of 40 mg/kg body weight. One of these cohorts also received the test peptide at a daily dose of 5 mg/kg body weight, and the fourth cohort received only the peptide at a daily dose of 5 mg/kg body weight. Starting on day 32 of the study, the concentration of RP-182 will be increased to 20 mg/kg body weight in the gemcitabine + RP-182 cohort. Tumor volume is assessed at various time points after initial cell administration. After 50 days, mice are sacrificed.

In a second experiment, xenografts of C42B prostate cancer cells are introduced into four cohorts of mice, and tumors are allowed to grow to approximately 100 m ³ before treatment. One cohort was treated with vehicle alone, a second cohort was treated with docetaxel at 2.5 mg/kg body weight once a week, and a third cohort was treated with docetaxel at 10 mg/kg body weight subcutaneously once a day. A fourth cohort is treated with both 2.5 mg/kg docetaxel once a week and 10 mg/kg once daily of the test peptide. Tumor volumes are assessed at various time points after initial cell administration, and mice are sacrificed 27 days later. .

Peptides of the present disclosure (e.g., selected peptides of Table 3) produce synergistic effects when administered with chemotherapeutic agents, including gemcitabine and docetaxel, as well as checkpoint inhibitor therapy and other immunotherapies. is expected. In particular, the peptides of the present disclosure may be particularly useful when used in combination with recently developed CAR-T (chimeric antigen receptor/T cell) therapies. Such therapy, while destroying tumor cells, results in a very high systemic burden of dead cell material, overstimulating the immune system and resulting in a "cytokine storm" that can be fatal to the patient.

Example 3: Investigation of selected peptides

Example 4: Selective effects of targeted RP peptides on scleroderma macrophage survival Peripheral blood-derived macrophages were cultured from healthy volunteers and scleroderma patients and treated with arginase 1 (M2 marker) or IFNg (M1 cells were cultured in MCS-F for 7 days before qPCR for markers) and then treated with RP peptides ranging in dose from 0 to 100 uM. After 48 hours, the medium was changed and cells were re-treated with peptide. After 96 hours, cell viability was assayed by PrestoBlue assay. (FIG. 4A) Macrophages from healthy controls with a low arginase:IFNg ratio of 3.6 were resistant to the effects of the targeted RP peptides ((RP182, RP185, RP832C, RP837) on viability. (Figure 4B): In a scleroderma (SSc) patient with an elevated arginase:IFNg ratio of 8.8, the RP peptides of interest (RP182, RP185, RP832C, RP837) were administered at 0.01 uM and also significantly reduced survival at 96 hours.

Example 5: Bleomycin Pulmonary Fibrosis Rescue Intratracheal instillation (IT) of bleomycin was used as a model of pulmonary fibrosis. The rescue of bleomycin-induced pulmonary fibrosis in mice by the subject peptides was studied. Experimental parameters: Four groups (n=8) of male C57BL6 mice were studied over a period of 6-8 weeks. Bleomycin at 2.5 U/kg was administered IT in a single bolus. After 72 hours, 1 mg/kg of the target peptides (RP182, RP185, RP832, RP837) were administered IT once every 2 days. In this bleomycin challenge experiment, the fibrosis measurement is the Ashcroft score after trichrome staining. Collagen score is a quantitative measurement after hydroxyproline staining. Figure 1 shows a graph of these results.

The subject peptides reduced fibrosis and collagen deposition. Hematoxylin and eosin (H/E) and Mason's trichrome staining of lung tissue sections was performed. The alveoli of the vehicle group appear to be surrounded by fibrotic tissue with increased collagen deposition, unlike the alveoli of the treated and naive lung groups. Body weight change: The body weight of the RP peptide treatment group showed no significant change compared to the body weight of the vehicle treatment group. Ashcroft Score Analysis: Unlike the peptide treatment group, which exhibited well-organized lung structure and was therefore assigned a lower score, lung specimens from the vehicle group showed deformed lungs with increased fibrosis and collagen deposition. It was assigned a score close to 6 because it showed structure.

Fibrosis and collagen deposition level assessment: There was a significant reduction in fibrosis and collagen deposition in the treatment group compared to the vehicle group as measured by ImageJ software. Lung weight changes: Vehicle group had higher lung weight compared to peptide treated group due to fibrosis and decreased collagen content in peptide treated group. IHC staining for TGFβ1 and αSMA: Lung tissue treated with peptides showed a significant decrease in fibrosis-related markers of TGFβ1 and αSMA.

These results demonstrate that the exemplary peptides provided reduction in bleomycin-induced pulmonary fibrosis in a mouse model of pulmonary fibrosis.

Example 6: Cooperation of targeted RP peptides with PD-1 checkpoint inhibitors in CT26 xenografts Figure 2 shows that targeted RP peptides collaborate with PD-1 checkpoint inhibitors in CT26 xenografts. The results of a study of the effect on tumor volume are shown. Peptides RP832C and RP185 were administered once daily at 10 mg/kg. Anti-PD1 antibody was administered intraperitoneally at 200 ug twice a week. The results provided in the examples demonstrate the effectiveness of the peptides of the invention.

Notwithstanding the appended claims, the following notes are provided to illustrate aspects of the disclosure.

An immunomodulatory peptide 1.5 to 30 (e.g., 6 to 30 or 6 to 18) amino acid residues in length,
two or more (e.g., three or more or four or more) hydrophobic modules and one or more (e.g., two or more or three or more) hydrophilic modules each comprising at least one cationic residue. (e.g., having a length of 6 to 12 or 6 to 10 amino acid residues), described by one of formulas 1 to 7, which adopts an amphipathic conformation under physiological conditions, including An immunomodulatory peptide that contains striapathic regions of hydrophilic and hydrophobic modules and specifically binds to CD206.
2. The striapathic region is that of Equation 5: [J _1a J _1b ]-[X _1a X _1b ]-[J _2a J _2b ]-[X _2a X _2b ]-[J _3a ]-[X _3a ] (Formula 5A), where J _1a , J _1b , J _2a , J _2b , and J _3a each independently represent a hydrophobic amino acid residue (e.g., phenylalanine, tryptophan , alanine, valine, and glycine), and X _1a , X _1b , X _2a , X _2b , and X _3a are each independently selected from hydrophilic amino acid residues (e.g., lysine, arginine, histidine, aspartic acid, 1).
3. J _1a , J _1b , J _2a , J _2b , and J _3a are each independently selected from phenylalanine, tryptophan, alanine, and glycine, and each of X _1a , X _1b , X _2a , X _2b , and X _3a is Immunomodulatory peptide according to appendix 2, independently selected from lysine and arginine.
4. J _1a , J _1b , J _2a , J _2b , and J _3a are each independently selected from phenylalanine, tryptophan, alanine, and valine, and each of X _1a , X _1b , X _2a , X _2b , and X _3a is Immunomodulatory peptide according to appendix 2, independently selected from ornithine, lysine and arginine.
5. _{Formula : FAX 1a X 1b FAX 2a _ _ _} The immunomodulatory peptide according to any one of appendices 2 to 4, selected from ornithine, lysine, and arginine.

6. The immunomodulatory peptide according to appendix 5, having the sequence FAOOFAOOFO (SEQ ID NO: 19) (RP850).
7. Supplementary Notes 2 to 4 having the sequence defined by the formula: FWKX _1b FVX _2a KWX _3a (SEQ ID NO: 31), where X _1b , X _2a , and X _3a are each independently lysine or arginine; The immunomodulatory peptide according to any one of.
8. The immunomodulatory peptide according to appendix 7, having a sequence selected from FWKRFVRKWR (SEQ ID NO: 4) (RP837) and FWKKFVKKWK (SEQ ID NO: 7) (RP841).
9. J _1a , J _1b , J _2a , J _2b , and J _3a are each tryptophan, and X _1a , X _1b , X _2a , X _2b , and X _3a are each independently derived from histidine, lysine, and arginine. The immunomodulatory peptide according to appendix 8, which is selected.
10. having the sequence defined by the formula: WWX _1a HWWHX _2b WX _3a (SEQ ID NO: 32), where X _1a , X _2b , and X _3a are each independently histidine, lysine, or arginine; 9. The immunomodulatory peptide according to 9.

11. The immunomodulatory peptide according to appendix 10, having a sequence selected from WWHHWWHHWH (SEQ ID NO: 13), WWRHWWHRWR (SEQ ID NO: 14), and WWKHWWHKWK (SEQ ID NO: 15) (RP847-849).
Appendix 12. The striapathic region is that of equation 6, with the equation: [J _1a ]-[X _1a X _1b ]-[J _2a J _2b ]-[X _2a ]-[J _3a ]-[X _3a X _3b ] - [J _4a J _4b ] (Formula 6A), where J _1a , J _2a , J _2b , J _3a , J _4a , and J _4b each independently represent a hydrophobic amino acid residue. groups (e.g., phenylalanine, tryptophan, alanine, isoleucine, valine, and glycine), and X _1a , X _1b , X _2a , X _3a , and X _3b are each independently selected from hydrophilic amino acid residues (e.g., 1. The immunomodulatory peptide according to claim 1, selected from lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, or glutamine.
13. The immunomodulatory peptide according to appendix 12, having the sequence GDRGIKGHRGF (SEQ ID NO: 8) (RP842).
14. The striapathic region is that of Equation 1, with the equations: [J _1a J _1b ]-[X _1a X _1b ]-[J _2a J _2b ] (Equation 1A) and [J _2b J _2a ]-[X _1b X _1a ]-[J _1b J _1a ] (Formula 1B), where J _1a , J _1b , J _2a , and J _2b are each independently a hydrophobic and wherein X _1a and X _1b are each independently selected from hydrophilic amino acid residues (e.g. lysine or arginine). immunomodulatory peptides.
15. The immunomodulatory peptide according to appendix 14, having the sequence FWKRFV (SEQ ID NO: 5) (RP837N).

16. The striapathic region is of Equation 2 and has an arrangement defined by the equation: [J _1a J _1b ]−[X _1a X _1b ]−[J _2a ]−[X _2a ] (Equation 2A). , where J _1a , J _1b , J _2a , and J _2b are each independently selected from hydrophobic amino acid residues (e.g., phenylalanine, tryptophan, and valine), and X _1a , X _1b , and X _2a 1. The immunomodulatory peptide of claim 1, wherein each is independently selected from hydrophilic amino acid residues such as lysine or arginine.
17. The immunomodulatory peptide according to appendix 16, having the sequence FVRKWR (sequence 6) (RP837C ¹ ).
18. The striapathic region is that of equation 4, and the equation: [J _1a J _1b ]-[X _1a X _1b ]-[J _2a J _2b ]-[X _2a X _2b ]-[J _3a J _3b ]( 4A) and [J _3a J _3b ]-[X _2a X _2b ]-[J _2b J _2a ]-[X _1b X _1a ]-[J _1b J _1a ] (Equation 4B). wherein J _1a , J _1b , J _2a , J _2b , J _3a , and J _3b are each independently selected from hydrophobic amino acid residues, such as phenylalanine, tyrosine, or leucine. 1, wherein X _1a , X _1b , X _2a , and X _2b are each independently selected from hydrophilic amino acid residues (eg, lysine or arginine).
19. The immunomodulatory peptide according to appendix 18, having the sequence LYKKIIKKLL (SEQ ID NO: 12) (RP846).
20. The striapathic region is that of Equation 4: [J _1a J _1b J _1c ]-[X _1a ]-[J _2a J _2b ]-[X _2a X _2b ]-[J _3a J _3b ]( 4C), where J _1a , J _1b , J _1c , J _2a , J _2b , J _3a , and J _3b are each independently a hydrophobic amino acid residue (e.g. phenylalanine, tyrosine, or proline), and X _1a , X _2a , and X _2b are each independently selected from hydrophilic amino acid residues (e.g., aspartic acid, lysine, or arginine). The immunomodulatory peptides described.

21. The immunomodulatory peptide according to appendix 20, having the sequence FYPDFFKKFF (SEQ ID NO: 10) (RP844).
22. The striapathic region is that of Equation 4: [J _1a J _1b ] − [X _1a X _1b ] − [J _2a ] − [X _2a X _2b X _2c ] − [J _3a J _3b ] ( 4D), where J _1a , J _1b , J _2a , J _3a , and J _3b each independently represent a hydrophobic amino acid residue (e.g., phenylalanine, serine, glycine, X _1a , X _1b , X _2a , X _2b , and X _2c are each independently selected from hydrophilic amino acid residues (e.g., glutamic acid, aspartic acid, lysine, or arginine) , the immunomodulatory peptide according to Supplementary Note 1.
23. The immunomodulatory peptide according to appendix 22, having the sequence FFRKSKEKIG (SEQ ID NO: 18) (RP853).
24. The striapathic region is that of Equation 4: [J _1a J _1b ] - [X _1a X _1b ] - [J _2a J _2b ] - [X _2a X _2b ] - [J _3a ] (Equation 4E ), where J _1a , J _1b , J _2a , J _2b , and J _3a are each independently derived from a hydrophobic amino acid residue (e.g., phenylalanine, alanine, or isoleucine) 1, wherein X _1a , X _1b , X _2a , X _2b , and X _2c are each independently selected from hydrophilic amino acid residues (e.g., ornithine, lysine, or arginine). Regulatory peptides.
25. The striapathic region is that of Equation 5, and the equation: [J _1a ]-[X _1a ]-[J _2a J _2b J _2c ]-[X _2a ]-[J _3a J _3b ]-[X _3a X _3b ] (Formula 5C), where J _1a , J _2a , J _2b , J _2c , J _3a , and J _3b each independently represent a hydrophobic amino acid residue (e.g. phenylalanine, leucine, glycine, or isoleucine), and X _1a , X _2a , X _3a , and X _3b are each independently selected from hydrophilic amino acid residues (e.g., glutamine, lysine, or histidine) , the immunomodulatory peptide according to Supplementary Note 1.

26. The immunomodulatory peptide according to appendix 25, having the sequence FQFLGKIIHH (SEQ ID NO: 17) (RP852).
27. _The striapathic _{region is} that of _equation 7 _{, with the} equation _: [X _1a (Formula 7A), where J _1a , J _2a , J _3a , J _3b , and J _3c each independently represent a hydrophobic amino acid residue (e.g., isoleucine, valine, leucine). , serine, or alanine), and X _1a , X _1b , X _2a , and X _3a are each independently selected from hydrophilic amino acid residues (e.g., lysine or arginine). Immunomodulatory peptides.
28. The immunomodulatory peptide according to appendix 27, having the sequence KKIRVRLSA (SEQ ID NO: 16) (RP851).
29. The striapathic region is that of equation 5, with the equation: [J _1a J _1b ]-[X _1a X _1b ]-[J _2a J _2b J _2c ]-[X _2b ]-[J _3a ]-[X _3a ] (Formula 5B), where J _1a , J _1b , J _2a , J _2b , and J _3a each independently represent a hydrophobic amino acid residue (e.g., phenylalanine, alanine , threonine, or leucine), and X _1a , X _1b , X _2a , X _2b , and X _3a are each independently selected from a hydrophilic amino acid residue (e.g., histidine, aspartate, lysine, or arginine). An immunomodulatory peptide according to supplementary note 1, which is selected.
30. The immunomodulatory peptide according to appendix 29, having the sequence FFRHFATHLD (SEQ ID NO: 11) (RP845).

31. _The striapathic region is that of Equation 3: [X _1a X _{1b ]} - _[ J _1a J _1b J _1c J _1d ] - [X _2a wherein J _1a , J _1b , J _1c , J _1d , J _2a , and J _2b each independently represent a hydrophobic amino acid residue (e.g., leucine, serine, alanine, or phenylalanine), and X _1a , X _1b , X _2a , and X _2b are each independently selected from hydrophilic amino acid residues (e.g., glutamic acid, aspartic acid, lysine, asparagine, or arginine), The immunomodulatory peptide according to Supplementary Note 1.
32. The immunomodulatory peptide according to appendix 31, having the sequence EKLSAFRNFF (SEQ ID NO: 9) (RP843).
33. The striapathic region comprises a dimer of the first and second polypeptides connected via a peptide linker connecting the C-terminus of the first polypeptide and the N-terminus of the second polypeptide. The immunomodulatory peptide according to Supplementary Note 1.
34. 34. The immunomodulatory peptide according to claim 33, wherein the hydrophilic module consists of amino acid residues selected from lysine, arginine, and ornithine, and the hydrophobic module consists of amino acid residues selected from phenylalanine and tryptophan.
35. The first and second polypeptides have the following formula: [X1]-[J1]-[X2]-[J2] (Formula 3) or [J1]-[X1]-[J2]-[X2]( 35. An immunomodulatory peptide according to claim 33 or 34, comprising one of formula 2).

36. The dimer has the following formula: [X1]-[J1]-[X2]-[J2]-T-[J2]-[X2]-[J1]-[X1] or [J1]-[X1] -[J2]-[X2]-T-[X2]-[J2]-[X1]-[J1], wherein T is a peptide linker, Supplementary notes 33 to 35 The immunomodulatory peptide according to any one of.
37. The dimer has the following formula: [X _1a ]-[J _1a ]-[X _2a ]-[J _2a ]-T-[J _2a ]-[X _2a ]-[J _1a ]-[X _1a ] (Formula 12A) or [J _1a ]-[X _1a ]-[J _2a ]-[X _2a ]-T-[X _2a ]-[J _2a ]-[X _1a ]-[J _1a ] (Formula 13A) 37, wherein T is a peptide linker, such as a polyglycine linker.
38. 38. The immunomodulatory peptide according to appendix 37, having a sequence selected from RWKFGGFKWR (SEQ ID NO: 1) (RP832C) and FKWRGGRWKF (SEQ ID NO: 3) (RP837C).
39. _The dimer _has the following formula: [X _1a X _1b ]-[J _1a J _1b ]-T-[ _{J 1b} J _1a ]-[X _1b [ _{X 1a} X _1b ] _-T- [ _{X 1b} , J _1a and J _1b are each independently selected from hydrophobic amino acid residues (e.g., tryptophan or phenylalanine), and X _1a and X _1b are each independently selected from hydrophilic amino acid residues (e.g., asparagine). or arginine), the immunomodulatory peptide according to appendix 33 or 34.
40. The immunomodulatory peptide according to appendix 39, having the sequence FWKRGGRKWF (SEQ ID NO: 4) (peptide 837A).

41. a) a sequence selected from the peptide sequences in Table 3;
b) a sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, or at least 95% sequence identity) with a sequence defined in a), or c) a) a sequence with one or two amino acid substitutions compared to a sequence defined in 41. An immunomodulatory peptide according to any one of appendices 1 to 40, comprising the sequence (substitution or highly conservative amino acid substitution).
42. The immunomodulatory peptide according to any one of appendices 1 to 41, consisting of a sequence selected from any one of the sequences in Table 3 (SEQ ID NOs: 1 to 19).

An immunomodulatory peptide having a length of 43.6 to 30 amino acid residues,
a) a peptide sequence selected from SEQ ID NO: (1-19) (e.g. RP832C, 837, 837A, 837C, 837N, 841-842, 843-850, and 853); or b) a sequence as defined in a). a sequence having one or two amino acid substitutions compared to an immunomodulatory peptide comprising the sequence (conservative amino acid substitutions).
44. One or two amino acid substitutions as defined in b) result from the substitution of a cationic amino acid of the sequence with an alternative cationic amino acid residue (e.g. K for O, O for K, K for R, etc.) The immunomodulatory peptide according to appendix 43.
45. 44. The immunomodulatory peptide of claim 43, comprising the peptide sequences selected from RWKFGGFKWR (RP832C) (SEQ ID NO: 1), FKWRGGRWKF (RP837C) (SEQ ID NO: 3), and FWKRGGRKWF (RP837A) (SEQ ID NO: 4).

46. 44. The immunomodulatory peptide of claim 43, comprising a peptide sequence selected from FWKRFV (RP837N) (SEQ ID NO: 5) and FVRKWR (RP837C ¹ ) (SEQ ID NO: 6).
47. 44. The immunomodulatory peptide of claim 43, comprising a peptide sequence selected from FAOOFAOOFO (RP850) (SEQ ID NO: 19), FWKRFVRKWR (RP837) (SEQ ID NO: 4), and FWKKFVKKWK and (RP841) (SEQ ID NO: 7).
48. 44. The immunomodulatory peptide of claim 43, comprising a peptide sequence selected from WWHHWWHHWH (SEQ ID NO: 13), WWRHWWHRWR (SEQ ID NO: 14), and WWKHWWHKWK (SEQ ID NO: 15) (RP847-849).
49. The immunomodulatory peptide according to appendix 43, comprising the peptide sequence GDRGIKGHRGF (RP842) (SEQ ID NO: 8).
50. The immunomodulatory peptide according to appendix 43, comprising the peptide sequence LYKKIIKKLL (RP846) (SEQ ID NO: 12).

51. The immunomodulatory peptide according to appendix 43, comprising the peptide sequence FYPDFFKKFF (RP844) (SEQ ID NO: 10).
52. The immunomodulatory peptide according to appendix 43, comprising the peptide sequence FFRKSKEKIG (RP853) (SEQ ID NO: 18).
53. The immunomodulatory peptide according to appendix 43, comprising the peptide sequence FFRHFATHLD (RP845) (SEQ ID NO: 11).
54. The immunomodulatory peptide according to appendix 43, comprising the peptide sequence EKLSAFRNFF (RP843) (SEQ ID NO: 9).

55. An immunomodulatory peptide (e.g., 12 amino acid residues or less in length),
An immunomodulatory peptide comprising a sequence selected from RWKFGGFKWR (RP832C) (SEQ ID NO: 1), FKWRGGRWKF (RP837C) (SEQ ID NO: 3), and FWKRGGRKWF (RP837A) (SEQ ID NO: 4).
56. The immunomodulatory peptide according to appendix 55, consisting of the sequence RWKFGGFKWR (RP832C) (SEQ ID NO: 1).
57. The immunomodulatory peptide according to appendix 55, consisting of the sequence FKWRGGRWKF (RP837C) (SEQ ID NO: 3).
58. The immunomodulatory peptide according to appendix 55, consisting of the sequence FWKRGGRKWF (RP837A) (SEQ ID NO: 4).

59. A pharmaceutical composition comprising an immunomodulatory peptide according to any one of appendices 1 to 58 and a pharmaceutically acceptable carrier.
60. 60. The pharmaceutical composition of clause 59, wherein the composition is formulated for oral administration, parenteral administration, administration via inhalation, or topical administration.
61. 61. A pharmaceutical composition according to clause 59 or 60, wherein the composition is formulated for intravenous or subcutaneous administration.
62. 61. The pharmaceutical composition of Clause 59 or 60, wherein the composition is formulated for oral administration and further comprises an enteric coating.
63. Pharmaceutical composition according to clause 59 or 60, wherein the composition is formulated for topical delivery in a form selected from the group consisting of gel suspension, cream, microneedles and injected into a bandage or topical patch. thing.

64. A method of modulating macrophage activity, the method comprising:
A method comprising contacting a macrophage with a CD206 binding agent to modulate macrophage activity.
65. 65. The method of clause 64, wherein the CD206 binding agent binds to a mannose binding site to modulate signal regulatory protein (SIRP)-mannose binding to CD206.
66. 66. The method of clause 64 or 65, wherein the CD206 binding agent binds to CD206 with a binding energy of at least -650 kcal/mol.
67. The CD206 binding agent is Phe-708, Thr-709, Trp-710, Pro-714, Glu-719, Asn-720, Trp-721, Ala-722, Glu-725, Tyr-729, Glu-733, Asn directly contacting at least one amino acid residue of CD206 selected from -747, Asp-748, Ser-1691, Cys-1693, Phe-1694, and Phe-1703, Method described.
68. 68. The method according to any one of clauses 64 to 67, wherein the macrophage activity modulated is macrophage polarization.
69. 69. The method according to any one of appendices 64 to 68, wherein the survival rate of macrophages is reduced.
70. 69. The method according to any one of appendices 64 to 69, wherein the macrophage is an M2 macrophage or a tumor-associated macrophage (TAM).

71. 71. The method according to any one of appendices 64 to 70, wherein the CD206 binding agent inhibits macrophage activity.
72. 72. The method according to any one of appendices 64 to 71, wherein the CD206 binding agent is an immunomodulatory peptide.
73. 72. The method according to any one of appendices 64 to 71, wherein the macrophage is in vitro.
74. 72. The method according to any one of clauses 64 to 71, wherein the macrophage is in vivo.
75. 75. The method according to any one of claims 64 to 74, wherein the CD206 binding agent is an immunomodulatory peptide according to any one of claims 1 to 58.

76. A method of treating a subject for a condition associated with chronic inflammation, the method comprising:
A method comprising administering to a subject an effective amount of a CD206 binding agent (eg, an immunomodulatory peptide according to any one of appendices 1-58) to treat the subject for a condition associated with chronic inflammation.
77. Conditions related to chronic inflammation include scleroderma or multiple sclerosis, irritable bowel disease, ulcerative colitis, colitis, Crohn's disease, idiopathic pulmonary fibrosis, scleroderma, asthma, keratitis, arthritis, Osteoarthritis, rheumatoid arthritis, autoimmune disease, feline or human immunodeficiency virus (FIV or HIV) infection, cancer, age-related inflammation and/or stem cell dysfunction, graft-versus-host disease (GVHD), keloids from obesity, diabetes, diabetic wounds, other chronic wounds, atherosclerosis, Parkinson's disease, Alzheimer's disease, macular degeneration, gout, gastric ulcers, gastritis, mucositis, toxoplasmosis, and chronic viral or microbial infections. 77. The method of claim 76, wherein the method is selected from the group consisting of:
78. 78. The method according to Clause 76 or 77, wherein the CD206 binding agent is an immunomodulatory peptide according to any one of Clauses 1 to 58.
79. The method according to appendix 77, wherein the pathological condition is cancer.
80. 80. The method of clause 79, further comprising administering to the subject an effective amount of an additional agent.

81. 81. The method of clause 80, wherein the additional agent is a chemotherapeutic agent.
82. Chemotherapy agents include gemcitabine, docetaxel, bleomycin, erlotinib, gefitinib, lapatinib, imatinib, dasatinib, nilotinib, bosutinib, crizotinib, ceritinib, trametinib, bevacizumab, sunitinib, sorafenib, trastuzumab, ad-trastuzumab emtansine, rituximab, Ipilimumab, rapamycin , temsirolimus, everolimus, methotrexate, doxorubicin, Abraxane, forfilinox, cisplatin, carboplatin, 5-fluorouracil, Teismo, paclitaxel, prednisone, levothyroxine, and pemetrexed.
83. 82. The method according to appendix 81, wherein the chemotherapeutic agent is Abraxane.
84. 82. The method of clause 81, wherein the chemotherapeutic agent is gemcitabine or docetaxel.
85. 81. The method of clause 80, wherein the additional agent is an immunotherapeutic agent.

86. 86. The method according to appendix 85, wherein the immunotherapeutic agent is an immune checkpoint inhibitor.
87. Claim 86, wherein the immune checkpoint inhibitor is selected from a cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) inhibitor, a programmed death 1 (PD-1) inhibitor, and a PD-L1 inhibitor. Method.
88. 88. The method of clause 87, wherein the immune checkpoint inhibitor is selected from ipilimumab, pembrolizumab, and nivolumab.
89. 77. The method according to appendix 76, wherein the pathological condition associated with chronic inflammation is fibrosis.
90. 77. The method according to appendix 76, wherein the pathological condition associated with chronic inflammation is scleroderma.

91. 91. The method according to any one of clauses 76 to 90, wherein the CD206 binding agent is an immunomodulatory peptide according to any one of clauses 1 to 58.
92. 92. The method of clause 91, wherein the CD206 binding agent consists of an immunomodulatory peptide of Table 3.

Claims (1)

An immunomodulatory peptide,
a) a peptide sequence selected from SEQ ID NO: (1-19); or b) a sequence having one or two amino acid substitutions compared to the sequence defined in a), wherein said one or two An immunomodulatory peptide comprising the sequence: wherein the amino acid substitutions are of the amino acids set forth in Table 2.

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