Modulation of mammalian cell lineage by synthetic immodulins

Abstract

This invention provides synthetic immodulin peptides and related compositions and methods. The peptides of this invention exhibit new and surprising biological activities, such as the expansion of specified differentiated mammalian lineages from precursor cell populations which play important roles in post-apoptotic clearance, autoimmunity, targeted vaccination, cancer and trauma, by contacting mammalian cells with the synthetic peptides. Furthermore, the peptides of the invention can be made significantly more potent by each of a series of modifications described in the invention, including a carboxyterminal tripeptide extension to the canonical immodulin core sequence, other peptide extensions with kinase-inhibiting and other domains, substitution with modified amino acids, conjugation to certain bioactive small molecules, complexation to metals or glycosaminoglycans, and co-administration with helper molecules. The invention also teaches compositions and methods for enhancing previously disclosed uses of immodulin peptides to boost the efficacy of immodulin peptides in trauma, immune imbalance, cancer and other medically relevant conditions.

Claims

1. A method for stimulating the relative abundance or biological activity of a differentiation marker in a mammalian hematopoietic cell lineage in vivo or ex vivo, comprising: (i) contacting one or more live mammalian cells in vivo or ex vivo with a synthetic immodulin peptide, 20-60 amino acids in length, comprising an amino acid sequence corresponding to any one of SEQ ID NOs: 1-7 wherein the synthetic immodulin peptide is covalently attached to a non-amino acid small molecule of molecular mass less than one thousand daltons; and (ii) following the contacting step, measuring the abundance or biological activity of a marker distinctive for the differentiated cell lineage, wherein said marker is CD169; (iii) optimizing the dose of the synthetic immodulin peptide based on said measurement; thereby demonstrating that the optimized dose of the synthetic immodulin peptide stimulated the relative abundance or biological activity of the differentiated cell lineage.

2. The method according to claim 1, wherein the synthetic immodulin peptide comprises an amino acid sequence corresponding to any one of SEQ ID NOs: 4-7.

3. The method according to claim 1, comprising administration to a mammal of a therapeutically effective dose of the synthetic immodulin peptide wherein said therapeutically effective dose of the synthetic peptide is from about 0.01 mg/kg/day to about 50 mg/kg/day.

4. A synthetic immodulin peptide, 20-60 amino acids in length, comprising an amino acid sequence corresponding to any one of SEQ ID NOs: 4-7.

5. The synthetic immodulin peptide according to claim 4, further comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-12.

6. The synthetic immodulin peptide according to claim 4 co-formulated with a metal selected from the group consisting of: ferrous iron, ferric iron, ferrocene, zinc, copper, vanadium, ruthenium, cobalt, titanium, manganese, and calcium.

7. The synthetic immodulin peptide according to claim 4 co-formulated with a molecule selected from the group consisting of: transferrin, gallic acid, methyl gallate, gallocyanine, heparin, chondroitin sulfate, keratan sulfate, dermatan sulfate, and hyaluronic acid.

Description

DETAILED DESCRIPTION

(1) The terms subject and individual, as used herein, refer to mammalian individuals, and more particularly to pet animals (e.g., dogs, cats), agricultural animals (e.g., cows, horses, sheep, and the like), and primates (e.g., humans).

(2) The term treatment is used herein as equivalent to the term alleviating, which, as used herein, refers to an improvement, lessening, stabilization, or diminution of a symptom of a disease or immune imbalance. Alleviating also includes slowing or halting progression of a symptom.

(3) As used herein, co-administration, in conjunction with, concurrent, or concurrently, as used interchangeably herein, refers to administration of one treatment modality in addition to another treatment modality. As such, in conjunction with refers to administration of one treatment modality just before, during or soon after delivery of the other treatment modality to the subject.

(4) The term synthetic immodulin peptide shall mean a peptide molecule 20-60 amino acids in length prepared by chemical synthesis and comprising any of SEQ ID NOs: 1-7.

(5) The term phytochemical shall include D-heptomannulose, trehalose, naringin, resveratrol, polydatin, plumbagin, quercetin, curcumin, berberine, alpha-mangostin, wogonin, and ursolic acid.

(6) The term rexinoid includes all ligands of RXRs, and conjugates thereof.

(7) The term bexarotene-class rexinoid shall include bexarotene, LG100268, SR11237, HX600, HX531, BRF110 and conjugates thereof.

(8) The term NSAID includes ibuprofen, sulindac (and its sulfide and sulfone derivatives), indomethacin, aspirin, naproxin, ketoprofen, ketorolac, diclofenac and etodolac, and conjugates thereof.

(9) The term RLR/STING/IFN-class agonist shall include cyclic dinucleotides such as 23 cGAMP and cyclic di-GMP, nucleotides such poly-I:C and double-stranded ppp-RNA, and small molecule agonists such as G10, KIN1400, KIN1408, KIN 1148, RO8191, MSA-2, SR-717, alpha-mangostin, DMXAA and conjugates thereof.

(10) The term NR4A-class ligand shall include spironolactone, haloperidol, cytosporone B, C-DIM5, C-DIM8, C-DIM12, cilostazol, PDNPA and conjugates thereof.

(11) The term immunomodulant-class molecule shall include GIT-27, Schisandrin A, resiquimod (R-848), hydroxychloroquine, pidotimod, itraconazole, homoharringtonine, salidroside, celastrol, zymostenol, 7-dehydrocholesterol and conjugates thereof.

(12) The term Wnt-class molecule shall include gallic acid, methyl gallate, gallocyanine, epigallocatechin gallate, XAV939, ethacrynic acid, leonurine and conjugates thereof.

(13) The term growth factor class molecule shall include nerve growth factor (NGF), fibroblast growth factor (FGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and colony stimulating factor 1 (CSF1).

(14) Significantly increasing the abundance of a differentiated mammalian cell lineage shall mean increasing the relative abundance of said differentiated cell lineage by at least fifteen percent over baseline via the methods of this invention as compared to an untreated population control such that the difference between the two populations is statistically significant, for example, with a calculated probability of p<0.05 using Student's T-test, or other comparable statistical test well known to those skilled in the art. Differentiated mammalian cell lineage means a reproducing mammalian cell population expressing markers characteristic of a differentiated cell type. In vivo treatment means treatment within a living animal. Ex vivo treatment means removal of a population of mammalian cells from an animal, treatment of said cells, and, optionally, re-introduction of said treated cells back into the animal. Precursor cell, in the context of the invention, means a living mammalian cell capable of cellular growth or differentiation. Circulatory cell lineage includes cells of the blood and immune system, including hematopoietic, bone marrow and thymic cells. Nervous cell lineage includes cells of the central and peripheral nervous systems, eyes and ears. Endocrine cell lineage means cells of the hormone system, including pituitary, parathyroid, thyroid, adrenal and pancreas. Integumentary cell lineage means cells of the skin hair and subcutaneous tissue. Musculoskeletal cell lineage means cells of the bone, cartilage, ligaments, tendons and muscles. Pulmonary cell lineage means cells of the respiratory system, including lungs, trachea, larynx, nasal cavities and pharynx. Cardiovascular cell lineage means cells of the circulatory system, including the heart and blood vessels. Gastrointestinal cell lineage means cells of the digestive system, including stomach, intestinal tract, liver, pancreas, esophagus and salivary glands. Lymphatic cell lineage means cells of the lymphatic system, including spleen, lymph nodes, thymus and lymphatic vessels. Reproductive cell lineage means ovaries, uterus, mammary glands, testes, prostate and genitalia. Urinary cell lineage means cells of the kidneys, bladder and urethra.

(15) Significantly altering the relative abundance or bioactivity of a marker means changing the abundance or biological activity of a protein, mRNA, carbohydrate, lipid, metabolite or other biological analyte whose changed abundance or bioactivity is measurable in a population of cells by practitioners skilled in the art using commercially available kits, wherein the change is shown to occur to a statistically significant degree compared to a control population of cells. The difference between the measured abundance or bioactivity of a marker is significantly different in a population of cells that received treatment via the methods of this invention as compared to an untreated population control if the difference between the two populations is statistically significant, for example, with a calculated probability of p<0.05 using Student's T-test, or other comparable statistical test well known in the art. The abundance or bioactivity of a marker, such as a protein or RNA known to be diagnostic for the differentiated cell population in question, can be readily measured using commercially available test kits, well-known and widely used in the field e.g. ELISA kits, qPCR kits, enzymatic activity kits, etc. Test kits can be purchased for cell surface markers such as CD169, Clec9a, Clec10a, Clec12a, CD205, CD207, CD209, CD209L and MHCII, secreted proteins such as IL-10, TGFbeta, TNFalpha, CCL22 and COL1A1 (collagen), and nuclear proteins such as FoxP3, Nur77/NR4A1, RXRs and their heterodimers with other nuclear receptors.

(16) RXR means retinoid X receptor, and can refer to either the RXR gene or the protein it specifies. Rexinoid means a ligand of an RXR receptor. RXRs means any of the RXR isoforms, such as RXR-alpha, RXR-beta, RXR-gamma, and also covers heterodimers formed between them and other nuclear receptors such as NR4As. NR4As includes the orphan nuclear receptors NR4A1, NR4A2 and NR4A3, and can refer to either the NR4A gene or the protein it specifies. RXR receptors can and do form functional heterodimers with a variety of other nuclear receptors such as retinoic acid receptors (RARs), thyroid receptors (TRs), vitamin D receptor (VDR), liver X receptors (LXRs), peroxisome proliferator-activated receptors (PPARs), and the aforementioned NR4As.

(17) CD169 (also known as Siglec-1) means sialoadhesin, a cell adhesion molecule found on the surface of macrophages. Orthologs of this molecule in other mammalian species are included in this definition.

(18) C-lectins means a C-type lectin such as Clec4a, Clec9a, Clec10a or Clec12a, and orthologs thereof.

(19) MHCII means Class II major histocompatibility molecules such as HLA-DR and HLA-DQ and orthologs.

(20) CCL22 (also known as MDC) means CC motif chemokine 22, and its orthologs.

(21) COL1A1 means alpha-1 Type I collagen.

(22) STING means stimulator of interferon genes. STING is also known as TMEM173.

(23) RLRs means retinoic acid-inducible gene-I-like receptors.

(24) GSK3b or GSK3beta refers to glycogen synthase kinase 3 beta. GSK3a or GSK3alpha refers to glycogen synthase kinase 3 alpha.

(25) Nur77 is the protein product of the NR4A1 gene. As an analyte, the two terms are here used interchangeably. Sometimes the gene product may be referred to as Nur77/NR4A1 or NR4A1/Nur77.

(26) TLR4 inhibitor means an inhibitor of toll-like receptor 4 function.

(27) The effects of synthetic immodulin peptides on mammalian cells are IGF-independent because, unlike the IGFBPs from which their core sequences are derived, the peptides of this invention do not bind IGFs.

(28) This invention provides a method for significantly increasing the relative abundance of a differentiated mammalian cell lineage in vivo or ex vivo, comprising: (i) contacting one or more live mammalian cells in vivo or ex vivo with a synthetic immodulin peptide, 20-60 amino acids in length, comprising an amino acid sequence corresponding to any one of SEQ ID NOs: 1-4; and (ii) thereby significantly altering the relative abundance or bioactivity of a marker selected from the group consisting of GSK3beta, GSK3alpha, FoxP3, RNR4A1/Nur77, RXRs, RXR heterodimer, CD169, Clec9a, Clec10a, Clec12a, CD205, CD207, CD209, CD209L, MHCII, CCL22, IL-10, TNFalpha, TGFbeta and COL1A1 in said live mammalian cells, wherein said marker is distinctive for said differentiated mammalian cell lineage. More preferably, the mammalian cell lineage of the invention is a hematopoietic cell lineage. Even more preferably, the mammalian cell lineage of the invention is a monocytic cell lineage.

(29) This invention also provides a peptide having an amino terminus formed by covalent linkage to a small molecule of molecular mass less than one thousand daltons, preferably less than five hundred daltons. Said small molecule is selected from the group consisting of oleic acid, eicosapentanoic acid, lauric acid, decanoic acid, lignoceric acid, docosahexanoic acid, 2-hydroxy-2-decenoic acid, phenolic acids, anthraquinones, pentacyclic triterpenoids, retinoic acids, adapalene, rexinoids (e.g. bexarotene, SR11237, BRF110, HX531, HX600, rhein, sulindac), proprionic acids, TLR4 inhibitors, keto acids, cinnamic acids, aromatic carboxylic acids, indoleacetic acids, xanthenes, xanthones, fenofibric acid, valproic acid, 2-hexyl-4-pentynoic acid, 2,7-dichlorodihydro-fluorescein diacetate, indolyl-carboxylic acids, ibuprofen, GIT-27, MSA-2, SR-717, artemisinic acid and bromopyruvic acid.

(30) The invention also provides a synthetic immodulin peptide comprising a core sequence selected from the group consisting of any of SEQ ID NOs: 1-7. In some embodiments, said core sequence is aminoterminally extended by a sequence selected from the group consisting of sequence IDs 8-12.

(31) In some embodiments the invention provides a synthetic immX peptide, 20-60 amino acids in length, comprising an amino acid sequence corresponding to any of SEQ ID NOs: 5-7, having Xaa-Val-Asp at the peptide carboxy terminus wherein Xaa is D-alanine, D-serine or glycine. In some embodiments the invention provides a synthetic immX peptide, 20-60 amino acids in length, comprising an amino acid sequence corresponding to any of SEQ ID NOs: 5-7, having Xaa-Val-Asp at the peptide carboxy terminus wherein Xaa is D-alanine or D-serine. These end-modifications are preferred embodiments of the invention.

(32) In some embodiments the invention provides kinase inhibitor peptide sequences to be used in conjunction with the core immodulin sequences of this invention. Kinase inhibitor peptides have been widely used by practitioners in the field for several decades. For example, U.S. Pat. No. 5,783,405 lists dozens of peptide sequences and teaches their use as protein kinase C inhibitors. Among them are the sequences AFNSYELGS (SEQ ID NO: 9) and SLNPEWNET (SEQ ID NO: 8), claimed to inhibit PKC-delta and PKC-beta, respectively.

(33) In some embodiments the invention provides a therapeutic immodulin peptide complexed with metal, wherein said metal is selected from the group consisting of ferrous iron, ferric iron, zinc, copper, vanadium, ruthenium, cobalt, titanium, manganese, and calcium, or metallocene compounds containing these metals. In other embodiments, the invention provides a therapeutic immodulin peptide complexed with a component of the extracellular matrix such as collagen, iron-binding proteins or a glycosaminoglycan such as heparin, heparan sulfate, chondroitin sulfate, keratan sulfate, dermatan sulfate, and hyaluronate.

(34) In another aspect, the invention provides a method for treating a mammal showing symptoms of immune dysfunction or imbalance comprising administering to said mammal via intramuscular, subcutaneous, parenteral, transdermal, intranasal, intravenous or intrathecal route of administration a pharmaceutical formulation comprising a therapeutically effective dose of an immodulin peptide according to the invention, and a pharmaceutically acceptable excipient, thereby alleviating said symptoms of dysfunction. In some embodiments, the immodulin peptide is administered in a therapeutically effective dose of peptide between about 0.01 mg/kg/day to about 50 mg/kg/day.

(35) In another aspect, the invention provides for co-administration of a helper molecule that modulates the activity of cellular antiviral defenses. Preferably, said helper molecule is a modulator of the activity of cellular antiviral defenses, said helper selected from the group consisting of modulators of RIG-I, MDA5, MAVS, STING, IRF3, STAT1, STAT3, TBK1, PACT, LGP2, NFkappaB, DNA methylases such as 5-azacytidine or SAHA and toll-like receptors. Demethylation of DNA can derepress endogenous retroviral sequences which then, in turn, trigger cellular antiviral defense mechanisms. Preferably, the helper molecule is an agonist of RIG-I or MDA5 such as poly(I:C). Many agonists and enhancers of helper molecules are well-known in the art such as KIN-1400, a synthetic RIG-I agonist commercially available from Cayman Chemical (Ann Arbor, MI).

(36) This invention envisages an in vitro method for measuring cell differentiating potency of a synthetic immodulin peptide, the method comprising measurement of the abundance of a marker selected from the group consisting of RXRs, NR4As, CD169, C-lectins, MHCII, CCL22, COL1A1, STING, interferons, RLRs, Wnt, RANK or toll-like receptors in cultured mammalian cells that have been treated with the synthetic peptide. As will be understood by those of skill in the art, the mode of detection of a diagnostic signal will depend on the detection system utilized in the assay. For example, if a fluorescent detection reagent is utilized, the signal may be measured using a technology capable of quantitating the signal from the sample, such as by the use of a fluorometer. If a chemiluminescent detection system is used, then the signal will typically be detected using a luminometer. Methods for detecting signal from detection systems are well known in the art and need not be further described here.

(37) Sequence identity and homology, as referred to herein, can be determined using BLAST, particularly BLASTp as implemented by the National Center for Biotechnology Information (NCBI), using default parameters. It will be readily apparent to a practitioner skilled in the art that sequences claimed hereunder include all homologous and trivial variants of an immodulin peptide, such as by conservative substitution, extension and deletion in amino acid sequence. Trivial substitution variants include swapping of an amino acid with another belonging to the same class, without such substitution resulting in statistically and functionally significant change. Classes of amino acids include positively charged amino acids (arginine, lysine, histidine), negatively charged amino acids (aspartic acid, glutamic acid), aromatic amino acids (tyrosine, phenylalanine, tryptophan), branched chain amino acids (valine, leucine isoleucine) and other natural groupings such as (serine, threonine) and (asparagine, glutamine). For the purposes of this invention, such conservative substitutions to immodulin sequences, if they do not create a significant change in function, are considered equivalent to the original and are covered by the scope of this invention.

(38) For testing efficacy of pharmaceutical composition containing an immodulin peptide, an effective amount of therapeutic agent is administered to a subject having a disease. In some embodiments, the agent is administered at about 0.001 to about 50 milligrams per kilogram total body weight per day (mg/kg/day). In some embodiments the agent is administered at about 0.001 to about 50 mg/kg/day, e.g., 0.01, 0.015, 0.02, 0.05, 0.1, 0.2, 0.5, 0.7, 1, 2, 4, 5, 7, 9, 10, 15, 20, 25, 30, 35 or another dose from about 0.001 mg/kg/day to about 50 mg/kg/day.

(39) Therapeutic agents are preferably administered via oral or parenteral administration, including but not limited to intravenous (IV), intra-arterial (IA), intraperitoneal (IP), intramuscular (IM), intracardial, subcutaneous (SC), intrathoracic, intraspinal, intradermal (ID), transdermal, oral, sublingual, inhaled, and intranasal routes. IV, IP, IM, and ID administration may be by bolus or infusion administration. For SC administration, administration may be by bolus, infusion, or by implantable device, such as an implantable minipump (e.g., osmotic or mechanical minipump) or slow release implant. The agent may also be delivered in a slow release formulation adapted for IV, IP, IM, ID or SC administration. Inhaled agent is preferably delivered in discrete doses (e.g., via a metered dose inhaler adapted for protein delivery). Administration of a molecule comprising an agent via the transdermal route may be continuous or pulsatile. Administration of agents may also occur orally. For parenteral administration, compositions comprising a therapeutic agent may be in dry powder, semi-solid or liquid formulations. For parenteral administration by routes other than inhalation, the composition comprising an agent is preferably administered in a liquid formulation. Compositions comprising an agent formulation may contain additional components such as salts, buffers, bulking agents, osmolytes, antioxidants, detergents, surfactants, and other pharmaceutical excipients as are known in the art.

(40) As will be understood by practitioners skilled in the art, the symptoms of disease alleviated by the instant methods, as well as the methods used to measure the symptom(s) will vary, depending on the particular disease and the individual patient.

EXAMPLES

Example 1. Enhanced Metal Binding by Immodulin Peptides with C-Terminal Tripeptide Extension

(41) N-terminally biotinylated versions of the peptides listed below were used in ferric iron-binding assays involving either an Alexa488-labeled streptavidin protocol (Binding Assay 1) or FITC-NTA assay (Binding Assay 2). Fluorescence was standardized to binding with immX3AVD control peptide (100) and the average of multiple experiments using both protocols is shown. P values are shown relative to the control immX3AVD peptide (**<0.05). D-amino acid residues are underlined in the sequences and the extension tripeptide of immX peptides is shown in bold font. Binding Assay 1 was done as follows: NTA coated 96-well plates (G-Biosciences, St. Louis, MO) were charged with ferric chloride and contacted with equimolar biotinylated peptide: streptavidin-A488 complex (400 ng peptide per well) for 60 min at room temperature, washed with phosphate-buffered saline (PBS) and read at 525 nM in a fluorescence counter. Binding Assay 2 was done as follows: Streptavidin coated 96-well plates (G-Biosciences, St. Louis, MO) were contacted with 400 ng peptide per well in PBS buffer for 60 minutes at room temperature, washed in PBS, then contacted with FITC-NTA (TRC, Toronto, Canada) complexed with equimolar ferric chloride (10 molar equivalent per well) for 60 min at room temperature, washed with PBS and read at 525 nM in a fluorescence counter. Results of ferric iron binding experiments using Assay 1 (n=number of experiments averaged) are shown in the table below:

(42) TABLE-US-00003 PEPTIDE SEQUENCE n AvgSD SEQIDNO: None(buffer) 1 3.23.6** imm1 KNGFYHSRQCETSMDGEAGLCW 2 2.51.6** 42 imm2 KHGLYNLKQCKMSLNGQRGECW 2 1.60.8** 43 imm3 KKGFYKKKQCRPSKGRKRGFCW 5 66.74.7** 44 imm4 RNGNFHPKQCHPALDGQRGKCW 5 3.31.5** 45 imm5 RKGFYKRKQCKPSRGRKRGICW 5 71.66.6** 46 imm6 HRGFYRKRQCRSSQGQRRGPCW 2 65.94.9** 47 immX3AVD KKGFYKKKQCRPSKGRKRGFCWAVD 5 100.0 13 immX4AVD RNGNFHPKQCHPALDGQRGKCWAVD 2 1.21.3** 48 immX5AVD RKGFYKRKQCKPSRGRKRGICWAVD 2 88.114.2 15 immX3dAVD KKGFYKKKQCRPSKGRKRGFCW(dA)VD 1 108.11.5 immX3dAdVdD KKGFYKKKQCRPSKGRKRGFCW(dA)(dV)(dD) 2 102.49.1 immX3dSdVdD KKGFYKKKQCRPSKGRKRGFCW(dS)(dV)(dD) 2 133.6 immX3FVS KKGFYKKKQCRPSKGRKRGFCWFVS 2 78.36.5** 49 immX3RVD KKGFYKKKQCRPSKGRKRGFCWRVD 4 97.55.1 50 immX3K1dAdVdD SLNPEWNETKGFYKKKQCRPSKGRKRGFCW(dA)(dV)(dD) 4 108.26.6

Example 2. Enhanced Mammalian Cell Differentiation by immX Peptides

(43) Mammalian cell differentiation assays using THP1-Dual monocytes reporter cell line (Invivogen Inc, San Diego, CA) seeded at 210e5 cells per well in 96-well plates and cultured at 37 degrees C. in RPMI-1640 growth medium plus 10% fetal bovine serum and 1% penicillin/streptomycin, then treated for 24 hours with either 100 ng/ml Phorbol 12-myristate 13-acetate (PMA protocol; Cayman Chemical Company, Ann Arbor, MI) or a mixture of IL-4 (100 ng/ml), GM-CSF (100 ng/ml), TNF-alpha (20 ng/ml) and ionomycin (200 ng/ml) (Cytokine protocol; Peprotech, Rocky Hill, NJ). Peptide (330 nM) was then added, and incubation continued for an additional 24 hours. Culture supernatants were then assayed for CCL22. Plates with adherent cells were washed with PBS and assayed for immunoreactivity of surface markers such as CD169, Clec9a, Clec12a or MHCII using fuorescent tag- or biotin-labeled anti-human antibodies purchased from Miltenyi Biotec (Auburn, CA) and a relevant secondary detection reagent, or by fluorescence counts. Results were expressed as arbitrary units relative to the control immX3AVD peptide, and p values were also calculated and shown relative to the control immX3AVD peptide (values significantly above background are shown in bold font; **p<0.05 versus immX3AVD control; AU=arbitrary immunoreactivity units relative to control, avgSD). These results show why synthetic immX peptides comprising an amino acid sequence corresponding to any of SEQ ID NOs: 5-7 and having Xaa-Val-Asp at the peptide carboxy terminus wherein Xaa is D-alanine or D-serine, are preferred embodiments of the invention.

(44) TABLE-US-00004 PEPTIDE CD169+ (AU)# CCL22 pg/ml # Clec12A(AU)## None (buffer) 1.2 1.1** 17.1 1.2** 0.5 3.2** imm1 3.1 2.2** n.d. n.d. imm2 3.0 0.5** n.d. n.d. imm3 27.8 3.0** 55.3 16.1** n.d. imm4 3.8 1.1** n.d. n.d. imm5 36.5 3.0** n.d. n.d. imm6 19.9 2.3** n.d. n.d. immX3AVD 100 282.4 18.4 100 immX4AVD 1.0 0.3** n.d. n.d. immX5AVD 100.9 3.5 228.6 8.1 n.d. immX3dAVD 104.3 6.1 247.3 16.1 n.d. immX3dAdVdD 183.3 6.4** n.d. 106.6 5.2 immX3dSdVdD 145.7 9.3** n.d. n.d. immX3FVS 109.4 4.5 251.3 5.8 n.d. immX3RVD 88.1 4.2 272.5 35.7 n.d. immX3K1dAdVdD 249.3 13.9** 135.8 24.2** 321.9 25.6** AU: arbitrary units (immunoreactivity); #PMA protocol; ##cytokine protocol; n.d. = not determined

Example 3. Adjuvant Effect of Other Molecules on immX Peptide Potency in THP1-Dual Assay

(45) The THP1-Dual cell differentiation assay was carried out as described above in Example 2, using immX3 peptide (330 nM) in all samples plus the indicated helper or inhibitor molecule. AU=arbitrary ELISA units. **p<0.01.

(46) TABLE-US-00005 HELPER CAS# CLASS [CONC] CD169 (AU) None (buffer) 100.0 4.9 Hyaluronic acid glycosaminoglycan 0.6 ug/ml 110.5 10.2 Heparin glycosaminoglycan 1.0 ug/ml 34.1 2.9** Transferrin Fe-binding protein 0.8 ug/ml 126.8 9.3** Toosendanin 58812-37-6 GSK3b agonist 2 uM 188.2 16.3** Supercinnamaldehyde 70351-51-8 C/EBPb inhibitor 2 uM 159.7 16.6** Bisindolylamide 138489-18-6 PKC inhibitor 2 uM 130.1 8.4** RIG-I agonist [a] RIG-I agonist 1 ug/ml 135.4 9.1** RIG-I agonist [b] 446826-86-4 RIG-I agonist 2 uM 131.6 4.6** G10 702662-50-8 STING agonist 2 uM 143.4 17.8** 23-cGAMP Cyclic dinucleotide 15 uM 168.5 10.9** Cyclic-di-GMP Cyclic dinucleotide 15 uM 142.9 9.7** D-mannoheptulose Phytochemical 50 uM 140.8 12.4** Calcitriol 32222-06-3 VDR agonist 2 uM 102.0 11.3 Spironolactone 52-01-7 RXR or NR4A ligand 2 uM 120.1 10.5** C-DIM12 178946-89-9 RXR or NR4A ligand 2 uM 119.0 3.7** C-DIM8 151358-47-3 RXR or NR4A ligand 2 uM 91.0 24.8 Clobetasol 25122-46-7 RXR or NR4A ligand 2 uM 98.5 5.5 Cilostazol 73963-72-1 RXR or NR4A ligand 2 uM 99.4 27.7 Cytosporone B 321661-62-5 RXR or NR4A ligand 2 uM 120.3 6.4** Dihydroergotamine 6190-39-2 RXR or NR4A ligand 2 uM 170.3 20.7** 6-mercaptopurine 6112-76-1 RXR or NR4A ligand 2 uM 104.9 4.0 Bexarotene 153559-49-0 RXR or NR4A ligand 2 uM 96.9 6.8 LG100268 153559-76-3 RXR or NR4A ligand 2 uM 76.8 11.9** HX600 172705-89-4 RXR or NR4A ligand 2 uM 110.5 19.0 HX531 188844-34-0 RXR or NR4A ligand 2 uM 90.4 6.2 Cyclosporine A NFAT inhibitor 2 uM 128.9 6.7** GM-CSF Growth factor class 0.1 ug/ml 147.0 15.3** CSF1 Growth factor class 0.1 ug/ml 108.0 32.3 Leptin Growth factor class 1.0 ug/ml 103.3 11.6 GIT27 6501-72-0 TLR4 inhibitor 2 uM 166.5 34.1** [a] Invivogen Inc. (San Diego, CA) Cat. # tlrl-3pmnalv [b] Cayman Chem. Co. (Ann Arbor, MI) Cat. # 22441

Example 4. Binding to RXRs and Nur77 (NR4A1)

(47) Experiment 4A. Binding of immodulin peptides to AA111-228 DNA-binding domain of RXR-alpha (RXRa-DBD). 1 ug/well recombinant RXRa-DBD (Abcam, Cambridge, MA) was adsorbed to wells of a 96-well plate for 60 minutes at room temperature, then blocked with 200 uL 1% bovine serum albumin (BSA) in PBS buffer overnight. Plate was washed and 800 ng/well Streptavidin-Alexa 488 conjugate (SA488)-labelled immX peptide was added. The plate was incubated for 60 min at room temperature, washed and counted in a standard fluorometer (excitation/emission 485/525 nm). Background (buffer alone) was subtracted. The results show that imm3, imm5 and imm6 bind RXRa-DBD. *p<0.05, **p<0.01 vs no peptide; (Peptide/Fluorescence) imm1: 67085*; imm2: 238; imm3: 6,572129**; imm4: 2731; imm5: 6,802336**; imm6: 587192*; No peptide: 8232.

(48) Experiment 4B. Binding of immodulin peptides to RXR isoforms and domains. 100 ng/well recombinant human RXRa-DBD or RXRa-LBD, or 400 ng/well full length RXR-alpha, RXR-beta, RXR-gamma or PPAR-gamma (Abcam, Cambridge, MA) was adsorbed to wells of a 96-well plate for 60 minutes at room temperature, then blocked with 200 uL 1% BSA in PBS buffer overnight. The plate was washed with PBS and 800 ng/well Streptavidin-Alexa 488 conjugate (SA488)-labelled peptide was added. The plate was incubated for 2 hours at room temperature, washed and counted in a fluorometer (excitation/emission 485/525 nm). The results are expressed relative to imm3 binding (=100) and are shown in the table below. Significant binding above background is also shown (**p<0.05). The results indicate that imm3, imm5 and imm6 bind RXR-alpha and -gamma (especially RXR-gamma). When tested against domains of RXR-alpha, these peptides bind the DNA-binding domain (DBD) better than the ligand-binding domain (LBD).

(49) TABLE-US-00006 RXR No peptide imm3 imm4 imm5 imm6 None 0.8 0.4 1.3 1.2 1.3 1.7 1.2 2.8 0.6 1.1 RXR-alpha 1.2 0.3 11.2 1.0** 3.6 1.3 17.2 0.3** 12.9 0.5** RXR-beta 2.3 0.7 2.9 1.1 0.3 1.2 4.0 0.8 4.7 0.4 RXR-gamma 2.6 0.3 100.0 16.5** 3.6 7.0 90.0 19.6** 42.0 11.6** PPAR-gamma 0.9 0.4 2.1 1.4 0.1 0.0 1.6 0.4 6.8 2.0 RXR-alpha DBD 2.2 0.4 22.2 2.4** 3.2 2.0 22.7 3.1** 22.9 2.1** RXR-alpha LBD 1.8 0.1 0.6 1.5 3.0 1.3 1.8 1.9 1.6 1.4
Experiment 4C. Binding of Immodulin Peptides to Full-Length Human Nur77 (NR4A1).

(50) 400 ng/well recombinant human Nur77 protein (NR4A1) purchased from Abcam Inc, Cambridge, MA, was adsorbed to wells of a 96-well plate for 90 minutes at room temperature, then blocked with 200 uL 1% BSA in PBS buffer overnight. The plate was washed with PBS and then 333 ng/well Streptavidin-Alexa 488 conjugate (SA488)-labelled imm3 or imm4 peptide was added. The plate was incubated for 60 min at room temperature, washed and counted in a standard fluorometer (excitation/emission 485/525 nm). Background (buffer alone) was subtracted. The results (normalized to imm3 binding=100) are shown in the table below. They show that imm3 but not imm4 binds Nur77. **p<0.01 vs no Nur77 control.

(51) TABLE-US-00007 Nur77 (NR4A1) imm3 imm4 None 1.7 2.7 2.9 3.3 400 ng 100.0 9.3** 0.3 8.9
Experiment 4D. Binding of Immodulin Peptides to Glycosaminoglycans.

(52) Streptavidin-coated 96-well plates (G-Biosciences, St. Louis, MO) were pre-treated with biotinylated peptides [see Example 1 for key] at 1 ug/well, at room temperature for 60 minutes. The plate was washed with PBS buffer and then 1 ug FITC-labelled heparin or hyaluronic acid in 100 uL PBS buffer was added per well (all tests in quadruplicate). Incubation proceeded for 90 minutes at room temperature, followed by two PBS washes. The plate was read in a standard laboratory fluorometer (488/525 nm exitation/absorbance) and the counts normalized for immX3 peptide binding=100. Values statistically above background are shown in bold font. **p<0.05 versus immX3AVD.

(53) TABLE-US-00008 PEPTIDE Heparin Hyaluronate imm2 27.4 22.9 3.1 9.8 imm3 104.2 22.0 57.2 5.7** imm4 6.8 24.1 14.6 22.6 imm5 95.1 25.7 60.0 8.6** imm6 97.2 17.3 44.9 14.1** immX3AVD 100.0 4.7 100.0 22.8 immX4AVD 7.3 17.1 61.1 34.1 immX5AVD 68.7 26.2 113.9 15.7 immX3dAdVdD 55.5 5.7** 104.2 14.7 immX3K1dAdVdD 56.0 7.3** 101.9 16.4

Example 5. Potentiating Effects of C-Terminal Tripeptide Extension on Peptide Stability and the Systemic Efficacy of sV-Nephrilin and Traumatin-3N1A immX Peptides in Burn Trauma

(54) Experiment 5A. V-nephrilin peptide (imm3N1V) is a designed inhibitor of Rictor complex, an evolutionarily conserved assembly believed to modulate responses to cellular stress. We previously demonstrated the ability of nephrilin peptide to suppress neuroinflammation, loss of body mass, glycemic control and kidney function in a rat scald model, as well as sepsis mortality in a mouse model. The tripeptide extension of V-nephrilin peptide is sV-nephrilin (tripeptide extension in sV-nephrilin is underlined):

(55) TABLE-US-00009 imm3N1V(V-nephrilin): (SEQIDNO:51) RGVTEDYLRLETLVQKVVSKGFYKKKQCRPSKGRKRGFCW immX3N1sV(sV-nephrilin): (SEQIDNO:52) RGVTEDYLRLETLVQKVVSKGFYKKKQCRPSKGRKRGFCWAVD

(56) Duplicate wells per dilution point in a 96-well NTA-coated plate (G-Biosciences, St. Louis, MO) charged with ferric chloride were adsorbed with 2 ug imm3N1V (V-nephrilin) or immX3N1 (sV-nephrilin) peptide per well for 30 min at room temperature, washed, then incubated with 100 ul/well of 8 or 2 g/ml protease (Sigma Catalog #P4630, Pancreatic protease Type I) for 20 minutes at 37 degrees C. Plates were washed with PBS buffer and the remaining peptide bound was measured using an in-house calibrated anti-immodulin ELISA (rabbit polyclonal #2501). The percentages of surviving peptide after 8 or 2 g/ml protease treatment were, respectively: V-nephrilin (imm3N1V): 57.62.3% and 79.00.7%; sV-nephrilin (immX3N1): 75.12.8% and 90.43.4% (p<0.001 at both concentrations). Thus, the immX tripeptide extension at the C-terminus using D-amino acids improved peptide stability to protease degradation.

(57) Experiment 5B. This study explores the effect of immX extensions on sV-nephrilin (SEQ ID NO:31) vs V-nephrilin, as well as in a longer peptide also containing the immX extension (Traumatin-3N1A: SEQ ID NO:35) on in vivo efficacy in a rat scald-endotoxemia model. Animals were treated with peptides using a widely used protocol previously described (see, for example, U.S. Pat. No. 10,369,191). Adult Sprague Dawley rats of both sexes (250-300 gm, Charles River Laboratories, Wilmington, MA, USA) were injected with peptide plus equimolar ferric iron once daily by subcutaneous bolus injection, days 1-7; Treatment group sizes were (n=6): group S=sham-treated; group B=burn+vehicle; group V=burn+1 mg/kg V-nephrilin (V); group sV=burn+1 mg/kg sV-nephrilin (immX3N1sV); group T=burn+1 mg/kg Traumatin-3N1A. The first dose was administered after completion of the scald procedure. Injection volume was 400 uL. Control animals received the same volume of vehicle. On Day 8, animals received a single intraperitoneal injection of 2.5 mg/kg lipopolysaccharide (LPS; Sigma Cat #L-2880) and animals were then monitored by oximeter (MouseOx, Starr Life Sciences) for 4 hours, when respiratory distress (breath distension*breaths/min) was assessed relative to baseline. This was followed by sacrifice and tissue collection as in the published protocol, except that bronchoalveolar lavage (BAL) was collected and cells were recovered from BAL by centrifugation. These cells were stained with fluorescently-labeled antibodies for flow cytometry in order to ascertain granulocyte counts, as shown below. Results of the peptide comparison are presented in the table below. *p<0.05 vs B control. #p<0.05 vs V group.

(58) TABLE-US-00010 Day Group S Group B Group V Group sV Group T 1 Plasma albumin mg/ml (24 hr IR) 3.6 1.1* 9.3 1.2 7.9 2.1 7.4 1.6* 7.6 0.9* 1 Plasma IL-6 (pg/ml) 24.6 12.5* 47.3 18.5 31.4 19 17.5 9.3* 22.5 11*.sup. 5 Change in body weight (Pct vs Day 1) 7.0 2.0* 7.4 1.2 9.8 2.9 5.1 3.6* 5.3 3.5* 8 Wet/dry lung weight (corrected for body weight) 5.54 0.64* 8.27 1.43 7.97 0.76 7.07 0.48* 6.74 0.61* 8 Respiratory distress (Gp. S = 100) 100 14*# 322 86 349 95 203 30*# 197 39*# 8 BAL fluid IL1-beta (pg/ml) 13.8 5*#.sup. 139.6 34.sup. 112.8 9.4 67.1 16*# 45.7 24*# 8 BAL His48+ granulocytes (FITC units) 26.1 4.9* 34.7 3.2 28.9 3.1* 26.8 4.0* ND 8 Plasma creatinine (mg/dL) 0.65 0.2* 0.92 0.1 0.91 0.3 0.73 0.2* 0.64 0.2* 8 Plasma OHDG (ng/ml) 1.17 0.8* 2.18 0.7 1.44 1.0 1.31 0.6* 1.24 0.4*

(59) Example 6. N-terminal modification of peptide with small molecules of molecular mass below 500 daltons that are not amino acids. N-terminal modification of peptides with biotin has been disclosed. This example shows the difficulty in predicting success for this type of modification for a previously untried small molecules. The data in this example disclose, amongst other facts: (i) efficient coupling to carboxylic acids that are not amino acids or biotin, is possible using normal peptide synthesis conditions; (ii) surprising results showing that yields of correctly coupled product (as ascertained by mass spectroscopy analysis) varies greatly, even within the same class of compound; and (iii) similar results were obtained for attachment to an immodulin peptide containing SEQ ID NO:2 or a generic D-tetrapeptide dLys-dAsp-dLys-dPro, with similar efficiencies of coupling to either peptide, thereby demonstrating the generality of the method. Peptides were synthesized according to a common Fmoc/tBu solid phase synthesis strategy well-known in the art. Synthesis may be manual of automated. After the peptide synthesis the resin was divided into batches of 20 umol. Each batch was treated with one of the organic compounds specified in the table shown immediately below. The coupling was carried out using 2 equivalents of the compound, 2.4 equivalents of activator HATU or HCTU, and 4 equivalents of NMM base. The reaction mixture was renewed after 2 hrs reaction time and allowed to react another 4 hrs or overnight. After washing the resin several times with DMF, and subsequently with DCM, the batches were dried. For the cleavage of the peptides from the resin the resins were treated with 1% DTT, 2% water and 3% TIPS in TFA for 3.5 hrs. The cleavage solution was separated from the resin and treated with diethylether/n-pentane (1:1). The resulting precipitate was centrifuged and the pellet washed three times in the same DEE/pentane mixture. The recovered peptide was air dried and stored at 20 degrees C. or further purified by HPLC using a 0-50% acetonitrile gradient, 0.1% trifuoroacetic acid (20 min). The results of the above conjugation experiments show that, both inter-class and intra-class, there is wide variation in conjugation efficiency from compound to compound. As the practicality and cost of synthesis can be dramatically affected when product yield is low, it is therefore not obvious that any untested carboxylic acid should be assumed to be a good candidate for this type of peptide modification. The use of most of the compounds tested here has never been reported for this kind of peptide modification. It appears that the chance of practical success (>80% correct yield, for instance) for each instantiation of this technology is less than 50% until tested.

(60) TABLE-US-00011 Class Compound CAS No. MW Yld T4* Yld IM3* fatty acid oleic acid 112-80-1 282.5 44.21% fatty acid eicosapentaenoic acid 10417-94-4 302.5 66.79% fatty acid lignoceric acid 557-59-5 368.6 89.20% fatty acid decanoic acid 1002-62-6 172.2 88.67% 98.0% fatty acid docosahexanoic acid 6217-54-5 368.6 57.77% fatty acid lauric acid 143-07-7 200.3 85.14% 96.7% fatty acid 10-hydroxy-2-decenoic acid 14113-05-4 186.3 44.38% phenolic acid ferulic acid 1135-24-6 194.2 26.58% phenolic acid isoferulic acid 537-73-5 194.2 55.80% 70.2% phenolic acid Aspirin 50-78-2 180.2 56.5% phenolic acid valeroyl salicylate 64206-54-8 222.2 11.76% pentacyclic betulinic acid 472-15-1 456.7 .sup.<1% anthraquinone Rhein 478-43-3 284.2 50.95% anthraquinone Diacerein 13939-02-1 368.3 43.2% xanthone 2,7-dichlorodihydro- 4091-99-0 487.3 91.2% proprionic acid (s)-ketoprofen 22161-81-5 254.3 77.86% proprionic acid Ibuprofen 15687-27-1 206.3 93.42% 98.0% carboxylic acid trans-cinnamic acid 140-10-3 148.2 93.12% 81.5% carboxylic acid (s)-()-perillic acid 23635-14-5 166.2 27.96% carboxylic acid fenofibric acid 42017-89-0 318.8 85.67% 99.9% indoleacetic acid Indomethacin 53-86-1 357.8 87.5% 85.2%# pentanoic acid valproic acid 1069-66-5 144.2 91.43% 84.9% alkynoic acid 2-hexyl-pentynoic acid 96017-59-3 182.3 85.1% indolylcarboxylic RG-108 48208-26-0 334.3 74.3%@ retinoid all-trans retinoic acid 302-79-4 300.4 13.1% rexinoid Bexarotene 153559-49-0 348.5 97.09% 94.4% *% yield by MS for T4 (tetrapeptide) and imm3 peptide (>80% in bold type); #lost p-chlorophenone group; @indole core oxidized by Arg (protecting gp);

Example 7. Anti-Cancer Actions of Immodulin Peptides

(61) Immune modulation functions of immodulin peptides have great potential untility in the field of cancer. A375 cell line was obtained from American Type Culture Collection (ATCC). They were grown in a T-75 flask in DMEM Medium containing 10% fetal bovine serum and penicillin-streptomycin at 37 C. in a humidified, 5% CO.sub.2 incubator. Cells (100 l, 2,000 cells/well) were plated in a 96-well plate and incubated overnight at 37 C. in a humidified, 5% CO.sub.2 incubator. Next day, 10 l/well of compounds were added (quadruplicate wells). After 72 hour incubation with the compound, cell viability was measured in a luminometer after the addition of 100 IL/well CellTiterGlo reagent (Promega Inc, Madison, WI) as recommended by the manufacturer. Anti-cancer activity of immodulin peptides on A375 cells: A 96-well plate was seeded with 2,000 A375 cells per well in DMEM medium containing 10% FBS and PenStrep. After 24 hrs at 37 deg C., compounds and peptides were added (each treatment done in quadruplicate). After a further 72 hours incubation, 100 L/well of CTG assay reagent purchased from Promega Inc. (Madison, WI) was added. Plate was read after 10 minutes, as recommended by the manufacturer. Peptides were added at 2 uM. The results of this experiment are shown below, expressed as percent survival of A375 cells. They show that anti-melanoma activity of various immodulin peptides are influenced by the core immodulin sequence, extension sequences, N-terminally conjugated carboxylic acids RIG-I agonist (4 uM). *p<0.05, **p<0.01 vs no peptide control; RIG-I ag=4 uM Cayman Chem. Co. Cat #22441; vlp=valproic; dec=decanoic; lau=lauric; rg108=RG108; h4p=h4-pentynoic; bpa=bromopyruvic; nd=not determined.

(62) TABLE-US-00012 Peptide Sequence Buffer +RIG-Iag SEQIDNO: Nopeptide 100 100.7 imm1 KNGFYHSRQCETSMDGEAGLCW 102.1 Nd 42 imm2 KHGLYNLKQCKMSLNGQRGECW 99.0 Nd 43 imm3 KKGFYKKKQCRPSKGRKRGFCW 100.3 62.2** 44 imm4 RNGNFHPKQCHPALDGQRGKCW 98.8 Nd 45 imm5 RKGFYKRKQCKPSRGRKRGICW 103.4 Nd 46 imm6 HRGFYRKRQCRSSQGQRRGPCW 102.2 Nd 47 imm3avd KKGFYKKKQCRPSKGRKRGFCWAVD 96.0 12.5** 13 imm3fvs KKGFYKKKQCRPSKGRKRGFCWFVS 96.1 22.6** 49 imm3vlp (vlp)-KKGFYKKKQCRPSKGRKRGFCW 93.9** 32.6** 44 imm3dec (dec)-KKGFYKKKQCRPSKGRKRGFCW 89.0** 53.8** 44 imm3lau (lau)-KKGFYKKKQCRPSKGRKRGFCW 96.6* 17.4** 44 imm3rg8 (rg108)-KKGFYKKKQCRPSKGRKRGFCW 89.9** 105.8 44 imm3h4p (h4p)-KKGFYKKKQCRPSKGRKRGFCW 91.7** Nd 44 imm3K1 SLNPEWNETKGFYKKKQCRPSKGRKRGFCW 92.5** 17.0** 55 imm3K1bpa (bpa)SLNPEWNETKGFYKKKQCRPSKGRKRGFCW 98.4 Nd 55 imm3K1.1 SLNPEWNETKKGFYKKKQCRPSKGRKRGFCW 95.3* Nd 54

Example 8. Collagen Stimulating Activity of Immodulin Peptides

(63) Immodulin peptides have potential untility in the field of cosmetics. HFF-1 human fibroblast cell line was obtained from the American Type Culture Collection (ATCC). Cells were grown in a T-75 flask in DMEM Medium containing 10% fetal bovine serum and penicillin-streptomycin at 37 C. in a humidified, 5% CO.sub.2 incubator. Cells (100 l, 2,000 cells/well) were plated in a 96-well plate and incubated overnight at 37 C. in a humidified, 5% CO.sub.2 incubator. Next day, 10 l/well of compounds were added (quadruplicate wells). After 72 hour incubation with the compound, supernatants were collected for Collagen-1 ELISA assay and cell viability was measured in a luminometer after the addition of 100 IL/well CellTiterGlo reagent (Promega Inc, Madison, WI) as recommended by the manufacturer. Collagen stimulating activity of immodulin peptides in HFF-1 dermal fibroblasts: Peptides were added to cells at 2 uM. COL1 immunoreactivity was measured in the supernatants of cultured cells by ELISA using a rabbit monoclonal anti-COL1 primary antibody (Abcam, Cambridge, MA). The results of this experiment are shown in Table above. Control (buffer) value of immunoreactivity was set to 100. The data show that collagen stimulating activity of various immodulin peptides are influenced by specific extension sequences and by N-terminally conjugated carboxylic acids. *p<0.05, **p<0.01 vs no peptide control; bex=bexarotene; isf=isoferulic; vlp=valproic; dec=decanoic; cin=cinnamic; rhn=rhein;

(64) TABLE-US-00013 Peptide Sequence COL1 SEQIDNO Nopeptide 100 imm1 KNGFYHSRQCETSMDGEAGLCW 98 42 imm2 KHGLYNLKQCKMSLNGQRGECW 109 43 imm3 KKGFYKKKQCRPSKGRKRGFCW 101 44 imm4 RNGNFHPKQCHPALDGQRGKCW 105 45 imm5 RKGFYKRKQCKPSRGRKRGICW 93 46 imm6 HRGFYRKRQCRSSQGQRRGPCW 112 47 imm3bex (bex)-KKGFYKKKQCRPSKGRKRGFCW 102 44 imm3isf (isf)-KKGFYKKKQCRPSKGRKRGFCW 131* 44 imm3vlp (vlp)-KKGFYKKKQCRPSKGRKRGFCW 157** 44 imm3dec (dec)-KKGFYKKKQCRPSKGRKRGFCW 114 44 imm3cin (cin)-KKGFYKKKQCRPSKGRKRGFCW 106 44 imm3rhn (rhn)-KKGFYKKKQCRPSKGRKRGFCW 68** 44 imm3K9 AFNSYELGSKGFYKKKQCRPSKGRKRGFCW 155** 56 imm3K9.1 AFNSYELGSKKGFYKKKQCRPSKGRKRGFCW 156** 57 imm3K9c AFNSYELGSKGFYKKKQCRPSKGRKRGFCWAVDKY 158** 58 imm3K8 FNSYELGSLKKGFYKKKQCRPSKGRKRGFCW 98 59

(65) While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

(66) Incorporation by Reference of Sequence Listing file: Sequence Listing is entered into the application as part of the disclosure. Date of Creation: Jul. 8, 2025; Size of txt file in bytes: 22,321 bytes.

(67) Name of file: 18264181_SEQfile_070825.txt