WO2023088287A1

WO2023088287A1 - Fusion proteins comprising ai-073 core peptide and use thereof

Info

Publication number: WO2023088287A1
Application number: PCT/CN2022/132188
Authority: WO
Inventors: Qunmin Zhou; Xianfeng FANG; Dongling Li; Libing MU; Jianying Zhou
Original assignee: Acroimmune Guangzhou Biotech Ltd
Priority date: 2021-11-17
Filing date: 2022-11-16
Publication date: 2023-05-25

Abstract

The compositions of a glycosylated and/or sialylated core peptide (073 core) and compositions of proteins based on fusion of one or more copy of the peptide to the Fc fragment of human immunoglobulin and their use in treating diseases propagated by inflammation associated with tissue injuries or invading pathogens.

Description

FUSION PROTEINS COMPRISING AI-073 CORE PEPTIDE AND USE THEREOF

BACKGROUND OF THE INVENTION

Inflammation is an innate immune response to foreign pathogen infection and self-tissue injury. The inducers of inflammation thus can be classified into two major categories. The first and perhaps more potent one is so called as pathogen-associated molecular pattern (PAMP) , and the second and less studied one is so called as damage (danger) -associated molecular pattern (DAMP) (Janeway CA Jr. Cold Spring Harbor Symposia on Quantitative Biology. 1989; 54: 1; Matzinger P. Annual Review of Immunology. 1994; 12: 991) .

PAMPs present on almost all microbial pathogens, and the survival of host organisms is dependent on their ability to recognize these PAMPs in invading microbial pathogens and to mount immune response or defense reactions (Janeway CA Jr, Medzhitov R. Innate immune recognition. Annu Rev Immunol. 2002; 20: 197-216) . Some examples of the well characterized PAMPs are Lipopolysaccharide (LPS) , poly (I: C) , Pam3Cys, CpG DNA and etc. Toll-like receptor (TLR) , an evolutionarily ancient family, plays a crucial role in the detection of PAMPs in microbial infection and alerts the host immune system to response to pathogen invasion (Medzhitov R, Preston-Hurlburt P, Janeway CA Jr. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature. 1997; 388: 394; Medzhitov R, Janeway CA Jr. The Toll receptor family and microbial recognition. Trends Microbiol. 2000; 8: 452-6) .

Unlike PAMPs, which are present only on invading microbial pathogens, DAMPs by nature, are host self-components which are released or exposed on cell surfaces by necrotic or damage cells/organs when they are under stress and/or face foreign microbial invasion. Some of the well characterized DAMP include a variety range of molecules such as the heat-shock proteins (HSP60, HSP70, HSP90) , uric acid crystals, cellular or mitochondrial DNA, stress-induced RNA-binding chaperone proteins (such as cold-inducible RNA-binding protein, CIRP) , high mobility group box-1 (HMGB1) and etc.

HMGB1, normally a nuclear located chromatin-binding non-histone protein of about 25 kDa, ubiquitous present in almost all eukaryotic cells. HMGB1 is actively released by innate immune cells in response to invading pathogens or passively released by damaged/injured cells in the absence of pathogen invasion. Released HMGB1 can further amplify or active innate immune responses by up-regulating chemotaxis to recruit more cells into the inflammation site and/or increasing release of cytokines or other inflammation mediators. Thus, HMGB1 acts as a multi-functional alarmin that stimulates or amplifies inflammation upon sterile or infectious insult. The binding partners or receptors for HMGB1 include a variety of molecules such as TLRs (TLR2, TLR4 and TLR9) , immunoglobulin mucin-containing protein-3 (TIM-3) , C-X-C chemokine receptor 4 (CXCR4) , triggering receptor expressed on myeloid cells-1 (TREM-1) , receptor for advanced glycation end products (RAGE) , syndecan-3, thrombomodulin and so on (Marco E. Bianchi and Angelo A. Manfredi: How macrophages ring the inflammation alarm. PNAS, 2014, 111: 2866-2867) . Interaction of PAMPs like LPS or DAMPs like HMGB1 with their binding partners or receptors like TLRs or RAGE has well been implicated in the pathogenesis of a broad range of diseases including atherosclerosis, sepsis, neurodegenerative diseases, and autoimmune-related diseases.

The host organisms also have developed nature defense systems or balance mechanisms to count-act or attenuate the over-immune response that might be induced by microbial pathogen infection and/or cellular DAMPs. For instance, invasion pathogens such as bacteria or viruses entering in the respiratory airway tract are first trapped by the airway surface fluid and epithelial cells and can be rapidly removed from the lung by mucociliary clearance before they post danger to the host.

One family member with this kind of important nature defense function in the host is called mucin. Mucins are heavily glycosylated/sialylated proteins of a large molecular mass (>200 kDa) that are widely expressed on the apical surface of most secretory epithelial cells, particularly in the respiratory, digestive gastrointestinal (GI) and genitourinary tracts, all of which are constantly exposed to the external environmental stresses. Based on their structure, mucins can be classified into two categories: transmembrane/cell-surface bound or secreted/gel-forming. Transmembrane/cell-surface bound mucins include MUC1, MUC3A, MUC3B, MUC4, MUC12, MUC16, MUC17 and etc., whereas secreted/gel-forming mucins include MUC2, MUC5AC, MUC5B, MUC6, MUC19 and etc. The best-characterized and most widely expressed mucin molecule is MUC1, which is normally present on the apical surface of a variety of epithelial cells, including the respiratory, gastrointestinal, and reproductive tracts and the mammary glands.

One key feature of the mucin molecules in general, and MUC1 in particularly, is that they have a variable number of a perfect or nearly a perfect tandem repeat (TR) sequence, which is rich in serine (S) , threonine (T) and proline (P) residues (also called as STP domain or motif) . The serine and threonine residues in this STP domain are predicted to be O-linked glycosylation sites. For example, the heavily glycosylated human MUC1 molecule has multiple tandem repeats (about 20 to 120) of a 20-amino acid long consensus sequence which contains 2 serine residues, and 3 threonine residues, all of which have been shown to be modified by O-linked glycosylation and/or sialylation in vivo.

In addition to the well-known mucin molecules, some of other so-called mucin-like molecules are also heavily glycosylated/sialylated and play an important role in host innate immune defense against microbial pathogens or DAMPs induced cell damage. One such type of heavily sialylated mucin-like glycoproteins (sialoglycoproteins) are CD24 and CD52, both of which are anchored into cell outside membrane/cell-surface by a glycosylphosphatidylinositol (GPI) -linker and widely expressed in both the epithelial cells and immune cells such as lymphocytes, granulocytes, monocytes, macrophages and dendritic cells. In particular, CD24 is highly expressed in B1 regulatory lymphocytes (Bregs) , whereas CD52 is highly expressed in regulatory T lymphocytes (Tregs) . Both Bregs and Tregs cells play an important role in controlling host immune response to PAMP or DAMP stimulation.

MUC1 or mucin-like molecules such as CD24 and CD52 all have been found to directly bind to DAMP molecules such as HMGB1, as well as other sugar-binding molecules such as selectins and siglecs. Siglecs, stand for sialic acid-binding immuno globulin-like lectin, are type I transmembrane proteins widely expressed in many innate immune cells (NK cells, DC cells and other myeloid cells) as well as in B and T lymphocytes. All siglec molecules contain an IgV-like sialic acid binding domain at their outmost N-terminal followed by a variable number (1, to 16) of IgC2-like domains in their extracellular region. In the intracellular region, most siglec molecules contain immunoreceptor tyrosine-based inhibitory motifs (ITIM) or ITIM-like motifs, which recruit the phosphatases such as SHP-1, SHP2 and leads to attenuation or suppression of immune response. Interaction of siglecs with their ligands such as mucins or mucin-like molecules CD24 and CD52 repress or diminish the overall inflammatory response induced by DAMPs (Chen GY, et al. CD24 and Siglec-10 selectively repress tissue damage-induced immune responses. Science. 2009; 323: 1722–1725; Chen GY, et al. Amelioration of sepsis by inhibiting sialidase-mediated disruption of the CD24-SiglecG interaction. Nat Biotechnol. 2011; 29: 428–435; Abdulrahman M. Shathili et al: Specific Sialoforms Required for the Immune Suppressive Activity of Human Soluble CD52. Front Immunol. 2019; 10: 1967)

More recently, nature derived or genetically engineered soluble form of mucins or mucin-like molecules such as CD24Fc or CD52Fc have also been found to have therapeutic effects in a number of diseases models including graft vs host diseases (GVHD) , rheumatoid arthritis, and pathological setting in which infections cause tissue injuries such as COVID-19, influenza pneumonia and sepsis. Nevertheless, there is still an urgent need to develop a safe and more potent product with biological properties of superior siglec-binding and enhanced anti-inflammation activities.

SUMMARY OF THE INVENTION

The present disclosure provides compositions of a glycosylated and/or sialylated core peptide (also referred to as AI-073 core-peptide, 073 core-fragment, 073 core-portion, or simply called as 073 core) and compositions of proteins based on fusion of the core peptide to the Fc fragment of human immunoglobulin and their use in treating diseases propagated by inflammations associated with tissue injuries or invading microbial pathogens.

The present disclosure provides an isolated AI-073-Fc fusion protein (hereafter also called as AI-073) , wherein the amino acid sequence of said AI-073 protein comprising 073-core peptide, said 073-core peptide comprises one or more copies of a first fragment and one or more copies of a second fragment, each of said first fragment comprises a sequence independently selected from those as set forth in SEQ ID NO: 1 and 22 to 38, SEQ ID NO: 56 and each of said second fragment comprises an amino acid sequence derived from Mucin1 protein. For example, each of said second fragment comprises a sequence independently selected from those as set forth in SEQ ID NO: 2 and 8 to 21.

The present disclosure provides an immunoconjugate, comprising the 073-core fragment of the present disclosure, or the protein of the present disclosure.

The present disclosure provides a nucleic acid, encoding the 073-core fragment of the present disclosure, or the protein of the present disclosure.

The present disclosure provides a vector, comprising the nucleic acid the present disclosure.

The present disclosure provides a cell, comprising and/or expressing the 073-core fragment of the present disclosure, the protein of the present disclosure, the immunoconjugate the present disclosure, the nucleic acid the present disclosure, and/or the vector the present disclosure.

The present disclosure provides a composition, comprising the 073-core fragment of the present disclosure, the protein of the present disclosure, the immunoconjugate the present disclosure, the nucleic acid the present disclosure, the vector the present disclosure, and/or the cell the present disclosure, and optionally a pharmaceutically acceptable carrier.

The present disclosure provides a method for preparing the 073-core fragment of the present disclosure, or the protein of the present disclosure, comprising culturing the cell the present disclosure under a condition enabling the expression of said 073-core fragment or said protein.

The present disclosure provides a method for regulating a Siglec related signaling, comprising administering to a subject in need thereof an effective amount of the 073-core fragment of the present disclosure, the protein of the present disclosure, the immunoconjugate the present disclosure, the nucleic acid the present disclosure, the vector the present disclosure, the cell the present disclosure, and/or the composition the present disclosure.

The present disclosure provides a method for regulating an immune response, comprising administering to a subject in need thereof an effective amount of the 073-core fragment of the present disclosure, the protein of the present disclosure, the immunoconjugate the present disclosure, the nucleic acid the present disclosure, the vector the present disclosure, the cell the present disclosure, and/or the composition the present disclosure.

The present disclosure provides a method for repressing an immune-mediated tissue damage mediated by danger-associated molecular patterns (DAMPs) , comprising administering to a subject in need thereof an effective amount of the 073-core fragment of the present disclosure, the protein of the present disclosure, the immunoconjugate the present disclosure, the nucleic acid the present disclosure, the vector the present disclosure, the cell the present disclosure, and/or the composition the present disclosure.

The present disclosure provides a method for preventing, ameliorating and/or treating a disease or condition caused by an inflammatory response arising from tissue injuries from infectious agents, comprising administering to a subject in need thereof an effective amount of the 073-core fragment of the present disclosure, the protein of the present disclosure, the immunoconjugate the present disclosure, the nucleic acid the present disclosure, the vector the present disclosure, the cell the present disclosure, and/or the composition the present disclosure.

The present disclosure provides a method for preventing, ameliorating and/or treating a disease or condition caused by acute tissue damage from wound, comprising administering to a subject in need thereof an effective amount of the 073-core fragment of the present disclosure, the protein of the present disclosure, the immunoconjugate the present disclosure, the nucleic acid the present disclosure, the vector the present disclosure, the cell the present disclosure, and/or the composition the present disclosure.

Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

INCORPORATION BY REFERENCES

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are employed, and the accompanying drawings (also “figure” and “FIG. ” herein) , of which:

FIG. 1A illustrates the amino acid sequence alignment of the human Muc1 tandem repeat and its variants. FIG. 1B illustrates the amino acid sequence alignment of the Muc1 tandem repeats from human and other species.

FIG. 2A illustrates amino acid sequence alignment of the CD24 middle core peptide from various species. FIG. 2B illustrates amino acid sequence alignment of CD24 middle core peptide variants

FIG. 3A illustrates the schematic structure of the AI-073 fusion protein, comprising 073-core peptide and IgG-Fc. The preferred formation of AI-073 fusion protein is a dimer, covalently linked via disulfate chains of the hinge region and non-covalent interactions between CH2 and CH3 domains of human IgG1. FIGs. 3B-3C illustrate SDS-PAGE and SEC-HPLC analysis result of the purified AI-073 fusion protein. FIG. 3B shows an SDS-PAGE gel of the purified AI-073 fusion protein. Two μg of purified AI-073 fusion protein, either in reducing (R) or non-reducing conditions (NR) , was loaded into SDS-PAGE gel. After electrophoresis, the gel was stained with Coomassie Brilliant Blue dye. The molecule weight (kDa) of protein marker (M) in the gel was indicated to the left. FIG. 3C shows a size exclusion chromatography (SEC) -high performance liquid chromatography (HPLC) separation profile of the purified AI-073 fusion protein.

FIGs. 4A-4C illustrate the binding of AI-073 or CD24Fc control protein to different anti-CD24 mAb in ELISA. AI-073, CD24Fc (CHO-cell derived) , CD24Fc-293 (HEK293 cell derived) or human IgG1-Fc control protein were dissolved in coating buffer at concentration of 10 μg/mL and added into 96-well plates. The plate-bound protein was then detected by adding anti-CD24 mAb followed by HRP-conjugated second antibody and the substrate o-Phenylenediamine (OPD) . FIG. 4A shows the binding profile of AI-073 or CD24Fc to a two-fold serial dilution of the mouse anti-CD24 SN3 mAb. The EC50 values of the binding are shown in the underneath table. FIG. 4B shows the binding profile of AI-073 or CD24Fc to a two-fold serial dilution of the humanized anti-CD24 H3L3 mAb. The EC50 values of the binding are shown in the underneath table. FIG. 4C shows the binding profile of AI-073 or CD24Fc to a two-fold serial dilution of the mouse anti-CD24 ML5 mAb. The EC50 values of the binding are shown in the underneath table.

FIG. 5 illustrates the binding of AI-073 protein or CD24Fc protein to human Siglec-10 in ELISA. A 96-well plate pre-coated with HEK293 cell derived recombinant human Siglec-10-mIgFc protein (at concentration of 200 ng/mL) was incubated with the binding buffer containing a two-fold serial dilution of either AI-073 or CD24Fc fusion protein (all starting at 1.5 mg/mL) , the bound AI-073 or CD24Fc protein was detected by adding HRP-labeled goat anti-human IgG-Fc antibody and the substrate tetramethylbenzidin (TMB) . The EC50 values of the binding are shown in the underneath table.

FIG. 6 illustrates the binding of AI-073 protein or CD24Fc protein to human HMGB1 in ELISA. A 96-well plate pre-coated with recombinant human HMGB1-His tag protein (at concentration of 200 ng/mL) was incubated with the binding buffer containing a two-fold serial dilution of either AI-073 or CD24Fc fusion protein (both starting at 1.5 mg/mL) , the bound AI-073 or CD24Fc protein was then detected by HRP-labeled goat anti-human Fc antibody and the substrate TMB. The EC50 values of the binding are shown in the underneath table.

FIG. 7 illustrates the association of AI-073 protein with human HMGB1 in a pull-down assay. Recombinant HMGB1-His protein sample was incubated with AI-073, human IgG-Fc, or none for 5 min, the mixture was then incubated with protein A-conjugated beads to capture (or pull-down) the bound proteins. The captured proteins were then separated in an SDS-PAGE gel and visualized by Coomassie Brilliant Blue dye staining. The left gel (A) shows the input samples, and the right gel (B) show the pull-down samples, as marked. As indicated on the top of each gel, lane 1 represents the sample containing HMGB1 only, lane 2 represents the sample containing HMGB1 and human IgG-Fc, and lane 3 represents the sample containing HMGB1 and AI-073, lane M is the protein molecule weight marker sample. The positions of AI-073 protein, HMGB1 protein in input or pull-down sample are indicated to the right side of each gel, whereas the molecule weight (kDa) of the protein marker is displayed to the left side of each gel.

FIGs. 8A-8D illustrate the therapeutic effects of AI-073 to DSS-induced inflammatory bowel diseases in mice. Fig. 8A illustrates the procedure of DSS-induced mouse inflammatory bowel diseases and the treatment schedule. AI-073 protein or vehicle control was administrated to model mice by i.p injection on day 0 and day 6. Model mice were then observed daily, with body weight recorded, and survival rate calculated until day 14. Fig. 8B illustrates the animal body change (gram) vs time (day) curve in AI-073 protein treated group or vehicle control treated group. Fig. 8C illustrates the body weight loss rate (%) vs time (day) curve in AI-073 protein treated group or vehicle control treated group. Fig. 8D illustrates the animal survival rate (%) vs time (day) curve in AI-073 protein treated group (n=10) or vehicle control treated group (n=10) .

[1]FIGs. 9A-9B illustrate the methods of testing activities of AI-073 to collagen antibody induced arthritis (CAIA) . FIG. 9A illustrates the methods of inducing CAIA model and the treatment schedule. Arthritis was induced by i. v. injecting mice with anti-collagen cocktail antibodies (a mixture of 5-clones, 1.5mg/mouse) on day 0, and followed by i. v. injecting 50μg LPS on day 3 and day 4. Mice were then randomly separated into two groups, receiving either AI-073 protein (50 mg/kg) or vehicle control on day 0. On day 14, each mouse was re-administered 0.8 mg of anti-collagen cocktail mAbs by i. v. injection, followed by i.p. injection of 35 μg LPS on day 16. On day 19, these mice were treated with either a second dose (1 mg) of AI-073 or saline control. Mice were monitored daily, with body weight recorded, disease scored and survival rate calculated up to day 48. FIG. 9B illustrates the disease score ratio (day/day19) change from starting from day 19 to day 48 in animal group treated with either AI-073 protein (n=10) or vehicle control (n=10) .

FIGs. 10A-10B show the gel pictures of SDS-PAGE analysis of different version of AI-073-Fc proteins. FIG. 10A shows the SDS-PAGE gel of the purified, different versions of AI-073-Fc proteins in non-reducing (NR) conditions. FIG. 10B shows SDS-PAGE gel of the purified, different versions of AI-073-Fc proteins in reducing (R) conditions. About 5 μg of purified fusion protein of each sample in DTT-reducing (R) or non-reducing conditions (NR) , was loaded into SDS-PAGE gel. After electrophoresis, the gel was stained with Coomassie Brilliant Blue dye to visualize the protein bands. The molecule weight (kDa) of protein marker (M) in the gel was indicated to the right. AI-073-X protein, and AI-071-Y protein contains one or two copies of the 32-amino acid long 073-core peptide, respectively. AI-073-Z protein contains one copy of a 32-amino acid long 073-core peptide valiant with a 12-mer core peptide (GLAPNPT NATTK, Sequence ID. No 56: ) from other region of human CD24 mature protein.

FIGs. 11A-B illustrate the binding of different versions of AI-073-Fc protein (AI-073X, AI-073-Y and AI-073Z) and AI-073 control to different anti-CD24 mAb in ELISA. The different versions of AI-073-Fc protein (AI-073X, AI-073-Y and AI-073Z) and AI-073 control protein were dissolved in coating buffer at concentration of 2 μg/mL and added into 96-well plates. The plate-bound protein was then detected by adding 2-fold serial dilutions of anti-CD24 mAbs followed by HRP-conjugated goat anti-mouse IgG-Fc antibody (1: 2000) and the substrate o-Phenylenediamine (OPD) . FIG. 11A shows the binding profile of AI-073X, AI-073-Y, AI-073Z and AI-073 to two-fold serial dilutions of the mouse anti-CD24 SN3 mAb (staring at 2 μg/mL) . FIG. 11B shows the binding profile of AI-073X, AI-073-Y, AI-073Z and AI-073 to two-fold serial dilutions of the mouse anti-CD24 ML5 mAb (staring at 2 μg/mL) .

[2] Figures 12A-12B show the binding of different versions of AI-073-Fc protein (AI-073X, AI-073-Y and AI-073Z) to human Siglec-10 or HMGB1 in ELISA. A control sample of AI-073 fusion protein was included in the assay and served for comparison. Fig 12A shows the binding of these 3 different versions of AI-073-Fc protein (AI-073X, AI-073-Y and AI-073Z) and control sample of AI-073 to the GST-tagged human Siglec-10 protein. Briefly, a 96-well plate was coated with recombinant GST-Siglec-10 fusion protein (at concentration of 200 ng/mL) . After washing and blocking, the plate was incubated with 3-fold serial dilutions of different versions of AI-073-Fc protein (AI-073X, AI-073-Y and AI-073Z) or AI-073 control sample (all starting at 1.0 mg/mL) , the bound protein was then detected by adding HRP-labeled goat anti-human (1: 1000, Invitrogen, A18829) , followed by the addition of TMB substrate. Fig. 12B shows the binding of these different versions of AI-073-Fc protein (AI-073X-Fc, AI-073Y-Fc and AI-073Z-Fc) and AI-073 control sample to his-tagged human HMGB1 protein pre-bound to the plate. Briefly, a 96-well plate was coated with 100 ng/mL HMGB1-His tag protein (AcroBiosystems, HM1-H5220, HEK293 cell derived) . The plate was then incubated with 3-fold serial dilutions of different version of AI-073-Fc protein (AI-073X-Fc, AI-073Y-Fc and AI-073Z-Fc) or AI-073 control sample (all starting at 1.0 mg/mL) ) in PBST-1%BSA solution containing 1mM MgCl2 and 1mM CaCl2. The bound protein was then detected by adding HRP-labeled goat anti-human IgG-Fc antibody (1: 1000, Invitrogen, A18829) , followed by the addition of TMB substrate.

DETAILED DESCRIPTION

[1] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may 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.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in the specification and the appended claims, the singular forms "a, " "an" and "the" include plural referents unless the context clearly dictates otherwise.

For recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the

numbers

7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6, 9, and 7.0 are explicitly contemplated.

The term "peptide" or "polypeptide" may refer to a linked sequence of amino acids and may be natural, synthetic, or a modification or combination of natural and synthetic.

The term "glycopeptide" or "glycoprotein" may refer to a modification of natural or synthetic peptide or protein with sugar or oligosaccharide attached or linked to the amino acid residues.

The term "Substantially identical" may refer to a first and second amino acid sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%over a region of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 amino acids.

"Treatment" or "treating, " when referring to protection of an animal from a disease, may refer to preventing, suppressing, repressing, or completely eliminating the disease. Preventing the disease may involve administering a composition of the present invention to an animal prior to onset of the disease. Suppressing the disease may involve administering a composition of the present invention to an animal after induction of the disease but before its clinical appearance. Repressing the disease may involve administering a composition of the present invention to an animal after clinical appearance of the disease.

A "variant" may refer to a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity. Representative examples of "biological activity" may include the ability to bind to a toll-like receptor and to be bound by a specific antibody. Variant may also mean a protein with an amino acid sequence that is substantially identical to a referenced protein with an amino acid sequence that retains at least one biological activity. A conservative substitution of an amino acid, i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, degree and distribution of charged regions) may be recognized in the art as typically involving a minor change. These minor changes can be identified, in part, by considering the hydropathic index of amino acids, as understood in the art. The hydropathic index of an amino acid may be based on a consideration of its hydrophobicity and charge. It may be known in the art that amino acids of similar hydropathic indexes can be substituted and still retain protein function. In one aspect, amino acids having hydropathic indexes of +2 may be substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a peptide may permit calculation of the greatest local average hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. Substitution of amino acids having similar hydrophilicity values can result in peptides retaining biological activity, for example immunogenicity, as is understood in the art. Substitutions may be performed with amino acids having hydrophilicity values within +2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids may be influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function may be understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties.

The term “Mucin” or “Muc” may refer to a protein or peptide. As used herein, Mucin may be a transmembrane/membrane-bound protein. Mucin may encompass Mucin proteins, protein fragments, protein analogs, oligopeptides, and/or a variant thereof. For example, the Mucin fragment may not include the full-length Mucin protein. For example, the Mucin may comprise MUC1, MUC3A, MUC3B, MUC4, MUC12, MUC16, MUC17 and etc. The UniProt No. for MUC1 may be P15941.

The term “CD24” may refer to a protein or peptide. As used herein, CD24 may be a glycosylphosphatidylinositol (GPI) -anchored protein with potential O-and N-glycosylation sites. CD24 may encompass CD24 proteins, protein fragments, protein analogs, oligopeptides, and/or a variant thereof. For example, the CD24 fragment may not include the full length CD24 protein. The UniProt No. for CD24 may be P25063.

The term “fusion” as used herein refers to a fused molecule. wherein the components of the fusion molecule may be linked to each other by bonds, like peptide bonds, either directly or via a peptide linker. The individual peptide chains of the fusion molecule may be linked non-covalently, for example by disulfide bonds.

073-core peptide or fragment

In the present disclosure, a series of recombinant polypeptides or fusion proteins are created, described with their amino acid sequence disclosed here.

For example, the present application provides a protein comprising one or more copies of a first fragment and one or more copies of a second fragment, each of said first fragment may comprise a sequence independently selected from those as set forth in SEQ ID NO: 1, 22 to 38, or SEQ ID NO: 56, and each of said second fragment may comprise an amino acid sequence derived from Mucin1 protein. For example, each of said second fragment may comprise a sequence independently selected from those as set forth in SEQ ID NO: 2 and 8 to 21.

For example, the present application provides a composition comprising one or more copies of a first fragment and one or more copies of a second fragment, each of said first fragment may comprise a sequence independently selected from those as set forth in SEQ ID NO: 1 and 22 to 38, SEQ ID NO: 56, and each of said second fragment may comprise an amino acid sequence derived from Mucin1 protein. For example, each of said second fragment may comprise a sequence independently selected from those as set forth in SEQ ID NO: 2 and 8 to 21.

For example, the present application provides a pharmaceutical product comprising one or more copies of a first fragment and one or more copies of a second fragment, each of said first fragment may comprise a sequence independently selected from those as set forth in SEQ ID NO: 1 and 22 to 38, SEQ ID NO: 56, and each of said second fragment may comprise an amino acid sequence derived from Mucin1 protein. For example, each of said second fragment may comprise a sequence independently selected from those as set forth in SEQ ID NO: 2 and 8 to 21.

For example, the protein, the composition or the product may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more copies of said first fragment. For example, the protein, the composition or the product may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more copies of said second fragment.

For example, wherein one or more copies of the first fragment is fused directly or indirectly to one or more copies of the second fragment. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more copies of the first fragment might be fused directly or indirectly to1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more copies of the second fragment. For example, 1 or more copies of the first fragment might be fused directly or indirectly to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more copies of the second fragment. For example, 1 or more copies of the first fragment might be fused directly to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more copies of the second fragment.

For example, wherein the C-terminus of said one or more copies of the first fragment may be fused directly or indirectly to the N-terminus of one or more copies of the second fragment.

For example, wherein the C-terminus of said one or more copies of the second fragment may be fused directly or indirectly to the N-terminus of one or more copies of the first fragment.

For example, the polypeptide or protein of the present disclosure may comprise one or more copies of a 073-core portion, said 073-core portion comprises said first fragment and said second fragment. For example, each of said first fragment may comprise a sequence independently selected from those as set forth in SEQ ID NO: 1 and 22 to 38, SEQ ID NO: 56, and each of said second fragment may comprise a sequence independently selected from those as set forth in SEQ ID NO: 2 and 8 to 21. For example, each of said first fragment may comprise a sequence as set forth in SEQ ID NO: 1, and each of said second fragment may comprise a sequence as set forth in SEQ ID NO: 2.

For example, wherein the said first fragment and said second fragment may be fused directly or indirectly. For example, wherein the said 073-core portion may be fused directly or indirectly to one or more copies of the first fragment and/or one or more copies of the second fragment. For example, said fused indirectly may comprise fused via a linker. For example, wherein said linker may be a peptide linker. For example, said first fragment and second fragment fused indirectly may mean that said first fragment and second fragment might be fused via a linker. For example, the linker may be a (GnS) n linker such as GGGGS, or GGGGSGGGGSGGGGS.

For example, the polypeptide or protein of the present disclosure may comprise a sequence, 073-core portion or AI-073 core-fragment independently selected from those as set forth in SEQ ID NO: 3 to 7.

These polypeptides or fusion proteins comprise a so called 073-core peptide (or 073-fragment) . This 073-core peptide comprises one or more copies of a first fragment (also called as fragment-A hereafter) and one or more copies of a second fragment (also called as fragment-B hereafter) , each of said first fragment comprises the amino acid sequence STSNSGLAPNPT (SEQ ID NO: 01) and each of said second fragment comprises the amino acid sequence PAPGSTAPPAHGVTSAPDTR (SEQ ID NO: 02) . In the 073-core peptide, the fragment-A can be located either at the N-terminal such as set forth in SEQ ID NO: 03, or at the C-terminal such as set forth in SEQ ID NO: 04 or at both ends such as set forth in SEQ ID NO: 05. Similarly, the fragment-B can also be located at either the N-terminal, the C-terminal or in both ends. The fragment-A or the fragment-B or both can also be located in the middle such as set forth in SEQ ID NO: 06 or SEQ ID NO: 07. In each case, a single copy of the fragment-A or the fragment-B each contains 5 potential mucin-like O-glycosylation sites (serine or threonine in STP motif) , and when two or more copies of the fragment-A linked with two or more copies of the fragment-B together and expressed in mammalian cells, they may be heavily glycosylated and/or sialylated.

In the present disclosure, variants or modifications of the 073-core peptide are also provided. These variants or modifications can be occurred either in the fragment-A or in the fragment-B or both. For examples, these variants may have one of the following amino-acid sequences in the fragment-B: PAPGSTAPPAHGVTSAP ESR (SEQ ID NO: 08) ; PAPGSTAP AAHGVTSAPDTR (SEQ ID NO: 09) ; PAPGSTAP TAHGVTSAPDTR (SEQ ID NO: 10) ; PAPGSTAPQAHGVTSAPDTR (SEQ ID NO: 11) ; PAPGSTAPPAHGVTSAPD NR (SEQ ID NO: 12) or PAPG XXAPPAHGV XXAPD XR (X=S or T, SEQ ID NO: 13) . Each copy of the modified fragment-B in these variants may still contain 5 mucin-like O-glycosylation sites and might be heavily glycosylated and/or sialylated when two or more copies of it linked together and expressed in mammalian cells. In another example, the fragment-B may be derived from other mammalian species Muc1 and have one of the following sequences: PTPGSTAPPAHGVTSAPDTR (SEQ ID NO: 14) from Gibbon Muc1; AAPGSAAPPAHDVTSAPGTS (SEQ ID NO: 15) from Baboon Muc1; AAPGSTAPPAHVVTSAPDTS (SEQ ID NO: 16) from monkey (Macaca fascicularis) Muc1; APVDSTSSPVHGGTSSPATS (SEQ ID NO: 17) from mouse Muc1, PPEDSTSTAVTSGTSSPATS (SEQ ID NO: 18) from rat Muc1, APATSPTSVSATSPVHEVTS (SEQ ID NO: 19) from rabbit Muc1, PAPSSTTSLGKHSSSSLTSS (SEQ ID NO: 20) from dog Muc1, and PAPSPAASPGHDGASTPTSS (SEQ ID NO: 21) from cattle Muc1. Each copy of these fragments may contain 5 or more mucin-like O-glycosylation sites and might be heavily glycosylated and/or sialylated when two or more copies of it linked together and expressed in mammalian cells.

For the modifications in the fragment-A in 073-core peptide variants, this fragment may have one of following amino-acid sequences: STSNSG FAPNPT (SEQ ID NO: 22) from chimpanzee (Gorilla gorilla or Pan troglodytes) CD24; SSQSTSAAPSPA (SEQ ID NO: 23) from marmoset monkey (Callithrix jacchus) CD24; SSQNTSTTPNPA (SEQ ID NO: 24) from monkey (Macaca fascicularis) CD24; SNQSISTAPNPT (SEQ ID NO: 25) from hamster CD24; SSQSTSTAPNPA (SEQ ID NO: 26) from dog (Canis lupus familiaris) CD24; SSQTTSPAPHPA (SEQ ID NO: 27) from cattle CD24; SSQTTSAIPNPA (SEQ ID NO: 28) from camel CD24; GNQNISASPNPS (SEQ ID NO: 29) from mouse CD24, and GNQSISAAPNPT (SEQ ID NO: 30) from rat CD24. Each one of these fragments may contain 3, 4 or more mucin like O-glycosylation sites and might be heavily glycosylated or sialylated when two or more copies of it linked together and expressed in mammalian cells.

Yet, in another example, variants or modifications of the 073-core peptide may have one of the following amino-acid sequences in the fragment-A: XTSNSGLAPNPT (X=S or T, SEQ ID NO: 31) ; STXNSGLAPNPT (X=S or T, SEQ ID NO: 32) ; STSNXGLAPNPT (X=S or T, SEQ ID NO: 33) ; STSNTGLAPNPX (X=S or T, SEQ ID NO: 34) ; XTSNTGLAPNPX (X=S or T, SEQ ID NO: 35) ; STSNTXXAPNPT (X=S or T, SEQ ID NO: 36) , XXSNTGLAPNPT (X=S or T, SEQ ID NO: 37) and XXXNXGLAPNPX (each X is independently=S or T, SEQ ID NO: 38 ) . Each copy of these fragments may contain 3, 4 or more mucin like O-glycosylation sites and might be heavily glycosylated or sialylated when two or more copies of it linked together and expressed in mammalian cells.

For one example of the protein, wherein said one, two or more 073 core fragments may be linked together and fused with human IgG-Fc. The present example provides one of such isolated fusion proteins namely AI-073-Fc, wherein the amino acid sequence of said AI-073-Fc fusion protein consists of the sequence as set forth in SEQ ID NO: 47, 49 or 51.

For another example of the protein, wherein at least two or more copies of said 073 core fragment may be indirectly linked to each other via a linker. For one example of the protein, wherein said linker may be a peptide linker. For example, the peptide linker may be a (GnS) n linker such as GGGGS, or GGGGSGGGGSGGGGS.

For one example of the protein, wherein said immunoglobulin fragment may comprise a Fc portion of said immunoglobulin. For one example of the protein, further comprising a second portion, said second portion may comprise an immunoglobulin fragment. For one example of the protein, wherein said immunoglobulin fragment may comprise a Fc portion of said immunoglobulin.

For one example of the protein, wherein said immunoglobulin fragment may comprise a hinge region of said immunoglobulin. For one example of the protein, wherein said immunoglobulin fragment may comprise a CH2 domain. For one example of the protein, wherein said immunoglobulin fragment may comprise a CH3 domain. For one example of the protein, wherein said immunoglobulin fragment may comprise a CH4 domain. For example, said immunoglobulin fragment may comprise hinge region and CH2 and CH3 domains of said Ig protein. For example, said Ig may be selected from the group consisting of IgG1, IgG2, IgG3, IgG4, and IgA. For example, said immunoglobulin fragment may comprise hinge region and CH3 and CH4 domains of said Ig protein. For example, said Ig may be IgM. For example, said immunoglobulin fragment may comprise hinge region and CH2, CH3 and CH4 domains of said Ig protein. For one example of the protein, wherein said immunoglobulin may be selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgM and IgA. For example, wherein said immunoglobulin may comprise a sequence selected from those as set forth in SEQ ID NO: 39 to 46.

For one example of the protein, wherein said second portion may be directly or indirectly linked to said 073 core-derived-region.

For one example of the protein, wherein said second portion may be indirectly linked to said 073 core-derived-region via a linker. For example, said second portion may be directly linked to said 073 core-derived-region. For example, said second portion may be directly linked to said 073 core-derived-region, and said second portion may not comprise hinge region. For example, said second portion may be directly linked to said 073 core derived-region, and said second portion may comprise CH2 and CH3 domains of said Ig protein. For example, said second portion may be directly linked to said 073 core-derived-region, and said second portion may comprise CH3 and CH4 domains of said Ig protein.

For one example of the protein, wherein said 073 core-derived-region may be linked directly or indirectly to the N-terminus of said second portion.

For one example of the protein, it may comprise the amino acid sequence as set forth in SEQ ID NO: 03, SEQ ID NO: 04, SEQ ID NO: 05, SEQ ID NO: 6 or SEQ ID NO: 7.

In this patent application, 073 core peptide-containing Fc-fusion proteins can be created in such a way that a polypeptide comprising one or more copies 073 core peptide is fused with the human IgG-Fc tail, which is ideally located at the C-terminal. The human IgG-Fc tail may consist of hinge-CH2-CH3 regions with the amino acid sequences shown as in SEQ ID NO: 45. The hinge region of the human Fc may come from IgG1, IgG4 or IgA1 and have one of the amino acid sequences as set in SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 44.

For one example of the 073 core-fragment or the protein, it may be glycosylated.

For one example of the 073 core-fragment or the protein, it may be capable of binding to one or more Siglecs. For one example of the 073 core-fragment or the protein, wherein said one or more Siglecs may comprise human Siglec. For one example of the 073 core-fragment or the protein, wherein said one or more Siglecs may comprise Siglec-10.

For one example of the 073 core-fragment or the protein, it may be capable of binding to High Mobility Group Protein B1 (HMGB1) .

For one example of the 073 core-fragment or the protein, wherein said 073 core may be derived from human protein. For example, said 073 core from other mammalian species are also provided.

The present disclosure provides an immunoconjugate, comprising the 073 core-fragment of the present disclosure, or the protein of the present disclosure.

The present disclosure provides a nucleic acid, encoding the 073 core-fragment of the present disclosure, or the protein of the present disclosure.

The present disclosure provides a vector, comprising the nucleic acid of the present disclosure.

The present disclosure provides a cell, comprising and/or expressing the 073 core-fragment of the present disclosure, the protein of the present disclosure, the immunoconjugate of the present disclosure, the nucleic acid of the present disclosure, and/or the vector of the present disclosure.

The present disclosure provides a composition, comprising the 073 core-fragment of the present disclosure, the protein of the present disclosure, the immunoconjugate of the present disclosure, the nucleic acid of the present disclosure, the vector of the present disclosure, and/or the cell of the present disclosure, and optionally a pharmaceutically acceptable carrier.

The present disclosure provides a method for preparing the 073 core-fragment of the present disclosure, or the protein of the present disclosure, comprising culturing the cell of the present disclosure under a condition enabling the expression of said 073 core-fragment or said protein.

The present disclosure provides a method for regulating a Siglec related signaling, comprising administering to a subject in need thereof an effective amount of the 073 core-fragment of the present disclosure, the protein of the present disclosure, the immunoconjugate of the present disclosure, the nucleic acid of the present disclosure, the vector of the present disclosure, the cell of the present disclosure, and/or the composition of the present disclosure. For example, the Siglec related signaling may comprise Siglec-mediated regulation of immune cell function. For example, the Siglec related signaling may comprise CD24-Siglec 10/G interaction. The method of the present disclosure, which may activate the Siglec related signaling. The method of the present disclosure, which may inhibit the Siglec related signaling.

The present disclosure provides the 073 core-fragment of the present disclosure, the protein of the present disclosure, the immunoconjugate of the present disclosure, the nucleic acid of the present disclosure, the vector of the present disclosure, the cell of the present disclosure, and/or the composition of the present disclosure, for use in regulating a Siglec related signaling. For example, the Siglec related signaling may comprise Siglec-mediated regulation of immune cell function. For example, the Siglec related signaling may comprise CD24-Siglec 10/G interaction. For example, activating the Siglec related signaling. For example, inhibiting the Siglec related signaling.

The present disclosure provides a use of the 073 core-fragment of the present disclosure, the protein of the present disclosure, the immunoconjugate of the present disclosure, the nucleic acid of the present disclosure, the vector of the present disclosure, the cell of the present disclosure, and/or the composition of the present disclosure in the preparation of a medicament, wherein said medicament is used for regulating a Siglec related signaling. For example, the Siglec related signaling may comprise Siglec-mediated regulation of immune cell function. For example, the Siglec related signaling may comprise CD24-Siglec 10/G interaction. For example, activating the Siglec related signaling. For example, inhibiting the Siglec related signaling.

The present disclosure provides a method for regulating an immune response, comprising administering to a subject in need thereof an effective amount of the 073 core-fragment of the present disclosure, the protein of the present disclosure, the immunoconjugate of the present disclosure, the nucleic acid of the present disclosure, the vector of the present disclosure, the cell of the present disclosure, and/or the composition of the present disclosure.

The present disclosure provides the 073 core-fragment of the present disclosure, the protein of the present disclosure, the immunoconjugate of the present disclosure, the nucleic acid of the present disclosure, the vector of the present disclosure, the cell of the present disclosure, and/or the composition of the present disclosure, for use in regulating an immune response.

The present disclosure provides a use of the 073 core-fragment of the present disclosure, the protein of the present disclosure, the immunoconjugate of the present disclosure, the nucleic acid of the present disclosure, the vector of the present disclosure, the cell of the present disclosure, and/or the composition of the present disclosure in the preparation of a medicament, wherein said medicament is used for regulating an immune response.

The present disclosure provides a method for repressing an immune-mediated tissue damage mediated by danger-associated molecular patterns (DAMPs) , comprising administering to a subject in need thereof an effective amount of the 073 core-fragment of the present disclosure, the protein of the present disclosure, the immunoconjugate of the present disclosure, the nucleic acid of the present disclosure, the vector of the present disclosure, the cell of the present disclosure, and/or the composition of the present disclosure. For one example of the method, wherein said immune-mediated tissue damage may be selected from the group consisting of graft vs host diseases, immunotherapy-related adverse events, rheumatoid arthritis, inflammatory bowel diseases (IBD) , and multiple sclerosis (MS) .

The present disclosure provides the 073 core-fragment of the present disclosure, the protein of the present disclosure, the immunoconjugate of the present disclosure, the nucleic acid of the present disclosure, the vector of the present disclosure, the cell of the present disclosure, and/or the composition of the present disclosure, for use in repressing an immune-mediated tissue damage mediated by danger-associated molecular patterns (DAMPs) . For example, wherein said immune-mediated tissue damage may be selected from the group consisting of graft vs host diseases, immunotherapy-related adverse events, rheumatoid arthritis, inflammatory bowel diseases (IBD) , and multiple sclerosis (MS) .

The present disclosure provides a use of the 073 core-fragment of the present disclosure, the protein of the present disclosure, the immunoconjugate of the present disclosure, the nucleic acid of the present disclosure, the vector of the present disclosure, the cell of the present disclosure, and/or the composition of the present disclosure in the preparation of a medicament, wherein said medicament is used for repressing an immune-mediated tissue damage mediated by danger-associated molecular patterns (DAMPs) . For example, wherein said immune-mediated tissue damage may be selected from the group consisting of graft vs host diseases, immunotherapy-related adverse events, rheumatoid arthritis, inflammatory bowel diseases (IBD) , and multiple sclerosis (MS) .

The present disclosure provides a method for preventing, ameliorating and/or treating a disease or condition caused by an inflammatory response arising from tissue injuries from infectious agents, comprising administering to a subject in need thereof an effective amount of the 073-core-fragment of the present disclosure, the protein of the present disclosure, the immunoconjugate of the present disclosure, the nucleic acid of the present disclosure, the vector of the present disclosure, the cell of the present disclosure, and/or the composition of the present disclosure. For one example of the method, wherein said disease or condition may be associated with viral infection. For one example of the method, wherein said disease or condition may be COVID-19. For one example of the method, wherein said disease or condition may be influenza. For one example of the method, wherein said disease or condition may be acquired immunodeficiency syndrome (AIDS) . For one example of the method, wherein said disease or condition may be associated with bacterial infection. For one example of the method, wherein said disease or condition may be bacterial pneumonia.

The present disclosure provides the protein of the present disclosure, the immunoconjugate of the present disclosure, the nucleic acid of the present disclosure, the vector of the present disclosure, the cell of the present disclosure, and/or the composition of the present disclosure, for use in preventing, ameliorating and/or treating a disease or condition caused by an inflammatory response arising from tissue injuries from infectious agents. For example, wherein said disease or condition may be associated with viral infection. For example, wherein said disease or condition may be COVID-19. For example, wherein said disease or condition may be influenza. For example, wherein said disease or condition may be acquired immunodeficiency syndrome (AIDS) . For example, wherein said disease or condition may be associated with bacterial infection. For example, wherein said disease or condition may be bacterial pneumonia.

The present disclosure provides a use of the protein of the present disclosure, the immunoconjugate of the present disclosure, the nucleic acid of the present disclosure, the vector of the present disclosure, the cell of the present disclosure, and/or the composition of the present disclosure in the preparation of a medicament, wherein said medicament is used for preventing, ameliorating and/or treating a disease or condition caused by an inflammatory response arising from tissue injuries from infectious agents or pathogen-associated molecular patterns (PAMPs) . For example, wherein said disease or condition may be associated with viral infection. For example, wherein said disease or condition may be COVID-19. For example, wherein said disease or condition may be influenza. For example, wherein said disease or condition may be acquired immunodeficiency syndrome (AIDS) . For example, wherein said disease or condition may be associated with bacterial infection. For example, wherein said disease or condition may be bacterial pneumonia.

The present disclosure provides a method for preventing, ameliorating and/or treating a disease or condition caused by acute tissue damage from wound, comprising administering to a subject in need thereof an effective amount of the 073 core-fragment of the present disclosure, the protein of the present disclosure, the immunoconjugate of the present disclosure, the nucleic acid of the present disclosure, the vector of the present disclosure, the cell of the present disclosure, and/or the composition of the present disclosure. For one example of the method, wherein said disease or condition may be septicemia, crush syndrome and/or ischemia reperfusion injury.

The present disclosure provides the 073 core-fragment of the present disclosure, the protein of the present disclosure, the immunoconjugate of the present disclosure, the nucleic acid of the present disclosure, the vector of the present disclosure, the cell of the present disclosure, and/or the composition of the present disclosure, for use in preventing, ameliorating and/or treating a disease or condition caused by acute tissue damage from wound. For example, wherein said disease or condition may be septicemia, crush syndrome and/or ischemia reperfusion injury.

The present disclosure provides a use of the 073 core-fragment of the present disclosure, the protein of the present disclosure, the immunoconjugate of the present disclosure, the nucleic acid of the present disclosure, the vector of the present disclosure, the cell of the present disclosure, and/or the composition of the present disclosure in the preparation of a medicament, wherein said medicament is used for preventing, ameliorating and/or treating a disease or condition caused by acute tissue damage from wound. For example, wherein said disease or condition may be septicemia, crush syndrome and/or ischemia reperfusion injury.

Examples

The following examples are set forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc. ) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair (s) ; kb, kilobase (s) ; pl, picoliter (s) ; s or sec, second (s) ; min, minute (s) ; h or hr, hour (s) ; aa, amino acid (s) ; nt, nucleotide (s) ; i. m., intramuscular (ly) ; i.p., intraperitoneal (ly) ; s. c., subcutaneous (ly) ; and the like.

Example 1 Generation and characterization of AI-073-Fc fusion protein

In the present disclosure, polypeptides or fusion proteins contains one, two or more copies of 073-core peptide were generated by using recombinant DNA methods. FIG. 3A shows the schematic structure of one of these fusion proteins, namely AI-073-Fc fusion protein (hereafter also just called as AI-073) . The amino acid sequence of AI-073 protein may be as set forth in SEQ ID NO: 51. The N-terminal portion of this fusion protein contains a 76-mer peptide which comprises 3 copies of a 12-mer fragment-A with the amino acid sequence as set forth in SEQ ID NO: 01 and two copies of a 20-mer fragment-B with the amino acid sequence as set forth in SEQ ID NO: 02. The DNA fragment encoding this 76-mer long polypeptide was synthesized in-vitro and fused with a DNA fragment encoding a 26 amino acid long signal peptide MGRAMVARLGLGLLLLALLLPTQIYS (SEQ ID NO: 53) at the N-terminal and a DNA fragment encoding a 232 amino acid long human IgG1-hinge-CH2-CH3 region (SEQ ID NO: 45) at the C-terminal. The recombinant DNA was cloned into expression plasmid vector pDNA3.1 by using stand recombinant DNA techniques. The recombinant plasmids were transferred into mammalian CHO cells by electroporation. After electroporation, cells were cultured in serum-free media for 6-7 days and supernatants were then collected, passed over a column of Protein A resin (MabSelect from GE Healthcare) at a concentration not exceeding 16 g/L of resin (based on ELISA) . The Fc-fusion protein bound on the column was eluted and collected by using low pH solution (0.1 M citric acid, pH 3.5) . Eluted protein was then re-suspended in 1x PBS (pH=7.4) and stored at either 4 ℃ or -20 ℃ until use.

Approximately 2 μg of purified AI-073-Fc fusion protein, either in DTT-reducing or non-reducing conditions was loaded into an SDS-PAGE gel. As shown in Fig. 3B, under DTT-reduced condition, the size of this fusion protein sample is about the half of that in non-reducing condition, this agrees with the natural dimer formation of Fc-fusion protein. Under either DTT-reducing or non-reducing conditions, the appearing molecule weight of AI-073-Fc fusion protein is much greater than that theory deducted by protein peptide sequence only. For instance, the monomer of AI-073 mature peptide has 308 amino acid residues, which would have a theory corresponding protein molecule weight of about 32kD. The additional gained molecule weight (about 20-25 kD, which contributes almost half of the molecule weight) is most likely from massively glycans attached to this glycoprotein.

The purified, intact AI-073-Fc protein was further subjected to a SEC-HPLC analysis (Fig. 3C) . As shown in Fig. 3C, a main peak with a retention time (RT) at 7.276 min and an area of 98.79%was detected by this SEC-HPLC analysis.

Example 2 The 073 core in AI-073 is sialylated: ELISA analysis of AI-073 protein with different anti-CD24 mAbs

The AI-073-Fc protein, as illustrated in Fig. 3A, comprising a 073 core-peptide and human Ig Fc region. The 073 core-peptide contains 3 copies of a 12 amino acid long fragment-A (SEQ ID NO: 01) which is derived from human CD24 middle core peptide region, and two copies of a 20 amino acid long fragment-B (SEQ ID NO: 02) which is derived from human Muc1-TR. Thus, the antigen identity of AI-073 protein can be determined by the binding to the anti-CD24 middle core antibodies and/or anti-human Muc1-TR antibodies. Furthermore, the glycosylation and/or sialylation modified fragment-A in the AI-073 protein can be recognized and detected by glycosylation and/or sialylation-dependent mAbs such as SN3 (ab134375 from Abcam) , while the hypo-glycosylation/unsialylated fragment-A can be recognized and detected by H3L3 (a humanized anti-CD24 mAb, as described in the United States Patent Application 20210214458) . The total amount of fragment-A can be measured by the binding to another mouse mAb ML5, which recognizes human CD24 middle core peptide backbone.

For this purpose, an antigen binding ELISA was developed. Briefly, 96-well plates were coated with 10 μg/mL of either AI-073 (produced in CHO cells) , CD24Fc (produced in CHO cells) , CD24Fc-293 (from AcroBiosystems, Catalog #CD4-H5254, produced in human HEK293 cells) or human IgG1-Fc control (produced in CHO cells) at 4 ℃ overnight. After blocking with PBS-0.1%Tween20 solution (PBST) , 100μL of two-fold serial dilutions of either SN3 (ab134375 from Abcam) , ML5 (ab278509 from Abcam) or humanized H3L3 were added into the plates. The bound mouse SN3 or ML5 antibodies were detected by biotin-labeled goat anti-mouse IgG-Fc followed by HRP-labeled Avidin, whereas the bound humanized H3L3 antibody was detected by HRP-labeled goat anti-human IgG-Fab specific antibodies. The plates were then incubated with o-Phenylenediamine (OPD) substrates. After a color development at room temp for 15 mins, 1N HCl stop solution was added into the plates. The OD values at a wavelength of 492 nm (OD492) in each well were then measured.

The representative ELISA results are showed in Figs 4A-C, and the EC ₅₀ values are summarized in the table 1.

Table 1 Summary data of EC ₅₀

Detecting mAb (EC ₅₀)	AI-073	CD24Fc (CHO)	CD24Fc (HEK293)
SN3 (EC ₅₀, M)	2.599E-9	No binding	8.192E-9
H3L3 (EC ₅₀, M)	2.473E-9	2.234E-9	2.529E-9
ML5 (EC ₅₀, M)	7.227E-10	2.977E-9	1.547E-9
SN3/ML5 binding ratio	0.278	None	0.189
H3L3/ML5 binding ratio	0.292	1.332	0.611

Fig. 4A shows the binding of AI-073 or CD24Fc to the glycosylation and/or sialylation dependent SN3 mAb. As shown in the Figure, both CHO-derived AI-073 and HEK293-derived CD24Fc bind to SN3, indicated that the core peptide is highly glycosylated and/or sialylated. However, CHO cell derived CD24Fc showed no binding at all.

H3L3 mAb binds to hypo-glycosylated or un-sialylated CD24 molecule (seen in the United States Patent Application 20210214458) and is thus a good indication for the extent of un-sialylated 073 core. As shown in Figs. 4B-4C, and in Table 1, although AI-073 and CHO cells derived CD24Fc both bind to H3L3 at comparable EC ₅₀, AI-073 contained less un-sialylated residues per 073 core, as AI-073 protein has three copies of fragment-A (which contains CD24 middle core-peptide epitope) with a much more potent binding to ML5, which shows all antibody-accessible CD24 middle core-peptide epitope.

As outlined above, the ability of these three mAbs binding to AI-073 allow the practioneer with ordinary skill to optimize the composition for increasing glycosylation and/or sialylation. For instance, one may generate constructs or culture conditions to increase the ratio of SN3/ML5 binding while decreasing the ratio of H3L3/ML5 binding, using the method disclosed herein, or other methods to measure antibody-antigen-binding.

In one embodiment, optimal sialyation vs total CD24 middle core epitope, one may choose 1 to 10 or more copies of the 073 core to achieve optimal sialylation using the principle disclosed above.

In another embodiment, one may replace the amino acids within the 073 core, either uses homologous amino acids from other CD24 species from chimpanzee, monkey, dog, sheep, mouse, rat and etc. In yet another embodiment, one may switch the position of serine (Ser or S) or threonine (Thr or T) or increase or alter the pattern of amino acids to increase O-linked glycosylation using known art in the field. In yet another embodiment, one may increase sialylation of the 073 core using culture conditions that favors O-linked glycosylation and/or sialylation. In yet another embodiment, one may modify the CHO cells genetically to increase sialyotransferase activity.

Example 3. Generation of Siglec super agonist using glycosylated/sialylated AI-073 core

Defective Siglec function exacerbates inflammation caused by tissues injuries or infections. Diseases associated with such inflammation includes classic sterile inflammation such as drug-induced liver damage, rheumatoid arthritis, inflammatory bowel diseases (IBD) , multiple sclerosis, and pathological setting in which infections cause tissue injuries such as COVID-19, influenza pneumonia and sepsis.

A super siglec-agonist that show enhanced and broad binding to multiple Siglecs may have therapeutic valuation for treating diseases arising from inflammation caused by tissue injuries or infections.

AI-073 protein has a superior binding to Siglec-10

To demonstrate that AI-073 protein binds to Siglec-10, an ELSIA assay was developed. In-brief, 96-well plates were coated (100 μL per well) with 0.2 μg/mL of Siglec10-mIgG2aFc fusion protein (AcroBiosystems, SI0-H525b, HEK293 cell derived) at 4 ℃ overnight. After blocking with SuperBlock (Thermo, 37515) at room temperature for 1 hour, 100 μL of 2-fold serial dilutions of either AI-73 or CD24Fc (all starting at 1.5 mg/ml) were added. The bound AI-073 or CD24Fc protein was then detected by HRP-labeled goat anti-human IgG-Fc antibody (1:5000, Invitrogen, A18829) followed by the addition of tetramethylbenzidin (TMB) substrates. After a color development at room temp for 15 min, 2N HCl stop solution was added into the plate. The OD values at a wavelength of 450 nm (OD 450nm) in each well were then measured.

Fig. 5 shows one of the representative ELISA results. As shown in Fig, AI-073 has about 20-folder higher Siglec-10 binding affinity (EC ₅₀ of 1.43E-7M) than CD24Fc (EC ₅₀: 2.846E-6M) . Thus, this data indeed demonstrated that AI-073 has a superior binding to Siglec-10.

Example 4 AI-073 protein has a superior binding to High Mobility Group Box 1 (HMGB1)

To demonstrate that AI-073 protein also binds to HMGB1, a similar ELISA assay was developed. In-brief, 96-well plates were coated (100 μL per well) with 0.1 μg/mL HMGB1-His tag protein (AcroBiosystems, HM1-H5220, HEK293 cell derived) at 4 ℃ overnight. After blocking with SuperBlock (Thermo, 37515) at room temperature for 1 hour, 100 μL of 2-fold serial dilutions of AI-073 or CD24Fc (all starting at 1.5 mg/mL, and diluted in PBST-1%BSA solution containing 1mM MgCl2 and 1mM CaCl2) were added. The bound AI-073 or CD24Fc protein was then detected by adding HRP-labeled goat anti-human IgG-Fc antibody (1: 1000, Invitrogen, A18829) , followed by the addition of tetramethylbenzidin (TMB) substrates. After a color development at room temp for 15 mins, 2N HCl stop solution was then added into the plate. The OD values at a wavelength of 450 nm in each well were then measured.

Fig. 6 shows one of the representative ELISA results. As shown in Figure, AI-073 has more than 100-fold higher HMGB1-binding activity (EC ₅₀: 5.042E-8M) than CD24Fc (EC ₅₀: 5.095E-5M) . Thus, this data demonstrated that AI-073 also has a superior binding to HMGB1.

Probe of AI-073 protein and HMGB1 interaction by pull-down assay

To further verify the superior binding of AI-073 to HMGB1, a protein pull-down assay was developed (Fig. 7) . In-brief, 8 μL of 500 μg/mL recombinant HMGB1-His protein (AcroBiosystems, HM1-H5220, HEK293 cell derived) was mixed with either about 3μg AI-073 or human IgG1-Fc control protein or none, and set at room temp for 5 min. The mixtures were then incubated with protein A-conjugated beads to capture (or pull-down) the bound proteins. The captured proteins were separated in an SDS-PAGE gel and visualized by Coomassie Brilliant Blue dye staining.

Fig. 7 shows one of the representative HMGB1 pull-down assay results. As shown in Fig, HMGB1 proteins are clearly pull-down (captured) by AI-073, but not by IgG1-Fc control sample. Thus, this dada further demonstrated that AI-073 has HMGB1-binding activities.

Example 5 AI-073 protect mice against lethal IBD

Dextran sulfate sodium (DSS) -induced inflammatory bowel diseases (IBD) in mice

The DSS-induced IBD model is shown in Fig. 8A and the testing results are shown in Figs 8B, 8C and 8D. As shown in Fig. 8A and the following table, C57BL/6N mice (6-8 weeks old) were fed with 3%DSS in the drinking water for 7 days, and were monitored daily, for weight loss, disease progression and survival. On day 0, mice were randomly divided into two groups (10 animals/group) : one group was administered with AI-073 fusion protein by i.p. injection (dose: 50 mg/kg) on day 0 and day 6, the other group was administered with vehicle control (0.9%NaCl) by same i.p. injection.

Treatment group (N)	Inducing agent	Dosage (mg/kg)	Dosing schedule
AI-073 (n=10)	DSS	50	Day 0 and Day 6
Vehicle (n=10)	DSS	50	Day 0 and Day 6

On day 7, DSS water was removed and mice were then fed with normal drinking water and continue to be monitored daily, for recovery and survival, up to day 14.

The colitis progression was measured by the Disease Activity Index (DAI) , and scored as in the following table.

Disease Activity Index (DAI) parameters

DAI is obtained by the sum of each individual score.

In our experimental setting, animals in each group started to show signs of disease and weight loss (Figs 8B and 8C) from days 3 to 5 after drinking 3%DSS water. Compared to those in the saline treated vehicle control group, animals in AI-073 treated group showed a relatively faster recovery in body weight gain (in grams, Fig. 8B) or a less body weight lose rate (%, Fig. 8C) after switched to the normal drinking water on day 7. Also shown in Fig. 8D, at the end of the study (i.e at day 14) , the AI-073 treated group had a higher survive rate (80%) than that saline treated vehicle control group (40%) , although the difference was not statistically significant (P>0.05, Log-rank test) . Nevertheless, these data indicate that administration of AI-073 protein to subjects might have a partial protection against lethal IBD, such as DSS-induced colitis in mice.

Example 6 Diagram for Testing Arthritis

For this propose, a collagen antibody induced arthritis (CAIA) model in mice was developed. The disease model and treatment schedule are shown in Fig. 9A. Briefly, 7-8 weeks old female Balb/c mice were administered a cocktail of 5 anti-collagen mAbs (1.5 mg/mouse) by i. v. injection on day 0, followed by i.p. injection of 50 μg LPS on day 3 and day 4. On day 0, mice were randomly divided into two different treatment groups (each group has 10 mice) : group one was treated with AI-073 (50 mg/kg, i. v. injection) , group two was treated with saline vehicle control. On day 14, in both groups, each mouse was re-administered with 0.8 mg of the anti-collagen mAb cocktail by i. v. injection, followed by i.p. injection of 35 μg LPS on day 16. On day 19, mice in AI-073 treated group were again administrated with a second dose (1 mg) of AI-073 by i.p. injection, whereas mice in saline vehicle control treated group were administrated with saline by i.p. injection. All of these mice were monitored daily from day 0, up to day 48.

In our experimental setting, animals in each group started to show signs of disease and/or body weight loss from days 5 to day 7 after 1 ^st administration of anti-collagen mAb cocktail. However, as shown in the Fig. 9B, compared to that in saline treated vehicle control group, AI-073 treated group showed a reduced disease score ratio (day/day19) from day 20 to day 40, and at one time point, day 24, the mean difference between the AI-073 treated and vehicle treated group is statistically significant (P<0.05 by two-tailed T-test, marked as *on the top bar of vehicle group) . These data demonstrated that AI-073 protein might also have therapeutic value in the treatment of subjects with arthritis.

Example 7 Generation of AI-073-Fc fusion protein variants containing either one copy or two copies of 073 core peptide.

Since the 12-amino acid long fragment-A (STSNSGLAPNPT, SEQ ID NO: 01) and the 20-amino acid long fragment-B (PAPGSTAPPAHGVTSAPDTR, SEQ ID NO: 02) in the 073-core peptide each contains 5 potential mucin-like O-linked glycosylation sites (serine or threonine in STP motif) and no N-linked glycosylation site, it is expected that adding just one or two repeats of this 073-core peptide to the Fc-tail of human IgG would still be able to generate a fusion protein with the features of heavily glycosylation and/or sialylation modifications when produced in mammalian cells. To test if this is the case, two variants of AI-73 Fc-fusion proteins, namely AI-073X and AI-073Y, which contains either one copy or two copies of 073-core peptide, respectively, were generated by using the similar method as shown in example 1.

Briefly, DNA encoding either one copy 073-core peptide (SEQ ID NO: 03) or two copies of 073-core peptide (SEQ ID NO: 06) was fused with a DNA fragment encoding the signal peptide of the human CD24 (SEQ ID NO: 32) at the N-terminal and a DNA fragment encoding the human IgG1-hinge-CH2-CH3 region (SEQ ID NO: 28) at the C-terminal. The full-length amino acid sequences of the AI-073X and AI-073Y are shown in SEQ ID NO: 54, and SEQ ID NO: 55, respectively. These DNA molecules were synthesized in-vitro and cloned into expression plasmid vector by using stand recombinant DNA techniques and the recombinant plasmids were transferred into CHO cells by electroporation. After electroporation, cells were cultured in serum-free media for 6-7 days and supernatants are then collected, passed over a column of Protein A resin (MabSelect from GE Healthcare) . The Fc-fusion protein bound on the column were eluted and collected by using low pH solution such (0.1 M acetic acid, pH 3.5) . These two fusion proteins were successfully produced and purified from CHO transfectants by protein A-column. The identity and quality of these two fusion proteins were analyzed by SDS-PAGE and their results are described in detail in example 9.

Example 8 Generation of AI-073-Fc fusion protein variants containing CD24 core peptides from other region.

AI-073-Fc fusion protein variants containing a 32-amiao acid peptide of a 12-mer core peptide with the sequence of GLAPNPTNATTK (SEQ ID NO: 56) , which is derived from other region of CD24 and contains 3 potential O-linked glycosylation sites and one N-linked glycosylation site, and the same 20 amino-acid long fragment-B (PAPGSTAPPAHGVTSAPDTR, SEQ ID NO: 02) was also generated. This fusion protein, named as AI-071-Z, has the amino acid sequence as shown in SEQ ID NO: 57.

Briefly, DNA fragments encoding the 1 ^st peptide (SEQ ID NO: 56) and the 2 ^nd peptide (SEQ ID NO: 06) were linked together and fused with a DNA fragment encoding the signal peptide of human CD24 (SEQ ID NO: 32) at the N-terminal and a DNA fragment encoding the human IgG1-hinge-CH2-CH3 region (SEQ ID NO: 28) at the C-terminal. The full-length amino acid sequence of the AI-073-Z fusion protein is shown in SEQ ID NO: 57. These DNA molecules were synthesized in-vitro and cloned into expression plasmid vector by using stand recombinant DNA techniques and the recombinant plasmids were transferred into CHO cells by electroporation. After electroporation, cells were cultured in serum-free media for 6-7 days and supernatants are then collected, passed over a column of Protein A resin (MabSelect from GE Healthcare) . The Fc-fusion protein bound on the column were eluted and collected by using low pH solution such (0.1 M acetic acid, pH 3.5) . The fusion protein was successfully produced and purified from CHO transfectants by a protein A-column. The identity and quality of this fusion protein was analyzed by SDS-PAGE and the results are described in detail in example 9.

Example 9 Characterization of the different versions of AI-073 fusion proteins by SDS-PAGE.

Figure 10A and 10B show one of the representative SDS-PAGE gel electrophoresis analysis results of the purified, different versions of AI-073-Fc protein (lanes 1 to 3: AI-073-X, AI-073-Y, AI-073-Z) , along with AI-073 (lane 4) which served as a control sample for a comparison. As shown in the Figure 10A, under non-reducing conditions, the appear molecule weight of each of these 4 fusion proteins is about twice of that in reducing conditions (Fig. 10B) , which is in-agreement with a disulfide-linked homodimer form of these fusion proteins in their nature conditions.

Under either reducing or non-reducing conditions, the protein bands detected in AI-071X (lane 1) , AI-071-Y (lane 2) or AI-071-Z (lane 3) samples all showed a heterogenous and smear pattern. Under reducing condition, the appear molecule weight of the major bands detected in AI-073X (monomer has 264 AA) and AI-073-Z (monomer has 264 AA) samples is about 45 kDa, and in AI-073Y (monomer, has 296 AA) and AI-073 (monomer, has 316 AA) samples is about 60 kDa, all significantly larger than their predicted molecular weight based on the monomer amino acid sequences. Similarly, under non-reducing condition, the appear molecule weight of the major bands detected in AI-073-X (dimer, has 528AA) and AI-073-Z (dimer, has 528AA) samples is about 90 kDa, and in AI-073-Y (dimer, has 592 AA) and AI-073 (dimer, has 632AA) samples is about 120 kDa, also all significantly larger than their predicted molecular weight based on their amino acid sequences of a dimer. The appear additional molecular mass gained in these fusion proteins is most likely contributed by the massive saccharides attached to the protein core-peptide backbone. These results indicate that like AI-073 protein, AI-073-Fc fusion protein variants containing either one repeat or two tandem repeats of the 32 AA-long 073-core peptide, when expressed in CHO cells, are also heavily glycosylated. In addition, AI-073-Fc fusion protein variant AI-073-Z, which contains a 12-amino acid long peptide with 3 potential O-linked glycosylation sites and one N-linked glycosylation site (GLAPNPT NATTK, Sequence ID. No 56) from other region of CD24 mature protein, is also heavily glycosylated.

Example 10 Further characterization of the new series of AI-073 fusion proteins: probing their interactions with glycosylation and/or sialylation-dependent or independent antibodies in ELSIA.

For this purpose, a same antigen binding ELISA as that in example 1 was used for the analysis of these AI-073 protein variants to the binding of glycosylation/sialylation-dependent SN3 mAb or glycosylation/sialylation-independent ML5 mAb. Briefly, 96-well plates were coated with 2 μg/mL of the different versions of AI-073-Fc protein (AI-073-X, AI-073-Y, AI-073-Z) , along with AI-073 (which served as a control sample for a comparison) at 4 ℃ overnight. After blocking with PBS-0.1%Tween20 solution (PBST) , 100 μL of two-fold serial dilutions of either ML5 (ab278509 from Abcam) or SN3 (ab134375 from Abcam) were added into the plates. The bound mouse ML5 or SN3 antibodies were detected by HRP-labeled goat anti-mouse IgG-Fc antibodies followed by the o-Phenylenediamine (OPD) substrate. The representative ELISA results are showed in Figs 11A-B. As shown in the figure, AI-073, AI-073-X and AI-073-Y (which contains either one copy or two copies of 073 core peptide) are almost equally detected by either the sialylation dependent anti-CD24 SN3 mAb (Fig. 11A) or sialylation in-dependent anti-CD24 ML5 mAb (Fig. 11B) , indicating that these valiant proteins as well as AI-073 are sialylated. Also, as shown in the figure, AI-073-Z protein was weakly detected by SN3 mAb but not by ML5 mAb. The reduction of SN3 binding and the absent ML5 binding seen in AI-073-Z protein sample may be due to the loss of the binding epitope as the 12-mer peptide sequence (GLAPNPTNATTK) in AI-073-Z is only partially overlapped with the 12-mer peptide sequence (STSNSGLAPNPT) in AI-073, AI-073X, or AI-073Y.

Example 11: Detection the binding of AI-073-Fc valiant proteins to human Siglec-10 or HMGB1

To test if the AI-073-Fc protein variants containing 1 or 2 repeats of 073-core, or AI-073-Fc protein variants containing core peptide from other regions of CD24 also interact with HMGB1 and Siglecs such as Siglec-10, a similar ELISA assay method as seen in the example 3 (Siglec-10 binding ELISA) or in the example 4 (HMGB1 binding ELISA) was used. The representative ELISA results are shown in Fig. 12A and 12B. As shown in Fig. 12A, like that of AI-073, AI-073-Fc proteins containing either 1 copy (AI-073-X-Fc) or 2 copies of 073-core (AI-073-Y-Fc) or AI-073-Fc protein variant containing a core peptide from other region of CD24 (AI-073-Z) have same in-vitro binding activity to Siglec-10 molecule. Similarly, as shown in Fig. 12B, like that of AI-073, AI-073-X-Fc, AI-073-Y-Fc and AI-073-Z-Fc also showed the in-vitro binding to HMGB1. The HMGB1-binding intensity detected in the AI-073-Z-Fc sample is a little higher than that seen in AI-073, AI-073X-Fc, or AI-073Y-Fc. Taken together, these results indicate that AI-073-Fc protein variants containing 1 or 2 repeats of 073-core or the core peptide from some other region of human CD24 maintain the interaction with HMGB1 and Siglec-10.

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. 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.

Claims

A protein, comprising 073-core portion, said 073-core portion comprises one or more copies of a first fragment and one or more copies of a second fragment, each of said first fragment comprises a sequence independently selected from those as set forth in SEQ ID NO: 1 and 22 to 38, SEQ ID NO: 56 and each of said second fragment comprises an amino acid sequence derived from Mucin1 protein.
The protein of claim 1, wherein each of said second fragment comprises a sequence independently selected from those as set forth in SEQ ID NO: 2 and 8 to 21.
The protein of any one of claims 1-2, wherein the protein comprises 1, 2, 3, or more copies of said first fragment.
The protein of any one of claims 1-3, wherein the protein comprises 1, 2, 3, or more copies of said second fragment.
The protein of any one of claims 1-4, wherein one or more copies of the first fragment is fused directly or indirectly to one or more copies of the second fragment.
The protein of any one of claims 1-5, wherein the C-terminus of said one or more copies of the first fragment is fused directly or indirectly to the N-terminus of one or more copies of the second fragment.
The protein of any one of claims 1-6, wherein the C-terminus of said one or more copies of the second fragment is fused directly or indirectly to the N-terminus of one or more copies of the first fragment.
The protein of any one of claims 1-7, wherein the protein comprises one or more copies of a 073-core portion, said 073-core portion comprises said first fragment and said second fragment.
The protein of claim 8, wherein the said first fragment and said second fragment are fused directly or indirectly.
The protein of any one of claims 8-9, wherein the said 073-core portion is fused directly or indirectly to one or more copies of the first fragment and/or one or more copies of the second fragment.
The protein of any one of claims 5-10, said fused indirectly comprise fused via a linker.
The protein of claim 11, wherein said linker is a peptide linker.
The protein of any one of claims 1-12, wherein said protein comprises a sequence independently selected from those as set forth in SEQ ID NO: 3 to 7.
The protein of any one of claims 1-13, further comprising a second portion, said second portion comprises a half-life extending portion.
The protein of claim 14, wherein said half-life extending portion comprises an immunoglobulin fragment.
The protein of claim 15, wherein said immunoglobulin fragment comprises a Fc portion of said immunoglobulin.
The protein of any one of claims 1-16, further comprising a second portion, said second portion comprises an immunoglobulin fragment.
The protein of any one of claims 15-17, wherein said immunoglobulin fragment comprises a Fc portion of said immunoglobulin.
The protein of any one of claims 15-18, wherein said immunoglobulin fragment comprises a hinge region of said immunoglobulin.
The protein of any one of claims 15-19, wherein said immunoglobulin fragment comprises a CH2 domain.
The protein of any one of claims 15-20, wherein said immunoglobulin fragment comprises a CH3 domain.
The protein of any one of claims 15-21, wherein said immunoglobulin fragment comprises a CH4 domain.
The protein of any one of claims 15-22, wherein said immunoglobulin is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgM and IgA.
The protein of any one of claims 15-23, wherein said immunoglobulin comprises a sequence selected from those as set forth in SEQ ID NO: 39 to 46.
The protein of any one of claims 14-24, wherein said second portion is directly or indirectly linked to said 073-core portion.
The protein of claim 25, wherein said second portion is indirectly linked to said 073-core portion via a linker.
The protein of claim 26, wherein said linker is a peptide linker.
The protein of any one of claims 14-27, wherein the C-terminus of said 073-core portion is linked directly or indirectly to the N-terminus of said second portion.
The protein of any one of claims 1-28, comprising a sequence selected from those as set forth in SEQ ID NO: 47 to 52.
The protein of any one of claims 1-29, which is a fusion protein.
The protein of any one of claims 1-30, which is glycosylated.
The protein of any one of claims 1-31, which is capable of binding to one or more Siglecs.
The protein of claim 32, wherein said one or more Siglecs comprises human Siglec.
The protein of any one of claims 32-33, wherein said one or more Siglecs comprises Siglec-10.
The protein of any one of claims 1-34, which is capable of binding to High Mobility Group Protein B1 (HMGB1) .
The protein of any one of claims 8-35, wherein said 073-core portion is derived from human protein.
An immunoconjugate, comprising the protein of any one of claims 1-36.
A nucleic acid, encoding the protein of any one of claims 1-36.
A vector, comprising the nucleic acid of claim 38.
A cell, comprising and/or expressing the protein of any one of claims 1-36, the immunoconjugate of claim 37, the nucleic acid of claim 38, and/or the vector of claim 39.
A composition, comprising the protein of any one of claims 1-36, the immunoconjugate of claim 37, the nucleic acid of claim 38, the vector of claim 39, and/or the cell of claim 40, and optionally a pharmaceutically acceptable carrier.
A method for preparing the protein of any one of claims 1-36, comprising culturing the cell of claim 40 under a condition enabling the expression of said protein.
A method for regulating a Siglec related signaling, comprising administering to a subject in need thereof an effective amount of the protein of any one of claims 1-36, the immunoconjugate of claim 37, the nucleic acid of claim 38, the vector of claim 39, the cell of claim 40, and/or the composition of claim 41.
The method of claim 43, which activates the Siglec related signaling.
The method of claim 43, which inhibits the Siglec related signaling.
A method for regulating an immune response, comprising administering to a subject in need thereof an effective amount of the protein of any one of claims 1-36, the immunoconjugate of claim 37, the nucleic acid of claim 38, the vector of claim 39, the cell of claim 40, and/or the composition of claim 41.
A method for repressing an immune-mediated tissue damage mediated by danger-associated molecular patterns (DAMPs) , comprising administering to a subject in need thereof an effective amount of the protein of any one of claims 1-36, the immunoconjugate of claim 37, the nucleic acid of claim 38, the vector of claim 39, the cell of claim 40, and/or the composition of claim 41.
The method of claim 47, wherein said immune-mediated tissue damage is selected from the group consisting of graft vs host diseases, immunotherapy-related adverse events, rheumatoid arthritis, inflammatory bowel diseases (IBD) , and multiple sclerosis (MS) .
A method for preventing, ameliorating and/or treating a disease or condition caused by an inflammatory response arising from tissue injuries from infectious agents or pathogen-associated molecular patterns (PAMPs) , comprising administering to a subject in need thereof an effective amount of the protein of any one of claims 1-36, the immunoconjugate of claim 37, the nucleic acid of claim 38, the vector of claim 39, the cell of claim 40, and/or the composition of claim 41.
The method of claim 49, wherein said disease or condition is associated with viral infection.
The method of any one of claims 49-50, wherein said disease or condition is COVID-19.
The method of any one of claims 49-51, wherein said disease or condition is influenza.
The method of any one of claims 49-52, wherein said disease or condition is acquired immunodeficiency syndrome (AIDS) .
The method of claim 49, wherein said disease or condition is associated with bacterial infection.
The method of claim 54, wherein said disease or condition is bacterial pneumonia.
A method for preventing, ameliorating and/or treating a disease or condition caused by acute tissue damage from wound, comprising administering to a subject in need thereof an effective amount of the protein of any one of claims 1-36, the immunoconjugate of claim 37, the nucleic acid of claim 38, the vector of claim 39, the cell of claim 40, and/or the composition of claim 41.