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WO2023088287A1 - Protéines de fusion comprenant un peptide noyau ai-073 et leur utilisation - Google Patents

Protéines de fusion comprenant un peptide noyau ai-073 et leur utilisation Download PDF

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WO2023088287A1
WO2023088287A1 PCT/CN2022/132188 CN2022132188W WO2023088287A1 WO 2023088287 A1 WO2023088287 A1 WO 2023088287A1 CN 2022132188 W CN2022132188 W CN 2022132188W WO 2023088287 A1 WO2023088287 A1 WO 2023088287A1
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protein
fragment
present disclosure
copies
seq
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PCT/CN2022/132188
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English (en)
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Qunmin Zhou
Xianfeng FANG
Dongling Li
Libing MU
Jianying Zhou
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Acroimmune Guangzhou Biotech Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4727—Mucins, e.g. human intestinal mucin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • 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) .
  • PAMP pathogen-associated molecular pattern
  • DAMP damage -associated molecular pattern
  • 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) .
  • DAMPs 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) .
  • PAMPs like LPS or DAMPs like HMGB1 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.
  • mucin 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.
  • secreted/gel-forming mucins include MUC2, MUC5AC, MUC5B, MUC6, MUC19 and etc.
  • 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.
  • mucin molecules in general and MUC1 in particularly, are 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) .
  • TR tandem repeat
  • S serine
  • T threonine
  • P proline residues
  • STP domain also called as STP domain or motif
  • 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.
  • 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 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.
  • GPI glycosylphosphatidylinositol
  • CD24 is highly expressed in B1 regulatory lymphocytes (Bregs)
  • 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 s ialic acid-binding i mmuno g lobulin-like lec tin, 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.
  • siglec molecules 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.
  • 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
  • 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.
  • AI-073-Fc fusion protein hereafter also called as AI-073
  • 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.
  • 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.
  • DAMPs danger-associated molecular patterns
  • 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.
  • 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 ⁇ g of purified AI-073 fusion protein, either in reducing (R) or non-reducing conditions (NR) , was loaded into SDS-PAGE gel.
  • 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 ⁇ g/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) .
  • TMB tetramethylbenzidin
  • 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.
  • lane 1 represents the sample containing HMGB1 only
  • lane 2 represents the sample containing HMGB1 and human IgG-Fc
  • 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. 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 50 ⁇ g 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.
  • 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 ⁇ g 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.
  • 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 N ATTK, 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 ⁇ g/mL and added into 96-well plates.
  • 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 ⁇ g/mL) .
  • 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 ⁇ g/mL) .
  • 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 ⁇ g/mL) .
  • 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.
  • a 96-well plate was coated with recombinant GST-Siglec-10 fusion protein (at concentration of 200 ng/mL) .
  • AI-073-Fc protein AI-073X, AI-073-Y and AI-073Z
  • 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.
  • 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.
  • each intervening number there between with the same degree of precision is explicitly contemplated.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Muc may refer to a protein or peptide.
  • Mucin may be a transmembrane/membrane-bound protein. Mucin may encompass Mucin proteins, protein fragments, protein analogs, oligopeptides, and/or a variant thereof.
  • the Mucin fragment may not include the full-length Mucin protein.
  • the Mucin may comprise MUC1, MUC3A, MUC3B, MUC4, MUC12, MUC16, MUC17 and etc.
  • the UniProt No. for MUC1 may be P15941.
  • CD24 may refer to a protein or peptide.
  • 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.
  • the CD24 fragment may not include the full length CD24 protein.
  • the UniProt No. for CD24 may be P25063.
  • fusion 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.
  • 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.
  • 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
  • each of said second fragment may comprise an amino acid sequence derived from Mucin1 protein.
  • 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.
  • 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
  • each of said second fragment may comprise an amino acid sequence derived from Mucin1 protein.
  • 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.
  • 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
  • each of said second fragment may comprise an amino acid sequence derived from Mucin1 protein.
  • 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.
  • 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.
  • 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.
  • one or more copies of the first fragment is fused directly or indirectly to one or more copies of the second fragment.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • each of said first fragment may comprise a sequence as set forth in SEQ ID NO: 1
  • each of said second fragment may comprise a sequence as set forth in SEQ ID NO: 2.
  • the said first fragment and said second fragment may be fused directly or indirectly.
  • 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.
  • said fused indirectly may comprise fused via a linker.
  • said linker may be a peptide linker.
  • said first fragment and second fragment fused indirectly may mean that said first fragment and second fragment might be fused via a linker.
  • the linker may be a (GnS) n linker such as GGGGS, or GGGGSGGGGSGGGGS.
  • 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.
  • 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) .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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, PAPS
  • this fragment may have one of following amino-acid sequences: STSNSG F APNPT (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 GNQSIS
  • Each copy of these fragments may contain 3, 4 or more mucin like O-glycosylation
  • 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.
  • the protein wherein at least two or more copies of said 073 core fragment may be indirectly linked to each other via a linker.
  • said linker may be a peptide linker.
  • the peptide linker may be a (GnS) n linker such as GGGGS, or GGGGSGGGGSGGGGS.
  • said immunoglobulin fragment may comprise a Fc portion of said immunoglobulin.
  • said second portion may comprise an immunoglobulin fragment.
  • said immunoglobulin fragment may comprise a Fc portion of said immunoglobulin.
  • said immunoglobulin fragment may comprise a hinge region of said immunoglobulin.
  • said immunoglobulin fragment may comprise a CH2 domain.
  • said immunoglobulin fragment may comprise a CH3 domain.
  • said immunoglobulin fragment may comprise a CH4 domain.
  • said immunoglobulin fragment may comprise hinge region and CH2 and CH3 domains of said Ig protein.
  • said Ig may be selected from the group consisting of IgG1, IgG2, IgG3, IgG4, and IgA.
  • said immunoglobulin fragment may comprise hinge region and CH3 and CH4 domains of said Ig protein.
  • said Ig may be IgM.
  • said immunoglobulin fragment may comprise hinge region and CH2, CH3 and CH4 domains of said Ig protein.
  • said immunoglobulin may be selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgM and IgA.
  • said immunoglobulin may comprise a sequence selected from those as set forth in SEQ ID NO: 39 to 46.
  • said second portion may be directly or indirectly linked to said 073 core-derived-region.
  • said second portion may be indirectly linked to said 073 core-derived-region via a linker.
  • said second portion may be directly linked to said 073 core-derived-region.
  • said second portion may be directly linked to said 073 core-derived-region, and said second portion may not comprise hinge region.
  • 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.
  • 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.
  • said 073 core-derived-region may be linked directly or indirectly to the N-terminus of said second portion.
  • the protein 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.
  • 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.
  • the 073 core-fragment or the protein it may be glycosylated.
  • the 073 core-fragment or the protein may be capable of binding to one or more Siglecs.
  • said one or more Siglecs may comprise human Siglec.
  • said one or more Siglecs may comprise Siglec-10.
  • the 073 core-fragment or the protein may be capable of binding to High Mobility Group Protein B1 (HMGB1) .
  • HMGB1 High Mobility Group Protein B1
  • said 073 core-fragment or the protein wherein said 073 core may be derived from human protein.
  • 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.
  • the Siglec related signaling may comprise Siglec-mediated regulation of immune cell function.
  • 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.
  • the Siglec related signaling may comprise Siglec-mediated regulation of immune cell function.
  • 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.
  • the Siglec related signaling may comprise Siglec-mediated regulation of immune cell function.
  • 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.
  • 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) .
  • 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) .
  • 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.
  • said disease or condition may be associated with viral infection.
  • said disease or condition may be COVID-19.
  • said disease or condition may be influenza.
  • said disease or condition may be acquired immunodeficiency syndrome (AIDS) .
  • AIDS acquired immunodeficiency syndrome
  • said disease or condition may be associated with bacterial infection.
  • 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.
  • a disease or condition caused by an inflammatory response arising from tissue injuries from infectious agents.
  • said disease or condition may be associated with viral infection.
  • said disease or condition may be COVID-19.
  • said disease or condition may be influenza.
  • said disease or condition may be acquired immunodeficiency syndrome (AIDS) .
  • AIDS acquired immunodeficiency syndrome
  • said disease or condition may be associated with bacterial infection.
  • 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) .
  • said disease or condition may be associated with viral infection.
  • said disease or condition may be COVID-19.
  • said disease or condition may be influenza.
  • said disease or condition may be acquired immunodeficiency syndrome (AIDS) .
  • AIDS acquired immunodeficiency syndrome
  • said disease or condition may be associated with bacterial infection.
  • 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.
  • 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.
  • a 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.
  • said disease or condition may be septicemia, crush syndrome and/or ischemia reperfusion injury.
  • 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.
  • 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.
  • the purified, intact AI-073-Fc protein was further subjected to a SEC-HPLC analysis (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.
  • 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.
  • 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)
  • glycosylation and/or sialylation-dependent mAbs such as SN3 (ab134375 from Abcam)
  • 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) .
  • 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.
  • an antigen binding ELISA was developed. Briefly, 96-well plates were coated with 10 ⁇ g/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 °C overnight. After blocking with PBS-0.1%Tween20 solution (PBST) , 100 ⁇ L of two-fold serial dilutions of either SN3 (ab134375 from Abcam) , ML5 (ab278509 from Abcam) or humanized H3L3 were added into the plates.
  • PBST PBS-0.1%Tween20 solution
  • 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.
  • OPD o-Phenylenediamine
  • Fig. 4A shows the binding of AI-073 or CD24Fc to the glycosylation and/or sialylation dependent SN3 mAb.
  • both CHO-derived AI-073 and HEK293-derived CD24Fc bind to SN3, indicated that the core peptide is highly glycosylated and/or sialylated.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • IBD inflammatory bowel diseases
  • 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
  • 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.
  • AI-073 has about 20-folder higher Siglec-10 binding affinity (EC 50 of 1.43E-7M) than CD24Fc (EC 50 : 2.846E-6M) .
  • Siglec-10 binding affinity EC 50 of 1.43E-7M
  • CD24Fc EC 50 : 2.846E-6M
  • Example 4 AI-073 protein has a superior binding to High Mobility Group Box 1 (HMGB1)
  • HMGB1-His tag protein (AcroBiosystems, HM1-H5220, HEK293 cell derived) at 4 °C overnight. After blocking with SuperBlock (Thermo, 37515) at room temperature for 1 hour, 100 ⁇ L 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.
  • AI-073 has more than 100-fold higher HMGB1-binding activity (EC 50 : 5.042E-8M) than CD24Fc (EC 50 : 5.095E-5M) .
  • HMGB1-binding activity EC 50 : 5.042E-8M
  • CD24Fc EC 50 : 5.095E-5M
  • HMGB1-His protein (AcroBiosystems, HM1-H5220, HEK293 cell derived) was mixed with either about 3 ⁇ g 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.
  • Dextran sulfate sodium (DSS) -induced inflammatory bowel diseases (IBD) in mice Dextran sulfate sodium (DSS) -induced inflammatory bowel diseases (IBD) in mice
  • mice 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.
  • the colitis progression was measured by the Disease Activity Index (DAI) , and scored as in the following table.
  • DAI Disease Activity Index
  • DAI Disease Activity Index
  • DAI is obtained by the sum of each individual score.
  • a collagen antibody induced arthritis (CAIA) model in mice was developed.
  • the disease model and treatment schedule are shown in Fig. 9A.
  • 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 ⁇ g LPS on day 3 and day 4.
  • 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.
  • mice in both groups were re-administered with 0.8 mg of the anti-collagen mAb cocktail by i. v. injection, followed by i.p. injection of 35 ⁇ g LPS on day 16.
  • 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.
  • 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) .
  • Example 7 Generation of AI-073-Fc fusion protein variants containing either one copy or two copies of 073 core peptide.
  • 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.
  • 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.
  • 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.
  • 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.
  • Protein A resin MobSelect from GE Healthcare
  • 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.
  • 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.
  • SEQ ID NO: 57 The full-length amino acid sequence of the AI-073-Z fusion protein is shown in SEQ ID NO: 57.
  • 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.
  • Protein A resin MobSelect from GE Healthcare
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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.
  • 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 N ATTK, 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.
  • AI-073 protein variants were 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.
  • 96-well plates were coated with 2 ⁇ g/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 °C overnight.
  • 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.
  • 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.
  • GLAPNPTNATTK 12-mer peptide sequence
  • STSNSGLAPNPT 12-mer peptide sequence
  • Example 11 Detection the binding of AI-073-Fc valiant proteins to human Siglec-10 or HMGB1
  • 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.
  • 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.
  • AI-073-Z AI-073-Z-Fc also showed the in-vitro binding to HMGB1.
  • 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.
  • 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.

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Abstract

L'invention concerne les compositions d'un noyau de peptide noyau glycosylé et/ou sialylé (noyau 073) et des compositions de protéines basées sur la fusion d'une ou de plusieurs copies du peptide au fragment Fc de l'immunoglobuline humaine et leur utilisation dans le traitement de maladies se propageant par une inflammation associée à des lésions tissulaires ou à des pathogènes envahissants.
PCT/CN2022/132188 2021-11-17 2022-11-16 Protéines de fusion comprenant un peptide noyau ai-073 et leur utilisation WO2023088287A1 (fr)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003025122A2 (fr) * 2001-08-13 2003-03-27 University Of Kentucky Research Foundation Biomarqueurs de profil d'expression genique et cibles therapeutiques destines au vieillissement cerebral et a la deficience intellectuelle liee a l'age
CN101490085A (zh) * 2006-06-12 2009-07-22 特鲁比昂药品公司 具有效应功能的单链多价结合蛋白
WO2010037395A2 (fr) * 2008-10-01 2010-04-08 Dako Denmark A/S Multimères de mhc dans des vaccins et la surveillance immunitaire contre le cancer
WO2014165707A2 (fr) * 2013-04-03 2014-10-09 Memorial Sloan-Kettering Cancer Center Génération efficace de lymphocytes t ciblant une tumeur dérivés de cellules souches pluripotentes
AU2014246410A1 (en) * 2010-08-03 2014-10-30 Abbvie Inc. Dual variable domain immunoglobulins and uses thereof
WO2015037000A1 (fr) * 2013-09-11 2015-03-19 Compugen Ltd Polypeptides vstm5 et leurs utilisations en tant que médicament pour le traitement du cancer, de maladies infectieuses et de maladies de type immunitaire
CN111630067A (zh) * 2017-12-29 2020-09-04 安进公司 针对muc17和cd3的双特异性抗体构建体

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003025122A2 (fr) * 2001-08-13 2003-03-27 University Of Kentucky Research Foundation Biomarqueurs de profil d'expression genique et cibles therapeutiques destines au vieillissement cerebral et a la deficience intellectuelle liee a l'age
CN101490085A (zh) * 2006-06-12 2009-07-22 特鲁比昂药品公司 具有效应功能的单链多价结合蛋白
WO2010037395A2 (fr) * 2008-10-01 2010-04-08 Dako Denmark A/S Multimères de mhc dans des vaccins et la surveillance immunitaire contre le cancer
AU2014246410A1 (en) * 2010-08-03 2014-10-30 Abbvie Inc. Dual variable domain immunoglobulins and uses thereof
WO2014165707A2 (fr) * 2013-04-03 2014-10-09 Memorial Sloan-Kettering Cancer Center Génération efficace de lymphocytes t ciblant une tumeur dérivés de cellules souches pluripotentes
WO2015037000A1 (fr) * 2013-09-11 2015-03-19 Compugen Ltd Polypeptides vstm5 et leurs utilisations en tant que médicament pour le traitement du cancer, de maladies infectieuses et de maladies de type immunitaire
CN111630067A (zh) * 2017-12-29 2020-09-04 安进公司 针对muc17和cd3的双特异性抗体构建体

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