Chimeric recombinant dimer psgl-1 proteins, its preparation process, DNA sequence, eukaryotic expression vector, conjugate compound, and pharmaceutical compositions

Abstract

The present invention relates to a recombinant protein (p-selectin glycoprotein ligand-1 and neural retina-specific leucine zipper) chimeric protein psgl-1-nrl comprising a selectin-binding domain and a non-covalent dimerization domain, which is a leucine zipper and is more preferably the leucine zipper domain of the human or mouse neural retina-specific leucine zipper. The chimeric protein further comprises a covalent dimerization domain with at least one cysteine ​​suitable for forming a disulfide bridge with another monomeric protein to form a homodimer. In the chimeric protein, the psgl-1 domain corresponds to the extracellular region of human psgl-1 and is more preferably the selectin-binding region of the mature protein. The chimeric protein is correctly modified after translation and is efficiently expressed in a mammalian system.This is sulfated, glycosylated, sialylated, and binds to α, β, and β selectin, allowing for in vivo and in vitro targeting for diagnostic or therapeutic purposes.

Description

1 / 103 Recombinant dimer PSGL-1 chimeric proteins, their preparation process, DNA sequence, eukaryotic expression vector, conjugate compound, and pharmaceutical compositions. Field of invention

[0001] The present invention relates to the preparation of a novel Selectin targeting protein for diagnostic and therapeutic uses. State of the Art

[0002] P-selectin glycoprotein ligand-1 (PSGL-1) is a leukocyte adhesion molecule that mediates the binding and rolling of cells onto activated endothelial cells under physiological blood flow. This activity is an important initial step in leukocyte extravasation. PSGL-1 was initially identified as a ligand for P-selectin, and subsequent work revealed that PSGL-1 is also a ligand for E-selectin and L-selectin (see, for example, US Patent No. 6,277,975).

[0003] Two members of the selectin family are particularly relevant in the context of molecular imaging: P-selectin and E-selectin. It is known that upregulation or upregulation of P- or E-selectin expression in the vascular endothelium occurs under inflammatory conditions, while the presence of endothelial selectins under resting conditions is generally low or absent. Disease states in which selectins are useful molecular imaging targets include post-ischemic injury, acute coronary syndrome, arthritis, inflammatory bowel disease including ileitis and colitis, atherosclerosis, myocarditis, thrombosis, and multiple sclerosis. However, molecular imaging with selectin can be useful for delineating and identifying tissues in which selectin expression occurs under normal conditions, such as the skin microvasculature. Petition 870260035468, dated 04 / 16 / 2026, page 6 / 233 2 / 103

[0004] It is known that upregulation of P-selectin (also called CD62P) occurs very rapidly (within minutes), making P-selectin a potential marker of early stages of inflammatory disease. P-selectin is also found on the surface of activated platelets, making it a marker of thrombosis. E-selectin (CD62E) is also expressed in inflamed vasculature, although generally later in the inflammatory response than P-selectin. E-selectin is assumed to be a useful marker of inflammation in late stages of the disease.

[0005] Among the ligands of selectins, PSGL-1 plays an important role in recruiting white blood cells to inflamed tissues. White blood cells do not normally interact with the endothelium of blood vessels. However, inflammation causes the expression of cell adhesion molecules (CAMs) such as P-selectin on the surface of the blood vessel wall. White blood cells present in the bloodstream can interact with CAMs. The first step in this interaction process is carried out through the interaction of PSGL-1 with P-selectin and / or E-selectin on endothelial cells and adherent platelets. This interaction results in the rolling of the white blood cell over the surface of the endothelial cells followed by stable adhesion and transmigration of the white blood cell into the inflamed tissue.

[0006] PSGL-1 Human (GenBank Accession No. Q14242.1; GI 2498904) is a homodimeric disulfide-linked glycoprotein similar to mucin that is expressed on the surface of most hematopoietic cells, including, for example, neutrophils, monocytes, lymphocytes, dendritic cells, and platelets.

[0007] The amino acid sequence of human PSGL-1 exhibits an amino-terminal signal peptide (amino acid residues 1-17) and a Petition 870260035468, dated 04 / 16 / 2026, page 7 / 233 3 / 103 propeptide (amino acid residues 18-41) with a consensus cleavage site for paired basic amino acid conversion enzymes (PACE). The N-terminal extracellular region of the mature protein begins at residue 42. The extracellular domain of the PSGL-1 molecule contains several serine / threonine-rich decameric repeats that contain several O-glycosylation binding sites and also some N-glycosylation binding sites. This region of the molecule, which folds into a rod-like structure, is responsible for the mucin-like characteristics of PSGL-1. In neutrophils, this rod-like structure and the location of PSGL-1 on the tips of microvilli facilitate the binding of PSGL-1 to selectin-expressing cells. The decameric repeat region of PSGL-1 is followed by the transmembrane region (residues 268-292) and the cytoplasmic domain (residues 293-361).

[0008] The expression of recombinant PSGL-1 was first achieved by Sako et al.(Cell, 1993, 75(6), 1179-1186) allowed defining regions and modifications relevant to selectin binding, which have been reported, just to mention a few: Liu et al J. Biol. Chem. 1998, 12:7078-7087, Cummings RD, Brazilian Journal of. Medical and Biological Research, 1999, 32: 519-528, Sako et al. Cell, 1995, 83: 323-331 etc., cited below. Based on general studies on PSGL-1, important regions for selectin binding have been mapped in the N-terminal portion of mature PSGL-1 and encompass residues 5-16 with the three tyrosine sulfation sites and the O-linked oligosaccharide carrying sLex localized at Thr 16.

[0009] The complex post-translational modification pattern of PSGL-1 (the protein requires two distinct post-translational modifications for Ca2+-dependent recognition by the Pselectin lectin domain: tyrosine sulfation and a specific O-linked glycosylation in core 2 by fucose and sialic acid) requires that this Petition 870260035468, dated 04 / 16 / 2026, page 8 / 233 4 / 103 of the molecule must be expressed in recombinant eukaryotic systems. Fugang Li et al. J. Biol. Chem, 1996, 271:3255-3264 describe the requirements for recombinant expression of the correctly glycosylated rPSGL-1 form. US 5,827,817 and US 6,277,975 describe numerous variants of the PSGL-1 protein, including the PSGL-1-Fc IgG1 fusion protein and its expression in CHO and COS cells in combination with a fucosyltransferase (FT) gene. Thus, PSGL-1 chimeras with portions of the immunoglobulin Fc fragment have already been expressed in CHO or COS cells carrying the appropriate enzyme(s) for correct glycosylation.

[0010] The same Applicant has already observed that a shorter variant of such PSGL-1 IgG1 Fc fusion protein, covalently linked to phospholipids of ultrasound imaging microvesicles, exhibits enhanced target binding and increases microbubble stability. These findings are disclosed in WO2012 / 020030 by the same Applicant of this invention.

[0011] This Patent Application discloses the recombinant expression of a chimeric PSGL-1 protein that relies on non-covalent dimerization domains to produce a homodimeric form of the PSGL-1 fusion protein, thereby avoiding the use of antibody Fc fragments and the inconveniences of their presence. In fact, the recombinant construct of the present invention exploits the use of functional fragments of DNA regulatory proteins, leucine zippers, which promote protein-protein interactions and homo- or heterodimeric / multimeric forms under which they act as Transcription Factors, capable of interacting with DNA and regulating its expression.

[0012] Leucine zippers are protein domains with leucine repeats at every 7a (sometimes 4a) amino acid position, capable of forming a right-handed α-helix through the Petition 870260035468, dated 04 / 16 / 2026, page 9 / 233 5 / 103 which promote oligomerization with identical or different corresponding(s), thus generating homo- or hetero dimers / multimers and the properties of regulating DNA expression.

[0013] The use of leucine zippers as dimerization domains in E. coli has been explored to produce dimeric antibodies or their functional fragments, ScFv, F(ab')2. Preparation of bifunctional ScFv in De Kruif, J. and Logtenberg T., J. Biol. Chem., 1996, 271:7630-7634.

[0014] GCN4, a yeast leucine zipper, was used in Chingwei V. Lee et al., J. Immunological Methods, 2004, 284: 119-132, for the display in phages of F(ab')2 fragments in the M13 system in E. coli.

[0015] WO2005 / 105840, which deals with recombinant CD40, proposes the use of so-called fusion partners to induce oligomerization of CD40 variants in eukaryotic cells. Mannose-binding protein, the collagen-binding domain of tetranectin, and leucine zippers, including the Neural Retina-specific leucine zipper, NRL (GenBank Accession No. M81840), are listed as possible fusion partners.

[0016] The Applicant has now discovered that when functional fragments of PSGL-1 are cloned upstream of an NRL sequence, they are not only correctly processed and expressed as functional homodimers, but their expression and secretion also occur very efficiently, much more so than is observed for PSGL-1 Fc-derived constructs with the standard IgG structure that carries the Hinge region and the Fc, commonly used for dimeric protein expression, which has become the reference standard for PSGL-1 homodimer expression.

[0017] The present invention now allows the preparation of a specific reagent for P / E Selectin in suitable quantities and Petition 870260035468, dated 04 / 16 / 2026, page 10 / 233 6 / 103 with a standard quality for in vivo use in therapeutic or diagnostic applications.

[0018] The chimeric protein can be expressed at high levels in the CHO cell system, recognized with properties of safe use for the production of recombinant proteins and can provide the glycosylation and post-translational processing necessary for selectin binding. The high levels of expression and functional post-translational processing now allow for the industrial scale-up of this reagent. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1. Western blot of the conditioned medium of the Variants 1-5 under reducing and non-reducing conditions. Ranges 1 and 6: variant 5, respectively under reducing and non-reducing conditions; ranges 2 and 7: variant 1, respectively under reducing and non-reducing conditions; ranges 3 and 8: variant 2, respectively under reducing and non-reducing conditions; ranges 4 and 9: variant 3, respectively under reducing and non-reducing conditions; ranges 5 and 10: variant 2, respectively under reducing and non-reducing conditions. PM markers are on the left.

[0020] Figure 2. SPR Biacore (protein binding response in P-selectin) comparison between Fragment 1 (Fr-1, solid line) and the chimeric protein of the invention (Variant 1A, dashed line) at 125 nM each. Y-axis: Response in Resonance Unit (RU) which includes association / dissociation / regeneration as a function of time (s) on the X-axis.

[0021] Figure 3. Ultrasound imaging of inflammatory hind limbs in rats. Images were obtained using Fixed Bubble Imaging (FBI), 10 minutes after injection of microbubbles containing Fragment-1 or Variant 1A.

[0022] Figure 4. Obtaining an optical image of the LPS model Petition 870260035468, dated 04 / 16 / 2026, page 11 / 233 7 / 103 Inflammatory lesions of the hind limbs of mice after injection of liposomes loaded with Variant 1A-liposomes-DIR (mice 1 to 6) or Fr-1-liposomes-DIR (mice 7 to 12).

[0023] Fluorescent images were obtained two hours after liposome injection (left paw: inflamed paw; right paw: contralateral paw).

[0024] Figure 5. RP-HPLC analysis of the final set of fractions after purification with HA described in Example 13 (negative). The Retention Time (Rt) of the target protein is approximately 9.7 min. SUMMARY OF THE INVENTION

[0025] The present invention discloses a Ligand-1 protein of Recombinant chimeric P-selectin glycoprotein (PSGL-1) comprising at least one selectin-binding domain, a leucine zipper domain, and a disulfide bond promoter region, wherein the selectin-binding region preferably comprises at least the 5-16 amino acids of SEQIDNO:11.

[0026] The leucine zipper domain comprises an amino acid sequence that is at least 90% homologous or identical to aa 187-208 of SEQIDNO:12 (Neural Retina-Specific Leucine Zipper) or more preferably at least 90% homologous or identical to aa 181-215 of SEQIDNO:12.

[0027] In the recombinant chimeric PSGL-1 protein of the invention, the region that promotes disulfide bonds (covalent dimerization domain) comprises an amino acid sequence defined by the following general formula: (X1)nC(X2)m-(X3) where:

[0028] - X1, X2 represents any amino acid or amino acid sequence excluding cysteine ​​(Cys),

[0029] - C is Cys Petition 870260035468, dated 04 / 16 / 2026, page 12 / 233 8 / 103

[0030] - X3 is any amino acid and

[0031] - n, m are integers from 1-6,

[0032] or it is preferably (SEQIDNO:20).

[0033] A recombinant chimeric protein is a chimeric protein comprising two recombinant chimeric PSGL-1 monomers that are defined above, covalently linked to each other through at least one disulfide bond and is preferably a homodimer.

[0034] It is expressed in a mammalian system where it is correctly modified post-translationally and secreted into the culture medium at high levels in the form of a soluble dimer.

[0035] The invention further comprises the DNA sequence encoding the recombinant chimeric protein defined above and eukaryotic expression vectors controlling its expression in a mammalian system, preferably CHO cells. The mammalian expression system further comprises the beta 1,6-acetylglucosaminyltransferase (C2GnT) and fucosyltransferase VII (FTVII) glycosylation enzymes in a different expression vector or in the same vector for appropriate glycosylation.

[0036] Consequently, the invention further comprises the mammalian cell transformed with DNA encoding the chimeric recombinant PSGL-1 protein or the vector defined above and the isolated purified protein secreted by the mammalian cell.

[0037] The isolated chimeric protein is homodimeric, glycosylated O-linked at least at Thr at position 16 and sulfated at least at Tyr at positions 5, 7 and 10 of SEQIDNO:11.

[0038] Protein is a useful targeting agent for diagnostic or therapeutic clusters to which it can be conjugated via a ligand or spacer comprising the amino acid Cys or Lysine, preferably present and placed in Petition 870260035468, dated 04 / 16 / 2026, p. 13 / 233 9 / 103 terminal C.

[0039] Diagnostically useful groups are preferably selected from the group consisting of: a radioactive label, an enzyme, a fluorescent label, a luminescent label, a metal chelating compound, a gas-filled lipid microvesicle and a combination of the diagnostically active groups and are most preferably metal chelating compounds or gas-filled microvesicles.These conjugates are useful for obtaining images through ultrasound, magnetic resonance imaging, optoacoustics, scintigraphy, Single Photon Emission Computed Tomography (SPECT), Positron Emission Tomography (PET), X-ray, radiofrequency acoustics and optics, and in pathological conditions characterized by the overexpression of a selectin, such as: Acute Coronary Syndrome (ACS), Inflammatory Bowel Disease (IBD), Ulcerative Colitis, Crohn's disease, tumor-associated neoangiogenesis, rheumatoid arthritis, ischemia-reperfusion injury, graft rejection, or, more generally, any organ or tissue that expresses P-selectin and / or E-selectin above physiological levels. Most preferably the pathological condition is IBD, ACS, tumor detection, or graft rejection.

[0040] More preferably, the chimeric protein of the invention is a useful targeting agent for IBD, SCA, tumor detection and graft rejection.

[0041] The therapeutically active group is preferably selected from the group consisting of: cytokine, cystostatic agent, toxin, anti-inflammatory agent, immunomodulator, antiplatelet agent, corticosteroid, monoclonal antibody, growth factor or radiotherapeutic agents comprising groups that chelate metal to carry a radionuclide. Petition 870260035468, dated 04 / 16 / 2026, page 14 / 233 10 / 103

[0042] Pharmaceutical compositions comprising the recombinant chimeric protein or its conjugates for diagnostic or therapeutic purposes, as defined above, are further included in the invention.

[0043] A further embodiment of the invention is a process for preparing the recombinant chimeric protein of the invention comprising transforming a eukaryotic cell with the DNA sequence encoding the same or the eukaryotic expression vector comprising said DNA sequence to obtain a recombinant system that stably expresses the chimeric protein, collecting the culture medium and purifying the recombinant protein from said culture medium.

[0044] The invention also relates to a process for purifying recombinant soluble protein, comprising hydroxyapatite as the final chromatographic step. Hydroxyapatite, preferably Ceramic Hydroxyapatite, is preferably prepared after strong anion exchange chromatography and hydrophobic interaction chromatography.

[0045] Additional embodiments of the invention are methods for the therapeutic treatment of a pathological condition characterized by the overexpression of a selectin, using targeting conjugates comprising the recombinant soluble chimeric protein according to the invention, and imaging for the diagnosis of a pathological condition characterized by the overexpression of a selectin, comprising the pre-administration of the diagnostic conjugates or pharmaceutical compositions to an individual and image recording by means of an imaging method. Such imaging methods are preferably selected from the group consisting of: ultrasound, magnetic resonance imaging, optoacoustics, scintigraphy, Single Photon Emission Computed Tomography (SPECT), Petition 870260035468, dated 04 / 16 / 2026, p. 15 / 233 11 / 103 Positron Emission Tomography (PET), X-ray, acoustic and optical radiofrequency.

[0046] Pathological conditions characterized by the overexpression of a selectin are preferentially selected from the group consisting of: Acute Coronary Syndrome (ACS), Inflammatory Bowel Disease (IBD), Ulcerative Colitis, Crohn's disease, tumor-associated neoangiogenesis, rheumatoid arthritis, ischemia-reperfusion injury, graft rejection, or, more generally, any organ or tissue that expresses P-selectin and / or E-selectin above physiological levels. Most preferentially, the pathological condition is IBD, ACS, cancer, or graft rejection. DETAILED DESCRIPTION OF THE INVENTION Definitions

[0047] Unless otherwise provided, the amino acid and nucleotide sequence of human PSGL-1 to which the present invention relates is identified by GenBank Accession Number Q14242.1. This Accession Number also identifies functional domains, such as the PSGL-1 signal peptide region, comprised by aa 1-17, the propeptide region comprised by aa 18 to 41, which identifies the start of the mature protein at aa 42 (corresponding to aa 1 of SEQIDNO:11), the N- and O-glycosylation sites and the sulfation sites.

[0048] The amino acid sequence of the neural retinal (NRL)-specific leucine zipper is identified by NCBI Accession Number NP_006168.1 (GI:1709348, human). In the present invention, reference to the neural retinal (NRL)-specific leucine zipper protein includes all amino acid sequences that are at least 90% homologous in the NRL leucine zipper region, including mouse NRL (P54846).

[0049] NRL, first isolated and characterized by Swaroop et Petition 870260035468, dated 04 / 16 / 2026, page 16 / 233 12 / 103 al. Proc. Natl. Acad. Sci., 1992, 89: 266-270, were identified Single Nucleotide Polymorphisms: These are included, if functional, in the present invention; in the sequence NP_006168.1, the leucine zipper domain was identified between amino acids 187208. The flanking amino acids at the N-terminus or C-terminus of this region, derived from the NRL sequence, may also be part of the functional domain (non-covalent dimerization motif).

[0050] As used herein, the term recombinant is used to describe unnaturally altered or manipulated nucleic acids, host cells transfected with exogenous nucleic acids or unnaturally expressed polypeptides, through manipulation of isolated DNA and transformation of host cells. Recombinant is a term that specifically encompasses DNA molecules that have been constructed in vitro using genetic engineering techniques, and the use of the term recombinant as an adjective to describe a molecule, construct, vector, transfected cell, polypeptide, or polynucleotide includes such molecules, constructs, vectors, cells, polypeptides, or polynucleotides even if they have a partially identical sequence to that of the naturally occurring molecule.

[0051] The term polypeptide is used to refer to a compound of two or more amino acids linked through the main chain (as opposed to the side chain) by a peptide amide bond (-C(:O)NH-). The term peptide is used interchangeably here with polypeptide, but is generally used to refer to polypeptides that have fewer than 40 and preferably fewer than 25 amino acids.

[0052] The term protein is used to refer to a compound of more than 40 amino acids linked through the main chain (as opposed to the side chain) by a peptide amide bond (Petition 870260035468, dated 16 / 04 / 2026, p. 17 / 233 13 / 103 C(:O)NH-).

[0053] The term chimeric protein or fusion protein or chimera used in the present invention comprises a PSGL-1 polypeptide operationally linked to at least one non-PSGL-1 polypeptide. A PSGL-1 polypeptide shares the amino acid sequence of PSGL-1, preferably human, while the non-PSGL-1 polypeptide has an amino acid sequence that is not substantially homologous to PSGL-1 and is derived from the same or different organisms. In a preferred embodiment, the PSGL-1 polypeptide comprises the extracellular portion of PSGL1 or shorter fragments thereof, which still bind to selectins (in particular P and E selectin), such as the PSGL-1 de1-47 fragment (SEQIDNO:11) encompassing the 5-16 amino acid region, which defines the selectin-binding region. The selectin-binding region may optionally comprise flanking amino acid(s) at the N- or C-terminus of amino acids 5-16 derived from the PSGL-1 sequence.

[0054] The fusion or chimeric protein is expressed and produced in a recombinant system through the operational linkage of the DNA sequence encoding the PSGL-1 polypeptide to the DNA sequence encoding the non-PSGL-1 polypeptide, fused together in a frame under the control of eukaryotic expression regulatory regions, such as a promoter or a polyadenylation site.

[0055] The term binding refers to the determination, through standardized assays, including those described herein, that a binding peptide recognizes and binds reversibly to a certain target. Such standardized assays include, but are not limited to, equilibrium dialysis, gel filtration, surface plasmon resonance, immunoaffinity assays, which include immunoassays. Petition 870260035468, dated 04 / 16 / 2026, p. 18 / 233 14 / 103 competitive and the monitoring of spectroscopic changes resulting from the binding.

[0056] A labeling group or a detectable label, as used herein, is a group or cluster capable of generating a detectable signal. Particularly preferred are labels useful for diagnostic imaging, i.e., labels detectable by magnetic resonance imaging, radioactive detection, ultrasound, X-ray, light (UV, infrared, fluorescent, etc.) or carrying a group, such as a radioactive metal or other entity, that can be used in radiotherapy or other forms of therapy. Specific labels will be detailed below.

[0057] The term specificity refers to the binding of the polypeptide that has a higher binding affinity for one target compared to another. Binding specificity can be characterized by an equilibrium dissociation constant (KD) or an equilibrium association constant (Ka) for the two target materials tested. In a preferred embodiment, the binding peptides of the invention have a dissociation constant for a desired target that is less than approximately 10 μM, more preferably less than approximately 1 μM, and most preferably less than approximately 0.5 μM or even lower. The term selectin specificity refers to a group that binds to PSGL-1 that has a higher affinity for at least one selectin among the P, L, E selectins than an irrelevant target.

[0058] The term patient as used here refers to any mammal, especially humans.

[0059] The term pharmaceutically acceptable carrier or excipient refers to a non-toxic carrier or excipient that can be administered to a patient along with a compound of this invention and that does not destroy the pharmacological activity of the compound. Petition 870260035468, dated 04 / 16 / 2026, p. 19 / 233 15 / 103

[0060] The term target or target molecule refers to any substance to which a linking group or a linking polypeptide can bind, such as proteins or polypeptides, cells, receptors, carbohydrates, lipids, etc. As used herein, target includes the Selectin family in isolated form or expressed within or on the surface of a cell, tissue, or organ. Consequently, targeting group as used herein refers to a PSGL-1 protein or functional fragments thereof capable of binding to selectins, preferably P and E selectins, unless otherwise specified.

[0061] The terms therapeutic agent or therapeutics refer to a compound or agent that has a beneficial, therapeutic, or cytotoxic (i.e., malignant) effect on cells in vivo. Therapeutic agents include those compositions referred to as, for example, bioactive agents, cytotoxic agents, drugs, chemotherapeutic agents, radiotherapeutic agents, genetic material, etc.

[0062] The term positively charged amino acid refers to amino acids in the following group: Arginine, Histidine, Lysine. These amino acids are generally considered interchangeable, meaning that the substitution of one residue for another in the same group is generally well tolerated within a protein or polypeptide, even if this cannot be applied when the residue is contained within a critical domain of the protein (such as an alpha-helix, a link pocket, a conformation-dependent constraint, etc.).

[0063] The term negatively charged amino acid refers to amino acids in the following group: aspartic acid and glutamic acid. These amino acids are generally considered interchangeable, meaning that one residue can be replaced by another in the same group. Petition 870260035468, dated 04 / 16 / 2026, p. 20 / 233 The 16 / 103 group is generally well tolerated within a protein or polypeptide, although this may not apply when the residue is contained within a critical domain of the protein (such as a binding pocket or a conformation-dependent constraint, etc.).

[0064] The term non-polar side-chain amino acid refers to amino acids in the following group: Serine, Threonine, Asparagine, and Glutamine. These amino acids are generally considered interchangeable, meaning that the substitution of one residue for another in the same group is generally well tolerated within a protein or polypeptide, with the exceptions mentioned earlier.

[0065] The term amino acid with hydrophobic side chain refers to amino acids in the following group: Alanine, Valine, Isoleucine, Leucine, Methionine, Phenylalanine, Tyrosine and Tryptophan. These amino acids are generally considered interchangeable, meaning that the substitution of one residue for another in the same group is generally well tolerated within a protein or polypeptide, even if this cannot be applied when the residue is contained within a critical domain of the protein (such as an alpha-helix, a link pocket, a conformation-dependent steric constraint, etc.).

[0066] The amino acids with specific functions in protein are those included in the following group: Cysteine, Glycine, and Proline. Depending on whether the function is performed due to the size of the residue, glycine can be replaced by other small residues, such as Serine or Alanine. In variance, Cysteine ​​cannot be replaced when involved in a disulfide bridge.

[0067] By covalent dimerization domain, the Applicant refers to an amino acid sequence comprising a Petition 870260035468, dated 04 / 16 / 2026, page 21 / 233 17 / 103 Cysteine ​​that may be involved in a disulfide bridge with another cysteine ​​in a different polypeptide chain (extrachain disulfide bridge), stable in the extracellular environment.

[0068] By non-covalent dimerization domain, the Applicant refers to an amino acid sequence capable of determining and / or favoring protein-protein interactions. Preferred non-covalent dimerization domains are structural alpha-helices, determined by the periodic repetition of leucine residues, generally referred to in the art as leucine zippers.

[0069] The amino acid regions of the leucine zipper that can be used according to the invention are preferably devoid of any lysine (K or Lys). Leucine zipper domains are generally easily identified in eukaryotic regulatory proteins by a periodic repetition of leucine amino acids. Among these, for example, the leucine zipper of Q9Y2D1, which corresponds to amino acids 236-250, or the leucine zipper of transcription activating factor 5 (GI:114678546).

[0070] However, the preferred leucine zippers according to the present invention are selected from the group consisting of:

[0071] a leucine zipper protein fragment specific to the neural retina, isolated from mammals and sharing a degree of homology > 90% with fragment 187-208 of the human NRL protein, even more preferentially the leucine zipper domains of mouse NRL (Q543Y0) or human NRL, isoform 2 (P54845-2) or bovine protein F1N4J1. Detailed Description

[0072] The invention relates to a recombinant chimeric PSGL-1 protein comprising a Selectin-Binding domain and a non-covalent dimerization domain, which is a leucine zipper and is more preferably the leucine zipper domain of the Zipper of Petition 870260035468, dated 04 / 16 / 2026, page 22 / 233 18 / 103 leucine specific for human or mouse neural retina, which preferably corresponds at least to region 187-208 of NP_006168.1 (SEQ ID NO: 12) or more preferably, at least to region 181-215 of SEQ ID NO:12. Alternatively, the non-covalent dimerization domain shares a degree of homology > 90% with fragment 187-208 of the human NRL protein, even more preferably with the leucine zipper domains of mouse NRL (Q543Y0) or human NRL, isoform 2 (P54845-2) or bovine protein F1N4J1.

[0073] By P-selectin Binding Domains, the present inventors hereby refer to peptides or polypeptides comprising an amino acid sequence with binding affinity for selectins (active sequences), particularly P-selectin; said active sequences comprise at least amino acids 5-16 (Cummings RD, Brazilian Journal of Medical and Biological Research, 1999, 32:519-528) or are preferably selected from fragments 1-19, 5-41 and 1-47 of SEQ ID NO: 11 in which amino acid 1 represents the first amino acid of the mature PSGL-1 protein and corresponds to aa 42 of GenBank Accession Number Q14242. 1.

[0074] The PSGL-1 protein is a transmembrane homodimer linked by a disulfide bond at Cys 320, near the transmembrane domain, which is defined in the GenBank registry.

[0075] Until now, dimerization of recombinant PSGL-1 has been achieved by introducing the IgG1 Fc domain downstream of the PSGL-1 selectin-binding domain. The Fc domain comprises a hinge region carrying at least two cysteines, wrapped in disulfide bridges to covalently associate the chimeric protein in homodimers. The immunoglobulin Fc region has been used in several recombinant systems to enhance or to Petition 870260035468, dated 04 / 16 / 2026, p. 23 / 233 19 / 103 facilitate dimming, which is essential for connection to selectins.

[0076] However, the presence of the Fc domain presents some drawbacks. Firstly, it has been shown that PSGL-1 fused to an Fc domain, attached to the surface of gas microvesicles, induces the formation of aggregates (WO2012 / 020030). In addition, the Fc domain could also activate the immune response through specific recognition of the Fc receptor expressed by macrophages. This could lead to the elimination of the molecule carrying the Fc protein fragment from the bloodstream or activate an immune response such as allergic reactions.

[0077] In fact, the Applicant of the present invention discovered that a shorter Fc region provides enhanced properties to the derivatives used for the preparation of microbubbles. Such drawbacks were resolved by the present invention, in which the human NRL leucine zipper domain replaces the Fc region. This region is preferably the leucine zipper domain of the Neural Retina-Specific Leucine Zipper, comprising at least amino acids 187-208 of NP_006168.1 or preferably amino acids 186-209, 185-210, 184-211, 183-212, 182-213, 181-214, 181-215, 186-208, 186-210, 187-208 or any fragment comprising at least amino acids 187-208 plus 1, 2, 3, 4, 5, 6 or 7 additional flanking amino acids at the N- or C-terminus or both, of region 181-215 of sequence NP_006168.1 (SEQIDNO:12).

[0078] The Applicant discovered that the leucine zipper domain of NRL DNA regulatory proteins not only allows efficient covalent dimerization of the chimeric monomer by favoring the formation of disulfide bridges, but also provides very efficient expression and secretion of the functional dimeric protein in recombinant mammalian systems. Petition 870260035468, dated 04 / 16 / 2026, page 24 / 233 20 / 103

[0079] Even more surprisingly, this new chimeric protein is correctly post-translationally modified, i.e., it is correctly O-glycosylated and sulfated at Tyr in the expected PSGL-1 region, is dimeric and covalently linked by disulfide bridge(s), and binds to selectin targets with an affinity superior to or at least equivalent to that of the optimized Fragment 1 (Fr-1), described in WO2012 / 020030. These conclusions were obtained from a number of data that were detailed further in the experimental section.

[0080] The findings cited above are quite surprising since the present chimeric protein (P-selectin glycoprotein ligand-1 and Neural Retina-specific leucine zipper), referred to here as sPSGL-1-NRL, is a combination of unrelated protein domains and is even more peculiar when compared with the expression levels of other chimeric proteins engineered to carry the PSGL-1 selectin-binding domain as the N-terminal region, fused in a frame with other dimerization domains commonly used in the technique, such as the Fc fragment of IgG1.

[0081] Specifically, in one of the preferred embodiments of the invention, domain 1-47 of PSGL-1 of SEQIDNO:11 was merged into a frame with:

[0082] - a covalent dimerization domain, comprising at least one cysteine ​​that is defined below, preferably followed or alternatively preceded and framed with a non-covalent dimerization domain (or stabilization domain), such as the NRL leucine zipper, in place of the Fc or CH3 domain of human IgG1;

[0083] - optionally and preferably a spacer on terminal C, to avoid any steric interference with groups or clusters that will be connected to terminal C and Petition 870260035468, dated 04 / 16 / 2026, page 25 / 233 21 / 103

[0084] - a signal peptide for secretion at the N-terminus of the chimeric protein, which is suitable for removal by cleavage in the mature chimeric protein and which is preferably not the endogenous PSGL-1 signal and propeptide sequence.

[0085] Among the various variants that were produced and tested for expression in a mammalian system: Variant 1 and 1A representing preferred embodiments of the chimeric protein PSGL-1NRL of the present invention (SEQIDNO:2 and SEQIDNO:37), Variant 2 (SEQIDNO:4), which corresponds to PSGL-1 fused in a frame with the Hinge and CH3 domains of human IgG1, used respectively as covalent and non-covalent dimerization (or stabilization) domains, or Variant 5 (SEQIDNO:10), which comprises the same functional domains as the optimized Fragment 1 described in WO2012 / 020030 prepared for comparison purposes. Note that in the protein variants of interest, the sequence listings mentioned above identify a signal peptide that is removed by cleavage in the expressed mature form.

[0086] In particular, it was observed that only Variant 1, which contains the leucine zipper of NRL, produces expression levels that are suitable for the production of a pharmaceutical agent. Comparative data on the relative expression of Variants 1-5, expressed in the same cell system, are provided in Figure 1, in which the expression level of labeled chimeric proteins was evaluated through transient expression, SDS-PAGe under denaturing and non-denaturing conditions, and Western blot with an antibody directed to a common label (i.e., the FLAG octapeptide) placed at the C-terminus.

[0087] Therefore, without being limited to a particular theory, it is believed that the presence of a dimerization domain does not Petition 870260035468, dated 04 / 16 / 2026, page 26 / 233 22 / 103 covalent, such as a leucine zipper, and more preferably the NRL leucine zipper that favors protein-protein interaction, in a protein that is physiologically a dimer (PSGL-1), results, through a favorable folding or stabilizing effect of the final chimeric protein of the present invention, in higher expression levels, which can be further optimized by isolating and stabilizing in culture a single clone. The titers obtained after a preliminary clonal selection are well above 0.1 g / L.

[0088] In the chimeric protein of the present invention, the covalent dimerization domain, alternatively called the disulfide bond-promoting region(s), comprises at least one Cys residue available to form a disulfide bond with another Cys in a corresponding monomeric chimeric protein, such that the two chimeric protein monomers are covalently linked through at least one disulfide bridge.

[0089] A general formula that covers the covalent dimerization domain is: (X1)nC(X2)m-(X3),

[0090] Where X1 and X2 represent any amino acid or sequence of amino acids excluding Cys; C is cysteine, X3 is any amino acid and are integers from 1-6. Xi preferably comprises a Proline, a Histidine or a Threonine; more preferably it comprises a Proline and a Histidine or a Histidine and a Threonine or a Proline and a Threonine. According to a preferred embodiment, X1 comprises a Proline, a Histidine and a Threonine, preferably in this order and is at most 5. X2 is any amino acid or sequence of amino acids excluding Cys and preferably comprises Proline. Preferably X2 is Pro Petition 870260035468, dated 04 / 16 / 2026, p. 27 / 233 23 / 103 Pro; X3 is preferentially Cysteine ​​and comprises at least one Proline. More preferably, the cysteine-carrying region is the IgG1 Hinge region or functional fragments thereof. A preferred disulfide bond promoter region is: PHTCPPCP (SEQIDNO:20).

[0091] According to a preferred embodiment, the chimeric protein further comprises a C-terminus spacer comprising a residue suitable for covalent bioconjugation with other peptide or chemical groups, such as imaging and / or therapeutic groups. Examples of amino acids for bioconjugation are cysteine ​​and lysine. The spacer is approximately 4-20 amino acids long and comprises one or more amino acids selected from the group consisting of: Gly, Ser, Pro, Ala, Val, Leu; it carries a cysteine ​​or a lysine at its C-terminus, preferably in the penultimate position. The spacer is preferably a polyglycine that encloses an alanine or other neutral amino acid, such as valine or similar, and carries a cysteine ​​or a lysine, preferably a lysine, wherein said conjugating amino acid is followed by Gly, Ser, Pro, Ala, Val, Leu, preferably at least one Gly.The spacer preferably has the sequence G4AG4KG (SEQIDNO:17). Alternatively, the chimeric protein comprises a Flag sequence at its C-terminus, i.e., for identification and purification purposes. Flag sequences are commonly used by experts in the art and known in the field. An example is the sequence DYDDDDK (SEQIDNO:35), which allows recognition and / or purification of the protein by immunoaffinity with suitable antibodies.

[0092] According to a preferred embodiment, the monomeric protein is translated into the form of a precursor with the signal peptide and is then processed and finally secreted into the culture medium. Petition 870260035468, dated 04 / 16 / 2026, page 28 / 233 24 / 103 (conditioned medium) in the form of a homodimer after cleavage of the signal peptide.

[0093] A signal peptide for secretion is present at the terminal N of the chimeric protein precursor. Preferably, the signal peptide is the mouse IgH signal peptide, which has the sequence: MEWSWWVFLFFLSVTTGVHS (SEQIDNO:18). Other signal peptides (or leader peptides) can be used, such as the endogenous signal peptide sequence of PSGL-1 or other heterologous signal peptide sequences commonly used in recombinant protein expression techniques, which allow the secretion of post-translationally processed recombinant proteins. An example of some known signal peptides (or leaders) in the field is provided in Table 1. Table 1. Leader signal peptide sequences. Accession Number Gene Name Species Sequence P01728 LV2A_CAMUNDON O Ig lambda-2 V chain region Mus musculus MAWTSLILSLLALCSGAS S SEQIDNO:21 P01758 HVM14_CAMUNDON GO Ig 108A heavy V chain region Mus musculus MGWSWIFLFLLSGTAGV HS SEQIDNO:22 P01750 HVM06_CAMUNDON GO Ig 102 heavy V chain region Mus musculus MGWSCIILFLVATATGVH S SEQIDNO:23 P01749 HVM05_CAMUNDON GO Ig 3 heavy V chain region Mus musculus MGWSCIILFLVATATGVH S SEQIDNO:24 P01821 HVM45_CAMUNDON GO Ig heavy V chain region Mus musculus MAVLGLLFCLVTFPSCVL S SEQIDNO:25 Petition 870260035468, dated 04 / 16 / 2026, page 29 / 233 25 / 103 Accession Number Gene Name Species Sequence MC101 P01748 HVM04_CAMUNDON GO Ig 23 heavy chain V region Mus musculus MGWSCIILFLVAAANGVH S SEQIDNO:26 Q61508 ECM1_CAMUNDONG O Extracellular matrix protein 1 Mus musculus MGTVSRAALILACLALAS A SEQIDNO:27 P01751 HVM07_CAMUNDON GO Ig B1-8 / 186-2 heavy chain V region Mus musculus MGWSCIMLFLAATATGV HS SEQIDNO:28 P01831 THY1_CAMUNDONG O Thy-1 membrane glycoprotein Mus musculus MN PAISVALLLSVLQVSR G SEQIDNO:29 Q03402 CRIS3 CAMUNDONG O Cysteine-rich secretory protein 3 Mus musculus MALMLVLFFLAAVLPPSL L SEQIDNO:30 P01746 HVM02_CAMUNDON GO Ig 93G7 heavy chain V region Mus musculus MGWSFIFLFLLSVTAGVH S SEQIDNO:31 P26262 KLKB1_CAMUNDON GO Plasma kallikrein Mus musculus MILFNRVGYFVSLFATVS C SEQIDNO:32 P11627 L1CAM_CAMUNDON GO Neural cell adhesion molecule L1 Mus musculus MVVMLRYVWPLLLCSPC LL SEQIDNO:33 P06327 HVM52_CAMUNDON GO Ig VH558 A1 / A4 heavy chain V region Mus musculus MGWRWIFLFLLSGTAGV HC SEQIDNO:34

[0094] As an alternative to SEQIDNO:18 any of Petition 870260035468, dated 04 / 16 / 2026, p. 30 / 233 26 / 103 signal peptides above SEQIDNO:24 to SEQIDNO:34 can be used to achieve recombinant protein secretion.

[0095] The recombinant chimeric protein PSGL-1-NRL of the present invention efficiently binds to P / E selectin in a dimeric form wherein each monomer is the recombinant PSGL-1 that is defined above and is produced in an appropriately glycosylated and sulfated form. Several studies published to date have reviewed the post-translational requirements of PSGL-1 for binding to P / E / L selectin, i.e., glycosylation, sulfation, and mapped the residues important for this processing and for binding to selectin (RD Cumming, Braz. J. Biol. Res., 1999, 32(5): 520-528 and D. Sako Cell, 1995, 83: 323-331).

[0096] In the preferred embodiment of the chimeric protein according to the present invention, sialylation (presence of sialic acid) was evaluated by Liquid Chromatography coupled to Mass Spectrometry (LC-MS) after a mild acid treatment. The sialyl residue content of the recombinant proteins of the invention can be comprised of approximately 5% to 30% w / w, 10% to 28% w / w, 15% to 25% w / w, more preferably 15% to 25%.

[0097] In order to characterize the recombinant chimeric protein, peptide mapping was performed with Asp-N and enzymatic digestion with chymotrypsin.

[0098] The cyclization of the N-terminal glutamine (Gln) into pyroglutamine (pGlu), the sulfation of Tyr, the O-glycosylation of Thr (presence of Core 2 SLeX) and the dimeric structure were also evaluated and detailed further in the experimental section.

[0099] The dimeric structure of the proteins was confirmed by cleavage with chymotrypsin, which provides a fragment (TCPPCPL)2 according to a preferred embodiment of the recombinant protein.

[00100] Sulfation of tyrosine residues 5, 7 and 10 (Y) N Petition 870260035468, dated 04 / 16 / 2026, page 31 / 233 27 / 103 terminals was confirmed by Asp-N digestion. O-glycosylation with an SLeX tetrasaccharide motif was confirmed at threonine 16 via chymotrypsin cleavage.

[00101] Based on all that has been described above, it can be concluded that, in chimeric proteins, the N-terminal domain of PSGL-1, important for binding to Selectin, is appropriately sulfated at Tyr residues corresponding to positions 5, 7, and 10 of the mature PSGL-1 protein and glycosylated, in particular O-linked glycosylation at the threonine residue at position 16; the O-linked glycans typically comprise sugar residues such as N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), fucose, glucose, galactose, mannose (Man), hexose, xylose, sialic acid, or mixtures thereof.

[00102] These post-translation modifications (PTMs) have been summarized in SEQIDNO:38.

[00103] The O-linked glycans preferably present in the PSGL-1 portion of the chimeric protein of the present invention are preferably GalNac, GlcNAc, fucose, sialic acid and galactose. The O-linked glycans in PSGL-1 are preferably sialylated and fucosylated and preferably consist of the sialyl Lewis X glycan structure (sLex, sialic acid-galactopyranosyl-fucose-N-acetylglucosamine) linked to threonine residues.

[00104] This type of post-translational modification has been described as essential for binding to P-selectin (RD Cumming, Braz. J. Biol. Res., 1999, 32(5): 520-528 and D. Sako Cell, 1995, 83: 323-331).

[00105] Dimers are formed and each monomer is covalently linked by at least one disulfide bridge to the other.

[00106] Correct post-translational processing of chimeric proteins was achieved through expression in cells of Petition 870260035468, dated 04 / 16 / 2026, p. 32 / 233 28 / 103 mammalian cells such as HEK-293, COS-1, or CHO cells, which have been used in the past for PSGL-1 expression. In any case, PSGL-1 is preferentially co-expressed in mammalian cells along with C2GnT (core 2 β 1-6-N acetylglucosaminyltransferase) and any fucosyltransferase enzyme such as one of the following: Fuc-TIII (Fuc-T: fucosyltransferase), Fuc-IV, or Fuc-TVII (Fugang Li et al. J. Biol. Chem, 1996, 271:3255-3264) or their functional fragments. Preferably, Fuc-TVII or functional fragments thereof are used.Cells, preferably CHO-adapted cells for growth in suspension, are allowed to grow for at least 7 days, generally up to 14 days, using OptiCHO™ medium (LifeTechnology) (other chemically defined serum-free media may be used successfully, e.g., ActiCHO™ from GE / PAA, FortiCHO™, CellVento™ CHO 200 and CellVento™ CHO 220 from Millipore, 83836C from SAFC, BalanCD™ from Irvine, EX-CELL® from Sigma-Aldrich and similar media) and in the absence of selection pressure. Glutamine (or the analogue GlutMax™) was supplemented at 1-10 mM, preferably 4-8 mM. OptiCHO™ and ActiCHO™ (LifeTechnology) are preferred for expression purposes.

[00107] The chimeric protein can be purified to the required purity level through a three-step chromatography comprising: an anion-exchange chromatography, a hydrophobic interaction chromatography, and a size-exclusion chromatography or hydroxyapatite (HA) chromatography. Purification comprising an HA column as the final purification step is preferred and provides a pure, therapeutic-grade chimeric protein.

[00108] Therefore, according to a further embodiment, the present invention comprises a purification process for the chimeric protein that is defined above, comprising Petition 870260035468, dated 04 / 16 / 2026, page 33 / 233 29 / 103 as the final step, chromatography with hydroxyapatite (HA). More preferably, the purification process comprises a first chromatography performed on a strong anion exchange (AE), a second chromatography performed on a solid phase with hydrophobic interaction (HI), and a third step performed on HA, preferably Hydroxyapatite Ceramic.

[00109] More generally, the present invention relates to a process for the purification of any fusion or chimeric protein containing soluble PSGL-1, wherein said PSGL-1 comprises at least the 5-16 amino acids of mature PSGL-1 (SEQIDNO:11), which further comprises one or more flanking amino acids at the N- or C-terminus of such 5-16 fragment.

[00110] More preferably, the PSGL-1 fragment in the chimeric protein comprises all flanking amino acids up to at least amino acids 1-47 of mature PSGL-1. Even more preferably, the chimeric protein further comprises at least amino acids 187-208 of SEQIDNO:12 (Neural Retina-Specific Leucine Zipper) or an amino acid sequence at least 90% homologous or identical to said 187-208 region and a covalent dimerization domain that is defined previously by the general formula. A general embodiment for the chimeric protein has SEQIDNO:39.

[00111] Meanwhile, the purification process of the invention comprising HA as the last step can be successfully applied to any chimeric or recombinant protein that carries in the N-terminal region of mature PSGL-1 comprising at least the 5-16 or 1-47 amino acids of PSGL-1.

[00112] The process that includes HA as the final purification step allows achieving PSGL-1 purity greater than 95%, preferably 96%, 97%, 98% or 99% of other proteins and Petition 870260035468, dated 04 / 16 / 2026, page 34 / 233 30 / 103 substantially free of contaminating DNA, which is measured, for example, by commercial DNA quantification assays, such as the DNA Quantitation Kit, Fluorescence Assay (Sigma).

[00113] As for the final yields, the target protein is recovered through the process described above with yields above 50% of the total chimeric target protein content, typically above 60% and generally approximately or above 70%. These yields represent a good result and, more importantly for an industrial process, are quite standardized and can be reproduced with very low variations.

[00114] Standardized commercial resins or columns with these characteristics can be used according to the manufacturer's instructions. Ceramic HA is commercially available, for example, from BIORAD. Purification and elution conditions can be adjusted as is known to the expert.

[00115] To facilitate scale-up, gradient elution can be advantageously replaced by stepwise elution in order to limit the number of analyses in the process and reduce the total processing time. The purified protein (purity > 90%), in the preferred embodiments of Variant 1 and 1A described below, was characterized and:

[00116] · Correct removal of the leader peptide has been confirmed;

[00117] · The N-terminal structure of the Variant 1A glycoprotein of PSGL is mainly in the pyroglutamic form pQATEYEYL. In fact, the use of the Asp N digestion enzyme allowed the detection of N-terminal Gln cyclization, the process through which the Gln residue present at the N-terminal tends to undergo spontaneous cyclization to form pyroglutamic acid;

[00118] · The dimeric characteristic of PSGL Variant 1A was confirmed by gel electrophoresis under reducing and non-reducing conditions. Petition 870260035468, dated 04 / 16 / 2026, p. 35 / 233 31 / 103 reducing and enzymatic digestion followed by fragment characterization using LC-MS. Experimental data show that Variant-1A of PSGL could be entirely in its dimeric form;

[00119] · The presence of the O-glycan group in Thr16 was confirmed and its structure was identified. The expected Sialyl-Lewis-X motif, a Core 2 structure comprising N-acetylgalactosamine, N-acetylglucosamine, galactose, fucose and sialic acid was demonstrated, as is detailed further in the experimental section;

[00120] · Sulfation of residues Y5, Y7, and Y10 of the mature protein, important for binding to both L- and P-selectin, was also confirmed. Monitoring of glycoprotein sulfation was performed by Mass Spectrometry. As is known to the expert, sulfation of Tyrosines 5, 7, and 10, demonstrated in the present chimeric target protein, is extremely important for the biological activity of PSGL-1 and Variant 1A.

[00121] Additional protein characterization data were reported in this Patent Application and detailed further in the experimental section.

[00122] Therefore, according to the main aspect, the invention relates to an isolated and purified chimeric PSGL-1 protein, which is defined above, capable of binding to P, L and E selectins, preferentially to P selectin.

[00123] The isolated recombinant protein is a homodimer in which each homodimer has a primary sequence comprising, or preferably consisting of, the following amino acid sequences, most preferably in this order:

[00124] - amino acids 1-47 of the mature PSGL-1 protein (SEQIDNO:11) at the N terminus;

[00125] - at least one amino acid sequence that Petition 870260035468, dated 04 / 16 / 2026, p. 36 / 233 32 / 103 comprises or consists of a cysteine ​​suitable for a disulfide bridge with formula: (X1)nC(X2)m-(X3), as defined above, more preferably SEQIDNO:20;

[00126] - at least articles 181-215 of the NRL (SEQIDNO:12);

[00127] - optionally, a spacer amino acid up to 15 aa in length, carrying: at least one or more Gly and / or Ala and preferably an amino acid such as Lys or Cys at the C-terminus or, more preferably, at the penultimate position. More preferably, such a spacer is a polyglycine, consisting of 4, 5, 6, 7, 8, 9 or 10 glycines, preferably comprising and fitting at least one alanine, more preferably further comprising a lysine at the penultimate position for additional chemical conjugation. Even more preferably, the spacer amino acid is SEQIDNO:17. Therefore, in a particularly preferred embodiment, the chimeric protein has the sequence SEQIDNO:37, in which the signal peptide, cleaved from the mature protein, is still represented. A chimeric target protein monomer modified after translation is represented in SEQIDNO:38 as a preferred embodiment.

[00128] A general formula of the chimeric variant protein with motifs or regions essential for:

[00129] linkage to selectin, as described in detail above,

[00130] covalent dimerization (namely, a Cys comprising motif with flanking regions),

[00131] non-covalent dimerization (namely, a motif comprising at least NRL amino acids 187-208), which preferentially comprises residue(s) for chimeric protein conjugation, preferably at the last or penultimate position (namely, Lys or Cys) was also reported in SEQIDNO:39.

[00132] The purity of the isolated PSGL-1 chimeric protein and Petition 870260035468, dated 04 / 16 / 2026, page 37 / 233 The purified 33 / 103 concentration can be determined by UPLC-UV or Surface Plasmon Resonance, i.e., on a Biacore instrument, as detailed further in the Experimental Section.

[00133] The present invention further relates to any DNA sequence encoding the aforementioned protein in monomeric form, preferably comprising the nucleotide sequence encoding amino acids 1-118 of SEQIDNO:2 of the chimeric protein as defined above. According to a particularly preferred embodiment, the nucleotide sequence encompasses nt 1-354 of SEQIDNO:36 and preferably consists of nucleotides 1-360.

[00134] Alternatively, the DNA sequence encoding the chimeric protein of the present invention comprises at least the nucleotide sequence encoding the P-selectin binding domain of PSGL-1, preferably aa 1-47 of SEQIDNO:11 and the nucleotide sequence encoding at least aa 187-208 of the neural retina-specific leucine zipper domain (SEQIDNO:12), which optionally comprise 1, 2, 3, 4, 5, 6 flanking amino acids at the N or C terminus or, more preferably, encoding at least aa 181-215 of NRL (SEQIDNO:12).

[00135] The redundancy of the genetic code allows different codons to be used for a single amino acid, so different combinations of codons, i.e., different DNA sequences, allow the expression of the protein with the same primary amino acid sequence and produce the same recombinant protein. Such different DNA sequences, which can be designed to optimize expression in the selected recombinant system through the use of preferred codons for each organism, are therefore all included within the scope of the present invention. Petition 870260035468, dated 04 / 16 / 2026, p. 38 / 233 34 / 103

[00136] A particularly preferred recombinant chimeric protein comprises SEQIDNO:2 aa 1-118, encoded by SEQIDNO:1 nt 1354. The amino acids 1-118 of SEQIDNO:2 optionally further comprise at the C-terminus at least one amino acid suitable for chemical conjugation with reactive groups of labeling or therapeutic groups, wherein said amino acid is Lysine or Cysteine. More preferably, such reactive amino acid is followed by at least one other uncharged amino acid, preferably Glycine. Even more preferably, the amino acid suitable for conjugation is placed at the C-terminus of an amino acid sequence of up to 15 aa and is followed by at least one neutral or uncharged amino acid, such as Glycine.

[00137] The invention further comprises an expression vector comprising said DNA sequence and the transfected cell clone carrying the recombinant sequence, transiently or stably integrated into the genome. Preferred cell clones are those carrying stably integrated DNA copies of the vector, such as those obtained from CHO cells, more preferably from CHO cells adapted for growth in suspension, which are commercially available, i.e., in the Freedom™ CHO-S™ kit from Lifescience (ThermoFisher Scientific) together with an expression vector used as a standard for mammalian cell expression.

[00138] Suspension-adapted CHO cells can be amplified and grown to a density of approximately 2.107 cells / L, to achieve high-efficiency secretion of the recombinant protein.

[00139] According to a further aspect, the invention comprises the process for preparing the recombinant chimeric protein defined above, which comprises Petition 870260035468, dated 04 / 16 / 2026, page 39 / 233 35 / 103 transformation of a eukaryotic cell with the DNA sequence encoding the same or the vector comprising the DNA sequence encoding the same to obtain a recombinant eukaryotic system, preferably a CHO cell system that stably expresses the chimeric protein and in which said chimeric protein is preferably secreted into the medium, collection of the culture medium and recovery of the recombinant protein from said culture medium by purification.

[00140] The purified recombinant chimeric protein can then be successfully conjugated via the C-terminal residue to groups for diagnostic and / or therapeutic purposes or used as is, i.e., in combination with suitable ingredients or excipients in pharmaceutical compositions. Chimeric protein conjugates for diagnostic and therapeutic applications.

[00141] The chimeric protein of the present invention is used to target the active groups bound to it for diagnosis and therapy, targeting tissues, cells or organs that express selectin. The specificity of the chimeric protein, which is provided by the PSGL-1 region or fragments thereof, directs it to selectins if appropriately modified after translation (Liu et al. J. Biol. Chem., 1998, 273:7078-7087). It has been confirmed in this expression system that the binding strength to the target is not altered by the presence of non-covalent dimerization domains such as leucine zippers that are maintained by a very peculiar secondary structure.

[00142] Therefore, according to one of the main embodiments, the present invention is directed to conjugates comprising the chimeric protein as the targeting agent for imaging cells, tissues, organs expressing selectin, etc. Petition 870260035468, dated 04 / 16 / 2026, p. 40 / 233 36 / 103

[00143] By image acquisition clusters, the present inventors refer to any cluster detectable through diagnostic imaging procedures, that is, any diagnostically efficient cluster capable of providing, enhancing, or otherwise advantageously modifying the signal detected by a currently used diagnostic imaging technique, which includes: ultrasound (US), computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), single-photon emission computed tomography (SPECT), X-ray imaging, photoacoustic imaging, fluorescence imaging, and optics, which, in the present invention, comprises imaging during surgery, that is, any technique that enables the recording of images useful for diagnosis, preferably with contrast.Hybrid imaging methods are also considered in the present invention, in which the chimeric protein is linked to at least two detectable clusters using different imaging methods. Examples of hybrid imaging include PET / CT, SPECT / CT, MR / PET, MR / SPECT; ultrasound and MR, ultrasound and CT; MR and CT.

[00144] The imaging assembly may comprise any possible combination of imaging assemblies for dual detection, for example, for MRI / PET it may comprise a paramagnetic metal chelating unit and a radionuclide chelating unit. Examples of efficient diagnostic assemblies according to the invention include, for example, chelated radionuclides that emit gamma rays or positrons; paramagnetic metal ions in the form of chelated or polychelated complexes, X-ray absorbing agents that include Petition 870260035468, dated 04 / 16 / 2026, p. 41 / 233 37 / 103 atoms having an atomic number greater than 20; a dye molecule; a fluorescent molecule; a phosphorescent molecule; a molecule that absorbs in the UV spectrum; a quantum dot; a molecule capable of absorption within near- or far-infrared radiation and, in general, all groups that generate a detectable signal or interact specifically with a detection system. Based on all that has been described above, it is known to those skilled in the art that the imaging method to be used must be selected according to the detectable imaging group to which the diagnostic compounds of the invention are attached. Thus, for example, for a fluorescent imaging group such as CyC5 attached to a chimeric protein, a fluorescent light detection system will be appropriate.

[00145] The table below provides some examples of contrast-producing agents and the preferred imaging mode. Table 2. Examples of contrast-producing agents and preferred imaging modality. Contrast-Producing Agent Modality Ultrasound Microbubble, acoustically active liposome CT / X-ray Gold nanoparticles, iodinated nanoparticles, MRI Hyperpolarized neon / xenon / helium, gadolinium, iron oxide PET 18F 11c SPECT 99mTc, 123I, 111In Optical imaging Methylene blue, fluorescent dye such as cyanine dyes (Cy7, CyC5, indocyanine green) NIR dyes, IRDye800®CW, GFP, AlexaFluor® dyes, microbubble, nanoparticle Petition 870260035468, dated 04 / 16 / 2026, page 42 / 233 38 / 103 such as liposomes comprising fluorescent dyes DiR, NIR dyes, IRDye800®CW, GFP, AlexaFluor® dyes. Imaging with photoacoustics. Single-walled carbon nanotubes (SWINT), indocyanine green, gold nanoparticles, zinc phthalocyanine. Conjugation of chimeric proteins to clusters for diagnostic and therapeutic use with molecular targeting.

[00146] The preparation of conjugates of the chimeric protein of the invention is generally carried out by chemical means.

[00147] The reactive groups of the conjugation partners, that is, the chimeric protein and the group(s) to be conjugated to it, are present or prepared in a protected form. In the present description, unless otherwise indicated, the term protecting group designates a protecting group adapted to preserve the characteristic chemical function of the functional group to which it is attached. Specifically, in the present context, protecting groups are used to preserve amino or carboxyl functions. Suitable protecting groups may therefore include, for example, Fmoc, benzyl, benzyloxycarbonyl or alkyl esters or other groups commonly intended for the protection of such functions and known to the expert.

[00148] Other chemical groups, capable of reacting chemically with the N-terminal (-NH2) or C-terminal (-COOH) group of a polypeptide unit, such as the chimeric protein of the invention that transforms such a group, through a chemical reaction, into a suitable derivative that maintains the specificity of the corresponding polypeptide / protein by the selectin, but incapable of reacting chemically with, respectively, a carboxyl or amino functionality in a different group, are called deactivating groups. Petition 870260035468, dated 04 / 16 / 2026, page 43 / 233 39 / 103 Deactivating groups should not be involved in carboxamide crosslinking reactions. An example is the acetyl group [also referred to as CH3(CO)- or Ac], used to deactivate the amino terminus of a peptide chain by converting it into the corresponding non-reactive acetylated AcHN- group.

[00149] On the other hand, the amino groups themselves and derivatives thereof, such as, for example, -NH2, -NH(CH3) or H2NOC-CH2-NH-, can be used as deactivating groups for the free carboxyl group, providing the corresponding non-reactive amides CONH2, -CONH(CH3) or -CONH-CH2-CONH2, respectively.

[00150] For example, if the chimeric protein includes a reactive amino group (e.g., a primary amino group of Lysine), this can be reacted with a diagnostic group, such as a microvesicle component containing a suitable corresponding reactive group, such as an isothiocyanate group (to form a thiourea linkage), a reactive ester (to form an amide linkage), a carbonyl group (to form an imine linkage, which can be reduced to an amine linkage), an activated hydroxyl group, for example, in the form of a tosylate, a tresylate or a cyanate, a vinyl sulfone or an epoxide.

[00151] Alternatively, when the targeting ligand of the present invention includes a reactive thiol group, the suitable complementary reactive group in the diagnostic or therapeutic group, i.e., a microvesicle component, may include haloacetyl derivatives, maleimides (to form a thioether linkage) or a mixed disulfide comprising a sulfide in the form of a 2-pyridylthio (PDT) group (which, after reaction with a thiol derivative of the targeting ligand, results in the formation of a linkage). Petition 870260035468, dated 04 / 16 / 2026, page 44 / 233 40 / 103 stable disulfide), an activated hydroxyl group, for example, in the form of a tosylate, a tresylate or a cyanate, a vinyl sulfone or an epoxide.

[00152] Alternatively, according to one embodiment of the invention, a targeting ligand containing a reactive amino group (e.g., a primary amino group, in particular the terminal NH2 group) can first be reacted with a sulfur-containing compound to introduce a reactive thiol group into the targeting ligand, which is then reacted with a corresponding complementary group in the diagnostic component, i.e., a microvesicle component as illustrated above. Examples of suitable sulfur-containing compounds useful for introducing a reactive thiol group into a targeting ligand containing a reactive amino group include, for example: thioimidate (such as Traut's reagent), N-succinimidyl-S-acetylthioacetate (SATA), N-succinimidyl-S-acetylthiopropionate (SATP) or N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP).A detailed description of S-containing agents and their respective thiolation reactions can be found, for example, in Greg T. Hermanson's book: Bioconjugate Techniques, Elsevier ed., 2nd ed. (Apr. 2008), chapter 1, section 4-1. For example, one can prepare a maleimide-derivatized phospholipid (e.g., phosphatidylethanolamine - PE - or pegylated PE) and react it with a targeting ligand (e.g., SEQ ID NO:3) in which a primary amino group (e.g., the -NH2 of the Lysine side chain) has been previously reacted with a sulfur-containing compound (such as those illustrated above) to introduce a reactive thiol group; the resulting compound can then be used in the preparation of targeted gas-filled microvesicles.

[00153] According to an additional alternative, when the ligand Petition 870260035468, dated 04 / 16 / 2026, p. 45 / 233 41 / 103 targeting includes a reactive carboxylic group, suitable reactive groups in the diagnostic or therapeutic group, i.e., the microvesicle component, may be amines and hydrazides (to form amide or N-acyl, N'-alkyl-hydrazide functions).

[00154] According to the previous preferred embodiment, the targeting ligand containing a reactive amino group (e.g., on a lysine residue) can first be reacted with a compound containing maleimide to introduce a reactive maleimide group into the targeting ligand, which is then reacted with a corresponding complementary group in the microvesicle component. Maleimide-containing agents useful for introducing a reactive maleimide group into a targeting ligand containing a reactive amino group and the respective maleimide group addition reaction are well known. in the art. Examples of suitable maleimide-containing compounds include, for example: AMAS (N-(α-maleimidoacetoxy)succinimide ester), BMPS (N-(3-maleimidopropoxyl)succinimide ester), EMCS (N-(ε-maleimidocaproyloxy)succinimide ester), GMBS (N-(γ- maleimidobutyryloxy)succinimide ester), LC-SMCC (succinimidyl-4-(Nmaleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproate)), MBS (mmaleimidobenzoyl-N-hydroxysuccimide ester), SMCC (succinimidyl-4(N-maleimidomethyl)cyclohexane-1-carboxylate), SMPB (succinimidyl-4(p-maleimidophenyl)butyrate), reagent SM(PEG)n (succinimidyl-(Nmaleimidopropionamido)-ethylene glycol) ester), SMPH (succinimidyl-6-((3maleimidopropionamido) hexanoate)), sulfo-EMCS (N-^maleimidocaproyloxy) sulfosuccinimide ester), sulfo-GMBS (N-(ymaleimidobutyroyloxy)sulfosuccinimide ester), sulfo-KMUS (N-(kmaleimidoundecanoyloxy)-sulfosuccinimide ester), sulfo-MBS (mmaleimidobenzoyl-N-hydroxysulfosuccinimide ester), sulfo-SMCC (4 Petition 870260035468, dated 04 / 16 / 2026, page 46 / 233 42 / 103 (sulfosuccinimidyl N-maleimidomethyl)-cyclohexane-1-carboxylate), sulfo-SMPB (sulfosuccinimidyl 4-(p-maleimidophenyl (butyrate)).

[00155] Other analogous reagents may contain sulfhydryl reactive groups other than maleimide, for example, LC-SPDP (succinimidyl 6-[3-2-pyridyldithio)propionamido]hexanoate, NHS-Bromoacetate (N-hydroxysuccinimidyl bromoacetate), NHS-Iodoacetate (N-hydroxysuccinimidyl iodoacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SULFO-LC-SPDP (sulfosuccinimidyl-6-[3-(2-pyridyldithio)propionamido]hexanoate).

[00156] According to the microvesicle modality for obtaining ultrasound images for diagnosis, a thio-containing phospholipid (e.g., thiolated phosphatidylethanolamine PE or pegylated PE) can be reacted with a targeting ligand in which a primary amino group (e.g., the NH2 of the lysine side chain) has been previously reacted with a reactive thiol compound (e.g., a maleimide such as those illustrated above), to introduce a reactive thiol group therein; the compound obtained can then be used in the preparation of microvesicles or other diagnostic or therapeutic conjugates.

[00157] In the chimeric protein of the invention, the conjugates are preferably prepared at the C-terminus of the chimeric protein, leaving the N-terminus available for binding with the target selectins. Efficient clusters for ultrasound diagnosis Microvesicles

[00158] A class of contrast agents, particularly useful for obtaining high-contrast ultrasound images, includes suspensions of nano- and / or micrometer-sized gas bubbles dispersed in an aqueous medium. Of particular interest are those formulations in which the gas bubbles are stabilized, for example, through the use of emulsifiers, oils, thickeners. Petition 870260035468, dated 04 / 16 / 2026, p. 47 / 233 43 / 103 or sugars or through the capture or encapsulation of the gas or a precursor thereof in a variety of systems. These stabilized gas bubbles are generally referred to in the art with various terminologies, typically depending on the stabilizing material employed for their preparation; these terms include, for example, microspheres, microbubbles, microcapsules, or microballoons. The term gas-filled microvesicles, or in short microvesicles, as used herein, includes any of the above terminologies. Gas-filled microvesicles

[00159] According to a preferred embodiment of the present invention, gas-filled microvesicles are prepared with lipids or phospholipids covalently associated with the chimeric protein of the present invention, as a selectin targeting ligand, and are preferably microbubbles. By microbubbles, the present inventors refer to a gas bubble suspended in an aqueous carrier, which, at the gas-liquid interface, has a thin envelope (film) with a stabilizing amphiphilic material. Examples of aqueous suspensions of gas microbubbles are disclosed, for example, in US 5,271,928, US 5,445,813, US 5,413,774, US 5,556,610, 5,597,549, US 5,827,504 and WO 04 / 069284. The term also includes microbubble precursors in the form of freeze-dried or spray-dried components, preferably comprising phospholipid dispersions.

[00160] In the microbubble variance according to the previous definition, the terms microballoons or microcapsules include suspensions in which gas bubbles are surrounded by an envelope of solid material of a lipid or of natural or synthetic polymers. Examples of microballoons and their preparation are disclosed, for example, in US 5,711,933 and US 6,333,021. Petition 870260035468, dated 04 / 16 / 2026, p. 48 / 233 44 / 103

[00161] Suitable components for forming a stabilized microbubble envelope include, for example, phospholipids; lysophospholipids; fatty acids, such as palmitic acid, stearic acid, arachidonic acid or oleic acid; lipid-bearing polymers, such as chitin, hyaluronic acid, polyvinylpyrrolidone or polyethylene glycol (PEG), also referred to as pegylated lipids; sulfonated mono-, di-, oligo- or polysaccharides carrying lipids; cholesterol, cholesterol sulfate or cholesterol hemisuccinate; tocopherol hemisuccinate; lipids with ether- or ester-linked fatty acids; polymerized lipids; diacetyl phosphate; dicetyl phosphate; ceramides;Polyoxyethylene fatty acid esters (such as polyoxyethylene fatty acid stearates), polyoxyethylene fatty alcohols, polyoxyethylene fatty alcohol ethers, polyoxyethylated sorbitan fatty acid esters, polyethylene glycol glycerol ricinoleate, ethoxylated soybean sterols, ethoxylated castor oil or block copolymers of ethylene oxide (EO) and propylene oxide (PO); aliphatic sterol acid esters including cholesterol butyrate, cholesterol isobutyrate, cholesterol palmitate, cholesterol stearate, lanosterol acetate, ergosterol palmitate or photosterol n-butyrate; Sterol esters of sugar acids, including cholesterol glucuronides, lanosterol glucuronides, 7-dehydrocholesterol glucuronide, ergosterol glucuronide, cholesterol gluconate, lanosterol gluconate, or ergosterol gluconate;Sugar acid esters and alcohol esters including lauryl glucuronide, stearoyl glucuronide, myristoyl glucuronide, lauryl gluconate, myristoyl gluconate or stearoyl gluconate; sugar esters with aliphatic acids including sucrose laurate, fructose laurate, sucrose palmitate, sucrose stearate, glucuronic acid, gluconic acid or polyuronic acid; saponins including; Petition 870260035468, dated 04 / 16 / 2026, page 49 / 233 45 / 103 sarsassapogenin, smilagenin, hederagenin, oleanolic acid or digitoxigenin; glycerol or glycerol esters including glycerol tripalmitate, glycerol distearate, glycerol tristearate, glycerol dimyristate, glycerol trimyristate, glycerol dilaurate, glycerol trilaurate, glycerol dipalmitate; long-chain alcohols including n-decyl alcohol, lauryl alcohol, myristyl alcohol, cetyl alcohol or n-octadecyl alcohol; 6-(5-cholesten-3e-yloxy)-1-thio-e-D-galactopyranoside; digalactosyldiglyceride; 6-(5-cholesten-3 β yloxy)hexyl-6-amino-6-deoxy-1-thio- β -D-galactopyranoside; 6-(5cholesten-3 β -yloxy)hexyl-6-amino-6-deoxyl-1-thio- β -D-mannopyranoside; 12-(((7'-diethylaminocoumarin-3-yl)carbonyl)methylamino)octadecanoic acid; N-[12-(((7'-diethylaminocoumarin-3-yl)carbonyl)methylamino)octadecanoyl]-2-aminopalmitic acid; N-succinyldioleylphosphatidylethanolamine; 1,2-dioleyl-sn-glycerol; 1,2-dipalmitoyl-sn-3-succinylglycerol;1,3-dipalmitoyl-2-succinylglycerol; 1-hexadecyl-2-palmitoylglycerophosphoethanolamine or palmitoylhylhomocysteine; alkylamines or alkylammonium salts, comprising at least one (C10-C20), preferably (C14-C18), alkyl chain, such as, for example, Nestearylamine, N,N'-diestearylamine, N-hexadecylamine, N,N'-dihexadecylamine, N-stearylammonium chloride, N,N'-diestearylammonium chloride, N-hexadecylamonium chloride, N,N'-dihexadecylamonium chloride, dimethyldioctadecylamonium bromide (DDAB), hexadecyltrimethylammonium bromide (CTAB); tertiary or quaternary ammonium salts comprising one or preferably two (C10-C20), preferably (C14-C18), acyl chains linked to the N atom via a (C3-C6) bridge, such as, for example, 1,2-diestearoyl-3-trimethylammonium-propane (DSTAP), 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP), 1,2-oleoyl-3-trimethylammonium-propane (DOTAP), 1,2-diestearoyl-3-dimethylammonium-propane (DSDAP); and mixtures or combinations thereof. Petition 870260035468, dated 04 / 16 / 2026, page 50 / 233 46 / 103

[00162] Depending on the combination of components and the manufacturing process of the microbubbles, the examples of compounds listed above can be used as the main compound to form the microbubble envelope or in the form of simple additives, thus being present only in smaller quantities.

[00163] According to a preferred embodiment, at least one of the compounds forming the microbubble envelope is an amphiphilic compound (i.e., an organic molecule comprising both a hydrophilic and a lipophilic group), preferably a phospholipid, optionally in a mixture with any of the other materials mentioned above. According to the present description, the term phospholipid is intended to encompass any amphiphilic phospholipid compound whose molecules are capable of forming a film of stabilizing material (typically in the form of a monomolecular layer) at the gas-water boundary interface in the final microbubble suspension. Consequently, these materials are also referred to in the art as film-forming phospholipids.

[00164] Amphiphilic phospholipid compounds typically contain at least one phosphate group and at least one, preferably two, long-chain lipophilic hydrocarbon groups.

[00165] Examples of suitable phospholipids include glycerol esters with one or preferably two (identical or different) fatty acid residues and with phosphoric acid, wherein the phosphoric acid residue is in turn linked to a hydrophilic group, such as, for example, choline (phosphatidylcholines - PC), serine (phosphatidylserines - PS), glycerol (phosphatidylglycerols - PG), ethanolamine (phosphatidylethanolamines - PE), inositol (phosphatidylinositol - PI). Phospholipid esters with only one fatty acid residue are generally referred to in the art as the smooth forms of the phospholipid. Petition 870260035468, dated 04 / 16 / 2026, page 51 / 233 47 / 103 or lysophospholipids. The fatty acid residues present in phospholipids are generally long-chain aliphatic acids, typically containing 12 to 24 carbon atoms, preferably 14 to 22; the aliphatic chain may contain one or more unsaturations or is preferably completely saturated. Examples of suitable fatty acids included in phospholipids are, for example, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, oleic acid, linoleic acid, and linolenic acid. Preferably, saturated fatty acids such as myristic acid, palmitic acid, stearic acid, and arachidic acid are used.

[00166] Additional examples of phospholipids are phosphatidic acids, that is, the diesters of glycerol-phosphoric acid with fatty acids; sphingolipids such as sphingomyelins, that is, the phosphatidylcholine analogs in which the glycerol diester residue with fatty acids is replaced by a ceramide chain; cardiolipins, that is, the esters of 1,3-diphosphatidylglycerol with a fatty acid; glycolipids such as GM1 (or GM2) gangliosides or cerebrosides; glycolipids; sulfatides and glycosphingolipids.

[00167] As used herein, the term phospholipids includes naturally occurring, semi-synthetically prepared, or synthetic products that may be used individually or in mixtures.

[00168] Examples of naturally occurring phospholipids are natural lecithins (derived from phosphatidylcholine (PC)) such as, typically, soy lecithins or egg yolk lecithins.

[00169] Examples of semi-synthetic phospholipids are partially or completely hydrogenated derivatives of naturally occurring lecithins. Preferred phospholipids are fatty acid diesters of phosphatidylcholine, ethylphosphatidylcholine, phosphatidylglycerol, Petition 870260035468, dated 04 / 16 / 2026, page 52 / 233 48 / 103 phosphatidic acid, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol or sphingomyelin.

[00170] Examples of preferred phospholipids are, for example, dilauroyl phosphatidylcholine (DLPC), dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), diaraquidoyl phosphatidylcholine (DAPC), distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), 1,2-Diestearoyl-sn-glycero-3-Ethylphosphocholine (Ethyl-DSPC), dipentadecanoyl phosphatidylcholine (DPDPC), 1-myristoyl-2-palmitoyl phosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl phosphatidylcholine (PMPC), 1-palmitoyl-2-stearoyl phosphatidylcholine (PSPC), 1-stearoyl-2-palmitoyl-phosphatidylcholine (SPPC), 1-palmitoyl-2-oleylphosphatidylcholine (POPC), 1-oleyl-2-palmitoylphosphatidylcholine (OPPC), dilauroyl-phosphatidylglycerol (DLPG) and its alkali metal salts, diaraquidoylphosphatidylglycerol (DAPG) and its alkali metal salts, dimyristoylphosphatidylglycerol (DMPG) and its alkali metal salts, dipalmitoylphosphatidylglycerol (DPPG) and its alkali metal salts,distearoylphosphatidylglycerol (DSPG) and its alkali metal salts, dioleoyl-phosphatidylglycerol (DOPG) and its alkali metal salts, dimyristoyl phosphatidic acid (DMPA) and its alkali metal salts, dipalmitoyl phosphatidic acid (DPPA) and its alkali metal salts, distearoyl phosphatidic acid (DSPA), diaraquidoylphosphatidic acid (DAPA) and its alkali metal salts, dimyristoyl-phosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoyl phosphatidylethanolamine (DSPE), dioleylphosphatidylethanolamine (DOPE), diaraquidoylphosphatidylethanolamine (DAPE), dilinoleylphosphatidylethanolamine (DLPE), dimyristoyl phosphatidylserine (DMPS), diaraquidoyl phosphatidylserine (DAPS), dipalmitoyl phosphatidylserine (DPPS), distearoyl phosphatidylserine (DSPS), dioleoyl phosphatidylserine (DOPS), dipalmitoyl sphingomyelin (DPSP) and distearoyl sphingomyelin (DSSP), dilauroyl phosphatidylinositol (DLPI), diaraquidoyl phosphatidylinositol (DAPI), dimyristoyl phosphatidylinositol (DMPI),dipalmitoylphosphatidylinositol, Petition 870260035468, dated 04 / 16 / 2026, page 53 / 233 49 / 103 (DPPI), distearoylphosphatidylinositol (DSPI), dioleoyl-phosphatidylinositol (DOPI).

[00171] Suitable phospholipids also include phospholipids modified by attaching a hydrophilic polymer, such as polyethylene glycol (PEG) or polypropylene glycol (PPG), to them. Preferred polymer-modified phospholipids include pegylated phospholipids, i.e., phospholipids linked to a PEG polymer. Examples of pegylated phospholipids are pegylated phosphatidylethanolamines (abbreviated PE-PEGs), i.e., phosphatidylethanolamines in which the hydrophilic ethanolamine group is attached to a PEG molecule of variable molecular weight (e.g., from 300 to 20000 daltons, preferably from 500 to 5000 daltons), such as DPPE-PEG (or DSPE-PEG, DMPE-PEG, DAPEPEG or DOPE-PEG). For example, DPPE-PEG2000 refers to DPPE that has a PEG polymer attached to it, which has a weight-average molecular weight of approximately 2000.

[00172] Particularly preferred phospholipids are DAPC, DSPC, DSPG, DPPA, DSPA, DMPS, DPPS, DSPS and Ethyl-DSPC. DSPG or DSPC are the most preferred.

[00173] Mixtures of phospholipids may also be used, such as, for example, mixtures of DSPE, DPPE, DPPC, DSPC and / or DAPC with DSPS, DPPS, DSPA, DPPA, DSPG, DPPG, Ethyl-DSPC and / or Ethyl-DPPC.

[00174] In preferred embodiments, phospholipid is the main component of the microbubble stabilizing envelope, constituting up to at least 50% (w / w) of the total amount of components that form the envelope of gas-filled microbubbles. In some of the preferred embodiments, substantially the entire envelope (i.e., at least 80% and up to 100% by weight) may be formed of phospholipids. Petition 870260035468, dated 04 / 16 / 2026, page 54 / 233 50 / 103

[00175] Phospholipids can be conveniently used in mixtures with any of the compounds listed above. Thus, for example, substances such as cholesterol, ergosterol, phytosterol, sitosterol, lanosterol, tocopherol, propyl gallate or ascorbyl palmitate, fatty acids such as myristic acid, palmitic acid, stearic acid, arachidic acid and derivatives thereof or butylated hydroxytoluene and / or other non-phospholipid compounds can optionally be added to one or more of the above phospholipids in proportions ranging from zero to 50% by weight, preferably up to 25%. Amphiphilic compounds, such as C10-C20 carboxylic acids, preferably palmitic acid, are particularly preferred.

[00176] According to a preferred embodiment, the microbubble envelope according to the invention includes a compound carrying a net total charge (positive or negative). Said compound may be a charged amphiphilic material, preferably a lipid or a phospholipid.

[00177] Examples of phospholipids carrying a net negative charge are derived, in particular, from fatty acid diester derivatives, of phosphatidylserine, such as DMPS, DPPS, DSPS; of phosphatidic acid, such as DMPA, DPPA, DSPA; of phosphatidylglycerol, such as DMPG, DPPG and DSPG; or of phosphatidylinositol, such as DMPI, DPPI or DPPI. Furthermore, modified phospholipids, in particular PEG-modified phosphatidylethanolamines, such as DPPE-PEG or DSPE-PEG, can be used as negatively charged molecules. Also, the lyso- form of the phospholipids mentioned previously, such as lysophosphatidylserine derivatives (e.g., lyso-DMPS, -DPPS, or -DSPS), lysophosphatidic acid derivatives (e.g., lyso-DMPA, -DPPA, or -DSPA), and lysophosphatidylglycerol derivatives (e.g., lyso-DMPG, -DPPG, or -DSPG), Petition 870260035468, dated 04 / 16 / 2026, page 55 / 233 51 / 103 can be advantageously used as negatively charged compounds. Other examples of negatively charged compounds are bile acid salts such as cholic acid salts, deoxycholic acid salts or glycocholic acid salts; and fatty acid salts (C12-C24), preferably (C14-C22) such as, for example, palmitic acid salts, stearic acid salts, 1,2-dipalmitoyl-sn-3-succinylglycerol salts or 1,3-dipalmitoyl-2-succinylglycerol salts.

[00178] Preferably, the negatively charged compound is selected from DPPA, DPPS, DSPG, DPPG, DSPE-PEG2000, DSPE-PEG5000 or mixtures thereof.

[00179] The negatively charged component is typically associated with a corresponding positive indicator ion, which may be mono- (e.g., an alkali metal or ammonium), di- (e.g., an alkaline earth metal), or trivalent (e.g., aluminum). Preferably, the indicator ion is selected from alkali metal cations, such as Na+ or K+, more preferably Na+.

[00180] Examples of phospholipids carrying a net positive charge are derived from ethylphosphatidylcholine, in particular diesters of ethylphosphatidylcholine with fatty acids, such as 1,2-diestearoyl-sn-glycero-3-ethylphosphocholine (Ethyl-DSPC or DSEPC), 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (Ethyl-DPPC or DPEPC). The negative indicator ion is preferably a halide ion, in particular a chloride or bromide ion. Examples of positively charged compounds that can be incorporated into the microbubble envelope are mono-, di-, tri-, or tetra-alkylammonium salts with a halide indicator ion (e.g., chloride or bromide) comprising at least one (C10-C20), preferably (C14-C18), alkyl chain, such as, for example, mono- or distearylammonium chloride, mono- or dihexadecylammonium chloride, bromide of Petition 870260035468, dated 04 / 16 / 2026, page 56 / 233 52 / 103 dimethyldioctadecylammonium (DDAB) or hexadecyltrimethylammonium bromide (CTAB). Additional examples of positively charged compounds that can be incorporated into the microbubble envelope are tertiary or quaternary ammonium salts with a halide indicator ion (e.g., chloride or bromide) comprising one or preferably two (C10-C20), preferably (C14-C18), acyl chains linked to the N atom via a (C3-C6) alkylene bridge, such as, for example, 1,2-diestearoyl-3-trimethylammonium-propane (DSTAP), 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP), 1,2-oleoyl-3-trimethylammonium-propane (DOTAP) or 1,2-diestearoyl-3-dimethylammonium-propane (DSDAP).

[00181] DSEPC, DPEPC and / or DSTAP are preferably used as positively charged compounds in the microbubble envelope.

[00182] The positively charged component is typically associated with a corresponding negative indicator ion, which may be mono- (e.g., halide), di- (e.g., sulfate) or trivalent (e.g., phosphate). Preferably the indicator ion is selected from among the halide ions, such as F- (fluorine), Cl- (chlorine) or Br- (bromine).

[00183] Mixtures of neutral and charged compounds, in particular phospholipids and / or lipids, can be satisfactorily employed to form the microbubble envelope. The amount of charged lipid or phospholipid can vary from approximately 95 mol% to approximately 0.1 mol%, relative to the total amount of lipid and phospholipid, preferably from 80 mol% to 0.5 mol%.

[00184] Preferred mixtures of neutral phospholipids and lipids or charged phospholipids are, for example, DPPG / DSPC, DSTAP / DAPC, DPPS / DSPC, DPPS / DAPC, DPPE / DPPG, DSPA / DAPC, DSPA / DSPC, DSPC / PA (Distearoylphosphatidylcholine / Acid Petition 870260035468, dated 04 / 16 / 2026, page 57 / 233 53 / 103 Palmitic) and DSPG / DSPC.

[00185] Any of the components illustrated above useful for forming the stabilizing envelope of the gas-filled microvesicle, in particular phospholipids, preferably pegylated phospholipids, may be modified by inserting a suitable reactive group therein, in order to allow the binding of suitable compounds, such as a targeting ligand comprising the sequence presented as SEQ ID NO:1 or more preferably a sequence comprising amino acids 1-118 of SEQIDNO:1. For example, a pegylated phospholipid (e.g., DSPE-PEG2000) may comprise a terminal reactive group (e.g., maleimide, abbreviated mal, thus forming a DSPEPEG-mal component) capable of reacting (covalently) with a corresponding reactive group in a compound comprising the preceding sequence. Examples of additional suitable reactive groups are illustrated below in this descriptive report.

[00186] According to an alternative embodiment, the targeting linker component can be associated with air-filled microcapsules. Preferred examples of microcapsules are those having a stabilizing envelope comprising a polymer, preferably a biodegradable polymer or a biodegradable water-insoluble lipid (such as tripalmitin), optionally in a mixture with a biodegradable polymer. Examples of suitable microcapsules and their preparation are disclosed, for example, in US 5,711,933 and US 6,333,021, which are incorporated herein by reference in their entirety. Microcapsules having a protein envelope, i.e., made of natural proteins (albumin, hemoglobin), such as those described in US-A-4,276,885 or EP-A-0 324 938 (incorporated herein) Petition 870260035468, dated 04 / 16 / 2026, page 58 / 233 54 / 103 as a reference), can also be used. The targeting ligand can be incorporated into the microcapsules, for example, by linking it to a microcapsule envelope-forming component, according to the preparation methods illustrated previously, or by mixing an amphiphilic component, such as those illustrated previously, covalently linked to the targeting ligand with the microcapsule envelope-forming components.

[00187] Other excipients or additives may be present in the dry formulation of the microvesicles or may be added along with the aqueous carrier used for their reconstitution, without necessarily being involved (or only partially involved) in the formation of the microvesicle stabilizing envelope. These include pH regulators (such as histidine), osmolarity adjusters, viscosity improvers, emulsifiers, bulking agents, etc., and may be used in conventional amounts. For example, compounds such as polyoxypropylene glycol and polyoxyethylene glycol, as well as copolymers thereof, may be used. Examples of viscosity improvers or stabilizers are selected compounds of linear and cross-linked poly- and oligosaccharides, sugars, and hydrophilic polymers such as polyethylene glycol.

[00188] Since the preparation of gas-filled microvesicles may involve a freeze-drying or spray-drying step, it may be advantageous to include a freeze-drying additive in the formulation, such as a cryoprotective and / or lyoprotective agent and / or a bulking agent, for example, an amino acid such as glycine; a carbohydrate, for example, a sugar such as sucrose, mannitol, maltose, trehalose, glucose, lactose or a cyclodextrin or a polysaccharide such as dextran; or a Petition 870260035468, dated 04 / 16 / 2026, page 59 / 233 55 / 103 polyoxyalkylene glycol such as polyethylene glycol. Typically, the amount of freeze-drying additive can vary from approximately 10 to approximately 1000 times (w / w) the amount of the components that form the microvesicles.

[00189] Any biocompatible gas, gas precursor or mixture thereof may be used to fill the aforementioned microvesicles (also subsequently identified as microvesicle-forming gas).

[00190] The gas may comprise, for example, air; nitrogen; oxygen; carbon dioxide; hydrogen; nitrous oxide; a noble or inert gas such as helium, argon, xenon or krypton; a radioactive gas such as Xe133 or Kr81; a hyperpolarized noble gas such as hyperpolarized helium, hyperpolarized xenon or hyperpolarized neon; a low molecular weight hydrocarbon (for example, containing up to 7 carbon atoms), for example, an alkane such as methane, ethane, propane, butane, isobutane, pentane or isopentane, a cycloalkane such as cyclobutane or cyclopentane, an alkene such as propene, butene or isobutene or an alkyne such as acetylene; an ether; a ketone; an ester; Halogenated gases, preferably fluorinated gases, such as low molecular weight halogenated, fluorinated or perfluorinated hydrocarbons (for example, containing up to 7 carbon atoms); or a mixture of any of the foregoing.When a halogenated hydrocarbon is used, preferably at least some, more preferably all, of the halogen atoms in said compound are fluorine atoms.

[00191] Fluorinated gases, particularly perfluorinated gases, are preferred, especially in the field of ultrasound imaging. Fluorinated gases include materials containing at least one fluorine atom, such as, for example, fluorinated hydrocarbons. Petition 870260035468, dated 04 / 16 / 2026, page 60 / 233 56 / 103 (organic compounds containing one or more carbon and fluorine atoms); sulfur hexafluoride; fluorinated ketones, preferably perfluorinated, such as perfluoroacetone; and fluorinated ethers, preferably perfluorinated, such as perfluorodiethyl ether. Preferred compounds are perfluorinated gases, such as SF6, or perfluorocarbons (perfluorinated hydrocarbons), that is, hydrocarbons in which all hydrogen atoms are replaced by fluorine atoms, which are known to form particularly stable microbubble suspensions, which are disclosed, for example, in EP 0554213, which is incorporated herein by reference.

[00192] The term perfluorocarbon includes saturated, unsaturated and cyclic perfluorocarbons. Examples of physiologically acceptable biocompatible perfluorocarbons are: perfluoroalkanes, such as perfluoromethane, perfluoroethane, perfluoropropanes, perfluorobutanes (e.g., perfluoro-n-butane, optionally in mixtures with other isomers such as perfluoroisobutane), perfluoropentanes, perfluorohexanes or perfluoroheptanes; perfluoroalkenes, such as perfluoropropene, perfluorobutenes (e.g., perfluorobut-2-ene) or perfluorobutadiene; perfluoroalkynes (e.g., perfluorobut-2-yne); and perfluorocycloalkanes (e.g., perfluorocyclobutane, perfluoromethylcyclobutane, perfluorodimethylcyclobutanes, perfluorotrimethylcyclobutanes, perfluorocyclopentane, perfluoromethylcyclopentane, perfluorodimethylcyclopentanes, perfluorocyclohexane, perfluoromethylcyclohexane, and perfluorocycloheptane). Preferred saturated perfluorocarbons include, for example, CF4, C2F6, C3F8, C4F8, C4F10, C5F12 and C6F12.

[00193] It may also be advantageous to use a mixture of any of the previous gases in any proportion. By Petition 870260035468, dated 04 / 16 / 2026, p. 61 / 233 57 / 103 For example, the mixture may comprise a conventional gas, such as nitrogen, air or carbon dioxide, and a gas that forms a stable microbubble suspension, such as sulfur hexafluoride or a perfluorocarbon as previously indicated. Examples of suitable gas mixtures may be found, for example, in WO 94 / 09829, which is incorporated herein by reference. Particularly preferred are the following combinations: a mixture of gases (A) and (B) in which gas (B) is a fluorinated gas, selected from those illustrated previously, which include mixtures thereof, and (A) is selected from air, oxygen, nitrogen, carbon dioxide or mixtures thereof. The amount of gas (B) may represent from approximately 0.5% to approximately 95% v / v of the total mixture, preferably from approximately 5% to 80%.

[00194] Particularly preferred gases are SF6, C3F8, C4F10 or mixtures thereof, optionally mixed with air, oxygen, nitrogen, carbon dioxide or mixtures thereof.

[00195] In certain circumstances it may be desirable to include a precursor for a gaseous substance (i.e., a material that is capable of being converted into a gas in vivo). Preferably, the gaseous precursor and the gas derived from it are physiologically acceptable. The gaseous precursor may be pH-activated, photoactivated, temperature-activated, etc. For example, certain perfluorocarbons can be used as temperature-activated gaseous precursors. These perfluorocarbons, such as perfluoropentane or perfluorohexane, have a liquid / gas phase transition temperature above room temperature (or the temperature at which the agents are produced and / or stored), but below body temperature; thus, they undergo a liquid / gas phase transition and are converted into a gas within the human body.

[00196] For use in MRI, microvesicles will Petition 870260035468, dated 04 / 16 / 2026, page 62 / 233 58 / 103 preferably contain a hyperpolarized noble gas such as hyperpolarized neon, hyperpolarized helium, hyperpolarized xenon or mixtures thereof, optionally in a mixture with air, carbon dioxide, oxygen, nitrogen, helium, xenon or any of the halogenated hydrocarbons defined above.

[00197] For use in scintigraphy, the microvesicle will preferably contain radioactive gases such as Xe133 or Kr81 or mixtures thereof, optionally mixed with air, carbon dioxide, oxygen, nitrogen, helium, krypton or any of the halogenated hydrocarbons defined above. Metal chelating agents for MRI imaging and therapy.

[00198] The most reliable and most frequently applied method of linking a metal ion, which may be the imaging probe or the radiotherapy effector, to a biological molecule such as the chimeric protein of the invention, is through bifunctional chelating agents, which carry the metal chelating cage and a reactive group to covalently link the biological molecule.

[00199] Metallic coordination cages can be divided into cyclic, such as DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) or TETA (1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid) or linear, such as EDTA (ethylenediaminetetraacetic acid) or DTPA (diethylenetriaminepentaacetic acid).

[00200] Once the most suitable metal chelating cage or chelating ligand has been selected, conjugation with the biological molecule of interest via a reactive group can be carried out through solid-phase or solution synthesis. Lattuada L. et al. Comments in Chem Soc. Rev, 2011, 40, 3019-3049 on synthetic approaches to conjugate metal chelating ligands to Petition 870260035468, dated 04 / 16 / 2026, p. 63 / 233 59 / 103 other portions, in particular biomolecules, to prepare chelating agents for target metals.

[00201] According to the modality disclosed in this paragraph, the metal is detectable through an imaging technique or a radionuclide useful for therapy. Metals suitable for imaging specifically include paramagnetic metal ions detectable through Magnetic Resonance Imaging (MRI) or radionuclides detectable through imaging techniques such as Scintigraphy, Single Photon Emission Computed Tomography (SPECT), and Positron Emission Tomography (PET).

[00202] In this context, the terms: chelating agent, chelating ligand or chelating agent comprise groups, agents, compounds or chemical molecules characterized by the presence of polar groups capable of forming a complex containing more than one coordinate bond with a transition metal or other metal. In a preferred aspect of the invention, said chelating ligand includes cyclic or linear polyaminopolycarboxylic or polyphosphonic acids and contains at least one amino, thiol or carboxyl group present as the free or optionally activated functionality, suitable for conjugation of the functional groups of the targeting protein or suitable bifunctional ligands.

[00203] Ligands can be used as spacers or to improve the pharmacokinetic properties of the molecule as a whole. Some of the most frequently used ligands were summarized in Liu S. and Edwards S. Bioconjugate Chem. 2001, 12: 7-34 as well as disclosed for peptide conjugation, in WO2008 / 071679.

[00204] With the term labeled or complexed as used herein, i.e., in the context of a metal-labeled chelating ligand, Petition 870260035468, dated 04 / 16 / 2026, p. 64 / 233 60 / 103 The present inventors refer to a ligand that is complexed with the metal, that is, a ligand that is in the form of a chelated or coordinated complex with the metallic element.

[00205] By the terms metallic entity or metallic element, the present inventors refer to a metallic ion that is detectable by means of an imaging technique or radionuclides for imaging or therapy. The term includes paramagnetic metallic ions detectable by Magnetic Resonance Imaging (MRI) and radiation-emitting metals such as radionuclides, detectable by scintigraphy imaging, Single Photon Emission Computed Tomography (SPECT) and Positron Emission Tomography (PET) or suitable for radiotherapy, as defined below.

[00206] Suitable chelating ligands are selected from the group consisting of: a polyaminopolycarboxylic acid and its derivative comprising, for example, diethylenetriaminepentaacetic acid (DTPA) and its derivative including benzo-DTPA, dibenzo-DTPA, phenyl-DTPA, diphenyl-DTPA, benzyl-DTPA and dibenzyl DTPA, N,N- Bis[2-[(carboxymethyl)[(methylcarbamoyl)methyl]ethyl]-glycine (DTPA-BMA), N-[2-[bis(carboxymethyl)amino]-3-(4-ethoxyphenyl)propyl)]-N-[2[bis(carboxymethyl) amino]ethyl]glycine (EOB-DTPA), 4-carboxy acid5,8,11 -tris(carboxymethyl)-1-phenyl-2-oxa-5,8,11-triazatridecan-13-oic acid (BOPTA), N,N-bis[2-[bis(carboxymethyl)amino]ethyl]L-glutamic acid (DTPA-GLU); DTPA-Lys (see compound 1 of Figure 3a); ethylenediaminetotraacetic acid (EDTA); 1,4,7,10-teraazacyclododecane 1,4,7-triacetic acid (DO3A) and derivatives thereof, including, for example, [10-(2-hydroxypropyl)-1,4,7,10-teraazacyclododecane 1,4,7-triacetic acid (HPDO3A); 1,4,7-triazacyclononane N,N',N-triacetic acid (NOTA); 6-[bis(carboxymethyl)amino]tetrahydro-6-methyl-1H-1,4-diazepine-1,4(5H) Petition 870260035468, dated 16 / 04 / 2026, page 65 / 233 61 / 103 diacetic acid (AAZTA) and derivatives thereof, for example, as disclosed in WO03 / 008390, incorporated herein by reference, 1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetraacetic acid (DOTA) and derivatives thereof including, for example, benzo-DOTA, dibenzo-DOTA, (a,a',a”,a”')-tetramethyl-1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetraacetic acid (DOTMA); or acid 1,4,8,11-tetraazacyclotetradecane-N,N',N,N'-tetraacetic acid (TETA); or corresponding compounds in which one or more of the carboxylic groups are replaced by a phosphonic and / or phosphinic group, which include, for example, N,N'-bis-(pyridoxal-5-phosphate)ethylenediamine-N,N'-diacetic acid (DPDP); ethylenedinitrilotetrakis(methylphosphonic) acid (EDTP), acid 1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetra(methylenephosphonic) (DOTP), macrocyclic phosphonoalkyl-polyaza compounds, for example, disclosed in US 5,362,476 and US 5,409,689 and linear phosphonoalkyl derivatives disclosed in US 6,509,324; or macrocyclic chelating agents such as texaphyrins, porphyrins, phthalocyanines.

[00207] Among those described above, particularly preferred are: DTPA, DTPA-Glu, DTPA-Lys, DOTA and DOTA derivatives, such as those disclosed in Price EW and Orvig, Chem. Soc. Rev. 2014, 43:260 and pyridyl-DO3A disclosed in Hermann et al, Dalton Trans., 2008, 3027-3047 or the multidentate AAZTA binder and its derivatives described, i.e., in WO03 / 008394 and WO2013 / 135750.

[00208] The preferred paramagnetic metallic elements for MRI are those with atomic numbers ranging from 20 to 31, 39, 42, 43, 44, 49 and from 57 to 83.

[00209] Even more preferred paramagnetic metal ions are selected from the following: Fe(2+), Fe(3+), Cu(2+), Ni(2+), Rh(2+), Co(2+), Cr(3+), Gd(3+), Eu(3+), Dy(3+), Tb(3+), Pm(3+), Nd(3+), Tm(3+), Ce(3+), Y(3+), Ho(3+), Er(3+), La(3+), Yb(3+), Mn(3+), Mn(2+; Gd(3+) Petition 870260035468, dated 04 / 16 / 2026, page 66 / 233 62 / 103 being the most preferred. Nuclear Imaging (Imaging with Radionuclides) and Radiotherapy

[00210] In another embodiment of the invention, the group to which the chimeric selectin targeting protein of the present invention is attached is a radionuclide, for obtaining radioimaging (diagnosis) or radiotherapy (therapeutic application).

[00211] The main characteristics of a radiometal chelating group relate to its use in vivo under extremely dilute conditions, i.e., concentrations in nM to pM; however, some of the most suitable combinations between chelating group and radiometal are known and summarized in Price EW and Orvig C Chem. Soc. Rev, 2014, 43, 260.

[00212] For radiographic imaging, the targeting agent can be linked to a detectable cluster for radiographic imaging, that is, a cluster that is detectable through imaging techniques such as scintigraphy, Single Photon Emission Computed Tomography (SPECT) or Positron Emission Tomography (PET).

[00213] Preferably, said detectable group for obtaining radioimaging comprises a radionuclide chelated to a chelating agent that is generally bifunctional and that comprises the chelating group with metal complexation properties and a functional group for binding to the biological molecule, such as the Selectin targeting agent of the present invention or, alternatively, is directly linked to the biological molecule (i.e., iodine).

[00214] Functional groups that form an amide, a thiourea, a urea, a Schiff base or thioether linkages can be prepared Petition 870260035468, dated 04 / 16 / 2026, p. 67 / 233 63 / 103 with amine or thiol groups in the proteins that carry the chelating agent, labeled with a radionuclide detectable through the aforementioned imaging techniques such as scintigraphy, SPECT or PET.

[00215] In any case, the most preferred chelating ligands are linear or more preferably macrocyclic chelating ligands and are those discussed previously, such as DTPA or, more preferably, DOTA which still represents a gold standard for a number of isotopes including 111In, 177Lu, 86 / 90Y, 225Ac and 44 / 47Sc and which was extensively used with 67 / 68Ga, although more recently superseded by the more stable NOTA and DOTA derived from it.

[00216] Additional suitable examples of chelating ligands for radionuclides may be selected from linear, macrocyclic, terpyridine and N3S, N2S2, N2S3, N2S4, N3S3 or N4 chelating ligands which include, for example, the ligands disclosed in US 5,367,080, US 5,364,613, US 5,021,556, US 5,075,099 and US 5,886,142 and other chelating ligands known in the art which include, but are not limited to, 6-Hydrazinopyridine-3-carboxylic acid (HYNIC) or 1,4,8,11-tetra-azacyclotetradecane-N,N',N,N'-tetraacetic acid (TETA); and bis-amino bis-thiol (BAT) chelating agents such as, for example, those disclosed in US 5,720,934 or phospho-derivatives of polyazamacrocyclic compounds, such as those described in WO2005 / 062828.

[00217] N4 chelating ligands are also described, for example, in US 5,608,110, US 5,665,329, US 5,656,254 and US 5,688,487. Certain N3S or N2S2 chelating agents are described, for example, in US 5,659,041, US 5,574,140, ​​US 5,780,006, US 5,662,885 and US 5,976,495. Chelating agents may also include derivatives of the mercapto-acetyl-acetylglycyl-glycine (MAG3) chelating ligand, which contains an N3S and N2S2 system such as MAMA. Petition 870260035468, dated 04 / 16 / 2026, p. 68 / 233 64 / 103 (monoamide monoaminadithiols), DADS (N2S diaminadithiols), CODADS and similar ligand systems. These ligand systems and a variety of others are described in Liu and Edwards, Chem Rev, 1999, 99, 2235-2268 and references cited therein.

[00218] The chelating agent may also include complexes containing ligand atoms that are not donated to the metal in a tetradentate arrangement. These include boronic acid adducts of technetium and rhenium dioximes, for example, described in US 5,183,653, US 5,387,409 and US 5,118,797.

[00219] In another embodiment, the disulfide bonds of the fusion protein or polypeptide of the invention are used as ligands for chelation of a radionuclide such as 99mTc. In this way, the peptide loop is expanded by the introduction of Tc (S-peptide altered to S-peptide-Tc-S-peptide).

[00220] Preferred radionuclides according to the present invention include, for example Example: 99mTc, 51Cr, 67Ga, 68Ga, 47Sc, 167Tm, 141Ce, 111In, 113In, 168Yb, 175Yb, 140La, 90Y, 88Y, 153Sm, 166Ho, 165Dy, 166Dy, 61Cu, 62Cu, 64Cu, 67Cu, 97Ru, 103Ru, 186Re, 188Re, 203Pb, 211Bi, 212Bi, 213Bi, 214Bi, 105Rh, 109Pd, 117mSn, 149Pm, 161Tb, 177Lu, 198Au, 111Ag, 199Au, 51Mn, 52mMn, 52Fe, 60Cu, 72As, 94mTc ou110In,142Pr,159Gd.

[00221] The choice of radionuclide will be based on the desired therapeutic or diagnostic application. For example, for therapeutic purposes (e.g., to deliver radiotherapy to primary tumors and metastases), preferred radionuclides may include 64Cu, 90Y, 105Rh, 111In, 117mSn, 149Pm, 153Sm, 161Tb, 166Dy, 166Ho, 175Yb, 177Lu, 186 / 188Re and 199Au, with 186 / 188Re, 177Lu and 90Y being particularly preferred. For diagnostic purposes (e.g., to locate inflammation and to monitor its development after therapy) preferred radionuclides may include 64Cu, 67Ga, 68Ga, 99mTc and 111In. 99mTc is particularly preferred. Petition 870260035468, dated 04 / 16 / 2026, page 69 / 233 65 / 103 isotope is used for diagnostic applications due to its low cost, availability, imaging properties, and highly specific activity. In particular, the nuclear and radioactive properties of 99mTc make this isotope an ideal scintigraphy imaging agent. This isotope, in fact, has a single photon energy of 140 keV and a radioactive half-life of approximately 6 hours and is readily available in a 99Mo-99mTc generator.

[00222] The preferred metallic radionuclides for use in PET imaging are positron-emitting metal ions such as, for example: 51Mn, 52Fe, 60Cu, 68Ga, 72As, 94mTc or 110In.

[00223] Preferred chelating ligands are para111In and radioactive lanthanides such as, for example, 177Lu, 90Y, 153Sm and 166Ho or para67Ga, 68Ga, 61Cu, 62Cu, 64Cu or 67Cu) selected from the group consisting of: 2 Bioconj. Chem (1999), 10, 137 WO 01 / 46207 HO2C— / \ y—CO2H Bioorg Med Chem Lett (2000), 10: 2133

[00224] In particular, for metallic entities that include111In and Petition 870260035468, dated 04 / 16 / 2026, p. 70 / 233 66 / 103 radioactive lanthanides such as, for example, 177Lu, 90Y, 153Sm and 166Ho, the following ligand residues are particularly preferred: where in formulas a) and b) above, R is alkyl, preferably methyl.

[00225] For the radioactive elements 99mTc, 186Re, 188Re, the following chelating groups d) al) are particularly preferred: Tr Tr Petition 870260035468, dated 04 / 16 / 2026, p. 71 / 233 67 / 103

[00226] These and other metal chelating groups are, for example, described in US 5,608,110, US 6,143,274, US 5,627,286, US 5,662,885, US 5,780,006 and US 5,976,495.

[00227] Additionally, the chelating group of formula c) above is described in US 6,143,274; the chelating groups of formulas h) and i) above are described in US 5,627,286 and US 6,093,382; and the chelating group of formula l) is described in US 5,662,885, US 5,780,006 and Petition 870260035468, dated 04 / 16 / 2026, page 72 / 233 68 / 103 US 5,976,495.

[00228] In formulas h) and i), X is CH2 or O, Y is branched or unbranched C1-C10 alkyl; Y is aryl, aryloxy, arylamino, arylaminoacyl; Y is arylalkyl wherein the alkyl group or groups attached to the aryl group are branched or unbranched C1-C10 alkyl groups, branched or unbranched C1-C10 hydroxy or polyhydroxyalkyl groups, or polyalkoxyalkyl or polyhydroxypolyalkoxyalkyl groups, J is >C(=O), -OC(=O)-, -SO2-, >NC(=O)-, >NC(=S), -N(Y)-, -NC(=NCH3)-, -NC(=NH)-, —N=N-, homopolyamides or heteropolyamines derived from synthetic or naturally occurring amino acids; wherein in all n is 1-100. Folate derivatives of these structures are described, for example, in US 6,093,382.

[00229] Contrast agents for obtaining radioimages comprising one of the above binding residues of a) al) labeled with 99mTc are preferred for scintigraphy as the detectable grouping for imaging. Obtaining images with PET using labeled sugars

[00230] In a further embodiment of the invention, the fusion protein is linked to a labeled sugar group for use in PET Imaging.

[00231] Preferably, the sugar group is labeled by halogenation with radionuclides such as, for example: 1241, 1251, 1311, 123I, 77Br, 76Br and 18F; 18F being particularly preferred. Optical imaging

[00232] In some embodiments, an imaging agent is conjugated to the chimeric protein of the invention. Among optical imaging agents, fluorescent dyes for in vivo, ex vivo and in vitro imaging applications are well known to those skilled in the art, and a wide range of fluorochromes are excellent for obtaining fluorescent images. Petition 870260035468, dated 04 / 16 / 2026, p. 73 / 233 69 / 103 in vivo has been developed and is also commercially available. These reagents maximize, to varying extents, the depth of tissue penetration, the light scattering and fluorescence emission properties of fluorescent chromophores to provide optimal signal-to-background ratios. In general, light absorption and scattering decrease with increasing wavelength; below approximately 700 nm, these effects result in penetration depths smaller than a few millimeters, while above 900 nm water absorption can interfere with the signal-to-background ratio. Therefore, fluorochromes with excitation / emission in the near-infrared (NIR) region (700-900 nm) have been primarily explored so far for in vivo imaging in small animals and, potentially, in humans.

[00233] Among these, the most commonly used are: Indocyanine Green, cyanine derivatives Cy3, Cy3.5, Cy5 and Cy5.5, Cy7 (cyanine dyes and derivatives are available, for example, from GE Healthcare), LS-287, LS-288, IRDye®800CW, IR-820, IR®-806, IR-786, IRDye® 800RS, IRDye®750, IRDye®650 (IRDye®s are available from Li-Cor Bioscience), Alexa Fluor®647, Alexa Fluor®350, Alexa Fluor®405, Alexa Fluor®430, Alexa Fluor®488, Alexa Fluor®514, Alexa Fluor®532, Alexa Fluor®546, Alexa Fluor®568, Alexa Fluor®594, Alexa Fluor®680, Alexa Fluor®750 (Alexa Fluor® dyes are available, for example, from Invitrogen), combinations thereof, and others whose chemical structures have been reported, for example, in WO2014 / 191467.

[00234] The structure of most compounds for image-guided surgery and their commercial availability have been described, for example, in Gibbs SL Quant Imaging Med Surg 2012, 2(3):177-187. Cyanine dyes and their are particularly preferred. Petition 870260035468, dated 04 / 16 / 2026, p. 74 / 233 70 / 103 chemical derivatives developed for NIR imaging, such as: Cy5.5, IRDye®800CW, IRDye® 800RS and IRDye®750. Uses

[00235] The targeted diagnostic agents described above are particularly useful for in vivo and ex vivo imaging. The chimeric targeting protein of the invention can additionally be used for therapeutic purposes, which include any method for treating a disease in a patient wherein the chimeric targeting protein of the invention is used, optionally in association with an imaging agent, to deliver an ex-vivo and / or in vivo therapeutic compound, i.e., a molecule that is capable of exerting or is responsible for exerting a biological effect, on the cell, tissue or organ that expresses selectin.

[00236] Typically, this use involves conjugating the chimeric protein of the invention with an agent / group / molecule possessing biological activity, such as a cytokine, a cystostatic agent (such as doxorubicin, methotrexate, cisplatin, vinblastine, vincristine, etc.), a toxin, an anti-inflammatory agent, an immunomodulator such as a cytokine inhibitor, an antiplatelet agent, a corticosteroid, a monoclonal antibody, a growth factor, and the radiotherapeutic agents described above comprising metal-chelating groups carrying a radionuclide, and is performed to target the biologically active group to the tissue / cell / organ expressing selectin, in which inflammation is observed or detected.

[00237] The pathological inflammatory condition, for diagnostic and / or therapeutic purposes, must be characterized by selectin expression levels above physiological levels; the selectin is preferentially E-selectin and / or P-selectin, more Petition 870260035468, dated 04 / 16 / 2026, p. 75 / 233 71 / 103 preferably P-selectin. In particular, the imaging agents of the invention are useful for detecting inflammatory conditions of the vascular endothelium such as those listed below.

[00238] More preferably, the pathological inflammatory condition is selected from the following, characterized by selectin expression levels above physiological levels: Acute Coronary Syndrome (ACS), Inflammatory Bowel Disease (IBD), Ulcerative Colitis, Crohn's disease, tumor-associated neoangiogenesis, rheumatoid arthritis, ischemia-reperfusion injury, graft rejection or, more generally, any organ or tissue expressing P-selectin and / or E-selectin above physiological levels.

[00239] More preferably, the chimeric protein of the invention is a useful targeting agent for IBD, SCA, tumor detection and graft rejection.

[00240] Furthermore, the targeted imaging agents according to the invention are employed as an efficient diagnostic tool during the (therapeutic) treatment of a patient suffering from an inflammatory disease or pathology, wherein includes any time before the start of treatment, during the course of said treatment and / or at the end of said treatment, to monitor and evaluate the same. For example, the targeted imaging agents of the invention can be advantageously employed in monitoring and / or tracking anti-inflammatory treatment (for example, of any of the diseases or pathologies mentioned above), for example, to determine or evaluate the effects of administering an anti-inflammatory drug or inflammation inhibitor on the disease or pathology.

[00241] For example, in DII, the targeted imaging agents of the invention can be used to track the Petition 870260035468, dated 04 / 16 / 2026, p. 76 / 233 72 / 103 response to therapeutic treatment with, for example, mesalamine, corticosteroids, methotrexate or infliximab and to stratify patients according to their response to therapeutic treatment or to monitor remission maintenance therapy. Preferably, the imaging technique is based on microbubbles directed to the chimeric soluble protein for detection with ultrasound.

[00242] In a preferred embodiment, during treatment a region of interest of the patient is subjected to the chosen image detection system after administration of the targeted imaging agents of the invention, for example, at regular time intervals, at a predetermined time interval after each drug administration or therapeutic intervention and / or after a selected number of drug administrations or treatments; a final image acquisition of the region of interest is then preferably performed at the end or conclusion of the treatment.

[00243] The targeted imaging agents of the invention can be further used in ultrasound-related techniques, i.e., in therapy-associated imaging, where they are advantageously associated with controlled localized destruction of gas-filled microvesicles, for example, by means of high acoustic pressure ultrasound waves (typically higher than that generally employed in non-destructive diagnostic imaging methods). This controlled destruction can be used, for example, for the treatment of blood clots (a technique also known as sonothrombolysis), optionally in combination with the delivery of a suitable therapeutic compound associated with the contrast agent. Alternatively, so-called therapy-associated imaging may include the delivery of a therapeutic agent in Petition 870260035468, dated 04 / 16 / 2026, p. 77 / 233 73 / 103 cells, as a result of transient membrane permeabilization at the cellular level induced by a burst or localized activation of microvesicles. This technique can be used, for example, for efficient delivery of genetic material into cells; alternatively, a drug can be delivered locally, optionally in combination with genetic material, thus allowing for combined pharmaceutical / genetic therapy of the patient (e.g., in the case of tumor treatment). The therapeutic agent can be associated with the gas-filled microvesicle according to conventional methods or can be administered as a separate compound from the composition. In addition, the targeting agent could be used to facilitate the delivery of the therapeutic agent into brain tissue through transient opening of the Blood-Brain Barrier after ultrasound exposure.

[00244] Typically, an effective amount of the targeted diagnostic agent is administered (e.g., by injection) to a patient who needs it, and the part of the patient's body or tissue from which images will be obtained or treated (region of interest) is subjected to the desired imaging method. Preferably, the contrast agent is administered intravenously. The term patient includes any individual (human or animal) who is undergoing administration of the contrast agent for diagnostic / therapeutic or experimental purposes (which include, for example, the use of a contrast agent in laboratory animals, for example, to monitor an experimental therapeutic treatment).

[00245] According to a preferred embodiment, an efficient quantity of targeted microvesicles is administered to a patient, typically by injection of a suspension of Petition 870260035468, dated 04 / 16 / 2026, page 78 / 233 74 / 103 same. Obtaining images of the region of interest will thus be enhanced by the presence of microvesicles bound to the target in the region of interest.

[00246] A variety of imaging techniques can be employed in ultrasound applications, for example, including fundamental and nonlinear (e.g., harmonic) B-mode imaging, pulsed or phase-inversion imaging, and fundamental and nonlinear Doppler imaging; if desired, three- or four-dimensional imaging techniques can be used. In addition, diagnostic techniques involving the destruction of gas-filled microvesicles (e.g., through high-pressure acoustic ultrasound waves) are also considered, as they are highly sensitive detection methods.

[00247] The microvesicles according to the invention can typically be administered at a concentration of approximately 0.01 to approximately 5.0 μL of gas (captured within the microvesicles) per kg of patient, depending, for example, on their respective composition, the tissue or organ from which images will be obtained, and / or the chosen imaging technique. This general concentration range can obviously vary depending on the specific imaging applications, for example, when signals can be observed at very low doses such as in color Doppler or pulse power inversion. Other possible diagnostic imaging applications include scintigraphy, optical imaging, photoacoustic imaging, magnetic resonance imaging, and X-ray imaging, which includes X-ray phase contrast imaging.

[00248] Ultrasound imaging methods Petition 870260035468, dated 04 / 16 / 2026, p. 79 / 233 75 / 103 can also be conveniently used in conjunction with MRI as mentioned earlier for combined (or fusion) imaging methods to achieve better mapping of the affected lesion. Pharmaceutical compositions

[00249] The invention further comprises pharmaceutical compositions comprising the previously disclosed chimeric protein, conjugated or modified according to the intended uses, which can be administered topically or parenterally, including intranasal, subcutaneous, intramuscular, intravenous, intra-arterial, intra-articular or intralesional administration. Ordinarily, intravenous (iv), intra-arterial, intra-articular, intracardiac administration is preferred.

[00250] The molecules of the present invention, such as the active ingredient, are dissolved, dispersed, or mixed in a diluent or excipient that is pharmaceutically acceptable and compatible with the active ingredient, as is well known in the art. Suitable excipients are, for example, water, saline solution, phosphate-buffered saline (PBS), dextrose, glycerol, ethanol, or similar substances and combinations thereof. Other suitable carriers are well known to those skilled in the art. In addition, if desired, the composition may contain smaller amounts of auxiliary substances such as wetting or emulsifying agents, stabilizing and / or pH buffering agents.

[00251] The pharmaceutical compositions according to the present invention can be manufactured through, for example, conventional processes of mixing, dissolving, granulating, grinding, pulverization, dragee production, levigation, emulsification, encapsulation, capture or lyophilization.

[00252] Pharmaceutical compositions for use according to Petition 870260035468, dated 04 / 16 / 2026, page 80 / 233 76 / 103 The present invention can thus be formulated using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate the processing of the active compounds into preparations that can be used pharmaceutically. The appropriate formulation depends on the chosen route of administration.

[00253] For injection, the compounds of the invention can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution or physiological saline buffer, additionally comprising suitable excipient or stabilizing compounds.

[00254] Oral administration can be achieved through liquid or solid compositions. Among the latter, dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, varnish solutions and suitable organic solvents or solvent mixtures. Dyes or pigments may be added to the tablets or dragee coatings for identification or to characterize different dose combinations of active compounds.

[00255] Solid compositions administered orally include snap-fit ​​capsules made of gelatin as well as soft-sealed capsules made of gelatin and a plasticizing agent, such as glycerol or sorbitol. Snap-fit ​​capsules may contain the active ingredients mixed with a filling such as lactose, binding agents such as starches, lubricants such as talc or magnesium stearate, and optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or Petition 870260035468, dated 04 / 16 / 2026, p. 81 / 233 77 / 103 liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration must be in dosages appropriate for the chosen route of administration. For buccal administration, the compositions may take the form of conventionally formulated tablets or lozenges.

[00256] In one embodiment according to the present invention, the peptides are administered orally (for example, in the form of a syrup, a capsule, or a tablet). In certain embodiments, the delivery of the peptides can be enhanced through the use of protective excipients. This is typically achieved by complexing the peptide with a composition to make it resistant to acid and enzymatic hydrolysis or by packaging the peptide in an appropriately resistant carrier such as a liposome. The means of protecting peptides for oral delivery are well known in the art.

[00257] Compressed tablets can be prepared by compressing the active peptide(s) in a free-flowing form such as a powder or granules in a suitable machine, optionally mixed with a binding agent (e.g., povidone, gelatin, hydroxypropyl methylcellulose), a lubricant, an inert diluent, a preservative, a disintegrating agent (e.g., sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose), a surfactant, or a dispersing agent. Molded tablets can be made by molding a mixture of the powdered peptide(s) moistened with an inert liquid diluent in a suitable machine. The tablets can optionally be coated or cut and can be formulated to provide slow or controlled release of the active ingredient contained therein, for example, hydroxypropyl methylcellulose in varying proportions to provide the Petition 870260035468, dated 04 / 16 / 2026, page 82 / 233 78 / 103 desired release profile.

[00258] A syrup can be made by adding the active peptide(s) to a concentrated aqueous solution of a sugar, for example, sucrose, to which any necessary ingredients may also be added. Such accessory ingredients may include flavorings, an agent to retard sugar crystallization, or an agent to increase the solubility of any other ingredients, such as a polyhydric alcohol, for example, glycerol or sorbitol.

[00259] For administration by inhalation, the variants for use according to the present invention are conveniently provided in the form of an aerosol spray presentation from a pressurized container or a nebulizer using a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a measured quantity. Capsules and cartridges of, for example, gelatin can be formulated for use in an inhaler or insufflators containing a powder mixture of the peptide and a suitable powder base such as lactose or starch.

[00260] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active ingredients in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily suspensions for injection. Suitable natural or synthetic carriers are well known in the art. The suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds or the stability of the chimeric protein or conjugate group, to allow the preparation of concentrated solutions. Petition 870260035468, dated 04 / 16 / 2026, page 83 / 233 79 / 103 Alternatively, the active ingredient may be in powder form for reconstitution with a suitable vehicle, for example, sterile, pyrogen-free water, prior to use.

[00261] The compounds of the present invention can also be formulated in rectal compositions such as suppositories or retention enemas, using, for example, conventional suppository bases such as cocoa butter or other glycerides.

[00262] The high half-life in serum can be maintained through the use of sustained-release protein or lipids in the form of packaging systems. Such sustained-release systems are well known to those skilled in the art and may comprise the formulation of the chimeric protein as is or in a conjugated form in nanospheres, nanovesicles, or liposomes.

[00263] The above formulations and methods of administration are intended to be illustrative and not limiting. It will be considered that, using the teaching provided here, other suitable formulations and modes of administration can be easily developed.

[00264] Pharmaceutical compositions suitable for use in the context of the present invention include compositions in which the active ingredients are contained in an amount efficient to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of a compound effective in preventing, delaying, alleviating, or improving the symptoms of a disease in the individual to be treated. Determining a therapeutically effective amount is well within the capabilities of those skilled in the art according to, for example, Goodman & Gilman, 9th ed., J.G. Hardman, A. Gilman, L.E. Limbird, Chapter I Pharmacokinetics, pp. 3-27.

[00265] Consequently, the present invention comprises Petition 870260035468, dated 04 / 16 / 2026, page 84 / 233 80 / 103 still methods for administering the previously disclosed chimeric protein as is, or preferably in the form of a conjugate, to persons who need it for therapeutic or, preferably, diagnostic purposes.

[00266] For diagnostic purposes, the pharmaceutical compositions of the invention can be pre-administered in an appropriate dosage, depending on the route of administration and the sensitivity of the diagnostic method, and then images are obtained using the most suitable technique. EXPERIMENTAL PART Example 1: Preparation and expression of recombinant fusion proteins tagged with a flag

[00267] A number of DNA constructs were prepared:

[00268] PSGL Variant 1: the sequence encodes amino acids 1-47 of the mature PSGL-1 protein, the hinge region of IgG1, the leucine zipper domain of NRL, a glycine spacer (G4SG4), and a FLAG sequence for affinity recognition. Mouse IgH signal peptide was used for secretion. The chimeric protein of Variant 1 has the amino acid sequence of SEQIDNO:2.

[00269] PSGL variant 2, which encodes amino acids 1-47 of the mature PSGL-1 protein, an IgG1 hinge region, an IgG1CH3 region with K->A sequence substitutions. Mouse IgH signal peptide was used for secretion. A FLAG sequence (SEQIDNO:35) at the C-terminus was used for purification purposes. The chimeric protein has SEQIDNO:4.

[00270] PSGL variant 3: which encodes amino acids 1-47 of the mature PSGL-1 protein, covalently linked to the hinge region of IgG1, to the 275-290 region of the mature PSGL-1 protein and to a FLAG sequence (SEQIDNO:35) at the C-terminus, used for the Petition 870260035468, dated 04 / 16 / 2026, p. 85 / 233 81 / 103 identification and purification purposes. Mouse IgH signal peptide was used for secretion. The chimeric protein has SEQIDNO:6.

[00271] PSGL Variant 4 encodes amino acids 1-47 of the mature PSGL-1 protein, covalently linked to the IgG1 hinge region and to amino acids 1-15 of the human IgG1 Fc region. Mouse IgH signal peptide was used for secretion. A C-terminus FLAG sequence (SEQIDNO:35) was used for purification purposes. The chimeric protein has SEQIDNO:8.

[00272] PSGL Variant 5 encodes amino acids 1-88 of the PSGL-1 protein (according to GI:2498904) with the endogenous signal and propeptide sequence covalently linked to the IgG1 hinge region and amino acids 1-15 of the human IgG1 Fc region. A C-terminus FLAG sequence (SEQIDNO:35) for affinity recognition was used for purification purposes. The chimeric protein has SEQIDNO:10. Small-Scale Transient Transfection

[00273] CHO-S, a Chinese hamster ovary cell line adapted for suspension (Freedom™ CHO-S™ Kit, GIBCO ThermoFisher Scientific) was cultured according to the manufacturer's instructions (July 2015) in a 5% CO2 humidified incubator at 37°C in chemically defined CD-CHO medium (Invitrogen, Carlsbad CA, Catalog # 12490-025, Lot # 1149771) supplemented with L-glutamine (Cellgro, Catalog # 61-030-RO, Lot # 61030158). Neither serum nor other animal-derived products were used in the culture of the CHO-S cells. 24 hours before transfection, cells were inoculated into a shaker flask and grown using serum-free chemically defined medium. Variant 1-5 expression constructs (250 μg of each plasmid) were transiently transfected into CHO cells. Petition 870260035468, dated 04 / 16 / 2026, page 86 / 233 82 / 103 in 0.05 liter suspension by electroporation. Briefly: 250 x 106 CHO cells were transfected using a Maxcyte Electroporator with 50% EP buffer and a QC400 cuvette. The cells were grown using serum-free medium for seven days before collection.

[00274] A Western blot performed on collection demonstrated the relative expression of PSGL-1 variants. Specific Western blot analysis for FLAG labeling in conditioned medium under non-reducing conditions confirmed protein dimerization. Variant 1 in conditioned medium exhibited the expected molecular weight of ~22 kDa under reducing conditions (Figure 1, Lane 2). This correctly formed a dimer under non-reducing conditions (Figure 1, Lane 7). The high molecular weight bands in bands 1-5 are most likely molecules non-specifically bound to anti-FLAG antibody (Anti-FLAG antibody, mouse monoclonal IgG1, Sigma-Aldrich, catalog # F1804).

[00275] In the case of band 3 (Variant 2), the bands with lower molecular weight could be a degradation product.

[00276] Western blotting in conditioned medium was performed with an antibody that recognizes the Flag epitope located at the C-terminus of the variants and is therefore indicative of the relative amount of different constructs of the chimeric protein secreted in the medium. Purification of FLAGGED Variants

[00277] Purification was performed via ion-exchange purification and anti-FLAG affinity purification. For each of the five variants, after clarification via 0.2 μM filtration, the conditioned medium was adjusted to pH 6 by adding 1 M HCl, and the volume was doubled with distilled water. The CM was then loaded onto a 1 mL anion-exchange chromatography column (Q column, GE) and eluted in 20 mM Tris pH 7.5, 1 M HCl. The elution fraction was applied to 0.5 mL of M2 affinity resin. Petition 870260035468, dated 04 / 16 / 2026, page 87 / 233 83 / 103 anti-FLAG resin was stirred at room temperature for 5 hours. The resin was washed with 10 mL of Tris-buffered saline. The protein was eluted with five volumes of the 100 pg / mL FLAG peptide column in Tris-buffered saline. For PSGL Variant 1, the eluted flow was re-incubated with antiFLAG M2 resin overnight at 4°C and elution was achieved using 0.25% acetic acid pH 3.5.

[00278] The silver-stained SDS-PAGE and DO220 analysis of purified PSGL Variants 1-5 suggest that Variant 1 has the highest levels of purity and expression among the five constructs.

[00279] The protein concentration in DO220, calculated by creating a standard curve with serial dilutions of bovine serum albumin and fitting the data from the PSGL variants to this curve, is provided below in Table 3. Table 3: Recovery of protein variants 1-5 after ion-exchange and affinity purification. Variant 1 Variant 2 Variant 3 Variant 4 Variant 5 Quantity (mg) 0.41 0.07 0.02 0.02 0.03

[0280] Among the five constructs, PSGL Variant 1 had the best yields: the highest expression level and resulting purity, at least one log higher, compared to the other variants. Both Western blot and silver-stained SDS-PAGE under reducing and non-reducing conditions suggested that PSGL Variant 1 underwent dimerization correctly. Example 2: Expression and purification of Variant 1A (without FLAG).

[00281] Stable CHO-S cell transformants, co-expressing the DNA sequence encoding core 2 beta-1,6-N-acetylglucosaminyltransferase (C2GnT-M), FTVII (fucosyltransferase) Petition 870260035468, dated 04 / 16 / 2026, page 88 / 233 84 / 103 VII) (Fugang Li et al. J. Biol. Chem, 1996, 271:3255-3264) and the chimeric protein encompassing aa 1-118 of SEQIDNO:1 without the FLAG sequence, were then produced according to the Freedom™ CHO-STM Kit Manual (Cat.N A13696-01 Lifescience Thermo Fisher Scientific, July 2015).

[00282] CHO-S clones were allowed to grow for at least 7 days, generally up to 14 days, using OptiCHO™ medium (other chemically defined whey-free media have been successfully used, e.g., ActiCHO™, CD FortiCHO™ and similar) and in the absence of selection pressure. Glutamine (or the analogue GlutMax) was supplemented at 1-10 mM, preferably 4-8 mM.

[00283] 100 mL of the supernatant from a set of 6 clones of Stable carbohydrates were collected, filtered through a 0.2 µm PES membrane, and loaded onto a Capto Q strong anion exchange column (GE 175316-02, 1.6 x 6 cm, 12 mL) previously equilibrated with 20 mM Tris-HCl pH 7.5.

[00284] The bound proteins were eluted with a linear gradient up to 1 M NaCl across 8 volumes of the column. The eluted fractions containing the target protein, also shown by SDS-PAGE analysis, were used for a second purification step by hydrophobic interaction chromatography of Phenyl.

[00285] The column was washed with 1 M NaOH, then equilibrated and stored at 4°C in 20% EtOH.

[00286] NaCl was dissolved in the pooled fractions to a concentration of 4 M. This pool was then loaded onto a 1 mL HIC column (HiTrap Phenyl HP™, GE Healthcare Life Sciences, Catalog # 17-1351-01) previously equilibrated with 20 mM TrisHCl pH 7.5, 4 M NaCl, according to the manufacturer's instructions for hydrophobic interaction chromatography. Elution was Petition 870260035468, dated 04 / 16 / 2026, p. 89 / 233 85 / 103 achieved by linearly decreasing the sodium chloride concentration to zero in 10 column volumes. Fractions containing the target protein, as shown by SDS-PAGE analysis, were pooled for a third size exclusion chromatography (SEC) step. The SEC column (HiLoad 16 / 600 Superdex 200™ pg, GE Healthcare Life Sciences, Catalog # 289893-35) was pre-equilibrated with 20 mM Tris-HCl pH 7.5, 150 mM NaCl, which was used as the mobile phase. Fractions containing the chimeric PSGL Variant 1, as shown by SDS-PAGE analysis, were pooled and concentrated (Amicon Ultra Centrifugal Filter Unit, EMD Millipore).

[00287] As a more sensitive alternative to SDS-PAGE, Western Blot analysis was performed with Phast-System (GE) as described in the instruction manual. The nitrocellulose membrane was saturated with PBS + 1% BSA (1 h at room temperature). The membrane was incubated 1 h at room temperature with PBS + 0.5% Triton X-100 + 1% BSA containing an anti-P-selectin Glycoprotein Ligand-1 antibody 1:1000 (anti-P-Selectin Glycoprotein Ligand-1 Antibody, clone KPL-1, EMD Millipore, cod. MAB4092).

[00288] After 3 washes with PBS + 0.5% Triton X-100, the membrane was incubated with anti-mouse IgG - HRP as the secondary antibody.

[00289] After 3 washes, the HRP signal was revealed with ECL, showing positive recognition of the anti-PSGL-1 antibody.

[00290] The chimeric PSGL variant 1A, from different subclones, appeared as a single band at approximately 60 kDa, which was altered to approximately 30 kDa under reducing conditions, as expected. No additional bands or degradation products were visible on Coomassie-stained SDS-PAGE gels of the purified product. Petition 870260035468, dated 04 / 16 / 2026, p. 90 / 233 86 / 103

[00291] One clone was selected for further subcloning, medium-scale growth, and purification on a 2 L culture medium scale. Example 3: Characterization of the purified Variant 1A

[00292] The recombinant protein of PSGL Variant 1A is highly heterogeneous due to different post-translational modifications, including sulfation and both N- and O-glycosylation. For its complete characterization and accurate quantification, a set of analytical methods was performed, including UPLC, MS, and peptide mapping procedures with specific enzymes. The structural characteristics of PSGL Variant 1A, essential for its biological activity, were also carefully monitored. 3.1. UPLC-UV

[00293] Reverse-phase chromatography was used to determine the target protein content and to quantify possible impurities or degradation products. For reverse-phase chromatography experiments, an Acquity UPLC BEH300 column (2.1x100 mm, 1.7 pm) at 50 °C was used in gradient mode. The mobile phases were (A) 0.1% TFA in water and (B) 0.1% TFA in acetonitrile. UV detection was performed at 216 nm. The use of UPLC technology combined with a dedicated sub 2 Dm column allowed for higher resolution, greater sensitivity, excellent peak shape, and a significant reduction in analysis times. The UPLC-UV method showed that the purity of PSGL Variant 1A achieved by the purification process was greater than 90%. 3.2. SEC-UV

[00294] Size exclusion chromatography (SEC), also called Gel Permeation Chromatography (GPC), coupled with UV detection, which allows the separation of proteins based on their size or effective shape (hydrodynamic radius), was used. Petition 870260035468, dated 04 / 16 / 2026, page 91 / 233 87 / 103 In this case, the method was used to measure possible aggregates and other size variants. For the size exclusion chromatography experiments, a TSK gel super SW mAb HTP 4.6x150 mm (Tosoh), 4 µm column was used at 30°C in isocratic mode. The mobile phase was 50 mM NaH2PÜ4, 50 mM Na2HPÜ4, 100 mM Na2SÜ4, and UV detection was performed at 216 nm. The use of UPLC instrumentation optimized to reduce extra-column band amplification allowed for high column efficiency and greater sensitivity. The SEC-UV method was applied to the analysis of the A-1 variant of PSGL: no aggregation was observed. 3.3. Sialic Acid Content

[00295] Sialylation is well known as a critical characteristic for the bioavailability, stability, metabolism, and immunogenicity of biopharmaceutical products. For this purpose, an HPLC MS method was developed for the determination of sialic acid from Variant 1A of the chimeric protein.

[00296] The analysis was based on a combination of chemical hydrolysis coupled with non-derivatization liquid chromatography interfaced with electrospray ionization tandem mass spectrometry (LC-MS / MS). Sialic acid (NAcetylneuraminic acid NANA) was first released from PSGL Variant 1A through acid hydrolysis under mild acidic conditions. Once the sialic acid was released, its quantification was performed using the LC-MS / MS method. The amount of sialic acid was determined by comparing the response in the sample with a standard reference calibration curve. In experiments performed with purified protein from other CHO subclones, sialylation was typically observed to be between 9.6 and 17.9% w / w. 3.4. MALDI-TOF-MS Petition 870260035468, dated 04 / 16 / 2026, page 92 / 233 88 / 103

[00297] Matrix-Assisted Laser Desorption-Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) ionization analyses were also performed to determine the molecular weight of PSGL Variant 1A. This technique involves mixing the sample with a matrix, which is then coated onto a plate or probe and subjected to a collimated focused laser beam, causing ionization and desorption. MALDI has the advantage of producing large mass ions with high sensitivity and little fragmentation. MALDI-TOF experiments were performed on PSGL Variant 1A in both intact and reduced forms. As determined by MALDI experiments, the average molecular weight of PSGL Variant 1A was measured at approximately 35.4 kDa. DTT reduction led to a 2-fold reduction in the mass of PSGL Variant 1A, indicating the dimeric nature of the purified protein. 3.5. Peptide Mapping (Enzymatic Digestion)

[00298] Peptide mapping (PMAP) was used to elucidate post-translational modifications (PTMs) of the chimeric glycoprotein, which include sulfation and N- and O-glycosylation. For this purpose, both chymotrypsin and Asp-N were applied for protein digestion, followed by LC-MS Liquid Chromatography-Mass Spectrometry. 3.5.1. Fragmentation with Chymotrypsin

[00299] Chymotrypsin fragmentation was performed following the protocol provided by the manufacturer (Chymotrypsin Endoproteinase MS Grade, Cat.N. 90056, Thermo Scientific). One vial of dried chymotrypsin was reconstituted in 25 µL of 0.1 M HCl and stored at -18 °C before use in the form of 2 µL aliquots in 500 µL Eppendorf tubes. To prepare the digestion buffer, consisting of 100 mM Tris-HCl pH (8), 10 mM CaCb, 3.03 g Tris Base (M 121.4 g / mol) and 368 mg CaCl2 (M 147.02 g / mol) were dissolved in Petition 870260035468, dated 04 / 16 / 2026, page 93 / 233 89 / 103 water. The pH was adjusted to 8.0 using 1.2 M HCl and the volume was made up to 250 mL.

[00300] Fifty (50) pL of a stock solution of PSGL-1 Variant 1A (0.5-1.0 mg / mL) were added to 2 pL of chymotrypsin and 48 pL of digestion buffer. The solution was incubated for 18 hours at 37 °C before injection into the LC-MS. 3.5.2. Fragmentation with Asp-N endoproteinase

[00301] Fragmentation with Asp-N endoproteinase was performed following the protocol provided by the manufacturer (Asp N sequencing grade Roche, Cat. N. 11054589001). One vial of dry Asp-N was reconstituted in 50 µL of water and stored at -18 °C before use in the form of 5 µL aliquots in 500 µL Eppendorf tubes. To prepare the digestion buffer, consisting of 50 mM Sodium Phosphate, 1.5 g of Na2HPO4 (M 119.98 g / mol) was dissolved in water. The pH was adjusted to 8.0 using 1 mL of NaOH and the volume was made up to 250 mL.

[00302] Ten (10) pL of a stock solution of PSGL-1 Variant 1A (0.5-1.0 mg / mL) were added to 5 pL of chymotrypsin and 35 pL of digestion buffer. The solution was incubated for 18 hours at 37 °C before injection into the LC-MS.

[00303] Similar LC-MS analyses were used for the characterization of the digestion products of Asp-N and chymotrypsin. These experiments were performed using a Waters Acquity® UPLC system consisting of a temperature-controlled sample manager, a binary solvent controller, and a heated column compartment. The column output was connected directly to the mass spectrometer. The analytical column was an Agilent® Poroshell® 120 EC-C18 2.1x 150 mm id, 2.7 µm particle size at 40°C. Elution of the compounds from the column was performed in gradient mode using a mobile phase consisting of water. Petition 870260035468, dated 04 / 16 / 2026, page 94 / 233 90 / 103 ultrapure with 0.1% TFA and acetonitrile with 0.1% TFA. The flow rate and injection volume were set at 0.3 mL / min and 10 DL, respectively. Detection was performed on a tandem quadrupole mass spectrometer equipped with a Waters Xevo TQ-S electrospray deionization source in positive and negative ionization modes according to the compound. Nitrogen and argon were used as nebulization and collision gases, respectively. Analyses were performed in scanning mode. Data acquisition and processing were performed using the MassLynx software package.

[00304] All previous tests have led to the following conclusions:

[00305] · the N-terminal structure of the Variant 1A glycoprotein PSGL-1 is dominated by the pyroglutamic form pQATEYEYL.

[00306] · The dimeric characteristic of PSGL Variant 1A was confirmed by the presence of (M+2H)+2= 728.5 assigned to the sequence (50TCPPCPL56)2. Ions corresponding to the monomeric form of this peptide were not detected, suggesting that PSGL Variant 1A is entirely in dimeric form.

[00307] · the structure of the O-glycan residues in Thr16 was confirmed: in fact, the expected Sialyl-Lewis-X motif, a Core 2 structure comprising N-Acetylgalactosamine, N-Acetylglucosamine, galactose, fucose and sialic acid was identified.

[00308] · Sulfation of Tyrosines 5, 7 and 10 was also demonstrated by mass spectrometry in negative scanning mode.

[00309] Most post-translational modifications (PTMs) were reported in SEQIDNO:38. Example 4: Affinity binding to P-selectin by Surface Plasmon Resonance (SPR) Petition 870260035468, dated 04 / 16 / 2026, p. 95 / 233 91 / 103

[00310] The experiments were performed with the purified protein Fr1 as described in Example 2 of WO2012 / 020030 and Variant 1A as described in Example 2 above.

[00311] The binding strength of PSGL Variant 1A was evaluated by Surface Plasmon Resonance (SPR) using a Biacore X100 from GE Healthcare Bio-Sciences AB (General Electric, Piscataway, NJ). SPR technology allows for real-time, label-free monitoring of the binding process between biological molecules. While P-selectin (target ligand) was bound to the surface of a sensor chip, proteins of interest circulated in solution over the surface using a microfluidic system to ensure reproducible sample delivery and low sample consumption. SPR detects binding events as changes in mass on the chip surface. As a result, a sensogram is generated by graphically representing the SPR response against time.

[00312] First, biotinylation of P-selectin / Fc (R&D Systems Europe Ltd) was performed in-house. Then, immobilization of P-selectin / Fc onto the SA Chip analysis sensor (Fc2) (Streptavidin chip, GE Healthcare Bio-Sciences) was performed as follows: A volume of 10x HBS-N buffer (GE Healthcare BioSciences AB) (50 mL) was diluted with 9 volumes of Milli-Q water (450 mL). Five hundred microliters of 1.5 M CaCl2 (Fluka), pH approximately 5.6, were added to the diluted buffer (1.5 mM final) and filtered through a 0.2 µm PES filter. The running buffer was prepared with 48.75 mL of HBS-N, 1.5 mM CaCl2 buffer, and 1.250 mL of P20 surfactant (GE Healthcare Bio-Sciences AB) in a 50 mL Falcon tube. Any unused HBS-N buffer and 1.5 mM CaCl2 buffer were stored at +4°C.

[00313] Each test sample was diluted in running buffer. Petition 870260035468, dated 04 / 16 / 2026, page 96 / 233 92 / 103 to achieve the indicated concentration of 125 nM. A manual run was initiated on the reference sensor (Fc1) and Fc2 with a flow rate of 30 pL / min. The 2-1 reference subtraction was selected. Once the baseline was stable, the analyte was injected for 30 s or longer if binding equilibrium had not been reached. To directly compare the binding of different analytes or different concentrations of the same analyte, it was necessary to program the same cycle parameters for each sample, for example: Wait 120 seconds Injection 30 s Wait 300 seconds

[00314] When the manual run was finished, the Biacore X100 Evaluation software version 1.0 was opened to create a coverage of each cycle.

[00315] Based on SPR experiments, the binding affinity for P-selectin was measured and the binding strength of the chimeric proteins to the target was found to be equivalent to or better than the binding of Fr1. Figure 2 shows the result of the Biacore run, in which the chimeric protein of the present invention (dashed line) showed greater binding to the P-selectin ligand compared to Fr. 1.

[00316] SPR was also used for titration of Variant 1A in cell supernatant, according to the method described in Chou TH. et al. Cytokine 51, 2010, 107-111. The sensor chip was coated with streptavidin and a biotinylated mouse α-PSGL-1 antibody (KPL-1, Abcam, cat. N. ab78188) in Bracco, used to quantify Variant 1A in the supernatant. Example 5: Preparation of Variant 1A-SMCC for conjugation with phospholipid

[00317] The process was carried out in accordance with example 9 of Petition 870260035468, dated 04 / 16 / 2026, page 97 / 233 93 / 103 WO2012 / 020030.

[00318] Briefly, Variant 1 (65 nmoles) of Example 2 was dissolved in 1 mL of 0.2 M Phosphate buffer pH 7.5 with 1 mM EDTA. A solution of Sulfo-SMCC (Sulfo-SMCC: 4-[N-maleimidomethyl]cyclohexane-1-carboxylate sulfosuccinimidyl) (Pierce) (55 mg / mL - 125 mM) was prepared in anhydrous Dimethyl Sulfoxide (DMSO, Fluka) and 52 µL of the solution were added to the Variant 1 solution. The solution was incubated at room temperature for 45 minutes. The solution was then centrifuged through a centrifuge column (Zeba spin-column 5 mL, Pierce # 89890) equilibrated in 20 mM phosphate buffer pH 6. Isopropanol was added to the solution to obtain a solution containing 35% isopropanol. Example 6: Preparation of the DSPE-PEG-SH conjugate

[00319] DSPE-PEG2000-SH (DSPE: PEG2000 modified distearoyl phosphatidylethanolamine) was prepared starting from DSPE-PEG2000-PDP (1,2-Diestearoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate (polyethylene glycol)-2000] ammonium salt) according to example 10 of WO2012 / 020030. The final DSPE-PEG2000-SH solution was diluted with isopropanol to obtain a solution containing 35% isopropanol. Example 7: Preparation of the DSPE-PEG-SH conjugate / Variant 1ASMCC

[00320] 1.7 mL of the Variant 1A-SMCC solution (65 nmoles) obtained in example 5 was added to a DSPEPEG2000-SH solution (325 nmoles - 5 equivalents) obtained in example 6. The solution was incubated for three hours at room temperature with agitation (spinning wheel).

[00321] The solution was then purified by anion-exchange chromatography with an ANX Sepharose gel (GE Healthcare). The solution containing the purified Variant 1 conjugate was centrifuged through Petition 870260035468, dated 04 / 16 / 2026, page 98 / 233 94 / 103 of a centrifuge column (Zeba spin column 10 mL, Pierce #89893) balanced in 20 mM TRIS buffer pH 7.5. Example 8: Preparation of microvesicles with the conjugates from Example 7.

[00322] 10 mg of a mixture of DSPC (DSPC: Distearolphosphatidylcholine) and palmitic acid (molar ratio of 80 / 20) were dissolved in cyclooctane (0.8 mL) at 70 °C.

[00323] Separately, the DSPE-PEGSH / Variant 1-SMCC conjugate solution prepared according to Example 7 (28 nmoles - 1.3 mL) was added to 8.7 mL of the 10% PEG4000 solution in distilled water. A DPPE-PEG5000 solution (DPPE: dipalmitoylphosphatidylethanolamine), (0.85 mg in 85 µL of water) was added to this aqueous phase.

[00324] The previously prepared organic and aqueous solutions were mixed using a high-speed homogenizer (Polytron PT3000) to obtain an emulsion. The resulting emulsion was heated with stirring at 60 °C for one hour and then cooled to room temperature (approximately 22 °C).

[00325] The emulsion obtained was diluted twice with a 10% PEG4000 solution in distilled water and sampled into DIN8R vials (0.5 mL emulsion / vial). The vials were frozen at -50 °C for 1 hour (Lyobeta 35 freeze dryer - TELSTAR), then freeze-dried at -20 °C and 0.2 mbar for 12 h. The lyophilized product was then exposed to an atmosphere of a mixture of perfluoro-n-butane and nitrogen (35 / 65 v / v) and the vials were sealed.

[00326] The product was dispersed in a volume of 150 mM saline solution (1 mL / vial) with gentle manual mixing. Example 9: In vitro binding activity of targeted microvesicles in a flow chamber setting. Petition 870260035468, dated 04 / 16 / 2026, p. 99 / 233 95 / 103

[00327] To test effective binding, targeted microvesicles prepared as described in WO2012 / 020030 (Example 6) and those prepared according to the present invention (Example 8) were injected into a flow chamber setup comprising a coating of mouse Fc P-Selectin (catalog number 737-PS, R&D Systems, Minneapolis, MN, USA) or human Fc P and E selectins (R&D catalog numbers 137-PS 50 and 724 ES 100) at 4 pg / mL. Microvesicles (equivalent to 80x106 / 400 pL of TBS++) were injected along the flow chamber (FCS2, Bioptech, USA) in cake form and their adhesion to the mouse P-selectin coating layer (or human P- or E-selectin) was evaluated over a period of 10 min at a flow rate of 1.0 mL / min (shear rate of 714 s-1) in the presence of 50% (v:v) human plasma (Stehelin & Cie AG) in TBS.A quantitative analysis of microvesicle accumulation was performed by counting the number of microvesicles adhering to the observed area at 2-minute intervals throughout the total 10-minute infusion, using the Analysis FIVE image processing program (SIS, Germany). After 10 minutes, five photos were randomly taken, and the number of bound microvesicles was evaluated and expressed as the number of bound bubbles (NBB) in 10 minutes. Each observed area measured 183 x 137 μm, which was measured with the aid of a stage micrometer. The measurement was taken between the center and the outlet of the chamber.

[00328] Similarly, targeted microvesicle suspensions prepared according to Example 8 (Variant 1A as targeting ligand) were injected into a flow chamber as previously described and their binding activity was determined according to the previous procedure.

[00329] The experiment was repeated on E-selectin coatings. Petition 870260035468, dated 04 / 16 / 2026, pp. 100 / 233 96 / 103 human and mouse P-selectin at the same concentration as above (4 μg / mL). The results are shown in Table 4. Table 4: Number of Microvesicles Bound in 10 min (NBM 10 min) NBM 10 min Preparation Mouse P selectin Human E selectin Human P selectin microbubble Fr-1 74.30 ± 4.27 54.1 ± 7.23 77.0 ± 4.83 microbubble Variant 1A 73.60 ± 5.87 53.00 ± 4.37 65.40 ± 4.93

[00330] As can be inferred from the previous results, the binding behavior of the newly prepared MB-PSGL-1 Variant 1A is similar to that of the microbubble carrying Fr 1: they exhibit firm binding without aggregation in plasma and bind to both human and mouse P-selectin as well as human E-selectin. Example 10: In vivo experiments of microvesicles with Fragment 1 (comparative) and Variant 1A

[00331] Microvesicles with Variant 1A prepared according to Example 8 above and those carrying Fragment 1 (Fr-1) prepared according to WO2012 / 020030, Example 6, were compared in a rat inflammation model. Inflammation was induced in the rat hind limb by injection of lipopolysaccharide (LPS, 0.26:B6 Sigma L-8274, 2.1 mg / kg). The effective binding of targeted microvesicles was assessed by contrast-enhanced ultrasound imaging 24 h after induction of the inflammatory process. Contrast-enhanced ultrasound imaging was performed using a Logiq E9 ultrasound scanner (General Electric Healthcare, Fairfield, Connecticut, USA) equipped with a 9L linear transducer (transmission frequency, 4.0 MHz (Res); dynamic range, 48 dB; depth, 20 Petition 870260035468, dated 04 / 16 / 2026, pp. 101 / 23397 / 103 mm; Time Gain Compensation (TGC), linear) operating in contrast mode. One image per second was recorded at a low mechanical index (MI=0.06) for 30 seconds, then one image every 15 seconds for up to ten minutes. Ten minutes after a single dose injection, the contrast-enhanced signal was collected for 10 seconds at a cadence of 4 Hz. The contrast-enhanced images were recorded as DICOM files and analyzed using dedicated software developed in the laboratory (VueBox, Bracco Suisse SA, Geneva, Switzerland). This software allows for quantitative analysis of the echographic signal after linearization of the log-compressed video sequences at the pixel level and provides contrast echo-power amplitude (expressed in the form of an arbitrary unit, AU) within an area of ​​interest (AOI).The fixed bubble imaging (FBI) algorithm integrated into the software developed in Bracco (VueBox commercial kit), which helps detect bound bubbles, was applied to the frames recorded over 10 minutes. FBI processing is based on a minimum intensity projection function applied to a subset of images that is dependent on the window length (40 frames). The resulting images (Figure 3) showed that the microbubble carrying Variant 1A is able to visualize inflammation in the rat's inflamed paw, and the observed signal is very similar to that observed with microbubbles carrying Fr-1. Example 11: Preparation of fluorescent liposomes with the conjugates from Example 7.

[00332] Cholesterol (28.25 mg - Merck #3672) was dissolved in 1 mL of chloroform. DSPE-PEG2000 (13.9 mg - Genzyme #LP-R4-039) was dissolved in 1 mL of chloroform. DiR (1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindotricarbocyanine Iodide - Molecular Probes #D12731) was dissolved in ethanol to obtain a 10 mg / mL solution. Petition 870260035468, dated 04 / 16 / 2026, page 102 / 233 98 / 103

[00333] DSPC (79.7 mg - 101 pmoles - Genzyme #LP-04-013) was weighed into a 100 mL rounded flask and dissolved in 30 mL of chloroform / methanol mixture (2 / 1 v / v). Samples of cholesterol solution (575 pL - 42 pmoles), DSPEPEG2000 solution (290 pL - 1.5 pmol), and DiR solution (100 pL - 1 pmol) were added to the DSPC solution. The resulting solution was stirred at 65°C for 10 min, and the solvents were removed under reduced pressure to obtain a lipid mixture. This mixture was dried at 65°C under 20 mmHg and then overnight at 25°C under 0.2 mBar.

[00334] The dried lipid mixture was again dispersed in 10 mL of 20 mM Tris buffer (pH 7.4) at 70°C with stirring (rotary evaporator) for 20 min. The resulting suspension was then extruded at 70°C several times through Nuclepore filters (1x1 µm, 1x0.6 µm and 4x0.4 µm). This extrusion step could be adapted to obtain various liposome sizes.The liposome suspension was then cooled to room temperature and stored at 4°C in the dark.

[00335] The incorporation of the conjugated variant was performed via a post-insertion procedure. Briefly, 500 pL of the liposome suspension were mixed with 1 mL of the Variant 1A conjugate solution prepared in Example 7 (22 nmoles) at room temperature for 16 hours in the dark. The resulting liposome suspension was used without purification for in vivo experiments.

[00336] Fr-1 liposomes were obtained using the same procedure described above, replacing the Variant 1A conjugates with Fr-1 conjugates (prepared as described in W02012 / 020030, Example 6). The characteristics of the two liposome suspensions were compared in Table 6.

[00337] The size and Zeta potential of liposomes were determined using a ZetaZSP Nanosizer (Malvern instruments). Petition 870260035468, dated 04 / 16 / 2026, page 103 / 233 99 / 103 The density of the ligand per liposome was determined after washing the liposomes (centrifugation 20000g / 30min) by determining the sialic acid content (according to example 3.3). Table 5 - Characteristics of control liposomes, Variant 1A and Fr-1 Liposome Size (nm) Zeta Potential (mV) Ligand Molecules / Liposome No post-insertion 268 -6.4 - Variant 1A conjugate post-insertion 278 -39.1 780 Fr-1 conjugate post-insertion 291 -34.7 700 Example 12. Optical imaging experiments using liposomes with Fragment 1 (comparative) and Variant 1A in the mouse LPS model.

[00338] Liposomes carrying Variant 1A were compared with liposomes carrying Fragment 1 in two groups of animals with hind limb inflammation. Inflammation was induced in the mouse hind limb by intramuscular injection of lipopolysaccharide (LPS, 0.26:B6 Sigma L-8274). Optical imaging was performed using the preclinical Fluobeam 700 system. The optical tip was positioned above the animal so that its hind limbs were centered in the camera's field of view (distance between the mouse and the camera 15 cm). Twenty-four hours after the induction of the inflammatory process, the targeted liposomes were injected and the fluorescent signal was monitored over time (up to 24 h). The injection and initial phase were recorded as a sequence with one image every 5 seconds for 16 minutes (as the time of Petition 870260035468, dated 04 / 16 / 2026, page 104 / 233 100 / 103 fixed exposure), followed by individual images at 20, 30, 45 min, 1, 2, and 4 hours. The images were analyzed using ImageJ software. In all capture frames, part of the area of ​​interest (AOI) was pulled to highlight the inflamed and contralateral paws. The fluorescence intensity per millisecond was calculated in the AOI of each image. The fluorescent images are presented in Figure 4, and the quantification results expressed as the ratio (Inflamed paw divided by contralateral paw) are presented in Table 7. The fluorescent images recorded 2 hours after injection of Variant 1A-liposomes-DIR showed a higher signal in the inflamed paw compared to the signal in the contralateral paw. In the case of animal 3, the signal observed in the inflamed paw is low, probably due to low LPS-induced inflammation in this mouse. Conversely, animal 6 shows a high signal indicative of high inflammation.The calculated ratio of inflamed paw to contralateral paw is higher for Variant 1A compared to that for Fragment-1 (Fr-1), indicating good agent specificity. Table 6: Relationship of the inflamed paw to the contralateral paw in the mouse model with LPS inflammation, 2h after injection of Variant A1-liposomes-DIR or Fr-1-liposomes-DIR Ratio of inflamed paw / contralateral paw (2h) Mouse N. Variant 1A Mouse N. Fr-1 1 13.2 7 4.3 2 23.0 8 6.0 3 3.9 9 3.5 4 18.0 10 4.1 5 6.6 11 4.7 6 12.8 12 5.3 Petition 870260035468, dated 04 / 16 / 2026, page 105 / 233 101 / 103 Average 12.9 4.7 SD 5.4 7.9 Example 13. Optimization of purification conditions by HA chromatography (negative or positive). Purification by AE / HI and HA (negative)

[00339] 600 mL of conditioned medium were filtered through a 0.22 μm membrane and loaded onto an AEX column (Capto Q™, GE, 2.6 x 7 cm, 37 mL) equilibrated with 20 mM Tris pH 7.5. Bound proteins were eluted by two-step elution at 30% and 100% with 20 mM Tris, 1 M NaCl, pH 7.5. The target protein was eluted in the 100% fraction. The column was washed with 1 M NaOH and stored at 4°C in 20% EtOH. The conductivity of the conditioned medium was less than 10 mS / cm.

[00340] Solid NaCl was added to the sample from the 100% elution step of the AEX purification to a final concentration of 4 M. The sample was split into two parts to avoid overloading the column. This was then loaded onto a hydrophobic interaction chromatographic column (Phenyl Sepharose™, GE, 1.6 x 9 cm, 18 mL volume) previously equilibrated with 20 mM Tris, 4 M NaCl, pH 7.5. Bound proteins were eluted in two steps at 50% and 100% of 20 mM Tris pH 7.5. The target protein was eluted in the 50% fraction.

[00341] After the second round, the column was washed with 0.2 M NaOH and stored at 4°C in 20% EtOH.

[00342] The groups from the first elution step in the two HIC runs were combined and the phosphate / CaCl2 concentrations were adjusted to 10 and 0.3 mM, respectively, by adding 500 mM phosphate pH 6.8 and 1 M CaCl2. The group was then diafiltered against 10 mM phosphate, 0.3 mM CaCl2, pH 6.8 in an Amicon™ (Merck) stirred ultrafiltration cell, fitted with a YM10 membrane, with nominal molecular weight limit. Petition 870260035468, dated 04 / 16 / 2026, page 106 / 233 102 / 103 of 10 kDa). Then, the diafiltrated group was loaded onto a Hydroxyapatite column (BioRad, 2.2 x 5 cm, 19 mL volume) previously equilibrated with 10 mM Phosphate, 0.3 mM CaCl2, pH 6.8. Variant 1A of PSGL was eluted in the FT fraction (negative chromatography).

[00343] The FT fraction was concentrated using an Amicon™ Centrifugal Filter Unit, according to the manufacturer's instructions. The purified protein was frozen at -40°C. The final concentration was 0.89 mg / mL for a total yield of 38.7 mg (73%) and a purity of 99.7% (Figure 5).

[00344] Residual DNA and protein contaminants were measured after each chromatographic step using a DNA quantification assay (DNA Quantitation Kit, Fluorescence Assay, Sigma, detection limit 2 mg / L) and RP-HPLC (purity vs other proteins) respectively.

[00345] The values ​​are summarized in the following table: Table 7: Purity and residual DNA content after 3-step chromatography Purification Step of PSGL Variant 1A Purity by RP-HPLC Residual DNA Content Anion Exchange (AE) <58% 97% Hydrophobic Interaction (HI) 72.6% 2% Hydroxyapatite (HA) 99.3% Below the detection limit Purification by AE / HI and HA (positive)

[00346] 1000 mL of Conditioned Medium were filtered through a 0.22 µm membrane and loaded onto an AE column (Capto Q™, GE, 2.6 x 7 cm, 37 mL) equilibrated with 20 mM Tris pH 7.5. Bound proteins were eluted by two-step elution at 30% and 100% of 20 Petition 870260035468, dated 04 / 16 / 2026, page 107 / 233 103 / 103 mM Tris, 1 M NaCl, pH 7.5. The target protein was eluted in the fraction at 100%. The column was washed with 1 M NaOH and stored at 4°C in 20% EtOH.

[00347] Solid NaCl was added to the sample from the 100% elution step of the AE purification to a final concentration of 4 M. The sample was split into two parts to avoid overloading the column. This was then loaded onto a hydrophobic interaction chromatographic column (Phenyl Sepharose™, GE, 1.6 x 9 cm, 18 mL volume) previously equilibrated with 20 mM Tris, 4 M NaCl, pH 7.5. Bound proteins were eluted in two steps at 50% and 100% of 20 mM Tris pH 7.5. The target protein was eluted in the 50% fraction.

[00348] After the third round, the column was washed with 0.2 M NaOH and stored at 4°C in 20% EtOH.

[00349] The groups from the first elution step of each of the HIC runs were combined and diluted 4 times with water. Then, the diluted group was loaded onto a Hydroxyapatite column (BioRad, 2.2 x 5 cm, 19 mL volume) previously equilibrated with 5 mM Phosphate, 1 mM MgCl2, pH 6.8. Bound proteins were eluted with a 0-12.5% ​​gradient of 500 mM phosphate buffer. The PSGL Variant 1A was eluted as the first peak.

[00350] The fractions containing the target protein were concentrated using an Amicon Centrifugal Filter Unit (Ultracel-10 membrane, 10 kDa MWCO), according to the manufacturer's instructions.

[00351] The purified protein was frozen at -40°C. The final concentration was 1.09 mg / mL for a total yield of 25 mg, corresponding to approximately 70% of the total target protein content and a purity of 98.7%. Petition 870260035468, dated 04 / 16 / 2026, page 108 / 233

Claims

1 / 5 CLAIMS 1.A dimeric protein, characterized in that it comprises two recombinant chimeric P-Selectin Glycoprotein Ligand-1 (PSGL-1) proteins, wherein the monomeric recombinant chimeric PSGL-1 protein comprises at least: a selectin-binding domain capable of specifically binding to a selectin protein and comprising at least amino acids 1-47 of SEQ ID NO: 11 (mature PSGL-1 sequence), a leucine zipper domain capable of forming a right-handed α-helix, thereby promoting dimerization through protein-protein interaction and comprising amino acids 187-208 of SEQ ID NO: 12 (Neural Retina-specific leucine zipper), and a disulfide bond promoter region comprising at least one cysteine ​​available to form a disulfide bond with another cysteine ​​in a chimeric monomeric protein counterpart, such that the two chimeric protein monomers are covalently linked to each other. another by at least one disulfide bond.

2. Dimeric protein comprising two recombinant chimeric PSGL1 proteins, according to claim 1, characterized in that said leucine zipper comprises amino acids 181-215 of SEQ ID NO:

12.

3. A dimeric protein comprising two recombinant chimeric PSGL1 proteins according to claim 1 or 2, characterized in that said disulfide bond promoter region comprises an amino acid sequence defined by the following general formula: (X1)nC(X2)m-(X3) wherein - X1, X2 represent any amino acid or amino acid sequence excluding cysteine ​​(Cys), Petition 870260035468, dated 04 / 16 / 2026, page 109 / 233 2 / 5 - C is Cys - X3 is any amino acid, and - n, m are integers from 1 to 6.

4. A dimeric protein comprising two recombinant chimeric PSGL1 proteins, according to claim 3, characterized in that: - X1 preferably comprises a Proline, a Histidine or a Threonine; - n is at most 5, and - X2 comprises at least one Proline.

5. Dimeric protein comprising two recombinant chimeric PSGL1 proteins, according to claim 4, characterized in that: - X2 is Pro-Pro; - X3 comprises a Cysteine ​​and at least one Proline.

6. Dimeric protein comprising two recombinant chimeric PSGL1 proteins, according to claim 5, characterized in that said disulfide bond promoter region is a hinge region of IgG1, of SEQ ID NO:

20.

7. Dimeric protein comprising two recombinant chimeric PSGL1 proteins, according to any one of claims 1 to 6, characterized in that it further comprises a polyglycine spacer comprising at least one lysine (Lys or K) or one cysteine ​​(Cys or C).

8. Dimeric protein comprising two recombinant chimeric PSGL1 proteins, according to claim 7, characterized in that said spacer is SEQ ID NO:

17.

9. Dimeric protein, according to any one of claims 1 to 8, characterized in that it is a homodimer. Petition 870260035468, dated 16 / 04 / 2026, page 110 / 233 3 / 5 10. A dimeric protein comprising two recombinant chimeric PSGL-1 proteins, according to any one of claims 1 to 8, characterized in that the monomeric recombinant chimeric PSGL-1 protein further comprises a signal peptide sequence suitable for secretion and for removal by cleavage prior to secretion.

11. Dimeric protein comprising two recombinant chimeric PSGL-1 proteins, according to claim 10, characterized in that said signal peptide is selected from the group consisting of: SEQ ID NO: 18 and SEQ ID NOs: 21-34.

12. Dimeric protein comprising two recombinant chimeric PSGL-1 proteins, according to claim 10 or 11, characterized in that said signal peptide is the mouse IgH signal peptide, of SEQ ID NO:

18.

13. DNA sequence, characterized in that it encodes the monomeric recombinant chimeric PSGL-1 protein comprised within the dimeric protein, as defined in any one of claims 1 to 8 and 10 to 12, wherein the monomeric recombinant chimeric PSGL-1 protein comprises a selectin-binding domain capable of specifically binding to the selectin protein and comprising at least amino acids 5-16 of SEQ ID NO: 11 (sequence of mature PSGL-1), a leucine zipper domain capable of forming a right-handed α-helix, thus promoting dimerization through protein-protein interaction and comprising amino acids 187-208 of SEQ ID NO: 12 (Neural Retina-specific leucine zipper), and a disulfide bond promoter region comprising at least one cysteine ​​available to form a disulfide bond with another cysteine ​​in a monomeric chimeric protein counterpart, wherein the DNA sequence encoding the protein Petition 870260035468, dated 04 / 16 / 2026, page.111 / 233 4 / 5 chimeric recombinant monomeric PSGL-1 is selected from SEQ ID NOs: 1 and 36.

14. Eukaryotic expression vector, characterized in that it comprises the DNA sequence as defined in claim 13.

15. Dimeric protein, according to any one of claims 1 to 12, characterized in that it is homodimeric, O-linked glycosylated at least at Thr at position 16 and sulfated at least at Tyr at positions 5, 7 and 10 of SEQ ID NO:

11.

16. Conjugated compound, characterized in that it comprises the recombinant dimeric protein, as defined in any one of claims 1 to 12 or 15, and a diagnostic or therapeutic group.

17. Conjugate, according to claim 16, characterized in that the diagnostically useful group is selected from the group consisting of: a radioactive label, an enzyme, a fluorescent label, a luminescent label, a metal chelating compound, a gas-filled lipid microvesicle and a combination of the diagnostically active groups.

18. Conjugate, according to claim 17, characterized in that the lipid of the gas-filled microvesicle is a phospholipid.

19. A device, according to any one of claims 16 to 18, characterized in that it is for use in an imaging process selected from the group consisting of: ultrasound, magnetic resonance imaging, optoacoustics, scintigraphy, Single Photon Emission Computed Tomography (SPECT), Positron Emission Tomography (PET), X-ray, acoustics and radiofrequency optics.

20. Pharmaceutical compositions, characterized by the fact that they comprise the dimeric recombinant chimeric protein, as defined in any one of claims 1 to 12 or 15, or the conjugates, as defined in any one of claims 16 to 19.

21. Process for preparing the dimeric recombinant protein, as defined in any one of claims 1 to 12 or 15, characterized in that it comprises transforming a eukaryotic cell with the DNA sequence, as defined in claim 13, or the vector, as defined in claim 14, to obtain a recombinant system that stably expresses the chimeric protein, collecting the culture medium and purifying the recombinant protein from said culture medium. Petition 870260035468, dated 04 / 16 / 2026, pp. 113 / 233

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