MAdCAM-1 POLYPEPTIDES AND USES THEREOF

MAdCAM-1 polypeptides are provided. Accordingly there is provided a polypeptide comprising an amino acid sequence of MAdCAM-1 capable of binding alpha4beta7 integrin, wherein said polypeptide is in association with a therapeutic or a detectable moiety. Also provided are methods of treating and diagnosing inflammation mediated by MAdCAM-1.

SEQUENCE LISTING STATEMENT

The XML file, entitled 94381SecondReplacementSequenceListing.xml, created on Jul. 10, 2023, comprising 82,682 bytes, submitted concurrently with the filing of this application is incorporated herein by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to MAdCAM-1 polypeptides and uses thereof.

Mucosal Vascular Addressin Cell Adhesion Molecule 1 (MAdCAM-1) is a cell-surface immunoglobulin (Ig) superfamily member composed of two extracellular Ig-like domains, followed by a mucin-like domain, a transmembrane domain, and a short cytoplasmic domain. The murine MAdCAM-1 comprises an additional Ig-like domain. MAdCAM-1 specifically binds the lymphocyte homing receptor α4β7 integrin.

The α4β7 integrin has several ligands, including MAdCAM-1, fibronectin and vascular cell adhesion molecule-1 (VCAM-1). Like other integrins, the functionality of α4β7depends on its conformational state12. That is, integrins change conformation upon specific stimuli (e.g. chemokines) which induces a specific “inside-out signaling” that changes the integrin's inactive conformation (a low-affinity bent conformation) into its active form, characterized by a high-affinity (HA) extended conformation which may be distinct for different ligands13. Thus, whether integrin α4β7has HA for MAdCAM-1 or e.g. VCAM-1 depends on the stimulus received; and while a large percentage of circulating lymphocytes express α4β7, only a fraction of these cells utilize a conformation enabling binding to MAdCAM-1.

MAdCAM-1 has been shown to be expressed at sites of lymphocyte extravasation. In particular, MAdCAM-1 expression was reported in vascular endothelial cells of mucosal tissues, including gut-associated tissues or lymphoid organs (e.g. Peyer's patches, Mesenteric lymph nodes and venules of the lamina propria of the small and large intestine) and the lactating mammary gland. MAdCAM-1 expression is upregulated on endothelia, especially high endothelial venules (HEV), in a variety of chronic inflammatory diseases, and may mediate increased leukocyte trafficking into inflamed tissues. Consequently, MAdCAM-1 binding to α4β7 was shown to mediate the progression of e.g. inflammatory bowel disease (IBD), Type I diabetes, chronic inflammatory liver disease and a chronic progressive form of EAE. IBD (such as ulcerative colitis and Crohn's disease), for example, is a chronic disease involving inflammation of the gastrointestinal tract. IBD is a growing problem with rising incidence since the 19thcentury, affecting an estimated two million people in the United States alone. Symptoms include abdominal pain, cramping, diarrhea and rectal bleeding. Current IBD treatments includes anti-inflammatory drugs (such as, corticosteroids and sulfasalazine), immunosuppressive drugs (such as, 6-mercaptopurine, cyclosporine and azathioprine), anti-TNFα and surgery (such as, colectomy).

Antibodies against α4β7 integrin or MAdCAM-1 and inhibitory polypeptides that interfere with α4β7 integrin binding to MAdCAM-1 in conformational sensitive or insensitive manner have been suggested in the art (see e.g. U.S. Pat. Nos. 7,147,851, 6,037,324, 8,277,808, US patent application Publication No. US20070202097; Yang et al. Scand J Immunol. 1995; 42: 235-47 and Qi et al. J Biol Chem. 2012 May 4; 287(19):15749-59).

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present invention there is provided a polypeptide comprising an amino acid sequence of MAdCAM-1 capable of binding α4β7 integrin, wherein the polypeptide is in association with a therapeutic moiety, wherein the therapeutic moiety is not a constant region of an antibody.

According to an aspect of some embodiments of the present invention there is provided a targeted particle comprising a therapeutic and/or a detectable moiety, the particle is attached to a polypeptide comprising an amino acid sequence of MAdCAM-1.

According to some embodiments of the invention, the amino acid sequence of MAdCAM-1 does not comprise a functional Mucin-like domain.

According to some embodiments of the invention, the amino acid sequence of MAdCAM-1 is more than 5 amino acids in length.

According to some embodiments of the invention, the therapeutic moiety is attached to the polypeptide via a linker.

According to some embodiments of the invention, the therapeutic moiety is attached to-, or encapsulated in a particle.

According to some embodiments of the invention, the particle is a lipid particle.

According to some embodiments of the invention, the therapeutic moiety is a polynucleotide, a small molecule or a polypeptide.

According to some embodiments of the invention, the therapeutic moiety is an RNA silencing agent.

According to some embodiments of the invention, the therapeutic moiety downregulates expression and/or activity of a pro-inflammatory cytokine.

According to an aspect of some embodiments of the present invention there is provided a method of treating a disease associated with inflammation mediated by MAdCAM-1 in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the polypeptide or the particle, thereby treating the disease associated with inflammation mediated by MAdCAM-1.

According to an aspect of some embodiments of the present invention there is provided the polypeptide or the particle, for use in treating a disease associated with inflammation mediated by MAdCAM-1.

According to some embodiments of the invention, the disease is inflammatory bowel disease (IBD).

According to an aspect of some embodiments of the present invention there is provided a polypeptide comprising an amino acid sequence of MAdCAM-1 capable of binding α4β7 integrin, wherein the polypeptide is in association with a detectable moiety, wherein the amino acid sequence of MAdCAM-1 is more than 5 amino acids long and does not comprise a functional Mucin-like domain.

According to some embodiments of the invention, the detectable moiety is attached to the polypeptide via a linker.

According to some embodiments of the invention, the MAdCAM-1 is human MAdCAM-1.

According to some embodiments of the invention, the amino acid sequence of MAdCAM-1 comprises an amino acid sequence of a first Ig-like domain (D1) located N to C in a full length MAdCAM-1.

According to some embodiments of the invention, the amino acid sequence of MAdCAM-1 comprises an amino acid sequence of a second Ig-like domain (D2) located N to C in a full length MAdCAM-1.

According to some embodiments of the invention, the amino acid sequence of MAdCAM-1 comprises an amino acid sequence of a first Ig-like domain (D1) and a second Ig-like domain (D2) located N to C in a full length MAdCAM-1.

According to some embodiments of the invention, the amino acid sequence of MAdCAM-1 comprises (SEQ ID NO: 21).

According to some embodiments of the invention, the amino acid sequence of MAdCAM-1 comprises SEQ ID NO: 22.

According to an aspect of some embodiments of the present invention there is provided a method of diagnosing inflammation mediated by MAdCAM-1 in a subject in need, the method comprising:(a) administering to the subject the polypeptide or the particle; and(b) determining an amount of the polypeptide or the particle in a suspected inflamed tissue of the subject following the administering,

wherein an amount above a predetermined threshold is indicative of presence of inflammation mediated by MAdCAM-1 in the tissue of the subject.

According to an aspect of some embodiments of the present invention there is provided a method of monitoring efficacy of treatment against inflammation mediated by MAdCAM-1 in a subject in need, the method comprising:(a) administering to the subject the polypeptide or the particle following the treatment; and(b) determining an amount of the polypeptide or the particle in a suspected inflamed tissue of the subject following the administering,

wherein a decrease in the amount following the treatment beyond a predetermined threshold is indicative of reduction in inflammation and efficaciousness of the treatment.

According to some embodiments of the invention, the method comprising treating the subject with the treatment prior to the (a).

According to some embodiments of the invention, the tissue is a gut tissue.

According to some embodiments of the invention, the inflammation is associated with inflammatory bowel disease (IBD).

According to some embodiments of the invention, the polypeptide comprises an amino acid sequence of a constant region of an antibody.

According to some embodiments of the invention, the constant region of an antibody is an Fc domain or a fragment thereof.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to MAdCAM-1 polypeptides and uses thereof.

Mucosal Vascular Addressin Cell Adhesion Molecule 1 (MAdCAM-1) is a cell-surface immunoglobulin (Ig) superfamily member which specifically binds the lymphocyte homing receptor α4β7 integrin. The α4β7 integrin has several ligands, including MAdCAM-1, fibronectin and vascular cell adhesion molecule-1 (VCAM-1). Like other integrins, the functionality of α4β7depends on its conformational state. Thus, whether integrin α4β7has high affinity (HA) for MAdCAM-1 or e.g. VCAM-1 depends on the specific stimulus received; and while a large percentage of circulating lymphocytes express αα4β7, only a fraction of these cells utilize a conformation enabling binding to MAdCAM-1.

Whilst reducing specific embodiments of the present invention to practice, the present inventors have conceived a novel strategy to targeted delivery of therapeutic or detectable moieties to cells expressing α4β7 integrin in a conformation-sensitive manner, using a polypeptide comprising MAdCAM-1 amino acid sequence as a targeting moiety.

As is illustrated hereinunder and in the examples section, which follows, the present inventors generated a recombinant protein comprising the two Ig-like domains (D1 and D2) of MAdCAM-1 fused to an FC domain of an antibody attached to a lipid nanoparticle (LNP) encapsulating an IFNγ siRNA as a therapeutic moiety or attached to a microPET/CT detectable moiety (64Cu) (Examples 1-3 of the Examples section which follows). Using these compositions the present inventors were able to knockdown IFNγ in a selective subset of leukocytes expressing the HA α4β7and induce a therapeutic effect in an inflammatory bowel (IBD) disease mouse model (Example 3 of the Examples section which follows); and to analyze the biodistribution of the composition using microPET/CT (Example 1 of the Examples section which follows).

Consequently, specific embodiments of the present invention suggest compositions comprising MAdCAM-1 polypeptides in association with therapeutic or detectable moieties and their use in treating, diagnosing and monitoring treatment efficacy in conditions associated with inflammation mediated by MAdCAM-1.

Thus, according to an aspect of the present invention, there is provided a polypeptide comprising an amino acid sequence of MAdCAM-1 capable of binding α4β7 integrin, wherein said polypeptide is in association with a therapeutic moiety, wherein said therapeutic moiety is not a constant region of an antibody.

According to an aspect of the present invention, there is provided a polypeptide comprising an amino acid sequence of MAdCAM-1 capable of binding α4β7 integrin, wherein said polypeptide is attached to a therapeutic moiety, wherein said therapeutic moiety is not a constant region of an antibody.

According to an additional or an alternative aspect of the present invention, there is provided a polypeptide comprising an amino acid sequence of MAdCAM-1 capable of binding α4β7 integrin, wherein said polypeptide is in association with a detectable moiety, wherein said amino acid sequence of MAdCAM-1 is more than 5 amino acids long and does not comprise a functional Mucin-like domain.

According to an aspect of the present invention, there is provided a polypeptide comprising an amino acid sequence of MAdCAM-1 capable of binding α4β7 integrin, wherein said polypeptide is attached to a detectable moiety, wherein said amino acid sequence of MAdCAM-1 is more than 5 amino acids long and does not comprise a functional Mucin-like domain.

As used herein, the term “polypeptide” or “peptide” encompasses native peptides (either degradation products, synthetically synthesized peptides or recombinant peptides) and peptidomimetics (typically, synthetically synthesized peptides), as well as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into cells. Such modifications include, but are not limited to N terminus modification, C terminus modification, peptide bond modification, backbone modifications, and residue modification. Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, C. A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which is incorporated by reference as if fully set forth herein.

The term “amino acid” or “amino acids” is understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine. Furthermore, the term “amino acid” includes both D- and L-amino acids.

When referring to “an amino acid sequence” the meaning is to the chemical embodiment of the term and not the literal embodiment of the term.

As used herein the term “MAdCAM-1” (also known as addressin and mucosal vascular addressin cell adhesion molecule 1) refers to the polypeptide of the MADCAM1 gene (Gene ID 8174). According to specific embodiments, the MAdCAM-1 is the mouse MAdCAM-1, such as provided in the following Accession Nos. BAA23364, NP_001345714, NP_038619 (SEQ ID NO: 23-25). According to specific embodiments, the MAdCAM-1 is the human MAdCAM-1, such as provided in the following Accession Nos. NP_570116, NP_570118 (SEQ ID NO: 26-27).

As used herein, the phrase “an amino acid sequence of MAdCAM-1” refers to full length MAdCAM-1 or a fragment thereof or a homolog thereof which maintains at least the α4β7 integrin binding capability of the full length MAdCAM-1.

The homolog (naturally occurring or synthetically/recombinantly produced) can be, for example, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical or homologous to the polypeptide provided in SEQ ID NO: 23-27 or a functional fragment thereof which exhibit the desired activity (i.e., at least binding a α4β7 integrin); or at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the polynucleotide sequence encoding same.

Sequence identity or homology can be determined using any protein or nucleic acid sequence alignment algorithm such as Blast, ClustalW, and MUSCLE.

The homolog may also refer to an ortholog, a deletion, insertion, or substitution variant, including a conservative and non-conservative amino acid substitution, as further described hereinbelow.

According to specific embodiments, the amino acid sequence of MAdCAM-1 may comprise conservative and/or non-conservative amino acid substitutions.

The term “conservative substitution” as used herein, refers to the replacement of an amino acid present in the native sequence in the peptide with a naturally or non-naturally occurring amino or a peptidomimetics having similar steric properties. Where the side-chain of the native amino acid to be replaced is either polar or hydrophobic, the conservative substitution should be with a naturally occurring amino acid, a non-naturally occurring amino acid or with a peptidomimetic moiety which is also polar or hydrophobic (in addition to having the same steric properties as the side-chain of the replaced amino acid).

As naturally occurring amino acids are typically grouped according to their properties, conservative substitutions by naturally occurring amino acids can be easily determined bearing in mind the fact that in accordance with the invention replacement of charged amino acids by sterically similar non-charged amino acids are considered as conservative substitutions.

For producing conservative substitutions by non-naturally occurring amino acids it is also possible to use amino acid analogs (synthetic amino acids) well known in the art. A peptidomimetic of the naturally occurring amino acid is well documented in the literature known to the skilled practitioner.

When affecting conservative substitutions the substituting amino acid should have the same or a similar functional group in the side chain as the original amino acid.

The phrase “non-conservative substitutions” as used herein refers to replacement of the amino acid as present in the parent sequence by another naturally or non-naturally occurring amino acid, having different electrochemical and/or steric properties. Thus, the side chain of the substituting amino acid can be significantly larger (or smaller) than the side chain of the native amino acid being substituted and/or can have functional groups with significantly different electronic properties than the amino acid being substituted. Examples of non-conservative substitutions of this type include the substitution of phenylalanine or cycohexylmethyl glycine for alanine, isoleucine for glycine, or —NH—CH[(—CH2)5—COOHF]—CO— for aspartic acid. Those non-conservative substitutions which fall under the scope of the present invention are those which still constitute an amino acid sequence capable of binding α4β7 integrin.

According to specific embodiments, the amino acid sequence of MAdCAM-1 does not comprise a mutation at a D62 residue corresponding to MAdCAM-1 SEQ ID NO: 25.

According to specific embodiments, the amino acid sequence of MAdCAM-1 does not comprise a D62A mutation corresponding to MAdCAM-1 SEQ ID NO: 25.

As used herein, the phrase “corresponding to SEQ ID NO:” intends to include the corresponding amino acid residue in the recited SEQ ID NO relative to any other MAdCAM-1 amino acid sequence.

According to specific embodiments, the amino acid sequence of MAdCAM-1 binds α4β7 integrin with a higher selectivity, as compared to the full length MAdCAM-1.

As used herein, the term “selectivity” refers to the ability to bind α4β7 integrin and not other proteins (e.g. L-selectin, VLA-4), which may be manifested as higher affinity (e.g., Kd) to α4β7 integrin as compared to other proteins. Increased affinity can be, for examples, of at least 5, 10, 100, 1000 or 10000 fold.

According to specific embodiment, the amino acid sequence of MAdCAM-1 is a fragment of MAdCAM-1 with reduced binding to L-selectin and/or VLA-4, as compared to the full length MAdCAM-1.

According to specific embodiment, the amino acid sequence of MAdCAM-1 is a fragment of MAdCAM-1 not capable of binding L-selectin, as compared to the full length MAdCAM-1.

According to specific embodiment, the amino acid sequence of MAdCAM-1 is a fragment of MAdCAM-1 not capable of binding VLA-4, as compared to the full length MAdCAM-1.

Assays for testing binding are well known in the art and include, but not limited to flow cytometry, BiaCore, bio-layer interferometry Blitz® assay, HPLC, surface plasmon resonance.

According to specific embodiments, the amino acid sequence of MAdCAM-1 binds α4β7 with a Kd≥10−5, 10−4or 10−3.

According to specific embodiments, the amino acid sequence of MAdCAM-1 binds α4β7 with a Kd of 1-100 nM.

As used herein, the term “α4β7 integrin (also known as LPAM-1)” refers to a cell membrane heterodimer protein composed of an α4 chain encoded by the ITGA4 gene (Gene ID 3676) and a β7 chain encoded by the ITGB7 gene (Gene ID 3695). According to specific embodiments, the α4β7 integrin refers to the mouse the α4β7 integrin. According to specific embodiments, the α4β7 integrin refers to the human α4β7 integrin.

Binding of MAdCAM-1 to α4β7 is conformation dependent. α4β7, like other integrins change conformation upon specific stimuli (e.g. chemokines) which induces a specific “inside-out signaling” that changes the integrin's inactive conformation (a low-affinity bent conformation) into its active form, characterized by a high-affinity (HA) extended conformation which may be distinct for different ligands. Hence, according to specific embodiments, testing binding to α4β7 is effected by assessing binding to cells (e.g. T cells e.g. TK-1 cells) following treatment that induces formation of a high affinity conformation e.g. Mn2+, CCL25 or PMA.

In this respect, according to specific embodiments, binding is not to a linear domain.

According to specific embodiments, the amino acid sequence of MAdCAM-1 is at least 4 amino acids in length, at least 5 amino acids in length, at least 6 amino acids in length, at least 7 amino acids in length, at least 8 amino acids in length, at least 9 amino acids in length, at least 10 amino acids in length, at least 15 amino acids in length, at least 20 amino acids in length, at least 30 amino acids in length at least 50 amino acids in length at least 80 amino acids in length, at least 100 amino acids in length, at least 150 amino acids in length.

According to a specific embodiment, the amino acid sequence of MAdCAM-1 is more than 5 amino acids in length.

According to specific embodiments, the amino acid sequence of MAdCAM-1 is less than 406 amino acids in length, less than 350 amino acids in length, less than 300 amino acids in length, less than 250 amino acids in length, less than 210 amino acids in length or less than 200 amino acids in length, each possibility represents a separate embodiment of the present invention.

According to a specific embodiment, the amino acid sequence of MAdCAM-1 is 6-199 amino acids in length.

According to a specific embodiment, the amino acid sequence of MAdCAM-1 is 6-195 amino acids in length.

MAdCAM-1 comprises an extracellular domain, a transmembrane domain and an intracellular domain. The extracellular domain of MAdCAM-1 comprises two Ig-like domains (D1 and D2) and Mucin-like domain. In some species, e.g. mouse, MAdCAM-1 also comprises an additional Ig-like domain (D3). A schematic representation is shown inFIG.1C.

According to specific embodiments, the amino acid sequence of MAdCAM-1 comprises an amino acid sequence of an extracellular domain of MAdCAM-1.

As used herein, the phrase “an amino acid sequence of an extracellular domain of MAdCAM-1” refers to the full length extracellular domain of MAdCAM-1 or a fragment thereof or a homolog thereof.

According to specific embodiments, the extracellular domain of MAdCAM-1 corresponds to amino acids coordinates 22-365 of SEQ ID NO: 25.

According to specific embodiments, the extracellular domain of MAdCAM-1 corresponds to amino acids coordinates 23-319 of SEQ ID NO: 26.

According to specific embodiments, the extracellular domain of MAdCAM-1 has at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 28-29.

According to specific embodiments, the amino acid sequence of MAdCAM-1 does not comprise an amino acid sequence of a transmembrane and/or an intracellular domain of MAdCAM-1.

As used herein, the phrase “an amino acid sequence of a transmembrane domain of MAdCAM-1” refers to the full length transmembrane domain of MAdCAM-1 or a fragment thereof or a homolog thereof.

According to specific embodiments, the amino acid sequence of a transmembrane domain of MAdCAM-1 refers to the full length transmembrane domain.

According to specific embodiments, the transmembrane domain of MAdCAM-1 corresponds to amino acids coordinates 366-385 of SEQ ID NO: 25.

According to specific embodiments, the transmembrane domain of MAdCAM-1 corresponds to amino acids coordinates 320 339- of SEQ ID NO: 26.

According to specific embodiments, the transmembrane domain of MAdCAM-1 has at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 30-31.

As used herein, the phrase “an amino acid sequence of an intracellular domain of MAdCAM-1” refers to the full length intracellular domain of MAdCAM-1 or a fragment thereof or a homolog thereof.

According to specific embodiments, the amino acid sequence of an intracellular domain of MAdCAM-1 refers to the full length intracellular domain.

According to specific embodiments, the intracellular domain of MAdCAM-1 corresponds to amino acids coordinates 386-405 of SEQ ID NO: 25.

According to specific embodiments, the intracellular domain of MAdCAM-1 corresponds to amino acids coordinates 340-382 of SEQ ID NO: 26.

According to specific embodiments, intracellular domain of MAdCAM-1 has at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 32-33.

According to specific embodiments, the amino acid sequence of MAdCAM-1 comprises an amino acid sequence of a first Ig-like domain (D1) located N to C in a full length MAdCAM-1.

As used herein, the phrase “amino acid sequence of a first Ig-like domain (D1)” refers to an amino acid sequence of the full length Ig-like domain of MAdCAM-1 which comprises a motif as set forth in RSCB PDB ENTRY ID: 1 gsm, and is the first Ig-like domain located N to C in a full length MAdCAM-1, or a fragment thereof or a homolog thereof.

According to specific embodiments, the amino acid sequence of the first Ig-like domain (D1) refers to the full length first Ig-like domain.

According to specific embodiments, the amino acid sequence of the first Ig-like domain (D1) is at least 4 amino acids in length, at least 5 amino acids in length, at least 6 amino acids in length, at least 7 amino acids in length, at least 8 amino acids in length, at least 9 amino acids in length, at least 10 amino acids in length, at least 20 amino acids in length, at least 30 amino acids in length, at least 50 amino acids in length, at least 70 amino acids in length, at least 80 amino acids in length or at least 90 amino acids in length, each possibility represents a separate embodiment of the present invention.

According to specific embodiments, the first Ig-like domain (D1) corresponds to amino acids coordinates 22-115 of SEQ ID NO: 25.

According to specific embodiments, the first Ig-like domain (D1) corresponds to amino acids coordinates 23-112 of SEQ ID NO: 26.

According to specific embodiments, the first Ig-like domain (D1) has at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 34-35.

According to a specific embodiment, the amino acid sequence of MAdCAM-1 comprises SEQ ID NO: 34.

According to a specific embodiment, the amino acid sequence of MAdCAM-1 comprises SEQ ID NO: 35.

According to a specific embodiment, the amino acid sequence of MAdCAM-1 comprises comprises (SEQ ID NO: 21).

According to specific embodiments, the amino acid sequence of MAdCAM-1 comprises an amino acid sequence of a second Ig-like domain (D2) located N to C in a full length MAdCAM-1.

As used herein, the phrase “amino acid sequence of a second Ig-like domain (D2)” refers to an amino acid sequence of the full length Ig-like domain of MAdCAM-1 which comprises a motif as set forth in RSCB PDB ENTRY ID: 1 gsm, and is the second Ig-like domain located N to C in a full length MAdCAM-1, or a fragment thereof or a homolog thereof.

According to specific embodiments, the amino acid sequence of the second Ig-like domain (D2) refers to the full length second Ig-like domain.

According to specific embodiments, the second Ig-like domain (D2) corresponds to amino acids coordinates 116-220 of SEQ ID NO: 25.

According to specific embodiments, the second Ig-like domain (D2) corresponds to amino acids coordinates 113-220 of SEQ ID NO: 26.

According to specific embodiments, the second Ig-like domain (D2) has at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 36-37.

According to a specific embodiment, the amino acid sequence of MAdCAM-1 comprises SEQ ID NO: 36.

According to a specific embodiment, the amino acid sequence of MAdCAM-1 comprises SEQ ID NO: 37.

According to specific embodiments, the amino acid sequence of MAdCAM-1 comprises an amino acid sequence of a first Ig-like domain (D1) and a second Ig-like domain (D2) located N to C in a full length MAdCAM-1.

According to specific embodiments, the amino acid sequence of MAdCAM-1 consists of an amino acid sequence of a first Ig-like domain (D1) and a second Ig-like domain (D2) located N to C in a full length MAdCAM-1.

According to specific embodiments, the amino acid sequence of MAdCAM-1 has at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 22.

According to a specific embodiment, the amino acid sequence of MAdCAM-1 comprises SEQ ID NO: 22.

According to a specific embodiment, the amino acid sequence of MAdCAM-1 consists of SEQ ID NO: 22.

According to specific embodiments, the amino acid sequence of MAdCAM-1 does not comprise an amino acid sequence of a third Ig-like domain (D3) of MAdCAM-1.

As used herein, the phrase “amino acid sequence of a third Ig-like domain (D3)” refers to an amino acid sequence of the full length Ig-like domain of MAdCAM-1 which comprises a motif as set forth in RSCB PDB ENTRY ID: 1 gsm, and is the third Ig-like domain located N to C in a full length MAdCAM-1, or a fragment thereof or a homolog thereof.

According to specific embodiments, the amino acid sequence of the third Ig-like domain (D3) refers to the full length third Ig-like domain.

According to specific embodiments, the third Ig-like domain (D3) corresponds to amino acids coordinates 258-365 of SEQ ID NO: 25.

According to specific embodiments, the third Ig-like domain (D3) has at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 38.

According to specific embodiments, the amino acid sequence of MAdCAM-1 does not comprise an amino acid sequence of a mucin-like domain of MAdCAM-1.

As used herein, the phrase “amino acid sequence of mucin-like domain” refers to an amino acid sequence of the full length mucin-like domain of MAdCAM-1 which comprises a motif as set forth in RSCB PDB ENTRY ID: 1 gsm, or a fragment thereof or a homolog thereof.

According to specific embodiments, the amino acid sequence of the mucin-like domain refers to the full length mucin-like domain.

According to specific embodiments, the amino acid sequence of the mucin-like domain refers to a functional mucin-like domain.

As use herein, the phrase “functional mucin-like domain” refers to a portion of the mucin-like domain which maintains at least the L-selectin binding activities of the full length mucin-like domain. Methods of determining binding are well known in the art and are further described hereinabove.

According to specific embodiments, the amino acid sequence of the mucin-like domain corresponds to amino acids coordinates 221-257 of SEQ ID NO: 25.

According to specific embodiments, the amino acid sequence of the mucin-like domain corresponds to amino acids coordinates 226-319 of SEQ ID NO: 26.

According to specific embodiments, the mucin-like domain has at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 39-40.

Besides containing the amino acid sequence of MAdCAM-1, the polypeptides of some embodiments of the present invention may further comprise additional elements to increase stability or penetration into cells, to aid in purification or in attachment to the therapeutic or the detectable moiety or a particle comprising same and/or to improve expression, pharmacokinetics, or bioactivity.

Non-limiting examples of such additional elements include polyethylene glycol (PEG), tags such as histidine tag, myc tag, flag tag, signal peptide and the like.

According to specific embodiments, the peptide further comprises an amino acid sequence of a constant region of an antibody.

According to specific embodiments, the antibody constant region is of an IgG antibody.

According to a specific embodiment, the antibody constant region is of an IgG1, IgG2 or IgG4.

According to a specific embodiment, the antibody constant region is IgG2 e.g. IgG2a.

According to specific embodiments, the antibody constant region is of human origin.

As used herein, the phrase “amino acid sequence of a constant region of an antibody” refers to an amino acid sequence of the full length constant region of an antibody, or a fragment thereof. Hence, the constant region of an antibody may be a light chain constant region, a heavy chain constant region or a portion thereof e.g. an Fc domain or a portion thereof.

According to specific embodiments, the constant region of an antibody is an Fc domain of an antibody or a fragment thereof.

According to specific embodiments, the polypeptide comprises a portion of an Fc domain of an antibody excluding the CH1 domain.

According to a specific embodiments, the polypeptide comprises a portion of the Fc domain as set forth in SEQ ID NO: 13.

According to specific embodiments, the amino acid sequence of the antibody constant region is located C terminally to the amino acid sequence of MAdCAM-1.

The polypeptides of some embodiments of the invention or the proteinaceous moieties further described herein below may be synthesized by any techniques that are known to those skilled in the art of peptide synthesis, such as, but not limited to, solid phase and recombinant techniques.

Large scale peptide synthesis is described by Andersson Biopolymers 2000; 55(3):227-50.

Alternatively or additionally, any of the polypeptides and proteinaceous moieties described herein can be encoded from a polynucleotide.

Thus, according to an aspect of the present invention there is provided an isolated polynucleotide encoding the polypeptide.

As used herein the term “polynucleotide” refers to a single or double stranded nucleic acid sequence which is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence and/or a composite polynucleotide sequences (e.g., a combination of the above).

To express exogenous peptide or agent in mammalian cells, a polynucleotide sequence encoding the peptide and/or the agent is preferably ligated into a nucleic acid construct suitable for mammalian cell expression.

Thus, according to an aspect of the present invention there is provided a nucleic acid construct comprising a polynucleotide encoding the polypeptide and a cis-acting regulatory element for directing expression of the polynucleotide in a cell.

Methods of recombinantly expressing a polypeptide are well known in the art.

As mentioned, the polypeptide is in association with a therapeutic moiety or a detectable moiety.

According to an embodiment, the polypeptide is attached to a therapeutic moiety or a detectable moiety.

As used herein, the phrase “in association with”, refers to direct or indirect (e.g. through a linker) binding of the polypeptide to the therapeutic or detectable moiety or to a particle comprising the therapeutic or detectable moiety.

According to specific embodiments, the therapeutic moiety or the detectable moiety is a heterologous therapeutic or detectable moiety.

The therapeutic moiety may be any molecule, including polynucleotides, small molecule chemical compounds and polypeptides.

Non-limiting examples of therapeutic moieties which can be used with specific embodiments of the invention include an anti-inflammatory cytokine (e.g IL4, IL-10, IL-13, IFNα, TGFβ), an anti-inflammatory drug, an immunosuppressive agent, steroids, an immunomodulatory agent, an enzyme.

According to specific embodiments, the therapeutic moiety is not a constant region of an antibody.

According to specific embodiments, the therapeutic moiety downregulates expression and/or activity of a gene of interest.

Downregulating expression and/or activity can be can be effected at the protein level (e.g., small molecules, inhibitory peptides, enzymes that cleave the polypeptide, aptamers and the like) or at the genomic (i.e. genome editing agent e.g. homologous recombination and site specific endonucleases) and/or the transcript level using a variety of molecules which interfere with transcription and/or translation (e.g., RNA silencing agents) of a target expression product described herein.

Non-limiting examples of downregulating agents are described in details hereinbelow.

Downregulation at the nucleic acid level is typically effected using a nucleic acid agent, having a nucleic acid backbone, DNA, RNA, mimetics thereof or a combination of same. The nucleic acid agent may be encoded from a DNA molecule or provided to the cell per se.

According to specific embodiments, the therapeutic moiety is an RNA silencing agent.

As used herein, the phrase “RNA silencing” refers to a group of regulatory mechanisms [e.g. RNA interference (RNAi), transcriptional gene silencing (TGS), post-transcriptional gene silencing (PTGS), quelling, co-suppression, and translational repression] mediated by RNA molecules which result in the inhibition or “silencing” of the expression of a corresponding protein-coding gene. RNA silencing has been observed in many types of organisms, including plants, animals, and fungi.

As used herein, the term “RNA silencing agent” refers to an RNA which is capable of specifically inhibiting or “silencing” the expression of a target gene. In certain embodiments, the RNA silencing agent is capable of preventing complete processing (e.g., the full translation and/or expression) of an mRNA molecule through a post-transcriptional silencing mechanism. RNA silencing agents include non-coding RNA molecules, for example RNA duplexes comprising paired strands, as well as precursor RNAs from which such small non-coding RNAs can be generated. Exemplary RNA silencing agents include dsRNAs such as siRNAs, miRNAs and shRNAs.

In one embodiment, the RNA silencing agent is capable of inducing RNA interference.

In another embodiment, the RNA silencing agent is capable of mediating translational repression.

Non-limiting examples of RNA silencing agents that can be used according to specific embodiments of the present invention include dsRNA, siRNA, shRNA, antisense, miRNA and miRNA mimics. Methods and algorithms of designing RNA silencing agents and predicting their efficiency are well known in the art.

According to specific embodiments, the RNA silencing agent is a siRNA.

Nucleic acid agents can also operate at the DNA level as summarized infra.

Downregulation can also be achieved by inactivating the gene via introducing targeted mutations involving loss-of function alterations (e.g. point mutations, deletions and insertions) in the gene structure.

As used herein, the phrase “loss-of-function alterations” refers to any mutation in the DNA sequence of a gene which results in down-regulation of the expression level and/or activity of the expressed product, i.e., the mRNA transcript and/or the translated protein. Non-limiting examples of such loss-of-function alterations include a missense mutation, i.e., a mutation which changes an amino acid residue in the protein with another amino acid residue and thereby abolishes the enzymatic activity of the protein; a nonsense mutation, i.e., a mutation which introduces a stop codon in a protein, e.g., an early stop codon which results in a shorter protein devoid of the enzymatic activity; a frame-shift mutation, i.e., a mutation, usually, deletion or insertion of nucleic acid(s) which changes the reading frame of the protein, and may result in an early termination by introducing a stop codon into a reading frame (e.g., a truncated protein, devoid of the enzymatic activity), or in a longer amino acid sequence (e.g., a readthrough protein) which affects the secondary or tertiary structure of the protein and results in a non-functional protein, devoid of the enzymatic activity of the non-mutated polypeptide; a readthrough mutation due to a frame-shift mutation or a modified stop codon mutation (i.e., when the stop codon is mutated into an amino acid codon), with an abolished enzymatic activity; a promoter mutation, i.e., a mutation in a promoter sequence, usually 5′ to the transcription start site of a gene, which results in down-regulation of a specific gene product; a regulatory mutation, i.e., a mutation in a region upstream or downstream, or within a gene, which affects the expression of the gene product; a deletion mutation, i.e., a mutation which deletes coding nucleic acids in a gene sequence and which may result in a frame-shift mutation or an in-frame mutation (within the coding sequence, deletion of one or more amino acid codons); an insertion mutation, i.e., a mutation which inserts coding or non-coding nucleic acids into a gene sequence, and which may result in a frame-shift mutation or an in-frame insertion of one or more amino acid codons; an inversion, i.e., a mutation which results in an inverted coding or non-coding sequence; a splice mutation i.e., a mutation which results in abnormal splicing or poor splicing; and a duplication mutation, i.e., a mutation which results in a duplicated coding or non-coding sequence, which can be in-frame or can cause a frame-shift.

According to specific embodiments loss-of-function alteration of a gene may comprise at least one allele of the gene.

The term “allele” as used herein, refers to any of one or more alternative forms of a gene locus, all of which alleles relate to a trait or characteristic. In a diploid cell or organism, the two alleles of a given gene occupy corresponding loci on a pair of homologous chromosomes.

According to other specific embodiments loss-of-function alteration of a gene comprises both alleles of the gene.

Methods of introducing nucleic acid alterations to a gene of interest are well known in the art using well known genome editing agents [see for example Menke D. Genesis (2013) 51: -618; Capecchi, Science (1989) 244:1288-1292; Santiago et al. Proc Natl Acad Sci USA (2008) 105:5809-5814; International Patent Application Nos. WO 2014085593, WO 2009071334 and WO 2011146121; U.S. Pat. Nos. 8,771,945, 8,586,526, 6,774,279 and UP Patent Application Publication Nos. 20030232410, 20050026157, US20060014264; the contents of which are incorporated by reference in their entireties] and include targeted homologous recombination (e.g. “Hit and run”, “double-replacement”), site specific recombinases (e.g. the Cre recombinase and the Flp recombinase), PB transposases (e.g. Sleeping Beauty, piggyBac, Tol2 or Frog Prince), genome editing by engineered nucleases [e.g. meganucleases, Zinc finger nucleases (ZFNs), transcription-activator like effector nucleases (TALENs) and CRISPR/Cas system (including the gRNA and/or the endonuclease e.g. Cas9)] and genome editing using recombinant adeno-associated virus (rAAV) platform. Agents for introducing nucleic acid alterations to a gene of interest can be designed by publically available sources or obtained commercially from e.g. Transposagen, Addgene and Sangamo Biosciences. Thus, according to specific embodiments, the therapeutic moiety is a genome editing system or a component in a genome editing system, e.g. a homologous polynucleotide comprising a mutation, a gRNA, a restriction enzyme, a nuclease or a polynucleotide encoding same e.g. meganuclease, TALEN, ZFN, Cas9.

Downregulation can also be affected at the polypeptide level using e.g. small molecules, peptides, antibodies and/or polynucleotides.

An example of such a downregulating agent would be any molecule which interferes with the target protein activity (e.g., catalytic or interaction) by binding the target protein and/or cleaving the target protein. Such molecules can be a small molecule, antagonists, antibody or inhibitory peptide.

Another inhibitory agent which can be used along with some embodiments of the invention is a molecule which prevents target activation or substrate binding.

Another downregulating agent which can be used along with some embodiments of the invention is an aptamer. As used herein, the term “aptamer” refers to double stranded or single stranded RNA molecule that binds to specific molecular target, such as a protein. Various methods are known in the art which can be used to design protein specific aptamers. The skilled artisan can employ SELEX (Systematic Evolution of Ligands by Exponential Enrichment) for efficient selection as described in Stoltenburg R, Reinemann C, and Strehlitz B (Biomolecular engineering (2007) 24(4):381-403).

It will be appreciated that a non-functional analogue of at least a catalytic or binding portion of the target can be also used as an inhibitory agent.

According to specific embodiments, the therapeutic moiety downregulates expression and/or activity of IFNγ.

According to a specific embodiment, the therapeutic moiety is an IFNγ siRNA, such as, but not limited to SEQ ID NO: 9-10.

According to other specific embodiments, the therapeutic moiety is an anti-inflammatory cytokine or a polynucleotide encoding same. Non-limiting examples of anti-inflammatory cytokines include IL4, IL-10, IL-13, IFNα, TGFβ.

Examples of detectable moieties that can be used in the present invention include but are not limited to radioactive isotopes, phosphorescent chemicals, chemiluminescent chemicals, fluorescent chemicals, enzymes, fluorescent polypeptides and a radioactive isotope (such as[125]iodine). The detectable moiety can be a member of a binding pair, which is identifiable via its interaction with an additional member of the binding pair, and a label which is directly visualized. In one example, the label is a fluorescent protein or an enzyme producing a colorimetric reaction.

According to specific embodiments, the detectable moiety is not a tag such as histidine tag, myc tag, flag tag and the like which aid in purification of the polypeptide.

Further examples of detectable moieties, include those detectable by Positron Emission Tomagraphy (PET) and Magnetic Resonance Imaging (MRI), all of which are well known to those of skill in the art.

According to specific embodiments, the therapeutic or detectable moiety is attached to or encapsulated in a cell-penetrating moiety.

As used herein the phrase “cell penetrating moiety” refers to a moiety which enhances translocation of the therapeutic or detectable moiety across a cell membrane. Non-limiting examples of cell penetrating moieties include cell penetrating peptides and lipid particles.

As used herein, a “cell-penetrating peptide” is a peptide that comprises a short (about 12-30 residues) amino acid sequence or functional motif that confers the energy-independent (i.e., non-endocytotic) translocation properties associated with transport of the membrane-permeable complex across the plasma and/or nuclear membranes of a cell. The cell-penetrating peptide used in the membrane-permeable complex of some embodiments of the invention comprises at least one non-functional cysteine residue, which is either free or derivatized to form a disulfide link with a double-stranded ribonucleic acid that has been modified for such linkage. Representative amino acid motifs conferring such properties are listed in U.S. Pat. No. 6,348,185, the contents of which are expressly incorporated herein by reference. The cell-penetrating peptides of some embodiments of the invention may include, but are not limited to, penetratin, transportan, pIs1, TAT(48-60), pVEC, MTS, and MAP.

Additional description on lipid particles is provided hereinbelow.

According to specific embodiments, the therapeutic or detectable moiety is directly or indirectly bound to the polypeptide.

Accordingly, according to specific embodiments, the therapeutic or detectable moiety can be attached to the polypeptide covalently or non-covalently.

The therapeutic or detectable moiety can be attached to the polypeptide directly or via a linker.

According to specific embodiments, the therapeutic or detectable moiety is attached to the polypeptide via a linker. Any linker known in the art can be used with specific embodiments of the invention, including but not limited to a polypeptide, a synthetic linker, a chemical moiety, a polymer, a particle.

According to some embodiments, when the therapeutic or detectable moiety is proteinaceous it can be attached to the polypeptide by translationally fusing the polynucleotide encoding the polypeptide with the nucleic acid sequence encoding the therapeutic or detectable moiety.

A therapeutic or detectable moiety can be attached, for example, to the polypeptide using standard chemical synthesis techniques widely practiced in the art [see e.g., hypertexttransferprotocol://worldwideweb (dot) chemistry (dot) org/portal/Chemistry)], such as using any suitable chemical linkage, direct or indirect, as via a peptide bond (when the functional moiety is a polypeptide), or via covalent bonding to an intervening linker element, such as a linker peptide or other chemical moiety, such as an organic polymer. Chimeric peptides may be linked via bonding at the carboxy (C) or amino (N) termini of the peptides, or via bonding to internal chemical groups such as straight, branched or cyclic side chains, internal carbon or nitrogen atoms, and the like. Description of fluorescent labeling of antibodies is provided in details in U.S. Pat. Nos. 3,940,475, 4,289,747, and 4,376,110.

According to other embodiments, the therapeutic or detectable moiety is attached to-, or encapsulated in- a particle.

Accordingly, according to specific embodiments, the polypeptide is attached to the particle which comprises (also referred to as loaded with) the therapeutic or detectable moiety via direct or indirect binding.

As used herein, “particle” refers to a nano to micro structures which are not biological cells.

The particle may be a synthetic carrier, gel or other object or material having an external surface which is capable of being loadable with (e.g., encapsulated in or attached to) a therapeutic or detectable moiety. The particle may be either polymeric or non-polymeric preparations.

Exemplary particles that may be used according to specific embodiments of the invention include, but are not limited to, polymeric particles, microcapsules, liposomes, microspheres, microemulsions, nanoparticles, nanocapsules, nano-spheres, nano-liposomes, nano-emulsions, lipid nanoparticles and nanotubes.

In one embodiment, the particle is a biological particle—e.g. an erythrocyte or a cell ghost.

In another embodiment, the particle is a non-biological particle—i.e. not a cell.

Suitable particles in accordance with some embodiments of the invention are preferably non-toxic.

According to a particular embodiment, the particle is a nanoparticle.

As used herein, the term “nanoparticle” refers to a particle or particles having an intermediate size between individual atoms and macroscopic bulk solids. Generally, nanoparticle has a characteristic size (e.g., diameter for generally spherical nanoparticles, or length for generally elongated nanoparticles) in the sub-micrometer range, e.g., from about 1 nm to about 500 nm, or from about 1 nm to about 200 nm, or of the order of 10 nm, e.g., from about 1 nm to about 100 nm. According to specific embodiments, the particle is a nanoparticle having a size of 40-120 nm.

The nanoparticles may be of any shape, including, without limitation, elongated particle shapes, such as nanowires, or irregular shapes, in addition to more regular shapes, such as generally spherical, hexagonal and cubic nanoparticles. According to one embodiment, the nanoparticles are generally spherical.

The particles may have a charged surface (i.e., positively charged or negatively charged) or a neutral surface.

Agents which are used to fabricate the particles may be selected according to the desired charge required on the outer surface of the particles.

According to specific embodiments, the particle if a lipid particle.

Thus, for example if a negatively charged surface is desired, the particles may be fabricated from negatively charged lipids, such as described herein below.

When a positively charged surface is desired, the particles may be fabricated from positively charged lipids, such as described herein below.

As mentioned, non-charged particles are also contemplated by the present invention. Such particles may be fabricated from neutral lipids such as phosphatidylethanolamine or dioleilphosphatidylethanolamine (DOPE).

In a specific embodiment, the particle is a liposome. As used herein and as recognized in the art, liposomes include any synthetic (i.e., not naturally occurring) structure composed of lipid bilayers, which enclose a volume. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. The liposomes may be prepared by any of the known methods in the art [Monkkonen, J. et al., 1994, J. Drug Target, 2:299-308; Monkkonen, J. et al., 1993, Calcif. Tissue Int., 53:139-145; Lasic D., Liposomes Technology Inc., Elsevier, 1993, 63-105. (chapter 3); Winterhalter M, Lasic D, Chem Phys Lipids, 1993 September; 64(1-3):35-43].

The diameter of the liposomes used preferably ranges from 20-200 nm and more preferably from 20-100 nm. For sizing liposomes, extrusion, homogenization or exposure to ultrasound irradiation may be used, Homogenizers which may be conveniently used include microfluidizers (produced by Microfluidics of Boston, MA, USA) or microfluidic micro mixer (Precision NanoSystems, Vancouver, BC, Canada). In a typical homogenization procedure, liposomes are recirculated through a standard emulsion homogenizer until selected liposomes sizes are observed. The particle size distribution can be monitored by conventional laser beam particle size discrimination. Extrusion of liposomes through a small-pore polycarbonate membrane or an asymmetric ceramic membrane is an effective method for reducing liposome sizes to a relatively well defined size distribution. Typically, the suspension is cycled through the membrane one or more times until the desired liposome size distribution is achieved. The liposomes may be extruded through successively smaller pore membranes to achieve a gradual reduction in liposome size.

The liposomes may be unilamellar or may be multilamellar. Unilamellar liposomes may be preferred in some instances as they represent a larger surface area per lipid mass. The liposomes may be fabricated from a single phospholipid or mixtures of phospholipids. The liposomes may also comprise other lipid materials such as cholesterol. For fabricating liposomes with a negative electrical surface potential, acidic phospho- or sphingo- or other synthetic-lipids may be used. Preferably, the lipids have a high partition coefficient into lipid bilayers and a low desorption rate from the lipid assembly. Exemplary phospholipids that may be used for fabricating liposomes with a negative electrical surface potential include, but are not limited to phosphatidylserine, phosphatidic acid, phosphatidylcholine and phosphatidyl glycerol.

Other negatively charged lipids which are not liposome forming lipids that may be used are sphingolipids such as cerebroside sulfate, and various gangliosides.

The most commonly used and commercially available lipids derivatized into lipopolymers are those based on phosphatidyl ethanolamine (PE), usually distearylphosphatidylethanolamine (DSPE).

The lipid phase of the liposome may comprise a physiologically acceptable liposome forming lipid or a combination of physiologically acceptable liposome forming lipids for medical or veterinarian applications. Liposomes are spherical bilayer structures composed of different kind of amphiphatic lipids. These lipids have glycerol backbone with two hydrophobic acyl chains and a hydrophilic head groups such as phosphate in combination or derivatives of the same may contain amine, imine, acid or alcohols.

Typically, the acyl chain is between 12 to about 24 carbon atoms in length, and has varying degrees of saturation being fully, partially or non-hydrogenated lipids. Further, the lipid matrix may be of natural source, semi-synthetic or fully synthetic lipid, and neutral, negatively or positively charged.

According to specific embodiments, the particle is a lipid nanoparticle.

Non-limiting examples of lipid nanoparticles that can be used with specific embodiments of the present invention and methods of producing same are further described hereinbelow and in the Examples section which follows, and also in e.g. Ramishetti et al. Adv Mater. 2020 Jan. 30:e1906128, International Patent Application publication Nos. WO2016/189532, WO2018/015881 and WO2018087753, WO2017194454 and US Patent Application Publication no. US20130245107, the contents of which are fully incorporated herein by reference.

The lipid nanoparticle may be prepared by any of the methods known in the art, such as disclosed in e.g. Jayaraman et al. Angew chem. July 2012, Semple et al. Nat Biotech. 2010, Kauffman et al. Nano Lett, Oct 2015; and in the Examples section which follows.

The core of the particle may be hydrophilic or hydrophobic. The core of the lipid nanoparticle may comprise some lipids, such that it is not fully hydrophilic.

According to specific embodiments, the core of the particle is hydrophobic.

According to specific embodiments, the core of the particle is hydrophilic.

According to specific embodiments, the core of the particle contains an amorphous lipid core.

It will be appreciated that combinations of different lipids may be used to fabricate the particles disclosed herein, including a mixture of more than one cationic lipid, a mixture of more than one anionic lipid, a mixture of more than one neutral lipid, a mixture of more than one ionizable lipid, a mixture of at least one cationic lipid and at least one anionic lipid, a mixture of at least one cationic lipid and at least one neutral lipid, a mixture of at least one anionic lipid and at least one neutral lipid, a mixture of at least one ionizable lipid and at least one neutral lipid, and additional combinations of the above.

According to some exemplary embodiments, the plurality of lipids of the lipid particles may be of natural or synthetic source and may be selected from, but not limited to: cationic lipids, phosphatidylethanolamines, ionizable lipids, membrane stabilizing lipids, phospholipids, and the like, or combinations thereof. Each possibility represents a separate embodiment of the present invention.

In some embodiments, the membrane stabilizing lipids may be selected from, but not limited to: cholesterol, phospholipids (such as, for example, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, diphosphatidylglycerols), cephalins, sphingolipids (sphingomyelins and glycosphingolipids), glycoglycerolipids, and combinations thereof. Each possibility represents a separate embodiment of the present invention.

In addition, according to specific embodiments, polymer-lipid based formulations may be used.

The polymers may be employed as homopolymers or as block or random copolymers.

The particles may also include other components. Examples of such other components includes, without being limited thereto, fatty alcohols, fatty acids, and/or cholesterol esters or any other pharmaceutically acceptable excipients which may affect the surface charge, the membrane fluidity and assist in the incorporation of the biologically active lipid into the lipid assembly. Examples of sterols include cholesterol, cholesterol hemisuccinate, cholesterol sulfate, or any other derivatives of cholesterol. Lipid assemblies according to specific embodiments of the invention include either those which form a micelle (typically when the assembly is absent from a lipid matrix) or those which form a liposome (typically, when a lipid matrix is present).

According to one embodiment, the lipid phase comprises phospholipids.

The phospholipids may be a glycerophospholipid. Examples of glycerophospholipid include, without being limited thereto, phosphatidylglycerol (PG) including dimyristoyl phosphatidylglycerol (DMPG); phosphatidylcholine (PC), including egg yolk phosphatidylcholine and dimyristoyl phosphatidylcholine (DMPC), phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylserine (PS) and sphingomyelin (SM) and derivatives of the same.

Another group of lipid matrix employed according to the invention includes cationic lipids (monocationic or polycationic lipids). Cationic lipids typically comprise a lipophilic moiety, such as a sterol or the same glycerol backbone to which two acyl or two alkyl, or one acyl and one alkyl chain contribute the hydrophobic region of the amphipathic molecule, to form a lipid having an overall net positive charge. Non-limiting examples of cationic lipids that may be used with specific embodiments of the invention include, 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP) 1,2-dioleyloxy-3-(trimethylanino) propane (DOTAP), N-[-1-(2,3,-ditetradecyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N-[1-(2,3,-dioleyloxy)propyl]-N,N-dimethyl-N-hydroxy ethyl-ammonium bromide (DORIE), N-[1-(2,3-dioleyloxy)propyl];-N,N,N-trimethylammonium chloride (DOTMA); 3 ;N-(N′,N′- dimethylaminoethane) carbamoly]; cholesterol (DC-Chol), and I dimethyldioctadecylammonium (DDAB), N-[2-[[2,5-bis[3aminopropyl)amino]-1-oxopentyl]amino ]ethyl]N,N dimethul-2,3 bis (1-oXo-9-octadecenyl) oX;-1 propanaminium (DOSPA), ceramide carbamoyl spermine (CCS), D-Lin-MC3-DMA (Cas No. 1224606-06-7) and the cationic lipids described in Ramishetti et al. Adv Mater. 2020 Jan. 30:e1906128 and International Application Publication No. WO201808775, the contents of which are fully incorporated herein by reference.

According to a specific embodiments, the cationic lipid is D-Lin-MC3-DMA.

The cationic lipids may be used alone, in combination with cholesterol, with neutral phospholipids or other known lipid assembly components. In addition, the cationic lipids may form part of a derivatized phospholipids such as the neutral lipid dioleoylphosphatidyl ethanolamine (DOPE) derivatized with polylysine to form a cationic lipopolymer.

According to specific embodiments, the polymer used for fabricating the particles is biocompatible and biodegradable, such as poly(DL-lactide-co-glycolide) polymer (PLGA). However, additional polymers which may be used for fabricating the particles include, but are not limited to, PLA (polylactic acid), and their copolymers, polyanhydrides, polyalkyl-cyanoacrylates (such as polyisobutylcyanoacrylate), polyethyleneglycols, polyethyleneoxides and their derivatives, chitosan, albumin, gelatin and the like.

The particles of the present invention may be modified to enhance their circulatory half-life (e.g. by PEGylation) to reduce their clearance, to prolong their scavenging time-frame and to allow antibody binding. The PEG which is incorporated into the particles may be characterized by of any of various combinations of chemical composition and/or molecular weight, depending on the application and purpose.

According to some embodiments, the particle include one or more PEG derivatives. According to specific embodiments, the PEG or PEG derivative may be conjugated to as, a lipid. Non-limiting examples of PEG derivative include PEG-DMG 3-N-(-methoxy poly(ethylene glycol)2000)carbamoyl-1,2-dimyrisyl glycerol, PEG-cDMA 3-N-(-methoxy poly(ethylene glycol)2000)carbamoyl-1,2-dimyristyloxy-propylamine; PEG-cDSA, 3-N-(-methoxy poly(ethylene glycol)2000)carbamoyl-1,2-distearyloxy-propylamine, DSPE-PEG, PEG-maleimide, DSPE-PEG-maleimide, or combinations thereof.

According to some embodiments, the lipid phase may comprise about 30-60% (mol) cationic lipids. For example, the cationic lipid(s) may comprise about 40-50% (mol) of the lipid phase.

According to some embodiments, the lipid phase may comprise about 20-70% (mol) membrane stabilizing lipids. For example, the membrane stabilizing lipids may comprise about 40-60% of the lipid phase. In some embodiments, more than one type of membrane stabilizing lipid may be used in the lipid phase. For example, the membrane stabilizing lipid may include cholesterol (being about 30-50% (mol) of the lipid phase), and a phospholipid (such as, for example, DSPC), that may be about 5-15% (mol) of the lipid phase.

According to some embodiments, the lipid phase may comprise about 0.01-3% (mol) of PEG-maleimide (optionally conjugated to a lipid). For example, the PEG-maleimide may comprise about 0.05-0.6% of the lipid mixture.

According to some embodiments, an additional PEG-derivative (conjugated to a lipid) may comprise about 0.5-10% of the lipid phase composition.

According to exemplary embodiments, the particles may be comprised of a cationic lipid (such D-Lin-MC3-DMA), cholesterol, 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), PEG derivative (such as DMG-PEG) and PEG-maleimide conjugated to a lipid (such as DSPE-PEG-maleimide); at various mol/mol ratios. For example, the lipid phase may be comprised of: cationic lipid (DLinMC3-DMA)/Chol/DSPC/DMG-PEG/DSPE-PEG-maleimide (mol/mol 50:10:38:1.5:0.5).

Attaching the therapeutic or detectable moiety to the particle can be effected concomitant with, or following particle assembly, by methods well known in the art such as disclosed in the Examples section which follows, and also in e.g. Ramishetti et al. Adv Mater. 2020 Jan. 30:e1906128, International Patent Application publication Nos. WO2018/015881 and WO2018087753, WO2017194454 and US Patent Application Publication no. US20130245107, the contents of which are fully incorporated herein by reference.

Any suitable particle (e.g. lipid particle): therapeutic or detectable moiety ratio that is efficacious is contemplated by some embodiments of the invention. According to specific embodiments, the particle (e.g. lipid particle): therapeutic or detectable moiety ratios (w/w) include about 1:1 to about 50:1, about 2:1 to about 30:1, about 5:1 to about 100:1, about 10:1 to about 40:1, about 15:1 to about 25:1. According to a specific embodiment the particle (e.g. lipid particle) : therapeutic or detectable moiety ratios (w/w) is 16:1.

The desired amount of the therapeutic or detectable moiety attached to- or encapsulated in the particle varies depending on the type of the therapeutic or detectable moiety. However, it is preferable that the therapeutic or detectable moiety can be attached to- or encapsulated in the particle at a high loading efficiency.

Methods of covalently binding a polypeptide to a particle are known in the art and disclosed in the Examples section which follows and for example in U.S. Pat. Nos. 5,171,578, 5,204,096 and 5,258,499, the contents of which are fully incorporated herein by reference. Methods of non-covalently binding a polypeptide to a particle are also known in the art and are also disclosed in the Examples section which follows and e.g. International Patent Application Publication NO. WO2018/015881, the contents of which are fully incorporated herein by reference.

According to a specific embodiment, the polypeptide is attached to the particle directly or via a linker.

According to specific embodiments, the polypeptide binds the particle via a linker. The linker may be attached to the particle and/or the polypeptide covalently or non-covalently.

According to specific embodiments, the linker is covalently attached to the particle.

According to specific embodiments, the linker is chemically conjugated (coupled) to the particle. Such methods are known in the art and are further described hereinabove and in the Examples section which follows. According to a specific embodiment, the linker is chemically conjugated to the particle using maleimide-thiol chemistry.

According to specific embodiments, the linker is non-covalently attached to the polypeptide.

Linkers that can be used with specific embodiments of the invention are known in the art and are further described hereinabove and below.

According to specific embodiments, the linker is a protein capable of forming an immune-complex with the polypeptide. Thus, according to specific embodiments, the polypeptide binds the particle via a protein capable of immune-complexing with an antibody constant region or a tag comprised in the polypeptide. Non-limiting examples of such proteins include a secondary anti-FC antibody or an anti-tag antibody (e.g. ScFv), Protein A, Protein G, Protein L, Protein Z, Protein LG, Protein LA and Protein AG, proteins described in Lombardi et al., Discovery Today, Volume 20, Number 10, Pages 1271-1283, October 201; Braisted et al., Proc. Natl. Acad. Sci. USA, Vol. 93, pp. 5688-5692, June 1996; and Y. J. Jeong et al., Peptides 31 (2010) 202-206, the contents of which are incorporated herein by reference.

According to a specific embodiments, the polypeptide binds the particle via a secondary antibody that binds the antibody constant region comprised in the polypeptide.

According to specific embodiments, the antibody is a ScFv.

According to specific embodiment, the antibody is a humanized antibody.

Thus, according to an aspect of the present invention there is provided a method for the preparation of a targeted particle, the method comprising attaching a therapeutic or a detectable moiety to a particle or encapsulating therapeutic or a detectable moiety in a particle and attaching the loaded particle to the polypeptide comprising an amino acid sequence of MAdCAM-1 disclosed herein to generate a targeted particle.

According to an aspect of the present invention, there is provided a targeted particle comprising a therapeutic and/or a detectable moiety, said particle is attached to a polypeptide comprising an amino acid sequence of MAdCAM-1 disclosed herein.

According to specific embodiments, the therapeutic or a detectable moiety is attached to said particle.

According to specific embodiments, the therapeutic or a detectable moiety is encapsulated in said particle.

As the polypeptides comprising an amino acid sequence of MAdCAM-1 and targeted particles disclosed herein are in association with a therapeutic or a detectable moieties, specific embodiments of the present invention contemplate their use in methods of treating, diagnosing and monitoring treatment efficacy in conditions associated with inflammation mediated by MAdCAM-1.

Thus, according to an aspect of the present invention, there is provided a method of treating a disease associated with inflammation mediated by MAdCAM-1 in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the polypeptide, thereby treating the disease associated with inflammation mediated by MAdCAM-1.

According to an additional or an alternative aspect of the present invention, there is provided the polypeptide for use in treating a disease associated with inflammation mediated by MAdCAM-1.

According to an additional or an alternative aspect of the present invention, there is provided method of treating a disease associated with inflammation mediated by MAdCAM-1 in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the targeted particle, thereby treating the disease associated with inflammation mediated by MAdCAM-1.

According to an additional or an alternative aspect of the present invention, there is provided the targeted particle for use in treating a disease associated with inflammation mediated by MAdCAM-1.

The term “treating” or “treatment” refers to inhibiting, preventing or arresting the development of a pathology (disease, disorder or medical condition) and/or causing the reduction, remission, or regression of a pathology or a symptom of a pathology. Those of skill in the art will understand that various methodologies and assays can be used to assess the development of a pathology, and similarly, various methodologies and assays may be used to assess the reduction, remission or regression of a pathology.

As used herein, the term “subject” includes mammals, e.g., human beings at any age and of any gender. According to specific embodiments, the term “subject” refers to a subject who suffers from the pathology (disease, disorder or medical condition).

According to specific embodiments, the subject is a human.

As used herein the phrase “inflammation mediated by MAdCAM-1” refers to pathological condition which involves inflammation in which MAdCAM-1 activity contributes to onset or progression.

As used herein, the phrase “disease associated with inflammation mediated by MAdCAM-1” refers to a disease in which inflammation mediated by MAdCAM-1 contributes to onset or progression.

Non-limiting examples of diseases associated with inflammation mediated by MAdCAM-1 include inflammatory bowel disease (IBD), such as ulcerative colitis, Crohn's disease, ileitis, Celiac disease, nontropical Sprue, enteropathy associated with seronegative arthropathies, microscopic or collagenous colitis, eosinophilic gastroenteritis, or pouchitis resulting after proctocolectomy, and ileoanal anastomosis, pancreatitis and insulin-dependent diabetes mellitus, mastitis (mammary gland), cholecystitis, cholangitis or pericholangitis (bile duct and surrounding tissue of the liver), chronic bronchitis, chronic sinusitis, asthma, and graft versus host disease (e.g., in the gastrointestinal tract), chronic inflammatory diseases of the lung which result in interstitial fibrosis, such as hypersensitivity pneumonitis, collagen diseases, sarcoidosis, autoimmune-mediated liver disease and other idiopathic conditions.

According to specific embodiments, the disease is IBD.

According to an additional or an alternative aspect of the present invention, there is provided a method of diagnosing inflammation mediated by MAdCAM-1 in a subject in need, the method comprising:(a) administering to the subject the polypeptide or the targeted particle; and(b) determining an amount of said polypeptide or said targeted particle in a suspected inflamed tissue of the subject following said administering,
wherein an amount above a predetermined threshold is indicative of presence of inflammation mediated by MAdCAM-1 in said tissue of said subject.

According to an additional or an alternative aspect of the present invention, there is provided a method of monitoring efficacy of treatment against inflammation mediated by MAdCAM-1 in a subject in need, the method comprising:(a) administering to the subject the polypeptide or the targeted particle following the treatment; and(b) determining an amount of the polypeptide or the targeted particle in a suspected inflamed tissue of the subject following said administering,
wherein a decrease in the amount following said treatment beyond a predetermined threshold is indicative of reduction in inflammation and efficaciousness of the treatment.

According to specific embodiments, the tissue is a gut tissue.

Determining the amount of the polypeptide or the targeted particle may be effected by any method known in the art and depends on the detectable moiety used. Non-limiting examples of such methods include PET/CT and in-vivo fluoresce imaging.

According to specific embodiments, the inflammation is associated with a disease, such as the ones disclosed hereinabove e.g. IBD.

Thus, according to specific embodiments, the method comprising treating the subject with said treatment prior to administration of the polypeptide or the targeted particle.

According to specific embodiments, treatment is with the polypeptide comprising the MAdCAM-1 amino acid sequence attached to a therapeutic moiety or the targeted particle described herein.

According to specific embodiments, the predetermined threshold is derived from a control subject, such as a healthy subject or a subject with a known disease.

Thus, the predetermined level can be experimentally determined by comparing the amount in a healthy subject with the amount in a subject having an inflammatory disease mediated by MAdCAM-1 (e.g. IBD) with known stage.

According to specific embodiments, the decrease beyond a predetermined threshold is statistically significant.

The polypeptide or targeted particle of some embodiments of the invention can be administered to an organism per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.

Herein the term “active ingredient” refers to the polypeptide in association with a therapeutic or detectable moiety accountable for the biological effect.

Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, intraperitoneal, intranasal, or intraocular injections.

Conventional approaches for drug delivery to the central nervous system (CNS) include: neurosurgical strategies (e.g., intracerebral injection or intracerebroventricular infusion); molecular manipulation of the agent (e.g., production of a chimeric fusion protein that comprises a transport peptide that has an affinity for an endothelial cell surface molecule in combination with an agent that is itself incapable of crossing the BBB) in an attempt to exploit one of the endogenous transport pathways of the BBB; pharmacological strategies designed to increase the lipid solubility of an agent (e.g., conjugation of water-soluble agents to lipid or cholesterol carriers); and the transitory disruption of the integrity of the BBB by hyperosmotic disruption (resulting from the infusion of a mannitol solution into the carotid artery or the use of a biologically active agent such as an angiotensin peptide). However, each of these strategies has limitations, such as the inherent risks associated with an invasive surgical procedure, a size limitation imposed by a limitation inherent in the endogenous transport systems, potentially undesirable biological side effects associated with the systemic administration of a chimeric molecule comprised of a carrier motif that could be active outside of the CNS, and the possible risk of brain damage within regions of the brain where the BBB is disrupted, which renders it a suboptimal delivery method.

Alternately, one may administer the pharmaceutical composition in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a tissue region of a patient.

Pharmaceutical compositions suitable for use in context of some embodiments of the invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., acute liver disease) or prolong the survival of the subject being treated.

Dosage amount and interval may be adjusted individually to provide levels of the active ingredient are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

The polypeptide attached to a therapeutic moiety or targeted particle of some embodiments of the invention can be administered to the subject as a single treatment or in combination with other established or experimental therapeutic regimen to treat an inflammatory disease mediated by MAdCAM-1 (e.g. IBD) (e.g., before, simultaneously or following) including, but not limited to anti-inflammatory drugs (such as, corticosteroids and sulfasalazine), immunosuppressive drugs (such as, 6-mercaptopurine, cyclosporine and azathioprine), anti-TNFα, surgery (such as colectomy) and other treatment regimens known in the art.

As used herein the term “about” refers to ±10%.

The term “consisting of” means “including and limited to”.

EXAMPLES

Materials and Methods

Cloning—Sequences of the primers used are listed in Table 1 hereinbelow. Murine MAdCAM-1 D1 and D2 domains (hereinafter “D1D2”) were synthesized as a gBlock gene fragment (Integrated DNA Technologies, USA) and cDNA of Rat IgG2awas obtained from an in-house hybridoma clone using RNeasy minkit (Qiagen, Netherlands) and qScript cDNA synthesis kit (Quantabio, USA). The Fc of Rat IgG2awas amplified from the obtained cDNA using primer pair F1 and R1, the primers added the required homology sequences for the Gibson assembly to the 5′ ends of the amplicon. Following, the murine MAdCAM-D1D2 and IgG Fc were assembled in the pCMV3-FLAG plasmid using Gibson Assembly (New England Biolabs, USA). In a later stage, the entire construct was reassembled in the pcDNA3.4 expression plasmid to improve protein yield using primer pair F2 and R2. The point mutation for the negative control, hereinafter “mD1D2”, was generated using site-directed mutagenesis with primers F3 and R3 used to amplify the entire plasmid while generating the point mutation. The resulting PCR product was DpnI digested, purified on a 0.8% agarose gel, phosphorylated using T4 Polynucleotide kinase (New England Biolabs, USA), circularized using T4 DNA Ligase (New England Biolabs, USA) and transformed into chemically competent bacteria. To improve flexibility in the context of the LNPs, the CH1 domain of Rat IgG2awas later removed by PCR amplification of the entire plasmid using primers F4 and R4. SeeFIG.11for the sequence of the entire construct.

Cell Culture—Cell lines used: Expi293 (ThermoFisher Scientific), TK-1 (ATCC), HEK293 (ATCC). All cell lines were tested every 2 months for mycoplasma and discarded when positive. Expi293 cells were grown in Expi293 Expression Medium (ThermoFisher Scientific, USA) in disposable Erlenmeyer flasks at 37° C. and 8% CO2on a shaker rotating at 125 rpm. Cells were grown at densities between 0.3×106-5×106cells per ml. At least 3 passages after thawing, cells were transfected with the expression plasmid encoding the D1D2-Fc using the Expifectamine293 transfection kit (ThermoFisher Scientific, USA). 18 hours post transfection, enhancer 1 and enhancer 2 from the Expifectamine293 transfection kit were added to boost recombinant protein expression levels. Five days post transfection the culture medium was harvested for purification of the secreted protein.

TK-1 cells were grown in RPMI1640 medium supplemented with 10% fetal bovine serum, L-glutamine and Pen-Strep-Nystatin (Biological Industries, Israel). Cells were grown at densities between 0.3×106-2×106cells per ml in either T25 or T75 cell culture flasks (Greiner bio-one, Austria).

Protein purification—Conditioned medium was separated from the Expi293 cells by centrifugation at 300 g for five minutes. The supernatant was centrifuged again at 5000 g for 20 minutes to remove cellular debris. Using the Äkta FPLC protein purification system (GE Healthcare, UK), the protein was purified with a 1 ml Histrap column (GE Healthcare, UK). The sample was adjusted to the composition of the binding buffer (20 mM NaPO4at pH 7.4 and 500 mM NaCl) and passed through a 0.2 μm syringe filter (Sartorius, Germany) prior to loading into the FPLC. Flow rate during binding and elution was 0.5 ml/min and during washes 1 ml/min. The columns were washed with 20 ml of 0.5 M NaOH and 20 ml deionized water before it was equilibrated with 10 ml of binding buffer and loaded with the sample. Protein elution was effected using binding buffer supplemented with 0.5 M imidazole. The elution was performed stepwise with incremental increases of ˜30 mM imidazole per fraction until a final concentration of 0.5 M was reached. Following, fractions were loaded on SDS-PAGE gel and stained with Coomassie to determine which fractions contained the D1D2-Fc at sufficient purity. Pooled fractions were buffer exchanged to PBS using PD-10 desalting columns (GE Healthcare, UK). The purified protein was concentrated to >1 mg/ml using Amicon ultra centrifugal filters (EMD Millipore, USA), snap frozen in liquid nitrogen and stored at −80° C.

SDS-PAGE gel analysis—As the presence of the hinge domain should dimerize the D1D2, protein sample was diluted in sample buffer with or without the presence of 500 mM DTT to compare between monomers and dimers. Samples were loaded on a 10% polyacrylamide gel and either stained with Coomassie (Bio-rad, USA) or transferred to a nitrocellulose membrane using iBlot 2 Dry Blotting System (ThermoFisher Scientific, USA). Membrane was blocked with 5% skim milk in PBS for 2 hours at RT and incubated with anti-rat IgG-HRP (Jackson Immunoresearch, USA). Following, membrane was washed with PBS+0.01% Tween20 and developed using SuperSignal West Pico chemiluminescent substrate (ThermoFisher Scientific, USA). Chemiluminescence was measured with the Amersham Imager 600 (GE Healthcare, USA).

In-vitro binding—TK-1 cells were activated according to Y. Yang et al.30. Briefly, cells were washed with PBS and resuspended in resuspension buffer (HBSS with 10 mM HEPES buffer, 2 mM CaCl2and 2 mM MgCl2). Non-activated cells were kept on ice while activated cells were resuspended in pre-incubation buffer (HBSS with 10 mM HEPES buffer and 2 mM EDTA), incubated at RT for 30 minutes with gentle rotation, washed with PBS and finally resuspended in activation buffer (HBSS with 10 mM HEPES buffer, 2 mM CaCl2and 2 mM MnCl2). MAdCAM-D1D2-Fc and the controls were added to both activated and non-activated cells and incubated for 30 minutes at 4° C. Cells were subsequently washed and stained with either anti-human IgG or anti-mouse IgG conjugated to AlexaFluor647 (Biolegend, USA). Binding of D1D2-Fc to TK-1 cells was assessed by analyzing the fluorescence of the cells by flow cytometry. Binding of D1D2-targeted Cy5-LNPs was detected by flow cytometry directly using the Cy5 fluorescence (without addition of another antibody). In the binding experiments with Cy5-LNPs, the HBSS in the activation buffer was replaced with RPMI+10% FBS due to a high background using LNPs.

Preparation of LNPs—LNPs were prepared as previously described31by using the Nanoassemblr microfluidic mixer (Precision Nanosystems, Canada). The current gold standard for LNP production, DLin-MC3-DMA, was used as an ionizable lipid with a pKaof 6.44 that obtains a positive charge under acidic conditions. LNPs were prepared at pH 4.5 to ensure that DLin-MC3-DMA is ionized and hence siRNA encapsulation is maximized. Lipid mixture (DLin-MC3-DMA, DSPC, Cholesterol, PEG-DMG and DSPE-PEG-Maleimide at 50:10:38:1.5:0.5 molar ratio) in ethanol was mixed with siRNA in acetate buffer, pH 4.5, at a combined flowrate of 2 ml/min. Lipid and siRNA were mixed at a 1:3 volume ratio (1:16 w/w siRNA to lipid). For Cy5-labeled LNPs, 20% Cy5-labeled siRNA (SEQ ID NO: 9-10) was mixed with 80% unlabeled siRNA. The resulting LNPs were dialyzed against PBS for 24 hours to remove the ethanol and restore the pH to neutral. The hydrodynamic diameter and Zeta potential of the LNPs were measured by dynamic light scattering using disposable cuvettes in the Malvern Zetasizer (Malvern Instruments, UK).

Determining the siRNA encapsulation efficiency—LNPs were either lysed with Triton X-100 or not and the total amount of siRNA in the sample was measured with the Quant-iT Ribogreen RNA assay kit (ThermoFisher Scientific, USA). After subtracting the blank measurement, the encapsulation efficiency (in percentage) was calculated by (1-Non-lysed LNPs/Lysed LNPs)*100.

Conjugation of RG7 to LNPs and gel filtration—RG7/1.3 antibody (BioXCell, USA) was reduced in PBS supplemented with 1 mM DTT and 5 mM EDTA by incubating for 1 hour at room temperature. The DTT was subsequently removed by buffer exchange to 5 mM EDTA in PBS using 7K Zeba spin desalting columns (ThermoFisher Scientific, USA). Immediately following buffer exchange, the reduced antibody was added to the LNPs at a ratio of 0.67 mg antibody per ml of LNPs. The mixture was incubated for 2 hours at room temperature with gentle shaking followed by overnight incubation at 4° C. The following day, LNPs were separated from the free antibody using sepharose CL4B beads on a gel filtration column with PBS as the mobile phase. Fractions containing pure LNPs were pooled and concentrated to the initial volume using 100K Amicon centrifugal filters (EMD Millipore, USA). The loss of LNPs during conjugation and gel filtration was estimated by lysing LNPs prior to and following conjugation with Triton X-100 followed by measuring the amount of released siRNA using Quant-iT Ribogreen RNA assay kit (ThermoFisher Scientific, USA). Following to attach the generated MAdCAM-D1D2-Fc or MAdCAM-mD1D2-Fc fusion proteins or a DATK32 antibody (BioXcell, cat no: BE0034), the RG7-LNPs were incubated with the fusion protein or the antibody for 30 minutes at room temperature.

Dot blot analysis—Dot blot analysis was performed using Minifold I system 96-wells device (GE Healthcare, UK). A nitrocellulose membrane was added on top of two filter papers (Whatman plc, UK) and loaded into the 96 dot blot device. Wells were filled with PBS and vacuum was applied to wet the membrane. Samples (conjugated LNPs, unconjugated LNPs and several different amounts of free antibody) were added to the wells in a 100 μl volume followed by a vacuum to pass the sample through the membrane. Wells were washed with PBS and the device was again applied to a vacuum. Following, the membrane was blocked with 5% skim milk in PBS for 2 hours followed by incubation with anti-mouse IgG antibody linked to HRP (diluted in PBS with 1% skim milk) for 1 hour at room temperature. Next, the membrane was washed 3 times with PBST (5 minutes per wash) and the samples were detected by adding the SuperSignal West Pico chemiluminescent substrate (ThermoFisher Scientific, USA). Chemiluminescence was measured with the Amersham Imager 600 (GE Healthcare, USA).

Confocal microscopy—Cells were stained with Hoechst (nucleus) and anti-CD45-AlexaFluor488 (cell membrane). Following, cells were resuspended in PBS and images were taken with a Zeiss confocal microscope. The images were created by merging 11 frames from a Z-stack with 0.3 μm per frame.

Ex-vivo binding—Leukocytes were extracted from the spleen and mesenteric lymph nodes (mLN) of both healthy C57B1/6 mice and IL-10 KO mice that spontaneously developed colitis. For the mLN, tissue was homogenized and cells were strained through a 70 μm cell strainer. Cells were washed with PBS, centrifuged and the pellet was washed again with PBS and resuspended as a single cell suspension. For the spleen, tissue was homogenized, cells were strained through a 70 μm cell strainer. Following a single wash with PBS, red blood cells were lysed with ddH2O for a few seconds followed by addition of 10× HBSS to restore the solution to physiological conditions. Cells were strained a second time to ensure a single-cell suspension. Next, cells were stained with the appropriate cell surface markers (CD4, CD8, CD19, CD11b (Biolegend, USA, catalog numbers 100414, 100723, 115509 and 101259, respectively) and allowed to bind to either D1D2-LNPs, DATK32-LNPs or the respective control mD1D2-LNPs or isotype-LNPs. Following a 20-minutes incubation at 4° C., cells were washed and resuspended in FACS buffer. LNP binding for each leukocyte subpopulation was determined by the level of Cy5 as measured by flow cytometry. SeeFIG.13for the gating strategy used for cells from the mesenteric lymph nodes. SeeFIG.14for the gating strategy used for cells from the spleen.

Animal experiments—The Tel Aviv Institutional Animal Care and Use Committee approved the animal protocols for all in-vivo studies in accordance with current regulations and standards of the Israel Ministry of Health.

Wild type C57BL/6 and IL-10 KO C57BL/6 mice were kept in a specific-pathogen-free animal facility at Tel Aviv University. For the PAC colitis model, piroxicam was administered to IL-10 KO mice in the chow at a concentration of 200 ppm for a total period of 11 days. Freshly prepared LNPs encapsulated with either siCD45 (SEQ ID NO: 20, 41) or siNC (SEQ ID NO: 18-19) were conjugated to RG7 and purified using CL4B resin, as described hereinabove. Note that, in a 5′ to 3′ direction, bases No. 1, 2, 5, 6, 10, 11, 12, 13 and 18 of SEQ ID NO: 20, bases No. 13 and 17 of SEQ ID No. 41, bases No. 1, 2, 4, 6, 12, 14, 16, 18, 24 and 25 of SEQ ID NO: 18 and bases No. 3, 9, 11, 13, 23, 25, 26 and 27 of SEQ ID NO: 19 are modified 2′-O-Methyl RNA bases which provide resistance against nucleases; nucleotides No. 20 and 21 of SEQ ID NO: 20 and nucleotides No. 20 and 21 of SEQ ID NO: 41 are deoxy-ribonucleotides (and not ribonucleotides) and are linked together with a phosphorothioate bond; and the last nucleotide of SEQ ID NO: 18 is also a deoxy-ribonucleotide (and not ribonucleotide).

The LNPs were injected 7 days following administration of piroxicam, and CD45 expression in various organs was assessed 4 days post-injection. For healthy mice, CD45 expression was assessed 4 days post-injection. After sacrificing the animals, organs were homogenized and single cell suspensions were obtained using 70 μm cell strainers (Corning, USA). Cells were stained with antibodies against CD3 (Brilliant Violet 421), CD4 (APC-Cy7), CD8a (AlexaFluor 488), CD19 (PE-Cy5.5), CD11b (Brilliant Violet 650), F4/80 (Brilliant Violet 605) and CD45 (AlexaFluor 647) and analyzed by flow cytometry. All antibodies were purchased from BioLegend, USA.

10-weeks-old C57B1/6 female mice (Harlan laboratories) were injected with LNPs at a dose of 1.5 mg/kg and sacrificed 24 hours later. Blood was collected and analyzed by A.M.L. Israel for complete blood count (Sysmex and Advida-120) and biochemistry (Cobas-6000). Liver samples were used for histology (Histospeck, Israel). Splenic TNF-α and IL-6 levels were measured by DuoSet ELISA kits (R&D Systems, USA).

Therapeutic Efficacy Studies with siIFNγ:

To test in-silico optimized siRNA sequences against murine IFNγ, HEK293 cells stably expressing murine IFNγ were generated by transfecting the cells with a pcDNA3 plasmid harboring the murine IFNγ gene. Stably expressing cells were selected with G418. Next, cells were transfected with each of the siRNA sequences and with a control sequence (siNC). 48 hours post-transfection, cells were lysed, RNA was extracted and cDNA was generated. Silencing efficiency was determined using qPCR with SYBR green (seeFIG.10). The sequence that most efficiently silenced IFNγ (SEQ ID NO: 9-10) was used for the subsequent efficacy experiments. Note that, in a 5′ to 3′ direction, bases No. 1, 2, 4, 6, 12, 14, 16, 18 and 24 of SEQ ID NO: 9 and bases No. 3, 9, 11, 13, 23, 26 and 27 of SEQ ID NO: 10 are modified 2′-O-Methyl RNA bases which provide resistance against nucleases; and nucleotide No. 25 of SEQ ID NO: 9 is a deoxy-ribonucleotide (and not ribonucleotide).

For the efficacy studies, colitis was induced in 9-weeks-old female C57BL/6 IL-10 KO mice by mixing piroxicam (200 ppm) in the food. Freshly prepared LNPs encapsulated with either siIFNγ (SEQ ID NO: 9-10) or siNC (SEQ ID NO: 18-19) were conjugated to RG7 and purified using CL4B resin, as described hereinabove. LNPs were injected intravenously at day 4, 6, 8 and 10, post piroxicam administration. The volume of administered LNPs was calculated with the Ribogreen assay (Thermo Fisher Scientific, USA) for each LNP preparation to ensure a consistent dose of 1.5 mg siRNA per kg body weight. 30 minutes prior to injection, LNPs were mixed with the D1D2-Fc or mD1D2-Fc (final protein concentration of 60 μg/ml) and the total volume was completed to 200 μl. As a positive control, a monoclonal antibody against TNF-α was used (Clone MP6-XT22) that blocks the pro-inflammatory cytokine TNF-α.

Animals were randomized before piroxicam treatment and the study was performed in a double-blinded fashion. The experiment was performed by a CRO to ensure that the investigator was blinded during group allocation.

Body weight was recorded daily and mice were sacrificed at day 11 of the experiment. The colon was harvested to measure the length, to analyze colonic cytokine levels and to perform colon histology. Blood samples were collected to measure IL-6 and IL-1β expression levels.

Molecular imaging studies—1,4,7-Triazacyclononane-1,4-bis-acetic acid-7-maleimidoethylacetamide (NOTA-mal: Macro cyclics; Dallas, TX) was conjugated to MAdCAM-D1D2-Fc or MAdCAM-mD1D2-Fc fusion protein using a previously described approach with minor modifications32. Briefly, to 500 μg of fusion protein in 150 μl of phosphate buffer (pH 7.0) was added to freshly prepared 2-iminothiolane and then NOTA-mal in phosphate buffer (10% dimethyl sulphoxide) and 2IT such that final concentration ratios were: fusion protein, 1:NOTA-mal, 20:2IT, 10. The reaction was mixed by gentle pipetting, briefly centrifuged, and then placed in a 37° C. water bath for 30 minutes. Unbound chelator was removed using centrifugal filter units (3 kDa MW cut-off; Centricon, Millipore, Billerica MA), and the immunoconjugate was concentrated into phosphate buffer (0.1 M, pH 7.0) and stored in aliquots at −80° C.

For the radiolabeling, 6 volume equivalents of sodium acetate buffer were added to 178 GBq (4.78 mCi)64Cu in 5 μL HCl (0.04 N). The MAdCAM-D1D2-Fc or MAdCAM-mD1D2-Fc fusion protein (177 μg in 61 μL phosphate buffer) was then added to 15.8 μl of64Cu solution (31.7 MBq (858 μCi)). Following a 30 minutes incubation at room temperature, the degree of radiolabeling was assessed by thin-layer chromatography (TLC; Whatman No. 1 paper eluted with phosphate buffer; 0.1 M, pH 8, 100 mM EDTA) and radiochemical purity was found to be >95%. The radioimmunoconjugate was diluted with saline and sterile filtered (0.2 μm) before injection.

For the imaging studies,64Cu-labeled MAdCAM-D1D2-Fc or MAdCAM-mD1D2-Fc fusion protein (22 μg antibody, 3.12 MBq (84.3 μCi)) was injected into the tail vein. The mice were then anesthetized using isoflurane (1-4% in oxygen) and placed in a Bruker Albira multimodality (PET/CT) small-animal imaging system (Bruker Corporation; Woodbridge CT) for imaging. PET/CT data were collected for 30 minutes at 1, 3 and 24 hours post-injection (p.i.). At 24 hours p.i., the mice were euthanized by CO2inhalation, and an ex-vivo biodistribution analysis was carried out. Tissues were collected and weighed, and the radioactivity was assayed. The large colon was excised, measured and weighed to confirm the presence of colitis in the piroxicam treated group. PET and CT images were registered manually using AMIDE software33. Data from volumes of interest (VOIs) were used to calculate the biodistribution in selected tissues for the small animal PET imaging studies.

Statistical analysis—Data in the bar charts is expressed as mean ±95% confidence interval (CI). Boxplots center line represents the median, box represents the interquartile range and whiskers represent minimum and maximum values. Statistical analysis was performed in Python and GraphPad Prism. In general, when comparing 2 groups (for instance mD1D2 to D1D2), Student's t-test was used, and a one-way ANOVA was used when comparing 3 or more groups. A more complex statistical model (2-way interaction mixed model) was used in the molecular imaging section to confirm the correlation between colitis severity and D1D2 uptake. More specifically, for the CD45 silencing, a two-sided student's t-test was performed between the mD1D2-LNPs and D1D2-LNPs groups. For the toxicity study, a one-way ANOVA test was performed to exclude significant differences between any of the groups. For the efficacy study, a one-way ANOVA with Dunnett's post hoc test was used to demonstrate a significant difference between the D1D2-IFNγ-LNPs group and the negative control groups (D1D2-siNC-LNPs, mD1D2-IFNγ-LNPs and mock-treated). The same post hoc test revealed a significant difference between the groups D1D2-IFNγ-LNPs and mAb TNF-α only in the colonic TNF-α expression levels (p<0.05). Group size for the in-vivo silencing and toxicity study was 5 mice per group. In the efficacy study, group size was 12 mice per group. In all figures * for p<0.05, ** for p<0.01 and *** for p<0.0001.

Generation of a Novel MAdCAM-1-Fc Fusion Protein

The present inventors have designed a recombinant fusion protein containing two domains of the intestinal endothelium ligand MAdCAM-1, namely the integrin binding domains D1 and D2, fused to an Fc domain, hereinafter D1D2-Fc. Specifically, domains D1 (SEQ ID NO: 11) and D2 (SEQ ID NO: 12) of murine MAdCAM-1 were fused to the N-terminus of the Fc domain of rat IgG2a(including the hinge, excluding CH1, SEQ ID NO: 13). In addition, a signal peptide for secretion and a FLAG-tag were added to the N-terminus of the construct and a 6× HIS-tag was added at the C-terminus for purification purposes (seeFIGS.1A-Cand11-12, SEQ ID NOs: 14-15). Purity and size (˜50 kDa) were confirmed by SDS-PAGE (FIG.2A).

A mutated version of the D1D2-Fc was also generated, hereinafter mD1D2-Fc, and served as a negative control. Specifically, the mutated fusion comprised a D62A mutation corresponding to SEQ ID NO: 25 (seeFIGS.11-12, SEQ ID NOs: 16-17); this mutation in the D1 domain, has been reported to severely affect the ability of MAdCAM-1 to bind α4β7integrin14,15.

Following purification, the functionality of the generated D1D2-Fc and mD1D2-Fc was tested in-vitro by assessing their binding to TK-1 cells using flow cytometry. TK-1 cells have been shown to express high levels of α4β7integrin10,16and are therefore an excellent in-vitro model to test the functionality of the recombinant MAdCAM-1 constructs. Cells were either treated with Mn2+to create the high affinity (HA)-α4β7or with Ca2+as a low affinity (LA)-α4β7control. To verify this specificity, exclusive adhesion was confirmed in the presence of Mn2+to a monolayer of HEK293 cells that were stably transfected with membrane-bound MAdCAM-1 (FIGS.17A-B). As expected, in the absence of Mn2+, no significant cell binding was visible with both D1D2-Fc and mD1D2-Fc. However, following Mn2+treatment, while the mD1D2-Fc did not bind the cells, the D1D2-Fc significantly bound the activated cells expressing the HA-α4β7(FIG.2B). This demonstrated that the generated D1D2-Fc is functional and can distinguish between the HA and LA α4β7conformation; and that the mD1D2-Fc is a suitable negative control.

After validating that the D1D2-Fc protein binds exclusively to the HA conformation of integrin α4β7, the present inventors evaluated its ability to facilitate specific imaging of inflammatory leukocytes. To this end, each of the generated D1D2-Fc and mD1D2-Fc proteins was conjugated directly to a chelator, NOTA, to enable labeling with the radioisotope64Cu. Notably, the conjugation of the D1D2-Fc to NOTA did not affect protein functionality, as was tested by binding to TK-1 cells in vitro with flow cytometry (FIG.3A). Following, the protein-NOTA-64Cu conjugate was injected to healthy mice and to mice with colitis and 24 hours post-injection the mice were imaged by PET/CT. Injection of the radiolabeled D1D2 protein did not affect the colitis severity (FIG.3C). As shown inFIG.3B, injection of the D1D2-NOTA-64Cu resulted in an increased uptake in the gut of colitic mice while the signal in the liver decreased. The transaxial image in particular clearly displays the preferential gut uptake of the D1D2-NOTA-64Cu in mice with colitis. The difference in gut uptake of the mD1D2-NOTA-64Cu conjugate between healthy and colitic mice was not as dramatic. This uptake was significantly correlated to colitis severity with n=5 mice/group (p=0.014). A detailed statistical analysis on the correlation of colitis severity and specific D1D2-Fc uptake is shown inFIG.3D.

Generation of Lipid Nanoparticles Comprising a MAdCAM-1-Fc Fusion Protein as a Targeting Moiety

In the next step the generated D1D2-Fc fusion protein was used to decorate the surface of lipid nanoparticles (LNPs) in order to produce targeted LNPs with the ability to deliver e.g. siRNA specifically to these leukocytes.

To generate uniformly-sized LNPs that have a high siRNA encapsulation efficiency and minimal batch-to-batch variation, the NanoAssemblr™ microfluidic mixing device19; and an LNP formulation that was previously reported4,5,21using the ionizable lipid, DLin-MC3-DMA, which aids in siRNA encapsulation20, were utilized. The generated LNPs had mean diameter of ˜40 nm and a zeta potential of ˜−10 mV (Table 2 hereinbelow) and siRNA encapsulation efficiencies were close to 90% (FIG.2D) with polydispersity index of less than 0.2 (Table 2 hereinbelow). The size and uniformity of the particles was confirmed using transmission electron microscopy (FIG.2C).

Next, the LNPs were conjugated to RG7, a monoclonal antibody against rat IgG2a, using maleimide-thiol chemistry. The presence of RG7 on the surface of the LNPs was confirmed by dot blot (FIG.9). In this way the targeting protein (e.g. D1D2-Fc) can be bound to the LNP surface through affinity by the RG7 linker. Thus, the D1D2-Fc or mD1D2-Fc were subsequently attached to the RG7-LNPs by adding the protein to the LNPs solution to generate D1D2-targeted and mD1D2-targeted LNPs. The optimal concentration of the targeting protein was optimized such that the minimal amount of protein that achieves the highest level of LNP binding to TK-1 cells was added. Further, this linkage was sufficiently stable in freshly isolated mouse blood plasma for at least 1 hour at 37° C., making this approach feasible for in vivo administration (FIG.15).

In addition, the RG7 conjugation strategy was compared with two other options: direct conjugation to the DSPE-PEG-maleimide lipid using reduced cysteine residues in the D1D2 protein or by using a previously published conjugation strategy that involves ASSET (Anchored Secondary ScFv Enabling Targeting), a lipidated scFv against rat IgG2athat readily incorporates in the LNPs [Kedmi, R. et al.Nat. Nanotechnol.13, 214-219 (2018)]. When ASSET is incorporated into the LNPs, it can bind the D1D2-Fc by affinity. As shown inFIGS.16A-C, the RG7-mediated conjugation was superior over the other two methods.

Following preparation, conjugation and purification of the LNPs, the functionality of the D1D2-targeted LNPs was tested. To this end, binding of targeted LNPs encapsulating Cy5-siRNA (SEQ ID NO: 9-10) to cells (Mn2+or Ca2+treated) was tested by flow cytometry. As shown inFIG.2E, LNPs' binding increased dramatically when targeted by D1D2 as compared to mD1D2. Furthermore, confocal microscopy indicated internalization of the Cy5-labeled, D1D2-targeted LNPs (FIG.2F). Next, primary leukocytes from spleen and mesenteric lymph nodes (mLN) were extracted from healthy mice or mice with colitis and binding of D1D2-targeted LNPs was compared to both mD1D2-targeted and DATK32-targeted (a conformational insensitive mAb against α4β7integrin) LNPs. As expected, the DATK32 antibody-targeted LNPs were unable to distinguish between HA and LA α4β7integrin (as it displays similar binding levels between the Mn2+-treated group and the control group). Surprisingly, the DATK32-targeted LNPs were unable to bind to CD4+ T-cells. D1D2-targeted LNPs, on the other hand, demonstrated a strong binding preference to Mn2+-activated cells (seeFIGS.7-8and18A-19B). Notably, D1D2-targeted LNPs strongly bound to CD4+T-cells in a conformation-dependent manner. When injected intravenously, the LNPs were also able to bind primary leukocytes. Splenocytes were extracted 1 hour post-injection and Cy5-labeled cells were identified by flow cytometry (FIG.20).

CCL25 specifically increases α4β7integrin's affinity for MAdCAM-1 by binding to the CCR9 receptor. As expected, CCL25 treatment of cells enhances D1D2-targeted LNP binding compared to CXCL10-treated control cells (CXCL10 increases α4β7integrin's affinity for VCAM-1), seeFIG.21.

MAdCAM-1-Fc Fusion Protein Target Specific Delivery of a Payload to Gut-Homing Leukocytes in a Colitis Mouse Model

The feasibility of in-vivo gene silencing using the generated D1-D2-targeted LNPs was evaluated. In the first step, the strategy was used to silence a surrogate marker, the pan-leukocyte gene CD45. A piroxicam-accelerated colitis (PAC) in IL-10 knock-out (KO) mice on a C57BL/6 background23,24was used as a model of experimental colitis. Of note, IL-10KO mice with active colitis have strongly increased levels of MAdCAM-1 in the gut (Connor, E. M., et al. doi:10.1002/j1b.65.3.349), pointing to the importance of MAdCAM-1 in IL-10 deficient mice. CD45 silencing in healthy or colitic mock-treated mice was compared to healthy or colitic mice, respectively, that were injected with either of three different LNP formulations: D1D2-LNPs loaded with negative control siRNA (siNC, SEQ ID NO: 18-19); D1D2-LNPs loaded with CD45 siRNA (siCD45, SEQ ID NO: 20, 41) and mD1D2-LNPs loaded with siCD45 (SEQ ID NO: 20, 41). Five days post intravenous injection of the targeted LNPs, cells from the mesenteric lymph nodes (mLNs) were collected as T-cells there are activated by gut-tropic APCs. Cells from the spleen were also collected as a control peripheral lymphoid organ. Cells were analyzed for their CD45 expression using flow cytometry (seeFIGS.13-14for the gating strategy). As shown inFIG.4C, following injection of siCD45 D1D2-targeted LNPs, silencing levels in the spleen were lower compared to the CD4 population in the mLN. In the mLNs, silencing levels in CD8+T-cells and B-cells were lower compared to the CD4+T-cell population. The CD4+population was most effectively silenced by the siCD45 D1D2-targeted LNPs. As a negative control, healthy mice were also injected with D1D2-targeted LNPs encapsulating siCD45 yielding no significant results in any cell type from any organ (FIG.4B). This result could be attributed to the fact that in healthy mice there are less circulating lymphocytes with α4β7integrin configured in the HA conformation. Silencing results in colonic CD4+T-cells were not statistically significant, probably due to the fact that these cells have the integrin reconfigured to the LA state as they use αEβ7for their retention (FIG.4D).

Injection of the different LNPs did not affect the colitis severity in the colon as was concluded from colon histology in mice from the different groups (FIG.22).

Following, the potential adverse effects of the targeted LNPs was investigated. The presence of liver damage or systemic immune activation upon intravenous injection of the LNPs would provide a barrier for future clinical translation and hence render therapeutic efficacy studies obsolete. Thus, a toxicity study was performed to ensure that D1D2-targeted LNPs are safe upon systemic administration. Specifically, the levels of common liver enzymes secreted in the blood were measured and potential damage to the liver was assessed by histology. Furthermore, the immunogenicity of the targeted LNPs was assessed by performing a whole blood cell count and by measuring the levels of the pro-inflammatory cytokines TNF-α and IL-6 in the spleen. Two therapeutically irrelevant siRNAs were encapsulated to avoid interference with the immune system, namely the siCD45 (SEQ ID NO: 20, 41) and the siNC (SEQ ID NO: 18-19). The toxicity parameters in healthy mock-treated mice were compared to healthy mice that were injected with either of three different LNP formulations: D1D2-LNPs loaded with siNC; D1D2-LNPs loaded with siCD45 and mD1D2-LNPs loaded with siCD45.

Upon injection of any of the LNP formulations, no elevation of the liver enzymes aspartate aminotransferase (AST), alanine transaminase (ALT) and alkaline phosphatase (ALP) was observed (FIG.5B). In addition, liver histology did not reveal any excessive bleeding or liver damage (FIG.5D). Thus, the injection of the LNPs did not cause any observable liver toxicity. Furthermore, none of the LNP formulations induced unwanted immune responses as measured by a change in blood count (FIG.5C) and splenic TNF-α and IL-6 expression levels (FIG.5A). There was no significant difference in counts of platelets, neutrophils and lymphocytes between the groups and counts of eosinophils and monocytes in the blood were undetectable in almost all groups.

Based on the CD45 silencing results, a therapeutic target gene related to CD4+T-cell biology was explored for therapeutic efficacy studies. Because IFNγ is secreted by inflammatory Th1 cells and as IFNγ is causatively involved in experimental colitis25, this gene was chosen as a therapeutic target gene for an efficacy study using the generated D1D2-Fc as a targeting moiety in PAC IL-10KO mice. An optimized IFNγ siRNA (siIFNγ) sequence was selected and tested in vitro and resulted in over 80% gene knockdown (FIG.10). To test the therapeutic efficacy, D1D2-targeted LNPs or mD1D2-targeted LNPs loaded with siIFNγ (SEQ ID NO: 9-10) were injected into mice with colitis at days 4, 6, 8 and 10 upon initiation of colitis. A control mouse group injected with D1D2-LNPs loaded with a control siNC (SEQ ID NO: 18-19) was used to correct for possible therapeutic effects unrelated to IFNγ. In addition, mice without piroxicam were used as a healthy control and as positive treatment control in colitic mice a validated mAb against TNF-α was used (administered at days 4, 6, 8 and 10). The antibody against TNF-α was used as a positive control for amelioration of colitis. Blocking of TNF-α with mAbs has been well validated and anti-TNF-α mAbs are currently used in the clinic [e.g. infliximab (Ferreiro, R. & Barreiro-de Acosta,Infliximab: Pharmacology, Uses and Limitations10, 39-74, Nova Science Publishers, Inc., 2012)]. At day 11, mice were sacrificed and colitis severity was assessed in all groups (FIGS.6A-H).

Starting from day 8, there was a significant difference in weight change in the D1D2-silFNγ group as compared to the negative control groups (FIG.6B). Colonic levels of IFNγ decreased dramatically (˜2.5-fold) in D1D2-targeted LNPs loaded with siIFNγ as compared to the mD1D2-targeted LNPs control (FIG.6D). Colonic IFNγ levels also decreased moderately in mice treated with anti-TNF-α, likely due to an overall decrease in intestinal inflammation. Because IFNγ affects TNF-α expression [Vila-del Sol, V., et al.J. Immunol.181, 4461-4470 (2008)] and because these two cytokines have synergistic effects on NF-κB signaling [Wesemann, D. R. & Benveniste, E. N.J. Immunol.171, 5313-5319 (2003)], an associated reduction of other pro-inflammatory cytokines is expected by silencing IFNγ. Mice treated with D1D2-targeted LNPs loaded with siIFNγ and mice treated with anti-TNF-α mAb showed a strong decrease in colonic TNF-α levels (FIG.6C). The colonic TNF-α levels in mice treated with the mAb against TNF-α (positive control) was even a bit lower than the D1D2-targeted LNPs loaded with siIFNγ group (p<0.05). Furthermore, blood IL-6 and IL-1β levels decreased dramatically and equally both in mice treated with D1D2-targeted LNPs loaded with siIFNγ and mice treated with anti-TNF-α mAb (FIGS.6E-F). Colon length (colon shortening is an important marker of colonic inflammation) was significantly increased (p<0.0001) in the D1D2-targeted LNPs loaded with siIFNγ group as compared to the mD1D2-targeted LNPs control (FIG.6G). A significantly lower colon histological score (p<0.0001) further supported the improved therapeutic outcome in mice treated with D1D2-targeted LNPs loaded with siIFNγ (FIG.6H). These results were also in accordance with visual examination of photomicrographs of sections of the colon (FIG.23). Taken together, the data demonstrated a strong therapeutic response in mice treated with D1D2-targeted LNPs loaded with siIFNγ while the mutated control (mD1D2) did not lead to any significant improved therapeutic outcome.

Generation of Lipid Nanoparticles Comprising a MAdCAM-1 Amino Acid Seuqence as a Targeting Moiety

In order to avoid use of rat IgG Fc regions which could render MadCAM-1-Fc fusion protein immunogenic in a human subject, alternative LNP conjugation strategies are evaluated. A non-limiting example include generating a MadCAM-1-Fc fusion protein using the Fc region of human IgG. Alternatively, a small non-immunogenic peptide is integrated into the MadCAM-1 recombinant protein and a humanized scFv against this tag is either chemically conjugated to the LNP or a lipidated humanized scFv against this tag is used (similarly to the ASSET described hereinabove).

REFERENCES

Other References are Cited Throughout the Application