WO2020104802A1 - Islet cell engraftment - Google Patents

Abstract

A composition of polymer particles, wherein the polymer particles are loaded with an agent that is capable of enhancing the binding of islet cells to liver tissue, and wherein (i) the polymer particles comprise linked molecules, wherein the linked molecules comprise:a) asialoglycoprotein receptor (ASGPR)-binding molecules that are capable of binding the asialoglycoprotein receptor (ASGPR) on a cell surface, orb) a binding- molecule that is specific for a cell surface marker that is predominantly present in liver tissue; and/or (ii) the polymer particles are between about 0.5 and about 100 microns in diameter.

Description

Islet Cell Engraftment

The invention relates to a composition of polymer particles for enhancement of islet cell engraftment, and related methods and treatments for diabetes.

Background

In Type I diabetes (T1D), destruction of pancreatic beta cells by autoimmune processes leads to an absolute requirement for insulin replacement. Hypoglycaemia is the most common side effect of insulin treatment affecting approximately 25% of patients with T1D. Severe hypoglycaemia (SH), is defined as a low blood glucose requiring external assistance. Severe hypoglycaemia has an annual prevalence of 30- 40 %, affects over 10% of those with T1D and leads to impaired awareness of hypoglycaemia (IAH) with associated increased morbidity and mortality. Human islet allotransplantation is a therapeutic option for the treatment of T1D, stabilising glycaemic control, decreasing the frequency of recurrent severe hypoglycaemia, and restoring awareness of hypoglycaemia where compromised. Recent multi-centre Phase III studies have confirmed the efficacy of islet transplantation in preventing severe hypoglycaemia and have recommended consideration of this therapy for patients with T1D and IAH.

A major problem limiting transplant success is that >60% of transplanted islets fail to engraft into the liver following transplantation. Due to poor engraftment, islets from 2-3 pancreas donors are required in each recipient to impact on glycaemic control. Islets are clusters of polyhormonal cells transplanted in an avascular state. Following islet transplantation, the blood vessel supply between islets and the liver starts to be established by day 3. The majority of islet loss occurs predominantly within the first 3 days post-transplant. Hypoxia secondary to the lack of a blood supply is a major contributing factor although other mechanisms including inflammation secondary to auto- and alloimmunity may contribute to this islet loss. A blood vessel supply that is established more rapidly between host and donor may diminish this early loss of islets. Furthermore a microenvironment in the liver which favours their retention may also ameliorate this early loss and potentially aid the subsequent engraftment of islets into the liver by preventing their escape into the systemic circulation. Preconditioning the host liver with growth factors (GFs) creates a proliferative “niche” for liver regeneration and repair (Shimoda, M., et al. 2010. Diabetologia 53, 1669-79, which is incorporated herein by reference). This process involves re modelling and proliferation of hepatocytes and associated cells. Scaffolds containing GFs and extracellular matrix induced hepatocyte and non-parenchymal cell proliferation in normal and regenerating rat liver (Hammond, J. S . et al. 201 1. J. Hepatol. 54, 279-287, which is incorporated herein by reference), this in turn promotes the retention of cells including macrophages in the liver ¹⁷. Tri iodothyronine (Forbes, S . et al. 1998. Gene Ther. 5, 552-555, and Alwahsh, S . M. et al. 2018. Cell. Mol. Life Sci. 75, 1307- 1324, which are incorporated herein by reference) and a deleted form of hepatocyte growth factor (Hammond, J. S . et al.

201 1. J. Hepatol. 54, 279-287) act synergistically to enhance liver proliferation and enable in vivo retroviral gene transfer via the peripheral venous system (Forbes, S . et al. 1998. Gene Ther. 5, 552-555) . In addition, keratinocyte growth factor (KGF) has been used to enhance rat liver proliferation (Forbes, S . J. et al. 2000.

Gastroenterology 118, 591-8, which is incorporated herein by reference). Such studies provide proof of principle that such techniques may promote the retention and engraftment of other cells including islets into the liver. KGF is a small polypeptide member of the fibroblast growth factor family (FGF-7), binds to the KGF receptor and has proliferative and anti-apoptotic effects on various epithelial cells such as hepatocytes.

There are several challenges of administering the growth factors systemically; as they have short half-lives, low tissue penetration and effects on multiple organs with low local concentrations in the targeted organ necessitating dose escalation with off-target effects (Alwahsh, S . M. et al. 2018. Cell. Mol. Life Sci. 75, 1307- 1324).

What is required is a method to enhance islet engraftment in the liver and to maintain long-term graft function. Therefore, an aim of the present invention is to provide improved methods and compositions for enhancing islet engraftment in the liver and maintaining long-term graft function. SUMMARY OF INVENTION

According to a first aspect of the present invention, there is provided a composition of polymer particles,

wherein the polymer particles are loaded with an agent that is capable of enhancing the binding of islet cells to liver tissue, and

wherein

(i) the polymer particles comprise linked molecules, wherein the linked molecules comprise:

a) asialoglycoprotein receptor (ASGPR)-binding molecules that are capable of binding the asialoglycoprotein receptor (ASGPR) on a cell surface, or

b) a binding-molecule that is specific for a cell surface marker that is predominantly present in liver tissue; and/or

(ii) the polymer particles are between about 0.5 and about 100 microns in diameter.

The invention herein has advantageously provided a composition that can specifically target polymer particles to the liver and promote hepatocyte proliferation, which enhance islet cell engraftment. In particular, the invention shows engineered polymer particles that can target the liver specifically to achieve selective agent delivery, such as growth factor, and can promote islet engraftment and normalise blood glucose levels. The polymer particles can protect the agent for delivery and provide a controlled and localised release. The targeted agent delivery can also increase polymer particle retention in the liver through exploiting asialoglycoprotein receptor (ASGPR)- mediated endocytosis. The number of ASGPR in the plasma membrane of the hepatocyte ranges from 16000 to 35000 with a specific binding affinity toward galactose moieties, for example that can be attached on the polymer particles according to the invention. It has been demonstrated herein that targeted growth factor delivery to the liver can advantageously enhance islet engraftment and improved metabolic control in a mouse model of T1D.

The agent

The agent that is capable of enhancing the binding of islet cells to liver tissue may comprise a growth factor. The growth factor may comprise or consist of fibroblast growth factor. In one embodiment, the agent comprises or consists of keratinocyte growth factor (KGF). The agent may comprise or consist of a functional variant or equivalent of keratinocyte growth factor, such as Palifermin (trade name Kepivance, marketed by Biovitrum). Palifermin is a truncated human recombinant keratinocyte growth factor (KGF) produced in, for example Escherichia coli. The skilled person will understand that natural and unnatural variants of keratinocyte growth factor may exist, which may substantially retain the function of keratinocyte growth factor. Variants may include truncated or mutated variants. Recombinant human KGF contains 164 amino acids and a 16 a. a. histidine-based tag for a total length of 180 a. a. and has a predicted molecular mass of 21.2 kDa including his-tag. As a result of glycosylation, the recombinant protein migrates as two bands with an apparent molecular mass of 26 and 28 kDa in SDS-PAGE. In one embodiment, the agent comprises or consists of human KGF (FGF-7) accession number NP_002000 (https://www.ncbi.nlm.nih.gov/protein/4503705). In one embodiment, the agent comprises or consists of human KGF (FGF-7) of the sequence: CNDMTPEQMATNVNCSSPERHTRSYDYMEGGDIRVRRLFCRTQWYLRIDKRGK VKGTQEMKNNYNIMEIRTVAVGIVAIKGVESEFYLAMNKEGKLYAKKECNEDC NFKELILENHYNTYASAKWTHNGGEMFVALNQKGIPVRGKKTKKEQKTAHFLP MAIT (SEQ ID NO: 1), or a functional variant thereof.

In another embodiment, the agent comprises human KGF (FGF-7) with a HQ binding tag (for purification) of the sequence:

ERHVHQHQHQHQHQHQRCNDMTPEQMATNVNCSSPERHTRSYDYMEGGDIRV RRLFCRTQWYLRIDKRGKVKGTQEMKNNYNIMEIRTVAVGIVAIKGVESEFYLA MNKEGKLYAKKECNEDCNFKELILENHYNTYASAKWTHNGGEMFVALNQKGIP VRGKKTKKEQKTAHFLPMAIT (SEQ ID NO: 2), or a functional variant thereof.

Without being bound by theory, the mechanism of action of KGF could be to promote angiogenesis or to promote hepatocyte proliferation that in turn promotes angiogenesis. Therefore, other agents having such activities may be used. In one embodiment the agent may comprise a molecule capable of promoting angiogenesis and/or hepatocyte proliferation. The molecule may be a biological molecule such as a protein or peptide. For example, the agent may comprise or consist of VEGF (vascular endothelial growth factor), or a functional variant thereof. In another example, the agent may comprise or consist of HGF (hepatocyte growth factor), or a functional variant thereof. Natural or synthetic variants, or functional equivalents, of VEGF and HGF may be provided.

A functional variant of the agent may comprise homologues thereof. A functional variant of the agent, for example KGF, may comprise a truncated or modified variant that substantially retains the function of the wild-type agent, such as wild-type KGF. A modified variant can comprise one or more amino acid substitutions, addition or deletions. In another embodiment, a modified variant can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions or deletions. A variant may comprise a molecule having at least 70% identity to SEQ ID NO: 1 or 2. In another embodiment, a variant may comprise a molecule having at least 75% identity to SEQ ID NO: 1 or 2. In another embodiment, a variant may comprise a molecule having at least 80% identity to SEQ ID NO: 1 or 2. In another embodiment, a variant may comprise a molecule having at least 85% identity to SEQ ID NO: 1 or 2. In another embodiment, a variant may comprise a molecule having at least 90% identity to SEQ ID NO: 1 or 2. In another embodiment, a variant may comprise a molecule having at least 95% identity to SEQ ID NO: 1 or 2. In another embodiment, a variant may comprise a molecule having at least 98% identity to SEQ ID NO: 1 or 2. In another embodiment, a variant may comprise a molecule having at least 99% identity to SEQ ID NO: 1 or 2.

The agent may be a small molecule that is functionally equivalent to KGF. The agent may be a small molecule that is capable of enhancing the binding of islet cells in the liver. Additionally or alternatively, the agent may be a small molecule that is capable of enhancing the engraftment of islet cells in the liver. The binding or engraftment may be enhanced by at least 2%, or about 4%, or about 5%.

A combination of different agents, such as different growth factors, may be provided. In one embodiment, KGF, KGF functional variants or equivalents may be provided in combination with HGF or HGF functional variants or equivalents. In another embodiment, KGF, KGF functional variants or equivalents may be provided in combination with VEGF or VEGF functional variants or equivalents. In another embodiment, HGF, HGF functional variants or equivalents may be provided in combination with VEGF or VEGF functional variants or equivalents. In another embodiment, KGF, KGF functional variants or equivalents may be provided in combination with HGF or HGF functional variants or equivalents, and VEGF or VEGF functional variants or equivalents. Triiodothyronine (T3) may further be provided in one of the above combinations, for example the agents may comprise HGF, T3 and KGF (or their functional variants or equivalents) may be provided.

The agent(s) may be encapsulated within the polymer of the polymer particles. For example, the agent may be encapsulated within the polymer of the polymer particles by blending the agent with the polymer prior to emulsion. Additionally or alternatively, the agent(s) may be loaded into the particles after the particle creation, for example by soaking the particles in a solution or suspension of the agent.

The agent, such as KGF, may be provided in a therapeutically effective amount that is sufficient to enhance islet cell engraftment in the liver. The skilled person will understand that there will be a therapeutic window for a given agent and the effective amount may be readily determined by empirical dose studies without undue burden.

The agent, such as KGF, may be provided in the composition in the amount of between about 0.05 pg/mg and about 0.15 pg/mg of polymer particle. In another embodiment, the agent, such as KGF, may be provided in the composition in the amount of between about 0.01 pg/mg and about 0.2 pg/mg of polymer particle. In another embodiment, the agent, such as KGF, may be provided in the composition in the amount of between about 0.05 pg/mg and about 0.5 pg/mg of polymer particle. In one embodiment, the agent, such as KGF, may be provided in the composition in the amount of about 0.1 pg/mg of polymer particle.

The agent may be for controlled release. The polymer particles may provide controlled release of the agent in an aqueous environment, for example in vivo. The polymer particles may provide controlled release of the agent in the liver of a subject.

The release rate of the agent may be zero, first or second order. In one embodiment, the release rate is first order. The skilled person will understand that the choice of polymer material, the polymer blend, the porosity, and degradation rate, of the polymer articles can influence the agent release rate.

In one embodiment the release rate of the agent from the polymer particles is at least about 10% w/w over about 1 hour. Additionally or alternatively, the release rate of the agent from the polymer particles may be at least about 50% w/w over about 24 hours. Additionally or alternatively, the release rate of the agent from the polymer particles may be at least about 80% w/w over about 3 days.

In one embodiment the release rate of the agent from the polymer particles is between about 5% and about 20% w/w over about 1 hour. Additionally or alternatively, the release rate of the agent from the polymer particles may be between about 30% and about 60% w/w over about 24 hours. Additionally or alternatively, the release rate of the agent from the polymer particles may be between about 60% and about 90% w/w over about 3 days.

The polymer particles

The polymer particles may comprise or consist of one or more polymers. The polymer(s) may be synthetic or natural polymer(s). The polymer particles may comprise one or more polymer selected from the group comprising poly (a- hydroxyacids) including poly (D,L-lactide-co-glycolide)(PLGA), poly D,L-lactic acid (PDLLA), polyethyleneimine (PEI), polylactic or polyglcolic acids, poly-lactide poly- glycolide copolymers, and poly-lactide poly-glycolide polyethylene glycol copolymers, polyethylene glycol (PEG), polyesters, poly (e-caprolactone), poly (3- hydroxy-butyrate), poly (s-caproic acid), poly (p-dioxanone), poly (propylene fumarate), poly (ortho esters), polyol/diketene acetals addition polymers, polyanhydrides, poly (sebacic anhydride) (PSA), poly

(carboxybiscarboxyphenoxyphosphazene) (PCPP), poly [bis (p-carboxyphenoxy) methane] (PCPM), copolymers of SA, CPP and CPM (as described in Tamat and Langer in Journal of Biomaterials Science Polymer Edition, 3, 315-353. 1992 and by Domb in Chapter 8 of The Handbook of Biodegradable Polymers, Editors Domb A J and Wiseman R M, Harwood Academic Publishers), poly (amino acids), poly (pseudo amino acids), polyphosphazenes, derivatives of poly [(dichloro) phosphazene], poly [(organo) phosphazenes], polyphosphates, polyethylene glycol polypropylene block co-polymers for example that sold under the trade mark Pluronics™, natural or synthetic polymers such as silk, elastin, chitin, chitosan, fibrin, fibrinogen, polysaccharides (including pectins), alginates, collagen, peptides, polypeptides or proteins, copolymers prepared from the monomers of any of these polymers, random blends of these polymers, any suitable polymer and mixtures or combinations thereof. The polymer particles may comprise polymer selected from the group comprising poly(a-hydroxyacids) such as poly lactic acid (PLA), polyglycolic acid (PGA), poly(D,L-lactide-co-glycolide)(PLGA), poly D, L-lactic acid (PDLLA), poly-lactide poly-glycolide copolymers, and combinations thereof. In one embodiment, the polymer particles comprise PLGA.

In one embodiment, the polymer particle comprises PLGA 95:5. Alternatively, the polymer particle may comprise PLGA 50:50. Alternatively, the polymer particle may comprise PLGA 85: 15. Alternatively, the polymer particle may comprise any PLGA between PLGA 85: 15 and PLGA 95:5. Alternatively, the polymer particle may comprise PLGA 65:35. Alternatively, the polymer particle may comprise PLGA 72:25. PLGA having monomer ratios between the above PLGA embodiments may also be considered.

The polymer particles may comprise galactosylated polymer, such as galactosyalated PLGA.

The polymer particles may be biocompatible and/or biodegradable. By controlling the polymers used in the polymer particles their rate of degradation may be controlled. The polymer particles may completely degrade in a moist environment (i.e. 100% humidity), for example in vivo, over a period of 28 days or less. In another embodiment, the polymer particles may completely degrade in a moist environment (i.e. 100% humidity), for example in vivo, over a period of 20 days or less. In another embodiment, the polymer particles may completely degrade in a moist environment (i.e. 100% humidity), for example in vivo, over a period of 15 days or less. In another embodiment, the polymer particles may completely degrade in a moist environment (i.e. 100% humidity), for example in vivo, over a period of 10 days or less. In another embodiment, the polymer particles may completely degrade in a moist environment (i.e. 100% humidity), for example in vivo, over a period of 7 days or less. In another embodiment, the polymer particles may completely degrade in a moist environment (i.e. 100% humidity), for example in vivo, over a period of between 7 and 28 days. In another embodiment, the polymer particles may completely degrade in a moist environment (i.e. 100% humidity), for example in vivo, over a period of between 7 and 10 days. In another embodiment, the polymer particles may completely degrade in a moist environment (i.e. 100% humidity), for example in vivo, over a period of between 7 and 15 days.

The polymer particles may be solid, that is with a solid outer surface, or they may be porous. The particles may be irregular or substantially spherical in shape.

The polymer particles may have a size in their longest dimension of between about 0.5 and about 50 pm. Alternatively, polymer particles may have a size in their longest dimension of between about 0.5 and about 100 pm. In another embodiment, the polymer particles may have a size in their longest dimension of between about 1 and about 50 pm. The polymer particles may have a size in their longest dimension of between about 5 and about 50 pm. In one embodiment, the polymer particles are about 26 pm in diameter. In one embodiment, the polymer particles are at least 10 pm in diameter. The size of the polymer particles may refer to the average size of a population of polymer particles.

The ASGPR-binding molecules

In one embodiment, the asialoglycoprotein receptor (ASGPR)-binding molecules that are capable of binding the asialoglycoprotein receptor (ASGPR) on a cell surface may comprise or consist of a galactose moiety.

In another embodiment, the ASGPR-binding molecules may comprise or consist of molecules that compete for binding to the ASGPR with galactose. In one embodiment, the ASGPR-binding molecules may comprise or consist of antibodies, antibody fragments, antibody variants, or antibody mimetics.

The ASGPR-binding molecules that are capable of binding the ASGPR on a cell surface may comprise or consist of synthetic glucose-derivative polymers. For example synthetic glucose-derivative polymers described by Kim and Akaike (2001. The Journal of Biological Chemistry 276, 35312-35319. DOI

10.1074/jbc.M009749200), which is herein incorporated by reference. Such synthetic glucose-derivative polymers can comprise amphiphilic poly-(p-N-vinylbenzyl-d- glucuronamide) (PV6Gna) modified at the 6-OH position of glucose for hepatocyte recognition. The skilled person will recognise that other synthetic or natural molecules having ASGPR-binding ability may be provided and linked to the polymer particles.

Reference to ASGPR-binding may comprise specific ASGPR-binding. In particular, the molecule may have specific binding affinity for ASGPR.“Specific” is used to refer to the situation in which one member of a specific binding pair will not show any significant binding to other molecules on the cell surface other than its specific binding partner, such as ASGPR, and, e.g., has less than about 30% cross reactivity with any other molecule on the cell surface. In other embodiments it has less than 20%, 10%, or 1% cross reactivity with other molecules on the cell surface. The term is also applicable where an antigen binding domain of the binding molecule, such as an antibody, is specific for a particular epitope on the cell surface, such as an epitope on the ASGPR.

The term “antibody” as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding domain that specifically binds an antigen, whether natural or partly or wholly synthetically produced. The term also covers any polypeptide or protein having a binding domain which is, or is homologous to, an antibody binding domain. These can be derived from natural sources, or they may be partly or wholly synthetically produced. Examples of antibodies are the immunoglobulin isotypes (e.g., IgG, IgE, IgM, IgD and IgA) and their isotypic subclasses; fragments which comprise an antigen binding domain such as Fab, scFv, Fv, dAb, Fd; and diabodies. An “antigen binding domain” is the part of an antibody which comprises the area which specifically binds to and is complementary to part or all of an antigen. An antigen binding domain may be provided by one or more antibody variable domains. An antigen binding domain may comprise an antibody light chain variable region (VF) and an antibody heavy chain variable region (VH).

Antibodies may be polyclonal or monoclonal. A monoclonal antibody may be referred to as a “mAh”. It is possible to take monoclonal and other antibodies and use techniques of recombinant DNA technology to produce other antibodies or chimeric molecules which retain the specificity of the original antibody. As antibodies can be modified in a number of ways, the term“antibody” should be construed as covering any specific binding member or substance having a binding domain with the required specificity. Thus, this term covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, humanised antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. A humanised antibody may be a modified antibody having the variable regions of a non human, e.g., murine, antibody and the constant region of a human antibody.

It has been shown that fragments of a whole antibody can perform the function of binding antigens. Examples of binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CHI domains; (ii) the Fd fragment consisting of the VH and CHI domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment which consists of a VH domain; (v) isolated CDR regions; (vi) F(ab’)2 fragments, a bivalent fragment comprising two linked Fab fragments; (vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site; (viii) bispecific single chain Fv dimers and; (ix) “diabodies”, multivalent or multispecific fragments constructed by gene fusion.

The ASGPR-binding molecule, such as the galactose moiety, may be anchored to the polymer directly or via a linker molecule. The ASGPR-binding molecule, such as the galactose moiety may be anchored covalently to the polymer. The linker molecule may comprise a polymer, such as PEG. In one embodiment, the linker molecule may comprise a synthetic polymer.

The ASGPR-binding molecule, such as the galactose moiety, may be provided as part of a larger molecule, such as lactose. For example, the polymer particles may be functionalised with lactobionic acid. In one embodiment, the particles comprise PLGA (such as PLGA 50:50) functionalised with lactobionic acid.

Particle carrier

The composition may comprise a liquid carrier. In particular, the polymer particles may be suspended in an liquid carrier. The liquid carrier may be aqueous. The liquid carrier may comprise water. The liquid carrier may comprise a buffer, such as PBS. The liquid carrier may comprise cell culture medium, such as chemically defined cell culture medium. The culture medium may comprise DMEM (Dulbecco's Modified Eagle Medium). The skilled person will understand that any pharmaceutically acceptable medium could be used.

Islet cells

In one embodiment, the composition may further comprise islet cells.

The skilled person will recognise that the islet cells may be from any suitable source that is compatible with an intended recipient subject, such they function therein to produce insulin. For example, in an embodiment comprising islet cells, the islet cells may comprise human islet cells. The human islet cells may be harvested from a human. In another embodiment, the islet cells may be from a non-human animal that has been genetically modified for human immune system compatibility. The islet cells may be CD matched with an intended recipient subject. In another embodiment, the islet cells may be derived/differentiated from stem cells, such as reprogrammed cells or induced pluripotent stem cells.. The stem cells may be derived from the intended recipient subject.

The islet cells may be functional such that they are capable of producing insulin. The amount of insulin produced may be an effective amount as determined by the ability of a subject to show glycemic control post islet transplantation. The skilled person will recognise the ability for glycemic control using standard tests and procedures. For example, glycemic control can be determined by a physiological mixed meal tolerance test and observation of a detectable C-peptide (e.g. >50pmol/L). Additionally or alternatively, a composite scoring system may be used to observe appropriate levels of HbAlc and/or glucose in the patient.

In an embodiment comprising islet cells, about 2xl0⁵ islet cells may be provided in the composition. In another embodiment comprising islet cells, about 1.5xl0⁵, lxlO⁵ 8xl0⁴, or 5xl0⁴ islet cells may be provided in the composition. In another embodiment comprising islet cells, at least about 5xl0⁴ islet cells may be provided in the composition. In another embodiment comprising islet cells, at least about 1.5xl0⁴ islet cells may be provided in the composition. In another embodiment comprising islet cells, at least about 2xl0⁵ islet cells may be provided in the composition. In another embodiment comprising islet cells, between about 5xl0⁴ and about 7xl0⁵ islet cells may be provided in the composition. In another embodiment comprising islet cells, between about 5xl0⁴ and about 5xl0⁵ islet cells may be provided in the composition. In another embodiment comprising islet cells, between about 5xl0⁴ and about 2xl0⁵ islet cells may be provided in the composition.

The composition of polymer particles may be contained in a container suitable for delivery of the composition into the liver or arterial delivery. In one embodiment, the composition of polymer particles is contained in a syringe.

Other aspects

According to another aspect of the present invention, there is provided a method of islet cell engraftment in liver tissue, the method comprising:

A) delivering the composition comprising islet cells according to the invention herein into the liver tissue;

B) delivering the composition according to the invention herein into the liver tissue, and subsequently or concurrently delivering islet cells into the liver tissue; or

C) delivering islet cells into the liver tissue and subsequently delivering the composition according to the invention herein into the liver tissue.

According to another aspect of the present invention, there is provided a method of treatment or prevention of diabetes in a subject, the method comprising:

A) delivering the composition comprising islet cells according to the invention herein into the liver of the subject;

B) delivering the composition according to the invention herein into the liver of the subject, and subsequently or concurrently delivering islet cells into the liver tissue; or

C) delivering islet cells into the liver tissue and subsequently delivering the composition according to the invention herein into the liver tissue.

According to another aspect of the present invention, there is provided the composition according to the invention for use as a medicament. According to another aspect of the present invention, there is provided the composition according to the invention herein for use in the treatment or prevention of diabetes in a subject.

According to another aspect of the present invention, there is provided the composition according to the invention herein for use in the treatment or prevention of liver disease in a subject. The liver disease may comprise haemophilia. The liver disease may comprise metabolic disease of the hepatocyte.

In one embodiment, the agent (i.e. for delivery) may comprise a gene therapy agent. In particular, the agent may comprise nucleic acid arranged to modify/edit, enhance, suppress or replace a gene, or expression thereof, in one or more liver cells such as hepatocytes or sinusoidal cells. For example, clotting factors (such as Factor VIII) are produced in liver sinusoidal cells. The present invention may deliver a gene therapy agent to such cells to increase expression of clotting factors, such as Factor VIII. In such an embodiment, the gene therapy agent may comprise nucleic acid encoding the clotting factor. The gene therapy agent may be delivered with a viral or non-viral transfection agent.

The liver disease may comprise a metabolic disease of the hepatocytes. Therefore, in one embodiment, the agent for delivery may comprise a cell for transplantation, such as a hepatocyte cell.

The composition may be for use in combination with delivering islet cells into the liver of the subject. The liver tissue may be in vivo. In particular, the liver tissue may be in a subject. The subject may be a patient in need of functional insulin-producing islet cells. The subject may be a diabetic subject. The subject may be mammalian, such as human.

The diabetes may be type I diabetes. The composition and/or islet cells may be delivered into hepatic portal vein of the subject. Advantageously, delivery into the hepatic portal vein of the subject can be used in embodiments wherein the particles are too large for intravenous delivery. In another embodiment, delivery may be intravenously (IV), for example when the particle sizes are sufficiently small (e.g. 5-8 microns). In another embodiment, the delivery may be arterial delivery.

The islet cells may be provided in a pharmaceutically acceptable carrier. The islet cells may be provided in an aqueous carrier, such as a buffer. In one embodiment, the islet cells are provided and/or stored in cell media

In an embodiment, about 2xl0⁵ islet cells may be administered. In another embodiment, about 1.5xl0⁵, lxlO⁵ 8xl0⁴, or 5xl0⁴ islet cells may be administered. In another embodiment, at least about 5xl0⁴ islet cells may be administered. In another embodiment, at least about 1.5xl0⁴ islet cells may be administered. In another embodiment, at least about 2xl0⁵ islet cells may be may be administered. In another embodiment, between about 5xl0⁴ and about 7xl0⁵ islet cells may be administered. In another embodiment, between about 5xl0⁴ and about 5xl0⁵ islet cells may be administered. In another embodiment, between about 5xl0⁴ and about 2xl0⁵ islet cells may be administered.

The composition may be delivered over a period of 10 minutes to 3 hours, for example via the hepatic portal vein. In another embodiment, the composition may be delivered over a period of 20 minutes to 1 hour, for example via the hepatic portal vein. The composition may be delivered over a period of 20 minutes to 50 minutes, for example via the hepatic portal vein.

The islet cells may be delivered over a period of 10 minutes to 3 hours. In another embodiment, the islet cells may be delivered over a period of 20 minutes to 1 hour, for example via the hepatic portal vein. The islet cells may be delivered over a period of 20 minutes to 50 minutes, for example via the hepatic portal vein.

In one embodiment, the islet cells are administered after the composition of polymer particles. In another embodiment, the islet cells are administered concurrently with the composition of polymer particles, as a separate composition or as a mixed composition. The administration of the islet cells may be between 1 hour and 3 days after administration of the composition of polymer particles. In another embodiment, the administration of the islet cells may be between 12 hours and 2 days after administration of the composition of polymer particles. In another embodiment, the administration of the islet cells may be about 24 hours after administration of the composition of polymer particles.

In one embodiment, the administration of the composition and/or islet cells may be repeated.

According to another aspect of the present invention, there is provided the use of composition according to the invention herein for enhancing islet cell engraftment in the liver of a subject.

According to another aspect of the present invention, there is provided a kit for islet cell engraftment in liver tissue, the kit comprising:

A) a composition of polymer particles according to the invention herein; and

B) a composition of islet cells.

The kit may further comprise a syringe for storing and/or delivering the composition according to the invention.

The particles according to any aspect of the invention may be labelled, for example for tracking in vivo. The label may comprise fluorescent label, such as rhodamine. Additionally or alternatively, the particles according to any aspect of the invention may be tagged, for example to aid isolation and/or sorting. The skilled person will be familiar with a number of standard labelling and tagging methods and molecules that can be applied to the particles.

The skilled man will appreciate that the optional features of the first aspect, or any aspect or embodiment, of the invention can be applied to all aspects of the invention.

Embodiments of the invention will now be described, by way of example only, with reference to the following examples. Figure 1: Cell proliferation in liver and other organs after injection of growth factors. Mice received x2 injections of the following growth factors or vehicle 48 hours apart and were culled 24 hours following the last injection of growth factor or vehicle. One hour before cull, BrdU was injected i.p. to detect cell proliferation. The number of BrdU positive cells (=proliferating cells) was evaluated by the Operetta system and Columbus software. (A) vehicle (100 pL saline) s.c., HGF 250 pg/kg IV, T3 4 mg/kg s.c, KGF 1.25 mg/kg s.c, or combination of all three GFs. (B) Immunofluorescence staining for BrdU in various organs in mice receiving KGF 1.25 mg/kg s.c. x2 doses or (C) vehicle 100 pL saline s.c x2 doses. Arrows indicate BrdU positive cells. DAPI (blue) indicate cell nuclei. Data represent the mean ± SEM.

Figure 2: Characterisation of galactosylated PLGA particles. (A) Representative scanning electron micrographs of particles. (B) Size distribution of particles with average size diameter and entrapment efficiency (%) for total protein. (C) Individual daily release percentage, and (D) Cumulative release kinetics for the total protein payload over a three week period. Data represent mean ± SD. EE - entrapment efficiency.

Figure 3: Hepatic portal vein injection of 26 pm galactosylated particles via the HPV provides specific hepatic localisation. PLGA particles uncoated (22 pm) and galactosylated (2, 10 and 26 pm) were injected into mice (1 mg, HPV) to determine particle distribution in organs. (A) Representative fluorescent images (^c40) of organs extracted 24 hours after injection of particle formulations. Cryosections (30 pm) were fixed and stained: DAPI (blue, cell nuclei); fluorescent particles (red epifluorescence) are highlighted (white arrows). Scale bar= 60pm. (B) Mean particle counts (logarithm, from 11 slides) quantified per tissue grouped in formulations indicated. Bars represent the mean ± SD, n=3 mice per group. (C) Percentage of galactosylated (26 pm) and uncoated (22 pm) PLGA-particle biodistribution in various organs. (D) Small particles (2 pm, white arrows) localise with F4/80 positive cells in liver tissue suggesting phagocytic uptake. Scale bars 60 pm. Figure 4: Dose response study of KGF-PLGA particles (26 pm) that promote liver proliferation safely. (A) Serum ALT activity, bilirubin and albumin levels from mice receiving KGF-PLGA particles (HPV) versus vehicle (30% FCS only) for 24 hours at indicated doses. (B) Levels of exogenous human KGF in serum samples collected from mice receiving KGF s.c. 1.25 pg/day s.c. x2 doses for 2 days, vehicle (30% FCS) (HPV), 0.01 mg KGF-PLGA and 0.1 mg KGF-PLGA (HPV). (C) Macroscopic view of the liver before KGF-PLGA administration and 24 hr after injection via HPV. (D) H-E staining for liver tissues. Mice received vehicle (FCS), O.Olmg/lOO pL or 0.1 mg/100 pLgalactoylated KGF -particles (26 pm) via HPV injection, 24 hrs prior to culling. P < 0.001 for vehicle and 0.01 mg vs. 0.1 mg KGF-PLGA particles, One-way ANOVA, Tukey’s post-hoc. Data are mean ± SEM, n=3 mice per group. HPV: hepatic portal vein.

Figure 5: Effects of targeted KGF-PLGA delivery via HPV versus KGF s.c. on liver cell proliferation. (A) KGF 1.25 mg/kg s.c. x2 doses, 0.1 mg KGF-PLGA (HPV) or PLGA alone (HPV), 30% FCS (HPV), 72 hr following the first injection. BrdU was administered lmg i.p before cull. Representative micrographs of dual immunostaining applied on liver sections for BrdU (green, cell proliferation), HNF4a (red, hepatocytes) and DAPI (blue, nucleus staining). In the upper panels, white arrows show BrdU⁺ non- parenchymal cells (HNF4a ), as magnified in the inset. In the middle row, mainly dual positive nuclei are observed (orange-yellow) (white arrows), while in the lower panel (PLGA alone treated mice), the proliferating cells were mainly non-parenchymal. Inset (X400) show higher magnified regions of liver sections of different treatments. (B) Percentage of the proliferating (BrdU⁺) cells in each mouse group. (C) Fraction of proliferating hepatocytes (BrdU⁺, HNF4a⁺) to the total proliferating cells (BrdU⁺).

Cell counting by Operetta system and Columbus. Scale 100 pm. * P<0.05 using one-way ANOVA Tukey’s post-hoc.

Figure 6: Islet detection in liver tissue 72 hrs post islet-transplantation. Marginal intraportal islet transplants with n=400 islets alone and islets (n=400) co-transplanted with galactosylated KGF-PLGA particles (O. lmg) were performed and mice (n=3 per group) culled 72 hours post-transplant. (A) Dual immunofluorescence staining for islets (insulin - b cells and glucagon- a cells) in liver tissues. Scale 100 pm. (B) Number of b- cells determined in > 8 liver sections at least 50 pm apart per mouse. Insulin-positive cells were counted in > 15 (^c20) fields; Mean ± SEM shown. *P<0.05 by unpaired t- test.

Figure 7: Biochemical assays and assessment of liver fibrosis in diabetic C57B1/6 mice transplanted with a marginal islet mass ± KGF-PLGA particles (O.lmg) monitored for 6 weeks. (A) Marginal mass intraportal islet transplantation ± KGF- PLGA particles (O. lmg). Daily blood glucose concentrations are shown. Islets alone vs islets + KGF-PLGA (O.lmg) particles * P= 0.03 One-way ANOVA Tukey’s post-hoc. (B) Picrosirius red (PSR)-stained liver tissues for fibrosis, (C) Percentage of collagen (red pixels) to liver tissue (yellow). Each value represents the mean of 8 (^c 10) fields per mouse liver section. Data were generated using InForm software after using automated slide Scanner.

Figure 8: Regeneration and insulin content of pancreases from diabetic C57B1/6 mice transplanted with a marginal islet mass ± KGF-PLGA particles (O.lmg) assessed at 6 weeks post transplant. (A) Dual immunofluorescence staining for insulin and BrdU on pancreatic tissue. Representative micrographs show basal proliferation in all groups. White arrows - BrdU⁺ nuclei (proliferating cells) in the pancreas. No dual positive cells (Insulin⁺ BrdU⁺ / Ki-67⁺) were detected by Operetta imaging. (B) Pancreatic insulin levels normalised to protein content. Data represents mean ± SEM, n= 6 mice per group.

Figure 9: Intravenous (tail vein) injection of 10pm galactosylated PLGA particles stay predominantly in the lung and spleen. PLGA particles (uncoated 2pm, 22pm and galactosylated 10pm) (1 mg, i.v.) and control vehicle (saline) were injected into mice to investigate particle distribution in tissues. Scale bar=60pm. (A) Representative fluorescent images (x40) of organs (indicated left) extracted 24 hour after injection of different bioparticle formulations. Cryosections (30 pm) were fixed and stained with DAPI (blue) to label cell nuclei. Fluorescent particles (red epifluorescence) are highlighted (white arrows). Galactosylated particles of 10 pm were trapped in the microcirculation of the lungs and spleen and therefore larger diameter particles were not tested. (B) Particles quantified per tissue grouped by formulation. Average particle counts (from 11 slides) quantified per tissue grouped in formulations indicated. (C) The percentage distribution of particles between tissues.

Figure 10: Administration of 1 mg and 5 mg KGF-PLGA particles via the HPV. Mice were transplanted with 1 mg and 5 mg KGF-PLGA particles along with PLGA alone via the HPV. (A) Percentage weight loss 24 hrs after particle administration. (B) Alanine aminotransferase, (C) bilirubin, and (D) albumin. (E) Macroscopic view of the liver at time of mouse cull for each treatment (scale=100pm), and corresponding microscopic H-E staining for liver sections (scale=200pm) (F). N=2 -3 mice / group. Blue arrows - patchy areas. Biochemical serum analysis for liver injury markers.

Figure 11 : Serum biochemistry 6 weeks post transplantation of islets ± 0. 1 mg KGF-PLGA particles. (A) ALT, (B) AST, (C) albumin. Data are mean± SEM, and statistical analysis was performed by one way ANOVA-Tukey’s post hoc.

Example 1 - Keratinocyte growth factor-releasing particles enhance islet engraftment and improve metabolic control in Type 1 diabetes

Summary

Pancreatic islet transplantation into the liver may stabilise glycaemic control in patients with Type 1 diabetes (T1D). However due to poor islet engraftment after intraportal infusion, up to three donor pancreases are required for each recipient to impact on glycemic control. This study aims to enhance islet engraftment in the liver and to maintain long-term graft function. Diabetic mice were transplanted with a marginal islet transplant (n=400 islet equivalents (IEQ)) via the hepatic portal vein with and without FDA-compatible, keratinocyte growth factor (KGF)-loaded galactoslyated polylactide-co-glycolic acid (PLGA) particles. These KGF-PLGA particles specifically target the liver, promoting hepatocyte proliferation. Glycaemic control was superior in mice receiving islets in combination with the KGF loaded PLGA particles with blood glucose concentrations returning to normal in 6 out of 8 mice versus 0 out of 8 mice in the islet transplant alone group (p<0.01), by day 30 post-transplant. On histology, proliferation of cells was confined to the liver and the number of b-cells was significantly higher in liver sections of mice receiving islets and KGF-loaded particles compared to mice receiving islets alone 72 hr post transplant (1.7 fold; p<0.05). This work shows liver targeted PLGA particles achieve selective KGF delivery to the liver promoting islet engraftment and normalising blood glucose levels.

Introduction

We hypothesised that targeted growth factor (GF) delivery to the liver would promote hepatocyte proliferation leading to enhanced islet engraftment and improved metabolic control in a mouse model of T1D.

We aimed to firstly examine the growth factor that caused the greatest cell proliferation in the liver. The second aim was to engineer a polymer that contained the growth factor for its targeted delivery to the liver to create a microenvironment suitable for islet engraftment in the liver and to identify their biodistribution and release kinetics in vivo. Our third aim was to co-transplant KGF-loaded galactosylated PLGA particles with a marginal mass of islets via the clinically relevant hepatic portal vein of diabetic mice and determine their glycemic control over a 6 week period.

Materials and Methods

Animals

Male C57B1/6 mice of 8-10 weeks old were purchased from Harlan Laboratories (UK). Mice were housed under standard conditions in a 14-hr light to 10-hr dark cycle and given standard chow and water ad libitum. Animal procedures and experiments were conducted in accordance with ARRIVE guidelines and University of Edinburgh Institutional Animal Care Use Committee Protocols. At the end of all experiments, animals were humanely culled by high C0₂ concentration.

Injection of growth factors and proliferation of cells within liver

Twelve week old C57B1/6 mice were administered a range of growth factors to determine the GF that caused the greatest proliferation of cells within the liver. In pilot experiments 5mg/kg KGF (Sobi Pharmaceuticals, Sweden) intravenously (i.v.) was administered. However, 3 out of 8 mice developed anorexia and weight loss over a 15 day period and the dose of KGF (Sobi Pharmaceuticals, Sweden) was subsequently reduced to 1.25mg/kg subcutaneously (s.c).

Twelve week old C57B1/6 mice (n=8/group) received the following GFs or vehicle: Group 1) KGF 1.25mg/kg s.c., Group 2) HGF 250 pg/kg i.v., Group 3) T3 4 mg/kg s.c., Group 4) all three GFs and Group 5) 100 pL saline (vehicle), at time point day -2 and day 0 of the experiment. Mice were pulsed with 5-Bromo-2'-Desoxyuridine (BrdU, 1 mg dissolved in PBS) via a single intraperitoneal (i.p.) injection 48hrs later and were culled lhr following BrdU administration.

The liver lobes (caudal, right, medial, and left) were harvested, stained with different guiding colours, and fixed for 24 hr by buffered 4% formaldehyde, embedded in paraffin and cut serially (5 pm). Superfrost™ Plus slides were blocked with protein block (Spring Bioscience, UK) for 1 hr at room temperature and incubated overnight at 4 °C with the antibodies listed in Table 1. Primary antibodies were detected using fluorescent conjugated secondary antibodies (Alexa 488 and Alexa 555; Invitrogen, UK). Nuclei were stained with DAPI and mounted with Fluoromount (SouthernBiotech, UK) before imaging via an Operetta high content imaging system (PerkinElmer, UK) at x lO magnification and subsequently analysed using the Columbus software (PerkinElmer, UK). An average of 20-30 images (0.5 mm² field) were taken per section (25-30 images/section for liver). Isotype IgG antibodies replaced primary antibodies to serve as negative controls.

Immunostainings were performed for hepatocyte nuclear factor (HNF4a) as a marker of hepatocyte proliferation and for BrdU as a marker of total cell proliferation. The growth factor that was associated with the greatest proliferation index within the liver was selected and given subcutaneously prior to administering islets via the hepatic portal vein (HPV), to determine if glycaemic control was improved. The growth factor associated with the greatest hepatocyte proliferation was subsequently incorporated into a galactosylated PLGA particle. PLGA particles preparation, assembly, and characterisation

PLGA 50:50 lactide :glycolide ratio (52 kDa, DL-lactide, Lakeshore Biomaterials, USA) was functionalised with lactobionic acid (LB, Sigma Aldrich, UK) according to a procedure published by Yoon et al. ²⁴. Particles were fabricated from 5.5% PLGA in dichloromethane (DCM, Fisher, UK) by a double emulsion method. The polymer solution, plus aqueous solution of IX phosphate buffer saline (PBS, Sigma Aldrich, UK) containing 0.1% w/v KGF (ORF Genetics, Iceland) and 0.9% human serum albumin (HSA, Sigma Aldrich, UK) were homogenised using a high speed Silverson L5M homogeniser (Silverson Machines, UK). Next, the particles were stirred for 4 hours, filtered and then freeze dried before harvesting. Characterisation by scanning electron microscopy (SEM) was performed with a JEOL 6060L SEM imaging system (JEOL Ltd., Hertfordshire, UK). The mean diameter and particle size distribution was determined with the Coulter LS230 particle size analyser (Beckman, UK).

Total protein release (HSA+KGF) from the microparticles was measured by bicinchoninic acid assay (Sigma Aldrich, UK). Briefly, 25 mg of the microparticles were suspended in 1 ml PBS and gently rocked on a 3-dimensional shaker (Gyrotwister, Fisher Scientific UK Ltd) at 5 rpm in a humidified incubator at 37°C. Samples of PBS supernatant were collected at specified time points up to day 21 and replaced with fresh PBS to the end point.

Biodistribution of particles via the HPV or tail vein

For biodistribution studies, a fraction of the PLGA particle formulations was rhodamine-labelled for detection by fluorescence. Mice (n=3) received a single injection of 1 mg PLGA particles in 100 pL 30% fetal calf serum (FCS). Non- galactosylated (uncoated) PLGA particles (2, 10, 22 pm mean diameter) and in separate experiments galactosylated PLGA particles (2, 10, 26 pm mean diameter) were injected using a 30 gauge needle via the HPV. Similarly non-galactosylated PLGA particles (2, 10, 22 pm mean diameter) and galactosylated PLGA particles (2, 10 pm mean diameter) were injected via the tail vein (i.v.); control experiments with vehicle injections were also run (FCS injection alone). Briefly, mice were placed on a heating pad under anaesthesia with 2-2.5% isoflurane delivered in oxygen. Analgesia was administered s.c. (0.015 mg/mL buprenorphine, 50 pL) peri-operatively and a laparotomy was performed to expose the HPV. A 30G syringe was loaded with 1 mg of PLGA particles, mixed well to form a suspension and particles administered directly into the HPV; bleeding was controlled by application of haemostatic gauze and pressure on the injection site. The abdominal muscle layer was stitched with 6/0 vicrylsuture and the skin clamped with metal clips or interrupted sutures. Mice were given 500 pL saline s.c. and were recovered in their home cage on a heat pad for at least 30 min. For intravenous PLGA delivery, mice were anaesthetised as before and particles injected into the tail vein. Mice were humanely culled 24 hours post injection, and blood samples collected by cardiac puncture. The liver, lung, kidney, heart and spleen were harvested for further analysis. Cryosections (8-30 pm) were fixed and stained with DAPI to label cell nuclei. Epifluorescent-labelled particles were detected using fluorescence microscopy using an AlexaFluor 546 nm filter set. Particles were counted from an average of eleven 10X fields per organ.

Liver frozen sections (8 pm) were fixed in methanol-acetone (1 : 1) and further used to detect rhodamine-labelled PLGA particles in non-parenchymal cells by anti-F4/80 (marker for Kupffer cells) before visualisation using anti-rat Alexa Fluor 488 secondary antibody. Imaging was performed on the Operetta system.

Safety and efficacy studies with PLGA particles

Based on the data of particle biodistribution using different sizes and types of galactosylated PLGA (26 pm) particles, the most suitable route of adminstration ie. HPV versus peripheral tail vein administration, was selected to perform further safety and dose response studies. For dose-response studies, 0.01 mg, 0.1 mg, 1 mg, 5 mg of galactosylated KGF-PLGA particles (26 pm) were administered versus vehicle control (30% FCS).

Twelve week old C57B1/6 mice (6 groups; n= 3-4 per group) received a single 100 pL HPV injection of galactosylated KGF- PLGA particles (0.01 mg, 0.1 mg, 1 mg, 5 mg), KGF s.c. (1.25 mg/kg for 2 days) or 30% FCS (vehicle via HPV route) alone. Serum samples were collected for liver function tests (ALT, Albumin, bilirubin) and KGF concentrations 72 hours after the first injection of KGF-particles, KGF s.c. or vehicle. Mice were culled ~72hrs after the first injection of particles, KGF or vehicle and livers were H and E stained.

To determine the effect of galactosylated KGF- PLGA particles (O. lmg) versus KGF s.c. (1.25 mg/kg for 2 days) on hepatocyte proliferation, 12 week old C57B1/6 mice (4 groups; n= 3-4 per group) were treated with galactosylated KGF- PLGA particles (O. lmg) injected via the HPV, KGF s.c. (1.25 mg/kg (IOOmI) for 2 days), PLGA particles injected via the HPV and 30% FCS x2 doses via HPV. At 72 hours post first injection, mice were pulsed with BrdU 1 mg i.p. 1 hour before cull. Hepatocyte proliferation was determined by immunofluorescence staining.

Mouse islet isolation

Pancreatic islets were isolated from 12 week-old male C57B1/6 mice by a collagenase digestion method as previously reported ²⁵. The islets (250 islet/mL) were cultured free floating (37-C, 5% C02) in RPMI 1640 medium (Bio-Whittaker, Walkersville, MD) supplemented with L-glutamine (Sigma, UK), penicillin-streptomycin (1000 U/mLYlO mg/mL; Sigma, UK) and 10% (vol/vol) fetal calf serum (HyClone, Celbio, Logan, UT) in 5% C0₂ incubator for 24 hours at 22°C before the transplant. Islet purity was >90%.

Induction of diabetes in mice

Animals (n=8- 10/group) were rendered hyperglycemic at 16-17 weeks old by administration of 180 mg/kg i.p. streptozotocin (STZ) (Sigma-Aldrich) in ice cold acetate buffer, pH 4.5, following a 4-hour fast. Daily tail blood glucose measurements were taken using a glucometer (One Touch Verio, LifeScan). Mice were classed as hyperglycaemic if their non-fasted glucose levels were >17.0 mmol/L for two consecutive days. Once diabetes was confirmed, daily subcutaneous injections (0.5- 2.5U) of insulin glargine (Lantus, Sanofi) were administered, until the day prior to islet transplantation. All islet transplantations took place within 4 weeks of administration of STZ.

Transplantation of islets with subcutaneous KGF and galactosylated KGF-PLGA particles

Diabetic C57B1/6 mice (n=6-8/group) were transplanted with: 1) a marginal islet transplant mass of 400 islets alone (in 200 pL RPMI 1640 medium (vehicle)) and 2) 400 islets plus KGF 1.25 mg/kg s.c. x2 doses, one dose 48 hours before transplant and one dose at the time of islet transplant. Glycaemic control was monitored and mice sacrificed 6 weeks post-transplantation. In further experiments mice were transplanted with 1) a marginal islet transplant mass of 400 islets alone and 2) 400 islets plus O. lmg KGF loaded particles delivered concurrently via the HPV. Mice (n=3/group) were sacrificed 72 hours post transplantation. Livers were harvested, paraffin embedded and step sectioned and islets detected by dual immunofluorescence for insulin and glucagon as described below.

A further group of diabetic C57B1/6 mice (n=8- 10/group) were transplanted with: 1) a marginal islet transplant mass of 400 islets alone and 2) 400 islets plus O. lmg KGF loaded particles delivered concurrently via the HPV. Glycaemic control was monitored and mice sacrificed 6 weeks post-transplantation.

Body weight and venous blood glucose (OneTouch Verio, LifeScan) were documented daily before and after transplantation.

In the experiments with the KGF-PLGA particles, at 6 weeks post-transplant, mice were fasted overnight, administered 1 mg BrdU i.p. following which a basal tail blood glucose reading was taken and 2g glucose/kg fasted body mass given by i.p injection and glucose measurements taken 15min, 30min, 60min, 90min and 120min post glucose injection. A blood sample was taken at the 60min time point (into EDTA) for stimulated plasma insulin analysis. Mice were subsequently culled and tissue fixed for 24 hr by immersion in buffered 4% formaldehyde before analysis for cell proliferation in the organs and insulin content of the pancreas.

Analyses

Biochemical analysis

Liver function tests: Serum and EDTA-plasma samples were analysed by commercial kits according to the manufacturer’s instructions. Activity of alanine transaminase (ALT), albumin and bilirubin (Alpha Laboratories Ltd., Eastleigh, UK) were adapted for use on a CobasFara centrifugal analyser (Roche).

Human KGF concentrations: Exogenous KGF concentrations were measured by Human FGF-7 (KGF) ELISA Kit (Thermo Scientific, USA) following manufacturer’s instructions. KGF concentrations were expressed in pg/mL. Insulin concentrations: Plasma insulin concentrations were measured (Mercodia Ultrasensitive mouse insulin ELISA, Uppsala, Sweden) with a detection range of 0.2- 6.5pg/L and no cross-reactivity with mouse C-peptide or proinsulin.

Insulin content of pancreas: the pancreas was weighed, homogenised in Azol (1 1.4% glacial acetic acid, 0.8% FBS, 87.8% dH20) then sonicated twice after two 24 hour incubation periods at 4°C before spinning at 1500rpm for lOmins at 4°C. Insulin concentrations were analysed from the supernatant as described above.

Fraction of proliferating hepatocytes to total proliferating cells

Liver sections were stained with BrdU and HNF4a to quantify total cell and hepatocyte proliferation respectively. The fraction of proliferating hepatocytes to total proliferating cells was expressed as a percentage.

Detection and counting of transplanted islets in the recipient liver

The number of b-cells was determined in >8 consecutive liver slices per mouse at least 50 pm apart. Briefly, immunofluorescence co-staining for insulin and glucagon was performed to detect pancreatic islets in paraffin-embedded liver sections. Tissues were blocked with Bloxall (Vector Laboratories, UK), and Avidin/Biotin block (Invitrogen, UK). To eliminate endogenous immunoglobulin, tissue sections were incubated for 1 hr in M.O.M. Mouse IgG Blocking Reagent (Vector Laboratories, UK) and protein block. Anti-glucagon antibody was detected using a species specific secondary goat biotinylated antibody (Vector Laboratories, UK), Vectastain R.T.U, ABC reagent (Vector Laboratories, UK), and a Perkin Elmer TSA Plus Fluorescein, signalling amplification, kit (NEL741001KT). Antigen retrieval was performed by incubating the slides in citrate buffer (pH 6.0) for 15 min at 350°C to denature any antibodies in the tissues and prevent cross-reaction with the next antibody application.

Detection of fibrosis and necrosis in liver

Heamatoxylin and Eosin (H&E) stains were automatically produced using a Shandon Varistain Automated Slide Stainer. To study collagen fibres in the liver tissues, Picrosirius red (PSR) staining was performed using 0.1% Direct Red 80 (Sigma, UK) in saturated picric acid as previously described ²⁶. Under polarised light, collagen bundles appear red, counterstained with yellow and were readily differentiated from the black background, allowing quantitative morphometric analysis. Slides were scanned by Vectra^® Polaris™ Quantitative Pathology Imaging System (PerkinElmer, UK). Quantification of PSR staining was performed by threshold analysis of 10 non overlapping randomly selected fields of view per slide at a magnification of x lO using InForm software, and expressed as the percentage of positive staining of the total area.

Statistical Analysis

Significance was determined by unpaired / tests or one-way ANOVA with Tukey’s post-hoc testing using Prism 5.0 software (GraphPad Software, La Jolla, CA). A p value of <0.05 was considered significant.

Results

KGF enhanced cell proliferation in the liver more than other GFs

KGF 1.25 mg /kg by s.c. injection was associated with the greatest total proliferation of cells in the liver (parenchymal and non-parenchymal) versus HGF 250 pg/kg IV, T3 4mg/kg s.c. and all three GFs in combination; all mice were given 2 injections of GFs 48 hours apart (Figure 1A). Mice receiving KGF (s.c.) exhibited a pronounced cell proliferation in all organs including the lungs, pancreas, heart and spleen, as demonstrated by BrdU immunofluorescence staining (Figure IB). Basal cell proliferation in these organs collected from saline-injected mice are shown in comparison (Figure 1C). Therefore, KGF was selected for further studies.

Subcutaneous KGF+islets did not control blood glucose levels more effectively than islets alone

Mice with diabetes transplanted with a marginal number of islets plus KGF 1.25mg/kg s.c, (x2 injections 48 hours preceding transplant and at transplant), did not demonstrate improved glycaemic control at any time point compared with mice transplanted with islets alone at 6 weeks, with no mice cured from their diabetes: glucose: 19.1±2.2 mmol/L vs 20.2±1.8 mmol/L, respectively.

Fabricated particles demonstrated sustained release of KGF over >21 day period

The fabricated galactosylated PLGA particles were regular and spherical in shape with porous surfaces (Figure 2A). The chemistry modification of PLGA backbone by the addition of a galactose moiety achieved targeted KGF delivery. The average diameter of the particle was (mean±SD) 26±6 pm with 57.4±2.3% KGF loading efficiency (Figure 2B). The release kinetics showed a typical initial burst release phase and released approximately one-third of the KGF payload on day 1, followed by a gradual decline to 8% release on day 2 then 3% on day 3. The release was maintained at 1% between days 4 to 6 followed by a gradual build-up of release from day 9 to day 21, approaching 8% (Figure 2C). The cumulative release profile in the particles is shown in Figure 2D. Table 2 demonstrates the KGF delivery dose in ng per mg particles over 3 weeks. For O. lmg PLGA particles the expected KGF content was 60ng and a 70% release of KGF over 21 days would be ~40ng KGF.

Galactosylated PLGA particles administered via HPV injection specifically target the liver

Non-modified PLGA particles (mean diameter 22 pm) did not localise to the liver, but were found exclusively in lung (Figure 3A). In contrast, we found that galactosylated PLGA particles were retained in the liver, with the larger average diameter particles (26 pm) conferring exclusive hepatic localisation. Smaller galactosylated PLGA- particles (average 2 pm and 10 pm diameters) showed hepatic retention but this was not exclusive with particles detected in the lung, kidney, heart and spleen (Figure 3A- C). Moreover, the smallest galactosylated PLGA particles (average diameter 2 pm), were engulfed by F4/80-positive liver resident macrophages (Figure 3D). In parallel with these studies, the biodistribution of PLGA-particles after injection into a peripheral vein (tail vein) was determined. A large proportion of the galactosylated PLGA-particles (10 pm diameter) were retained in the lung and some were detected in the liver ie. exclusive hepatic retention was not seen. Since a large proportion of the 10 pm galactosylated PLGA particles were mechanically trapped in the lungs and spleen, larger sizes (26 pm diameter) of galactosylated-PLGA particles were not tested for hepatic localisation (Fig 9A-C).

A galactosylated KGF-loaded PLGA particle dose of O.lmg injected via the HPV promoted liver proliferation was not associated with liver damage with detectable circulating levels of KGF

Mice receiving 0.01 mg and 0.1 mg KGF-PLGA particles via the HPV remained well with no demonstrable weight loss compared to those receiving FCS 30% vehicle control. No difference in serum levels of albumin (marker of hepatocyte function), ALT activity or bilirubin (markers of liver injury) was observed among the groups receiving 0.01 mg or 0.1 mg KGF-particles injected via HPV 72 hours after transplant versus vehicle (Figure 4A), an effect that was still apparent 6 weeks post-transplant (Figure 11). Human KGF serum levels were detected after 24 hours in mice administered with 0.1 mg KGF-particles (Figure 4B). Macroscopically blood vessels in the liver appeared milky white 24 hour after particle injection via the HPV (Figure 4C). H-E staining exhibited normal liver architectures in the vehicle and 0.01 mg KGF-particles groups, while necrotic areas were very occasionally visible in the group administered 0.1 mg particles (Figure 4D).

Higher doses of KGF-particles (1 mg and 5 mg) were also trialled. The decrease in body weight was not significantly different between the groups 24 hours after treatment with the particles (Fig 10A). An increase in liver injury markers ALT, bilirubin and albumin was observed in mice receiving 1 mg and 5 mg KGF-particles (Fig. l02B-D) with patchy liver necrosis (Fig. lOE-F). Of note, one mouse in each of the groups (n=3 per group) receiving 1 mg or 5 mg KGF-particles became unwell and were humanely euthanised by cervical dislocation, 3 hours post injection of the particles.

Effect of galactosylated KGF-PLGA particles (O.lmg) on liver proliferation

Based on the dose response studies, 0.1 mg of particles made from galactosylated PLGA ± KGF were administered via the HPV in mice and liver proliferation (total and hepatocyte) examined at 72 hours. Co-localisation of BrdU⁺ and HNF4a⁺ cells in liver sections of mice that received KGF-PLGA particles (Fig. 5A) was 1.5 fold greater than in mice receiving KGF 1.25mg/kg s.c.x2 doses. The greatest cell proliferation overall was observed in the liver of mice treated with KGF-PLGA particles (Fig. 5B); 55% of proliferating cells were hepatocytes (P<0.05, Fig. 5C).

Galactosylated KGF-PLGA particles (O.lmg) transplanted with islets promoted islet engraftment and improved glycaemic control compared to mice receiving islets alone with no evidence of liver fibrosis

Greater numbers of islets were seen in the livers of hyperglycaemic mice transplanted intraportally with islets and KGF-PLGA particles versus islets alone in hyperglycaemic mice as evidenced by greater numbers of insulin-positive cells in the liver 72 hours post-transplant (Fig. 6A and Fig. 6B, P<0.05). Mice receiving islets and KGF-PLGA particles had tighter glycaemic control versus those receiving islets alone with blood glucose levels normalising by day 30 post-transplant (Fig. 7A, P<0.05 versus islet group) and with a greater proportion achieving a cure from their diabetes (75% vs. 0%; p<0.001). There was no difference collagen content in the liver tissues between the groups at day 42 (Fig. 7B-C). Serum biomarkers for liver injury and function were comparable among the groups (Fig.113A-C).

No evidence of pancreas regeneration 6 weeks post-transplantation of islets ± galactosylated KGF-PLGA particles (O.lmg)

No proliferating b-cells (insulin⁺) were detected in the pancreases of mice treated with galactosylated KGF-PLGA particles (Fig. 8A). There was no significant difference in pancreatic insulin content between the groups (Fig. 8B).

Discussion

Transplantation of islets into patients with T1D stabilises glycaemic control, reducing the incidence of hypoglycaemia ²’⁹’¹⁰’³⁰, but due to poor engraftment of islets into the liver ³¹’³², islets from 2 to 3 donor pancreases, a scarce resource, are required. Unfortunately islet transplantation still falls short of a cure and the supply of pancreases outstrips demand, emphasising the need to improve islet transplantation procedures. Previous studies in animal models had demonstrated that partial hepatectomy improved islet engraftment into the liver ³³’³⁴, mediated potentially by remodelling of the liver niche and, or, improved revascularisation of islets into the liver ³⁵. However, partial hepatectomy is not a clinically applicable procedure in man, but it does lead to the release of growth factors from the liver, which we hypothesised, may then modulate the liver niche increasing islet engraftment and improving glycaemic control. Previously, we had been shown that systemic administration of KGF plus T3 ¹⁹ as well as HGF plus T3 ³⁶ increased liver cell proliferation and subsequent retroviral gene transfer into the liver.

Our results demonstrate improved islet engraftment coupled with improved glycaemic control when the liver is targeted by the growth factor KGF packaged into “customised” PLGA particles: histologically the numbers of beta cells within the liver were greater in the mice receiving portal injection of O. lmg PLGA-KGF particles plus islets versus those receiving islets alone. Furthermore the majority of mice that were administered these particles were cured following islet transplantation with just a marginal number of islets. Importantly, this was in the context of no regeneration of beta cells in the native pancreas and KGF even at high doses did not affect the secretion of insulin, results which are again consistent with the effect being mediated by the islet mass transplanted into the liver. Although subcutaneous KGF was associated with increased proliferation of liver cells that were mainly non- parenchymal, this was not associated with improved glycaemic control following islet transplantation. KGF was not detectable systemically 24 hours following subcutaneous administration and these short term effects may not be sufficient to increase islet engraftment. Islet engraftment where blood vessels form between the islets and the liver, commences around day 3-4 and is largely complete by day 28 ³⁷. Furthermore KGF delivered subcutaneously increases cell proliferation in other organs including the lungs, pancreas, kidney, heart and spleen. In the context of translating this therapy into man, this carries the potential of a number of deleterious off target effects, therefore limiting its clinical translation.

When galactosylated PLGA particles were studied over a 21 day period, the route of administration, particle size and galactosylation influenced its sequestration within organs. The 26pm galactosylated PLGA particles delivered via the HPV route were associated with sequestration confined to the liver. In contrast, smaller galactosylated particles (2 pm and 10pm) delivered via the HPV route, were associated with sequestration not only within the liver but also within the capillary beds of the lungs and spleen and other organs. Control PLGA particles of 22pm without the galactosylated moiety were sequestered in the lung only, demonstrating that galactosylation is required for localisation of the particle in the liver via ASGPR- mediated endocytosis. When the 10pm galactosylated PLGA particles were delivered peripherally, a large proportion were trapped in the lung with only a proportion reaching the liver; hence this would not be a feasible way to translate this therapy into man.

The liver demonstrated hepatocyte proliferation with KGF 0.1 pg packaged into 0.1 mg PLGA particles with, importantly, no liver associated injury. With this dose of KGF, 55% of proliferating cells were hepatocytes, contrasting with 22% when two doses of 1.25mg/kg KGF was administered subcutaneously. Liver injury including patchy necrosis of the liver was demonstrable with 1 mg and 5 mg KGF-PLGA particles.

Current factors known to impact favourably on islet transplant outcomes are numbers of islets transplanted per se ³⁸, along with younger donor age ³⁹. This study is the first to show that modulation of the liver niche by galactosylated KGF-PLGA particles is a potential therapeutic strategy for increasing engraftment of islets in man and therefore represents an alternative approach, not described before, for improving islet transplant outcomes in man. Potential mechanisms of action that may promote the retention and engraftment of islets within the liver are not only the proliferation of hepatocytes with subsequent“trapping” of islets but also protection of hepatocytes from TNF induced apoptosis observed in animal models ⁴⁰. Islet transplantation is associated with inflammation in the liver and if protection of TNF induced apoptosis operates in man this would be advantageous. Indeed TNF-a inhibitors including etanercept are utilised as induction agents preceding transplantation and are associated with improved outcomes ⁴¹. KGF may also increase engraftment of islets by stimulating angiogenesis via VEGF induction indirectly ^42,43

In this study 0.1 mg of galactosylated KGF-PLGA particles would release 40 ng of KGF in a mouse over a three week period leading to increased islet engraftment in the liver. KGF has FDA approval and held a license for severe oral mucositis in patients with hematologic malignancies receiving myelotoxic therapy in the setting of autologous hematopoietic stem cell support as kepivance. The recommended dose is ~25 mg intravenously for a 70kg person over a 6 day period ⁴⁴. Extrapolating the dose of KGF administered in a mouse via particles direct to the liver to humans on a weight for weight basis we would anticipate that the dose used in man via the HPV would be 250 fold lower than the licensed dose for treating oral mucositis.

Such a strategy would mean that islets isolated from just one donor pancreas may be sufficient to diminish hypoglycaemia and stabilise glycaemic control in patients with T1D. This would enable more patients to be transplanted, diminish the overall morbidity from the procedure and be associated with significant cost savings.

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Palifermin (Rx) https://reference.medscape.com/drug/kepivance-palifermin-

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Tables

Table 1 Primary antibodies used in immunofluorescence staining

Table 2 Release of KGF (% and ng/ lmg particle) at the selected time points up to day

21

Claims

1. A composition of polymer particles, wherein the polymer particles are loaded with an agent that is capable of enhancing the binding of islet cells to liver tissue, and

wherein

(i) the polymer particles comprise linked molecules, wherein the linked molecules comprise:

a) asialoglycoprotein receptor (ASGPR)-binding molecules that are capable of binding the asialoglycoprotein receptor (ASGPR) on a cell surface, or

b) a binding-molecule that is specific for a cell surface marker that is predominantly present in liver tissue; and/or

(ii) the polymer particles are between about 0.5 and about 100 microns in diameter.

2. The composition according to claim 1, wherein the agent that is capable of enhancing the binding of islet cells to liver tissue comprises a growth factor.

3. The composition according to claim 1 or claim 2, wherein the agent comprises a molecule capable of promoting angiogenesis and/or hepatocyte proliferation.

4. The composition according to any preceding claim, wherein the agent comprises or consists of fibroblast growth factor.

5. The composition according to any preceding claim, wherein the agent comprises or consists of keratinocyte growth factor (KGF), or a functional variant or equivalent of keratinocyte growth factor.

6. The composition according to any preceding claim, wherein the agent comprises or consists of VEGF (vascular endothelial growth factor) or HGF (hepatocyte growth factor), or a functional variants thereof.

7. The composition according to any preceding claim, wherein the polymer particles provide controlled release of the agent in an aqueous environment.

8. The composition according to any preceding claim, wherein the polymer particles comprise PLGA.

9. The composition according to any preceding claim, wherein the polymer particles comprise galactosylated polymer.

10. The composition according to any preceding claim, wherein the asialoglycoprotein receptor (ASGPR)-binding molecules comprise or consist of:

i) a galactose moiety;

ii) molecules that compete for binding to the ASGPR with galactose;

iii) ASGPR-binding antibodies, antibody fragments, antibody variants, or antibody mimetics; or

iv) synthetic glucose-derivative polymers.

11. The composition according to any preceding claim, wherein the ASGPR-binding molecule is anchored to the polymer via a linker molecule.

12. The composition according to claim 11, wherein the linker molecule comprises a polymer.

13. The composition according to claim 11 or 12, wherein the linker molecule comprises PEG.

14. The composition according to any preceding claim, wherein the composition further comprise islet cells.

15. A method of islet cell engraftment in liver tissue, the method comprising:

A) delivering the composition comprising islet cells according to the invention herein into the liver tissue;

B) delivering the composition according to the invention herein into the liver tissue, and subsequently or concurrently delivering islet cells into the liver tissue; or

C) delivering islet cells into the liver tissue and subsequently delivering the composition according to the invention herein into the liver tissue.

16. A method of treatment or prevention of diabetes in a subject, the method comprising:

A) delivering the composition comprising islet cells according to the invention herein into the liver of the subject;

B) delivering the composition according to the invention herein into the liver of the subject, and subsequently or concurrently delivering islet cells into the liver tissue; or

C) delivering islet cells into the liver tissue and subsequently delivering the composition according to the invention herein into the liver tissue .

17. A composition according to any one of claims 1 - 14 for use as a medicament.

18. A composition according to any one of claims 1 - 14 for use in the treatment or prevention of diabetes in a subject.

19. A composition according to any one of claims 1 - 14 for use in the treatment or prevention of liver disease in a subject.

20. Use of the composition according to any one of claims 1 - 14 for enhancing islet cell engraftment in the liver of a subject.

21. A kit for islet cell engraftment in liver tissue, the kit comprising:

i) a composition of polymer particles according to any one of claims 1 - 14; and ii) a composition of islet cells.

22. The kit according to claim 21 further comprising a syringe for storing and/or delivering the composition.