Disease state controlled release of binding molecules

Abstract

The present invention relates to means to and methods for removing biological molecules from the direct surroundings. More specifically, the present invention relates to a single domain antibody that binds a biological molecule, wherein at least one hydrogel-forming polymer is conjugated to the single domain antibody, wherein the at least one hydrogel-forming polymer is degradable by an extracellular matrix degrading enzyme and / or the at least one hydrogel-forming polymer is conjugated to the single domain antibody via an amino acid sequence comprising an extracellular matrix degrading enzyme cleavage site. The invention particularly relates to an in situ forming hydrogel that, controlled by disease state, can release the single domain antibody that can bind and sequester a biological molecule. The hydrogel can be used to reduce or inhibit the bioactivity of a biological molecule. Preferably, the hydrogel is injectable, such as an intra-articular injectable hydrogel neutralizing pro-inflammatory cytokines in a diseased joint.

Description

[0001] DISEASE STATE CONTROLLED RELEASE OF BINDING MOLECULES

[0002] Technical field

[0003] The present invention relates to means to and methods for removing biological molecules from the direct surroundings. More specifically the invention relates to a polymer comprising a binding molecule that can bind and sequester a biological molecule. The polymer can preferably form a hydrogel. The hydrogel can be used to reduce or inhibit the bioactivity of a biological molecule. Preferably, the hydrogel is injectable, such as an intra-articular injectable hydrogel neutralizing pro-inflammatory cytokines in a diseased joint.

[0004] Background of the invention

[0005] Therapeutic proteins including antibodies, growth factors, cytokines and enzymes are a rapidly expanding drug class in clinical use. Their high specificity and potency for the target provides therapeutic benefits for the treatment of cancer, cardiovascular and metabolic diseases. However, protein drugs often exhibit short circulating half-life in vivo when delivered by conventional routes like oral or intravenous administration. This requires high dosages or frequent injections to achieve a therapeutic effect, resulting in poor patient comfort and even unexpected adverse events. To address these limitations, tremendous efforts have been made over recent decades to develop long-acting drug delivery systems (DDS), including liposomes, nano- and microparticles and hydrogels. These drug delivery systems can enhance drug bioavailability and safety by maintaining drug levels over a long period of time at the disease site within the therapeutic window, thus substantially decreasing required dosage and administration frequency.

[0006] Hydrogels offer an attractive platform for controlled protein delivery due to their capability to easily encapsulate bioactive therapeutics and protect labile proteins against premature degradation. Hydrogels are water-swollen polymer networks, which can be either physically or chemically crosslinked. Their soft nature and the high water content in the polymer networks shows physical similarity to natural extracellular matrices, giving the hydrogels excellent biocompatibility. Hydrogels can be made from different types of natural and synthetic polymers (e.g. dextran, hyaluronic acid, gelatin, polyethylene glycol (PEG), polyvinyl alcohol (PVA)) by forming a three dimensional network of polymer chains in often mild reaction conditions. The physicochemical properties and macroscopic architecture, such as mesh size, of the formed hydrogel are generally driven by the modification of the polymers, crosslink density, polymer concentration, or crosslinking chemistry, which governs how drugs diffuse inside and are released from the hydrogel network Proteins can be physically entrapped in the hydrogel or covalently conjugated to the polymer backbone of the hydrogel via a stable or cleavable covalent linkage. In some hydrogel-based protein delivery systems, the release of protein is driven by multiple mechanisms, such as diffusion, degradation, or a combination of both. When the hydrodynamic diameter of the protein is smaller than the hydrogel mesh size, the release process of the protein is dominated by diffusion, resulting in a short retention time owing to their quick transport within the network, ranging from hours to days. Additional interactions, such as electrostatic interactions and hydrophobic associations, between the drug and the hydrogel matrix can extend drug release up to days. However, initial burst release, which in general is observed in both above mentioned diffusion-dependent release systems, is one of main limitations to maintain drug levels within the therapeutic window for a long period of time. An alternative strategy to minimize burst effect is to covalently conjugate drugs to the polymers that can form a hydrogel network, either through highly stable bonds or linkages. The drugs immobilized inside the biodegradable hydrogel via stable linkages can be released at a steady rate over weeks, which is governed primarily by the degradation of the network. Over the past decades, in-situ forming injectable hydrogels prepared by the enzymatic crosslinking of polymers chains have received considerable interest in the designing and development of various useful drug delivery systems to treat osteoarthritis (OA).

[0007] Osteoarthritis (OA) is characterized by degeneration of articular cartilage, the development of osteophytes and mild to moderate inflammation, resulting in pain and loss of joint function. With advances in the understanding of the role of inflammation in OA, it is believed that inflammatory cytokines (such as I L-1 p, IL-6 and TNF-a) produced by chondrocytes and synoviocytes are major mediators in the pathophysiology of OA while the growth factor NGF is an important mediator of joint pain in OA. These cytokines are involved in increasing catabolic and inflammatory responses and the downregulation of anabolic events, resulting in structural damage to the OA joint. Therefore, a variety of biologicals targeting these cytokines have been considered.

[0008] Enzymatically-crosslinked hydrogels incorporating such biologicals are generally formed under mild gelation conditions, which is beneficial for the preservation of the integrity of incorporated bioactive agents or cells. In the art, injectable hydrogel systems have been described which are based on hyaluronic acid-tyramine (HA-TA) and dextran-tyramine (Dex- TA) conjugates or combinations of both. These hydrogels can be used as an injectable scaffold for mesenchymal stem cells (MSCs) and / or chondrocytes delivery in the joint or as a carrier platform for bioactive agents delivery. This platform exhibits a great potential for cartilage tissue engineering. These hydrogels are formed by enzyme-mediated crosslinking of tyramine moieties upon mixing of the polymer conjugates with horseradish peroxidase (HRP) as a catalyst and hydrogen peroxide (H2O2) as oxidant. These hydrogels can form in situ using a minimally invasive procedure allowing a mixture of hydrogel precursors and bioactive agents or cells to be administered using a syringe. This makes this platform an attractive biomaterial for local drug delivery.

[0009] However, a limitation associated with this in situ forming hydrogel platform is the uncontrolled rapid release of physically entrapped drugs at the initial stage after injection due to the still incomplete crosslinking of hydrogel networks. This so-called “burst release” causes an overdose of drugs to diffuse away from the injection site, which can result in undesired side effects.

[0010] It is an objective of the present disclosure to solve one or more of the above-mentioned or other problems in the art. More specifically, it is an objective of the present disclosure to provide new or improved means for removing and / or neutralizing biologicals, preferably over a prolonged period of time while minimizing initial burst effect.

[0011] Summary of the invention

[0012] The present inventors have developed hydrogel-based systems for local delivery of therapeutic molecules (e.g. single domain antibodies) with sustained-release profiles.

[0013] More specifically, the present disclosure provides for a single domain antibody that binds a cytokine, a cytokine receptor, a soluble antigen or an auto-antibody, wherein at least one hydrogel-forming polymer is conjugated to the single domain antibody, wherein the at least one hydrogel-forming polymer is degradable by an extracellular matrix degrading enzyme and / or the at least one hydrogel-forming polymer is conjugated to the single domain antibody via an amino acid sequence comprising an extracellular matrix degrading enzyme cleavage site. In other words, the extracellular matrix degrading enzyme and the extracellular matrix degrading enzyme cleavage site may be used separately or in combination, as release systems.

[0014] Accordingly, upon injection and in situ formation of a hydrogel, the release kinetics of the single domain antibody are determined by the degradation of the hydrogel network under influence of degrading enzymes, such as extracellular matrix degrading enzymes, particularly hyaluronidases and matrix metalloproteinases. In the human body, hyaluronidases are expressed in many different tissues with very high expression in the joint. Thus release kinetics are determined in the body by the local expression of hyaluronidases or matrix metalloproteinases providing distinct release profiles depending on the site of injection.

[0015] Many diseases are characterized by extensive break down and / or remodeling of the extracellular matrix which is mediated by the upregulation of extracellular matrix degrading enzymes such as matrix metalloproteinases (degrading ECM proteins) and hyaluronidases (degrading hyaluronic acid). The present inventors considered to exploit the upregulation of these enzymes as a disease trigger that subsequently releases single domain antibodies from a hydrogel network by network degradation. In this way, initial burst effect is avoided and the single domain antibodies have a release profile that follows disease severity, in that a more severe disease state is associated with a higher concentration of extracellular matrix degrading enzymes, leading to upregulated release of the single domain antibodies.

[0016] The degradation rate can furthermore be controlled by varying the weight to volume ratio of the polymers used for making the hydrogels, wherein a smaller weight to volume ratio will display more rapid degradation and thus release of the single domain antibodies.

[0017] The degradation rate can also be controlled by the ratio of HA(-TA) and Dex(-TA) polymers, with slower degradation in networks containing more Dex(-TA) polymer. The weight ratio of HA(-TA) and Dex(-TA) preferably is between 25:75 and 75:25, more preferably between 40:60 and 60:40, most preferably about 50:50.

[0018] In addition, by mixing HA functionalized polymers of which VHH release is controlled by presence of hyaluronidase and Dex functionalized polymers of which VHH release is controlled by cleavage by protease activity (e.g. by MMP13), dual responsive injectable release systems can be created of which rate of release is controlled by the enzymatic activity which is related to disease severity.

[0019] Detailed description of the invention

[0020] The present disclosure relates to a therapeutic compound, particularly a binding molecule, which preferably binds one or more biological molecule, wherein the one or more biological molecule can be a cytokine, a cytokine receptor, a soluble antigen or an auto-antibody. Preferably at least one hydrogel-forming polymer is conjugated to the therapeutic compound. At least part of the at least one (or a further) hydrogel-forming polymer may be degradable by an enzyme, preferably an extracellular matrix degrading enzyme. In addition or alternatively, the at least one (or a further) hydrogel-forming polymer is conjugated to the therapeutic compound via an amino acid sequence, said amino acid sequence preferably comprising an enzyme cleavage site, even more preferably comprising an extracellular matrix degrading enzyme cleavage site. There may be one or two or three or four or five or more hydrogelforming polymer(s) conjugated to the therapeutic compound.

[0021] In an embodiment, the therapeutic compound according the present disclosure is a protein, preferably a binding peptide, more preferably an antibody and / or functional fragment thereof. In a particularly preferred embodiment, the therapeutic compound according to the present disclosure is a single domain antibody. Herein, the term ‘antibody’ refers to a protein, preferably belonging to the immunoglobulin class of proteins, that may exhibit binding specificity to a specific target and may induce molecular or cellular responses. Binding specificity as used herein refers to the ability of antibodies to discriminate between its target (epitope) and (any) another molecule or protein in the given context. As used herein, the term ‘functional fragment’ refers to a portion of an antibody, which may be smaller in size and / or may lack certain parts of the full antibody, yet retains the ability to bind its target. Single domain antibody typically refers to a (functional) fragment of a (mammalian / human) antibody. A single domain antibody can also refer to a single monomeric variable antibody domain, such as a Variable domain of a Heavy chain only (VHH) antibody (which is devoid of light chains). VHHs can be found in Camelidae or cartilaginous fish (also referred to as VHH and VNAR respectively).

[0022] Herein, a ‘hydrogel-forming polymer’ refers to any polymer capable of forming a hydrogel, e.g. under suitable conditions. Hydrogels are water-swollen polymer networks, which can be either physically or chemically crosslinked. Hydrogels can be made of different types of natural and / or synthetic polymers (e.g. dextran, hyaluronic acid, gelatin, polyethylene glycol (PEG), polyvinyl alcohol (PVA)) by forming a three dimensional network of such polymer chains in often mild reaction conditions. The physicochemical properties and macroscopic architecture, such as mesh size, of a formed hydrogel (e.g. a hydrogel formed from the at least one (or a further) hydrogel-forming polymer according to the present disclosure) are generally driven by the modification of the polymers, crosslinking density, polymer concentration, and / or crosslinking chemistry, which govern(s) directly and / or indirectly how e.g. the therapeutic compound of the present disclosure is released from the hydrogel network and the degradation of the hydrogel. Herein, the term ‘hydrogel’ may be interchanged with any polymer network, such as water-swollen polymer network and / or with any (high molecular weight) polymer, wherein the polymer can preferably form a hydrogel.

[0023] Herein, the term ‘conjugate(d)’ may be used interchangeable with the term ‘couple(d)’ and refers to any means in which two or more components may be attached and / or combined and / or interact, for example by covalent bonds, ionic bonds, hydrogel bonds, van der Waals forces, polar bonds, non-polar bonds and / or hydrophobic interactions, preferably covalent bonds. Whereas the at least one (or a further) hydrogel-forming polymer of the present disclosure may be conjugated to the therapeutic compound by any means known in the art, preferably the at least one hydrogel-forming polymer of the present disclosure is covalently conjugated to the therapeutic compound. Advantageously, the current inventors found that presence of enzymes and / or enzymatic activity, particularly the presence and / or enzymatic activity of extracellular matrix degrading enzymes, is directly involved in releasing the therapeutic compound that is covalently conjugated to the polymer of the present disclosure. Accordingly, the release of the therapeutic compound according to the present disclosure is a consequence of the presence and / or enzymatic activity of one or more (matrix degrading) enzyme(s). In addition or alternatively, it is preferred that release of the therapeutic compound according to the present disclosure does not substantially take place in absence of (enzymatic activity) of the (extracellular matrix degrading) enzyme(s). In addition or alternatively, it is preferred that the release of the therapeutic compound according to the present disclosure is not a consequence of solely other environmental factors than presence of (extracellular matrix degrading) enzymes and / or (extracellular matrix degrading) enzymatic activity, such as pH change (e.g. acidity) and / or temperature change.

[0024] In an embodiment, the at least one hydrogel-forming polymer of the present disclosure may be selected from any natural and / or synthetic polymer(s) capable of forming a three dimensional network of polymer chains. Preferably, the at least one hydrogel-forming polymer is selected from the list consisting of dextran, hyaluronic acid, gelatin, (multi-arm) polyethylene glycol (PEG), polyvinyl alcohol and / or (functionalized) derivatives thereof. As used herein ‘functionalized derivatives’ refers to the at least one hydrogel-forming polymer, which has been exposed to any chemical modification, resulting in incorporation of one or more functional groups, wherein the one or more functional groups facilitate specific characteristics (depending on the introduced group(s)), wherein the preferred specific characteristic is hydrogel formation capability with diverse crosslinking methods (e.g., homo-, hetero-, enzymatic and / or photoreactive crosslinking). Particularly preferred functionalized derivatives of the at least one hydrogel-forming polymer relate to the at least one hydrogelforming polymer comprising one or more functional groups, preferably one or more phenolic group(s) (e.g. tyramine, tyrosine or dopamine) and / or one or more maleimide group(s). In addition or alternatively, the at least one hydrogel-forming polymer according to the present disclosure preferably comprises one or more phenolic group(s) and / or one or more maleimide group(s). In a particularly preferred embodiment, the at least one hydrogel-forming polymer is chosen from the list consisting of hyaluronic acid, hyaluronic acid-tyramine, hyaluronic acid- tyramine-maleimide, hyaluronic acid-maleimide, dextran, dextran-tyramine, dextran-tyramine- maleimide, dextran-maleimide and / or polyethylene glycol, polyethylene glycol-tyramine, polyethylene glycol-tyramine-maleimide or polyethylene glycol-maleimide. In addition or alternatively, the at least one hydrogel-forming polymer according to the present disclosure comprises between 10-10000 (repeating) monomer or disaccharide units, even more preferably between 15-8000 (repeating) monomer or disaccharide units, even more preferably between 20-6000, 30-5000, 40-3000, 50-1500, 60-1000, 70-300 or 20-200 (repeating) monomer or disaccharide units. In addition or alternatively, the at least one hydrogel-forming polymer ideally is in the range of 1-1000 kDa, more preferably 5-500 kDa, more preferably 10-166 kDa, more preferably 12-50 kDa, most preferably 25-45 kDa. Smaller polymers were found to improve injectability, while gel forming can still be achieved, for example by using polymers functionalized with phenolic groups (e.g. tyramine) and using a curing system.

[0025] The at least one hydrogel-forming polymer according to the present disclosure may also be a co-polymer, preferably a co-polymer of hyaluronic acid grafted with dextran-tyramine.

[0026] In addition or alternatively, the present disclosure also provides for a (composition capable of forming a) hydrogel which preferably has an average mesh size that prevents diffusion of the biological molecule(s) into the hydrogel. In addition or alternatively, a (composition capable of forming a) hydrogel according to the present disclosure is preferably not penetrable for biological molecules with a molecular weight between 5-500 kDa, preferably 10-400 kDa, more preferably 20-300 kDa, yet even more preferably 25-200, most preferably 30-100 kDa. In addition or alternatively, a (composition capable of forming a) hydrogel according to the present disclosure preferably allows release of the therapeutic compound upon presence of (enzymatic activity) of the (matrix degrading) enzyme(s). The mesh size of a hydrogel formed from the at least one (or a further) hydrogel-forming polymer of the present disclosure can be determined using any known method used in the art, but is preferably determined by measuring the diffusion of molecules with a known (average) hydrodynamic radius. In addition or alternatively, the mesh size of a hydrogel formed from the at least one (or a further) hydrogel-forming polymer of the present disclosure may be determined mechanically or on the basis of the molecule release (Grassi, Mario, et al. Molecules 14.8 (2009): 3003- 3017). Yet another way of determining pore size is theoretically using the so-called “single- pore-radius” model described by Russell Shawn M. et al. (Industrial & engineering chemistry research 44.22 (2005): 8213-8217).

[0027] In an embodiment, the (extracellular matrix degrading) enzyme according to the present disclosure is chosen from any enzyme which is more abundant in a subject with a disease, preferably a diseased joint, when compared to a healthy subject. In a preferred embodiment, the (extracellular matrix degrading) enzyme is a protease, more preferably chosen from any hyaluronidase(s), dextranase and / or matrix metalloproteinase(s) (MMPs) known in the art. Hyaluronidases are a family of enzymes that catalyze the degradation of hyaluronic acid. Dextranase is an enzyme that hydrolyzes a-(1 ,6)-glycosidic linkages in dextran. Matrix metalloproteinase(s) are a large family of calcium-dependent zinc-containing endopeptidases, which are involved in extracellular matrix remodeling, but can also act intracellularly. In a particularly preferred embodiment, the (extracellular matrix degrading) enzyme is chosen from the list consisting of hyaluronidase-1 , hyaluronidase-2, hyaluronidase-3 and / or hyaluronidase-4, and / or MMP1 , MMP3, MMP9 and / or MMP13, most preferably MMP13. Preferably, the (extracellular matrix degrading) enzyme is a human enzyme. Hyaluronidase-1 is present in major organs such as the liver, kidney, spleen, and heart, as well as in serum and urine. Hyaluronidase-2 exerts weaker enzymatic activity than hyaluronidase 1 and mainly breaks down high-molecular weight hyaluronic acid. Hyaluronidase-3 is found in the testis and bone marrow, and its role is yet to be elucidated. MMP1 is synthesized by macrophages, fibroblasts, and dendritic cells, is involved in promoting cell survival. MMP1 can degrade collagen I, II, III. MMP3 is secreted by fibroblasts, lymphocytes, endothelial, and dendritic cells and involved in the digestion of certain molecules in the extracellular matrix and basement membrane. MMP3 is also involved in modulating cell migration. MMP9 is secreted by dendritic, hematopoietic, macrophage, neutrophil, fibroblast, and lymphocyte cells can degrade collagen IV. MMP13 can also degrade collagen I, II, III. MMP-13 (collagenase 3), is secreted by epithelial cells, neuronal cells, connective tissue (Cartilage and Bone) and is one of the key enzymes responsible for the degradation of type II collagen. MMP-13 plays a crucial role during skeletal growth and long bone maturation.

[0028] In an embodiment, the therapeutic compound according to the present disclosure binds one or more biological molecule, preferably a peptide and / or protein. In a particularly preferred embodiment, the one (or a further) biological molecule is a cytokine, a cytokine receptor, a soluble antigen or an auto-antibody, most preferably a cytokine. Preferred cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors. Cytokines of the present disclosure can be produced by a broad range of cells, including immune cells like macrophages, B lymphocytes, T lymphocytes and mast cells, as well as endothelial cells, fibroblasts, and various stromal cells. The cytokine(s) of the present disclosure may also be produced by more than one type of cell. Said cytokine(s) typically act(s) through a receptor, and is / are especially important in the immune system and in regulation of tissue homeostasis and / or inflammation. Said cytokine(s) can, for instance modulate the balance between humoral and cell-based immune responses. Preferably, cytokine(s) according to the present disclosure is / are involved in joint-inflammation and / or joint disease. More preferably, said cytokine(s) is / are selected from the list consisting of Tumor Necrosis Factor-alpha (TNF-a), Nerve Growth Factor (NGF), Interleukin i p (I L-1 P), Interleukin 6 (IL-6), MMP9, Interleukin i p receptor (IL1 R) and / or IL-6 receptor (IL6R), most preferably TNF-a, NGF, 111 R and / or MMP9. In a particularly preferred embodiment, the therapeutic compound according to the present disclosure is a single domain antibody, more preferably a Variable domain of a Heavy chain only (VHH) antibody (which is devoid of light chains). The single domain antibody according to the present disclosure is preferably a variable domain of a single chain heavy chain only antibody of the type that can be found in Camelidae or cartilaginous fish (also referred to as VHH and VNAR respectively), which are naturally devoid of light chains. The antibody according to the present disclosure may be one single domain antibody, preferably one VHH, but is preferably a (genetic) fusion protein of two or more single domain antibodies, but preferably two single domain antibodies, most preferably two VHHs. Said (genetic) fusion protein may be monospecific (i.e. genetic fusion of two identical VHHs), but can also be bispecific (i.e. two distinct VHHs each recognizing their own antigen, which may be located in same (biological) molecule or located in different (biological) molecules). In addition or alternatively, the therapeutic compound according to the present disclosure has a molecular weight between 1-200 kDa, preferably between 2-100 kDa, 3-90, 4-80, 5-70, 6-60, 8-50, IQ- 40 kDa. The VHH according to the present disclosure may be recombinantly engineered and / or humanized.

[0029] In an embodiment, the antibody, preferably the VHH, of the present disclosure preferably has the following structure with complementarity determining regions (CDR) and framework regions (FR):

[0030] (N-terminus) FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 (C-terminus), wherein FR1 , FR2, FR3 and / or FR4 may be optional. Herein, the term ‘-’ is merely used to indicate the different CDRs and FRs and how these are positioned (i.e. are located) with respect to each other. The terms ‘(N-terminal)’ and ‘(C-terminal)’ are merely used to indicate the two ends of the (amino acid) sequence of the antibody, particularly the end with a free amino group (N- terminus) and the end with a free carboxyl group (C-terminus). The skilled person will understand that the ‘N-terminal’ and ‘C-terminal’ are used to described the structure of a typical antibody, preferably a typical VHH. The (genetic) fusion protein of two VHHs preferably has the following structure: (N-terminus) FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 (C-terminus) - linker - (N-terminus) FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 (C-terminus), wherein the linker can be any linker known in the art, but is preferably a linker comprising glycine-serine. Hereafter, the VHH that is N-terminal from the linker is referred to as ‘VHH-1’ and the VHH that is C-terminal from the linker is referred to as ‘VHH-2’.

[0031] Preferably, the at least one hydrogel-forming polymer is conjugated to the antibody by coupling to a (engineered) cysteine, preferably an (engineered) unpaired cysteine, preferably in FR4. Most preferably, the (engineered) (unpaired) cysteine is introduced C-terminally to CDR3, such as in FR4, preferably at the C-terminal end of FR4, more preferably within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20 or more amino acids of the C-terminal end of FR4, even more preferably within 5, 6, 7, 8, 9, 10 amino acids of the C-terminal end of FR4, most preferably within 5-10 amino acids of the C-terminal end of FR4.

[0032] More preferably the at least one hydrogel-forming polymer is conjugated to the antibody by coupling to a linker comprising glycine-serine-cysteine in the C-terminal end in FR4 (preferably within 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20 or more amino acids of the C- terminal end of FR4), most preferably by coupling to a linker comprising glycine-serine- cysteine-cysteine in the C-terminal end in FR4 (preferably within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20 or more amino acids of the C-terminal end of FR4), wherein preferably the coupling to cysteine, a linker comprising glycine-serine-cysteine, or a linker comprising glycine-serine-cysteine-cysteine is through reaction with a maleimide comprised in the at least one hydrogel-forming polymer.

[0033] In a preferred embodiment, the at least one hydrogel-forming polymer is preferably conjugated to the antibody by coupling to a thiol group in the (unpaired) cysteine. Said (unpaired) cysteine may be introduced using recombinant DNA technology, which the skilled person will be familiar with. Most preferably, the coupling to (an unpaired) cysteine is through reaction with thiol reactive groups, preferably a maleimide, comprised in the at least one hydrogel-forming polymer.

[0034] In addition or alternatively, a(n) (flexible) amino acid linker sequence is preferably incorporated C-terminally to CDR3, such as in FR4, preferably in FR4 and N-terminally to the (unpaired) cysteine. Said linker sequence may be introduced using recombinant DNA technology. Preferably, the (flexible) amino acid linker sequence is a hydrophilic linker. In addition or alternatively, the (flexible) linker preferably comprises small and / or polar amino acids, such as alanine, glycine and / or serine, preferably glycine and / or serine. The length of said (flexible) linker may be optimized to achieve appropriate separation of the antibody, but preferably comprises between 1-50, 1-45, 1-40, 1-35, 1-30, 2-30, 3-30, 3-25, 4-25 amino acids, most preferably between 5-25 amino acids.

[0035] In addition and alternatively, one or more additional (unpaired) cysteines are preferably introduced C-terminally to CDR3, such as in FR4, preferably between the (unpaired) cysteine and the C-terminal end of FR4. When in combination with the (flexible) amino acid linker sequence, the one or more additional (unpaired) cysteines are preferably incorporated C- terminally to the (flexible) amino acid linker, most preferably between the C-terminal end of the (flexible) amino acid linker and the (unpaired) cysteine. In a particularly preferred embodiment, the at least one hydrogel-forming polymer is conjugated to the antibody by coupling to the unpaired cysteine, through reaction with a maleimide, wherein the maleimide is comprised in the at least one hydrogel-forming polymer. Most preferably, a(n) (flexible) amino acid linker sequence is incorporated C-terminally to CDR3, such as in FR4, preferably between the C-terminal end of FR4 and the (unpaired) cysteine.

[0036] In an embodiment, in case the antibody according to the present disclosure is a (genetic) fusion protein of two or more, but preferably two VHHs, the (unpaired) cysteine, a(n) (flexible) amino acid linker sequence and / or the one or more additional (unpaired) cysteines are introduced at the same location as described herein for one VHH, however preferably in VHH-2. In addition or alternatively, when the antibody according to the present disclosure is a (genetic) fusion protein of two or more, but preferably two, VHHs, the (unpaired) cysteine may be introduced to the linker.

[0037] In an embodiment, the (extracellular matrix degrading enzyme) cleavage site according to the present disclosure is engineered in the primary sequence of the single domain antibody (i.e. the sequence including the framework regions (FRs) and the complementarity-determining regions (CDRs) of the VHH). Preferably , the (extracellular matrix degrading enzyme) cleavage site is positioned C-terminally to CDR3, such as in FR4, preferably between the N- terminal end of FR4 of the antibody and the (unpaired) cysteine, wherein preferably the (unpaired) cysteine is within 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20 or more amino acids of the C-terminal end of FR4. In a particular preferred embodiment, the (extracellular matrix degrading enzyme) cleavage site is N-terminal to the (unpaired) cysteine, most preferably in FR4 and N-terminal to the (unpaired cysteine). Said (extracellular matrix degrading enzyme) cleavage site may be introduced using recombinant DNA technology.

[0038] In addition and alternatively, both a (flexible) amino acid linker and one or more additional (unpaired) cysteine(s) may be introduced C-terminally to CDR3, such as in FR4, preferably in FR4 and N-terminally of the (unpaired) cysteine. In a particularly preferred embodiment, the (unpaired) cysteine is C-terminally of the (extracellular matrix degrading enzyme) cleavage site, C-terminally of the (flexible) amino acid linker and C-terminally of the one or more additional (unpaired) cysteine(s). In addition or alternatively, the one or more additional (unpaired) cysteines are preferably incorporated between the (flexible) amino acid linker and / or the (extracellular matrix degrading enzyme) cleavage site and the (unpaired) cysteine, preferably in the following structure: (N-terminus) FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - (N-terminal part of) FR4 - flexible linker and / or (extracellular matrix degrading enzyme) cleavage site - one or more additional (unpaired) cysteines - (unpaired) cysteine - (C-terminal part of) FR4. Said(flexible) amino acid linker and one or more additional (unpaired) cysteine(s) may be introduced using recombinant DNA technology.

[0039] In a particularly preferred embodiment, the (extracellular matrix degrading enzyme) cleavage site according to the present disclosure is between the C-terminal to CDR3, such as in FR4, preferably between the N-terminal end of FR4 and the (unpaired) cysteine. In addition, the at least one hydrogel-forming polymer is preferably conjugated to the (unpaired) cysteine, through reaction with a maleimide, wherein the maleimide is comprised in the at least one hydrogel-forming polymer.

[0040] In an embodiment, when the antibody according to the present disclosure is a (genetic) fusion protein of two or more, but preferably two, VHHs, the (extracellular matrix degrading enzyme) cleavage site is introduced at the same location as described herein for one VHH, however preferably in VHH-2.

[0041] The present disclosure also relates to the use of a (flexible) linker, such as disclosed herein, to bind a moiety to an antibody, preferably to a VHH. Herein the moiety can be any moiety, preferably a drug, radionuclide, toxin, peptide, photosensitizer and / or (hydrogel-forming) polymer.

[0042] The present disclosure also foresees a combination. In an embodiment, the therapeutic compound according to the present disclosure, i.e. the first therapeutic compound according to the present disclosure, is preferably in combination with a second therapeutic compound, preferably a single domain antibody or VHH, that may (also) bind a biological molecule preferably a cytokine, a cytokine receptor, a soluble antigen or an auto-antibody, wherein at least one hydrogel-forming polymer is conjugated to the first therapeutic compound according to the present disclosure, wherein at least part of the at least one hydrogel-forming polymer is degradable by an extracellular matrix degrading enzyme and / or wherein at least one hydrogel-forming polymer is conjugated to the second therapeutic compound, preferably via an amino acid sequence comprising an (extracellular matrix degrading) enzyme cleavage site.

[0043] In an embodiment, the therapeutic compound according to the present disclosure is comprised in a composition, wherein the composition preferably further comprises (a suitable amount of) hydrogen peroxide and / or (a suitable amount of) peroxidase. For example, the composition further comprises peroxidase, to which hydrogen peroxide may be added, e.g. at most 1 hour or at most 10, 5, 1 minute or at most 10, 5, 1 second before injection into a subject in need thereof. Whereas the composition may comprise any peroxidase, horse radish peroxidase (HRP) is preferred. In a wt.% hydrogen peroxide, most preferably at least 0.003 wt.% hydrogen peroxide, wherein the wt.% is based on the total weight of the composition. In addition or alternatively, the composition preferably comprises at most 0.01, 0.03, 0.1, 0.3, 0.4, 0.5, 0.7, 1, 2, 3, 4, 5 wt.% hydrogen peroxide, most preferably at most 0.5 wt.% hydrogen peroxide, wherein the wt.% is based on the total weight of the composition. In addition or alternatively, the composition preferably comprises between 0.001-10, 0.002-5, 0.003-3, 0.003-0.3, 0.003-0.03 wt.% hydrogen peroxide, most preferably between 0.003-0.03 wt.% hydrogen peroxide, wherein the wt.% is based on the total weight of the composition. In addition or alternatively, the composition preferably comprises at least 0.00008, 0.00009, 0.0001 , 0.0002, 0.0003, 0.0004 wt.% peroxidase, most preferably at least 0.0002 wt.% peroxidase, wherein the wt.% is based on the total weight of the composition. In addition or alternatively, the composition preferably comprises at most 0.05, 0.1 , 0.2, 0.4, 0.6, 0.8, 1, 2 wt.% peroxidase, most preferably at most 0.2 wt.% peroxidase, wherein the wt.% is based on the total weight of the composition. In addition or alternatively, the composition preferably comprises between , 0.00008-2, 0.00009-1, 0.001-0.5, 0002-0.2 wt.% peroxidase, most preferably between 0.0002-0.2 wt.% peroxidase wherein the wt.% is based on the total weight of the composition, preferred embodiment, the composition comprises at least 0.001 , 0.003, 0.007, 0.01

[0044] In an embodiment, the therapeutic compound according to the present disclosure is preferably comprised in a composition, wherein the composition preferably comprises at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 w / v.% of the at least one hydrogel-forming polymer, wherein the weight is drawn on the total volume of the composition.

[0045] In addition or alternatively, the composition according to the present disclosure preferably as a viscosity of 1-10,000 mPa.s, more preferably 1-1000 mPa.s, more preferably 1-500 mPa.s, more preferably 1-250 mPa.s, more preferably 1-100 mPa.s, more preferably 1-50 mPa.s, most preferably 5-50 mPa.s. Preferably this is determined by using MCR 301 rheometer (Anton-Paar) equipped with Peltier temperature control (C-PTD200), and e.g. by using DG 26.7 at 20 °C (+ / - 0.2 °C) and parallel plates (PP; 0 25 mm, 1 mm gap) at 25 °C (+ / - 0.2 °C) respectively.

[0046] In addition or alternatively, the composition of the present disclosure preferably comprises at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4 w / v.% of the at least one hydrogel-forming polymer, wherein the weight is drawn on the total volume of the composition. In addition or alternatively, the composition of the present disclosure preferably comprises 1-30, 2-20, 4-15, 6-10, 4-7 w / v.% of the at least one hydrogel-forming polymer, wherein the weight is drawn on the total volume of the composition. The higher the w / v.% of the at least one hydrogel-forming polymer, the slower the hydrogel degradation (e.g. due to extracellular matrix degrading enzymes). Other factors that may affect the degradation of the at least one hydrogel-forming polymer include the molecular weight of the at least one hydrogel-forming polymer, the crosslinking density and / or the degree of substitution of the functional group (if present) e.g. tyramine. Generally, a higher molecular weight, crosslinking density and / or degree of substitution of the functional group (if present) result in slower hydrogel degradation. As will be understood by the skilled person, these factors (potentially) affecting the degradation of the at least one or more hydrogel-forming polymer may be varied to tailor the physicochemical properties of the formed hydrogel.

[0047] In addition or alternatively, the composition may further comprise at least one hydrogelforming polymer that is not degradable by an extracellular matrix degrading enzyme and / or and that is not conjugated to the therapeutic compound. Preferably, the composition of the present disclosure comprises between 10-90 wt.%, preferably between 10-90, 15-80, 20-70, 25-60, 30-50, 10-60, 10-50, 10-40, 10-30, 10-20, 50-90, 60-90, 70-80, 80-90 wt.% of at least one hydrogel-forming polymer that is not degradable by an extracellular matrix degrading enzyme and / or and that is not conjugated to the therapeutic compound relative to the total weight of the hydrogel forming polymer in the composition. More preferably the wt.% ratio between the at least one hydrogel-forming polymer that is not degradable by an extracellular matrix degrading enzyme (and / or and that is not conjugated to the therapeutic compound) and the at least one hydrogel-forming polymer is between 9:1-1 :9, more preferably between 8:1-1:8, 7:1-1 :7, 6:1-1:6, 5:1-1:5, 4:1-1 :4, 3:1-1:3, 2:1-1:2, most preferably 1 :1.

[0048] Preferably, the at least one hydrogel-forming polymer that is not degradable by an extracellular matrix degrading enzyme is preferably dextran-tyramine, preferably wherein the number of tyramines per 100 monosaccharide units is between 2-30, preferably between 3- 20, more preferably between 5-15. In addition or alternatively, the composition according to the present disclosure preferably comprises hydrogel-forming polymer, wherein the crosslinking density in the composition is between 10-100%, preferably between 20-90, 30- 80, 40-60, 50-100, 10-60, 10-50, 10-40, 60-100, 70-100, 80-100% with respect to the amount of crosslinked tyramine units relative to the total amount of crosslinkable tyramine units. In addition or alternatively, the amount of therapeutic compound according to the present disclosure per the total amount of hydrogel-forming polymer in the composition is preferably between 0.001-20, 0.002-15, 0.003-10 wt.%, more preferably between 0.001-10, 0.002-8, 0. 003-4, 0.01-3, 0.05-0.3 wt.%, most preferably between 0.05-3 wt.%, wherein the wt.% is drawn on the wt. of the therapeutic compound with respect to the wt. of the at least one or more hydrogel-forming polymer. Preferably the wt.% ratio between the (at least one) therapeutic compound (preferably single domain antibody) and the at least one hydrogelforming polymer is between 1 :100 and 1:1 , more preferably between 1:50-1 :5, 1:20-1 :5, 1:15- 1 :3, most preferably 1:8.

[0049] Foreseen herein is a therapeutic compound wherein at least one hydrogel-forming polymer is conjugated. The at least one hydrogel-forming polymer may also be optional and / or replaced by any other moiety, for example a drug, radionuclide, toxin, peptide, photosensitizer.

[0050] In an embodiment, the composition according to the present disclosure is preferably in the form of an injectable hydrogel and / or an injectable composition capable of (in situ) forming a hydrogel. The composition according to the present disclosure preferably facilitates local accumulation of the composition at the injection site and / or is particularly suitable for injection at discrete injection sites such as a tumor, a site of inflammation, a synovial or other cavity, and within engineered tissues. The composition according to the present disclosure is preferably particularly suited for intra-articular injection. In addition, the composition may be injected using any suitable means, such as a container with a closeable orifice, for instance a printing nozzle or a printing device. Preferably, the composition of the present disclosure can pass through a hollow needle that is used to administer fluids to the body of a human in the medical profession. The needle may be a hypodermic needle which is a thin, hollow tube with a sharp tip that contains a small opening at the pointed end. Needle-free systems also exist and these also use injectable aqueous compositions as described herein. The needle is commonly used with a syringe, a hand-operated device with a plunger, to inject substances into the body. Preferably said hydrogel is formed in situ in the preferred injection site. Said hydrogel is preferably formed within 2 hours, more preferably within 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5 minute(s) after injection.

[0051] In an embodiment, therapeutic compound according to the present disclosure may be used in the prevention and / or treatment of disease, preferably auto-immune disease, cancer, osteoarthritis, inflammation, preferably in the treatment of joint-inflammation or degenerative joint disease, most preferably in the prevention and / or treatment of joint(-related) inflammation and / or joint(-related) disease. Optimal use of the therapeutic compound may be determined by those skilled in the art, and will vary depending on e.g., the strength of the preparation, the mode of administration, and the advancement of the disease condition. Additional factors relating to a particular subject being treated including subject age, weight, gender, diet, and time of administration, will result in a need to adjust the use. The therapeutic compound according to the present disclosure may be for use in combination therapy, preferably for use in co-delivery of multiple therapeutic agents that target simultaneously multiple pathological processes, preferably cartilage breakdown by inhibition of, for example, MMP-13 or ADAMTS5, synovial inflammation by blocking IL-1 p, TNF or IL-6, blocking pain using anti-NGF VHH and / or promoting cartilage repair by facilitating the delivery of FGF18, TGFp and BMP7.

[0052] As disclosed herein, the term ‘subject’ may preferably refer to a human.

[0053] The present disclosure further relates to an in vitro method for reducing the bioavailability of a biological molecule such as a cytokine, a cytokine receptor, a soluble antigen and / or an autoantibody, in a biological system, the method comprising providing said system with the therapeutic compound according to the present disclosure, preferably with the therapeutic compound according to the present disclosure, more preferably with the single domain antibody according to the present disclosure. In a preferred embodiment, the biological system may be an in vitro cell culture system. Such systems are inherently closed and provide a local environment to the therapeutic compound. The therapeutic compound is particularly suited to deplete the culture medium from one or more undesired soluble biological molecules such as proteinaceous factors.

[0054] The present disclosure further relates to a method of producing a hydrogel, said method comprising the step of curing a composition comprising the therapeutic compound (wherein at least one hydrogel-forming polymer is conjugated to the therapeutic compound), wherein said composition preferably comprises a suitable amount of a curing system. The term "suitable amount" with respect to the curing system as referred to herein, should be interpreted as an amount capable of curing said composition. In a preferred embodiment, the curing system according to the present disclosure comprises enzymatic curing, preferably using (a suitable amount of) peroxide, preferably hydrogen peroxide, and (a suitable amount of) peroxidase. The skilled person will understand that alternative curing systems based on curing radicals, including oxygen radicals, may be used and are envisioned as embodiments of the present disclosure. In another preferred embodiment, the composition comprises at least 0.001 , 0.003, 0.007, 0.01 wt.% hydrogen peroxide, most preferably at least 0.003 wt.% hydrogen peroxide, wherein the wt.% is based on the total weight of the composition. In addition or alternatively, the composition preferably comprises at most 0.01, 0.03, 0.1, 0.3, 0.4, 0.5, 0.7, 1 , 2, 3, 4, 5 wt.% hydrogen peroxide, most preferably at most 0.5 wt.% hydrogen peroxide, wherein the wt.% is based on the total weight of the composition. In addition or alternatively, the composition preferably comprises between 0.001-10, 0.002-5, 0.003-3, 0.003-0.3, 0.003- 0.03 wt.% hydrogen peroxide, most preferably between 0.003-0.03 wt.% hydrogen peroxide, wherein the wt.% is based on the total weight of the composition. In addition or alternatively, the composition comprises at least 0.00008, 0.00009, 0.0001 , 0.0002, 0.0003, 0.0004wt.% peroxidase, most preferably at least 0.0002 wt.% peroxidase, wherein the wt.% is based on the total weight of the composition. In addition or alternatively, the composition comprises preferably at most 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2 wt.% peroxidase, most preferably at most 0.2 wt.% peroxidase, wherein the wt.% is based on the total weight of the composition. In addition or alternatively, the composition comprises preferably between 0.00008-2, 0.00009-1, 0.001-0.5, 0002-0.2 wt.% peroxidase, most preferably between 0.0002-0.2 wt.% peroxidase, most preferably between 0.0002-0.2 wt.% peroxidase, wherein the wt.% is based on the total weight of the composition. Preferably, crosslinking is done via a phenolic compound, preferably a tyramine group. The amount of curing system may also depend on the amount of the at least one hydrogel-forming polymer in the composition, preferably a functional derivative of said hydrogel-forming polymer (i.e. hydrogel-forming polymer with one or more functional groups e.g. tyramine) in the composition. Preferably the ratio between peroxidase and the phenolic moieties on the at least one hydrogel-forming polymer is between 3:1-1:200 mg / mmol, more preferably between 2:1-1:150, most preferably between 1.5:1-1 :100 mg / mmol. Preferably, the molar ratio between peroxide and the phenolic moieties on the at least one hydrogel-forming polymer is between 1:16-1:2 mmokmmol, more preferably between 1 :8-1 :2.2, most preferably between 1:6-1 :2.2. In addition or alternatively, curing of a composition comprising the therapeutic compound may occur spontaneously (i.e. wherein said composition preferably does not comprise a curing system), particularly when the molecular weight of the at least one hydrogel-forming polymer is at least 1MDa and / or the composition comprises at least 2.5 w / v.% of the at least one hydrogel-forming polymer, wherein the weight is drawn on the total volume of the composition.

[0055] The present disclosure further relates to a hydrogel obtainable by the method as disclosed herein. Said hydrogel may be in any form, such as a macroscopic hydrogel, a microgel or nanogel, but is preferably a macroscopic hydrogel. The hydrogel obtainable by the method as disclosure herein may be both in s / tu-gelating (i.e. administered in liquid form to the desired site of administration, where the hydrogel subsequently gelates) or shear-thinning (i.e. preformed hydrogel which is administered the desired site of administration), but is preferably in s / tu-gelating. General definitions

[0056] In the following description and examples, a number of terms are used. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided. Unless otherwise defined herein, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0057] In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

[0058] Methods of carrying out the conventional techniques used in methods of the present invention will be evident to the skilled worker.

[0059] Brief description of the figures

[0060] Figure 1 Schematic showing the function of the injectable retention and triggered release system. After intra-articular injection, the VHHs neutralizing pro-inflammatory cytokines will be released in the joint due to proteolytic activity of the MMPs which are upregulated in the OA joint due to the activity of pro-inflammatory cytokines. Subsequently, the VHHs will capture and neutralize cytokines present in the joint cavity. Due to the reduced size of the VHH-cytokine complexes, these can be cleared from by the synovial membrane, removing the trigger that leads to MMP activity and thereby restoring the joint homeostasis.

[0061] Figure 2. Biological activity of HA-TA-VHH conjugates and VHH-loaded hydrogels. A) The effect of VHH and HA-TA-VHH conjugates on blocking TNFa signaling using the NF-KB reporter (Luc) HEK293 cell line, compared to the commercial antibody Adalimumab (Ada). B) Schematic illustration of trans well experimental design (top) and the effect of the hydrogel and VHH- loaded hydrogel on blocking TNFa signaling using the N F-KB reporter (Luc) HEK293 cell line. The Luciferase activity was normalized to the total DNA content, and expressed as relative induction with respect to the control (non-treated cells). Positive controls were treated with TNFa in the absence of VHH. Data are reported as average ± standard deviation.

[0062] Figure 3. A) Schematic illustration of tethered-VHH release triggered by enzymatic degradation. B) Enzymatic degradation of HA-TA hydrogels and C) VHH release profile from hydrogels exposed to a buffer containing 5.0 or 50 U / rnL Hyaluronidase at 37 °C as determined by ELISA .

[0063] Figure 4. Effects of VHH-loaded hydrogels on ex vivo cartilage explants after stimulation with TNFa. A) Bovine cartilage explants harvest. The cartilage explants were removed from the condyles with 3 mm biopsy punch and a scalpel. B) Schematic of the cartilage explants cultured in wells and incubated with VHH-loaded hydrogel. C) Cumulative GAG release from cartilage explants into the culture medium over a time period of 14 days.

[0064] Figure 5. Design and characterization of a VHH construct with a MMP cleavage sequence. A) Schematic illustration of the used VHH constructs. Cys stands for unpaired Cysteine. The cleavage site in MMPcleav-13B1 VHH can be cleaved by MMP-13. B) SDS- PAGE analysis of the VHH before and after introducing a MMP cleavage sequence. Protein ladder (lane 1), 13B1 VHH (lane 2), MMPcleav-13B1 VHH (lane 3). C) MMP cleavage test of MMPcleav-13B1 VHH by Western blot after incubation with a concentration range of activated MMP-13 for 2 hours at 37°C. The graph showed the dose-response relationship between the concentration of MMP-13 and the cleavage of MMPcleav-13B1.

[0065] Figure 6. Conjugation of MMPcleav-13B1 VHH to Dex A) Schematic illustration of the formation and cleavage of Dex-VHH conjugates. B) SDS-PAGE results of free MMPcleav-13B1 (lane 2), Dex-MMPcleav-13B1 conjugate (lane 3), cleaved MMPcleav-13B1 from conjugate (lane 4) with protein ladder (lane 1).

[0066] Figure 1. VHH-loaded microgel production using diffusion-based enzymatic crosslinking. A) Photographs of the assembled PDMS microfluidic chip. B) Schematic illustration of the process of VHH-loaded microgel production. This microfluidic platform consisted of a PDMS microfluidics chip as a microdroplet generator and a semipermeable silicone tube submerged in a H2O2 diffusion bath for enzymatic crosslinking. C) Microphotograph of VHH-loaded microgels. D) Size distribution of VHH-loaded microgels.

[0067] Figure 8. VHH release profile from VHH loaded microgels in vitro. A) Schematic representation - release of tethered VHHs from microgels was triggered by MMP-13 through cleavage of the MMP-cleavable sequence engineered in the C-terminus of the VHHs. B) Cumulative release of VHH from microgels. VHH-loaded microgels were incubated with (red circles) or without (black squares) 10 nM activated MMP-13. MMP-13 was refreshed at every time point to maintain enzyme activity.

[0068] Figure 9. A) Schematic overview of the creation of HA-VHH conjugate, Dex-VHH conjugate and HMW Dex-VHH conjugates. B) Optical images of two resulting hydrogels formed by using HRP as a catalyst and H2O2 as an oxidant. C) Protein analysis, i) SDS- PAGE results of HA-VHH conjugate (left), Dex-VHH conjugate (middle) and HMW Dex-VHH conjugate (right); The star (*) highlighted HMW Dex-VHH conjugate which was retained in the slot due to its high molecular weight, ii) Western blot results of HA-VHH conjugate using an anti-VHH antibody.

[0069] Figure 10. Schematic of the formulation and handling of the four conditions evaluated in the in vivo retention time study in Wistar Rats (Created with BioRender). In all conditions the VHH was NIR labelled, the conditions consisted of; 1) VHH only condition, 2) HMW Dex- VHH conjugate, 3) Dex / HA-VHH in situ crosslinked and 4) Dex-VHH in situ crosslinked.

[0070] Figure 11. Retention time of plain VHH coupled to a NIR label and VHH tethered to the biomaterials. The graph shows the relative fluorescent units in percentage over 91 days (13 weeks), the insert shows the fluorescent half-life of the VHH and the images below corresponds with the graphs. Fluorescent intensity of the mean signal (n=6) of three images per joint per rat until 5 weeks, after which the rats were euthanized and from 6-13 weeks n=3. In each image both a background ROI was measured and a ROI around the knee joint.

[0071] Figure 12. Conjugation efficiency of various 65E5 VHHs to Dex-Mai and HA-Mal. The graph shows improved conjugation of the VHH to both HA-Mal and Dex-Mai after introducing a GS linker into the C-terminus of the VHH.

[0072] Figure 13. The stability of the VHH is not affected by the enzymatic activity of Hyaluronidase and Dextranase as shown on Western Blot.

[0073] Figure 14. The stability of the VHH is not affected by the enzymatic activity of Hyaluronidase and Dextranase. The VHH is not degraded into smaller fragments as shown on SDS-PAGE. Figure 15. Conjugation of MMPcleav-B1 VHH to Dex A) Schematic illustration of conjugation strategy and VHH cleavage. B) SDS-PAGE results of Dex-VHH conjugate and cleaved VHH from conjugate. Free MMPcleav-B1 (lane 2), Dex-MMPcleav-B1 conjugate (lane 3), cleaved MMPcleav-B1 from conjugate (lane 4). C) Binding kinetics of Dex- MMPcleav-B1 conjugate and cleaved MMPcleav-B1 from conjugate targeting IL1-R.

[0074] Figure 16. The degradation profiles of 5 w / v% hydrogel discs with various compositions ranging from pure Dex-TA (•) to pure HA-TA (°) incubated with 5 ll / rnl Hyaluronidase for 29 days.

[0075] Figure 17. Shear dependent viscosity of 10 %w / v LMW Dex-TA / HA-TA (50 / 50), 5 %w / v MMW HA-TA, and 10 %w / v LMW Dex-TA / MMW HA-TA (50 / 50) polymer solutions. LMW Dex = 40 kDa, LMW HA-TA = 30 kDa, MMW = 300 kDa. Injectability ranges are indicated.

[0076] The following Examples illustrate the different embodiments of the invention. VHH binding to NGF, MMP9 and IL1 R were kindly provided by Orthros Medical b.v. Raalte, the Netherlands. VHH binding to TNFa were kindly provided by Verlin b.v, Apeldoorn, the Netherlands. DNA constructs with engineered protease cleavage site and / or C-terminally introduced cysteine were ordered at GeneScript. EXAMPLE 1

[0077] This example describes a method to achieve site-specific VHH conjugation to maleimide functionalized polysaccharides, including dextran-maleimide (Dex-Mai) and hyaluronic acid- maleimide (HA-Mal), with a high conjugation efficiency. In this example, an additional genetic engineering step is used to incorporate readily accessible cysteine residues within the structure of a VHH, which makes it possible to conjugate the VHHs to polymer backbones.

[0078] The inventors have shown successful conjugation of the VHH to the polymer backbone via thiol-maleimide chemistry. Using an NF-KB luciferase reporter cell line, the inventors demonstrated that the hydrogel, functionalized with the VHH, efficiently inactivated TNFa. In addition, a nearly linear VHH release from hydrogel was observed in vitro in presence of 5 U / mL of hyaluronidase. Furthermore, the VHH-loaded hydrogel could significantly reduce cartilage degradation activated by exposure to TNF-a. The results demonstrate that this type of VHH-loaded hydrogels have great potential for the neutralization of pro-inflammatory catabolic cytokines after intra-articular injection by increasing the retention time of neutralizing antibody fragments in the joint cavity.

[0079] Materials

[0080] Sodium hyaluronate (HA, Mw 27 kDa, pharmaceutical grade) was purchased from Contipro Pharma, Dolni Dobrouc, Czech Republic. Dextran-tyramine (Dex-TA) containing 15 tyramine moieties per 100 saccharide units, was synthesized as described in literature [Fu, Y.; Zoetebier, B.; Both, S.; Dijkstra, P.J.; Karperien, M. Engineering of Optimized Hydrogel Formulations for Cartilage Repair. Polymers 2021, 13, 1526. https: / / doi.org / 10.3390 / polym13091526] . Tyramine hydrochloride (TA HCI, >98%), NaCI (>99.0%), horseradish peroxidase (HRP, 325 units / mg solid), hyaluronidase, 1,9-dimethyl- methylene blue, chondroitin sulfate from bovine trachea, Tween-20, DMSO-de (99.9%), D2O (99.9%), tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and Amicon® Ultra-0.5 centrifugal filter (3 kDa MWCO) were purchased from Sigma-Aldrich. 4-(4,6-Dimethoxy-1 ,3,5- triazin-2-yl)-4-methylmorpholinium chloride (DMTMM, 97%) was purchased from Fluorochem Ltd., Hadfield, UK. n-(2-Aminoethyl)maleimide hydrochloride (MEA HCI , >93.0%) was purchased from TCI EUROPE N.V., Zwijndrecht, the Netherlands. Hydrogen peroxide (H2O2, 30%), ethanol (>99.9%) and diethyl ether (>99.7%) were purchased from Merck, Kenilworth, NJ, USA. Phosphate buffered saline (PBS) were obtained from Lonza, Basel, Switzerland. Milli-Q water was used from the Milli-Q Advantage A10 system (Merck KGaA, Darmstadt, Germany) equipped with a 0.22 pm Mi II ipak®-40 Express filter. Synthesis of hyaluronic acid-tyramine-maleimide (HA-TA-Mal)

[0081] First, HA-TA was prepared by amidation of the HA carboxyl groups with tyramine. Briefly, Sodium hyaluronate (5.00 g, 12.5 mmol repeating units (r.u.)) was dissolved in 500 mL Milli-Q water in a 1 L round bottom flask equipped with a stirrer bar. While stirring at room temperature, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM, 3.46 g, 12.5 mmol) and tyramine hydrochloride (TA HCI, 2.17 g, 12.5 mmol) were added subsequently. After 24 hours, 40 ml saturated NaCI solution was added to the reaction mixture and the reaction mixture was poured into 2.5 L cold ethanol. The crude product was isolated by centrifugation at 5000 rpm, washed several times with ethanol, and dried in vacuum. The crude product was dissolved in 75 mL Milli-Q water and dialyzed against Milli-Q water for 3 days (MWCO 3500 Da). Lyophilization yielded the product as a white foam. The successful syntheses of HA-TA were confirmed using1H NMR in D2O,1H-NMR (400 MHz, D2O): 6(ppm) = 7.16 (d, 2H); 6.77 (d, 2H); 4.73 - 4.71 (m, 1 H); 4.45 (d, 1 H); 4,36 (s, 1 H); 3.76-3.22 (m, 6H); 2,79 (s, 2H); 2.61 (s, 2H); 1.93 (s, 3H). (Fig. S1A).

[0082] Next, HA-TA (1 ,5g, 3.5 mmol r.u.) was dissolved in 50 mL of MES solution (20mM, pH6.8) in an 100 mL round bottom flask equipped with a stirrer bar. While stirring at room temperature, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM, 0.653 g, 2.36 mmol) and N-(2-Aminoethyl)maleimide hydrochloride (AEM HCI, 0.417 g, 2.36 mmol) were added subsequently. The mixture was stirred at room temperature for 10 h. The reaction mixture purified by extensive dialysis (MWCO 3500 Da) against Milli-Q water, followed by neutralization with NaOH and lyophilization yielded the product as a white foam. The successful synthesis of HA-TA-Mal was confirmed by1H-NMR in D2O.1H-NMR (400 MHz, D2O): b(ppm) = 7.16 (d, 2H); 6.79 (s, 2H); 6.77 (d, 2H); 4.73 - 4.71 (m, 1 H); 4.45 (d, 1H); 4.36 (s, 1 H); 3.76-3.22 (m, 6H); 2.79 (s, 2H); 2.61 (s, 2H); 1.93 (s, 3H). The number of tyramine or maleimide per 100 disaccharide units was calculated based on the methyl group at 1.93 ppm (corresponding to the 3 methyl protons from HA, compared with the integral of the 2 aromatic protons of tyramine at 6.77 ppm and 2 aromatic protons of tyramine at 7.16 ppm or 2 maleimide protons at 6.79 ppm. The resulting HA-TA-Mal contained 5 tyramine moieties and 12 maleimide moieties per 100 disaccharide units (Fig. S1 B).

[0083] Preparation of HA-VHH conjugates.

[0084] VHH 65E5 containing an unpaired free cysteine in its C-terminal tail binding with high affinity to TNFa was provided by Orthros Medical bv (Raalte, the Netherlands). HA-VHH conjugates were prepared as follows; the VHHs were reduced in PBS by the addition of a 10 fold molar excess of TCEP and incubation for 1 hour at 37 °C, followed by the removal of TCEP through ultrafiltration units with a 3 kDa MWCO (Amicon Ultra, Millipore). Afterwards, the reduced VHHs were added to an HA-TA-Mal solution in PBS (pH 7.4), in a 1 :10 molar ratio (VHH:Polymer) to produce HA-VHH conjugates. The reaction mixtures were gently shaken overnight at 4 °C. The unreacted maleimide moieties were blocked by addition of excess cysteine. Finally, excess cysteine was removed through ultrafiltration units with a 3 kDa MWCO at 4 °C with PBS buffer (pH 7.4). The unreacted VHH and successful HA-VHH conjugates were separated on a SDS-PAGE gel under reducing conditions and subsequently identified using Western blot. The conjugation efficiency of VHH to HA-TA-Mal was analyzed by AlphaView software and calculated as follows:

[0085] Control VHH — Unreacted VHH Conjuqation efficiency = - - - x 100%

[0086] J J JControl VHH

[0087] Enzyme-linked immunosorbent assay (ELISA)

[0088] Nunc MaxiSorp flat-bottom 96-well plates (442404; Thermo Fisher Scientific, MA, USA) were coated with TNFa (210-TA, R&D systems, MN, USA) in PBS overnight at 4 °C, after which they were blocked with 2% BSA in PBS for two hours at RT in order to block non-specific binding sites. Subsequently, a serial dilution of VHHs (ranging from 10 pM to 1.0 pM) in 1% BSA in PBS was incubated in the TNFa coated wells for two hours at RT. The wells were washed with PBST (PBS containing 0.05% Tween-20) and PBS. Bound VHHs were detected by incubation with a rabbit anti-VHH antibody (1 :1000 dilution) (K1612, QVQ, Utrecht, the Netherlands) and a goat anti-rabbit antibody coupled to a peroxidase (1 :2000 dilution) (PO448, Dako, CA, USA). The reactions were carried out by the addition of a substrate reagent and stop solution (DY999, R&D systems, MN, USA ), and the absorbance was measured at 450 nm (Micro Plate Reader). The ECso (the concentration of antibody that gives half-maximal binding) were calculated using the curve fitting model (MyCurveFit.com, MyAssays Ltd, Brighton, UK).

[0089] Luciferase Reporter assay

[0090] The stably transfected NF-KB reporter (Luc) HEK293 cell line was used for analysis of cellular responses that result in modulation of NF-KB activities triggered by pro-inflammatory cytokines. Cells were seeded at a density of 7500 cells / cm2and cultured in DMEM supplemented with 10% FBS, 100 U / mL penicillin and 100 U / mL streptomycin, and incubated at 37°C with 5% CO2 for 48 hours. Afterwards, the cells were cultured in starvation medium (DMEM, 1% FBS, 100 U / mL penicillin and 100 U / mL streptomycin) for 24 hours and subsequently stimulated with TNFa (2 ng / mL) (R&D systems, MN, USA) in the presence or absence of VHH or HA-TA-VHH conjugates with different molar ratios of TNFa to VHH. As a positive control, a commercial anti-TNFa antibody (Adalimumab) was applied in this experiment. After 24 hours, cells were washed with PBS and lysed with lysis buffer (Promega, Wl, USA). To evaluate luciferase activity, cell lysate was added to luciferase assay reagent (Promega, Wl, USA) and luminescence was measured with a Vector microplate reader (Promega, Wl, USA. The luminescence units were corrected for DNA content. The DNA concentration was determined the CyQuant Cell Proliferation Assay Kit, Invitrogen, MA, USA according to the manufacturer's instructions.

[0091] Hydrogel formation and gelation time

[0092] Enzymatically crosslinked hydrogels of the tyramine-conjugated polymers (Dex-TA, HA-TA and HA-TA-Mal) at a total polymer concentration of 5.0% w / v were prepared in vials at room temperature. In a typical procedure, after dissolving the tyramine-conjugated polymers in PBS, HRP (40 units / mL) was added to the polymer solution and incubated overnight at 4 °C. Subsequently, a freshly prepared PBS solution of H2O2 (0.15 % w / v ) was added to the HRP containing polymer solution and immediately mixed using a vortex mixer. The final gel concentrations were a 5% w / v polymer, 4 U / mL HRP and 0.015% H2O2. The time to form a gel was determined using the vial tilting method. The experiment was performed in triplicate.

[0093] Rheological analysis

[0094] Cylindrical hydrogels (8 mm diameter and 1.5 mm height) of tyramine-conjugated polymers for rheological experiment were prepared in Teflon molds at a final gel concentrations of 5% w / v polymer, 4 U / mL HRP and 0.015% H2O2. After equilibrating overnight in PBS the rheological properties of the gels were measured.

[0095] Rheological experiments were carried out with a MCR 301 rheometer (Anton-Paar) using parallel plates (08mm, 0°) set to 25 °C in the oscillatory mode. In a typical experiment, preformed gel samples were placed at the center of ground plate, while the upper plate was lowered to achieve an initial normal force of 0.05 N. The storage (G’) and loss (G”) moduli were recorded under oscillatory shear at a strain of 0.5% and frequency of 1 Hz.

[0096] Enzymatic degradation of HA hydrogel and VHH release

[0097] In degradation experiments, the cylindrical hydrogels of HA-TA at a concentration of 5% w / v were prepared in 8 mm diameter, 1.5 mm high molds and the initial weight of the hydrogels (Wi) was measured after equilibrating overnight in PBS. 1.0 mL of PBS containing 5.0 or 50 U / mL hyaluronidase was placed on top of the prepared hydrogels and the samples were incubated at 37 °C. At regular time intervals, the hydrogels were weighed. The incubation buffer (including hyaluronidase) was replaced every 2-3 days and the experiments were performed in triplicate. The remaining gel (%) was calculated from the original gel weight after preparation (Wi) and remaining gel weight after exposure to the hyaluronidase containing buffer (Wt), expressed as Wt / Wi x 100%.

[0098] For VHH release tests, VHH-loaded cylindrical hydrogels (0.46 nmol VHH) consisting of HA- VHH conjugate and HA-TA at a total of polymer concentration of 5% w / v were prepared in 8 mm diameter, 1.5 mm high molds. Subsequently, the VHH-loaded hydrogels were incubated with a release buffer containing hyaluronidase and 0.1% BSA at 37 °C. At regular time intervals, the buffer was collected from the sample and the concentration of released VHH in the buffer was determined using an indirect ELISA. The experiments were performed in triplicate.

[0099] Bovine cartilage explant harvest and culture

[0100] Bovine knee joints were obtained from the local slaughterhouse (Haaksbergen, the Netherlands), and cartilage was harvested from the femoral condyles and cultured. Briefly, cylindrical cartilage disks were isolated from the load-bearing area of the femoral condyles using a 3 mm biopsy punch and then sliced to obtain the top 1 mm of cartilage explants with intact superficial zone. Before the start of the experiment, the cartilage explants were left in culture to equilibrate for 2 days in DMEM medium supplemented with 10% FBS, 10mM HEPES, 0.1 mM nonessential amino acids, 0.4 mM proline, 100 U / rnL penicillin and 100 U / rnL streptomycin at 37°C with 5% CO2. After equilibration to in vitro environment, the cartilage explants were cultured in the same medium in 24-well plates with various treatment conditions for 14 days.

[0101] Treatment conditions were divided into four groups: no treatment (control), 5 ng / ml of TNFa treatment (TNFa), 5 ng / ml of TNFa and 50 ng / ml of free VHH treatment (TNFa+VHH), 5 ng / ml TNFa and VHH-loaded (0.8 pg per gel) hydrogel treatment (TNFa+VHH-loaded gel), and VHH-loaded (0.8 pg per gel) hydrogel treatment (VHH-loaded gel).

[0102] Glycosaminoglycan (GAG) loss assay.

[0103] During the in vitro culture of cartilage explants, the culture medium was refreshed and collected every 2 or 3 day. The cumulative GAG release from explants was defined as the GAG released into the medium divided by the sum of GAG release in culture medium during 14 days culture. GAG levels in the culture medium were determined by reacting with 1,9- dimethyl-methylene blue (DMMB), using chondroitin sulfate from bovine as standard. After incubating with DMMB, the samples were examined using a plate reader at 525 nm. Results and discussion

[0104] Synthesis and characterization of HA-TA-Mal

[0105] HA is a linear natural polysaccharide containing primary and secondary hydroxyl, carboxyl, and N-acetyl groups within each disaccharide repeating unit. The chemical modification of HA is mainly focused on its two functional groups, i.e. the carboxylic acid group and hydroxyl groups. Here, the synthesis of HA-TA-Mal involved the modifications of the carboxylic group on the glucuronic acid part of HA, which was achieved by subsequent reacting HA with TA HCI and AEM HCI via DMTMM-activated amidation, respectively.1H-NMR spectrum of HA-TA-Mal showed that the number of substituted tyramine (TA) and maleimide (Mai) moieties per 100 disaccharides in HA was 5 and 12, respectively. In the present work, injectable hydrogels were obtained by enzymatic crosslinking via the formation of tyramine- tyramine bonds under physiological conditions by mixing the polymer conjugates with HRP as a catalyst and H2O2 as an oxidant. The storage modulus (G') and loss modulus (G") reveal that highly elastic hydrogels were formed at non-toxic amounts of HRP and H2O2. The rheological properties are important in keeping the integrity of network structures, thus containing drugs within the hydrogels and releasing them to target sites for a long period of time. The gelation time of HA-TA was around 7 min and slightly slower gelation was observed after modifying HA-TA with maleimide. On the other hand, mixing HA-TA-Mal with Dex-TA, which had a high substitution degree of TA (DS=15), largely decreased the gelation time. The gelation time can be controlled by changing the HRP concentration from seconds up to minutes, facilitating the handling and in situ formation of hydrogels, giving the hydrogel system great potential as injectable scaffolds for various applications.

[0106] Conjugation of VHHs to HA-TA-Mal

[0107] Many bioconjugation strategies are developed to create polymer-protein conjugates. An ideal modification should be capable of targeting a uniquely reactive amino acid while retaining the biological activity of the proteins. Among the 21 natural amino acids, free cysteine provides a unique opportunity to achieve the synthesis of site-specific polymer-protein conjugates due to its low natural abundance in native proteins. However, many proteins do not contain free cysteines that are accessible for modification, because most of the wild-type cysteine residues are generally tied up in disulfide linkages. To overcome this limitation, incorporating a free cysteine residue in the C-terminus of VHHs using genetic engineering enabled the inventors to develop a biocompatible and efficient way to couple VHHs to HA. The hyaluronic acid, intended for bioconjugation, was modified with Mai groups considering their specificity at neutral pH for the reaction with thiols over primary amines. The conjugation of VHHs to HA is illustrated where HA-TA-Mal is incubated with the free cysteine-containing VHH 65E5 (anti- TNFa) overnight at 4 °C to produce the HA-TA-VHH conjugate. Residual maleimides were quenched by excess of cysteine. Characterization of the HA-TA-VHH conjugate was done by western blot analysis. The free VHH-65E5 resulted in one band, indicating no molecular modifications while the HA-TA-VHH conjugate vaguely appears as a smear with a larger effective molecular weight than free VHH-65E5, leaving behind a small amount of nonconjugated VHH-65E5 at the position (Mw) of the control VHH. Quantification showed that 95% of the VHH 65E5 was tethered onto the backbone of HA via the thiol-maleimide reaction. The binding affinity of the HA-TA-VHH conjugate for TNFa was characterized using ELISA. The conjugated VHH showed a low ECso (the concentration of antibody that gives half- maximal binding) value of 2.6 nM corresponding to a high affinity. The affinity of the polymer- VHH complex was slightly lower than the binding affinity of the native VHH-65E5 (ECso= 1.2 nM). Nevertheless the HA-TA-VHH conjugate exhibited sufficient ability to bind TNFa. The HA-TA-VHH was further tested by preparing hydrogels based on a mixture of HA-TA-VHH conjugate complemented with HA-TA which was subsequently allowed to crosslink (using HRP / H2O2) and form a hydrogel in situ in a bone-shape mold, within 10 minutes. Prior to conjugation the VHH was labeled with FITC enabling visualization of the VHH in the hydrogel using fluorescence. This property makes the hydrogel suitable for injection and defect filling, showing the opportunity for cartilage repair by resorting joint homeostasis when loaded with therapeutic biologies.

[0108] Functional analysis of HA-TA-VHH conjugates and VHH-loaded hydrogels The VHHs in the hydrogel should be available and remain active after coupling and crosslinking over a prolonged period of time. Previously has been demonstrated the efficacy in neutralizing activity of DKK1 and FRZB VHHs in vitro using hMSCs as a cell model. Here, to study if the VHH after coupling is able to capture and deactivate TNFa, the inventors used an NFKB luciferase reporter cell line. The expression of luciferase in cells is upregulated after stimulating with TNFa by activating NFKB signaling. Dose-response experiments were performed to investigate the capacity of the VHH in blocking the TNFa signaling. The results showed that both non-conjugated and conjugated VHHs can efficiently block the effect of TNFa. 100-fold molar excess of both VHHs completely restored the luciferase expression to its original levels. At the same concentration, there were no significant differences between non-conjugated and conjugated VHHs in capturing and deactivation of TNFa (Fig 2A). To prove that the VHHs remain active after gelation, the inventors made a thin hydrogel layer consisting of equimolar HA-TA-VHH conjugate either mixed with HA-TA or Dex-TA on the bottom of transwell inserts. As shown in the schematic, TNFa was added to the transwell insert to stimulate the luciferase reporter cells which were plated in the bottom well plate (Fig 2B). The inventors hypothesized that the VHH-loaded gel layer can capture TNFa and prevent them from activating NFKB signaling of the luciferase reporter cells. The result showed that the VHH-loaded hydrogel could indeed efficiently capture TNFa, resulting in a low expression level of luciferase, similar as the control. Interestingly, a pure HA hydrogel without VHH loading was also able to capture TNFa to limited extent. HA has a negative charge and through electrostatic interactions may reduce diffusion of TNFa through the hydrogel. Indeed, when a neutral Dex-TA instead of HA-TA gel was applied stimulation with TNFa resulted in similar activation of the reporter construct compared to control. When VHH were conjugated to Dex-TA-Mal polymers, the corresponding functionalized hydrogels were equally effective in blocking TNFa activity as the functionalized HA-TA-Mal hydrogels (Fig 2B).

[0109] Enzymatic degradation-triggered VHH release

[0110] HA is a naturally occurring polysaccharide that is found natively in many tissues (e.g. cartilage), and is degraded by hyaluronidases (HAase) that are for instance present in the joint. HA is widely used in tissue engineering and drug delivery due to its excellent biocompatibility and degradation profile. In this study, the degradation profiles of HA-TA hydrogels at polymer concentrations of 5% w / v was examined in presence of low (5.0 U / rnL) and high (50 U / rnL) concentrations of hyaluronidase. The hydrogels were kept at 37 °C and their weights were measured at regular time intervals. The results presented in Fig 3A showed that the gels were nearly degraded within 14 and 28 days at high and low concentration of hyaluronidase, respectively. In many hydrogel delivery systems, drugs are physically entrapped inside the hydrogel network and then passively released over time. Under these conditions, diffusion is the dominant mechanism for drug release from the hydrogel, which usually results in rapid release of the drug from the gel after administration often with an initial burst release. To overcome this issue, a promising approach in the present work was to conjugate VHHs directly to the polymer backbone of the hydrogel through stable covalent bonds. Thus the release of VHHs loaded in the HA hydrogel was governed primarily by network degradation followed by the release of VHH conjugated to small HA fragments (Fig 3B). To examine the release profile of the tethered VHH, VHH- loaded HA hydrogels were prepared using a mixture consisting of HA-TA-VHH conjugate and HA-TA in the presence of HRP and H2O2 which were subsequently treated with a low and high concentration of HAase. The release was followed over a period of 14 days (Fig 3C). In accordance with the degradation profile of the HA hydrogel, the VHH-loaded hydrogel exposed to 50 U / ml HAase exhibited fast release with nearly 90% VHH released over 10 days. Interestingly, an almost linear drug release over a period of 2-14 days was observed with 5.0 U / ml HAase. In addition, it is worth mentioning that an initial release was found for VHH-loaded hydrogels without HAase of approximately 20% of the entrapped VHH over a time course of 2 to 3 days, indicating that a small portion of the VHH was not conjugated to the backbone of polymer resulting in a diffusion-dependent release. To avoid the initial burst release, a thorough washing step is preferred to remove non-conjugated VHH from the hydrogel, resulting in a hydrogel delivery system which will be able to achieve close to zeroorder kinetics, without an initial burst release and a relatively constant release over time. This strategy to control the release of VHH is based on regulating network degradation in response to environmental cues, which is dependent on the actual local enzyme activity.

[0111] VHH-loaded hydrogel reduces cartilage degradation induced by TNFa

[0112] Recent progress on understanding of pathogenesis of OA, suggesting a locally elevated inflammatory state is believed to be linked to the progression of OA in the early stages of the disease. Pro-inflammatory cytokines such as I L-1 p, TNFa and IL-6 are critical mediators in the disturbed metabolism and enhanced catabolism of tissue in the OA joint. In order to study the effects of VHH-loaded hydrogels on cartilage tissue in the presence of the cytokine TNFa, the inventors studied the loss of extracellular matrix compounds in bovine cartilage explants. The changes of ECM composition in the cartilage was profiled by measuring glycosaminoglycan (GAG) release into the culture medium. Cartilage explants were isolated from the femoral condyles of the bovine knee joint, cultured and treated for 2 weeks. The culture medium, including TNFa was exchanged 3 times per week, and compared with explants cultured without TNFa (Fig 4). The loss of GAG content into the culture medium from the cartilage explants was assessed, showing TNFa increased the GAG loss compared to the control. When adding free VHH 65E5, the loss of GAG was decreased to similar values as the control, indicating the catabolic effect of TNFa on cartilage was blocked. Treating the cartilage explant with a VHH loaded hydrogel in the absence of TNFa did not affect GAG release. Interestingly, in the presence of TNFa, the VHH loaded hydrogel neutralized the effect of the cytokine on GAG release. The level of GAG loss was slightly higher than the control and the group treated with free VHH. This could be due to the fact that the hydrogel with tethered VHH was passively lying next to the explant, having less access to capture the TNFa in the culture medium compared to the free VHH. The inventors cannot exclude, however, that in the course of 14 days stimulation TNFa induced the degradation of the hydrogel itself via upregulation of hyaluronidase activity and that the subsequently released HA fragments resulted in a small increase in cumulative GAG release. In practicality, the in vivo degradation of the HA hydrogel will lead to VHH release from the hydrogel. In addition, increasing VHH loading in the hydrogel may also improve the capacity of VHH-loaded hydrogel to capture its targeting pro-inflammatory cytokines in vivo. Consequently, the results demonstrated that VHH-loaded hydrogels have great potential for capturing cytokine TNFa and reducing cartilage tissue degradation induced by TNFa. Conclusion

[0113] The inventors successfully synthesized tyramine and maleimide functionalized hyaluronic acid (HA-TA-Mal) and developed a biocompatible and efficient way to couple VHHs to hyaluronic acid, preserving the biological activity of the VHH. These conjugates could be used for in situ generation of VHH-loaded hydrogels capable of capturing the cytokine TNFa. In addition, the inventors have identified that the VHH, immobilized covalently into a hydrogel, was released at a rate dictated by the enzymatic degradation of the hydrogel network in presence of hyaluronidase. This strategy will enable a relatively constant release to enhance VHH therapeutic lifetime. An ex vivo study using cartilage explants indicated that a VHH- loaded hydrogel efficiently prevented the progression of cartilage degradation induced by TNFa. These results demonstrate the efficacy of this functionalized injectable hydrogel in neutralizing its targeting pro-inflammatory cytokine, which provides proof of concept that intraarticular injection of hydrogels loaded with VHHs can be used for the long term release of neutralizing VHHs in the joint cavity. The study also opens up new possibilities for testing the same concept with potential other therapeutic VHHs to treat osteoarthritis.

[0114] EXAMPLE 2

[0115] As an alternative strategy for controlled release of VHH, Example 2 presents an enzyme- responsive drug delivery system from which the release of VHHs is triggered and then modulated through matrix metalloproteinases (MMPs) present in the synovial fluid. To achieve this, a genetically engineered VHH with an MMP-cleavable sequence was covalently conjugated to dextran and this conjugate was subsequently used to form VHH-loaded microgels using microfluidics. Upon exposure to the MM P-13, the sustained release of VHH from microgels was observed over the period of one week.

[0116] In this Example, the inventors developed an enzyme-responsive drug delivery system triggering the release of a cytokine neutralizing VHH from microgels by matrix metalloproteinases (MMPs). To achieve this, dextran-tyramine (Dex-TA) was functionalized with maleimide to obtain Dex-TA-Mal. Maleimide chemistry was subsequently used for the covalent bonding of the polymer with a variable domain of a single chain, heavy chain only antibody (VHH) genetically engineered with an MMP cleavable peptide sequence and an unpaired free cysteine residue at its C-terminus. The inventors show that the MMP cleavage site of the VHH was still accessible after conjugating to dextran resulting in the release of the VHH in the presence of active MMP-13. The Dex-TA-VHH conjugates were subsequently used to form stable microgels on a microfluidic chip by inducing tyramine - tyramine crosslinks in the presence of horseradish peroxidase and hydrogen peroxide. Upon exposure to active MMP-13, sustained release of VHH from microgels was observed over a period of one week. Our results suggest that the covalent tethering of VHH engineered with an MMP- sensitive peptide linker to hydrogel matrices can be used for creating injectable MMP triggered release systems of disease modifying antibody fragments.

[0117] Materials

[0118] Dextran 40 (40 kDa, pharmaceutical grade) was purchased from Pharmacosmos, Denmark. Tyramine (99%), DMF (anhydrous, 99.8%), LiCI (99.0%), p-nitrophenyl chloroformate (96%), N-Boc-1 ,4-diaminobutane (>97.0%), pyridine (anhydrous, 99.8%), triethylamine (Et3N, >99.5%), DMSO-d6(99.9%), sodium hydroxide(NaOH, >97.0%), tris(2- carboxyethyl)phosphine hydrochloride (TECP) trifluoroacetic acid (TFA, >99.0%), horseradish peroxidase (HRP, 325 units / mg solid), bovine serum albumin (BSA, >95.0%), p- Aminophenylmercuric acetate and hydrogen peroxide (30%) were purchased from Sigma- Aldrich. Brij® 35, Span® 80, coomassie brilliant blue R 250, n-hexadecane (>99.0%), ethanol (>99.9%) and diethyl ether (>99.7%) were purchased from Merck. N-(a- maleimidoacetoxy)succinimide ester was purchased from Fluorochem Ltd. UK Milli-Q water was used from Milli-Q Advantage A10 system equipped with an 0.22pm Millipak®-40 Express filter. 13B1 and MMPcleav-13B1 VHHs binding with high affinity to IL1-R were produced in yeast and kindly provided by Orthros Medical (Raalte, the Netherlands). The following MMP13 cleavable peptide sequence was used GPQGIWGQ.

[0119] SDS-PAGE analysis of VHH

[0120] For the VHHs analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE) was carried out in a Mini-PROTEAN Tetra Cell system (Bio-Rad). The VHH samples were treated with 4x Laemmli sample buffer (Bio-Rad), adding 10 wt% 2-mercaptoethanol to the buffer prior to mixing with the sample. Samples were then heated to 95°C for 5 minutes to denature the protein sample and ensure reduction of any disulfide bonds. Samples were loaded onto commercially available 10-well 4-15% Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad). Gels ran for 45 minutes at 160 V in a running buffer (25 mM Tris, 192 mM Glycine, 0.1% w / v SDS, pH 8.3). Protein bands were subsequently visualized by incubating the gels in a staining solution (0.1% Coomassie Brilliant Blue R 250, 40% MeOH, 10% acetic acid in water) with gentle agitation for 1 hour. This was followed by a destaining protocol in which the gels were gently agitated in a destaining solution (40% MeOH, 10% acetic acid in water) for several hours, refreshing the solution every hour until the background of the gel became fully destained. Finally, the gels were imaged by FluorChem M system (ProteinSimple). Synthesis of Dex-TA-Mal

[0121] The synthesis of Dex-TA-Mal involved three steps. In short, firstly, 5.0 g of dextran (30.9 mmol repeating units (r.u.)) was activated with 2.0 g of PNC (9.9 mmol) to yield Dex-PNC with DS value of 16. Next, Dex-TA-bNH2 with DS 6 of -NH2 group was synthesized via Dex- PNC after subsequently reacting with / V-BOC-1,4-diaminobutane (0.415g, 2.21 mmol) and tyramine (0.606g, 4.41 mmol) and . Lastly, 0.741g of AMAS ( 2.94mmol) was added to react with Dex-TA-bNH2 to yield Dex-TA-Mal and the DS of TA and Mai are 2 and 4.8, respectively.

[0122] Preparation of the Dex-VHH conjugate.

[0123] Before conjugation of the VHH to Dex-TA-Mal, the VHH were first reduced by addition of a 10-fold molar excess of TCEP in PBS and incubation for 1 hour at 37 °C, followed by the removal of TCEP via 3 kDa MWCO centrifugal filters. A typical conjugation was carried out as follows, the reduced VHH was added to a Dex-TA- Mai solution in PBS (pH7.4), in a 1:10 molar ratio (VHH:polymer) to produce the Dex-VHH conjugate. The reaction mixture was gently shaken overnight at 4 °C. The unreacted reactive maleimide moieties were blocked by the addition of an excess cysteine. Finally, the excess of unreacted cysteine was removed through 3 kDa MWCO centrifugal filters (Amicon Ultra, Millipore, USA) at 4 °C with PBS buffer (pH 7.4).

[0124] MMP cleavage test

[0125] Before cleavage tests with recombinant human MMP-13 protein, the proMMP-13 protein (R&D Systems, Minneapolis, USA) was activated with p-aminophenylmercuric acetate (APMA) in a MMP assay buffer (50 mM Tris, 10 mM CaCh, 150 mM NaCI, 0.05% Brij35 (w / v), pH 7.4). Typically, proMMP13 was diluted to 50pg / mL in the assay buffer and activated by adding APMA to a final concentration of 1.0 mM at 37°C for 2 hours. VHHs were diluted in the assay buffer and treated with a concentration range of MMP-13 (0.3125, 0.625, 1.25, 2.5, and 5.0 nM). After incubating at 37°C for 2 hours, samples were analyzed by western blot and the percentage of cleaved VHH was quantified using Imaged.

[0126] Similarly, Dex-VHH conjugates were diluted in assay buffer and treated with 5.0 nM of MMP- 13. After incubating at 37°C for 2 hours, samples were anaylsed by SDS-PAGE followed by coomassie blue staining.

[0127] Surface Plasmon Resonance imaging (SPRi)

[0128] For immobilization purposes recombinant IL-1 R (Sino Biological, Beijing, China ) was diluted to concentrations ranging from 8.125-130 nM in sodium acetate immobilization buffer with a pH of 4.5. The immobilization on G-type Easy2Spot sensors was carried out using a continuous flow spotter (Wasatch Microfluidics, Salt Lake city, UT, US), where 48 spots were printed in 30 minutes. To reduce non-specific interaction, the sensor was blocked by two subsequent quenching steps of 7 minutes, which consisted of 1% (w / v) BSA solution in sodium acetate buffer at pH 4.5 followed by 0.2 M ethanolamine at pH 8.5. After immobilization and deactivation of the sensor, the analytes were diluted in a system buffer containing 0.075% tween-80 in PBS. Regeneration was carried out using 200 mM phosphoric acid with a pH of 2.5. The IBIS MX96 (IBIS Technologies, Enschede, the Netherlands) was used for SPRi measurements. Back and forth flow was set to 10pL min-1in a flow cell containing 12 pL of sample. Sprint software was used for data collection and referencing.

[0129] SPRi kinetic titration assay

[0130] The affinity of the VHH and VHH-polymer constructs to recombinant IL-1 R was determined by the kinetic titration method with Rmax o. Recombinant IL-1 R was immobilized on an Easy2Spot sensor in quadruple as described above, followed by a blocking step. For the SPRi measurements, first an analyte injection with blanks (running buffer only, PBS containing 0.075% tween-80) was injected that provided background for interaction signals. The next analyte injection consisted of a blank followed by an increasing concentration (8.125 - 130 nM) of each VHH in running buffer. The initial baseline time was set to 1 min, association time of the interactions was 15 min followed by 12 min dissociation. After each analyte injection, the sensor was regenerated using a double regeneration pulse for 30 seconds.

[0131] Fabrication of microfluidic droplet generator

[0132] A PDMS droplet generator with a 34G inlet, 32G outlet and 18G side channel as oil inlet was prepared in a flow focusing design to generate droplets. The outlet channel was connected to silicon tubing, which has a transparent and semi-permeable nature, enabling in-line monitoring and diffusion of H2O2 to induce enzymatic crosslinking. See Fig 7A and B for more details.

[0133] Microgels production and characterization Hydrogel precursor solution (1.5 mL) that consisted of 5.0 % (w / v) Dex-TA-Mal, 150 pg VHH and 50 U / rnL HRP in PBS was emulsified with an oil phase, containing n-hexadecane supplemented with 1.0% Span 80 as surfactant, at a 1 :10 precursor / oil flow ratio. Note here that the VHH was mixed with Dex-TA-Mal and incubated for 1 .0 hour at 4 °C prior to addition of HRP. The precursor droplets were then transported through a silicone tubing that was immersed in an H2O2 bath containing 30% of H2O2. Thus, H2O2 was diffused through the tubing, the oil and into the precursor droplets activating enzymatic crosslinking of the polymers in microgels. The produced microgels were collected in n-hexadecane, and the emulsion was broken by three consecutive washes using n-hexadecane, removing the surfactant, and a subsequent wash with PBS, transferring the microgels to an aqueous phase. For assessment of the microgels’ size and monodispersity, brightfield microphotographs were taken and analyzed using Imaged.

[0134] VHH Release test

[0135] To study the VHH release profile from dextran microgels, 50 pl of suspension containing VHH loaded-microgels were pipetted into 1.5 mL Eppendorf tubes. 200 pl of activated MMP-13 containing assay buffer (10 nM) was added into the tube and the mixture was incubated at 37 °C on a shaking plate with 50 rpm. Additionally, 0.5% BSA was added in the buffer to prevent adsorption onto the tube surface. At pre-determined time points, the supernatant in the tubes was collected and replaced with freshly activated MMP-13 containing assay buffer. Samples that were treated with assay buffer without MMP-13 were set as a control in this release test. In addition, the microgels were fully digested with 10 U / rnL of dextranase after 12.5 days, to determine the quantity of unreleased VHH. The released VHH from the microgels was quantified using an indirect ELISA.

[0136] Results and discussion

[0137] Design and characterization of VHH constructs with a MMP cleavage sequence

[0138] Here, VHH clones that bind to IL-1 R were selected from a phage VHH immune library which was obtained after immunization of Llamas with recombinant IL-1R. The phage display library was constructed from cDNA synthesized from mRNA which was isolated from lymphocytes of the immunized Llama. After affinity selection, the strongest binding VHH clone (13B1) was subcloned in an expression plasmid and produced in yeast. Using recombinant DNA technology the inventors introduced an unpaired cysteine in the C-terminus of this VHH and subsequently used this amino acid for directed conjugation of the VHH to the polymeric backbone of the microgel. In this clone also a peptide sequences containing an MMP cleavage site (Fig 5A) was introduced to obtain a MMP cleavable VHH (MMPcleav-13B1). As shown in Fig 5B, the molecular weight of MMPcleav-13B1 was clearly higher than its original VHH(13B1). To determine whether the engineered MMP cleavage site at the C-terminal of the VHH can be cleaved by MMPs in vitro, a concentration range of activated MMP-13 was incubated with the VHH at 37°C for 2 h. After separation by SDS-PAGE and Western blot analysis, the cleaved VHH with a lower molecular weight was identified by comparing to the uncleaved VHH. This cleavage was shown to be MMP-13 concentration-dependent, with full cleavage achieved at 2.5 nM after 2 hours (Fig 5C), indicating that MMP-13 efficiently and specifically cleaved the engineered MMP cleavage site of the VHH. To test whether the engineered VHH containing the MMP cleavable peptide sequence at its C-terminus influenced the binding the VHH to the IL-1 R, the binding kinetics of the VHH 13B1 and VHH MMPcleav-13B1 to IL-1 R were characterized using surface plasmon resonance imaging (SPRi) measurements. The equilibrium dissociation coefficient (KD), which is inversely proportional to binding affinity between antibody and antigen, was calculated by dividing the measured dissociation rate (KOff) by the measured association rate (Kon). The MMPcleav-13B1 binding toward IL-1 R in SPRi showed a low KD value of 0.9 nM, corresponding with high affinity (Table 1). When the binding kinetics of the MMPcleav-13B1 are compared to 13B1 (KD= 0.4 nM), it can be observed that the KOff value is similar, while the Konvalue for MMPcleav-13B1 is slightly lower which resulted in a slightly higher KD. This indicated that the genetic engineering marginally affected the affinity of the VHH for IL-1 R. The inventors next determined whether cleavage of VHH MMPcleav-13B1 with active MMP13 affected the affinity of the VHH for the IL-1 R. Cleavage of the VHH increased both the Kon and KOff value resulting in an overall decrease in affinity of the cleaved VHH to IL-1 R. Remarkably, incubating 13B1 with MMP13 did not obviously affect the affinity, indicating that the change in Konand KOff for MMPcleav-13B1 is likely caused by the loss of peptide sequences at the C- terminal tail of the VHH after cleavage.

[0139] Conjugation of VHH to dextran

[0140] Table 1 Binding kinetics of 13B1 and MMPcleav-13B1 VHHs.

[0141] Konand K and dissociation constant (K ) of VHH binding to IL 1 -R are determined by SPRi measurements;

[0142] *: VHHs were incubated with activated MMP- 13 containing buffer for 2 hours at 37°C before measurements; #: MMPcleav- 13B 1 VHH was conjugated onto the backbone of dextran to form Dex-MMPcleav- 13B 1 conjugate.

[0143] Dextran is a nontoxic and highly water-soluble polysaccharide that has been used clinically for over 50 years as an antithrombotic and blood volume expander. Recently, dextran is widely under investigation for use as a polymeric carrier of drugs. Dextran contains a large number of hydroxyl groups, making it suitable for chemical modification and subsequent crosslinking to yield hydrogels. For conjugation of VHHs to dextran, maleimide was chosen as a thiol reactive group that could be used to specifically couple the cysteine-containing VHH to the polymeric backbone via stable thioether bonds. In addition, tyramine moieties were introduced to dextran resulting in a crosslinkable polymer, which yields a stable macromolecular hydrogel networks through an enzymatic crosslinking reaction in the presence of horse radish peroxidase and hydrogen peroxide. The inventors have successfully developed a method for chemical modification of dextran with tyramine and maleimide groups to produce Dex-TA-Mal. Here, successful synthesis of Dex-TA-Mal was confirmed using1H NMR. The number of tyramine and maleimide moieties per 100 monosaccharides in dextran were 2 and 4.8 respectively. Next, the efficient generation of Dex-VHH conjugates was achieved through the direct coupling of the maleimide to cysteine sulfhydryl groups of the free cysteine engineered in the C-terminal tail of the VHH in PBS (Fig 6). Successful conjugation of the VHH to Dextran was confirmed using SDS-PAGE analysis. As shown in Fig 6B , an obvious decrease of free VHH (Fig 6B, lane 3, Dex-VHH) in contrast to untreated control (Fig 6B, lane 2, pure VHH) and a smeared band at higher molecular weights corresponding to Dex-VHH conjugates was found in the SDS-PAGE gels stained with coomassie blue, which indicated efficient conjugation of the VHH via the thiol-maleimide reaction under mild conditions.

[0144] After incubating the Dex-VHH conjugates with activated MMP-13, a clear protein band corresponding to cleaved MMPcleav-13B1 was presented in lane 4 of the SDS-PAGE gel (Fig 6B, lane 4 ). Its apparent molecular weight was slightly lower than protein MMPcIeav- 13B1 in lane 2 due to loss of a small protein fragment, which means that the VHH was successfully cleaved off from the polymer. This proves that the MMP cleavage site of the VHH was still accessible after coupling to dextran and cleavable in the presence of active MMP-13. The inventors next determined the binding affinity for the Dex-MMPcleav-13B1 conjugate before and after cleaving with active MMP13 using SPRi. As shown in table 1, conjugation of the VHH to the polymer reduced the affinity of the VHH for the IL-1 R approximately 50-fold. After cleaving the VHH from the dextran polymer, the affinity improved as a result of a marked increased Konvalue. Remarkably, it was also seen that the Koff value of the cleaved VHH was increased compared to the conjugated VHH. This might be explained by the multivalency of Dex-VHH; i.e. multiple VHHs are conjugated to one dextran molecule. In conclusion, coupling VHH to dextran via thiol-maleimide chemistry affected the affinity of the VHH in a negative way, but its affinity recovered to some extent after cleavage by activated MMP13.

[0145] VHH loaded microgels production

[0146] In recent years, many fabrication techniques have been explored to produce microgels with varying sizes. A single-step microfluidic strategy has been reported that enabled the high- throughput production of monodisperse microgels via enzymatic outside-in crosslinking of tyramine-conjugated polymer droplets. Inspired by the design of needle-based microfluidic device, the inventors developed a simple, cost-effective and more reliable microfluidic device fabricated in polydimethylsiloxane (PDMS) with dispensing needles (Fig 7A). This device design consisted of a central channel and one side channel and could be operated in a flow focusing manner to produce monodispersed microdroplets with good control over the size. A schematic of the microfluidic platform for microgels production is presented in Fig 7B, which consists of a PDMS based microfluidic chip as a microdroplet generator and a semipermeable silicone tube submerged in an H2O2 diffusion bath for enzymatic crosslinking. To generate homogeneous microdroplets, the oil phase was injected from side inlets at a flow rate of 100 pL min-1and the hydrogel precursor was delivered using a 60 pm inner diameter dispensing needle at a flow rate 10 pL min-1. Dex-TA-Mal, VHH MMP13cleav-13B1 and HRP containing microdroplets were subsequently crosslinked after flowing through the semipermeable silicone tube, which allowed the diffusion of H2O2 into the microdroplets triggering crosslinking of the tyramine conjugated polymers. Fig 7C and D show the formation of monodisperse microgels with a mean diameter of 55 pm with a narrow size distribution (Coefficient of variation, CV = 3.3).

[0147] MMP triggered VHH release from microgels

[0148] Often drug delivery from hydrogel carriers is based on passive means (e.g., physical encapsulation and subsequent release), which is strongly diffusion-dependent. However, the diffusion-controlled mechanism typically yields a relatively short release time and a large burst release, resulting in potential side effects and off-target toxicity due to increasing drug exposure to dangerous levels in the target tissue. Here, an alternative strategy was employed by tethering VHHs to the hydrogel network (Fig 8A). The VHHs can be released from microgels at a rate, controlled by the cleavage of an engineered MMP cleavable sequence. To assess the tethered VHH release profile from microgels in the presence of active MMPs in vitro, VHH-loaded microgels were incubated with 10 nM active MMP-13. As shown in Fig 8B the VHH-loaded microgels exposed to activated MMP13 showed sustained release of VHH over a period of one week with an initial burst release (around 29 % ) during the first 12 h. In contrast, in absence of activated MMP13, the microgels showed a burst release of VHH during the first 12 h, which subsequently plateaued for the remaining 13 days. Incubating the VHH-loaded microgels with activated MMP-13 released a total of 77 (±1.3)% of loaded VHH over the same period. It is important to note that an initial burst release occurred both with and without MMP-13 treatment. The burst release is likely caused by non-conjugated VHHs still present in the microgels. This can be reduced by optimizing the procedure of making the VHH-loaded microgels, for example by first conjugating the VHH to the polymer-TA backbone prior to microdroplet and microgel formation.

[0149] Conclusion

[0150] The inventors report the design of an MMP-responsive VHH delivery system. Specifically, the genetic engineering of a MMP-specific and cleavable sequence into the C-terminal tail of a VHH conjugated to the dextran backbone of an enzymatically crosslinked microgel allows the release of the VHH from the microgel in an MMP13 dependent manner. Based on SPRi measurements the VHH retained its bioactivity after conjugation and subsequent release after cleavage by active MMP13. The dextran-tyramine conjugates with VHH can be used in the production of VHH-loaded hydrogels. Traditionally, hydrogels are crosslinked into bulk hydrogels at the millimeter scale or larger, which limits their intended application, particularly in cases in which injection is needed or smaller sizes are required. As an alternative approach, microscale hydrogel particles, so called microgels, were produced using a simple microfluidic device. The size of these microgels enables their application by simple injection. The inventors demonstrated the sustained release of the conjugated VHHs over a period of one week in an MMP13 dependent manner. Our system has great potential as an intraarticular disease triggered drug delivery system for treatment of osteoarthritis. Cartilage matrix degradation by MMP activity is a hall mark of degenerative joint diseases like osteoarthritis. By selecting the proper target for neutralization by a VHH, MMP13 mediated release of such VHH may interfere with the vicious cycle of cartilage matrix degradation in osteoarthritis in a disease dependent manner. Our study provides validation that covalent conjugation of therapeutic proteins engineered with protease-cleavable sequences to a hydrogel network is viable approach for the protease triggered delivery of therapeutic proteins. The approach could be utilized to develop a promising protein delivery system in response to environmental cues such as expression of a specific proteases at the disease site to achieve disease-specific release of a potential therapeutic protein.

[0151] EXAMPLE 3

[0152] In vivo experiments were performed to show the capability of different VHH formulations, including free VHH, high molecular weight (HMW) Dex-VHH conjugates, VHH-loaded Dex-TA hydrogels and VHH-loaded Dex / HA-TA hydrogels, to keep VHHs inside the joint cavity for a therapeutic relevant timespan. To develop VHH into a therapeutic option for local intra-articular treatment the residence time of the VHH in the joint needs to be improved. The inventors hypothesized that this could be achieved by the covalent coupling to the polymer backbone of tyramine conjugated polysaccharides such as dextran and hyaluronic acid. These polymers can cross link in situ in a hydrogel after mixing with the enzyme horseradish peroxidase (HRP) and hydrogen peroxide as initiator. Two different strategies were explored to couple the VHH to these materials. For HA-TA extra maleimide groups were introduced using EDC / NHS chemistry. These maleimide groups can react with a free cysteine which was engineered in the C- terminal tail of the VHH resulting in targeted and directed conjugation of the VHH to the HA- TA backbone. For the conjugation of this VHH to low and high molecular weight (LMW and HMW, respectively) Dex-TA, a novel bio-conjugation method was used via direct coupling of tyramine residues to cysteine sulfhydryl groups using the enzyme tyrosinase. Both methods resulted in a highly efficient conjugation of the VHH to the respective backbone. Mixing HA- TA-VHH with LMW Dex-TA in a 1 :1 ratio or LMW Dex-TA-VHH with HRP and hydrogen peroxide yielded stable hydrogels in vitro and in vivo after intra-articular injection in the knee joint of the rat. Depending on the composition, VHH laden hydrogels or polymer conjugates remained in the joint for a period of at least 13 weeks.

[0153] Materials

[0154] Dex (40 kDa, pharmaceutical grade, Dex) and high molecular weight Dex (500 kDa, pharmaceutical grade, HMW Dex) were purchased from Pharmacosmos (Holbaek, Denmark). Sodium hyaluronate (27 kDa, pharmaceutical grade) was purchased from Contipro Pharma (Dolni Dobrouc, Czech Republic). Tyramine (TA, 99%), Tyramine hydrochloride (TA HCI >98%), p-nitrophenyl chloroformate (PNC, 96%), LiCI (>99.0%), anhydrous dimethylformamide (DMF, 99.8%), pepsin, Eukitt mounting medium, pyridine (anhydrous, 99.8%), NaHCO3(99.7%, NaCI (>99.0%), DMSO-d6(99.9%), D2O (99.9 atom % D), hydrogen peroxide (H2O2, 30 wt% in H2O), horseradish peroxidase (HRP, 325 units / mg solid), tyrosinase from mushroom (>1000 unit / mg), tris(2-carboxyethyl)phosphine hydrochloride (TCEP) were purchased from Sigma Aldrich (St. Louis, MO, USA). 4-(4,6-Dimethoxy-1 ,3,5- triazin-2-yl)-4-methylmorpholinium chloride (DMTMM, 97%) was purchased from Fluorochem Ltd. (Hadfield, UK). N-(2-Aminoethyl)maleimide Hydrochloride (MEA HCI, >93.0%) was purchased from TCI Chemicals (Zwijndrecht, Belgium). IRDye 800CW-NHS ester was purchased from LI-COR Biosciences (Lincoln, NE, USA). Ethanol (>99.9%) and diethyl ether (>99.7%) were purchased from Merck (Darmstadt, Germany). Milli-Q water was used from the Milli-Q Advantage A10 system equipped with a 0.22 pm Millipak®-40 Express filter. Synthesis of hyaluronic acid-tyramine-maleimide (HA-TA-Mal)

[0155] First, HA-TA was prepared by amidation of the HA carboxyl groups with tyramine by using a procedure adapted from Rydergren and D’Este et al. Briefly, Sodium hyaluronate (5.00 g, 12.5 mmol repeating units (r.u.)) was dissolved in 500 mL Milli-Q water in an 1 L round bottom flask equipped with a stirrer bar. While stirring at room temperature, 4-(4,6-dimethoxy- 1 ,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM, 3.46 g, 12.5 mmol) and tyramine hydrochloride (TA HCI, 2.17 g, 12.5 mmol) were added subsequently. After 24 hours, 40 ml saturated NaCI solution was added to the reaction mixture and the reaction mixture was poured into 2.5 L cold ethanol. The crude product was isolated by centrifugation at 5000 rpm followed by drying in vacuum. The crude product was dissolved in 75 mL Milli-Q water and dialyzed against Milli-Q water for 3 days (MWCO 3500 Da). Filter sterilization and lyophilization yielded the product as a white foam. The successful synthesis of HA-TA was confirmed using1H NMR in D2O. The resulting HA-TA contained 5 tyramine moieties per 100 disaccharide units.

[0156] Next, HA-TA (1 ,5g, 4.0 mmol r.u.) was dissolved in 50 mL of MES solution (20mM, pH6.8) in an 100 mL round bottom flask equipped with a stirrer bar. While stirring at room temperature, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM, 0.653 g, 2.36mmol) and N-(2-Aminoethyl)maleimide hydrochloride (AEM HCI, 0.417g, 2.36 mmol) were added subsequently. The mixture was stirred at room temperature for 10 h. The reaction mixture was purified by extensive dialysis (MWCO 3500 Da) against Milli-Q water, followed by neutralization with NaOH. Subsequently, sterile filtration and lyophilization yielded the product as a white foam. The successful synthesis of HA-TA-Mal was confirmed using1H-NMR in D2O.1H-NMR (400 MHz, D2O): b(ppm) = 1.93 (methyl protons, s, 3H); 6.77, 7.16 (aromatic protons, d, 2H); 6.79 (maleimide protons, s, 2H). The number of tyramine or maleimide per 100 disaccharide units was calculated based on the methyl group at 1.93 ppm (corresponding to the 3 methyl protons from HA, compared with the integral of the 2 aromatic protons of tyramine at 6.77ppm or 2 maleimide protons at 6.79ppm. The resulting HA-TA-Mal contained 5 tyramine moieties and 12 maleimide moieties per 100 disaccharide units.

[0157] Synthesis of Dex-T A and HMW Dex-TA

[0158] Dex was functionalized with tyramine. In short, Dex was activated with PNC, which was subsequently substituted with tyramine. Typically, Dex (5.00 g, 30.8 mmol repeating units (r.u.)) and LiCI (4.0 g, dried at 115 °C) was dissolved in 200 mL of anhydrous DMF at 95 °C under nitrogen atmosphere. Once the Dex was completely dissolved, the solution was cooled to 0 °C and anhydrous pyridine (2.0 ml, 25.8 mmol) was added. Subsequently, freshly sublimed para-nitrophenyl chloroformate (2.5 g, 12.4 mmol) was added in small portions, keeping the temperature below 2 °C. After 1 hour, the reaction mixture was poured into 1 L of ice-cold ethanol. The precipitate was filtered off (Por 4) and washed with copious amounts of cold ethanol and diethyl ether. After drying under vacuum, the product (denoted as Dex-PNC) was obtained as a white powder (DS 25).1H-NMR (400 MHz, DMSO-de): b(ppm) = 3.0-4.0 (saccharide ring protons, m, 6H); 4.2-5.8 (anomeric and hydroxyl protons, m, 4H); 7.58, 8.34 (aromatic protons, d, 2H).

[0159] Next, Dex-PNC (5.00 g, 24.0 mmol r.u., 6.02 mmol p-nitrophenyl carbonate) was dissolved in 100 mL of anhydrous DMF and tyramine (1.649g, 12.04 mmol) was added under a nitrogen flow. After 1 hour, the reaction mixture was poured into 1.0 L of ice-cold ethanol. The precipitate was filtered off (Por 4) and washed with copious amounts of cold ethanol and diethyl ether. After drying under vacuum, the crude product was obtained as a white powder. The crude product was dissolved in water and dialyzed against Mi II i-Q water for 3 days (MWCO 3500 Da), followed by filter sterilization and freeze-drying. The yield of the product (denoted as Dex-TA, DS 15) was a white foam.1H-NMR (400 MHz, DMSO-de): b(ppm) = 3.0- 4.0 (saccharide ring protons, m, 6H); 4.2-5.8 (anomeric and hydroxyl protons, m, 4H); 6.67, 6.99 (aromatic protons, d,2H).

[0160] The same synthesis method was used to generate HMW Dex-TA. The degree of substitution (DS), defined as the number of p-nitrophenyl carbonate or tyramine per 100 saccharide units, was calculated based on the integrals of 4.2-5.8 ppm (corresponding to the 4 anomeric protons from Dex), compared with the integral of the aromatic protons of tyramine (6.60-6.75 and 6.90-7.07). The resulting Dex-TA and HMW Dex-TA contained 15 and 3.5 tyramine moieties per 100 saccharide units, respectively.

[0161] Preparation of VHH-IRDye 800CW

[0162] IRDye 800CWwas coupled covalently to VHH according to the following procedure. The VHH B1 used in this study, binding with high affinity to the IL-1 R, is kindly provided by QVQ Holding b.v (Utrecht, the Netherlands). First the concentration of VHH was determined by measuring absorbance by using a NanoDrop 2000 UV-Vis spectrophotometer. VHH was next concentrated to 2 mg / ml in 0.1 M NaHCOa (pH 8.2) using 10-kDa centrifugal filters (Amicon Ultra, Millipore, Sigma Aldrich). Subsequently, 0.5 ml of the concentrated VHH was incubated with 940 pg of IRDye 800CW-NHS ester (0.8 pmol in 100 pl DMSO, which corresponded to a 6-fold molar excess over VHH) and the mixture was incubated for 2 h at room temperature. After 2 h, the unreacted IRDye 800CW-NHS esters were blocked by addition of excess glycine. Finally, the reaction mixture was purified by centrifugal filtration through 10-kDa centrifugal filters, and washing with phosphate buffered saline (PBS) buffer (pH 7.4) was repeated until no noticeable IRDye800CWwas detected in the filtrate. VHH coupled to IRDye 800CW is further referred to as NIR labelled VHH or VHH-NIR. Preparation of HA-VHH, Dex-VHH and HMW Dex-VHH conjugates.

[0163] Before conjugation of VHH-NIR to polymers (HA-TA-Mal, Dex-TA and HMW Dex-TA), a reduced VHH-NIR in PBS was prepared by addition of 10-fold molar excess of TCEP and incubation for 1h at 37 °C, by following by removal of unreacted TCEP via 3 kDa MWCO centrifugal filters.

[0164] HA-VHH conjugate’. A typical conjugation was carried out as follows, the reduced VHH-NIR was added to HA-TA-Mal solution in PBS (pH7.4), in a 1:10 molar ratio to produce HA-VHH conjugate. The reaction mixture was gently shaken overnight at 4 °C. The unreacted reactive maleimide moieties were blocked by addition of excess cysteine. Finally, excess unreacted cysteine was removed through 3 kDa MWCO centrifugal filters (Amicon Ultra, Millipore) at 4°C with PBS buffer (pH 7.4).

[0165] Dex-VHH and HMW Dex-VHH conjugates: the reduced VHH-NIR was added to Dex-TA or HMW Dex-VHH solution in PBS (pH 7.4) in the presence of 10 U / ml Tyrosinase, in a 1:10 molar ratio to produce low molecular weight (LMW) Dex-VHH conjugate. LMW Dex-VHH conjugate will be further referred to as Dex-VHH. The reaction mixture was gently shaken at room temperature for 1 hour. The same conjugation method was used to generate HMW Dex-VHH conjugates as well.

[0166] Hydrogel formation

[0167] Enzymatically crosslinking of HA-TA and Dex-TA was achieved by mixing 10% w / v polymer solution, 40 unit HRP and 0.15 wt. % H2O2 in PBS. Gelation was confirmed using the vial tilting method. After the (in situ) gelation in the in vivo study Dex-TA + VHH is further referred to as Dex-VHH and Dex / HA-TA + VHH as Dex / HA-VHH.

[0168] Gel electrophoresis

[0169] For polymer-conjugates (HA-VHH, Dex-VHH and HMW Dex-VHH) analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out in a Mini-PROTEAN Tetra Cell system (Bio-Rad). This was connected to a PowerPac Basic (BioRad, Hercules, CA, USA) programmable power supply. The polymer-conjugates samples were treated with 4x Laemmli sample buffer (BioRad), to which 10 wt. % 2-mercaptoethanol was added prior to mixing with the sample, Samples were then heated to 95 °C for 5 minutes to denature the protein sample and ensure reduction of any disulphide bonds. Samples were loaded onto commercially available 10-well 4-15% Mini-PROTEAN TGX Precast Protein Gels (BioRad). Gels ran for 45 minutes at 160 mV in a running buffer (25 mM Tris, 192 mM Glycine, 0.1% (w / v) SDS, pH 8.3). Protein bands were subsequently visualized by incubating the gels in a staining solution (0.1% Coommasie Brilliant Blue R 250, 40% MeOH, 10% acetic acid in water ) with gentle agitation for 1 hour. This was followed by a destaining protocol in which the gels were gently agitated in a destaining solution (40% MeOH, 10% acetic acid) for several hours, replenishing the solution every hour until the background of the gel became fully destained. Finally, the gels were imaged by FluorChem M system and analyzed by AlphaView software (ProteinSimple).

[0170] In vivo safety and retention time study

[0171] All animal procedures were performed in accordance with the Utrecht University Medical Ethical Committee for Animal Studies (study license number: AVD1150020185184). Ten-to twelve -week-old (-300 g) male Wistar Han rats (Charles River, Netherlands BV) were housed with a 12h light-dark regime and had access to food pellets (SDS Diets, 801722) and tap water ad libitum. After an acclimatization period of one week, the experiments started. To study in vivo retention time of the conditions were studied by bilateral IA injection into the knee joint of the animals of four 4 experimental groups; 1) plain VHH; 2) Dex-VHH; 3) Dex / HA-VHH and 4) HMW Dex-VHH. All VHH used in these conditions were NIR labelled VHH. From the 12 animals, 24 knee joints were randomized into the four treatment groups (n=6 joints per group). Half of the animals (n=3 joints per group) were euthanized after 35 days (5 weeks) and the other half (n=3 joints per group) were kept until 91 days (13 weeks). Before IA injection, a baseline scan at 800 nm was measured and after IA injection, scans were made at pre-determined time points for 91 days, using the Pearl small animal imager (LI-COR, Biosciences, Lincoln, NE, U.S.A.). Afterwards, images were processed using Image Studio lite (LI-COR).

[0172] Histological analysis in vivo study

[0173] Knee joints were harvested and fixated in 4% buffered formaldehyde (pH = 7) for 1 week and then decalcified in 0.5M EDTA set to pH 7.0 with NaOH for 6 weeks. Every week the samples were re- fixated in 4% neutral buffered formaldehyde for 24 hours. The decalcified tissue was dehydrated in a series of ethanol (70-100% ethanol), cleared in xylene, and paraffin infiltrated. Knee joints were paraffin embedded and 5 pm sections were made. Sections were stained with Gill 3 Hematoxylin, Fast Green and Safranin-0 and an immunohistochemistry staining for CD68 and anti-VHH were performed. For this, slides were blocked for endogenous peroxidase using 0.3% H2O2 for 10 minutes at room temperature. Antigen retrieval was performed using 0.1% pepsin in 0.02M HCI solution for 30 minutes at 37°C and all slides were blocked in 5% PBS-BSA for 30 minutes at room temperature. Primary antibody incubation was done overnight at 4°C with 0.75 pg / ml CD68 mouse monoclonal antibody (ab31630, Abeam) or with 0.75 pg / ml normal mouse lgG1 as negative control (sc3877, Santa Cruz). The next day slides were incubated with anti-mouse HRP (Envision, Dako) for 30 minutes at room temperature and subsequently incubated with liquid DAB+ 2-component system (Agilent) for 5 minutes. Sections were counterstained with hematoxylin, dehydrated, cleared in xylene and covered with a glass slide using Eukitt mounting medium.

[0174] For the anti-VHH immunohistochemistry the protocol was slightly different. The VHH in the sections were detected using 2.5 pg / ml HRP-linked rabbit anti-camelid VHH monoclonal antibody (A01861-200, GenScript, Piscataway, NJ, USA). After this step, incubation with DAB was carried out followed by counterstaining, dehydration, clearing in xylene and covered with a glass slide using Eukitt mounting medium.

[0175] Histological analysis using near-infrared fluorescent imaging

[0176] Sections (5 pm) were made which were used for near-infrared fluorescent (NIRF) imaging. Sections containing the released or bare VHH-NIR were mounted in an Axio Scan.ZI slide scanner (Carl Zeiss AG, Jena, Germany) and sections were scanned using a Colibri5 / 7 light source, a 20x objective, and shading correction. Channel settings were as follows: DAPI was imaged using a 49 DAPI filter cube together with 385 nm LEDs at 20 % power and 22 ms exposure time and NIR was imaged using a Cy7 filter cube together with 735 nm LEDs at 100 % power and 300 ms exposure time. Stitching of scanned tiles was performed online using ZEN 3.3 (blue edition; Carl Zeiss AG, Jena, Germany). Afterwards, images were processed using ZEN 3.3 (blue edition).

[0177] Results and Discussion

[0178] Site-Specific Conjugation of VHH to HA, Dex and HMW Dex

[0179] The strategies of polymer-VHH conjugation are illustrated in Fig 9A. Prior to conjugation of VHHs to polymers, VHHs were labelled with NIR for non-invasive longitudinal follow up after IA injection. Two different natural polymers, HA and Dex with low and high molecular weight, have been utilized to create three distinct polymer-VHH conjugates. In order to conjugate a cysteine-containing VHH to HA, maleimide was chosen as a thiol reactive group that could be used to specifically couple the cysteine-containing VHH to the polymer backbone via stable thioether bonds. In addition, the tyramine moiety was introduced to HA backbone which facilitates the formation of stable macromolecular hydrogel networks after an enzymatic crosslinking reaction involving horse radish peroxidase (HRP) and hydrogen peroxide (H2O2) as initiator (Fig 9B). Modification of HA with tyramine and maleimide groups was achieved via amidation of carboxyl groups of HA. The number of modified tyramine and maleimide moieties per 100 disaccharides in hyaluronic acid were 5 and 12 respectively. The HA-TA-Mal was then incubated with cysteine-containing VHH overnight at 4°C to produce HA-VHH conjugates. Unreacted maleimide units were quenched by incubating the conjugates with cysteine. Successful conjugation was confirmed using SDS-PAGE. Free VHH resulted in one band of approximately 15 kDa. The HA-VHH conjugates vaguely appeared as a smear with a larger effective molecular weight than free VHH (Fig 9C_i) and significantly less amount of unconjugated VHH after incubation with HA-TA- maleimide was found. Quantification of these bands revealed a coupling efficiency of > 90%, clearly indicating that the conjugation chemistry via the thiol-maleimide reaction is efficient under physiologic conditions. To confirm successful conjugation of VHH to HA, western blot analysis was used to detect specifically VHH. As shown in Fig 9C_ ii a clear smeared band, which means HA-VHH conjugates, at higher molecular weight than free VHH was detected.

[0180] For conjugation of VHH to Dex, firstly Dex of average molecular weight 40 kDa and 500 kDa were modified with tyramine to generate Dex-TA and HMW Dex-TA, respectively.1H NMR spectrum indicated that the number of modified tyramine moieties per 100 saccharide units in Dex and HMW Dex were 15 and 3.5 respectively. Next, the efficient generation of Dex-VHH and HMW Dex-VHH was achieved through the direct coupling of tyramine residues to cysteine sulfhydryl groups in the presence of 10 ll / rnl tyrosinase and adventitious oxygen. Briefly, phenols of tyramine moiety are oxidized by tyrosinase to yield o-quinone intermediates that are able to couple with cysteine sulfhydryl group on VHH. Both Dex-VHH and HMW Dex-VHH reaction mixtures were analyzed by SDS-PAGE (Fig. 9C_ i ). Efficient conjugation of VHH to these two types of Dex was shown with coupling efficiencies of >90%. An obvious decrease of free VHH in contrast to untreated control and a clear smeared band corresponding to Dex-VHH conjugate was found in the SDS-PAGE for Dex-VHH. However, for HMW Dex-VHH, the conjugates retained in the slot of the gel most likely due to its large molecular weight of >500 kDa. In addition, the remaining o-quinone intermediates on the oxidized Dex backbone could still be enzymatically crosslinked to yield a crosslinked polymer network in the presence of HRP and H2O2, which means the tyrosinase-catalyzed sitespecific protein-polymer coupling is still compatible with the aforementioned enzymatic crosslinking of tyramine-functionalized polymer.

[0181] In vivo retention time study of VHH coupled to biomaterials

[0182] Six healthy knee joints of Wistar Han rats per group (total n = 24 joints) were used. Bilateral IA injections were performed using the biomaterials conjugated with NIR labelled VHH and the retention of the VHH in the joint was studied using non-invasive longitudinal imaging. HMW Dex-VHH was injected unmodified in the knee joint. In contrast, HRP mediated crosslinking was used to create hydrogels in situ for Dex-VHH or for a combination of Dex-TA mixed with HA-TA and HA-VHH (1:1 ratio). The hydrogels were created in situ upon intraarticular (IA) injection, by pre-mixing Dex-TA solution with Dex-VHH conjugate to create Dex- VHH or Dex-TA / HA-TA (50 / 50) solution complemented with HA-VHH conjugate with HRP and H2O2 (Fig 10). The latter is referred to as Dex / HA-VHH. Both HMW Dex-TA and the VHH only control were injected directly.

[0183] Prior to IA injection a 800 nm baseline scan of the rats was made using the Pearl small animal imager, after which the inventors directly IA injected one condition in each joint and made a scan directly after injection. Swollen joints or other adverse reactions did not occur during the study.

[0184] Directly after IA injections the signal in the conditions was 52.1 % ± 11.0, 39.3% ± 11.4, 67.4% ± 33.0, 43.7% ± 15.6 for respectively the VHH only, HMW Dex-VHH, Dex-VHH and Dex / HA-VHH (Fig 11). Just as seen previously, the fluorescent signal went up for all conditions in the first 24 h after which the signal gradually decreased. The increase in signal in the first 24hrs is likely due to the concentration of label at specific sites in the joint. The inserts of figure 3 show the fluorescence half-life of the NIR-labelled VHH, either as plain VHH or as tethered to the different materials. As seen for all conditions the half-life is around 5-6 days.

[0185] For all four conditions, signal was still present after 91 days (13 weeks) post IA injections (Fig. 3). The signals found at this timepoint were 4.1% ± 3.1 , 2.1% ± 0.8, 3.7% ± 0.3 and 3.1% ± 2.1 for respectively the VHH only, HMW Dex-VHH, Dex-VHH and Dex / HA-VHH. After 13 weeks rats were sacrificed and knee joints were measured again after removal of the skin. The lowest fluorescence intensity was found in the HMW Dex-VHH and Dex / HA-VHH groups. Any moiety smaller than 10 nm is readily reabsorbed by blood capillaries and moieties larger than 10 nm are introduced to the lymphatic system. Since VHH are smaller than 10 nm it was expected that they would be rapidly cleared, which is well-known for VHH and other smaller moieties.

[0186] Surprisingly, the bare VHH conjugated with a NIR label showed clear presence in the joint cavity even after 13 weeks post injection. Another variable that influences the transport of a moiety is charge. The VHH used in this approach has an isoelectric point of 7.9 and was therefore considered slightly positively charged. It is possible that electrostatic interactions of positively charged VHH with negatively charged tissues, such as cartilage could prolong retention time. In addition, it was seen that this specific VHH had a very high binding affinity of KD = 29.8 pM for IL-1 R. For this reason, it is possible that the VHH was firmly attached to IL- 1 R in the joint. As a follow-up study the efficacy of the VHH in the joint should be evaluated, to see if besides presence in the joint it is still biologically active in vivo.

[0187] Previously, it has been shown that the short retention time of 10 kDa Dex was significantly enhanced when a much larger Dex polymer was used. In this study the inventors used Dex with a Mw of 500 kDa as well for conjugation of the VHH. The inventors postulated that this molecule can move freely throughout the joint and might be cleared by the lymphatic system, where the rate of clearance is dependent on the size of the polymer. Still the Mw of this Dex network is much lower than Mw of the crosslinked Dex-VHH macromolecular network. These hydrogels had a higher fluorescent intensity at time of sacrifice (Fig 11). Next to the larger Mw of this network entanglement of the crosslinked network may also contribute to the increased retention time.

[0188] HA is a naturally occurring polysaccharide that is degraded by enzymes (e.g., hyaluronidase) that are present in the joint. Dex-VHH can be degraded as well, but this occurs via hydrolysis of the ether bonds which is a slow process. The inventors therefore reasoned that the degradation of a Dex-VHH hydrogel can be accelerated by mixing in HA, which has previously been shown for macromolecular networks of Dex-TA and HA. Indeed as shown in figure 3, the decay in signal of the Dex / HA-VHH hydrogels was much faster than for the Dex- VHH hydrogel. This was also confirmed by histology which will be discussed in the next section.

[0189] Histological assessment

[0190] Both the safety and biocompatibility of the IA injected material was evaluated using histological analysis, which was carried out after 5 weeks and 13 weeks post injection. In the histological analysis, the inventors focused on the presence of structural changes, the tissue reaction and the presence of the NIR fluorescent signal. None of the conditions showed any signs of structural damage to the cartilage as determined by the Safranin O / Fast green staining.

[0191] The potential inflammatory response of the polymers or hydrogels was determined using an anti-CD68 IHC staining which stains macrophages. CD68+ cells surrounding the constructs were present in both hydrogel conditions after 5 weeks post injection (anti-CD68). No CD68 + staining was detected after 13 weeks. The CD68+ expression was slightly higher for the Dex- VHH. However, in both hydrogel conditions it was suggested that there was a mild and transient foreign body response to both hydrogels, based on the presence of a thin capsulelike layer surrounding the constructs after 5 weeks post injection and the absence of a notable tissue reaction after 13 weeks.

[0192] Since the VHH-NIR was conjugated to both types of hydrogel, it was possible to look into the degradation of the material (NIRF). The amount of fluorescence present for Dex-VHH condition versus the Dex / HA-VHH condition shows that the latter was degraded faster. Fluorescence signal was present at the same location as the material. In addition, an anti- VHH stain was carried out to see if the VHH could be localized as well (anti-VHH). Here it was seen that the VHH also co-localized at the same position as the NIRF imaging and the hydrogel material, confirming the VHH was still present in the hydrogel. Looking at the position of the material and the NIRF signal after 5 weeks post injection throughout the joint, most of the materials were found at the infrapatellar fat pad, in the joint cavity and next to the synovial membrane.

[0193] Previously it was seen that in vitro Dex / HA-VHH hydrogels were actively degraded by hyaluronidase. In vivo these hydrogels are also expected to be degraded by hyaluronidase depending on its presence. Next to enzymatic degradation, hydrolysis of the ether bonds of the hydrogel material also takes part in the degradation process. As a results, the in vivo degradation is often faster in comparison to in vitro degradation. As seen in the conditions of Dex-VHH, there was almost no material degradation. This is in line what was previously found.

[0194] To conclude this research, both hydrogel materials seem to be promising as a drug delivery system for VHH and Dex / HA-VHH offers a great opportunity to tune the degradation time of such gels depending on the ratio between Dex-TA and HA-TA. The use of Dex can be seen as an advantage, mainly due to its great biocompatibility and therefore has a large contribution in tissue engineering. Moreover, previously it was shown within an osteoarthritic knee of the rat that the clearance within the joint of larger constructs of Dex was even slower in comparison to a native joint. This may also account for the Dex-VHH containing hydrogels. However, this most likely only applies to Dex-VHH only hydrogels, due to the expectation that the secretion of hyaluronidase in a diseased knee is increased.

[0195] A remarkable finding of this study was the prolonged retention time of VHH-NIR which was directly injected in the knee joint space. The reason for this increased retention time and the lack of an additive effect of conjugation of the NIR labelled VHH to HA or to Dex to increase retention time is presently unknown. It is contrary to the notion that relatively small molecules with a hydrodynamic radius of < 10nm might be rapidly cleared from the knee joint. Whether this is a consistent feature of VHH in general or is depending on the identity and amino acid sequence of the VHH is presently unknown. In pilot experiments much shorter, but also highly variable retention times were observed using different VHH (data not shown).

[0196] Moreover, previously the inventors showed various other strategies using Dex-TA and Dex / HA-TA, for instance by the creation of spherical particles and spatiotemporal material functionalization using avidin and biotin analogs. In addition, the inventors also found that in these spherical particles mesenchymal stem cells (MSCs) could be encapsulated which resulted in a prolonged in vivo retention time after intra-articular injection in Wistar Rats. This all offers some great future perspectives for hydrogel materials composed of both Dex-TA and Dex / HA-TA. Conclusion

[0197] The inventors showed the successful synthesis of HA-TA-mal and the use of the mal for conjugation to a free cysteine engineered in the C-terminus of the VHH and the subsequent implementation of these conjugates in a Dex / HA-VHH hydrogel. Furthermore, the inventors showed the ability to conjugate VHH to tyramine groups introduced in the back bone of Dex molecules using the enzyme tyrosinase. By both the creation of Dex-TA and Dex / HA-TA hydrogel conjugated with VHH, the inventors created a dual orthogonal injectable hydrogel. Lastly, to proof the concept of this dual orthogonal injectable hydrogel, the inventors showed in a small animal study the safety and prolonged retention time of the hydrogels and the VHH that were conjugated to the polymer backbone. Overall, this shows the potential of having a DDS composed of Dex with great future perspective.

[0198] EXAMPLE 4

[0199] Here the inventors describe a method for creating polymer-VHH conjugates based on thiol- maleimide chemistry. To achieve site-selective modification, an unpaired cysteine was genetically engineered into the C-terminus of VHH, and two maleimide functionalized polysaccharides, Dex-Mai and HA-Mal, were synthesized for conjugation. By incorporating a hydrophilic flexible GS linker between the variable domain of the VHH and the unpaired cysteine, to form VHH-GS-Cys, significantly improved its conjugation efficiency to the Dex- Mai and HA-Mal from 4.8% to 74.6% and 7.2% to 70.7%, respectively (Figure 12).

[0200] Incorporation of an additional unpaired cysteine at the C-terminus of VHH-GS could also significantly increase the accessibility of the cysteines for the coupling reaction resulting in a high conjugation efficiency to Dex-Mai (92.5%) and HA-Mal (91.3%).

[0201] EXAMPLE 5

[0202] VHH intact after incubation with Hyaluronidase and Dextranase

[0203] Figure 13 shows that the stability and activity of the VHH is not affected by the enzymatic activity of Hyaluronidase and Dextranase as shown on Western Blot.

[0204] Figure 14 also shows that the stability and activity of the VHH is not affected by the enzymatic activity of Hyaluronidase and Dextranase. The VHH is not degraded into smaller fragments as shown on SDS-PAGE.

[0205] Both figures confirm that the VHH is intact after release from the hydrogels by treatment with Hyaluronidase and Dextranase, which is crucial for the function of the VHHs. Methods

[0206] Gel electrophoresis and western blot

[0207] Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis were used to confirm VHH stability after 14 days incubation with Hyaluronidase and Dextranase. VHHs were reduced with b-mercaptoethanol (BioRad) and denatured by heating at 95°C for 5 min. Samples were loaded onto 4-15% Mini- PROTEAN TGX Precast Protein Gels (BioRad) in running buffer containing 25 mM Tris-HCI, 192 mM glycine and 3.5 mM SDS in water.

[0208] For protein staining gels were fixated for 30 min in 50% methanol (MeOH), 10% acetic acid and subsequently stained in 0.25% w / v Coomassie Brilliant Blue R250 in 50% MeOH, 10% acetic acid for 2 to 4 hours. Gels were then destained using 5% MeOH and 7.5% acetic acid for several hours under gentle shaking and regularly refreshing the solution. Protein bands were imaged with a FluorChem M system (ProteinSimple) and band intensities were analysed using Imaged.

[0209] For specifically staining the VHH, the sample in the electrophoresed protein gels were transferred with a TransBlot turbo system (BioRad) onto PVDF membranes. PVDF membranes were then blocked with 5% w / v BSA in PBS with 0.1% Tween 20 for 1 hour and subsequently incubated with anti-VHH primary antibody (1000x dilution) (QE19, QVQ Holding) in blocking buffer at 4°C overnight. Membranes were washed and incubated in goat anti-rabbit-HRP secondary antibody (2000x dilution) (Invitrogen) and StrepTactin-HRP (BioRad) at RT for 1 hour. After washing, immunoreactive bands were developed with the SuperSignal Pico PLUS chemiluminescent substrate (Thermo Fischer) and membranes were imaged with a FluorChem M system.

[0210] MMP Cleavage Test - affinity of VHH retained after cleavage with MMP

[0211] Figure 15 shows conjugation of MMPIeav-B1 VHH to Dex A) Schematic illustration of conjugation strategy and VHH cleavage. B) SDS-PAGE results of Dex-VHH conjugate and cleaved VHH from conjugate. Free MMPcleav-B1 (lane 2), Dex-MMPcleav-B1 conjugate (lane 3), cleaved MMPcleav-B1 from conjugate (lane 4). C) Binding kinetics of Dex- MMPcleav-B1 conjugate and cleaved MMPcleav-B1 from conjugate targeting IL1-R.

[0212] This figure shows more specifically that the affinity of a VHH with an MMP-cleavable sequence in the C-terminus is not affected after cleaving said sequence. The affinity of the VHH was measured by SPR both for the VHH coupled to dextran and after release by cleavage of the MMP-cleavable sequence by MMP13 (Fig 15C). To make the conjugation and release of the VHH possible, the MMP-cleavable sequence should ideally be positioned between CDR3 and the engineered cysteine at the end of FR4, or the glycine-serine-cysteine at the end of FR4, or the glycine-serine-cysteine-cysteine at the end of FR4

[0213] Methods

[0214] Surface Plasmon Resonance imaging (SPRi)

[0215] For immobilization purposes recombinant IL-1 R (Sino Biological, Beijing, China ) was diluted to concentrations ranging from 8.125-130 nM in sodium acetate immobilization buffer with a pH of 4.5, prepared as described previously.26 The immobilization on G-type Easy2Spot sensors was carried out using a continuous flow spotter (Wasatch Microfluidics, Salt Lake city, UT, US), where 48 spots were printed in 30 minutes. To reduce non-specific interaction, the sensor was blocked by two subsequent quenching steps of 7 minutes, which consisted of 1% (w / v) BSA solution in sodium acetate buffer at pH 4.5 followed by 0.2 M ethanolamine at pH 8.5. After immobilization and deactivation of the sensor, the analytes were diluted in a system buffer containing 0.075% tween-80 in PBS. Regeneration was carried out using 200 mM phosphoric acid with a pH of 2.5. The IBIS MX96 (IBIS Technologies, Enschede, the Netherlands) was used for SPRi measurements. Back and forth flow was set to 10pL min-1 in a flow cell containing 12 pL of sample. Sprint software was used for data collection and referencing.

[0216] SPRi kinetic titration assay

[0217] The affinity of the VHH and VHH-polymer constructs to recombinant IL-1R was determined by the kinetic titration method with RmaxlOO proposed by Schasfoort et al27. Recombinant IL- 1 R was immobilized on an Easy2Spot sensor in quadruple as described above, followed by a blocking step. For the SPRi measurements, first an analyte injection with blanks (running buffer only, PBS containing 0.075% tween-80) was injected that provided background for interaction signals. The next analyte injection consisted of a blank followed by an increasing concentration (8.125 - 130 nM) of each VHH in running buffer. The initial baseline time was set to 1 min, association time of the interactions was 15 min followed by 12 min dissociation. After each analyte injection, the sensor was regenerated using a double regeneration pulse for 30 seconds. Hydrogel degradation, dependence on composition Dex-TA / HA-TA

[0218] Figure 16 shows the degradation profiles of 5 w / v% hydrogel discs with various compositions ranging from pure Dex-TA (•) to pure HA-TA (°) incubated with 5 ll / rnl Hyaluronidase for 29 days.

[0219] This figure shows the relation between the degradation profile and the hydrogel composition. It was found that 50 / 50 Dex-TA / HA-TA provides in general the best degradation profile, in that that the hydrogel is still structurally reasonably intact after 30 days (without mechanical stress).

[0220] Methods

[0221] In degradation experiments, the cylindrical hydrogels of HA-TA / Dex-Ta at a concentration of 5% w / v were prepared in 8 mm diameter, 1.5 mm high molds and the initial weight of the hydrogels (Wi) was measured after equilibrating overnight in PBS. 1.0 mL of PBS containing 5.0 U / rnL hyaluronidase was placed on top of the prepared hydrogels and the samples were incubated at 37 °C. At regular time intervals, the hydrogels were weighed. The incubation buffer (including hyaluronidase) was replaced every 2-3 days and the experiments were performed in triplicate. The remaining gel (%) was calculated from the original gel weight after preparation (Wi) and remaining gel weight after exposure to the hyaluronidase containing buffer (Wt), expressed as Wt / Wi x 100%.

[0222] Viscosity of low and medium molecular weight HA - 1 njectabil ity

[0223] Figure 17 shows shear dependent viscosity of 10 %w / v LMW Dex-TA / HA-TA (50 / 50), 5 %w / v MMW HA-TA, and 10 %w / v LMW Dex-TA / MMW HA-TA (50 / 50) polymer solutions. LMW Dex = 40 kDa, LMW HA-TA = 30 kDa, MMW = 300 kDa. Injectability ranges are indicated: Dark grey = High Viscosity Fluids; intermediate grey = Moderate Viscosity Fluids; Light grey = Low Viscosity Fluids: Injectable Viscosity Range:

[0224] •Low-Viscosity Fluids: Easily injectable through small needles, with viscosities up to rt

[0225] Examples include saline and water-like solutions.

[0226] •Moderate-Viscosity Fluids: Injectable through larger needles (e g 18-22 gauge) with viscosities in the range of Examples include certain oils or polymeric solutions.

[0227] •High-Viscosity Fluids: Require specialized equipment or very large needles for injection. Fluids with viscosities up ti l.ooo-io.ooc • P-i-s might still be injectable with sufficient pressure and the right setup.

[0228] Figure 17 shows the viscosity of LMW HA-TA compared with MMW (medium molecular weight) HA-TA. The first 2 lines overlap; the addition of LMW Dex-TA has (almost) no influence on the viscosity of LMW and MMW HA-TA. Also the w / vol % has minor influence although it may become more influential at higher MW. It was also found that the Dex-TA makes a minimal contribution to the viscosity compared to HA-TA.

[0229] The best injectability is achieved by using LMW: It can be inferred that the hydrogel-forming polymer ideally is in the range of 10-166 kDa, preferably 12-50 kDa, or in other words, in the range of 60-1000, more preferably 70-300 (repeating) monomer or disaccharide units. In prior art no such small polymers are used, because they do not form hydrogels there, which means that the polymer VHH would be quickly clarified. Or they prefer HMW polymers because they spontaneously form hydrogels.

[0230] Methods

[0231] Shear sweep measurements

[0232] The rheological properties on non-crosslinked LMW Dex-TA supplemented with LMW and MMW HA-TA hydrogel solution was carried out with an MCR 301 rheometer (Anton-Paar) equipped with Peltier temperature control (C-PTD200). The viscosity measurements of LMW Dex-TA / HA-TA and LMW Dex-TA / MMW HA-TA were carried out using DG 26.7 at 20 °C (+ / - 0.2 °C) and parallel plates (PP; 0 25 mm, 1 mm gap) at 25 °C (+ / - 0.2 °C) respectively. The shear sweep measurement of LMW Dex-TA / HA-TA hydrogel solution was carried out in a previous study.

Claims

CLAIMS1. Single domain antibody that binds a cytokine, a cytokine receptor, a soluble antigen or an auto-antibody, wherein at least one hydrogel-forming polymer is conjugated to the single domain antibody, wherein the at least one hydrogel-forming polymer is degradable by an extracellular matrix degrading enzyme and / or the at least one hydrogel-forming polymer is conjugated to the single domain antibody via an amino acid sequence comprising an extracellular matrix degrading enzyme cleavage site, wherein the at least one hydrogel-forming polymer comprises 20-1000 monomer units, wherein the hydrogel-forming polymer is chosen from: hyaluronic acid, hyaluronic acid- tyramine, hyaluronic acid-tyramine-maleimide, hyaluronic acid-maleimide, dextran, dextran- tyramine, dextran-tyramine-maleimide, dextran-maleimide, (multi-arm) polyethylene glycol, polyethylene glycol- tyramine, polyethylene glycol - tyramine - maleimide, and / or polyethylene glycol - maleimide, wherein the extracellular matrix degrading enzyme is chosen from hyaluronidase, dextranase and protease, wherein the hyaluronidase is preferably chosen from hyaluronidase-1 , hyaluronidase -2, hyaluronidase -3 and / or hyaluronidase-4, and / or wherein the protease preferably is a matrix metalloproteinase (MMP), and wherein the MMP preferably is chosen from MMP1, MMP3, MMP9 and / or MMP13, and wherein wherein the single domain antibody binds Tumor Necrosis Factor-alpha (TNF-a), Nerve Growth Factor (NGF), Interleukin ip (I L-1 P), Interleukin 6 (IL-6), MMP9, Interleukin ip receptor (IL1 R) and / or IL-6 receptor (IL6R).

2. Single domain antibody according to claim 1, wherein the at least one hydrogel-forming polymer comprises 20-300 monomer units.

3. Single domain antibody according to any one of the previous claims, wherein at least one hydrogel-forming polymer comprises one or more functional groups, preferably one or more phenolic group(s) and / or one or more maleimide group(s), wherein the phenolic group(s) are preferably chosen from tyramine, tyrosine or dopamine, most preferably tyramine.

4. Single domain antibody according to any one of the previous claims, wherein the single domain antibody is a Variable domain of a Heavy chain only (VHH) antibody.

5. Single domain antibody according to claim 4, wherein the VHH antibody has the following structure with complementarity determining regions (CDR) and framework regions (FR): (N-terminus) FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 (C-terminus)wherein the at least one hydrogel-forming polymer is conjugated to the VHH antibody by coupling to a cysteine in FR4, more preferably by coupling to a linker comprising glycine- serine-cysteine in FR4, most preferably by coupling to a linker comprising glycine-serine- cysteine-cysteine in FR4, wherein preferably the coupling to cysteine, a linker comprising glycine-serine-cysteine, or a linker comprising glycine-serine-cysteine-cysteine is through reaction with a maleimide comprised in the at least one hydrogel-forming polymer.

6. Single domain antibody according to claim 5, wherein the extracellular matrix degrading enzyme cleavage site is between CDR3 and the cysteine in FR4, the glycine-serine-cysteine in FR4, or the glycine-serine-cysteine-cysteine in FR4.

7. Single domain antibody according to any one of the previous claims, wherein at least one hydrogel-forming polymer is conjugated to the single domain antibody, wherein the at least one hydrogel-forming polymer is degradable by an extracellular matrix degrading enzyme, and wherein the single domain antibody is in combination with a second single domain antibody that binds a cytokine, a cytokine receptor, a soluble antigen or an auto-antibody, wherein at least one hydrogel-forming polymer is conjugated to the second single domain antibody via an amino acid sequence comprising an extracellular matrix degrading enzyme cleavage site.

8. Single domain antibody according to any one of the previous claims, comprised in a composition, preferably further comprising hydrogen peroxide and / or peroxidase.

9. Single domain antibody according to claim 8, wherein:- a total of hydrogel forming polymer in the composition has a weight / volume percentage of between 1w / v% and 10w / v% or between 4w / v% and 7w / v%;- the composition further comprises hydrogel forming polymer that is not degradable by an extracellular matrix degrading enzyme and that is not conjugated to a single domain antibody, preferably in an amount of between 10 wt.% and 90 wt.% or between 40 wt.% and 60 wt.% relative to a total of hydrogel forming polymer in the composition;- the composition further comprises hydrogel forming polymer that is not degradable by an extracellular matrix degrading enzyme and that is not conjugated to a single domain antibody, being dextran-tyramine, preferably wherein the number of tyramines per 100 saccharide unit is between 5 and 15;- a total of hydrogel forming polymer in the composition has a crosslinking density of between 10% and 100% with respect to the total of crosslinkable tyramine units; and / or- the amount of single domain antibody in the composition per total of hydrogel forming polymer in the composition is between 0.001-20 wt.%, preferably between 0.05-3 wt.%, wherein the wt.% is drawn on the wt. of the single domain antibody with respect to the wt. of the at least one or more hydrogel-forming polymer.

10. Single domain antibody according to claim 8 or 9, wherein the composition is in the form of an injectable hydrogel.

11. Single domain antibody according to any one of claims 8-10, wherein the composition comprises 1-30 w / v.%, more preferably 2-20 w / v.%, most preferably 4-15 w / v.% of the at least one hydrogel-forming polymer, wherein the weight is drawn on the total volume of the composition.

12. Single domain antibody according to any one of the previous claims, for use in the treatment of auto-immune disease, cancer, osteoarthritis, inflammation, preferably in the treatment of joint-inflammation or degenerative joint disease.

13. In vitro method for reducing the bioavailability of a cytokine, a cytokine receptor, a soluble antigen and / or an auto-antibody in a biological system, the method comprising providing said system with a single domain antibody according to any one of the previous claims.

14. Method of producing a hydrogel, said method comprising the step of curing a composition according to any one of claims 9-11, wherein said composition comprises hydrogen peroxide and a peroxidase.

15. Hydrogel obtainable by the method of claim 14.

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