WO2022157373A1

WO2022157373A1 - Compositions and kits for in vivo imaging of cardiac sarcoidosis

Info

Publication number: WO2022157373A1
Application number: PCT/EP2022/051556
Authority: WO
Inventors: Henri BAUDHUIN; Karine CLAUWAERT; Tony Lahoutte
Original assignee: Vrije Universiteit Brussel
Priority date: 2021-01-25
Filing date: 2022-01-25
Publication date: 2022-07-28

Abstract

The present invention relates to immunoglobulin single variable domains directed against human macrophage mannose receptor (MMR) and MMR-binding fragments thereof, and their use in non-invasive in vivo medical imaging for the detection, diagnosis and/or prognosis of cardiac sarcoidosis. Further provided are methods for diagnosis and/or prognosis of cardiac sarcoidosis, in particular by non-invasive in vivo medical imaging. Also provided are kits comprising such immunoglobulin single variable domains and fragments thereof for the production of a diagnostic for the detection of cardiac sarcoidosis.

Description

COMPOSITIONS AND KITS FOR IN VIVO IMAGING OF CARDIAC SARCOIDOSIS

FIELD OF THE INVENTION

The present invention relates to immunoglobulin single variable domains directed against human macrophage mannose receptor (MMR) and their uses in the field of cardiology. More specifically, it concerns immunoglobulin single variable domains directed against human MMR and their use in non-invasive in vivo medical imaging, in particular for the detection, diagnosis and/or prognosis of cardiac sarcoidosis.

BACKGROUND

Sarcoidosis is an inflammatory granulomatous disease that can affect any organ. Sarcoidosis is characterized by epithelioid cell-rich, noncaseating granulomas caused by an exaggerated immune response. The clinical manifestations of cardiac sarcoidosis (CS) include heart block, atrial and ventricular arrhythmias, and heart failure. While sarcoidosis can be a systemic disease, pulmonary involvement is the most common presentation. Historically, only 5% of the patients were found to have clinical manifestation of cardiac sarcoidosis. However, there is increasing understanding that CS may be underdiagnosed. Based on an autopsy study from Japan, only about a quarter of patients with CS had an antemortem clinical diagnosis of sarcoidosis.

The diagnosis of CS can be challenging given the patchy infiltration of the myocardium but, with the increased availability of advanced cardiac imaging, more cases of CS are being identified. While ¹⁸F-fluorodeoxyglucose (FDG) positron emission tomography (PET) is a well-established modality to image inflammation and diagnose CS, there are limitations to its specificity and reproducibility. FDG PET relies on persistent uptake of glucose by the granulomatous inflammatory cells, in a metabolic setting where cardiac myocytes have switched from glucose to fatty acid metabolism (Randle cycle) as an energy substrate. If the conversion to the Randle cycle is incomplete, the specificity of FDG PET scan is poor. Therefore, an adequate dietary preparation before cardiac PET is essential. Moreover, the presence of FDG uptake is not specific to CS and may be seen in myocarditis or in another inflammatory cardiomyopathy. Although useful, FDG does not target a specific molecule or receptor on the surface of the cells involved in the CS disease process. Therefore, there is a need for probes that allow a more specific molecular characterization of inflamed or diseased tissue using CS-related membrane antigens.

Elevated expression of M2 macrophage-associated markers have been noted in diseased sarcoidosis tissues, including CD206 and CD163, and their expression correlated with disease severity.

The inventors have found that immunoglobulin single variable domains directed against or specifically binding to human macrophage mannose receptor (MMR) are useful in the diagnosis and/or prognosis of cardiac sarcoidosis by in vivo medical imaging.

SUMMARY OF THE INVENTION

In a first aspect, the present invention according to claim 1 provides an immunoglobulin single variable domain directed against or specifically binding to human macrophage mannose receptor (MMR) or an MMR-binding fragment thereof for use in the diagnosis and/or prognosis of cardiac sarcoidosis, wherein said immunoglobulin single variable domain or MMR-binding fragment thereof is coupled to a detectable label.

Preferred embodiments of the immunoglobulin single variable domain or MMR- binding fragment thereof for use according to claim 1 are defined in claims 2 to 20.

In a second aspect, the present disclosure provides a method for diagnosing and/or prognosing cardiac sarcoidosis according to claim 21.

Preferred embodiments of the method according to claim 21 are defined in claims 22, 23, and 25 to 29.

In a third aspect, the present disclosure provides a method of imaging cardiac sarcoidosis in a subject according to claim 24, comprising administering to the subject an immunoglobulin single variable domain directed against or specifically binding to human MMR or an MMR-binding fragment thereof coupled to a detectable label, and imaging the immunoglobulin single variable domain directed against or specifically binding to human MMR or an MMR-binding fragment thereof in the subject so as to image cardiac granulomata in the subject. Preferred embodiments of the method according to claim 24 are defined in claims 25 to 29.

In a fourth aspect, the present disclosure provides the use of a kit comprising an immunoglobulin single variable domain directed against or specifically binding to human MMR or an MMR-binding fragment thereof and one or more detectable labels for the production of a diagnostic for the detection of cardiac sarcoidosis, according to claim 30.

DESCRIPTION OF FIGURES

Figure 1 depicts a schematic overview of a Phase II clinical trial in patients with cardiac sarcoidosis using amongst others the ⁶⁸Ga-NOTA-anti-MMR sdAb for use according to the current invention.

Figure 2 shows a graphical representation of the distribution of sized particles in the final concentrated formulation according to an embodiment of the current invention. A particle size analysis was performed via Dynamic Light Scattering on the final concentrated formulation to analyze the distribution of particles in the solution according to an embodiment of the current invention. The solution was tested in triplicate. Panel 1A shows that the majority of particles has a maximum hydrodynamic diameter of 3 nm. Panel IB confirms that no other particle sizes are present in the solution.

Figure 3 illustrates a MDSC thermogram to determine the Tg' of a 5% AA-2G solution according to an embodiment of the current disclosure.

DEFINITIONS

Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the present invention. The terms and definitions used herein are provided solely to aid in the understanding of the invention.

As used herein, the following terms have the following meanings: "A", "an", and "the" as used herein refers to both singular and plural referents unless the context clearly dictates otherwise. By way of example, "a compartment" refers to one or more than one compartment.

"About" as used herein referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of +/- 20% or less, preferably +/-10% or less, more preferably +/-5% or less, even more preferably +/-1% or less, and still more preferably +/-0.1% or less of and from the specified value, in so far such variations are appropriate to perform in the disclosed invention. However, it is to be understood that the value to which the modifier "about" refers is itself also specifically disclosed.

"Comprise", "comprising", and "comprises" and "comprised of" as used herein are synonymous with "include", "including", "includes" or "contain", "containing", "contains" and are inclusive or open-ended terms that specifies the presence of what follows e.g., component and do not exclude or preclude the presence of additional, non-recited components, features, element, members, steps, known in the art or disclosed therein.

The terms first, second and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order, unless specified. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within that range, as well as the recited endpoints.

Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the following claims, any of the claimed embodiments can be used in any combination.

Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, definitions for the terms used in the description are included to better appreciate the teaching of the present invention. The terms or definitions used herein are provided solely to aid in the understanding of the invention.

As used herein, the terms "polypeptide", "protein", "peptide" are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.

"Antibody" as used herein refers to antibodies which comprise two heavy chains H, each comprising a constant region and a variable region. These heavy chains are linked by disulfide bridges at the so-called hinge region. In conventional antibodies, as opposed to heavy-chain-only antibodies, each heavy chain is linked to a light chain L (each also comprising a constant region and variable region) through further disulfide bridges in an arrangement that is often referred to as forming an overall "Y" shape. Thus, a conventional antibody has the overall configuration (HL)2. Each heavy chain and light chain variable region has three complementary determining regions (CDRs). Together the variable region of a light chain and a heavy chain define the binding specificity of the antibody for the target.

Full-sized monoclonal antibodies (mAbs) have a number of disadvantages that have so far limited their effective use in the clinic. mAbs are macromolecules with a relatively poor penetration into solid and isolated tissues such as tumors and heart tissues. In addition, complete mAbs feature a long residence time in the body and a potential increase in background signals because of binding to Fc receptors on nontarget cells, making them less suitable for molecular imaging applications. Indeed, for imaging the most important properties of a tracer are: rapid interaction with the target, fast clearing of unbound molecules from the body and low non-specific accumulation, especially around the area of interest. These requirements have led to the development of a myriad of antibody-derived probe formats, like scFv, trying to combine specificity with a small size for favorable pharmacokinetics.

"Antibody fragment" as used herein refers to an entity which is less than a full antibody, for example a variable region from a heavy and/or light chain, a single chain variable region, a Fab fragment, a F(ab')2 fragment, a variable region and a portion of a constant region, a heavy chain, a light chain, a single chain or the like and including conjugates of each of the same. A variable region from a heavy chain or a light chain can be considered as a basic functional binding unit of antibody and is sometimes referred to as a domain antibody. Alternatively, a variable region from a heavy chain and light chain can be associated together, for example by covalent bonds to provide what is referred to as a "single chain variable fragment (scFv)", and comprises three CDRs from the heavy and three CDRs from the light chain (nominally referred to as Hl, H2, H3 for the heavy chain and LI, L2 and L3 for the light chain), in the same way as a complete antibody.

"Immunoglobulin single variable domain" as used herein defines molecules wherein the antigen-binding site is present on, and formed by, a single immunoglobulin domain. Although in conventional immunoglobulins typically two immunoglobulin variable domains interact to form a functional antigen binding site, the term "immunoglobulin single variable domain" also includes fragments of conventional immunoglobulins wherein a functional antigen binding site is formed by a single variable domain.

Generally, an immunoglobulin single variable domain will have an amino acid sequence comprising 4 framework regions (FR1 to FR4) and 3 complementarity determining regions (CDR1 to CDR3), preferably according to the following formula (1):

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1), or any suitable fragment thereof (which will then usually contain at least some of the amino acid residues that form at least one of the complementarity determining regions). It should be clear that framework regions of immunoglobulin single variable domains may also contribute to the binding of their antigens. The delineation of the CDR sequences (and thus also the FR sequences) can be based on the IMGT unique numbering system for V-domains and V-like domains. Alternatively, the delineation of the FR and CDR sequences can be done by using the Kabat numbering system as applied to VHH domains from Camelids. Immunoglobulin single variable domains comprising 4 FRs and 3 CDRs are known to the person skilled in the art and have been described.

Typical, but non-limiting, examples of immunoglobulin single variable domains include light chain variable domains (e.g., a VL domain) or a suitable fragment thereof, or heavy chain variable domains (e.g., a VH domain derived from a conventional four-chain antibody or VHH domain derived from a heavy-chain-only antibody) or a suitable fragment thereof, as long as it is capable of forming a functional antigen binding site. The immunoglobulin single variable domain may be a domain antibody, or a single domain antibody, or a "dAB" or "dAb", or a VHH domain or another immunoglobulin single variable domain, or any suitable fragment of any one thereof.

In the context of the present invention, "suitable fragment" or "functional fragment" or "antigen-binding fragment" of an immunoglobulin single variable domain means a fragment of the immunoglobulin single variable domain which has the same or similar binding specificity of the immunoglobulin single variable domain from which it is derived. Thus, a suitable fragment of an immunoglobulin single variable domain directed against or specifically binding to particular antigen also binds to that particular antigen.

It should be noted that the immunoglobulin single variable domains as binding domain moiety in their broadest sense are not limited by a specific biological source or by a specific method of preparation. The term "immunoglobulin single variable domain" encompasses variable domains of different origin, comprising mouse, rat, rabbit, donkey, human, shark, camelid variable domains. According to specific embodiments, the immunoglobulin single variable domains are derived from shark antibodies (the so-called immunoglobulin new antigen receptors or IgNARs), more specifically from naturally occurring heavy chain shark antibodies, devoid of light chains, and are known as VNAR domains. Preferably, the immunoglobulin single variable domains are derived from camelid antibodies. More preferably, the immunoglobulin single variable domains are derived from naturally occurring heavy- chain-only camelid antibodies, devoid of light chains, and are known as VHH domains.

The terms "VHH domain", "VHH antibody" and "VHH" as used herein interchangeably with the term "single domain antibody fragment (sdAb)" and refer to a single domain antigen-binding fragment. It particularly refers to a single variable domain derived from naturally occurring heavy-chain-only antibodies and is known to the person skilled in the art. VHH domains are usually derived from heavy-chain-only antibodies (devoid of light chains) seen in camelids and consequently are often referred to as VHH antibody or VHH. Camelids comprise old world camelids Camelus bactrianus and Camelus dromedarius') and new world camelids (for example Vicugna pacos, Lama glama, Lama guanicoe and Lama vicugna). The small size and unique biophysical properties of VHH domains excel conventional antibody fragments for the recognition of uncommon or hidden epitopes and for binding into cavities or active sites of protein targets. VHH domains are stable, survive the gastro-intestinal system and can easily be manufactured. Therefore, VHH domains can be used in many applications including drug discovery and therapy, but also as a versatile and valuable tool for purification, functional study and crystallization of proteins.

The VHH domains of the invention generally comprise a single amino acid chain that can be considered to comprise 4 "framework regions" or FR's and 3 "complementarity determining regions" or CDR's, according to formula (1) (as defined above). The term "complementarity determining region" or "CDR" refers to regions in VHH domains and contains the amino acid sequences capable of specifically binding to antigenic targets. These CDRs account for the basic specificity of the VHH domains for a particular antigenic determinant structure. Such regions are also referred to as "hypervariable regions." The VHH domains have 3 CDR regions, each non-contiguous with the others (termed CDR1, CDR2, CDR3). The delineation of the FR and CDR sequences is often based on the IMGT unique numbering system for V-domains and V-like domains. Alternatively, the delineation of the FR and CDR sequences can be done by using the Kabat numbering system as applied to VHH domains from Camelids. As will be known by the person skilled in the art, the VHH domains can in particular be characterized by the presence of one or more Camelidae hallmark residues in one or more of the framework sequences (according to Kabat numbering). Such VHH domains can generally be generated or obtained by suitably immunizing a species of camelid with a desired target, (i.e., so as to raise an immune response and/or heavy chain antibodies directed against a desired target), by obtaining a suitable biological sample from said camelid (such as a blood sample, or any sample of B-cells), and by generating VHH domains directed against the desired target, starting from said sample, using any suitable technique known per se. Such techniques will be clear to the skilled person. Alternatively, such naturally occurring VHH domains against the desired target can be obtained from naive libraries of camelid VHH domains, for example by screening such a library using the desired target or at least one part, fragment, antigenic determinant or epitope thereof using one or more screening techniques known per se. Such libraries and techniques are for example described in W09937681, W00190190, W003025020 and WO03035694. Alternatively, improved synthetic or semi-synthetic libraries derived from naive VHH libraries may be used, such as VHH libraries obtained from naive VHH libraries by techniques such as random mutagenesis and/or CDR shuffling, as for example described in W00043507. Yet another technique for obtaining VHH domains directed against a desired target involves suitably immunizing a transgenic mammal that is capable of expressing heavy chain antibodies (i.e. so as to raise an immune response and/or heavy chain antibodies directed against a desired target), obtaining a suitable biological sample from said transgenic mammal (such as a blood sample, or any sample of B-cells), and then generating VHH domains directed against the desired target starting from said sample, using any suitable technique known per se. For example, for this purpose, the heavy chain antibody-expressing mice and the further methods and techniques described in WO02085945 and in WO04049794 can be used.

A particularly preferred class of immunoglobulin single variable domains of the invention comprises VHH domains with an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VHH domain, but that has been "humanized" , i.e. by replacing one or more amino acid residues in the amino acid sequence of said naturally occurring VHH domain (and in particular in the framework sequences) by one or more of the amino acid residues that occur at the corresponding position(s) in a VH domain from a conventional 4-chain antibody from a human being. This can be performed in a manner known per se, which will be clear to the skilled person and on the basis of the prior art on humanization. Again, it should be noted that such humanized VHH domains of the invention can be obtained in any suitable manner known per se and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring VHH domain as a starting material. Humanized VHH domains may have several advantages, such as a reduced immunogenicity, compared to the corresponding naturally occurring VHH domains. Such humanization generally involves replacing one or more amino acid residues in the sequence of a naturally occurring VHH with the amino acid residues that occur at the same position in a human VH domain, such as a human VH3 domain. The humanizing substitutions should be chosen such that the resulting humanized VHH domains still retain the favorable properties of VHH domains as defined herein. The skilled person will be able to select humanizing substitutions or suitable combinations of humanizing substitutions which optimize or achieve a desired or suitable balance between the favorable properties provided by the humanizing substitutions on the one hand and the favorable properties of naturally occurring VHH domains on the other hand.

Also within the scope of the invention are natural or synthetic analogs, mutants, variants, alleles, homologs and orthologs of the antibody, antibody fragment, immunoglobulin single variable domain or antigen-binding fragment thereof, of the invention as defined herein, e.g. generated by substitution of one or more amino acids in the sequence of the antibody, antibody fragment, immunoglobulin single variable domain or antigen-binding fragment thereof, of the invention as defined herein.

By means of non-limiting examples, a substitution may for example be a conservative substitution and/or an amino acid residue may be replaced by another amino acid residue. Thus, any one or more substitutions, deletions or insertions, or any combination thereof, that either improve the properties of the antibody, antibody fragment, immunoglobulin single variable domain or antigen-binding fragment thereof of the invention or that at least do not detract too much from the desired properties or from the balance or combination of desired properties of the antibody, antibody fragment, immunoglobulin single variable domain or antigen-binding fragment thereof of the invention (i.e. to the extent that the antibody, antibody fragment, immunoglobulin single variable domain or antigen-binding fragment thereof is no longer suited for its intended use) are included within the scope of the invention. A skilled person will generally be able to determine and select suitable substitutions, deletions or insertions, or suitable combinations of thereof, based on the disclosure herein and optionally after a limited degree of routine experimentation, which may for example involve introducing a limited number of possible substitutions and determining their influence on the properties of the antibody, antibody fragment, immunoglobulin single variable domain or antigenbinding fragment thereof thus obtained.

Further, depending on the host organism used to express the antibody, antibody fragment, immunoglobulin single variable domain or antigen-binding fragment thereof of the invention, such deletions and/or substitutions may be designed in such a way that one or more sites for post-translational modification (such as one or more glycosylation sites) are removed, as will be within the ability of the person skilled in the art. Alternatively, substitutions or insertions may be designed so as to introduce one or more sites for attachment of functional groups, for example to allow sitespecific pegylation.

Examples of modifications, as well as examples of amino acid residues within the antibody, antibody fragment, immunoglobulin single variable domain or antigenbinding fragment thereof, that can be modified (i.e., either on the protein backbone but preferably on a side chain), methods and techniques that can be used to introduce such modifications and the potential uses and advantages of such modifications will be clear to the skilled person. For example, such a modification may involve the introduction (e.g., by covalent linking or in another suitable manner) of one or more functional groups, residues or moieties into or onto the antibody, antibody fragment, immunoglobulin single variable domain or antigen-binding fragment thereof, and in particular of one or more functional groups, residues or moieties that confer one or more desired properties or functionalities to the antibody, antibody fragment, immunoglobulin single variable domain or antigen-binding fragment thereof of the invention.

Examples of such functional groups and of techniques for introducing them will be clear to the skilled person, and can generally comprise all functional groups and techniques mentioned in the general background art cited hereinabove as well as the functional groups and techniques known per se for the modification of pharmaceutical proteins, and in particular for the modification of antibodies, antibody fragments, immunoglobulin single variable domains or antigen-binding fragments thereof (including single domain antibody fragments) for which reference is for example made to Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, PA (1980). Such functional groups may for example be linked directly (for example covalently) to an antibody, antibody fragment, immunoglobulin single variable domain or antigen-binding fragment thereof of the invention, or optionally via a suitable linker or spacer, as will again be clear to the skilled person.

One of the most widely used techniques for increasing the half-life and/or reducing immunogenicity of pharmaceutical proteins comprises attachment of a suitable pharmacologically acceptable polymer, such as poly(ethyleneglycol) (PEG) or derivatives thereof (such as methoxypoly(ethyleneglycol) or mPEG). Generally, any suitable form of pegylation can be used, such as the pegylation used in the art for antibodies and antibody fragments. Preferably, site-directed pegylation is used, in particular via a cysteine-residue. For example, for this purpose, PEG may be attached to a cysteine residue that naturally occurs in an antibody, antibody fragment, immunoglobulin single variable domain or antigen-binding fragment thereof of the invention, an antibody, antibody fragment, immunoglobulin single variable domain or antigen-binding fragment thereof of the invention may be modified so as to suitably introduce one or more cysteine residues for attachment of PEG, or an amino acid sequence comprising one or more cysteine residues for attachment of PEG may be fused to the N- and/or C-terminus of an antibody, antibody fragment, immunoglobulin single variable domain or antigen-binding fragment thereof of the invention, all using techniques of protein engineering known per se to the skilled person. Another, usually less preferred modification comprises N-linked or O-linked glycosylation, usually as part of co-translational and/or post-translational modification, depending on the host cell used for expressing an antibody, antibody fragment, immunoglobulin single variable domain or antigen-binding fragment thereof of the invention.

As used herein, the term "specifically binding to" in the context of an antibody, antibody fragment, immunoglobulin single variable domain or antigen-binding fragment thereof, refers to the ability of this antibody or immunoglobulin single variable domain (fragment) to preferentially bind to a particular antigen that is present in a homogeneous mixture of different antigens and does not necessarily imply high affinity (as defined further herein). In certain embodiments, a specific binding interaction will discriminate between desirable and undesirable antigens in a sample, in some embodiments more than about 10 to 100-fold or more (e.g., more than about 1000- or 10,000-fold). The term "affinity", as used herein, refers to the degree to which an antibody or fragment thereof binds to an antigen so as to shift the equilibrium of antigen and an antibody, antibody fragment, immunoglobulin single variable domain or antigen-binding fragment thereof toward the presence of a complex formed by their binding. Thus, for example, where an antigen and antibody or immunoglobulin single variable domain (fragment) are combined in relatively equal concentration, an antibody or immunoglobulin single variable domain (fragment) of high affinity will bind to the available antigen so as to shift the equilibrium toward high concentration of the resulting complex. The dissociation constant is commonly used to describe the affinity between the antibody or immunoglobulin single variable domain (fragment) and the antigenic target. Typically, the dissociation constant is lower than 10'⁵ M. Preferably, the dissociation constant is lower than 10'⁶ M, more preferably, lower than 10'⁷ M. Most preferably, the dissociation constant is lower than 10'⁸ M.

"Lyophilizing" in this document refers to freeze-drying a liquid or pre-lyophilization formulation. Freeze-drying is performed by freezing the formulation and then subliming ice from the frozen content at a temperature suitable for primary drying. Under this condition the product temperature is below the collapse temperature of the formulation. A secondary drying stage may then be carried out, which produces a suitable lyophilized cake.

A "lyophilization excipient" in this document refers to a compound added to the lyophilization process to serve a specific function. They are added to increase bulk, aid manufacturing, improve stability, enhance drug delivery and targeting, and modify drug safety or pharmacokinetic profile.

"Reconstitution" in this document refers to dissolving a lyophilized protein formulation in a diluent such that the protein is dispersed in the reconstituted formulation. The reconstituted formulation should be suitable for administration (e.g. parenteral administration) to a subject to be treated with the antibody or antibody fragment of interest.

As used herein, the term "medical imaging" refers to the technique and process that is used to visualize the inside of an organism's body (or parts and/or functions thereof), for clinical purposes (e.g., disease diagnosis, prognosis or therapy monitoring) or medical science (e.g., study of anatomy and physiology). Examples of medical imaging methods include invasive techniques, such as intravascular ultrasound (IVUS), as well as non-invasive techniques, such as magnetic resonance imaging (MRI), ultrasound (US) and nuclear imaging. Examples of nuclear imaging include positron emission tomography (PET) and single photon emission computed tomography (SPECT). Thus, in one embodiment, the anti-MMR immunoglobulin single variable domains, as described hereinbefore, are coupled to a radionuclide.

As used herein, the term "diagnosing" or grammatically equivalent wordings, means determining whether or not a subject suffers from a particular disease or disorder. As used herein, "prognosing" or grammatically equivalent wordings, means determining whether or not a subject has a risk of developing a particular disease or disorder.

DETAILED DESCRIPTION OF THE INVENTION

The current invention is directed to methods and compositions for the diagnosis and/or prognosis of cardiac sarcoidosis. Sarcoidosis is a multi-system inflammatory disorder of unknown etiology resulting in formation of non-caseating granulomas. Cardiac involvement— which is associated with worse prognosis— has been detected in approximately 25% of individuals based on autopsy or cardiac imaging studies. Advanced cardiac imaging is useful in identifying patients who have higher risk of adverse events such as ventricular tachycardia or death, in whom preventive therapies such as defibrillators should be more strongly considered.

In a first aspect, the current invention is directed to an immunoglobulin single variable domain directed against or specifically binding to human macrophage mannose receptor (MMR) or an MMR-binding fragment thereof for use in the diagnosis and/or prognosis of cardiac sarcoidosis, wherein said immunoglobulin single variable domain or MMR-binding fragment thereof is coupled to a detectable label.

The term "macrophage mannose receptor" (MMR), as used herein, is known in the art and refers to a type I transmembrane protein, first identified in mammalian tissue macrophages and later in dendritic cells and a variety of endothelial and epithelial cells. Macrophages are central actors of the innate and adaptive immune responses. They are disseminated throughout most organs to protect against entry of infectious agents by internalizing and most of the time, killing them. Among the surface receptors present on macrophages, the mannose receptor recognizes a variety of molecular patterns generic to microorganisms. The MMR is composed of a single subunit with N- and O-linked glycosylation and consists of five domains: an N- terminal cysteine-rich region, which recognizes terminal sulfated sugar residues; a fibronectin type II domain with unclear function; a series of eight C-type, lectin-like carbohydrate recognition domains (CRDs) involved in Ca²⁺-dependent recognition of mannose, fucose, or N-acetylglucosamine residues on the envelop of pathogens or on endogenous glycoproteins with CRDs 4-8 showing affinity for ligands comparable with that of intact MR; a single transmembrane domain; and a 45 residue-long cytoplasmic tail that contains motifs critical for MR-mediated endocytosis and sorting in endosomes. In particular, the human macrophage mannose receptor is known as Mrcl or CD206 (accession number nucleotide sequence: NM_002438.2; accession number protein sequence: NP_002429.1).

The immunoglobulin single variable domains directed against or specifically binding to human macrophage mannose receptor (MMR) or an MMR-binding fragment thereof of the invention are able to selectively bind to or target MMR-expressing cells, such as MMR-positive TAMs linked to a hypoxic region of a solid tumor or M2 macrophages associated with cardiac sarcoidosis-associated granulomata.

The immunoglobulin single variable domains directed against or specifically binding to human macrophage mannose receptor (MMR) or an MMR-binding fragment thereof of the invention are used as targeting vehicles in non-invasive molecular imaging techniques or for targeted therapeutic applications.

Non-invasive molecular imaging is aimed at tracking cellular and molecular events in their native environment in the intact living subject. In its broadest sense, molecular imaging entails the administration of a tracer molecule labeled with a detectable label for visualization.

In an embodiment, said immunoglobulin single variable domain or antigen-binding fragment thereof is coupled to a detectable label.

In a preferred embodiment, the detectable label is selected from the group consisting of a radionuclide, a fluorescent moiety, a phosphorescent label, a chemiluminescent label, a metal, a metal chelate, a metallic cation, a chromophore, an enzyme or a combination of one of the aforementioned labels.

In a particularly preferred embodiment, said detectable label is a radionuclide. "Radionuclide" as used herein refers to an atom that has excess nuclear energy, making it unstable. This excess energy can be used in one of three ways: emitted from the nucleus as gamma radiation; transferred to one of its electrons to release it as a conversion electron; or used to create and emit a new particle (alpha particle or beta particle) from the nucleus. The terms "radionuclide" or "radioisotope" can be used interchangeably. As used herein, a molecule viz. a immunoglobulin single variable domain or MMR-binding fragment thereof, is referred to as "radiolabeled" when it is coupled or fused to or labeled with a radionuclide.

Said radionuclide may be chosen from the group of fluor 18 (¹⁸F), scandium 44 (⁴⁴Sc), lutetium 177 (¹⁷⁷Lu), zirconium 89 (⁸⁹Zr), indium 111 (^mln), yttrium 90 (⁹⁰Y), copper 61 (⁶¹Cu), copper 64 (⁶⁴Cu), actinium 225 (²²⁵Ac), bismuth 213 (²¹³Bi), gallium 67 (⁶⁷Ga), gallium 68 (⁶⁸Ga), technetium 99m (^99mTc), iodine 123 (¹²³I), iodine 124 (¹²⁴I), iodine 125 (¹²⁵I), iodine 131 (¹³¹I).

In an embodiment, the radionuclide is a gallium radioisotope solution obtained directly from a gallium radionuclide generator. In a most preferred embodiment, the radionuclide is gallium 68 (⁶⁸Ga).

These radionuclides are suitable for medical applications, such as in vivo nuclear imaging or Targeted Radionuclide Therapy (TRNT). In an embodiment, the immunoglobulin single variable domain or MMR-binding fragment thereof is coupled or fused directly to said radionuclide. In another embodiment, the immunoglobulin single variable domain or antigen-binding fragment thereof is coupled or fused to said radionuclide through a linker. As used herein, "linker molecules" or "linkers" are peptides of 1 to 200 amino acids length, and are typically, but not necessarily, chosen or designed to be unstructured and flexible.

Primarily, radioactively labeled biomolecules are used in combination with positronemission tomography (PET) or single photon-emission computed tomography (SPECT)-based imaging techniques.

For radiopharmaceuticals, radiolytic damage during radiolabeling of the immunoglobulin single variable domain or MMR-binding fragment thereof can be a serious problem. Such radiolytic damage can cause, for example, release of the radionuclide or it can damage the immunoglobulin single variable domain or MMR- binding fragment thereof. Significant radiolytic damage induced by the radioactive label can occur if labeling of the immunoglobulin single variable domain or MMR- binding fragment thereof occurs without concomitant or subsequent addition of one or more radioprotectants (compounds that protect against radiolytic damage). Hence, it is critical to find inhibitors of radiolysis that can be used to prevent both methionine oxidation and other radiolytic decomposition routes in radiopharmaceuticals. For this purpose, compounds known as radical scavengers or antioxidants are typically used. These are compounds that react rapidly with, e.g., hydroxyl radicals and superoxide, thus preventing them from reacting with the radiopharmaceutical of interest or reagents for its preparation. There has been extensive research in this area. Most of it has focused on the prevention of radiolytic damage in radiodiagnostic formulations, and several radical scavengers have been proposed for such use.

In order to identify suitable antioxidant radical scavengers that might be useful as rad io protectants, the inventors performed several studies. Ideally, the rad io protectant should be able to be added directly to the formulation without significantly decreasing the radiochemical purity (RCP) of the product. Radiochemical purity (RCP) may be defined as "the proportion of the total radioactivity in the sample which is present as the desired radiolabelled species". Radiochemical purity is important in radiopharmacy since it is the radiochemical form which determines the biodistribution of the radiopharmaceutical. RCP can be determined by any method known from the prior art, such as instant Thin Layer Chromatography (iTLC) or Size Exclusion Chromatography (SEC). By assessing RCP, one can determine the compatibility of the radioprotectant with the radiolabeling reaction. In addition, SEC and iTLC allow to measure the amount of radiolysis and the amount of free radionuclide. High activity tests allow to assess the efficiency of the radioprotectant.

The impact of the radioprotectant on the profiling and the functionality of the immunoglobulin single variable domain or MMR-binding fragment thereof can be assessed by various protein analysis techniques known from the state of the art.

Ascorbic acid (AA), also known as vitamin C, plays key roles in a variety of biological processes like collagen formation, carnitine synthesis, iron absorption, drug metabolism and the function of the immune system. Furthermore, ascorbic acid is also a well-known and potent natural antioxidant and has the ability to protect other molecules (e.g., DNA, proteins...) from highly reactive or oxidizing agents, such as free radicals. Therefore, ascorbic acid has been proposed as alternative buffer system for metalloradiopharmaceuticals. Indeed, with a pKa of 4.2, ascorbic acid can offer, along with its salt-form sodium ascorbate, ideal buffering capacity in the pH range of 3.5 - 5.0, which is the typical range in which radiolabelings of metallic radionuclides is carried out, for instance ⁶⁸Ga-NOTA radiolabeling.

The current disclosure provides a method for labeling a biomolecule such as the immunoglobulin single variable domain or MMR-binding fragment thereof, with one or more detectable labels, wherein at least one of said detectable labels is a radionuclide, said labeling occurs in the presence of vitamin C and optionally ethanol.

The concentration of vitamin C in the labeling reaction is between 0.1 mg/ml and 10 mg/ml, more preferably between 0.2 mg/ml and 5 mg/ml, more preferably between 0.2 mg/ml and 2.5 mg/ml. If ethanol is present, the concentration of ethanol in the labeling reaction is between 5% v/v and 15% v/v.

However, as discussed above, vitamin C has a low stability in solution and high amounts of vitamin C interfere with the radiolabeling reaction.

The inventors found that a subgroup of ascorbyl glucosides, namely 2-O-a-D- glucopyranosyl ascorbic acid (AA-2G), 2-O-p-D-glucopyranosyl ascorbic acid (AA- 2G), 5-O-a-D-glucopyranosyl ascorbic acid (AA-5G), 6-O-a-D-glucopyranosyl ascorbic acid (AA-6G), 3-O-glycosyl-L-ascorbic acid and 6-O-acyl-2-O-a-D- glucopyranosyl ascorbic acid, have a potent anti-oxidant activity, are soluble in aqueous solvents and, in contrast to vitamin C, have a high stability in solution. Their high stability in solution is caused by the fact that a glucose group protects the hydroxyl groups, which prevents the degradation of these ascorbic acid derivatives. The inventors found that, besides the excellent stabilizing properties during lyophilization, the aforementioned selected ascorbyl glucosides offer a radioprotective effect during labeling with a radionuclide, enhancing the radiochemical purity (RCP) and reducing radiolysis.

Thus, the present disclosure also provides a method for labeling the immunoglobulin single variable domain or MMR-binding fragment with one or more detectable labels, wherein at least one of said detectable labels is a radionuclide, said labeling occurs in the presence a derivative of vitamin C, wherein said derivative is an ascorbyl glucoside chosen from the list of 2-O-a-D-glucopyranosyl ascorbic acid, 2-O-p-D- glucopyranosyl ascorbic acid, 5-O-a-D-glucopyranosyl ascorbic acid, 6-O-a-D- glucopyranosyl ascorbic acid, 3-O-glycosyl-L-ascorbic acid, 6-O-acyl-2-O-a-D- glucopyranosyl ascorbic acid or a mixture thereof and optionally ethanol. If present, the concentration of ethanol in the labeling reaction is between 5% v/v and 15% v/v and the concentration of said derivative of vitamin C in the labeling reaction is between 5 mg/ml and 150 mg/ml.

In an embodiment, the immunoglobulin single variable domain or MMR-binding fragment is conjugated to a chelating agent. Chelating agents are bifunctional linkers, since they have a metal binding moiety function and also possess a chemically reactive functional group. The former provides for the sequestration of the metallic radionuclide while the latter aspect provides the requisite chemistry for covalent attachment to a targeting vector of interest, such as an antibody or antibody fragment.

The chelating agent may be any chelating agent which is effective at moderate temperatures, for example from 10-30°C, and suitably at ambient temperature, and at moderate pHs, for example of from 3-8 and at low concentrations (for example from l-10pM) and reaching acceptable yield in a relatively short time. The chelation may be achieved at moderate temperatures and in particular at ambient temperature, so that heating steps or stages may be avoided, thus simplifying the procedure and ensuring that the radioactivity of the radionuclide remains at a good level. Versatile chelating agents of this type, which are effective at neutral pHs as well as at low pH, are known in the art.

In an embodiment, the immunoglobulin single variable domain or MMR-binding fragment is coupled to a chelating agent chosen from the group of DTPA (diethylentriaminepentaacetic acid) and derivatives (including 1B4M-DTPA derivatives and CHX-A"-DTPA derivatives), DOTA (1,4,7, 10-tetraazacyclododecane-

1.4.7.10-tetraacetic acid) and derivatives (including DOTA-GA derivatives, DOTAM derivatives, DO3A and derivatives, DO2A and derivatives, CB-DO2A derivatives and DO3AM derivatives), NOTA (l,4,7-triazacyclononane-l,4,7-triacetic acid) and derivatives (including NODA derivatives, NODA-GA derivatives, NO2A derivatives, NOTAM derivatives, NOPO derivatives and TRAP derivatives), HBED (N,N-bis(2- hydroxybenzyl)ethylenediamine-N,N-diacetic acid) and derivatives (including HBED-CC derivatives, HBED-CI derivatives, HBED-CA derivatives, HBED-AA derivatives and SHBED derivatives), DEPA (7-[2-(bis-carboxymethyl-amino)-ethyl]-

4.10-bis-carboxymethyl-l,4,7,10-tetraaza-cyclododec-l-yl-acetic acid) and derivatives, picolinic acid (PA) based chelators and derivatives (including H2dedpa, H4octapa, H2azapa and HSdecapa and derivatives), HEHA (1,2,7,10,13- hexaazacyclooctadecane-l,4,7,10,13,16-hexaacetic acid) and derivatives, TETA (l,4,8,ll-tetraazacyclotetradecane-l,4,8,ll-tetraacetic acid) and derivatives (including TE2A derivatives, CB-TE2A derivatives, CB-TE1A1P derivatives, CB-TE2P derivatives, MM-TE2A derivatives and DM-TE2A derivatives), NETA ([2-(4,7-bis- carboxymethyl-[l,4,7]triazacyclononan-l-yl-ethyl]-2-carbonylmethyl-amino]- tetraacetic acid) and derivatives (including C-NETA derivatives and NE3TA derivatives), AAZTA (l,4-bis(carboxymethyl)-6-[bis(carboxymethyl)]amino-6- methylperhydro-l,4-diazepine) and derivatives, DATA (6-amino-l,4-diazepine- triacetic acid) and derivatives, TCMC (1,4,7, 10-tetraaza-l, 4,7, 10-tetra(2- carbamoylmethyl)cyclododecane) and derivatives, PCTA (3,6,9, 15-tetraazabicyclo [9.3.1]pentadeca-l(15),ll,13-triene-3,6,9-triacetic acid) and derivatives, Macropa (6-[[16-[(6-carboxypyridin-2-yl)methyl]-l,4,10,13-tetraoxa-7,16 diazacyclooctadec-7-yl]methyl]-4-isothiocyanatopyridine-2-carboxylic acid) and derivatives, THP (tris(hydroxypyridinone)) and derivates, DFO (deferoxamine) and derivatives, BCPA (N, N'-l,4-butanediylbis[3-(2-chlorophenyl)acrylamide]) and derivatives, MAG-2 (2-mercaptoacetyldiglycyl) and derivatives, MAG-3 (2- mercaptoacetyltriglycyl) and derivatives, MAS-3 (mercaptoacetyltriserine) and derivatives, HYNIC (hydrazinonicotinic acid) and derivatives and RESCA (Restrained Complexing Agent).

In a preferred embodiment, the immunoglobulin single variable domain or MMR- binding fragment are conjugated to NOTA. In another preferred embodiment, the immunoglobulin single variable domain or MMR-binding fragment are conjugated to DOTA.

In an embodiment, functional groups, such as maleimide, NCS and NHS, are added to the chelating agent in order to allow various conjugation methods. For instance, the isothiocyanate function (R-NCS) allows formation of stable thiourea bonds at alkaline pH with free amines. NHS is another example of an amine-reactive linker.

In a preferred embodiment, the radionuclide is coupled to the aforementioned chelating agent. In an embodiment, the radionuclide is coupled to the chelating agent before conjugation of the chelating agent to the biomolecule. In another embodiment, the radionuclide is coupled to the chelating agent after conjugation of the chelating agent to the biomolecule. In another or further embodiment, the antibody or fragment thereof further comprises a fluorescent moiety as detectable label.

In an embodiment, the aforementioned fluorescent moiety is selected from the group consisting of xanthene (e.g., fluorescein, rhodamine), cyanine (e.g., Cy5, Cy5.5, IRdye800CW etc), squaraines, dipyrromethene, tetrapyrrole, naphthalene, oxadiazole, naphthalene, coumarin, oxazine derivatives and fluorescent metals such as europium or others metals from the lanthanide series.

Combined use of radioactive and fluorescence signals can help strengthen in vivo medical imaging applications, such as image-guided surgery. This type of image guidance can come in 2 forms. In a first embodiment, separate radioactive and fluorescent tracers can be used, for instance for pre- and intraoperative imaging. To ensure surgical accuracy, in such a dual-tracer application one has to make sure both tracers independently allow delineation of the same lesions. Although such an approach supports the use of existing radiotracers, it is chemically extremely challenging to create fluorescent tracers that behave in an identical manner (on a molecular scale, fluorescent dyes are inherently different from radiolabels).

Said radioactive and fluorescent signature can be integrated in a single bimodal/hybrid tracer. Integration ensures colocalization of the two signatures and promotes an advanced form of symbiosis (the best of both worlds) that empowers surgeons for instance surgeons to improve intraoperative target delineation. Hybrid tracers come in many forms; not only can the biomolecule or targeting vehicle on which they are based vary from small molecules to nanoparticles (including proteins and nanocolloids), but they also may use different radionuclides (e.g., p or y emission) or fluorescent moieties (e.g., light with different wavelengths). Although each individual hybrid tracer and administration route has been designed to serve a specific purpose, conceptually all use revolves around the notion that both signatures can be used to (for instance intraoperatively) depict complementary features of the same target. Despite differences in signal intensities, there is a high level of overlap in the way multiplexing of the different imaging signatures occurs. In the context of surgical guidance, the radioactive signal allows identification and localization of a lesion by means of its radioactive signature (even in deeper tissue layers), whereas the fluorescent signal allows direct lesion visualization and delineation in exposed tissue in the surgical field or provides high-resolution pathologic identification of the tracer accumulation. Labeling of the immunoglobulin single variable domain or MMR-binding fragment thereof occurs in the presence of a labeling buffer. Also, in the case the immunoglobulin single variable domains or MMR-binding fragments thereof are lyophilized, the immunoglobulin single variable domains or MMR-binding fragments thereof should be reconstituted prior to labeling. To reconstitute the immunoglobulin single variable domain or MMR-binding fragment thereof, a labeling buffer is added to dissolve the lyophilizate such that the immunoglobulin single variable domain or MMR-binding fragment thereof is dispersed in the reconstituted formulation.

The labeling buffer can be any buffer known from the state of the art suited for this purpose. In an embodiment the lyophilized precursor sample, comprising the immunoglobulin single variable domain or MMR-binding fragment thereof, is reconstituted with a certain volume of the labeling buffer and labeled with an equal volume of a detectable label in solution.

Suitable pharmaceutically acceptable buffers include inorganic and organic buffers. Examples of inorganic buffers include phosphate buffers, such as sodium phosphate, sodium phosphate dibasic, potassium phosphate and ammonium phosphate; bicarbonate or carbonate buffers; succinate buffers such as disodium succinate hexahydrate; borate buffers such as sodium borate; cacodylate buffers; citrate buffers such as sodium citrate or potassium citrate; sodium chloride, zinc chloride or zwitterionic buffers. Examples of organic buffers include Tris(hydroxymethyl)aminomethane (Tris base) buffers, such as Tris HCI, Tris EDTA, Tris acetate, Tris phosphate or Tris glycine, 3-(N-morpholino)propanesulfonic acid (MOPS), N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), dextrose, lactose, tartaric acid, formate, arginine or acetate buffers such as ammonium, sodium or potassium acetate.

In a preferred embodiment, the labeling buffer is a phosphate, succinate, formate or an acetate buffer, such as a sodium acetate buffer.

This buffer is used to dilute or reconstitute the immunoglobulin single variable domain or MMR-binding fragment thereof prior to labeling. Acetate buffers are recognized as a substance for pharmaceutical use and human use and are thus ideal candidates to use during labeling of radiopharmaceuticals. In an embodiment the acetate buffer is a sodium acetate buffer. In an embodiment the acetate buffer is a IM sodium acetate buffer with pH 5. The current disclosure also describes a method of lyophilizing a biomolecule, such as the immunoglobulin single variable domain directed against or specifically binding to human macrophage mannose receptor (MMR) or an MMR-binding fragment thereof as described herein.

Lyophilization is commonly used in the production of pharmaceutical compounds to increase the stability of the Active Pharmaceutical Ingredient (API) by removing solvents. Lyophilization offers many advantages as it allows the processing and development of pharmaceutical compounds, otherwise unstable in solution, hence improving their shelf life. This technique can facilitate development, usage, distribution and commercialization of new drugs. It is therefore understandable that the growing market of biopharmaceuticals is associated with an increased interest in lyophilization of products for medical use. One additional advantage lyophilization offers to radiopharmaceutical precursors is the possibility of a kit development with the previously described advantages, further enhancing the practicality of these tracers and favoring their usage in clinic.

As discussed above, a concentration of vitamin C between 0.2 mg/ml and 2.5 mg/ml in the labeling reaction shows the best results in a radiolabeling study. However, vitamin C has a low stability in solution. To circumvent this problem, vitamin C may in an embodiment be co-lyophilized with the biomolecule. In a preferred embodiment, vitamin C is co-lyophilized in an amount between 1 mg and 7 mg, more preferably between 2 mg and 6 mg, such as 5 mg, with said biomolecule.

In another embodiment the labeling occurs in the presence of a vitamin C derivative, wherein a concentration of the vitamin C derivative between 5 mg/ml and 150 mg/ml in the labeling reaction shows the best results in a radiolabeling study. In an embodiment, the vitamin C derivative is co-lyophilized with the immunoglobulin single variable domain or MMR-binding fragment. In an embodiment, the amount of said vitamin C derivative in the lyophilizate is between 20 mg and 150 mg, more preferably between 20 mg and 100 mg, more preferably between 20 mg and 80 mg, such as 50 mg.

In an embodiment, additional lyophilization excipients can be added to the lyophilization solution. In an embodiment, D-mannitol, sucrose, polysorbate 80 or a combination thereof is added as additional lyophilization excipients to the lyophilization solution. The vitamin C (derivative) can however interfere with the labeling reaction. Hence, a sufficient amount of immunoglobulin single variable domain or MMR-binding fragment should be present in the labeling reaction in order to yield a sufficient amount of labeled immunoglobulin single variable domain or MMR-binding fragment.

In an embodiment, the amount of the immunoglobulin single variable domain or MMR-binding fragment in the lyophilizate is between 7.5 nmoles and 700 nmoles. In an embodiment, when the composition is intended for radiolabeling, said immunoglobulin single variable domain or MMR-binding fragment is present in the composition in a quantity of between 7.5 nmoles and 75 nmoles, more preferably of between 15 nmoles and 55 nmoles, more preferably of between 20 nmoles and 40 nmoles, such as 30 nmoles. In an embodiment, when said immunoglobulin single variable domain or MMR-binding fragment further comprises a fluorescent moiety as detectable label, said immunoglobulin single variable domain or MMR-binding fragment is present in the composition in a quantity of between 75 nmoles and 750 nmoles.

Providing such an amount of immunoglobulin single variable domain or MMR-binding fragment is able to overcome the possible interference caused by the vitamin C (derivative) on the labeling reaction.

In an embodiment, the lyophilized composition is prior to labeling with a detectable label reconstituted with a buffer. In an embodiment, the buffer comprises ethanol (EtOH). Ethanol has since long been used as co-solvent in the production of [¹⁸F]- FDG for anti-radiolytic purposes and has several interesting properties. The most relevant in this context is its ability to prevent or reduce radiolysis even further. Moreover, ethanol is low toxic for injection (at low doses), does not cause immunoreactivity issues with proteins and does not interfere with radiolabeling reactions. Additionally, ethanol has other positive properties, such as improved solubility of lipophilic compounds and can, at low concentration, even improve the stability of proteins. Finally, ethanol seems to have another remarkable, intriguing, and potentially highly valuable characteristic, namely, that it can even significantly improve labeling efficiencies of radiometals.

In an alternative embodiment, the lyophilized composition is not reconstituted with a buffer prior to labeling, but is immediately reconstituted with the radionuclide solution in a single step 'reconstitution and labeling' procedure. In this way, the reconstitution is executed by the same liquid which is added for the labeling and in which the detectable label resides. In an embodiment, the reconstitution is executed by the eluate of a radionuclide generator, such as a germanium-68/gallium-68 generator. As the ascorbic acid derivative has a pKa of 4.2, this allows buffer capacity in the ideal pH range (pH 4-5), making reconstitution with a stabilizing buffer prior to labeling unnecessary. Additionally, in the case of radiolabeling, the inventors have shown that the aforementioned ascorbic acid derivatives have some complexing capacity towards radiometals, which will prevent the formation of colloids, hereby taking over the role of the stabilizing buffer normally used for reconstitution prior to addition of the radiometal.

In a preferred embodiment, said diagnosis and/or prognosis of cardiac sarcoidosis is by non-invasive in vivo medical imaging. In another preferred embodiment, said immunoglobulin single variable domain or MMR-binding fragment thereof is used as contrast agent in non-invasive in vivo medical imaging. In a more preferred embodiment, said non-invasive in vivo medical imaging is positron-emission tomography (PET) imaging or single photon-emission computed tomography (SPECT) imaging.

In an embodiment, said immunoglobulin single variable domain has the formula

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 wherein FR1 to FR4 are framework regions and wherein CDR1 to CDR3 are complementarity determining regions. In a preferred embodiment, CDR1 comprises the amino acid sequence FSLDYYAIG (SEQ ID NO. l), CDR2 comprises the amino acid sequence CISYKGGST (SEQ ID NO.2), and CDR3 comprises the amino acid sequence GFWCYKYDY (SEQ ID NO.3). In a more preferred embodiment, said immunoglobulin single variable domain comprises a heavy chain variable domain, the amino acid sequence of which comprises the sequence 4 or the sequence 5:

QVQLQESGGGLVQPGGSLRLSCAASGFSLDYYAIGWFRQAPGKEREGISCISYKGGSTTYAD SVKGRFTISKDNAKNTAYLQMNSLKPEDTGIYSCAAGFWCYKYDYWGQGTQVTVSS (SEQ ID NO.4). DVQLQESGGGLVQPGGSLRLSCAASGFSLDYYAIGWFRQAPGKEREGISCISYKGGSTTYAD SVKGRFTISKDNAKNTAYLQMNSLKPEDTGIYSCAAGFWCYKYDYWGQGTQVTVSS (SEQ ID NO.5).

In an even more preferred embodiment, said immunoglobulin single variable domain is a VHH domain.

The immunoglobulin single variable domain or MMR-binding fragment thereof of the present invention can be administered by an appropriate route. Suitable routes of administration include, but are not limited to, orally, intraperitoneally, subcutaneously, intramuscularly, topically e.g. transdermally, rectally, sublingualis, intravenously, buccally, or inhalationally. In a preferred embodiment, the immunoglobulin single variable domain or MMR-binding fragment thereof is administered intravenously.

In an embodiment, the immunoglobulin single variable domain or MMR-binding fragment thereof is administered at a dose between 1 pg and 1000 pg, more preferably between 10 pg and 500 pg, more preferably between 10 pg and 100 pg, more preferably between 20 pg and 70 pg, more preferably between 40 pg and 60 pg, such as 50 pg.

Dose and route of administration will in general depend on the nature of the disease (type, grade, and stage of the tumor etc.) and the patient (constitution, age, gender etc.), and will be determined by the skilled medical expert responsible for the diagnosis. With respect to the possible doses for the compounds which are described below, it is clear that the medical expert responsible for the treatment will carefully monitor whether any dose-limiting toxicity or other severe side effects occur and undertake the necessary steps to manage those.

Generally, for diagnostic, prognostic and predictive use, the immunoglobulin single variable domain directed against or specifically binding to human macrophage mannose receptor (MMR) or an MMR-binding fragment thereof as envisaged herein may be formulated as a preparation or compositions comprising at least one immunoglobulin single variable domain directed against or specifically binding to human macrophage mannose receptor (MMR) or an MMR-binding fragment thereof as envisaged herein and at least one acceptable carrier, diluent or excipient and/or adjuvant, and optionally one or more further polypeptides and/or compounds. Such a formulation may be suitable for intraperitoneal, intravenous or other administration. Thus, the immunoglobulin single variable domain directed against or specifically binding to human macrophage mannose receptor (MMR) or an MMR- binding fragment thereof as envisaged herein and/or the compositions comprising the same can for example be administered systemically, locally or topically to the tissue or organ of interest, and preferably intraperitoneally or intravenously, depending on the specific pharmaceutical formulation or composition to be used.

The dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form must be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof.

The amount of the immunoglobulin single variable domain directed against or specifically binding to human macrophage mannose receptor (MMR) or an MMR- binding fragment thereof as envisaged herein required for use in diagnosis and/or prognosis of cardiac sarcoidosis may vary not only with the particular immunoglobulin single variable domain directed against or specifically binding to human macrophage mannose receptor (MMR) or an MMR-binding fragment thereof selected but also with the route of administration and will be ultimately at the discretion of the attendant physician or clinician. Also the dosage of the immunoglobulin single variable domain directed against or specifically binding to human macrophage mannose receptor (MMR) or an MMR-binding fragment thereof envisaged herein may vary.

In particular, the immunoglobulin single variable domain directed against or specifically binding to human macrophage mannose receptor (MMR) or an MMR- binding fragment thereof as envisaged herein will be administered in an amount which will be determined by the medical practitioner. Typically, for each disease indication an optimal dosage will be determined specifying the amount to be administered per kg body weight, per m² body surface area or for defined patient categories. The clinician will generally be able to determine a suitable dose, depending on the factors mentioned herein. It will also be clear that in specific cases, the clinician may choose to deviate from these amounts, for example on the basis of the factors cited above and his expert judgment.

Useful dosages of the immunoglobulin single variable domain directed against or specifically binding to human macrophage mannose receptor (MMR) or an MMR- binding fragment thereof as envisaged herein can be determined by determining their in vitro activity, and/or in vivo activity in animal models.

In certain embodiments, the present invention provides a radiolabeled immunoglobulin single variable domain directed against or specifically binding to human macrophage mannose receptor (MMR) or an MMR-binding fragment thereof as disclosed herein for use in the diagnosis and/or prognosis of cardiac sarcoidosis by administering to a subject in need thereof the radiolabeled immunoglobulin single variable domain directed against or specifically binding to human macrophage mannose receptor (MMR) or an MMR-binding fragment thereof at a dose of between 1 pg and 1000 pg of immunoglobulin single variable domain directed against or specifically binding to human macrophage mannose receptor (MMR) or an MMR- binding fragment thereof. In further particular embodiments, the present invention provides a radiolabeled immunoglobulin single variable domain directed against or specifically binding to human macrophage mannose receptor (MMR) or an MMR- binding fragment thereof as disclosed herein for use in the diagnosis and/or prognosis of cardiac sarcoidosis by administering to a subject in need thereof the radiolabeled immunoglobulin single variable domain directed against or specifically binding to human macrophage mannose receptor (MMR) or an MMR-binding fragment thereof at a dose of between 10 pg and 500 pg of radiolabeled immunoglobulin single variable domain directed against or specifically binding to human macrophage mannose receptor (MMR) or an MMR-binding fragment thereof, such as in particular between 10 and 100 pg of radiolabeled immunoglobulin single variable domain directed against or specifically binding to human macrophage mannose receptor (MMR) or an MMR-binding fragment thereof, preferably between 20 and 70 pg of radiolabeled immunoglobulin single variable domain directed against or specifically binding to human macrophage mannose receptor (MMR) or an MMR- binding fragment thereof, such as between 40 and 60 pg of radiolabeled immunoglobulin single variable domain directed against or specifically binding to human macrophage mannose receptor (MMR) or an MMR-binding fragment thereof, more preferably but not limited to about 50 pg of radiolabeled immunoglobulin single variable domain directed against or specifically binding to human macrophage mannose receptor (MMR) or an MMR-binding fragment thereof. In certain embodiments, the present invention provides a radiolabeled immunoglobulin single variable domain directed against or specifically binding to human macrophage mannose receptor (MMR) or an MMR-binding fragment thereof as disclosed herein for use in the diagnosis and/or prognosis of cardiac sarcoidosis by administering to a subject in need thereof the radiolabeled immunoglobulin single variable domain directed against or specifically binding to human macrophage mannose receptor (MMR) or an MMR-binding fragment thereof at a dose of between 100 pg and 200 pg of immunoglobulin single variable domain directed against or specifically binding to human macrophage mannose receptor (MMR) or an MMR-binding fragment thereof, preferably but not limited to about 100 pg of radiolabeled immunoglobulin single variable domain directed against or specifically binding to human macrophage mannose receptor (MMR) or an MMR-binding fragment thereof.

In further particular embodiments, the radiolabeled immunoglobulin single variable domain directed against or specifically binding to human macrophage mannose receptor (MMR) or an MMR-binding fragment thereof as disclosed herein have a specific activity of from about 1 to about 1000 mCi/mg, or from about 5 to about 250 mCi/mg, preferably about 20 to about 50 mCi/mg, preferably about 15 to about 45 mCi/mg, preferably about 25 to about 35 mCi/mg, and most preferably 30 mCi/mg.

Methods for the calculation of (mean) effective dose to be expected in humans, based on probe biodistribution data in humans are known to the skilled person and may include software programs, such as for example but not limited to OLINDA 1.0 software. Assuming identical biodistribution of the same compound, labeled with different isotopes, a (mean) effective dose can be calculated for multiple radioisotopes.

In particular the present inventors have found that the radiolabeled immunoglobulin single variable domain directed against or specifically binding to human macrophage mannose receptor (MMR) or an MMR-binding fragment thereof, can be used to effectively diagnose and/or prognose cardiac sarcoidosis, at a calculated mean effective dose of between 0.002 and 0.1 mSv/MBq, more preferably between 0.01 and 0.08 mSv/MBq. Estimated mean effective dose calculations based on the biodistribution data of the ⁶⁸Ga-labeled radiolabeled immunoglobulin single variable domain directed against or specifically binding to human macrophage mannose receptor (MMR) or an MMR- binding fragment thereof obtained from the first-in-human phase I trial, when using different radio-isotopes as parameter for the OLINDA calculations yield the following for different radio-isotopes:

For 68-Ga: between 0.01 and 0.08 mSv/MBq, more preferably between 0.03 and 0.05 mSv/MBq, and more preferably 0.0427 mSv/MBq.

For 124-1: between 0.01 and 0.08 mSv/MBq, more preferably between 0.02 and 0.04 mSv/MBq, and more preferably 0.0304 mSv/MBq.

For 131-1: between 0.001 and 0.02 mSv/MBq, more preferably between 0.01 and 0.02 mSv/MBq and more preferably 0.0188 mSv/MBq.

In certain embodiments, diagnosis of cardiac sarcoidosis is achieved by administering radiolabeled immunoglobulin single variable domain directed against or specifically binding to human macrophage mannose receptor (MMR) or an MMR- binding fragment thereof as disclosed herein to a subject in need thereof, characterized in that the radiolabeled immunoglobulin single variable domain directed against or specifically binding to human macrophage mannose receptor (MMR) or an MMR-binding fragment thereof has a calculated mean effective dose of between 0.001 and 0.05 mSv/MBq in a subject, such as but not limited to a calculated mean effective dose of between 0.02 and 0.05 mSv/MBq, more preferably between 0.02 and 0.04 mSv/MBq, most preferably between 0.03 and 0.05 mSv/MBq.

The subject or patient to which the immunoglobulin single variable domain directed against or specifically binding to human macrophage mannose receptor (MMR) or an MMR-binding fragment thereof described herein may be administered can be any warm-blooded animal, but is in particular a mammal, and more in particular a human suffering from, or at risk of, cardiac sarcoidosis.

The detection efficiency and specificity of the VHH sequences or functional fragments thereof and polypeptides described herein, and of compositions comprising the same, can be tested using any suitable in vitro assay, cell-based assay, in vivo assay and/or animal model known per se, or any combination thereof, depending on the specific disease or disorder involved. Suitable assays and animal models will be clear to the skilled person.

The skilled person will generally be able to select a suitable in vitro assay, cellular assay or animal model to test the VHH sequences or functional fragments thereof and polypeptides described herein for binding to the tumor-specific molecule; as well as for their diagnostic efficiency in respect of cardiac sarcoidosis.

In an embodiment, the immunoglobulin single variable domain or MMR-binding fragment thereof is formulated in a pharmaceutical composition. In a preferred embodiment, said pharmaceutical composition further comprises a buffer selected from the group consisting of phosphate buffers, such as sodium phosphate, sodium phosphate dibasic, potassium phosphate and ammonium phosphate, bicarbonate or carbonate buffers, succinate buffers such as disodium succinate hexahydrate, borate buffers such as sodium borate, cacodylate buffers; citrate buffers such as sodium citrate or potassium citrate, sodium chloride, zinc chloride or zwitterionic buffers, Tris(hydroxymethyl)aminomethane (Tris base) buffers, such as Tris HCI, Tris EDTA, Tris acetate, Tris phosphate or Tris glycine, 3-(N-morpholino)propanesulfonic acid (MOPS), N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), dextrose, lactose, tartaric acid, formate, arginine, and acetate buffers such as ammonium, sodium or potassium acetate. In a more preferred embodiment, said pharmaceutical composition further comprises vitamin C or a derivative thereof. In another more preferred embodiment, said pharmaceutical composition further comprises ethanol.

In a second aspect, the present disclosure provides methods for diagnosing and/or prognosing cardiac sarcoidosis.

In a preferred embodiment, said method comprising the steps of administering to a subject an immunoglobulin single variable domain directed against or specifically binding to human macrophage mannose receptor (MMR) or an MMR-binding fragment thereof, wherein said immunoglobulin single variable domain or MMR- binding fragment thereof is coupled to a detectable label. In a more preferred embodiment, said diagnosis and/or prognosis is by non-invasive in vivo medical imaging. In an even more preferred embodiment, said non-invasive in vivo medical imaging is positron-emission tomography (PET) imaging or single photon-emission computed tomography (SPECT) imaging. In a preferred embodiment, said detectable label is a radionuclide. In a more preferred embodiment, the radionuclide is selected from the group consisting of fluor 18 (¹⁸F), scandium 44 (⁴⁴Sc), lutetium 177 (¹⁷⁷Lu), zirconium 89 (⁸⁹Zr), indium 111 (^mln), yttrium 90 (⁹⁰Y), copper 61 (⁶¹Cu), copper 64 (⁶⁴Cu), actinium 225 (²²⁵Ac), bismuth 213 (²¹³Bi), gallium 67 (⁶⁷Ga), gallium 68 (⁶⁸Ga), technetium 99m (^99mTc), iodine 123 (¹²³I), iodine 124 (¹²⁴I), iodine 125 (¹²⁵I), iodine 131 (¹³¹I). In an even more preferred embodiment, the radionuclide is gallium 68 (⁶⁸Ga).

In a fourth aspect, the present disclosure provides the use of a kit comprising an immunoglobulin single variable domain directed against or specifically binding to human macrophage mannose receptor (MMR) or an MMR-binding fragment thereof and one or more detectable labels for the production of a diagnostic for the detection of cardiac sarcoidosis.

Developing kits brings important advantages regarding the Chemistry, Manufacturing 8<. Controls (CMC) and economical aspects, as they allow standardized and simplified preparation protocols and the ability for any center to prepare the biopharmaceutical with minimal GMP license. As such, they allow multi-center studies in development phase and international distribution and commercialization upon market approval.

In addition, in the specific case a radionuclide is used as a detectable label, most of these resulting radiotracers have a relatively short half-life and consequently have to be produced in situ, for example in the radiopharmacy section of the relevant hospital, under sterile conditions. Some hospitals have difficulty with this if they do not have specialist radiochemistry laboratories and therefore their ability to offer treatments such as PET may be restricted. To solve this problem, so-called 'cold kits' have been produced which are relatively simple to use and do not require significant handling of the radionuclide.

The invention provides a kit comprising one or more of the aforementioned immunoglobulin single variable domain directed against or specifically binding to human macrophage mannose receptor (MMR) or an MMR-binding fragment thereof. In an embodiment, the kit further comprises a stabilizing buffer. In an embodiment, the kit is used in the production of a diagnostic for the detection of cardiac sarcoidosis. The stabilizing buffer can be any buffer known from the state of the art suited for this purpose. In an embodiment the lyophilized precursor sample, comprising the immunoglobulin single variable domain directed against or specifically binding to human macrophage mannose receptor (MMR) or an MMR-binding fragment thereof and a vitamin C derivative, is reconstituted with a certain volume of the stabilizing buffer and labeled with an equal volume of a detectable label in solution.

Suitable pharmaceutically acceptable buffers for incorporation into the kit include inorganic and organic buffers. Examples of inorganic buffers include phosphate buffers, such as sodium phosphate, sodium phosphate dibasic, potassium phosphate and ammonium phosphate; bicarbonate or carbonate buffers; succinate buffers such as disodium succinate hexahydrate; borate buffers such as sodium borate; cacodylate buffers; citrate buffers such as sodium citrate or potassium citrate; sodium chloride, zinc chloride or zwitterionic buffers. Examples of organic buffers include Tris(hydroxymethyl)aminomethane (Tris base) buffers, such as Tris HCI, Tris EDTA, Tris acetate, Tris phosphate or Tris glycine, 3-(N-morpholino)propanesulfonic acid (MOPS), N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), dextrose, lactose, tartaric acid, formate, arginine or acetate buffers such as ammonium, sodium or potassium acetate.

In an embodiment of the kit, the stabilizing buffer is chosen from an acetate, phosphate, succinate, formate or a HEPES buffer.

This buffer is used to dilute or reconstitute the immunoglobulin single variable domain directed against or specifically binding to human macrophage mannose receptor (MMR) or an MMR-binding fragment thereof prior to labeling. Acetate buffers are recognized as a substance for pharmaceutical use and human use and are thus ideal candidates to use during labeling of biopharmaceuticals. In an embodiment the acetate buffer is a sodium acetate buffer. In an embodiment the acetate buffer is a 0.5 M - IM sodium acetate buffer with pH 5.

In an embodiment of the kit, said buffer comprises ethanol at a concentration between 10% v/v and 30% v/v. In an embodiment, the ethanol concentration in the stabilizing buffer is between 12% v/v and 28% v/v, more preferably between 15% v/v and 25% v/v, such as 20% v/v. In an embodiment of the kit, the immunoglobulin single variable domain directed against or specifically binding to human macrophage mannose receptor (MMR) or an MMR-binding fragment thereof is coupled to NOTA or DOTA as chelating agent.

EXAMPLES

EXAMPLE 1 - IN VIVO IMAGING OF CARDIAC SARCOIDOSIS BY MEANS OF A ⁶⁸GA-LABELED SINGLE DOMAIN ANTIBODY FRAGMENT DIRECTED AGAINST HUMAN MACROPHAGE MANNO ACCORDING TO THE CURRENT INVENTION: A PHASE

Hypothesis

There is emerging evidence that M2 polarization plays a role in sarcoidosis (little is known in cardiac sarcoidosis (CS)) and this anti-inflammatory/pro-fibrotic pathway is responsible for fibrosis. There is also ample evidence that myocardial fibrosis or scar (as evidenced by cardiac magnetic resonance (CMR) imaging) is associated with increased odds of both all-cause mortality and arrhythmogenic events. Imaging sarcoid granulomas by targeting the pro-inflammatory and anti-inflammatory/pro- fibrotic pathway using both ¹⁸F-FDG positron emission tomography/computed tomography (PET/CT) and a ⁶⁸Ga-NOTA-anti-MMR single domain antibody fragment (sdAb) for PET/CT according to the current invention may provide valuable insights in M1/M2 polarization balance during the disease course and may guide treatment.

Brief summary

A prospective imaging study investigating the ability of a ⁶⁸Ga-NOTA-anti-MMR sdAb for PET/CT to detect cardiac involvement in patients with endomyocardial biopsy proven cardiac sarcoidosis (CS) or suspected CS according to HRS consensus recommendation (2014).

Description

Prospective study including 15 patients. All patients will undergo ⁶⁸Ga-NOTA-anti- MMR PET/CT according to the current invention, ¹⁸F-FDG PET/CT and ¹³N-NH3 PET/CT as well as cardiac magnetic resonance (CMR) imaging (late gadolinium enhancement, T1 and T2 mapping) within 1-week time interval. At 6 months the imaging studies will be repeated. Interventions

Included patients (n = 15) will undergo whole-body and cardiac/respiratory gated chest PET/CT acquisition for ⁶⁸Ga-NOTA-anti-MMR and ¹⁸F-FDG PET/CT, cardiac/respiratory gated chest ¹³N-NH3 PET/CT as well as cardiac magnetic resonance imaging. In addition, a blood sample (at time of ⁶⁸Ga-NOTA-anti-MMR PET/CT) will be taken to assess inflammatory activity/biomarkers (ESR and CRP), angiotensin converting enzyme and plasma cytokine profile (IFN-y, TNF-a, IL-lb, IL- 6, IL-12 for Ml and IL-4, IL-10, IL-13, TGF-p and CD206 for M2). Immunohistochemistry will be done on cardiac biopsies for CD68 (Ml) and CD206/MMR (M2) to determine M1/M2 ratio. Multimodal imaging and biochemical evaluation will be performed 6 months later/after treatment initiation to evaluate disease activity and temporal changes. Figure 1 depicts a schematic overview of the Phase II clinical trial in patients with cardiac sarcoidosis using amongst others the ⁶⁸Ga-NOTA-anti-MMR sdAb for use according to the current invention.

Outcome measures

Primary outcome measures:

1. Sensitivity of a ⁶⁸Ga-NOTA-anti-MMR sdAb for PET/CT according to the current invention versus CMR and combined ¹⁸F-FDG/¹³N-NH3 PET/CT to detect cardiac involvement

2. Specificity of a ⁶⁸Ga-NOTA-anti-MMR sdAb for PET/CT according to the current invention versus CMR and combined ¹⁸F-FDG/¹³N-NH3 PET/CT to detect cardiac involvement

Secondary outcome measures:

1. Sensitivity of a ⁶⁸Ga-NOTA-anti-MMR sdAb for PET/CT according to the current invention versus ¹⁸F-FDG PET/CT for detecting extracardiac lesions

2. Correlation of ⁶⁸Ga-NOTA-anti-MMR PET/CT uptake (qualitative and quantitative) according to the current invention with CD206 (MMR) expressing macrophages on EMB biopsies as assessed by immunohistochemistry

3. Comparison between a ⁶⁸Ga-NOTA-anti-MMR sdAb for PET/CT according to the current invention and ¹⁸F-FDG PET/CT during time course/treatment

4. Physician confidence in study interpretation: survey will be performed to ask physician's confidence level to interpret the PET/CT images 5. Patient satisfaction: patient surveys will be performed to evaluate if patients preferred ⁶⁸Ga-NOTA-anti-MMR PET/CT study protocol according to the current invention or ¹⁸F-FDG PET/CT-¹³N-NH3 PET/CT study protocol

6. Interobserver reproducibility of assessment of ⁶⁸Ga-NOTA-anti-MMR PET/CT according to the current invention and ¹⁸F-FDG PET/CT-¹³N-NH3PET/CT in a fully blinded setting.

Eligibility

Inclusion criteria

• All patients > 18 years at time of imaging

• Compliance with study protocol and provide written informed consent

• Patients with endomyocardial biopsy proven cardiac sarcoidosis

• Patients with suspected CS according to HRS consensus recommendation (2014)

Exclusion criteria

• Pregnancy or breastfeeding

• Contra-indication for CMR (metallic implants, non-MRI compliant pace-maker or other cardiac device, claustrophobia, history of renal insufficiency)

• Recent or current immunosuppressive treatment

• Contraindication to the cardiac preparation diet for ¹⁸F-FDG PET/CT

EXAMPLE 2 - BIODISTRIBUTION, DOSIMETRY AND SAFETY OF A ⁶⁸GA- LABELED SINGLE DOMAIN ANTIBODY FRAGMENT DIRECTED AGAINST HUMAN MACROPHAGE MANNOSE RECEPTOR (MMR) PRODUCED FROM A LYOPHILIZED KIT FORMULATION FOR USE ACCORDING TO THE CURRENT INVENTION: COMPARABILITY WITH THE SINGLE DOMAIN ANTIBODY FRAGMENT PRODUCED FROM LIQUID FORMULATION.

Hypothesis

The production process and formulation of GMP grade lyophilized single domain antibody fragment (sdAb) directed against human macrophage mannose receptor (MMR) is different from the initial production process and formulation of the GMP grade 'liquid' sdAb used in the initial Phase I academic clinical trial and the Phase II study described above. From regulatory standpoint these diagnostic drug products are different although the drug substance is biologically identical (same amino acid sequence). This requires a new Phase I study in 6 subjects to show the safety, biodistribution and dosimetry in human subjects. Data will be compared to the Phase I study results using the ⁶⁸Ga-sdAb directed against human macrophage mannose receptor (MMR) produced from liquid formulation.

Description

Healthy volunteers (n = 6) will be included. All subjects will undergo one ⁶⁸Ga-NOTA- anti-MMR sdAb tracer injection followed by 3 whole body PET/CT scans.

Interventions

Included subjects (n = 6) will undergo whole-body ⁶⁸Ga-NOTA-anti-MMR PET/CT at three consecutive time points post injection (lh, 2h, 4h). Blood samples will be taken for safety analysis and PK determination.

Primary objectives:

To evaluate the human safety and tolerability, biodistribution and dosimetry of ⁶⁸Ga- sdAb directed against human macrophage mannose receptor (MMR) produced from lyophilized formulation.

Eligibility

Inclusion criteria

• All subjects > 18 years

• Compliance with study protocol and provide written informed consent

• Healthy subjects based on medical history, clinical examination, ECG, haematology and biochemistry

• Women of child-bearing potential: non-pregnant and use of adequate and medical approved contraception method to avoid pregnancy for at least 1 month before enrollment through 1 month after dosing and they should have a negative serum pregnancy test within 24 hrs prior to activity administration

Exclusion criteria

• Active or chronic medical illness

• Drugs or substance abuse

• Chronic medical treatment at time of scan

• Current participation in other clinical trials EXAMPLE 3 - PREPARATION OF A LABELED NANOBODY DIRECTED TO MMR

Material and Methods

All commercially obtained chemicals were of analytic grade. The recombinant anti- MMR sdAb-proteins were produced without terminal tags by the VIB Protein Service Facility in Pichia pastoris and were formulated in PBS during the final batch purification. p-SCN-Bn-NOTA was purchased from Macrocyclics (Macrocyclics, Inc., Plano, TX, USA). ⁶⁸Ga was obtained from a ⁶⁸Ge/⁶⁸Ga Galli EoTM generator (IRE, Belgium). High purity water (TraceSELECT™, for trace analysis (Riedel-de Haen), Honeywell) and ethanol (Ethanol ENSURE®, Ph..Eur., Merck, Darmstadt, Germany) were used in the preparation of any buffer or solution. High grade ascorbic acid (99.7

- 100.5%, puriss. p.a., Ph.Eur., Sigma-Aldrich, St. Louis 63103, MO, USA), gentisic acid (99%, Acros Organics, part of Thermo Fisher Scientific, Geel, Belgium), Polyvinylpyrrolidone (average MW 10000 g/mol, Sigma-Aldrich, St. Louis, MO, USA) sucrose (> 99.5%, Sigma-Aldrich, St. Louis, MO, USA), D-mannitol (> 98%, Sigma- Aldrich, St. Louis, MO, USA) and polysorbate 80 (Ph.Eur., Aca Pharma, Nazareth, Belgium) were used in the respective buffer/solution preparation.

Conjugation of p-SCN-Bn-NOTA to sdAb protein sdAb proteins (Anti-MMR: 10 - 16 mg, 0.79 - 1.26 pmol) were buffer-exchanged to 0.5M sodium carbonate/0.15M NaCI buffer (Sodium carbonate anhydrous - Sodium hydrogen carbonate - Sodium Chloride, VWR Chemicals, Leuven, Belgium), pH 8.8

- 8.9, using PD-10 size exclusion disposable columns (GE Healthcare, Buckinghamshire, UK). Protein solution (2.2 - 2.4 mg/ml) was added to a thirty-fold molar excess p-SCN-Bn-NOTA. After 2 h incubation at room temperature (RT), the NOTA-sdAb protein solution was concentrated, if necessary, with Vivaspin 2 concentrator (MW cut-off 5kDa) (Sartorius Stedim Lab, Stonehouse, UK) and loaded on a SEC column. The collected fractions containing the monomeric NOTA-sdAb protein were pooled and the solution was passed through a 0.22 pm - 13mm filter (Millex, Merck Millipore, Tullagreen Carrigtwohill County Cork, Ireland). The protein concentration is determined by UV absorption at 280 nm (NOTA-anti-MMR sdAb: E = 40660 M’Tcrrr¹, MW = 13130 g/mol).

Preparation of [⁶⁸Ga ]Ga-NOTA -sdAb

The NOTA-sdAb precursor sample (100 pg or 200 pg, as specified) was first diluted or reconstituted with 1.1 ml (unless stated otherwise) of the respective IM NaOAc buffer (Sodium acetate trihydrate, > 99.5%, puriss. p.a., Ph.Eur., Sigma-Aldrich Chemie, Steinhelm, Germany - Acetic acid, > 99.8%, puriss. p.a., Ph.Eur., Sigma- Aldrich Chemie, Steinhelm, Germany) pH 5, after which the full ⁶⁸Ga eluate (1-1.1 ml) was added. In case of higher radiolabeling volumes, the ⁶⁸Ga eluate was further diluted accordingly with 0.1N HCI (Hydrochloric acid, > 37% puriss. p.a., Ph.Eur., Sigma-Aldrich Chemie, Steinhelm, Germany). The sample was incubated for 10 minutes at RT, filtered, where specified, and then analyzed for radiochemical purity by iTLC and SEC (SEC: [⁶⁸Ga]Ga-NOTA-sdAb Rt = 4.6 min, a = 0.10 min; ⁶⁸Ga- citrate Rt = 7.6 min, o = 0.34 min; Radiolytic product Rt = 8.3, o = 0.25 min; iTLC- SG: [⁶⁸Ga]Ga-NOTA-sdAb Rf = 0.05 o = 0.01, Radiolytic product Rf = 0.72, o = 0.08 ⁶⁸Ga-citrate Rf = 1.13, o = 0.06).

Chromatographic Analysis

Size exclusion chromatography (SEC) purification of NOTA-sdAb was conducted on an NGC Chromatography system (Bio-Rad Laboratories, USA) using a Superdex 30 pg HiLoad 16/600 column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) at a flow rate of 1 ml/min flow rate (mass > 6 mg) or a Superdex Peptide 10/300GL column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) at a flow rate of 0.5 ml/min (mass < 6 mg) and 0.1M sodium acetate pH 7 (Sodium acetate trihydrate, > 99.5%, puriss. p.a., Ph.Eur., Sigma-Aldrich Chemie, Steinhelm, Germany) as mobile phase. The latter was also used for quality control of NOTA-sdAbs.

Quality control of [⁶⁸Ga]Ga-NOTA-sdAbs was performed on a Hitachi Chromaster Chromatography system (VWR, Leuven, Belgium) using SEC on a Superdex Peptide 3.2/300 column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) at a flow rate of 0.15 ml/min flow rate and 0.02 M PBS/0.28M NaCI pH 7.4 (PBS Tablets, Merck, Darmstadt, Germany) as mobile phase, and by instant thin layer chromatography (iTLC) on silica gel (SG) paper (Agilent Technologies, Folsom, CA, USA) with 0.1 M sodium citrate pH 5 (Citric acid, trisodium salt, dihydrate - Citric acid monohydrate, Acros Organics, part of Thermo Fisher Scientific, Geel, Belgium) as mobile phase. The iTLC strips were measured via a miniGita Single TLC-scanner (Elysia-Raytest, Belgium). pH

The pH of solution was measured with a pH electrode Blueline 14 on a Lab 855 digital pH meter (SI Analytics, Mainz, Germany). Measurement of radiolabeling solutions was measured after decay (typically the next day). The meter is calibrated once a month with 3 calibration solutions at pH 4.01, 6.87 and 9.18 (SI Analytics, Mainz, Germany).

Surface Plasmon Resonance

Surface Plasmon Resonance (SPR) was performed on a Biacore T200 (GE Healthcare) system. Briefly, a CM5 chip was coated with either recombinant hMMR via 1-ethyl- 3-(-3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) chemistry. The affinity was determined by flowing different concentrations of precursor over the immobilized protein. The obtained curves were fitted with a 1 : 1 sdAb:antigen binding model to calculate the binding parameters. A reference sample containing anti-MMR-(HIS)6 sdAb, stored at -20°C, was added during each run.

SDS-PAGE

SDS-PAGE was performed on NOVEX Wedgewell 16% 10-well gel (Thermo Fischer Scientific, Carlsbad, CA, USA), where 10 and 2 pg of NOTA-sdAb was loaded in both reducing and non-reducing conditions. The gel was run at 80V for 10 minutes, then at 150V for 65, after which a Coomassie Blue staining was performed for detection. Gels were visualized with the Amersham 680RGB Imager (GE Healthcare BioSciences AB, Uppsala, Sweden) and analyzed via the GE ImageQuant TL ID v 8.2.0 analysis software.

Osmolality

The osmolality of the formulations was measured using an Advanced® MicroOsmometer (Model 3300, Advanced Instruments Inc., Norwood, MA, US) based upon the freezing point depression method. Calibration of the device was performed using Clinitrol™ 290 reference solution (Advanced Instruments Inc., Norwood, MA, US). As the osmolality of some formulations was higher than the upper range value of 2000 mOsm/kg, all measured samples were diluted (1 : 1) with milliQ water and the result was multiplied by two. The measurements were conducted in triplicate (on 20- pL aliquots) and mean values were reported.

Particle Size Analysis

Dynamic Light Scattering (DLS) was applied to evaluate the presence of particles in the formulations. Measurements were conducted in triplicate at 25°C using a Zetasizer Nano ZS apparatus (Malvern Instruments Ltd., Mavern, UK) with attenuator index 11, i.e., 100% transmission of the light through the sample. Results

1. Ascorbic acid

Ascorbic acid is being tested for its compatibility with ⁶⁸Ga radiolabeling and as potential alternative buffer system to the current sodium acetate buffer.

1.1. Compatibility test

AA was tested for radiolabeling compatibility by performing a radiolabeling with different concentrations in the buffer. The RCP was analyzed 10 min and 3h post labeling.

The results showed high and comparable RCP with increasing concentrations of AA, suggesting a good compatibility of AA with the ⁶⁸Ga radiolabeling.

1.2. AA buffer system

Based on the positive outcome of the AA compatibility test, we assessed the potential of using AA as alternative buffer system to the acetate buffer. To this purpose, a 0.5M ascorbic acid buffer system pH 5 was tested for ⁶⁸Ga radiolabeling. RCP was analyzed 10 min post labeling.

Although the 0.5M AA buffer system could successfully buffer the HCI solution from the generator in a 1: 1 ratio (end pH ~ 4.5), remarkably, and in contrast to the first compatibility testing, almost no incorporation of ⁶⁸Ga ions in the NOTA chelator occurred, suggesting that the AA at such high concentration does interact with the ⁶⁸Ga ions.

1.3. Ascorbic acid stability testing

To confirm the compatibility of AA at 5 mg/ml with the ⁶⁸Ga radiolabeling, 3 independent radiolabelings were performed. RCP was analyzed 10 min post labeling. However, these provided inconsistent results.

A factor of variability, not taken into account, was the dissolution time of the AA. To investigate the influence of the dissolution time on the RCP, two additional radiolabelings were performed, where the incubation time of AA was varied. In a first labeling, the labeling was carried out within 5 minutes after dissolution, while for a second labeling the AA was incubated overnight (approximately 12 hours) in the sodium acetate buffer at 2 - 8°C. A massive decrease in labeling efficiency was observed when leaving the buffer with the dissolved ascorbic acid resting overnight. From this experiment, we confirmed that the ascorbic acid can influence the radiolabeling and that, more specifically, it is the time the AA is dissolved in solution that greatly affects the interference with the radiolabeling. Oxidation of AA over time might induce a form of AA with increased chelating capacity towards ⁶⁸Ga ions.

2. Ethanol

2.1. Compatibility test

In a first test, two labelings were performed to assess the compatibility of ethanol with the radiolabeling. One labeling was performed with standard IM NaOAc pH 5 buffer, while the second was performed with IM NaOAc/20%EtOH pH 5 buffer. RCP was analyzed 10 min post labeling.

The RCP of both solutions was > 99% after 10 minutes, showing that ethanol is compatible with the ⁶⁸Ga radiolabeling.

2.2. High activity test

A first high activity test (starting activties > lGBq) was performed with different amounts of ethanol (expressed as VEtOH/Vbuffer%) on lyophilized samples, to assess the efficiency of ethanol as radioprotectant and compatbility with additional excipients from the freeze-drying formulation (Table 1).

These results seem to indicate a direct correlation between the rad io protectant effect and the amount of ethanol. The difference in RCP between the lyophilized and the reference non-lyophilized samples is due to a difference in mass caused by an erroneous measurement of concentration by UV spectrometry.

3. In-depth compatibility assessment of ethanol

In this next section a more in-depth compatibility assessment is presented for ethanol to verify the impact of ethanol on the protein profiling and functionality.

3.1. sdAb protein compatibility testing

SDS-PAGE was performed for the MMR base protein, where the protein was exposed to varying amounts of ethanol to estimate which maximal ethanol content (up to 60 v/v%)) could be used without causing protein-aggregation (Table 2). A first SDS- PAGE was performed within 30 minutes of preparing the samples, where, at any ethanol content, no precipitation upon visual inspection could be observed nor aggregation, as a single, major band was detected between 10 and 15 kDa, corresponding with monomeric sdAb (Molecular weight MMR sdAb = 12678 Da; Molecular weight HER2 sdAb = 12628 Da). A second gel was run the next day, while having stored samples overnight in the fridge at 2 - 8 °C. Here, visible precipitation was observed upon inspection of the samples for the MMR sdAb starting at 50% ethanol content. Surprisingly, however, no aggregation or precipitation could be observed on the gel, while again a single-major band was detected between 10 and 15 kDa at all ethanol contents.

The observed precipitation could be reversible upon dilution with sample buffer (samples were diluted at least 1 :2), upon heating of the samples at 95°C or by the combination of both.

3.2. Compatibility confirmation test

To confirm that ethanol contents of up to 40% did not cause aggregation/precipitation upon radiolabeling, the radiolabeling solutions were filtered through a 0.22 pm filter. The experiment was repeated in triplicate for each ethanol content.

The remaining activity on filters is presented as % compared to the initial activity in the vial minus the remaining activity in the vial after uptake of the solution, all decay corrected to timepoint of activity measurement of the solution after 10 minutes of incubation (Table 3).

Carrying out a radiolabeling with 40% ethanol in the buffer shows a high precipitation of the compound. More than 65% of the activity remained on the filter for the NOTA- MMR compound. At a 30% ethanol content, low precipitation could still be observed, while no precipitation is observed at a 20% ethanol content. As such, the ethanol content was set to 20% V(EtOH)/V(buffer)% in the IM NaOAc radiolabeling buffer (which results in 10% ethanol content upon radiolabeling) for further development and testing.

Noteworthy, since 40% ethanol causes precipitation of the protein, it is likely that radiolytic product also precipitates at such high V/V% ethanol. This might in turn cause a false readings of analytic methods, such as the iTLC, as radiolytic product would remain at the application point and be measured as intact compoud (cf. section 2.3.2)

3.3. Precursor functionality testing

To investigate the effect of ethanol on the functionality of the precursor, NOTA-sdAb samples were diluted in a 20% ethanol/O.lM NaOAc solution and tested for affinity via Surface Plasmon Resonance (SPR) (Table 4). The affinity (a measurement for functionality) is represented as dissociation constant kD (k_Off rate/k_on rate), where a lower kD is correlated with a higher affinity and vice versa.

The affinity of the NOTA-MMR exposed to 20% ethanol was comparable to the affinity of the non-exposed NOTA-MMR and MMR.HIS reference compound. No difference in affinity was observed either between the lyophilized and non-lyophilized NOTA-MMR precursor, suggesting that ethanol does not affect functionality even in presence of the lyophilization excipients.

In summary, these results indicate that exposing the NOTA-sdAbs to a 20% V/V% ethanol content does not impact the potency of the precursors.

3.4. Combination study

From the first high activity experiments it was clear that ethanol, at 20% V/V%, as stand-alone was not potent enough to minimize radiolysis to acceptable levels. Therefore, an additional combination study was performed to assess the potential of combining 20% ethanol with AA in different amounts (Table 5). Additionally, the mass of NOTA-sdAb was increased from 100 pg to 200 pg per sample to overcome the interference of AA on the radiolabeling.

From this first study, we found that combining ethanol with AA had no adverse effect on the radiolabeling, while increasing the NOTA-sdAb mass to 200 pg provided RCP > 99% even at 5 mg/ml of AA or GA. Reducing the pH to 4 in an AA setting, which could reduce the oxidation of AA in solution and therefore increase its stability, however, lead to precipitation of the tracer, as about 10% of activity remained on the filter after filtration of the solution.

Other derivatives of AA such as 2-O-a-D-glucopyranosyl ascorbic acid, 2-O-p-D- glucopyranosyl ascorbic acid, 5-O-a-D-glucopyranosyl ascorbic acid, 6-O-a-D- glucopyranosyl ascorbic acid, 3-O-glycosyl-L-ascorbic acid, 6-O-acyl-2-O-a-D- glucopyranosyl ascorbic acid were equally found to be effective (data not shown).

4. Development final formulation

In the first step towards a final formulation, the combination of 20% ethanol - 5 mg/ml AA was tested in increasing radiolabeling volumes. Based on these results, an iteration of the formulation was performed. Finally, the formulation was confirmed at high activity for both NOTA-sdAbs. 4.1. Volume study

The previously mentioned combination of ethanol and AA was tested at different radiolabeling volumes to simulate conditions of other ⁶⁸Ga-generators and to in this manner test potential compatibility with other commercially available ⁶⁸Ge/Ga generators, such as Eckert & Ziegler's GalliaPharm (0.1N HCI - 5 ml elution volume) or ITG's ⁶⁸Ga generator (0.05N HCI - 4 ml elution volume).

For each condition, 3 labelings were performed and tested for RCP 10 min and 3 hours after radiolabeling (Table 6). The 5 mg/ml AA was dissolved in IM NaOAc/20% Ethanol pH5 buffer 20 to 28h prior to radiolabeling, along with the excipients of a previously designed freeze-drying formulation. The final buffer was then stored at 2 - 8°C.

From this first volume study, the interfering effect of AA on the labeling became apparent at a radiolabeling volume of 7.5 ml and 10 ml after 10 min incubation. After 3 hours an RCP > 99% is obtained, suggesting only a mild chelating capacity of AA towards ⁶⁸Ga, reducing the ⁶⁸Ga-NOTA complexation rate, but not preventing the reaction. However, since a fixed concentration of AA is used in this formulation, a higher volume is correlated with a higher mass of AA for a same amount of precursor (e.g. a 2.2 ml final volume yields 5.5 mg of AA, while a 10 ml volume yields 25 mg of AA). This increase in mass of AA, along with the dilution factor for the precursor, results in a lower RCP after 10 min of incubation.

To avoid a varying AA to precursor ratio, a fixed amount of AA (5 mg) was tested in a following study (Table 7).

Changing to fixed amount of AA, resulted in an increase in RCP, reaching > 99% even at 10 ml, the highest radiolabeling volume tested.

4.2. High activity study

The 20% ethanol - 5 mg AA (fixed) formulation was evaluated at high activities in both the 2.2 ml and 10 ml final radiolabeling volumes to verify its potency to prevent radiolysis, while still obtaining a RCP > 95% (Table 8). The excipients for the lyophilization were added again, taking the dilution factor of the radiolabeling volume into consideration. The high activity results show a high RCP >95% for both NOTA-sdAb precursors, while no radiolysis could be observed even 3 hours after radiolabeling. This confirms the validity of the formulation, which could be implemented in an on-going kit development.

5. Characterization final formulation

Further characterization of the final formulation was performed to ensure compatibility for clinical use. Firstly, the osmolality of the final solution, that would be injected in a patient, was determined and secondly, a particle size analysis was performed to verify potential presence of microprecipation or other unexpected particles.

5.1. Osmolality

The osmolality of different solutions was analyzed to investigate the impact of different compounds on the osmolality, while mimicking the conditions as if the solution would be injected as final solution, taking the dilution with the ⁶⁸Ga eluate into account. A 1: 1 dilution occurs of the IM NaOAc buffer with the ⁶⁸Ga eluate, resulting in a final concentration of 0.5M NaOAc and 10% ethanol (where applicable). This study allows us to define a range for the final product specifications.

Following conditions were analyzed:

• Reference: 0.5 M NaOAc pH 5 buffer

• Basic condition: 0.5M NaOAc pH 5 buffer + excipients lyophilization

• Intermediate condition: 0.5M NaOAc/10% Ethanol pH 5 buffer + excipients lyophilization

• Final condition: 0.5M NaOAc/10% Ethanol pH 5 buffer + excipients lyophilization + 5 mg VitC

Additionally, the final condition was tested in a concentrated and diluted form, simulating the 2.2 and 10 ml radiolabeling volume. Each sample was further diluted 1 on 2 with milliQ water to prevent saturation of the osmometer and each solution was measured in triplicate (Table 9).

The reference solution, containing solely sodium acetate and precursor, already shows a relatively high osmolality of 811 mOsm/kg (a solution of 300 mOsm/kg is considered isotonic). The addition of the excipients for lyophilization has a minor impact on the osmolality, while ethanol greatly increases the osmolality to nearly 2500 mOsm/kg. Addition of VitC further increase the osmolality slightly to nearly 2700 mOsm/kg. Strangely, no difference is observed between the concentrated and diluted formulation 2. However, this confirms the strong influence of ethanol on the osmolality of the solutions.

5.2. Particle Size Analysis

A particle size analysis was performed via Dynamic Light Scattering on the final concentrated formulation to analyze the distribution of particles in the solution. The solution was tested in triplicate.

The mean hydrodynamic diameter (Dh) for each run is 0.78, 1.11 and 0.82 nm, respectively, resulting in an overall average of 0.90 nm with o = 0.15 (Fig 2A). No particles above 3 nm Dh were measured, which suggests a clear and pure solution and no microprecipitation of any of the compounds (Fig 2B).

The present invention is in no way limited to the embodiments described in the examples and/or shown in the figures. On the contrary, methods according to the present invention may be realized in many different ways without departing from the scope of the invention.

6. Ascorbic acid glucoside as radioprotectant

A first exploratory study was performed to assess the potency of AA-2G as radioprotectant and its potential interference with ⁶⁸Ga labeling. Different conditions, including the combination with ethanol, were tested. The RCP was analyzed via iTLC and SEC 10 minutes and 3 hours post labeling (Table 10).

From this study, we learned that the radioprotective effect of AA-2G is inferior to the native ascorbic acid, as the molar equivalent mix of 20%Ethanol - 9.6 mg AA-2G to 20% Ethanol - 5 mg VitC was unable to maintain a RCP above 95% after 3 hours in a 10 ml labeling volume, while radiolysis was barely below 5% in the 2.2 ml labeling volume (compared to a RCP > 99% in both labeling volumes for the 20%Ethanol - 5 mg VitC mix). This is in concordance with other studies, who found AA-2G to be less effective as radical scavenger compared to natural VitC. Larger amounts of AA- 2G were able to reduce radiolysis, however, using 50 mg increases 'free' ⁶⁸Ga fraction 10 minutes after labeling. The mix of 10% Ethanol with 25 mg AA-2G still results in a 3 - 5% radiolysis 3h post labeling, while 10% Ethanol - 50 mg AA-2G could almost entirely prevent radiolysis.

The ethanol content can be further increased to 20%, to obtain a 20% Ethanol - 50mg AA-2G mix, which might prove ideal to prevent radiolysis. However, due to its interaction with ⁶⁸Ga, the incubation time might have to be prolonged by 2 - 5 minutes, to ensure an RCP above 95%.

7. Ascorbic acid glucoside as lyo-/cryoprotectant

Since a relatively high amount of AA-2G is required to reduce radiolysis in ⁶⁸Ga labeling, we investigated if AA-2G could have any stabilizing properties regarding lyophilization. If so, designing a proper lyophilization formulation could facilitate the implementation of AA-2G in a cold kit form, where it would serve as stabilizing excipient in the dry NOTA-sdAb product and as radioprotectant upon reconstitution with the radiolabeling buffer.

A first characterization of AA-2G was performed which serve as a basis to design a new lyophilization formulation. A 5% AA-2G solution was analyzed via MDSC to determine its Tg' (Figure 3), which showed to be between -30 and -35°C. This Tg' potentially allows us to apply a previously developed drying cycle (Table 11). As such, samples containing NOTA-anti-MMR precursor were lyophilized with a 1 ml 5% AA-2G formulation for a first stability study. Additionally, a few blanc samples with 1 ml of a 5% AA-2G/5% mannitol formulation were also lyophilized.

Visually, the dried samples provided an elegant white cake. The 5% AA-2G formulation showed some shrinking upon drying, leading to a detached pellet, while the samples containing mannitol provided a slightly more appealing cake structure without shrinking. MDSC analysis showed a high Tg of ~ 65°C for both formulations and a residual moisture of 2.3% and 2.7% for the 5% AA-2G and 5% AA-2G/5% mannitol, respectively. These results indicate that both formulations have the potential to act as freeze-dry formulation for the NOTA-sdAb.

8. One-step labeling procedure

An immunoglobulin single variable domain conjugated to a NOTA chelator (50 nmoles) is lyophilized with 100 mg of a vitamin C derivative as lyophilization excipient. This lyophilized sample is reconstituted and labeled by direct elution of a ⁶⁸Ga eluate in the lyophilized vial, without prior reconstitution with a buffer.

9. Two-step labeling procedure

An immunoglobulin single variable domain conjugated to a NOTA chelator (40 nmoles) is lyophilized with 50 mg of a vitamin C derivative as lyophilization excipient. This lyophilized sample is reconstituted with 1.1 ml of IM NaOAc buffer comprising 20% ethanol (pH 5), after which the full ⁶⁸Ga eluate (1-1.1 ml) was added for labeling.

Table 1: Efficiency test ethanol

Table 2: Effect of ethanol on protein aggregation via SDS-PAGE

Table 3: Effect of ethanol on protein aggregation

Table 4: Effect of ethanol on NOTA-sdAb precursor functionality via SPR

Table 5: Combination study Ethanol - AA

Table 6: Volume study Ethanol 20% - 5 mg/ml AA

Table 7: Volume study Ethanol 20%- 5 mg AA (fixed)

Table 9: Osmolality of different conditions

Table 10: AA-2G exploratory study with high activity

Table 11: Freeze-drying cycle applied for AA-2G-based formulations

Claims

1. An immunoglobulin single variable domain directed against or specifically binding to human macrophage mannose receptor (MMR) or an MMR-binding fragment thereof for use in the diagnosis and/or prognosis of cardiac sarcoidosis, wherein said immunoglobulin single variable domain or MMR- binding fragment thereof is coupled to a detectable label.

2. The immunoglobulin single variable domain or MMR-binding fragment thereof for use according to claim 1, wherein said detectable label is selected from the group consisting of a radionuclide, a fluorescent moiety, a phosphorescent label, a chemiluminescent label, a metal, a metal chelate, a metallic cation, a chromophore, an enzyme or a combination of one of the aforementioned labels.

3. The immunoglobulin single variable domain or MMR-binding fragment thereof for use according to claim 1 or 2, wherein said detectable label is a radionuclide, said radionuclide is selected from the group consisting of fluor 18 (18F), scandium 44 (⁴⁴Sc), lutetium 177 (¹⁷⁷Lu), zirconium 89 (⁸⁹Zr), indium 111 (^mln), yttrium 90 (⁹⁰Y), copper 61 (⁶¹Cu), copper 64 (⁶⁴Cu), actinium 225 (²²⁵Ac), bismuth 213 (²¹³Bi), gallium 67 (⁶⁷Ga), gallium 68 (⁶⁸Ga), technetium 99m (^99mTc), iodine 123 (¹²³I), iodine 124 (¹²⁴I), iodine 125 (¹²⁵I), iodine 131 (¹³¹I).

4. The immunoglobulin single variable domain or MMR-binding fragment thereof for use according to claim 3, wherein said radionuclide is gallium 68 (⁶⁸Ga).

5. The immunoglobulin single variable domain or MMR-binding fragment thereof for use according to any of claims 1 to 4, wherein said immunoglobulin single variable domain or MMR-binding fragment thereof is coupled to a chelating agent selected from the group consisting of DTPA and derivatives, DOTA and derivatives, NOTA and derivatives, HBED and derivatives, DEPA and derivatives, picolinic acid based chelators and derivatives, HEHA and derivatives, TETA and derivatives, NETA and derivatives, AAZTA and derivatives, DATA and derivatives, TCMC and derivatives, PCTA and derivatives, Macropa and derivatives, THP and derivates, DFO and derivatives, BCPA and derivatives, MAG-2 and derivatives, MAG-3 and derivatives, MAS-3 and derivatives, HYNIC and derivatives and RESCA.

6. The immunoglobulin single variable domain or MMR-binding fragment thereof for use according to claim 1 or 2, wherein said detectable label is a fluorescent moiety, said fluorescent moiety is selected from the group consisting of xanthene, cyanine, squaraines, dipyrromethene, tetrapyrrole, naphthalene, oxadiazole, naphthalene, coumarin, oxazine derivatives and fluorescent metals such as europium or others metals from the lanthanide series. The immunoglobulin single variable domain or MMR-binding fragment thereof for use according to claim 1 or 2, wherein said detectable label is a bimodal label comprising a radionuclide and a fluorescent moiety. The immunoglobulin single variable domain or MMR-binding fragment thereof for use according to any of claims 1 to 7, wherein said diagnosis and/or prognosis of cardiac sarcoidosis is by non-invasive in vivo medical imaging. The immunoglobulin single variable domain or MMR-binding fragment thereof for use according to any of claims 1 to 8 wherein said immunoglobulin single variable domain or MMR-binding fragment thereof is used as contrast agent in non-invasive in vivo medical imaging. The immunoglobulin single variable domain or MMR-binding fragment thereof for use according to claim 8 or 9, wherein said non-invasive in vivo medical imaging is positron-emission tomography (PET) imaging or single photonemission computed tomography (SPECT) imaging. The immunoglobulin single variable domain or MMR-binding fragment thereof for use according to any of claims 1 to 10, wherein said immunoglobulin single variable domain has the formula FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein FR1 to FR4 are framework regions and wherein CDR1 to CDR3 are complementarity determining regions. The immunoglobulin single variable domain or MMR-binding fragment thereof for use according to claim 11, wherein CDR1 comprises the amino acid sequence FSLDYYAIG (SEQ ID NO. l), CDR2 comprises the amino acid sequence CISYKGGST (SEQ ID NO.2), and CDR3 comprises the amino acid sequence GFWCYKYDY (SEQ ID NO.3). The immunoglobulin single variable domain or MMR-binding fragment thereof for use according to any of claims 1 to 12, wherein said immunoglobulin single variable domain comprises a heavy chain variable domain, the amino acid sequence of which comprises the sequence QVQLQESGGGLVQPGGSLRLSCAASGFSLDYYAIGWFRQAPGKEREGISCISYKGGS TTYADSVKGRFTISKDNAKNTAYLQMNSLKPEDTGIYSCAAGFWCYKYDYWGQGTQV TVSS (SEQ ID NO.4) or the sequence DVQLQESGGGLVQPGGSLRLSCAASGFSLDYYAIGWFRQAPGKEREGISCISYKGGS TTYADSVKGRFTISKDNAKNTAYLQMNSLKPEDTGIYSCAAGFWCYKYDYWGQGTQV TVSS (SEQ ID NO.5). The immunoglobulin single variable domain or MMR-binding fragment thereof for use according to any of claims 1 to 13, wherein said immunoglobulin single variable domain is a VHH domain. The immunoglobulin single variable domain or MMR-binding fragment thereof for use according to any of claims 1 to 14, wherein said immunoglobulin single variable domain or MMR-binding fragment thereof is administered intravenously. The immunoglobulin single variable domain or MMR-binding fragment thereof for use according to any of claims, wherein said immunoglobulin single variable domain or MMR-binding fragment thereof is administered at a dose of between 1 and 1000 pg. The immunoglobulin single variable domain or MMR-binding fragment thereof for use according to any of claims 1 to 14, wherein said immunoglobulin single variable domain or MMR-binding fragment thereof is formulated in a pharmaceutical composition. The immunoglobulin single variable domain or MMR-binding fragment thereof for use according to claim 17, wherein said pharmaceutical composition further comprises a buffer selected from the group consisting of phosphate buffers, such as sodium phosphate, sodium phosphate dibasic, potassium phosphate and ammonium phosphate, bicarbonate or carbonate buffers, succinate buffers such as disodium succinate hexahydrate, borate buffers such as sodium borate, cacodylate buffers; citrate buffers such as sodium citrate or potassium citrate, sodium chloride, zinc chloride or zwitterionic buffers, Tris(hydroxymethyl)aminomethane (Tris base) buffers, such as Tris HCI, Tris EDTA, Tris acetate, Tris phosphate or Tris glycine, 3-(N- morpholino)propanesulfonic acid (MOPS), N-(2-hydroxyethyl)piperazine-N'- (2-ethanesulfonic acid) (HEPES), dextrose, lactose, tartaric acid, formate, arginine, and acetate buffers such as ammonium, sodium or potassium acetate. The immunoglobulin single variable domain or MMR-binding fragment thereof for use according to claim 18, wherein said pharmaceutical composition further comprises vitamin C or a derivative thereof. The immunoglobulin single variable domain or MMR-binding fragment thereof for use according to claim 18 or 19, wherein said pharmaceutical composition further comprises ethanol. A method for diagnosing and/or prognosing cardiac sarcoidosis, the method comprising the steps of administering to a subject an immunoglobulin single variable domain directed against or specifically binding to human macrophage mannose receptor (MMR) or an MMR-binding fragment thereof, wherein said immunoglobulin single variable domain or MMR-binding fragment thereof is coupled to a detectable label.

22. The method according to claim 21, wherein said diagnosis and/or prognosis is by non-invasive in vivo medical imaging.

23. The method according to claim 22, wherein said non-invasive in vivo medical imaging is positron-emission tomography (PET) imaging or single photonemission computed tomography (SPECT) imaging.

24. A method of imaging cardiac sarcoidosis in a subject, the method comprising :

- administering to the subject an immunoglobulin single variable domain directed against or specifically binding to human macrophage mannose receptor (MMR) or an MMR-binding fragment thereof, wherein the immunoglobulin single variable domain directed against or specifically binding to human macrophage mannose receptor (MMR) or an MMR-binding fragment thereof is coupled to a detectable label, and

- imaging the immunoglobulin single variable domain directed against or specifically binding to human macrophage mannose receptor (MMR) or an MMR-binding fragment thereof coupled to a detectable label in the subject so as to image cardiac granulomata in the subject.

25. The method according to any of claims 21 to 24, wherein said detectable label is a radionuclide.

26. The method according to claim 25, wherein said radionuclide is selected from the group consisting of fluor 18 (¹⁸F), scandium 44 (⁴⁴Sc), lutetium 177 (¹⁷⁷Lu), zirconium 89 (⁸⁹Zr), indium 111 (^X11ln), yttrium 90 (⁹⁰Y), copper 61 (⁶¹Cu), copper 64 (⁶⁴Cu), actinium 225 (²²⁵Ac), bismuth 213 (²¹³Bi), gallium 67 (⁶⁷Ga), gallium 68 (⁶⁸Ga), technetium 99m (^99mTc), iodine 123 (¹²³I), iodine 124 (¹²⁴I), iodine 125 (¹²⁵I), iodine 131 (¹³¹I).

27. The method according to claim 26, wherein said radionuclide is gallium 68 (⁶⁸Ga).

28. The method of any of claims 21 to 27, wherein said immunoglobulin single variable domain or MMR-binding fragment thereof is coupled to a chelating agent selected from the group consisting of DTPA and derivatives, DOTA and derivatives, NOTA and derivatives, HBED and derivatives, DEPA and derivatives, picolinic acid based chelators and derivatives, HEHA and derivatives, TETA and derivatives, NETA and derivatives, AAZTA and derivatives, DATA and derivatives, TCMC and derivatives, PCTA and derivatives, Macropa and derivatives, THP and derivates, DFO and derivatives, BCPA and derivatives, MAG-2 and derivatives, MAG-3 and derivatives, MAS-3 and derivatives, HYNIC and derivatives and RESCA. The method according to any of claims 21 to 28, wherein said immunoglobulin single variable domain directed against or specifically binding to human macrophage mannose receptor (MMR) or an MMR-binding fragment thereof is a contrast agent in non-invasive in vivo medical imaging. Use of a kit comprising an immunoglobulin single variable domain directed against or specifically binding to human macrophage mannose receptor (MMR) or an MMR-binding fragment thereof and one or more detectable labels for the production of a diagnostic for the detection of cardiac sarcoidosis.