WO2022156907A1

WO2022156907A1 - Method and kit for labeling a biomolecule with one or more detectable labels, including a radiolabel

Info

Publication number: WO2022156907A1
Application number: PCT/EP2021/051571
Authority: WO
Inventors: Henri BAUDHUIN; Philippe VANWOLLEGHEM; Catarina XAVIER
Original assignee: Vrije Universiteit Brussel
Priority date: 2021-01-25
Filing date: 2021-01-25
Publication date: 2022-07-28

Abstract

The current invention relates to a method for labeling an antibody or a fragment thereof with a detectable label, wherein at least one of said detectable labels is a radionuclide. The labeling occurs either in the presence of ethanol and vitamin C, wherein the concentration of ethanol in the labeling reaction is between 5% v/v and 15% v/v and the concentration of vitamin C in the labeling reaction is between 0.2 mg/ml and 2.5 mg/ml. Or the labeling occurs in the presence of ethanol and a derivative of vitamin C, wherein said derivative is an ascorbyl glucoside chosen from the list of 2-O-α-D-glucopyranosyl ascorbic acid, 2-O-β-D-glucopyranosyl ascorbic acid, 5-O-α-D-glucopyranosyl ascorbic acid, 6-O-α-D-glucopyranosyl ascorbic acid, 3-O-glycosyl-L-ascorbic acid, 6-O-acyl-2-O-α-D-glucopyranosyl ascorbic acid or a mixture thereof, wherein 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. The current invention also relates to kits for labeling an antibody or antibody fragment with a radionuclide and specific uses of those kits.

Description

METHOD AND KIT FOR LABELING A BIOMOLECULE WITH ONE OR MORE DETECTABLE LABELS, INCLUDING A RADIOLABEL

FIELD OF THE INVENTION

The present invention relates to a method for labeling a biomolecule with one or more detectable labels. In a second aspect, the present invention also relates to a kit for labeling a biomolecule with one or more detectable labels.

BACKGROUND

In the area of radiopharmaceuticals, where a radionuclide is to be delivered at a specific site in the body, the correct preparation of the radiopharmaceutical compound is critical and stumbles upon several challenges. One of the issues in radiopharmaceutical preparations, is the potential damage that can be brought to the targeting vehicle to which the radionuclide is linked during or after the radiolabeling. Indeed, ionizing radiation coming from radionuclides can directly induce damage due to the emitted particles by the isotope. Indirectly, the ionizing radiation can induce damage by the formation of free radicals or other highly reactive compounds in solutions. Such highly reactive compounds tend to degrade organic compounds, such as peptide, proteins, DNA-sequences etc. Especially protein-based targeting vehicles, such as antibodies or fragments thereof, are particularly sensitive to such radiolytic degradation or radiolysis, with loss of immunoreactivity or binding capability consequently. This can render the radiotracer unusable and can cause increased radiation toxicity to certain organs when injected, as these radio metabolites might accumulate more easily in non-target organs.

It is evident that radiolysis is an undesired occurrence, which can be prevented or diminished by adding antioxidants or scavenger molecules in the reaction mixture. These molecules will interact with and neutralize the formed radicals, therefore protecting the targeting vehicle. It has, in that regard, become common to develop anti-radiolytic formulations comprising a rad io protectant when carrying out a radiolabeling.

Scott et al describe in their research article a number of additives that inhibit radiolysis and that can be safely added to the synthesis procedures and reformulation steps to provide suitable clinical formulations. US20070269375 further describes methods of making and using stabilized radiopharmaceutical formulations. More specifically, US '375 discusses the use of stabilizers useful in the preparation and stabilization of targeted radiodiagnostic and radiotherapeutic compounds.

Fukumura et al discloses the use of additives to prepare “C-radiopharmaceuticals with good radiochemical purity even with high levels of radioactivity and specific activity.

AU2016201367 discloses radiopharmaceutical compositions and related methods, useful for medical imaging. AU '367 describes the use of the antioxidant ascorbic acid, under buffered conditions in a particular pH range, to stabilize a radiopharmaceutical composition useful for medical imaging, and thereby enhancing the shell life of the composition, while maintaining the composition as suitable for administration to a human, and other mammalian subjects.

US20200030465 discloses the use of various stabilizers against radiolytic degradation of a complex formed by a radionuclide and a cell receptor binding organic moiety linked to a chelating agent.

EP3242691 discloses a method of imaging cancer comprising amongst others preparing a molecule that is dual-labeled with a radionuclide and a fluorescent probe, in the presence of a radioprotective agent.

The stabilizers disclosed in the above-mentioned documents aid in preventing radiolysis of the radiopharmaceuticals. However, undesired effects such as precipitation of the targeting vehicle or interference with the radiolabeling reaction occur when these stabilizers are not provided in the correct concentration range.

The aim of the invention is to provide an improved method for labeling an antibody or fragment thereof, by using radioprotectants that enhance radiochemical purity and reduce radiolysis.

SUMMARY OF THE INVENTION

The present disclosure serves to provide a solution to one or more of above- mentioned disadvantages. To this end, the present disclosure provides a method according to claim 1. More in particular, the present disclosure provides a method for labeling a biomolecule with one or more detectable labels, wherein at least one of said detectable labels is a radionuclide, said labeling occurs in the presence of ethanol and vitamin C, wherein the concentration of ethanol in the labeling reaction is between 5% v/v and 15% v/v and the concentration of vitamin C in the labeling reaction is between 0.2 mg/ml and 2.5 mg/ml.

In addition, the present disclosure provides a method according to claim 2. More in particular, the present disclosure provides a method for labeling a biomolecule with one or more detectable labels, wherein at least one of said detectable labels is a radionuclide, said labeling occurs in the presence of ethanol and 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, 3-0- glycosyl-L-ascorbic acid, 6-O-acyl-2-O-a-D-glucopyranosyl ascorbic acid or a mixture thereof, wherein 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.

Preferred embodiments of these methods are shown in any of the claims 4 to 19.

In a second aspect the present invention relates to a kit according to claim 20. More particular, the present disclosure provides a kit for labeling a biomolecule with one or more detectable labels, wherein at least one of said detectable labels is a radionuclide, said kit comprises lyophilized biomolecules in a lyophilizate, wherein said lyophilizate comprises vitamin C, wherein the amount of vitamin C in the lyophilizate is between 1 mg and 7 mg, said kit further comprises a stabilizing buffer, wherein said buffer comprises ethanol at a concentration between 10% v/v and 30% v/v.

In addition, the present invention relates to a kit according to claim 21. More particular, the present disclosure provides a kit for labeling a biomolecule with one or more detectable labels, wherein at least one of said detectable labels is a radionuclide, said kit comprises lyophilized biomolecules in a lyophilizate, wherein said lyophilizate comprises a derivative of vitamin C, wherein the amount of the vitamin C derivative in the lyophilizate is between 20 mg and 150 mg, said kit further comprises a stabilizing buffer, wherein said buffer comprises ethanol at a concentration between 10% v/v and 30% v/v.

Preferred embodiments of these kits are shown in any of the claims 22 to 27.

In a third aspect the present invention relates to specific uses of the kit according to claims 28-39.

The method according to the present disclosure is able to diminish or avoid radiolytic damage during radiolabeling of a biomolecule. Furthermore, by providing a kit which is relatively simple to use and does not require significant handling of the radionuclide, the present disclosure facilitates development, usage, distribution and commercialization of lyophilized biomolecules which could be used as radiopharmaceuticals.

DESCRIPTION OF FIGURES

Figure 1 shows a graphical representation of the distribution of sized particles in the final concentrated formulation according to an embodiment of the current invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention concerns a method for labeling a biomolecule with one or more detectable labels, wherein at least one of said detectable labels is a radionuclide, wherein said labeling occurs either in the presence of ethanol and vitamin C or in the presence of ethanol and a derivative of vitamin C. In addition, the present invention concerns a kit for labeling a biomolecule with one or more detectable labels, wherein at least one of said detectable labels is a radionuclide, said kit comprises lyophilized biomolecules in a lyophilizate, wherein said lyophilizate comprises vitamin C or a derivative thereof and a stabilizing buffer.

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.

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.

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, each comprising a constant region and a variable region. These heavy chains are linked by disulfide bridges at the so-called hinge region. In addition, each heavy chain is linked to a light chain (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. Each variable region has three complementary determining regions (CDRs). Together the variable region of a light chain and heavy chain define the binding specificity of the antibody for the target.

"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 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 H1, H2, H3 for the heavy chain and L1, 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 (which is different from conventional immunoglobulins or their fragments, wherein typically two immunoglobulin variable domains interact to form an antigen binding site). It should however be clear that the term "immunoglobulin single variable domain" does comprise fragments of conventional immunoglobulins wherein the 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). 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 domain sequences (e.g. a VL domain sequence) or a suitable fragment thereof, or heavy chain variable domain sequences (e.g. a VH domain sequence or VHH domain sequence) or a suitable fragment thereof, as long as it is capable of forming a single antigen binding unit. Thus, according to a preferred embodiment, the immunoglobulin single variable domain is a light chain variable domain sequence (e.g. a VL domain sequence) or a heavy chain variable domain sequence (e.g. a VH domain sequence); more specifically, the immunoglobulin single variable domain is a heavy chain variable domain sequence that is derived from a conventional four-chain antibody or a heavy chain variable domain sequence that is derived from a heavy chain antibody. The immunoglobulin single variable domain may be a domain antibody, or a single domain antibody, or a "dAB" or "dAb", or a VHH domain sequence or another immunoglobulin single variable domain, or any suitable fragment of any one thereof. The immunoglobulin single variable domains, generally comprise a single amino acid chain that can be considered to comprise 4 "framework sequences" or FR's and 3 "complementary determining regions" or CDR's (as defined herein). 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.

It should be noted that the immunoglobulin single variable domains as binding domain moiety in their broadest sense are not limited to a specific biological source or to 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 domain sequences. 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 camelid antibodies, devoid of light chains, and are known as VHH domain sequences.

The term "VHH domain sequence" is, as used herein, is interchangeably with the term "single domain antibody fragment (sdAb)" and refers to a single domain antigen binding fragment. It particularly refers to a single variable domain derived from naturally occurring heavy chain antibodies and is known to the person skilled in the art. VHH domain sequences 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 sequence. Camelids comprise old world camelids (Camelus bactrianus and Camelus dromedarius) and new world camelids (for example Lama paccos, Lama glama, Lama guanicoe and Lama vicugna). The small size and unique biophysical properties of VHH domain sequences excel conventional antibody fragments for the recognition of uncommon or hidden epitopes and for binding into cavities or active sites of protein targets. VHH domain sequences are stable, survive the gastro-intestinal system and can easily be manufactured. Therefore, VHH domain sequences 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 domain sequences 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 variable regions in VHH domain sequences and contains the amino acid sequences capable of specifically binding to antigenic targets. These CDR regions account for the basic specificity of the VHH domain sequences for a particular antigenic determinant structure. Such regions are also referred to as "hypervariable regions." The VHH domain sequences 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 domain sequences 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).

"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 "cancer" refers to any neoplastic disorder, including such cellular disorders as, for example, renal cell cancer, Kaposi's sarcoma, chronic leukemia, breast cancer, sarcoma, ovarian carcinoma, rectal cancer, throat cancer, melanoma, colon cancer, bladder cancer, mastocytoma, lung cancer, mammary adenocarcinoma, pharyngeal squamous cell carcinoma, and gastrointestinal or stomach cancer.

"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.

"Biomolecule" as used herein can refer to peptides, small molecules, scaffold proteins, antibodies or antibody fragments.

Furthermore, 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.

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.

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.

For radiopharmaceuticals, radiolytic damage during radiolabeling of an antibody or antibody fragment can be a serious problem. Such radiolytic damage can cause, for example, release of the radionuclide or it can damage the biomolecule.

Significant radiolytic damage induced by the radioactive label can occur if labeling of the biomolecule occurs without concomitant or subsequent addition of one or more radioprotectants (compounds that protect against radiolytic damage). Radiolytic damage to the methionine (Met) residue in for instance antibodies or antibody fragments is the most facile mode of decomposition, possibly resulting in a methionine sulfoxide derivative. 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 radioprotectant 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 an antibody or the antibody fragment can be assessed by various protein analysis techniques known from the state of the art.

In a first aspect, the invention provides a method for labeling a biomolecule with one or more detectable labels, wherein at least one of said detectable labels is a radionuclide, said labeling occurs in the presence of ethanol and vitamin C, wherein the concentration of ethanol in the labeling reaction is between 5% v/v and 15% v/v and the concentration of vitamin C in the labeling reaction is between 0.2 mg/ml and 2.5 mg/ml.

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, vitamin C is 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. To this purpose, Vitamin C has proven be an attractive candidate as additive during reactions with radioactive compounds. Vitamin C is an FDA approved pharmaceutical suitable for human injection, with a low toxicity profile and its addition in formulations is easily justified. Furthermore, Vitamin C is compatible with lyophilization and has a buffering capacity in the desired range.

In addition, 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. Thus, the glucose function stabilizes the molecules, which will be less reactive to degradation reactions. In addition, the present invention provides a method for labeling a biomolecule with one or more detectable labels, wherein at least one of said detectable labels is a radionuclide, said labeling occurs in the presence of ethanol and 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, 3-0- glycosyl-L-ascorbic acid, 6-O-acyl-2-O-a-D-glucopyranosyl ascorbic acid or a mixture thereof, wherein 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, said labeling occurs only in the presence of a derivative of vitamin C, wherein said derivative is an ascorbyl glucoside chosen from the list of 2-O-a-D- glucopyranosyl ascorbic acid, 2-0-β-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, wherein the concentration of said derivative of vitamin C in the labeling reaction is between 5 mg/ml and 150 mg/ml.

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. Moreover, ethanol is low toxic for injection (at low doses), does not cause immunoreactivity issues with proteins and does not interfere with the radiolabeling reaction of various radionuclides such as ⁶⁸Ga. 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.

Studies assessing the compatibility of ethanol in the labeling reaction, showed that an ethanol concentration between 5% v/v and 15% v/v has an excellent rad io protectant effect, showing a high RCP. In a further embodiment, the ethanol concentration in the labeling reaction is between 8% v/v and 12% v/v, more preferably between 9% v/v and 11% v/v, such as 10% v/v.

It was found that higher concentrations of ethanol cause precipitation of antibodies or antibody fragments. In an embodiment sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is used to assess the amount of protein aggregation. In another embodiment, protein aggregation is quantified by filtering the radiolabeling solution and determining the activity remaining on the filter after the filtering step. In an embodiment the radiolabeling solution is filtered through a 0.22 pm filter. In an embodiment the remaining activity on the filter is presented as % compared to the initial activity in the vial minus the remaining activity in the vial after uptake of the solution. In addition, ethanol concentrations in the labeling reaction exceeding 20% v/v induce precipitation of the biomolecule, such as an antibody or antibody fragment. In an embodiment the effect of the radioprotectant on the functionality of the biomolecule, such as an antibody or antibody fragment is assessed via Surface Plasmon Resonance (SPR). SPR is able to measure the affinity of an antibody or antibody fragment by determining the dissociation constant (kD), where a lower kD is correlated with a higher affinity and vice versa. However, a high activity test indicated that ethanol as stand-alone is not potent enough to minimize radiolysis to acceptable levels.

The inventors have unexpectedly observed that combining ethanol and vitamin C (or one of the aforementioned vitamin C derivatives) has a strong radioprotective effect with no adverse effects on the radiolabeling reaction, showing a low degree of precipitation and a high RCP. If vitamin C is used, a concentration of vitamin C in the labeling reaction between 0.2 mg/ml and 2.5 mg/ml, more preferably between 0.5 mg/ml and 2.27 mg/ml is used. If one of the aforementioned vitamin C derivatives is used, a concentration in the labeling reaction between 5 mg/ml and 150 mg/ml, more preferably between 5 mg/ml and 100 mg/ml, more preferably between 5 mg/ml and 50 mg/ml, such as 22.7 mg/ml shows the best results in the radiolabeling study.

Higher concentrations negatively affect the RCP. By combining ethanol and vitamin C (or one of the aforementioned vitamin C derivatives) at those concentration ranges, radiolysis could be maximally prevented, whilst no negative effects of the radioprotectants could be observed. A too high concentration of EtOH would lead to precipitation of the antibody or antibody fragment, whilst a too high concentration of vitamin C (or one of the aforementioned vitamin C derivatives) interferes with the radiolabeling reaction. Obviously, if concentrations were too low, no radioprotective effect was observed. In an embodiment the RCP, the amount of free radionuclide and the amount of radiolysis are assessed by iTLC. In an embodiment the RCP, the amount of free radionuclide and the amount of radiolysis are assessed by SEC. In an embodiment, the radionuclide used for labeling is chosen from the group of fluor 18 (¹⁸F), lutetium 177 (¹⁷⁷Lu), zirconium 89 (⁸⁹Zr), indium 111 (^mln), yttrium 90 (⁹⁰Y), copper 64 (⁶⁴Cu), actinium 225 (²²⁵Ac), bismuth 213 (²¹³Bi), gallium 67 (⁶⁷Ga), gallium 68 (⁶⁸Ga), technetium 99m (^99mTc), iodium 123 (¹²³I), iodium 124 (¹²⁴I), iodium 125 (¹²⁵I), iodium 131 (¹³¹I).

These radionuclides are suitable for medical applications, such as in vivo nuclear imaging or Targeted Radionuclide Therapy (TRNT). In an embodiment, the antibody or antibody fragment is coupled or fused directly to said radionuclide. In another embodiment, the antibody or antibody fragment 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.

In an embodiment, the radionuclide is a gallium radioisotope solution obtained directly from a gallium radionuclide generator.

Fluorescent labeling is the process of binding fluorescent dyes to functional groups contained in biomolecules so that they can be visualized by fluorescence imaging.

In an embodiment, the antibody or fragment thereof further comprises a fluorescent moiety as detectable label.

In an embodiment, the aforementioned fluorescent moiety is chosen from the group 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).

In a second embodiment, a 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., β 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.

In an embodiment, the biomolecule 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 biomolecule 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 (1,4,7-Triazacyclononane- 1,4,7-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- ca rboxymethyl-amino)-ethyl]-4, 10-bis-carboxymethyl- 1,4,7, 10-tetraaza- cyclododec-l-yl-acetic acid) and derivatives, picolinic acid (PA) based chelators and derivatives (including H2dedpa, H4octapa, H2azapa and H5decapa and derivatives), HEHA (l,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 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 biomolecules are conjugated to NOTA. In another preferred embodiment, the biomolecules are conjugated to DOTA.

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 an embodiment, the radionuclide is linked to a fluorescent moiety, forming a bimodal label. In an embodiment the biomolecules are lyophilized and reconstituted with a labeling buffer prior to labeling.

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 favouring 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 biomolecule. 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 antibody or antibody fragment should be present in the labeling reaction in order to yield a high enough amount of labeled biomolecules.

In an embodiment, the amount of the biomolecule fragment in the lyophilizate is between 7.5 nmoles and 700 nmoles. In an embodiment, when the composition is intended for radiolabeling, said biomolecule 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 biomolecule further comprises a fluorescent moiety as detectable label, said biomolecule is present in the composition in a quantity of between 75 nmoles and 750 nmoles.

Providing such an amount of biomolecules is able to overcome the possible interference caused by the vitamin C (derivative) on the labeling reaction.

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. 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 non-target 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 scFvs, trying to combine specificity with a small size for favorable pharmacokinetics.

Immunoglobulin single variable domains are such antibody derived molecules wherein the antigen binding site is present on, and formed by, a single immunoglobulin domain (which is different from conventional immunoglobulins or their fragments, wherein typically two immunoglobulin variable domains interact to form an antigen binding site). In an embodiment, the biomolecule being labeled is an immunoglobulin single variable domain.

One preferred class of immunoglobulin single variable domains corresponds to the VHH domains of naturally occurring heavy chain antibodies, also called "VHH domain sequences" or "single domain antibody fragment (sdAb)".

Such VHH domain sequences 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 sequences 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 sequences, 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 domain sequences 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 domain sequences 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 domain sequences 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 sequence (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 domain sequences 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 domain sequences 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 domain sequences still retain the favourable properties of VHH domain sequences 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 favourable properties provided by the humanizing substitutions on the one hand and the favourable 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 or antibody fragment of the invention as defined herein.

By means of non-limiting examples, a substitution may for example be a conservative substitution (as described herein) 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 or antibody fragment 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 or antibody fragment of the invention (i.e. to the extent that the antibody or antibody fragment 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 antibodies or antibody fragments thus obtained.

Further, depending on the host organism used to express the antibody or antibody fragment 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 site-specific pegylation.

Examples of modifications, as well as examples of amino acid residues within the antibody or antibody fragment sequence, 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 or antibody fragment, and in particular of one or more functional groups, residues or moieties that confer one or more desired properties or functionalities to the antibody or antibody fragmentof 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 or antibody fragments (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 or antibody fragment 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 or antibody fragment of the invention, an antibody or antibody fragment 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 or antibody fragment 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 antibody or antibody fragment of the invention.

Labeling of the biomolecule occurs in the presence of a labeling buffer. Also, in the case the biomolecules are lyophilized, the biomolecules should be reconstituted prior to labeling. To reconstitute the biomolecule, a labeling buffer is added to dissolve the lyophilizate such that the biomolecule 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 biomolecules, 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) buffers, such as Tris HCI, Tris EDTA, Tris Acetate, Tris phosphate or Tris glycine, morpholine propanesulphonic acid (MOPS), and 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 prefered 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 biomolecule 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.

In an embodiment, ethanol is comprised in the buffer at a concentration between 10% v/v and 30% v/v, more preferably between 12% v/v and 28% v/v, more preferably between 15% v/v and 25% v/v, more preferably between 18% v/v and 23% v/v, such a 20% v/v. Studies have indicated that ethanol at this concentration range in the acetate buffer is able to offer radioprotection during the radiolabeling reaction. Lower concentrations are not sufficient to offer anti-radiolytic protection, whereas higher concentrations of ethanol cause precipitation of the biomoleculeas measured by SDS-PAGE and by determining the activity remaining on a filter after a filtering step. In addition, higher ethanol concentrations cause precipitation of the biomolecule, such as an antibody or antibody fragment.

It will be clear to a skilled person that the biomolecule used in the context of the current invention may be able to bind to any target that is considered useful for the context of the current invention.

In an embodiment, the biomolecule is directed against and/or specifically binds to one or more targets being linked to a disease or a pathology.

As used herein, the term "specifically binding to" in the context where the biomolecule is an antibody or antibody fragment, refers to the ability of this antibody (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 antibody or antibody fragment toward the presence of a complex formed by their binding. Thus, for example, where an antigen and antibody (fragment) are combined in relatively equal concentration, an antibody (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 (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.

In an embodiment, said biomoleculecan be directed against and/or specifically bind to one or more targets being linked to the development and progression of cancer (such as proliferation and survival of cancer cells), cancer metastasis, development and progression of cardiovascular diseases, development and progression of inflammatory disorders or to proteins specifically expressed by cell types involved in one or more of the aforementioned processes, for instance the expression of MMR by tumor-associated macrophages in hypoxic regions of the tumor.

In specifically preferred embodiments, the biomoleculeis directed against and/or specifically binds to human epidermal growth factor receptor type 2 (HER2).

HER2 is a transmembrane protein and a member of erbB family of receptor tyrosine kinase proteins. HER2 is a well-established tumor biomarker that is over-expressed in a wide variety of cancers, including breast, ovarian, lung, gastric, and oral cancers. Therefore, HER2 has great value as a molecular target and as a diagnostic or prognostic indicator of patient survival, or a predictive marker of the response to antineoplastic surgery.

In an embodiment, the amino acid sequence of a heavy chain variable domain that has been raised against HER2, comprises the following sequence: "QVQLQESGGGSVQAGGSLKLTCAASGYIFNSCGMGWYRQSPGRERELVSRISGDGDTWH KESVKGRFTISQDNVWKKTLYLQMNSLKPEDTAVYFCAVCYNLETYGQGTQVTVSS".

In another embodiment, the amino acid sequence of a heavy chain variable domain that has been raised against HER2, comprises the following sequence: "D VQLQESGGGSVQAGGSLKLTCAASGYIFNSCGMGWYRQSPGRERELVSRISGDGDTWHKE SVKGRFTISQDNVWKKTLYLQMNSLKPEDTAVYFCAVCYNLETYGQGTQVTVSS".

It is within the scope of the invention that the biomolecules as disclosed herein can only bind to HER2 in monomeric form, or can only bind to HER2 in multimeric form, or can bind to both the monomeric and the multimeric form of HER2.

In specifically preferred embodiments, the biomolecule is directed against and/or specifically binds to MMR. 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 glycosylations 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, y or /V-acetylglucosamine residues on the envelop of pathogens or on endogenous glycoproteins with C Ds 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).

In an embodiment, the amino acid sequence of a heavy chain variable domain that has been raised against MMR, comprises the following sequence: "QVQLQESGGGLVQPGGSLRLSCAASGFSLDYYAIGWFRQAPGKEREGISCISYKGGSTTYA DSVKGRFTISKDNAKNTAYLQMNSLKPEDTGIYSCAAGFWCYKYDYWGQGTQVTVSS".

Tumor-associated macrophages (TAMs) are an important component of the tumor stroma, both in murine models and human patients. TAMs can promote tumorgrowth by affecting angiogenesis, immune suppression and invasion and metastasis. The plasticity of macrophages offers perspectives for using them as in vivo sensors for the tumor microenvironment they are exposed to. As a matter of fact, at the tumor site, these cells are confronted with different tumor microenvironments, leading to different TAM subsets with specialized functions and distinct molecular profiles. For example, in mammary tumors, at least two distinct TAM subpopulations have been described, based on a differential expression of markers such as the macrophage mannose receptor (MMR or MHC II), differences in pro-angiogenic or immunosuppressive properties and intratumoral localization (normoxic/perivascular tumor areas versus hypoxic regions). In an embodiment, the biomolecule specifically targets MMR-positive tumor- associated macrophages (TAMs) inside a tumor.

MMR-high TAMs are associated with hypoxic regions in the tumor, as illustrated in human breast cancer samples. This finding demonstrates the clinical relevance of targeting MMR-positive TAM subpopulations in the tumor stroma.

Most radiotracers have a relatively short half-life and so 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.

In a second aspect, the invention provides a kit for labeling a biomolecule with one or more detectable labels, wherein at least one of said detectable labels is a radionuclide, said kit comprises lyophilized biomoleculesthereof in a lyophilizate, wherein said lyophilizate comprises vitamin C, wherein the amount of vitamin C in the lyophilizate is between 1 mg and 7 mg, said kit further comprises a stabilizing buffer, wherein said buffer comprises ethanol at a concentration between 10% v/v and 30% v/v.

In addition, the invention provides a kit for labeling a biomolecule with one or more detectable labels, wherein at least one of said detectable labels is a radionuclide, said kit comprises lyophilized biomolecules in a lyophilizate, wherein said lyophilizate comprises a derivative of vitamin C, wherein the amount concentration of the vitamin C derivative in the lyophilizate is between 20 mg and 150 mg, said kit further comprises a stabilizing buffer, wherein said buffer comprises ethanol at a concentration between 10% v/v and 30% v/v.

Developing kits brings important advantages regarding the Chemistry, Manufacturing & Controls (CMC) and economical aspects, as they allow standardized and simplified preparation protocols and the ability for any center to prepare the radiopharmaceutical with minimal GMP license. As such, they allow multi-center studies in development phase and international distribution and commercialization upon market approval. In embodiments of the aforementioned kits, said stabilizing buffer is an acetate, phosphate, succinate, formate or a HEPES buffer.

In embodiments of the aforementioned kits, the biomolecule further comprises a fluorescent moiety as detectable label.

In specifically preferred embodiments of the aforementioned kits, said biomolecule is an immunoglobulin single variable domain.

In a further embodiment, said biomolecule is an immunoglobulin single variable domain directed against and/or specifically binding to HER2.

In another embodiment, said biomolecule is an immunoglobulin single variable domain directed against and/or specifically binding to MMR.

In a further embodiment, the biomolecule is an immunoglobulin single variable domain directed against and/or specifically binding to HER2 or MMR, wherein said immunoglobulin single variable domain is coupled to NOTA or DOTA as chelating agent and wherein the composition is suited for labeling with gallium 68.

In an embodiment the lyophilizate is reconstituted and labeled with a radionuclide to obtain a final solution, said solution being administered to a subject. The solution may be administered by any suitable method within the knowledge of the skilled man. It is clear that the final solution should be compatible with use in the clinic. For this purpose, the osmolality of the final solution should be as low as possible to avoid discomfort during injection. In an embodiment the final solution can be diluted prior to injection to decrease the osmolality of the final solution. Secondly, it is important that no microprecipitation of antibodies or antibody fragments or other unexpected particles are present in the final solution. To ensure that no microprecipitates or other unexpected particles are present, the hydrodynamic diameter of the particles in the final solution can be determined. In an embodiment the particle size is analysed using Dynamic Light Scattering.

In an embodiment, the biomolecules are as described above. Said radiolabel is preferably a radiolabel as described above, being chosen from the group of fluor 18 (¹⁸F), lutetium 177 (¹⁷⁷Lu), zirconium 89 (⁸⁹Zr), indium 111 (¹¹¹ln), yttrium 90 (⁹⁰Y), copper 64 (⁶⁴Cu), actinium 225 ( ²²⁵Ac), bismuth 213 (²¹³Bi), gallium 67 (⁶⁷Ga), gallium 68 (⁶⁸Ga), technetium 99m (^99mTc), iodium 123 (¹²³I), iodium 124 (¹²⁴I), iodium 125 (¹²⁵I), iodium 131 (¹³¹I). These radionuclides are suitable for medical applications, such as in vivo nuclear imaging. In an embodiment, the antibody or antibody fragment is coupled or fused directly to said radionuclide. In another embodiment, the antibody or antibody fragment is coupled or fused to said radionuclide through a linker.

In a further embodiment, the aforementioned biomolecule comprised in the kit is coupled to a chelating agent selected from the group of DTPA and derivatives (including 1B4M-DTPA derivatives and CHX-A"-DTPA derivatives), DOTA and derivatives (including DOTA-GA derivatives, DOTAM derivatives, DO3A and derivatives, DO2A and derivatives, CB-DO2A derivatives and DO3AM derivatives) NOTA and derivatives (including NODA derivatives, NODA-GA derivatives, NO2A derivatives, NOTAM derivatives, NOPO derivatives and TRAP derivatives), HBED and derivatives (including HBED-CC derivatives, HBED-CI derivatives, HBED-CA derivatives, HBED-AA derivatives and SHBED derivatives), DEPA and derivatives, picolinic acid (PA) based chelators and derivatives (including H2dedpa, H4octapa, H2azapa and H5decapa and derivatives), HEHA and derivatives, TETA and derivatives (including TE2A derivatives, CB-TE2A derivatives, CB-TE1A1P derivatives, CB-TE2P derivatives, MM-TE2A derivatives and DM-TE2A derivatives), NETA and derivatives (including C-NETA derivatives and NE3TA 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 .

Further embodiments of the kit are as described above in the context of the labeling methodology.

In an embodiment, the aforementioned kit is used in non-invasive in vivo medical imaging.

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). In an embodiment of the aforementioned use, the kit comprises a biomoleculedirected against and/or specifically binding to HER2, wherein HER2- expressing cells are visualized.

Amplification of the HER2 gene and/or overexpression of the protein have been identified in approximately 20% of invasive breast cancers. Next to the heterogeneity in breast cancer, also possible discordance exists in HER2 status between primary tumors and distant metastases. In this respect, assessment of HER2 expression by non-invasive in vivo medical imaging may become an important complement to immunohistochemistry or fluorescence in situ hybridization analyses of biopsied tissues. A large number of patients who are diagnosed HER2 negative according to the biopsy results still show some degree of HER2 expression.

Non-invasive molecular imaging of HER2 expression using various imaging modalities has been extensively studied. These modalities include radionuclide imaging with Positron Emission Tomography (PET) and Single Photon Emission Tomography (SPECT). PET and SPECT imaging of HER2 (HER2- PET and HER2- SPECT, respectively) provide high sensitivity and high spatial resolution. Hence, the development of a kit for use in non-invasive in vivo medical imaging of HER2 is of great interest.

In another embodiment of the aforementioned use, the kit comprises a biomolecule directed against and/or specifically binding to MMR, wherein MMR-expressing cells, such as Tumor-Associated Macrophages, are visualized.

In an embodiment, the lyophilized biomolecule is able to selectively bind to or target MMR-expressing cells, such as MMR-positive TAMs linked to a hypoxic region of a solid tumor. In this way, the relative percentage of the MMR-positive TAMs can be determined or the impact of a cancer therapy on the relative percentage of the MMR- positive TAMs can be assessed by in vivo medical imaging. In an embodiment, an antibody or antibody fragment specifically binding to MMR can be administered to a subject, and the presence and/or relative percentage of MMR-positive TAMs in the subject can be determined in order to diagnose cancer or prognose cancer aggressiveness in the subject according to the relative percentage of the MMR- positive TAMs.

In particular embodiments, determining the presence and/or relative percentage of MMR-positive TAMs or HER2-expressing cells can be done on a sample from an individual comprising cancer cells or suspected to comprise cancer cells. A sample may comprise any clinically relevant tissue sample, such as a tumor biopsy or fine needle aspirate, or a sample of bodily fluid, such as blood, plasma, serum, lymph, ascitic fluid, cystic fluid, urine or nipple exudate. The sample may be taken from a human, or, in a veterinary context, from non- human animals such as ruminants, horses, swine or sheep, or from domestic companion animals such as felines and canines. The sample may also be paraffin-embedded tissue sections. It is understood that the cancer tissue includes the primary tumor tissue as well as an organ-specific or tissue-specific metastasis tissue.

In an embodiment, the aforementioned kit is used in the diagnosis, prognosis and/or treatment of a disease or pathology.

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.

In an embodiment of the aforementioned use, the kit is used in the diagnosis, prognosis and/or treatment of cancer.

In the context of the present invention, prognosing an individual suffering from or suspected to suffer from cancer refers to a prediction of the survival probability of individual having cancer or relapse risk which is related to the invasive or metastatic behavior (i.e. malignant progression) of tumor tissue or cells.

In an embodiment, the biomolecule comprised in the kit is directed against and/or specifically binds to a tumor-associated antigen (also called a "solid tumor-specific antigen", a "tumor-specific antigen", "tumor antigen", "target protein present on and/or specific for a (solid) tumor", "tumor-specific target (protein)". Such a tumor- associated antigen includes any protein which is present only on tumor cells and not on any other cell, or any protein, which is present on some tumor cells and also on some normal, healthy cells. Non-limiting examples of tumor antigens include tissue differentiation antigens, mutant protein antigens, oncogenic viral antigens, cancertestis antigens and vascular or stromal specific antigens. It is expected that the antibodies or antibody fragments as disclosed herein will bind to at least to those analogs, variants, mutants, alleles, parts and fragments of the tumor-associated antigen that (still) contain the binding site, part or domain of the natural tumor antigen to which those antibodies or antibody fragments bind. By "tumor(s)" are meant primary tumors and/or metastases (wherever located) such as but not limited to gliomas, pancreatic tumors; lung cancer, e.g. small cell lung cancer, breast cancer; epidermoid carcinomas; neuroendocrine tumors; gynaecological and urological cancer, e.g. cervical, uterine, ovarian, prostate, renalcell carcinomas, testicular germ cell tumors or cancer; pancreas cancer (pancreatic adenocarcinoma); glioblastomas; head and/or neck cancer; CNS (central nervous system) cancer; bones tumors; solid pediatric tumors; haematological malignancies; AIDS-related cancer; soft-tissue sarcomas, and skin cancer, including melanoma and Kaposi's sarcoma.

In a specific embodiment it should be clear that the therapeutic method of the present invention against cancer can also be used in combination with any other cancer therapy known in the art such as irradiation, chemotherapy or surgery.

In an embodiment, the lyophilized biomoleculecomprised in the kit is an immunoglobulin single variable domain. These antibody fragments have desirable properties, resulting in high tumor uptake values, low healthy tissue uptake values and fast clearance from the blood and healthy tissues in a subject in need thereof, in particular in human patients in need thereof. Furthermore, through their high specificity and thus their high sensitivity for tumor cells, the immunoglobulin single variable domains as disclosed herein suggest a potential for either a lower dosage and/or a more accurate detection at the same dose, implying a reduction of unwanted side-effects and reduced toxicity, compared to known diagnostic imaging agents for determining cancer.

In an embodiment of the aforementioned use, the composition or kit comprises a biomolecule directed against and/or specifically binding to HER2, for the diagnosis, prognosis and/or treatment of HER2 overexpressing tumors, such as HER2 overexpressing breast cancer and/or HER2 overexpressing brain metastasis.

As used herein, the term "HER2 overexpressing" refers to cancerous or malignant cells or tissue characterized by HER2 gene amplification or HER2 protein overexpression and thus have abnormally high levels of the HER2 gene and/or the HER2 protein compared to normal healthy cells. HER2 overexpressing breast cancer characterized by cancerous breast cells is characterized by HER2 gene amplification or HER2 protein overexpression. In about 1 of every 5 breast cancers, the cancer cells make an excess of HER2, mainly caused by HER2 gene amplification due to one or more gene mutations. The elevated levels of HER2 protein that it causes can occur inmany types of cancer - and are thus not limited to breast cancer.

"Metastasis" is the term for the spread of cancer beyond its originating site in the body. Thus, metastatic lesions are cancerous tumors that are found in locations apart from the original starting point of the primary tumor. Metastatic tumors occur when cells from the primary tumor break off and travel to distant parts of the body via the lymph system and blood stream. Alternately, cells from the original tumor could seed into new tumors at adjacent organs or tissues.

The importance of HER2 as a prognostic, predictive, and therapeutic marker for certain types of cancer, and in particular, for invasive breast cancer, is well recognized, and therefore, it is critical to validate and standardize testing techniques in order to make an accurate assessment of the HER2 status. There are however significant contradictions among the outcomes of known available tests.

Therefore, by providing a kit for improved labeling of biomolecules directed against and/or specifically binding to HER2, the present invention meets the high need for a reproducible, high-throughput and highly sensitive diagnostic tools and assays for diagnosis and prognosis of HER2 related cancers.

PET imaging of HER2 provides strong quantification ability. Information regarding HER2 expression not only in primary tumors but also in distant metastases not amenable to biopsy may for instance reduce problems with false negative results and help in the diagnosis and prognosis of cancer. Real-time assays of overall tumor HER2 expression in patients allows to more accurately stratify patients and adjust therapy accordingly. HER2-PET and HER2-SPECT are particularly useful in real-time assays of overall tumor HER2 expression in patients, identification of HER2 expression in tumors over time, selection of patients for HER-targeted treatment (e.g., trastuzumab-based therapy), prediction of response to therapy, evaluation of drug efficacy, and many other applications. In an embodiment, the presence and/or relative percentage of HER2-expressing cells is determined to diagnose or prognose cancer. In an embodiment, the presence and/or relative percentage of HER2- expressing cells is determined to diagnose or prognose HER2 overexpressing breast cancer. In an embodiment, the presence and/or relative percentage of HER2- expressing cells is determined to diagnose or prognose HER2 overexpressing brain metastasis.

In another embodiment, the biomolecule comprised in the kit is directed against/specifically binds to HER2 in order to suppress the HER2 pathway and treat HER2 overexpressing tumors, such as HER2 overexpressing breast cancer and/or HER2 overexpressing brain metastasis.

Mechanisms to treat HER2 overexpressing tumors include for instance activation of antibody-dependent cellular cytotoxicity, inhibition of extracellular domain cleavage, abrogation of intracellular signaling, reduction of angiogenesis, and decreased DNA repair. These effects lead to tumor cell stasis and/or death. Targeting both HER2, with various approaches, and other pathways may enhance the clinical benefit and overcome potential resistance. In an embodiment, HER2 overexpressing tumors are targeted by a combination of mechanisms, such as inhibition of HER2 dimerization, HER1/HER2 tyrosine kinase inhibition, antiangiogenic mechanisms, heat shock protein inhibition and antiestrogen therapies. In an embodiment, an antibody-drug conjugate is used to target HER2 overexpressing tumors.

In another embodiment of the aforementioned use, the kit comprises a biomolecule directed against and/or specifically binding to MMR, for the targeting of MMR-positive tumor-associated macrophages (TAMs) inside a tumor.

Tumor-associated macrophages (TAMs) are an important component of the tumor stroma, both in murine models and human patients. TAMs can promote tumorgrowth by affecting angiogenesis, immune suppression and invasion and metastasis. The plasticity of macrophages offers perspectives for using them as in vivo sensors for the tumor microenvironment they are exposed to. As a matter of fact, at the tumor site, these cells are confronted with different tumor microenvironments, leading to different TAM subsets with specialized functions and distinct molecular profiles. For example, in mammary tumors, at least two distinct TAM subpopulations have been described, based on a differential expression of markers such as the macrophage mannose receptor (MMR or MHC II), differences in pro-angiogenic or immunosuppressive properties and intratumoral localization (normoxic/perivascular tumor areas versus hypoxic regions).

MMR-high TAMs are associated with hypoxic regions in the tumor, as illustrated in human breast cancer samples. This finding demonstrates the clinical relevance of targeting MMR-positive TAM subpopulations in the tumor stroma. Hence, the development of a labeling method, which facilitates the development, usage, distribution and commercialization of such a kit comprising a biomolecule directed against and/or specifically binding to MMR, is of great interest. In an embodiment of the aforementioned use, the kit is used in the diagnosis, prognosis and/or treatment of a cardiovascular disease.

In an embodiment, after the lyophilized biomolecule is reconstituted and labeled with a detectable label, for instance a radionuclide or a bimodal label, the final solution thus obtained can be administered to a subject suffering from or suspected to suffer from cardiovascular disease in order to diagnose or prognose cardiovascular disease.

Within the context of the disclosure, the term "cardiovascular disease," refers to an illness, injury, or symptoms related to an atherogenic process affecting the cardiovascular system. This includes the different stages marking the development of atherosclerotic plaques (different stages of plaques are classified according to guidelines such as those from the American Heart Association: neo-intimal, atheromatous, fibroatheromatous and collagen-rich lesions), as well as complications arising from the formation of an atherosclerotic plaque (stenosis, ischemia) and/or the rupture of an atherosclerotic plaque (thrombosis, embolism, myocardial infarction, arterial rupture, acute ischemic stroke). Cardiovascular disease refers, for example, to atherosclerosis, atherosclerotic plaques, especially the vulnerable plaques, coronary heart disease, thrombosis, stroke, myocardial infarction, vascular stenosis. Cardiovascular disease also refers to downstream complications of myocardial infarction or "post-infarction" complications due to ruptured plaques, including cardiac remodeling and cardiac failure.

In an embodiment, the kit is used in the diagnosis, prognosis and/or treatment of atherosclerosis.

"Atherosclerosis" herein refers to a disease affecting arterial blood vessels. Atherosclerosis can be characterized by a chronic inflammatory response in the walls of arteries, mainly due to the accumulation of macrophages and promoted by low density lipoproteins. The appearance of atherosclerotic plaques is a marker of atherosclerosis (also known as arteriosclerotic vascular disease or ASVD), which in itself is a typical cardiovascular disease and may lead to different cardiovascular complications, as described further herein.

According to one embodiment, the diagnosing and/or prognosing of a cardiovascular disease, in particular atherosclerosis, will preferably be done by detecting the presence or absence of atherosclerotic plaques, in particular vulnerable atherosclerotic plaques. In an embodiment, the aforementioned kit is used in targeting and/or detecting vulnerable atherosclerotique plaques.

As used herein, the term "atherosclerotic plaque," refers to a deposit of fat and other substances that accumulate in the lining of the artery wall. The terms "vulnerable atherosclerotic plaque" or "unstable atherosclerotic plaque" are used interchangeably herein and refer to atherosclerotic plaques with high likelihood of rapid progression and cardiovascular disease manifestations, including myocardial infarction and/or acute ischemic stroke. Unstable plaques are characterized by a large, soft lipid core that contains extracellular lipids and is covered by a thin fibrous cap, as well as an abundance of invasive inflammatory cells such as macrophages. In contrast, stable plaques have a small lipid core, thick fibrous caps, and little or no macrophage invasion with the development of fibrous tissue resulting in intimal thickening of the vessel. Atherosclerotic plaques formed by lipid accumulation in vessel lesions have a variety of characteristics, ranging from stable to unstable. Unstable plaques are prone to rupture followed by thrombus formation, vessel stenosis, and occlusion and frequently lead to acute myocardial infarction (AMI) and brain infarction. Thus, the specific diagnosis of unstable plaques would enable preventive treatments for AMI and brain infarction and represents a promising diagnostic target in clinical settings.

The anti-MMR immunoglobulin single variable domains, as described hereinbefore, are particularly useful as contrast agent in non-invasive in vivo medical imaging, in particular for the targeting and/or detection of vulnerable atherosclerotique plaques.

Preferably, a nuclear imaging approach is used. According to one specific embodiment, positron emission tomography (PET) is used for in vivo imaging with labeled anti-MMR immunoglobulin single variable domains. Alternatively, single photon emission computed tomography (SPECT) is used as in vivo imaging approach. Thus, in one embodiment, the anti-MMR immunoglobulin single variable domains, as described hereinbefore, are coupled to a radionuclide. It may be of additional advantage that the evolution of the degree of vulnerability of atherosclerotic plaques can be monitored in function of time. More specifically, the disclosure allows to monitor progression or regression of vulnerability of atherosclerotic plaques in function of time. Hereby, different stages of plaques are classified according to guidelines such as those from the American Heart Association: neo-intimal, atheromatous, fibroatheromatous and collagen-rich lesions. A further advantage of the disclosure is the possibility to assess the impact of a therapy on atherosclerosis and/or the degree of vulnerability of atherosclerotic plaques and/or the evolution in function of time of the degree of vulnerability of atherosclerotic plaques, by making use of the anti-MMR immunoglobulin single variable domains, as described hereinbefore.

In another preferred embodiment, the biomolecule as used in the present invention is coupled to or fused to a moiety, in particular a therapeutically active agent, either directly or through a linker. As used herein, a "therapeutically active agent" means any molecule that has or may have a therapeutic effect (i.e. curative or stabilizing effect) in the context of treatment of a cardiovascular disease, in particular of atherosclerosis, preferably vulnerable plaques, or of a postinfarction event such as cardiac remodeling or heart failure.

Preferably, a therapeutically active agent is a disease-modifying agent, which can be a cytotoxic agent, such as a toxin, or a cytotoxic drug, or an enzyme capable of converting a prodrug into a cytotoxic drug, or a radionuclide, or a cytotoxic cell, or which can be a non-cytotoxic agent. Even more preferably, a therapeutically active agent has a curative effect on the disease.

Alternatively, a therapeutically active agent is a disease-stabilizing agent, in particular a molecule that has a stabilizing effect on the evolution of a cardiovascular disease, in particular atherosclerosis, and more specifically, a stabilizing effect on vulnerable atherosclerotic plaques. Examples of stabilizing agents include antiinflammatory agents, in particular non-steroid anti-inflammatory molecules. According to one specific embodiment, the therapeutically active agent is not a cytotoxic agent.

In an embodiment of the aforementioned use, the kit is used in the diagnosis, prognosis and/or treatment of a viral disease.

In an embodiment, the biomolecule (such as an immunoglobulin single variable domain) is directed against a viral antigen. Antibodies are an important component in host immune responses to viral pathogens. Because of their unique maturation process, antibodies can evolve to be highly specific to viral antigens. Strategies for generation of therapeutic antibodies for viral infections are known from the state of the art and include for instance phage displayed antibody libraries, the isolation of mAbs from single-memory B cells, cloning IgG from single-antibody-secreting plasma B cells, proteomics-directed cloning of mAbs from serum and deep sequencing of paired antibodies encoding genes from B cells. By "viral disease" is meant a disease caused by a viral infectious agent such as but not limited to human cytomegalovirus, influenza, human immunodeficiency virus, respiratory syncytial virus, ebola, zika, rabies, hepatitis B virus and dengue. In an embodiment of the aforementioned use, the kit is used in the diagnosis, prognosis and/or treatment 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.

The invention is further described by the following non-limiting examples which further illustrate the invention, and are not intended to, nor should they be interpreted to, limit the scope of the invention

EXAMPLES

Material and Methods

All commercially obtained chemicals were of analytic grade. The recombinant anti- HER2 and 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.

4.1 Conjugation of p-SCN-Bn-NOTA to sdAb protein

SdAb proteins (Anti-HER2: 3 - 13mg, 0.24 - 1.03 pmol; 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 twenty-fold (anti-HER2) or thirty-fold (anti-MMR) 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-HER2 sdAb: E = 49690 M-l.cm-1, MW = 13310 g/mol; NOTA-anti-MMR sdAb: E = 40660 M-l.cm-1, MW = 13130 g/mol). 4.2 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, o = 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).

4.3 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).

4.4 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).

4.5 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 HER2Fc or recombinant hMMR via l-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-HER2-(HIS)6 or anti-MMR-(HIS)6 sdAb, stored at -20°C, was added during each run.

4.6 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.

4.7 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. 4.8 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.

DESCRIPTION OF FIGURES

Figure 1 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.

Examples

2.1 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.

2.1.1 Compatibility test

AA was tested for radiolabeling compatibility by performing a radiolabeling with different concentrations in the buffer (Table 1). The RCP was analyzed 10 min and 3h post labeling.

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

2.1.2 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 (Table 2). 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.

2.1.3 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 (Table 3).

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 (Table 4), 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.

2.2 Ethanol

2.2.1 Compatibility test

In a first test, two labelings were performed to assess the compatibility of ethanol with the radiolabeling (Table 5). 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.

2.2.2 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 6).

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.

2.3 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.

2.3.1 sdAb protein compatibility testing

SDS-PAGE was performed for both the HER2 and 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 7). 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 HER2 sdAb at 60% ethanol content and 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.

2.3.2 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 8).

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, while more than 40% remained for the NOTA-HER2 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)

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 9). The affinity (a measurement for functionality) is represented as dissociation constant kD (koff rate/kon 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. Comparable results were obtained for the NOTA-HER2 precursor, confirming the compatibility of these precursors with this ethanol content. The increased kD of the NOTA-HER2 exposed to 20% ethanol is within the error margin of the measurement.

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. 2.3.4 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 10). 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-β-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).

2.4 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.

2.4.1 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 11). 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 12).

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

2.4.2 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 13). 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.

2.5 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.

2.5.1 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 14).

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.

2.5.2 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 1A). No particles above 3 nm Dh were measured, which suggests a clear and pure solution and no microprecipitation of any of the compounds (Fig IB). 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.

Table 1 : Compatibility test of AA

Table 2: Ascorbic acid stability testing

Table 3: Consistency study AA

Table 4: Effect of AA incubation time on radiolabeling

Table 5: Compatibility test ethanol

Table 7: Effect of ethanol on protein aggregation via SDS-PAGE

Table 8: Effect of ethanol on protein aggregation

Table 9: Effect of ethanol on NOTA-sdAb precursor functionality via SPR

Table 10: Combination study Ethanol - AA

Table 11: Volume study Ethanol 20% - 5 mg/ml AA

Table 12: Volume study Ethanol 20%- 5 mg AA (fixed)

Table 13: High activity study Ethanol 20% - 5 mg AA (fixed)

Table 14: Osmolality of different conditions.

CH

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Claims

1. Method for labeling a biomolecule, with one or more detectable labels, wherein at least one of said detectable labels is a radionuclide, said labeling occurs in the presence of ethanol and vitamin C, characterized in that the concentration of ethanol in the labeling reaction is between 5% v/v and 15% v/v and the concentration of vitamin C in the labeling reaction is between 0.2 mg/ml and 2.5 mg/ml.

2. Method for labeling a biomolecule, with one or more detectable labels, wherein at least one of said detectable labels is a radionuclide, said labeling occurs in the presence of ethanol and 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, 3-0- glycosyl-L-ascorbic acid, 6-O-acyl-2-O-a-D-glucopyranosyl ascorbic acid or a mixture thereof, characterized in that 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.

3. Method for labeling a biomolecule, with one or more detectable labels, wherein at least one of said detectable labels is a radionuclide, said labeling occurs in the presence of 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, characterized in that the concentration of said derivative of vitamin C in the labeling reaction is between 5 mg/ml and 150 mg/ml.

4. Method according to claim 1 or 2, wherein the ethanol concentration in the labeling reaction is between 8% v/v and 12% v/v.

5. Method according to any one of the previous claims, said radionuclide being chosen from the group of fluor 18 (¹⁸F), lutetium 177 (¹⁷⁷Lu), zirconium 89 (⁸⁹Zr), indium 111 (^mln), yttrium 90 (⁹⁰Y), copper 64 (⁶⁴Cu), actinium 225 (²²⁵Ac), bismuth 213 (²¹³Bi), gallium 67 (⁶⁷Ga), gallium 68 (⁶⁸Ga), technetium 99m (^99mTc), iodium 123 (¹²³I), iodium 124 (¹²⁴I), iodium 125 (¹²⁵I), iodium 131 (¹³¹I).

6. Method according to any one of the previous claims, wherein said biomolecule further comprises a fluorescent moiety as detectable label.

7. Method according to claim 6, wherein said fluorescent moiety is chosen from the group 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.

8. Method according to any one of the previous claims, wherein said biomolecule is coupled to a chelating agent chosen from the group 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 .

9. Method according to claim 8, wherein a radionuclide is coupled to said chelating agent.

10. Method according to any one of the previous claims 1 and 4-9, wherein said biomolecule is lyophilized and reconstituted with a labeling buffer prior to labeling, wherein vitamin C is co-lyophilized in an amount between 1 mg and 7 mg with said antibody or antibody fragment.

11. Method according to any one of the previous claims 2 to 9, wherein said biomolecule is lyophilized and reconstituted with a labeling buffer prior to labeling, wherein said derivative of vitamin C is co-lyophilized in an amount between 20 mg and 150 mg with said biomolecule.

12. Method according to any of the claims 10-11, wherein the biomolecule consists of an antibody or antibody fragment and wherein the amount of the antibody or the antibody fragment in the lyophilizate is between 7.5 nmoles and 750 nmoles.

13. Method according to any one of the previous claims, wherein said biomolecule is an immunoglobulin single variable domain.

14. Method according to any one of the previous claims, wherein a labeling buffer is used during the labeling reaction, wherein the labeling buffer is an acetate, phosphate, succinate, formate or a HEPES buffer.

15. Method according to claim 14, wherein ethanol is comprised in the labeling buffer at a concentration between 10% v/v and 30% v/v.

16. Method according to any one of the previous claims, wherein said biomolecule is directed against and/or specifically binds to one or more targets being linked to a disease or a pathology.

17. Method according to any one of the previous claims, wherein said biomolecule is directed against and/or specifically binds to HER2.

18. Method according to any one of the previous claims, wherein said biomolecule is directed against and/or specifically binds to MMR.

19. Method according to claim 18, wherein said biomolecule specifically targets MMR-positive tumor-associated macrophages (TAMs) inside a tumor.

20. Kit for labeling a biomolecule with one or more detectable labels, wherein at least one of said detectable labels is a radionuclide, said kit comprises lyophilized biomolecules in a lyophilizate, wherein said lyophilizate comprises vitamin C, wherein the amount of vitamin C in the lyophilizate is between 1 mg and 7 mg, said kit further comprises a stabilizing buffer, wherein said buffer comprises ethanol at a concentration between 10% v/v and 30% v/v.

21. Kit for labeling a biomolecule with one or more detectable labels, wherein at least one of said detectable labels is a radionuclide, said kit comprises lyophilized biomolecules in a lyophilizate, wherein said lyophilizate comprises a derivative of vitamin C, wherein the amount of the vitamin C derivative in the lyophilizate is between 20 mg and 150 mg, said kit further comprises a stabilizing buffer, wherein said buffer comprises ethanol at a concentration between 10% v/v and 30% v/v.

22. Kit according to claim 20 or 21, wherein said stabilizing buffer is an acetate, phosphate, succinate, formate or a HEPES buffer.

23. Kit according to any of the previous claims 20 to 22, wherein said biomolecule further comprises a fluorescent moiety as detectable label.

24. Kit according to any of the previous claims 20 to 23, wherein said biomolecule is an immunoglobulin single variable domain.

25. Kit according to any of the previous claims 20-24, wherein said biomolecule is an immunoglobulin single variable domain directed against and/or specifically binding to HER2.

26. Kit according to any of the previous claims 20-24, wherein said biomolecule is an immunoglobulin single variable domain directed against and/or specifically binding to MMR.

27. Kit according to any of the previous claims 20-26, wherein the biomolecule is an immunoglobulin single variable domain directed against and/or specifically binding to HER2 or MMR, wherein said immunoglobulin single variable domain is coupled to NOTA or DOTA as chelating agent and wherein the composition is suited for labeling with gallium 68.

28. Kit according to any of the previous claims 20-27, for use in non-invasive in vivo medical imaging.

29. Kit for use according to claim 28, wherein said kit comprises a biomolecule directed against and/or specifically binding to HER2, wherein HER2- expressing cells are visualized.

30. Kit for use according to claim 28, wherein said kit comprises a biomolecule directed against and/or specifically binding to MMR, wherein MMR-expressing cells, such as Tumor-Associated Macrophages, are visualized.

31. Kit according to claims 20-27 for use in the diagnosis, prognosis and/or treatment of a disease or pathology.

32. Kit for use according to claim 31, wherein said composition or kit is used in the diagnosis, prognosis and/or treatment of cancer.

33. Kit for use according to claim 32, wherein said kit comprises a biomolecule directed against and/or specifically binding to HER2, for the diagnosis, prognosis and/or treatment of HER2 overexpresssing tumors, such as HER2 overexpressing breast cancer and/or HER2 overexpressing brain metastasis.

34. Kit for use according to claim 32, wherein said kit comprises a biomolecule directed against and/or specifically binding to MMR, for the targeting of MMR- positive tumor-associated macrophages (TAMs) inside a tumor.

35. Kit for use according to claim 31, for use in the diagnosis, prognosis and/or treatment of a cardiovascular disease.

36. Kit for use according to claim 35, wherein said cardiovascular disease is atherosclerosis.

37. Kit for use according to claim 36, for use in targeting and/or detecting vulnerable atherosclerotique plaques.

38. Kit for use according to claim 31, for use in the diagnosis, prognosis and/or treatment of a viral disease.

39. Kit for use according to claim 31, for use in the diagnosis, prognosis and/or treatment of cardiac sarcoidosis.