WO2022156908A1

WO2022156908A1 - Method for preparing a lyophilized composition

Info

Publication number: WO2022156908A1
Application number: PCT/EP2021/051573
Authority: WO
Inventors: Henri BAUDHUIN; Philippe VANWOLLEGHEM
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 preparing a composition of biomolecules, selected from peptides, small molecules, scaffold proteins, antibodies or antibody fragments, wherein said composition is lyophilized, characterized in that a vitamin C derivative is used as a lyophilization excipient, wherein the vitamin C derivative is an ascorbyl glucoside selected from the group of 2-O- -D- glucopyranosyl ascorbic acid, 2-O- -D-glucopyranosyl ascorbic acid, 5-O- -D- glucopyranosyl ascorbic acid, 6-O- -D-glucopyranosyl ascorbic acid, 3-O-glycosyl-L- ascorbic acid, 6-O-acyl-2-O- -D-glucopyranosyl ascorbic acid or a mixture thereof. The current invention also relates to a composition or a kit of lyophilized biomolecules, said biomolecules are chosen from the list of peptides, small molecules, scaffold proteins, antibodies or antibody fragments, said lyophilizate further comprises a vitamin C derivative, wherein said vitamin C derivative is chosen from the group of 2-O- -D-glucopyranosyl ascorbic acid, 2-O- -D-glucopyranosyl ascorbic acid, 5-O- -D-glucopyranosyl ascorbic acid, 6-O- -D-glucopyranosyl ascorbic acid, 3-O-glycosyl-L-ascorbic acid, 6-O-acyl-2-O- -D-glucopyranosyl ascorbic acid or a mixture thereof.

Description

METHOD FOR PREPARING A LYOPHILIZED COMPOSITION

FIELD OF THE INVENTION

The present invention relates to a method for preparing a lyophilized composition of biomolecules. In a second aspect, the present invention also relates to a composition or kit comprising lyophilized biomolecules.

BACKGROUND

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.

Lyophilization excipients are compounds added to the lyophilization process to serve a specific function. They can for instance increase bulk, aid manufacturing, improve stability, enhance drug delivery and targeting, and modify drug safety or pharmacokinetic profile.

These biopharmaceuticals comprise biomolecules, for instance antibodies, as described in US20030068416.

A specific example of biopharmaceuticals that can be lyophilized are radiopharmaceuticals. Lyophilization of these radiopharmaceutical precursors offers the possibility of a kit development with the previously described advantages, further enhancing the practicality of these tracers and favoring their usage in clinic.

US20130310537 describes a method for preparing a radiopharmaceutical by eluting a ⁶⁸Ge/⁶⁸Ga-Generator and feeding the resulting eluate into a precursor mixture. This precursor mixture is prepared by mixing a lyophilized precursor mixture and the buffer salt using a solvent. In the case of radiopharmaceuticals, lyophilization excipients can, in addition to their cryo-and lyoprotective effect during and after the lyophilization process, also offer protection from radiolytic degradation during the labeling reaction with the radioactive compound.

Both in US '416 and US '537, the precursor mixture comprises a stabilizer for preventing radiolytic degradation of the radiopharmaceutical. Ascorbic acid, also known as 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 stabilizer during reactions with radioactive compounds.

However, vitamin C has a low stability in solution and high amounts of vitamin C can interfere with the labelling reaction, both of which complicate the use of vitamin C as stabilizer in a lyophilization formulation.

The aim of the invention is to provide an improved method for preparing a lyophilized composition of biomolecules.

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 preparing a composition of biomolecules, selected from peptides, small molecules, scaffold proteins, antibodies or antibody fragments, wherein said composition is lyophilized, wherein a vitamin C derivative is used as a lyophilization excipient, wherein the vitamin C derivative is an ascorbyl glucoside selected from the group of 2-O-o-D-glucopyranosyl ascorbic acid, 2-O-p-D-glucopyranosyl ascorbic acid, 5-O-o-D-glucopyranosyl ascorbic acid, 6-O-o-D-glucopyranosyl ascorbic acid, 3-O-glycosyl-L-ascorbic acid, 6-O-acyl-2-O- o-D-glucopyranosyl ascorbic acid or a mixture thereof. Preferred embodiments of the method are shown in any of the claims 2 to 14.

In a second aspect, the present disclosure relates to a composition according to claim 15. More in particular, the present disclosure provides a composition of lyophilized biomolecules, said biomolecules are chosen from the list of peptides, small molecules, scaffold proteins, antibodies or antibody fragments, said lyophilizate further comprises a vitamin C derivative, wherein said vitamin C derivative is chosen from the group of 2-O-o-D-glucopyranosyl ascorbic acid, 2-O-p-D-glucopyranosyl ascorbic acid, 5-O-o-D-glucopyranosyl ascorbic acid, 6-O-o-D-glucopyranosyl ascorbic acid, 3-O-glycosyl-L-ascorbic acid, 6-O-acyl-2-O-o-D-glucopyranosyl ascorbic acid or a mixture thereof. Preferred embodiments of the composition are shown in any of the claims 16 to 26.

In a third aspect, the present invention relates to a kit according to claim 27. More in particular, the present disclosure provides a kit comprising one or more of the aforementioned compositions. Preferred embodiments of the kit are shown in any of the claims 28-29.

In a last aspect, the present invention relates to specific uses of the kit or composition according to claims 30-39.

The method according to the present disclosure is able to improve the lyophilization of a composition of biomolecules, such as antibodies or antibody fragments. Furthermore, by providing a composition and a kit which are relatively simple to use, the present disclosure facilitates development, usage, distribution and commercialization of lyophilized biomolecules.

DESCRIPTION OF FIGURES

Figure 1 illustrates a MDSC thermogram to determine the Tg' of a 5% AA-2G solution according to an embodiment of the current disclosure.

DETAILED DESCRIPTION OF THE INVENTION

The present invention concerns a method for preparing a composition of biomolecules, such as antibodies or antibody fragments, wherein said composition is lyophilized and wherein a vitamin C derivative is used as a lyophilization excipient. In addition, the present invention concerns a composition and a kit comprising the lyophilizate of the aforementioned method.

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.

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.

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

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 Hl, H2, H3 for the heavy chain and LI, L2 and L3 for the light chain), in the same way as a complete antibody.

"Immunoglobulin single variable domain" as used herein defines molecules wherein the antigen binding site is present on, and formed by, a single immunoglobulin domain (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 finished drug products 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.

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.

A practical limitation of many biomolecules such as antibodies and antibody fragments is their relatively low stability in solution, which requires storage at low temperature (-20°C) to obtain a reasonable shelf-life. A commonly applied solution for this limitation is to lyophilize these biomolecules. Lyophilization, a method to remove solvents (typically water) from solutions, is well-known to increase stability of pharmaceutical compounds, including peptides and proteins. 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.

Ascorbic acid (AA), also known as vitamin C, plays key roles in a variety of biological processes like collagen formation, carnitine synthesis, iron absorption, protection against cellular oxidative stress, drug metabolism and the function of the immune system.

Vitamin C is an FDA approved pharmaceutical suitable for human injection, with a low toxicity profile and its addition in formulations is easily justified. Additionally, vitamin C is compatible with lyophilization, which makes it a good candidate to function as lyophilization excipient.

However, vitamin C has a low stability in solution and high amounts of vitamin C interfere with various forms of labeling, such as radiolabeling, which complicates the use of vitamin C as stabilizer in the lyophilization formulation. In water, the degradation mechanism of ascorbic acid is well known in the literature. Ascorbic acid has two acidic protons on the C2-and C3-enolic hydroxyl groups conjugated with its Cl-carbonyl group. In acidic solutions, ascorbic acid (pKa = 4) can be deprotonated leading to the ascorbate. Ascorbate is an excellent reducing agent and readily undergoes two consecutive one-electron oxidations to form ascorbate radical then dehydroascorbic acid (DHA).

In an attempt to solve this instability issue, various AA derivatives, such as ascorbyl (acyl-, -phosphate, and -sulfate), ascorbyl palmitate, ascorbyl methyl and ascorbyl glucoside have been chemically or biologically synthetized. Ascorbyl glucoside or ascorbic acid glucoside is a derivative, where ascorbic acid is linked with a sugar molecule.

The inventors found that certain ascorbyl glucosides, namely 2-O-o-D- glucopyranosyl ascorbic acid (AA-2G), 2-O-p-D-glucopyranosyl ascorbic acid (AA- 2G), 5-O-o-D-glucopyranosyl ascorbic acid (AA-5G), 6-O-o-D-glucopyranosyl ascorbic acid (AA-6G), 3-O-glycosyl-L-ascorbic acid and 6-O-acyl-2-O-o-D- glucopyranosyl ascorbic acid, can be used as a stabilizing excipient for various biomolecules during lyophilization.

In addition, in contrast to vitamin C, these selected ascorbyl glucosides have a high stability in solution. Their high stability in solution is caused by the fact that a glucose group protects the hydroxyl groups; therefore, the degradation mechanism leading to the DHA is not be possible, 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 a first aspect, the invention provides a method for preparing a composition of biomolecules, selected from peptides, small molecules, scaffold proteins (such as DARPins, affibodies, monobodies, knottins), antibodies or antibody fragments, , wherein said composition is lyophilized and wherein a vitamin C derivative is used as a lyophilization excipient, wherein the vitamin C derivative is an ascorbyl glucoside selected from the group of 2-O-o-D-glucopyranosyl ascorbic acid, 2-O-p-D- glucopyranosyl ascorbic acid, 5-O-o-D-glucopyranosyl ascorbic acid, 6-O-o-D- glucopyranosyl ascorbic acid, 3-O-glycosyl-L-ascorbic acid, 6-O-acyl-2-O-o-D- glucopyranosyl ascorbic acid or a mixture thereof.

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.

In a preferred embodiment, the vitamin C derivative used as a lyophilization excipient is 6-O-acyl-2-O-o-D-glucopyranosyl ascorbic acid. In another preferred embodiment, 3-O-glycosyl-L-ascorbic acid is used as a lyophilization excipient. In another preferred embodiment, 2-O-o-D-glucopyranosyl ascorbic acid is used as a lyophilization excipient.

Often these biomolecules are used as targeting vehicles in non-invasive molecular imaging techniques or for targeted therapeutic applications.

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

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

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 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. In another embodiment, said detectable label is a radionuclide and said radionuclide is chosen from the group of fluor 18 (¹⁸F), lutetium 177 (¹⁷⁷Lu), zirconium 89 (⁸⁹Zr), indium 111 (ⁱⁿln), 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 biomolecule is coupled or fused directly to said radionuclide. In another embodiment, the biomolecule 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.

However, 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).

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.

Ascorbic acid is also a well-known and potent natural antioxidant and has the ability to protect other molecules (e.g. DNA, proteins...) from highly reactive or oxidizing agents, such as free radicals. Therefore, ascorbic acid has been proposed as alternative buffer system for metalloradiopharmaceuticals. Indeed, with a pKa of 4.2, ascorbic acid can offer, along with its salt-form sodium ascorbate, ideal buffering capacity in the pH range of 3.5 - 5.0, which is the typical range in which radiolabelings of metallic radionuclides is carried out, for instance 68Ga-NOTA radiolabeling. However, as discussed above, vitamin C has a low stability in solution and high amounts of vitamin C interfere with the radiolabeling reaction.

The inventors found that, besides the excellent stabilizing properties during lyophilization, the aforementioned selected ascorbyl glucosides have anti-oxidant properties and offer a radioprotective effect during labeling with a radionuclide, enhancing the radiochemical purity (RCP) and reducing radiolysis.

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

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

In an embodiment, the biomolecules are conjugated to a chelating agent before lyophilization. Chelating agents are bifunctional linkers, linking the biomolecule with the detectable label. For instance, chelating agents for radiolabeling 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 the 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. 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, wherein the chelating agent is selected from the group of DTPA (Diethylentriaminepentaacetic acid) and derivatives (including 1B4M-DTPA derivatives and CHX-A"-DTPA derivatives), DOTA (l,4,7,10-Tetraazacyclododecane-l,4,7,10-tetraacetic acid) and derivatives (including DOTA-GA derivatives, DOTAM derivatives, DO3A and derivatives, DO2A and derivatives, CB-DO2A derivatives and DO3AM derivatives) NOTA (l,4,7-Triazacyclononane-l,4,7-triacetic acid) and derivatives (including NODA derivatives, NODA-GA derivatives, NO2A derivatives, NOTAM derivatives, NOPO derivatives and TRAP derivatives), HBED (N,N-bis(2- hydroxybenzyl)ethylenediamine-N,N-diacetic acid) and derivatives (including HBED-CC derivatives, HBED-CI derivatives, HBED-CA derivatives, HBED-AA derivatives and SHBED derivatives), DEPA (7-[2-(bis-carboxymethyl-amino)-ethyl]- 4,10-bis-carboxymethyl-l,4,7,10-tetraaza-cyclododec-l-yl-acetic acid) and derivatives, picolinic acid (PA) based chelators and derivatives (including H2dedpa, H4octapa, H2azapa and H5decapa and derivatives), HEHA (1,2,7,10,13- hexaazacyclooctadecane-l,4,7,10,13,16-hexaacetic acid) and derivatives, TETA (l,4,8,ll-tetraazacyclotetradecane-l,4,8,ll-tetraacetic acid) and derivatives (including TE2A derivatives, CB-TE2A derivatives, CB-TE1A1P derivatives, CB-TE2P derivatives, MM-TE2A derivatives and DM-TE2A derivatives), NETA ([2-(4,7-bis- carboxymethyl-[l,4,7]triazacyclononan-l-yl-ethyl]-2-carbonylmethyl-amino]- tetraacetic acid) and derivatives (including C-NETA derivatives and NE3TA derivatives), AAZTA (l,4-bis(carboxymethyl)-6-[bis(carboxymethyl)]amino-6- methylperhydro-l,4-diazepine) and derivatives, DATA (6-amino-l,4-diazepine- triacetic acid) and derivatives, TCMC (l,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, ared 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 an embodiment, the detectable label is a bimodal label comprising a radionuclide and a fluorescent moiety.

A radioactive and fluorescent signature can be integrated in a single bimodal/hybrid label. Integration ensures colocalization of the two signatures and promotes an advanced form of symbiosis (the best of both worlds) that empowers 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., 0 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 a preferred embodiment, the vitamin C derivative is present in the lyophilizate in an amount of between 20 mg and 150 mg, more preferably between 20 mg and 120 mg, more preferably between 20 mg and 80 mg, such as 50 mg. This amount showed the best results in an optimization study regarding the lyophilization formulation and protocol, whereby the dried samples provided an elegant white cake. In addition, Modulated Differential Scanning Calorimetry (MDSC) analysis showed a high glass transition temperature (Tg') and Karl Fischer titration revealed a low residual moisture (RM) when the vitamin C derivative was present in said amount. In addition, the lyophilized samples with the aforementioned lyophilization formulation were stored at 2 - 8°C and analysis over time demonstrated an excellent stability.

In addition, by using this amount of the vitamin C derivative in a lyophilization buffer, radiolysis in the radiolabeling studies could be maximally prevented, whilst no negative effects of the vitamin C derivative could be observed. A too high amount of the vitamin C derivative interferes with the radiolabeling reaction in radiolabeling studies. Obviously, if the amount was too low, no radioprotective effect was observed.

In addition, a radiolabeling study revealed that much higher amounts of the aforementioned vitamin C derivatives could be used compared to native vitamin C before significantly reducing the radiochemical purity (RCP). 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 rad io protectant 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.

In an embodiment the RCP, the amount of free radionuclide and the amount of radiolysis in a radiolabeling study are assessed by iTLC. In an embodiment the RCP, the amount of free radionuclide and the amount of radiolysis in a radiolabeling study are assessed by SEC. The presence of the vitamin C derivative in the reconstituted lyophilized composition prevents radiolytic damage induced by the radioactive label during and after labeling of the biomolecule with the radionuclide. The aforementioned vitamin C derivatives show an enhanced stability in solution compared to native vitamin C and are thus more adequate for long term radioprotection of these biomolecules. This property is especially useful for radioprotection when a long-lived isotope is used for radiolabeling. In an embodiment, the radionuclide is a gallium radioisotope solution obtained directly from a gallium radionuclide generator.

Radiopharmaceuticals, such as these radioactively labeled tracers, often rely on peptides as targeting vehicles for the delivery of the radionuclide. In an embodiment, the biomolecule is an antibody or an antibody fragment, such as an immunoglobulin single variable domain.

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).

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 fragments 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. 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.

In the case wherein the biomolecule is an antibody or antibody fragment, "specifically binding to" 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 biomolecules can 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 an embodiment, the biomolecule, preferably an antibody or an antibody fragment, is 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 HER.2, comprises the following sequence: "DVQLQESGGGSVQAGGSLKLTCAASGYIFNSCGMGWYRQSPGRERELVSRISGDGDTWH KESVKGRFTISQDNVWKKTLYLQMNSLKPEDTAVYFCAVCYNLETYGQGTQVTVSS".

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 an embodiment, the biomolecule, preferably an antibody or an antibody fragment, 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 va riety 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".

In a second aspect, the invention provides a composition of lyophilized biomolecules, said biomolecules are chosen from the list of peptides, small molecules, scaffold proteins, antibodies or antibody fragments, said lyophilizate further comprises a vitamin C derivative, wherein said vitamin C derivative is chosen from the group of 2-O-o-D-glucopyranosyl ascorbic acid, 2-O-p-D-glucopyranosyl ascorbic acid, 5-0- o-D-glucopyranosyl ascorbic acid, 6-O-o-D-glucopyranosyl ascorbic acid, 3-0- glycosyl-L-ascorbic acid, 6-O-acyl-2-O-o-D-glucopyranosyl ascorbic acid or a mixture thereof. In a preferred embodiment, said vitamin C derivative is present in said composition in an amount of between 20 to 150 mg.

In an embodiment, said biomolecule is coupled to a chelating agent, wherein the chelating agent is 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.

In an embodiment, the biomolecule is an antibody or antibody fragment, such as an immunoglobulin single variable domain.

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 antibodies or antibody fragments.

In an embodiment, said antibodies or antibody fragments are present in the composition in a quantity of between 7.5 nmoles and 750 nmoles. In an embodiment, when the composition is intended for radiolabeling after reconstitution, said antibodies or antibody fragments are 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 the composition is intended to be labeled with a fluorescent moiety after reconstitution, said antibodies or antibody fragments are present in the composition in a quantity of between 75 nmoles and 750 nmoles.

Providing such an amount of antibodies or antibody fragments is able to overcome the possible interference caused by the vitamin C derivative on the labeling reaction.

In an embodiment of the composition, the biomolecule is labeled with a detectable label. In an embodiment of the composition, said detectable label is a fluorescent label 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.

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

Since immunoglobulin single variable domains are derived from antibodies, they can be generated to virtually any target. This provides the potential for a wide field of applications, which lead, along with their favorable characteristics and compatibility with versatile (radio)chemistry to the development of a variety of immunoglobulin single variable domain-based tracers for both imaging (molecular characterization, diagnosis, image-guided surgery) and targeted radionuclide therapy (TRNT).

It will be clear to a skilled person that the antibody or antibody fragment used in the context of the current invention may be able to bind to any epitope that is considered useful for the context of the current invention. In an embodiment of the composition, the biomolecule, preferably an antibody or an antibody fragment such as an immunoglobulin single variable domain, is directed against and/or specifically binds to HER2.

In another embodiment of the composition, the biomolecule, preferably an antibody or an antibody fragment such as an immunoglobulin single variable domain, is directed against and/or specifically binds to MMR.

In an embodiment of the composition, the biomolecule is an immunoglobulin single variable domain, wherein this immunoglobulin single variable domain is conjugated to a chelating agent and wherein the composition is suited for labeling with a radionuclide, such as gallium 68.

In an embodiment of the composition, 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.

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 biopharmaceutical with minimal GMP license. As such, they allow multi-center studies in development phase and international distribution and commercialization upon market approval.

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

In a third aspect, the invention provides a kit comprising one or more of the aforementioned compositions. In an embodiment, the kit further comprises a stabilizing buffer. The stabilizing buffer can be any buffer known from the state of the art suited for this purpose. In an embodiment the lyophilized precursor sample, comprising the biomolecules and a vitamin C derivative, is reconstituted with a certain volume of the stabilizing buffer and labeled with an equal volume of a detectable label in solution.

Suitable pharmaceutically acceptable buffers for incorporation into the kit include inorganic and organic buffers. Examples of inorganic buffers include phosphate buffers, such as sodium phosphate, sodium phosphate dibasic, potassium phosphate and ammonium phosphate; bicarbonate or carbonate buffers; succinate buffers such as disodium succinate hexahydrate; borate buffers such as sodium borate; cacodylate buffers; citrate buffers such as sodium citrate or potassium citrate; sodium chloride, zinc chloride or zwitterionic buffers. Examples of organic buffers include tris (hydroxymethyl) aminomethane (TRIS) 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 an embodiment of the kit, the stabilizing buffer is chosen from an acetate, phosphate, succinate, formate or a HEPES buffer.

This buffer is used to dilute or reconstitute the 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 biopharmaceuticals. 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 of the kit, said buffer comprises ethanol at a concentration between 10% v/v and 30% v/v. In an embodiment, the ethanol concentration in the stabilizing buffer is between 12% v/v and 28% v/v, more preferably between 15% v/v and 25% v/v, such as 20% v/v.

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 the context of radiolabeling 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 can even significantly improve labeling efficiencies of radiometals.

Studies have indicated that ethanol at this concentration range in the buffer is able to offer high radioprotection during the radiolabeling reaction.

In the case wherein the biomolecule is an antibody or an antibody fragment, lower concentrations are not adequate to offer sufficient anti-radiolytic protection, whereas higher concentrations of ethanol cause precipitation of the antibody or antibody fragment. 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 stabilizing buffer exceeding 20% v/v induce precipitation of the antibody or antibody fragment. In an embodiment the effect of the radioprotectant on the functionality of the antibody or antibody fragment is assessed via Surface Plasmon Resonance (SPR). SPR is able to measure the affinity of the antibody or antibody fragment by determining the dissociation constant (kD), where a lower kD is correlated with a higher affinity and vice versa.

Further embodiments of the composition and kit are as described above in the context of the lyophilization methodology.

In an embodiment the lyophilizate is reconstituted and labeled with a detectable label, such as 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, preferably below 1000 mOsm/kg. 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 aforementioned composition or kit is used in non-invasive in vivo medical imaging or Targeted Radionuclide Therapy (TRNT).

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 composition or kit comprises a biomolecule, preferably an immunoglobulin single variable domain, directed 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, high spatial resolution. Hence, the development of a lyophilization method, which facilitates the development, usage, distribution and commercialization of such an antibody or antibody fragment for use in non-invasive in vivo medical imaging of HER2, is of great interest.

In addition, immunoglobulin single variable domains 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, preferably an immunoglobulin single variable domain directed against and/or specifically binding to MMR, wherein MMR-expressing cells, such as Tumor- Associated Macrophages, are visualized.

In an embodiment, the lyophilized antibody fragment 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 immunoglobulin single variable domain 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 composition or 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 composition or 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 is directed against and/or specifically binds to a tumor-associated antigen (also called a "solid tumor-specific antigen", a "tumorspecific 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, cancer-testis antigens and vascular or stromal specific antigens. It is expected that the biomolecules 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 biomolecules 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 of the aforementioned use, the composition or kit comprises a biomolecule, preferably an immunoglobulin single variable domain, directed against and/or specifically binding to HER.2, for the diagnosis, prognosis and/or treatment of HER.2 overexpressing tumors, such as HER.2 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 in many 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, with the biomolecule 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 specifically targets HER2 overexpressing tumors, such as HER2 overexpressing breast cancer and/or HER2 overexpressing brain metastasis in order to suppress the HER2 pathway and treat HER2 overexpressing tumors.

Mechanisms to target 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 anti-estrogen therapies. In an embodiment, an antibody-drug conjugate is used to target HER2 overexpressing tumors.

In an embodiment of the aforementioned use, the composition or kit comprises a biomolecule, preferably an immunoglobulin single variable domain, 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.

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

In an embodiment, after the lyophilized biomolecule, preferably an antibody or an antibody fragment, is reconstituted and labeled with a detectable label, for instance a radionuclide, 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. 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.

"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. 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 "instable 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 herein before, 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 composition or kit is used in the diagnosis, prognosis and/or treatment of a viral disease. In an embodiment, the biomolecule in the composition is an antibody or an antibody fragment (such as an immunoglobulin single variable domain) 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 singlememory 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

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.

EXAMPLE 1: Ascorbic acid glucoside as radioprotectant

A first exploratory study was performed to assess the potency of AA-2G as radioprotectant and its potential interference with ⁶⁸Ga labeling. Different conditions, including the combination with ethanol, were tested. The RCP was analyzed via iTLC and SEC 10 minutes and 3 hours post labeling (Table 1).

Table 1 AA-2G exploratory study with high activity.

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

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

EXAMPLE 2: Ascorbic acid glucoside as lyo-/cryoprotectant

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

A first characterization of AA-2G was performed which serve as a basis to design a new lyophilization formulation. A 5% AA-2G solution was analyzed via MDSC to determine its Tg' (Figure 1), which showed to be between -30 and -35°C. This Tg' potentially allows us to apply a previously developed drying cycle (Table 2). As such, samples containing NOTA-anti-HER2 precursor were lyophilized with a 1 ml 5% AA- 2G formulation for a first stability study. Additionally, a few blanc samples with 1 ml of a 5% AA-2G/5% mannitol formulation were also lyophilized.

Table 1 Freeze-drying cycle applied for AA-2G-based formulations

Visually, the dried samples provided an elegant white cake. The 5% AA-2G formulation showed some shrinking upon drying, leading to a detached pellet, while the samples containing mannitol provided a slightly more appealing cake structure without shrinking. MDSC analysis showed a high Tg of ~ 65°C for both formulations and a residual moisture of 2.3% and 2.7% for the 5% AA-2G and 5% AA-2G/5% mannitol, respectively. These results indicate that both formulations have the potential to act as freeze-dry formulation for the NOTA-sdAbs. The NOTA-anti-HER2 samples with the 5% AA-2G formulation were stored at 2 - 8°C and analyzed over time for stability (Table 3/ Table 4). Some analytical tests, however, such as UV spectrophotometry, SDS-PAGE or Western Blot cannot be performed, due to interference of AA-2G.

Table 3 Stability analyses NOTA-anti-HER2: Non-radioactive tests

Table 2 Stability analyses NOTA-anti-HER2: Radioactive tests

EXAMPLE 3: One-step labeling procedure

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

EXAMPLE 4: Two-step labeling procedure

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

DESCRIPTION OF FIGURES

Figure 1 illustrates an MDCS analysis to determine the Tg' of a 5% AA-2G solution according to the current invention. The Tg' showed to be between -30 and -35°C. Based on this Tg' it was decided to apply a previously developed drying cycle (table 2). Following the determination of the Tg', samples containing a NOTA-anti-HER2 precursor were lyophilized with a 1 ml 5% AA-2G formulation for a first stability study.

Claims

42

1. A method for preparing a composition of biomolecules, selected from peptides, small molecules, scaffold proteins, antibodies or antibody fragments, wherein said composition is lyophilized, characterized in that a vitamin C derivative is used as a lyophilization excipient, wherein the vitamin C derivative is an ascorbyl glucoside selected from the group of 2-O-o-D- glucopyranosyl ascorbic acid, 2-O-p-D-glucopyranosyl ascorbic acid, 5-O-a- D-glucopyranosyl ascorbic acid, 6-O-o-D-glucopyranosyl ascorbic acid, 3-0- glycosyl-L-ascorbic acid, 6-O-acyl-2-O-o-D-glucopyranosyl ascorbic acid or a mixture thereof.

2. Method according to claim 1, wherein said vitamin C derivative is present in the lyophilizate in an amount of between 20 mg and 150 mg.

3. Method according to any of the previous claims, wherein said biomolecule is labeled with a detectable label.

4. Method according to claim 3, wherein the detectable label is chosen from the group of a radionuclide, a fluorescent moiety, a phosphorescent label, a chemiluminescent label, a metal, a metal chelate, a metallic cation, a chromophore, an enzyme or a combination of one of the aforementioned labels.

5. Method according to claim 4, wherein said label is a fluorescent moiety 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.

6. Method according to any of the previous claims 3 to 5, wherein the lyophilized composition is prior to said labeling with said detectable label reconstituted with a buffer.

7. Method according to any of the previous claims 3 to 5, wherein the lyophilized composition is reconstituted in a buffer comprising said detectable label.

8. Method according to any of the claims 3 to 7, wherein said detectable label is a radionuclide, said radionuclide is chosen from the group of fluor 18 (¹⁸F), lutetium 177 (¹⁷⁷Lu), zirconium 89 (⁸⁹Zr), indium 111 (ⁱⁿln), 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).

9. Method according to any of the previous claims 3-7, wherein the detectable label is a bimodal label comprising a radionuclide and a fluorescent moiety. 43 Method according to any of the previous claims, wherein the biomolecule is coupled to a chelating agent, wherein the chelating agent is selected 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. Method according to any of the previous claims, wherein the biomolecule is an antibody or an antibody fragment, such as an immunoglobulin single variable domain. Method according to any of the previous claims, wherein the biomolecule is directed against and/or specifically binds to one or more targets being linked to a disease or a pathology. Method according to any of the previous claims, wherein the biomolecule, preferably an antibody or an antibody fragment, is directed against and/or specifically binds to HER2. Method according to any of the previous claims 1-12, wherein the biomolecule, preferably an antibody or an antibody fragment, is directed against and/or specifically binds to MMR. A composition of lyophilized biomolecules, said biomolecules are chosen from the list of peptides, small molecules, scaffold proteins, antibodies or antibody fragments, said lyophilizate further comprises a vitamin C derivative, wherein said vitamin C derivative is chosen from the group of 2-O-o-D-glucopyranosyl ascorbic acid, 2-O-p-D-glucopyranosyl ascorbic acid, 5-O-o-D-glucopyranosyl ascorbic acid, 6-O-o-D-glucopyranosyl ascorbic acid, 3-O-glycosyl-L-ascorbic acid, 6-O-acyl-2-O-o-D-glucopyranosyl ascorbic acid or a mixture thereof. Composition according to claim 15, wherein said vitamin C derivative is present in said composition in an amount of between 20 to 150 mg. Composition according to claim 15 or 16, wherein said biomolecule is coupled to a chelating agent and wherein the chelating agent is selected 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 44 derivatives, PCTA and derivatives, Macropa and derivatives, THP and derivates, DFO and derivatives, BCPA and derivatives. Composition according to any of the previous claims 15-17, wherein the biomolecule is an antibody or antibody fragment, such as an immunoglobulin single variable domain. Composition according to claim 18, wherein said antibodies or antibody fragments are present in the composition in a quantity of between 7.5 nmoles and 750 nmoles. Composition according to any of the previous claims 15-19, wherein said biomolecule is labeled with a detectable label. Composition according to claim 20, wherein said label is a fluorescent label chosen from the group of Xanthene, Cyanin, squaraines, dipyrromethene, tetrapyrrole, naphthalene, oxadiazole, naphthalene, coumarin, oxazine derivatives and fluorescent metals such as europium or others metals from the lanthanide series. Composition according to any of the previous claims 15-21, wherein the biomolecule is directed against and/or specifically binds to one or more targets being linked to a disease or a pathology. Composition according to any of the previous claims 15-22, wherein the biomolecule, preferably an antibody or an antibody fragment such as an immunoglobulin single variable domain, is directed against and/or specifically binds to HER2. Composition according to any of the previous claims 15-22, wherein the biomolecule, preferably an antibody or an antibody fragment such as an immunoglobulin single variable domain, is directed against and/or specifically binds to MMR. Composition according to any of the previous claims 15-22, wherein the biomolecule is an immunoglobulin single variable domain, wherein this immunoglobulin single variable domain is conjugated to a chelating agent and wherein the composition is suited for labeling with a radionuclide, such as gallium 68. Composition according to claim 25, 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. A kit comprising one or more compositions according to any of the claims 15

28. Kit according to claim 27 , further comprising a stabilizing buffer, wherein the stabilizing buffer is chosen from an acetate, phosphate, succinate, formate or a HEPES buffer.

29. Kit according to claim 28, wherein said buffer comprises ethanol at a concentration between 10% v/v and 30% v/v.

30. Composition or kit according to any one of the previous claims 15 to 29, for use in non-invasive in vivo medical imaging.

31. Composition or kit for use according to claim 30, wherein said composition or kit comprises a biomolecule, preferably an immunoglobulin single variable domain, directed against and/or specifically binding to HER.2, wherein HER2- expressing cells are visualized.

32. Composition or kit for use according to claim 30, wherein said composition or kit comprises a biomolecule, preferably an immunoglobulin single variable domain directed against and/or specifically binding to MMR, wherein MMR- expressing cells, such as Tumor-Associated Macrophages, are visualized.

33. Composition or kit according to any of the previous claims 15-29, for use in the diagnosis, prognosis and/or treatment of a disease or pathology.

34. Composition or kit for use according to claim 33, wherein said composition or kit is used in the diagnosis, prognosis and/or treatment of cancer.

35. Composition or kit for use according to claim 34, wherein said composition or kit comprises a biomolecule, preferably an immunoglobulin single variable domain, directed against and/or specifically binding to HER.2, for the diagnosis, prognosis and/or treatment of HER2 overexpressing tumors, such as HER2 overexpressing breast cancer and/or HER2 overexpressing brain metastasis.

36. Composition or kit for use according to claim 34, wherein said composition or kit comprises a biomolecule, preferably an immunoglobulin single variable domain, directed against and/or specifically binding to MMR, for the targeting of MMR-positive tumor-associated macrophages (TAMs) inside a tumor.

37. Composition or kit for use according to claim 33, wherein said composition or kit is used in the diagnosis, prognosis and/or treatment of a cardiovascular disease.

38. Composition or kit for use according to claim 33, wherein said composition or kit is used in the diagnosis, prognosis and/or treatment of a viral disease.

39. Kit for use according to claim 33, for use in the diagnosis, prognosis and/or treatment of cardiac sarcoidosis.