Materials and Methods for Medical Imaging

Technical field

The present invention relates generally to methods and materials for use in medical imaging, particularly Positron Emission Tomography (PET).

Background art

Positron Emission Tomography (PET) is a medical imaging technique that uses positron emitter labelled agents in trace amounts to image and probe the metabolism of tissue. PET provides a high level of spatial resolution and can be used to quantify, in-vivo, factors such as receptor density, proliferation and metabolism.

Proteins, including antibodies and antibody fragments, specifically recognize molecular disease markers such as growth factor receptors, which are over-expressed by tumour cells so there is great interest in producing radio-labelled proteins for disease diagnosis. The most commonly used PET isotope for labelling proteins is ¹²⁴ I. However ¹²⁴ I has several disadvantages, including a relatively low positron yield (23%), poor resolution due to the relatively high energy and so longer range of the positron in tissue and the presence of γ-ray emissions.

¹⁸ F is the most ideal PET isotope for imaging due to its simple decay and emission properties with high (97%) positron abundance and one of the lowest positron emission energies (0.635MeV max). The latter means that ¹⁸ F has one of the shortest positron linear ranges in tissues, hence producing very high resolution in PET imaging. In addition, all PET units with cyclotrons produce ¹⁸ F, so it is consequently the most readily available PET isotope. With the rapid expansion in PET centres in Europe currently underway, the development of simplified procedures for labelling targeted PET tracers is timely.

Monoclonal antibodies and derivatives such as diabodies have a high avidity for their target molecules but often are too large to be rapidly cleared from the bloodstream so unsuitable for direct labelling with ¹⁸ F which has a half-life of only 110min.

Although ¹⁸ F has a half-life of only 110min, which is short compared with the blood clearance rate of large proteins, affinity molecules such as affibodies and single chain

fragment variable (scFv) antibodies with molecular weight (mwt) of <60 kDa (or 15 nm when referring to nanoparticles) which is the renal clearance weight limit are cleared very rapidly from the blood ¹ (essential to enable localization of specifically bound antibody during imaging). Wu et al ¹ concluded fragments of this size are suitable for labelling with 1 ⁸ F. Recently, low mwt (7kDa) affinity proteins (< 60 amino acids), have been developed that show very rapid blood clearance and high target binding affinity ² . For example, anti HER-2 binding affibodies are commercially available (www.affibody.com).

Presently radio-labelling of proteins with ¹⁸ F requires the initial preparation of a ¹⁸ F- labelled group (e.g. p-[ ¹⁸ F]-fluorobenzoic acid), involving a multi-step procedure in which the ¹⁸ F ^" is introduced into the prosthetic component early. The preparation of these prosthetic groups is generally low yielding ( ^3"5 and refs therein), time consuming and requires expensive specialist equipment. Moreover, each protein molecule has only a single site to which ¹⁸ F can be attached. The high excess of substrate (i.e. unlabelled protein) inevitably decreases specific activity of the labelled protein (i.e. Radioactivity/unit mass).

An alternative method of radio-labelling of proteins using silicon-based fluoride acceptors ⁶ and methods for radio-labelling biotin using silanes or boroaryls ⁷ have been proposed. Similarly to the above methods requiring initial preparation of a ¹⁸ F-labelled group, each protein or other molecule has only a single site to which ¹⁸ F can be attached, inevitably decreasing the specific activity of the labelled protein.

The most commonly used PET tracer in oncology is [ ¹⁸ F]-FDG (2-[18F]fluoro-2-deoxy-D- glucose), a non-specific tracer that has been shown to be an effective tumour-imaging agent. Although FDG-PET is invaluable in the imaging of a number of tumours, it is not suitable for detection of all tumour types ⁸⁹ due to high uptake by normal tissues producing high background signal. It is also unable to differentiate some low/medium grade tumours from benign lesions ¹⁰ and its use is limited in the diagnosis of some recurrent tumours ¹¹ . Further, inflammation accumulates FDG so making it indistinguishable from tumour tissue. Other PET tracers are therefore required in oncology.

The HER-2 (erbB2/neu) gene, which codes for an epidermal growth factor receptor associated tyrosine kinase, is over expressed in 25% of breast cancer and is strongly associated with poor prognosis ¹² . Herceptin is very expensive (£20,000 per patient/year).

HER-2 negative cells have little or no response to the drug ¹³ whereas patients with breast cancers with high HER-2 expression derive clinical benefit from Herceptin ¹⁴ . Consequently the American Society of Clinical Oncology recommends over expression of HER2 should be evaluated on every primary breast cancer ¹⁵ to determine which patients may benefit from Herceptin.

Currently, determination of HER-2 requires invasive tumour biopsy. HER-2 status is then determined by one of several methods including fluorescent in-situ hybridisation (FISH) and immunohistochemistry. Herceptin is only effective against tumours that over-express the cell surface receptor ¹⁴ . Although FISH is commonly used to detect over expression of the HER-2 gene, it will not identify the 5-12% of breast tumours that do not exhibit gene amplification but over express HER-2 receptor and could benefit from Herceptin ¹⁶ . Thus accurate, non-invasive, ways of detecting cell surface receptor expression are required. PET may provide a solution.

Thus it can be seen that novel methods and materials for the convenient provision of radio-labelled conjugates with high specific activity would provide a contribution to the art.

Disclosure of the invention

The present invention now provides materials and methods suitable for the provision of high yielding radio-labelled conjugates which can serve as labelling moieties, for example for detecting diagnostic or prognostic markers.

In essence the labelling moiety is provided by use of a relatively large labelling 'platform' (such as a nanoparticle or dendrimer) providing a number of prosthetic groups. Some of these groups are conjugated to a target binding moiety (such as a peptide having binding affinity for the target) while others serve as positron emitter attachment sites (i.e. provide groups such as boronate groups that are readily fluorinated with ¹⁸ F ^' .).

Crucially, compared with current protein and peptide ¹⁸ F ^' radio-labelling techniques, the preparation of the labelling moiety does not require a multi-step synthesis of ¹⁸ F- prosthetic groups which must then be attached to the protein. In the present invention the platform is pre-prepared (and indeed preferably pre-attached to the target binding moiety) and the ¹⁸ F fluorination can be performed as a simple final step, which is a particular advantage when using short half-life isotopes.

Further, the use of the platform with multiple prosthetic groups can increase the specific activity of the final product composition, since each binding moiety can present multiple 1 ⁸ F binding sites. Thus a higher proportion of the binding moieties can be labelled on addition of ¹⁸ F than would be where the target binding moiety presented fewer ¹⁸ F binding sites. This in turn means that there is less dilution of PET-labelled target binders by unlabelled target binders.

Thus in preferred embodiments one or more of the disadvantages of the methods used in the art, as discussed above, may be obviated.

In various aspects the invention provides materials for preparing the radio-labelled conjugates, the conjugates themselves, and processes for preparing and using the same.

Some of these aspects will now be discussed in more detail.

Labelled moieties

Accordingly, one aspect of the invention provides a labelled platform conjugate, comprising

(i) a labelling platform, and

(ii) a target binding moiety, wherein the labelling platform has at least one positron emitter attached thereto.

Preferably the labelled platform conjugate is water-soluble.

Preferably the labelling platform conjugate has a molecular weight or size of less than the renal clearance limit so that the labelling platform conjugate may be rapidly cleared from the blood. By "renal clearance limit" is meant the maximum molecular weight or size at which molecules are filtered out of the blood in the kidney.

Preferably the labelling platform conjugate has a molecular weight less than 100 kDa, more preferably less than 90, 80, 70, 60 or 50 kDa, most preferably less than 40 or 30 kDa. However even higher molecular weights may be acceptable provided if the conjugates do not exceed the renal clearance size limit. Thus conjugates less than 15, 10, 9 , 8, 7, 6 nm may be preferred for rapid clearance, especially less than 6 nm.

By "labelling platform" is meant a moiety to which it is possible to attach, directly or via a linker, one or more positron emitters (via attachment sites, as described below). The labelling platform may be, for example, a nanoparticle or a dendrimer.

By "nanoparticle" is meant an inorganic, organic or inorganic/organic hybrid molecular particle whose size is comprised between 1 nm and 100 nm.

By "dendrimer" is meant a molecule such as a hyperbranched polymer possessing a dendritic (tree-like) structure.

By "target binding moiety " is meant any molecule which binds to a target. Preferably the target binding moiety selectively binds to a target. In preferred embodiments, the target may be diagnostic or prognostic marker associated, for example, with a pathology such as cancer. In other embodiments the target binding moiety may be affinity molecule which is one member of a binding pair such as streptavidin and biotin. Preferred targets of the invention are discussed in more detail hereinafter.

Some particular embodiments of the invention will now be described in more detail:

Labelling platform

Preferably the labelling platform is water-soluble. Preferably the labelling platform is a nanoparticle or a dendrimer. Preferably the platform has a minimum diameter of 2 nm.

In embodiments wherein the labelling platform is a nanoparticle, preferably the nanoparticle has a diameter of less then 15 nm, more preferably less than 10, 8 or 6 nm, most preferably less than 5 nm, in each case prior to coating or functionalisation.

The nanoparticle may be a TiO ₂ nanoparticle. TiO ₂ is preferred because it is relatively cheap, non-toxic, and generally easy to synthesise in the correct nanosize range. It is also easy to tune the size of the nanoparticles as desired. However alternative nanoparticles include ZnO (<9 nm; P. D. Cozzoli, A. Komowski and H. Weller, J. Phys. Chem. B 109, 2638 (2005)); Au nanoparticles (J. D. Gibson, B. P. Khanal and E. R. Zubarev, J. Amer. Chem. Soc, 129, 11653 (2007)); Fe ₂ O ₃ (A. K. Gupta and M. Gupta, Biomaterials, 26, 3995 (2005)).

In embodiments wherein the labelling platform is a dendrimer, preferably the dendrimer is water-soluble. The dendrimer may be a glycodendrimer i.e. a carbohydrate-containing molecules which can be grown generationwise following an iterative repetitive synthesis.

Dendrimers are formed by reiteration of formation steps to produce first generation, then second, third and fourth etc generation dendrimers, generally from a point like core (see below).

Most preferably the dendrimer is a poly(amidoamine) (PAMAM) dendrimer. These are water-soluble, biocompatible ¹⁸ and bioavailable ¹⁹ ' ²⁰ .

Table 1 shows how the size and number of attachment sites (e.g. positron emitter attachment sites) varies with generation for PAMAM dendrimers.

Table 1:

Preferably the dendrimer is a first, second, third, fourth, fifth, sixth, seventh or eighth generation dendrimer, more preferably a fourth, fifth, sixth or seventh generation dendrimer, most preferably a sixth generation dendrimer.

Other dendritic polymers such as diaminobutane polypropylene imine) (DAB)(Kobayashi, H., Kawamoto, S., Saga, T., Sato, N., Hiraga, A., Ishimori, T., Akita, Y., Mamede, M. H., Konishi, J., Togashi, K., and Brechbiel, M. WNovel liver macromolecular MR contrast agent with a polypropylenimine diaminobutyl dendrimer core: comparison to the vascular MR contrast agent with the polyamidoamine dendrimer core. Magn. Reson. Med. 2001 , 46, 795-802; Sideratou, Z., Tsiourvas, D., Paleos, C. M. Solubilization and release

properties of PEGylated diaminobutane poly(propyleneimine) dendrimers. J. Colloid Interface ScL 2001 , 242, 272-276); polyglycerol (PG)( Sunder, A.; Kramer, M.; Hanselmann, R.; Muhlaupt, R.; Frey, H. Molecular Nanocapsules Based on Amphiphilic Hyperbranched Polyglycerols. Angew. Chem., Int. Ed. 1999, 38, 3552-3555. Sunder, A.; Quincy, M. F.; Mu ^' ϊhaupt, R.; Frey, H. Hyperbranched Polyether Polyols with Liquid Crystalline Properties. Angew. Chem., Int. Ed. 1999, 38, 2928-2930. Sunder, A.; Mu ^' ϊhaupt, R.; Frey, H. Hyperbranched Polyether-

Polyols Based on Polyglycerol: Polarity Desigh by Block Copolymerisation with Propylene Oxide. Macromolecules 2000, 33, 309-314. Frey, H.; Haag, R. Dendritic polyglycerol: A new versatile biocompatible material. Rev. MoI. Biotechnol. 2002, 90, 257-267); poly(ethylene imine) (PEI)(Multiarm star nanocarriers containing a poly(ethylene imine) core and polylactide arms. J. Polym. ScL Part A: Polym. Chem. 2006, 44, 5740-5749; Wang, C. H.; Hsiue, G. H. Polymer-DNA hybrid nanoparticles based on folate- polyethylenimine-block-poly(L-lactide). Bioconjugate Chem. 2005, 16, 391-396.); polyesters (PE)( Ihre, H. R.; De Jesus, O. L. P.; Szoka, F. C; Frechet, J. M. J. Polyester dendritic systems for drug delivery applications: Design, synthesis, and characterization. Bioconjugate Chem. 2002, 13, 443-452; De Jesu's, O. L. P.; Ihre, H. R.; Gagne, L.; Fre'chet, J. M. J.; Szoka, F. C, Jr. Polyester dendritic systems for drug delivery applications: In vitro and in vivo evaluation. Bioconjugate Chem. 2002, 13, 453-461. Muscat, D.; van Benthem, R. A. T. M. Hyperbranched polyesteramides: New dendritic polymers. Top. Curr. Chem. 2001 , 212, 41-80).

Such dendrimers are well known to those skilled in the art, as evidenced for example Constantinos M. Paleos, Dimitris Tsiourvas, and ZiIi Sideratou. Molecular Engineering of Dendritic Polymers and Their Application as Drug and Gene Delivery Systems. MoI. Pharm. 2007, 4, 169-188. Svenson, S.; Tomalia, D. A. Dendrimers in biomedical applications: Reflections on the field. Adv. Drug Delivery Rev. 2005, 57, 2106- 2129. Bosman, A. W.; Janssen, H. M.; Meijer, E. W. About Dendrimers: Structure, Physical Properties, and Applications. Chem. Rev. 1999, 99, 1665-1688.

Target binding moiety

The target binding moiety binds a target as described herein. Preferably the target binding moiety selectively binds to one or more of the targets described below in relation to imaging methods. Target binding moieties may, for example, bind a target with an affinity of at least 10 ^"9 M, 10 ^~10 M, 10 ^'11 M or 10 ^"12 M.

A preferred target binding moiety is an immunoreactive specific binding member, which would include antibodies and binding fragments of antibodies (e.g., single chain antibodies, Fab' fragments, F(ab)'2 fragments, and scFv proteins and Affibodies® (Affibody, Teknikringen 30, floor 6, Box 700 04, Stockholm SE-10044, Sweden, U.S. Pat. No. 5,831,012)). Most preferable are binding fragments of antibodies e.g. Affibodies.

Molecules such as scFv with this molecular weight exhibit very rapid blood clearance ¹⁷ .

As well as such immunoreactive specific binding members, also included are other proteins, peptides, nucleic acids, or other biological or non-biological molecules.

In some preferable embodiments the target binding moiety is an affinity molecule or specific binding member that it is a member of a specific binding pair. That is, two different molecules where one of the molecules through chemical or physical means specifically binds to the second molecule. Such pairs include hapten-anti-hapten systems such as biotin or anti-biotin, avidin or biotin, a carbohydrate or a lectin, a complementary nucleotide sequence, an effector or a receptor molecule, an enzyme cofactor and an enzyme, an enzyme inhibitor or an enzyme, and the like. Alternatively the pair can be an antibody and antigen forming an antibody/antigen complex

In some other preferable embodiments, the target binding moiety is biotin. Biotin is a resilient molecule that can be exposed to more extreme conditions than can most proteins. Particular advantages of biotin are discussed in more detail below in relation to processes for making labelling (and labelled) platform conjugates of the present invention.

In some embodiments it is preferable that the target binding moiety has a molecular weight of less than 100 kDa, more preferably less than 80, 70, 60, 50, 40, 30 or 20 kDa and most preferably less than 10 kDa.

Labelling platforms conjugated to more than one target binding moiety may be below renal clearance limit, and it may be desirable also to produce multivalent labelling platforms. The mean number of target binding moieties per labelling platform can be adjusted by varying the target binding moiety to labelling platform ratio during conjugation (see below). Accordingly, the labelling platform conjugate may have more than one

target binding moiety attached thereto. Preferably it has 2,3,4 or 5 target binding moieties.

In embodiments wherein the target binding moiety is a protein, preferably the protein consists of less than 200, 150, 120, 100, 80 and most preferably less than 60 amino acids.

In some embodiments it may be desirable that the target binding moiety selectively binds to a target which requires a large target binding moiety. As discussed herein, large target binding moieties may not be desirable, for example as a large target binding moiety would lead to a large labelling platform conjugate. In such situations, a pre-targeting method may be employed, as described below. In these embodiments the target binding moiety may be an affinity molecule which is one member of a binding pair such as streptavidin and biotin.

In embodiments wherein the labelling platform is a dendrimer, it may be preferable that the target binding moiety is biotin. In embodiments wherein the labelling platform is a nanoparticle, it may be preferable that the target binding moiety is an affinity protein, more preferably that it is an anti-HER-2 Affibody ®.

Optional linkers for target binding moiety

The target binding moiety may be attached to the labelling platform via a linker. Preferably the linker is water-soluble. The linker may comprise one or more moieties selected from the group consisting of ether (including polyether such as polyethylene glycol), thioether, ester, amine, amide, imine, imide (e.g. succinimide), oxime, saturated or unsaturated, alkyl or cycloalkyl having 1 to 10 carbon atoms which may be substituted or unsubstituted, and substituted or unsubstituted aryl or heteroaryl. Other linkers include cyclic amines (e.g. aminopiperidines).

It may be preferable that the linker is of sufficient length that it overcomes potential steric hindrance by the labelling platform during binding of the target binding moiety to the target (e.g. in imaging methods as described below). For example, a linker comprising a polyethylene glycol moiety having 5 glycol subunits should be sufficiently ( 2nm) long to overcome steric hindrance, for example by the dendrimer during biotin binding. Preferably the linker is between 1 and 5 nm long.

In embodiments where the platform is pre-targeted, the molecular weight of the linker could vary from maybe a 100 Da to 100,000 Da. Varieties of PEG with specific mwt ranges e.g. PEG _1O ooo are readily commercially available.

In the light of the present disclosure, those skilled in the art will be able to provide different lengths of linker as appropriate to the relevant situation.

Positron emitters and attachment sites

A "positron emitter" is capable of emitting positrons. Preferably the positron emitter is an atom or ion. The positron emitter may be referred to herein as a PET isotope. Preferably the positron emitter is an ¹⁸ F atom, or an ¹⁸ F ^" anion.

Preferably, the positron emitters are atoms or ions, preferably ¹⁸ F. By " ¹⁸ F" is meant an ¹⁸ F atom, or an ¹⁸ F ^" anion. Most preferably the positron emitters are ¹⁸ F ^" .

Preferably the labelling platform has a plurality of positron emitters attached thereto. These will be attached to the labelling platform at positron emitter attachment sites.

By "positron emitter attachment site" is meant a site at which a positron emitter can be conveniently attached, preferably in a single reaction step, and these are described in more detail hereinafter. Thus the positron emitters may be provided at one or more "labelled positron emitter attachment sites".

In some embodiments, the labelled positron emitter attachment site may have more than one positron emitter attached thereto, e.g. 2, 3, 4 or 5 positron emitters.

The positron emitter attachment site may comprise one or more moieties to serve as linkers for linking the labelled positron emitter attachment site to the labelling platform. These may, for example, be selected from the group consisting of ether (including polyether such as polyethylene glycol), thioether, ester, amine, amide, imine, imide (e.g. succinimide), oxime, saturated or unsaturated, substituted or unsubstituted alkyl or cycloalkyl having 1 to 30 carbon atoms, substituted or unsubstituted aryl or heteroaryl, and saturated or unsaturated fatty acid having 1 to 30 carbon atoms. Other linkers include cyclic amines (e.g. aminopiperidines).

In embodiments wherein the labelling platform is a nanoparticle, preferably it is linked to the positron emitter attachment site by a moiety comprising a fatty acid having 15 to 25 carbon atoms, attached to the nanoparticle via its acid functionality. Preferably the fatty acid chain is attached to the positron emitter attachment site via an amide, ester, amine, an oxime or an ether.

In embodiments wherein the labelling platform is a dendrimer, preferably it is linked to the positron emitter attachment site by a moiety comprising an amide, an oxime or both. Other suitable moieties include esters, amines, or ethers.

The labelled positron emitter attachment sites may comprise a moiety having one of the following structures:

/ B ^" ( ¹⁸ F) ₃

R tBu

R ^' -iβpBu

In the above embodiments it will be understood that tBu could be replaced with any other alkyl or aryl group.

H

R ^{' 18} F

R^ ⁸ F

R' ¹⁸ F

wherein R is a moiety which may serve as a linker for attaching the labelled positron emitter attachment site to the labelling platform. Preferably this moiety is as described herein for (unlabelled) positron emitter attachment sites. Me is methyl and tBu is tertiary butyl.

In embodiments wherein the labelling platform is a dendrimer, preferably the dendrimer has at least 3 positron emitter attachment sites, preferably at least 4, 16, 32 ,64 or 100 positron emitter attachment sites, most preferably at least 128 positron emitter attachment sites. In embodiments wherein the labelling platform has 128 positron emitter attachment sites, the amount of precursor required for positron n emitter attachment could be decreased by a factor of up to 128 so improving specific activity.

In embodiments wherein the labelling platform is a nanoparticle, preferably the nanoparticle has at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 or more positron emitter attachment sites. However it will be appreciated that several hundred or more potential attachment sites may be provided.

The provision of multiple positron emitter attachment sites reduces the amount of precursor required for positron emitter attachment and so improves specific activity.

It is not necessary that all of the positron emitter attachment sites are labelled, and indeed generally speaking only one will be in any given conjugate. Therefore the labelling platform of the labelled platform conjugate may further have one or more (or many) positron emitter attachment sites (i.e. unlabelled) attached thereto. This is shown for example in Figure 2 which is a schematic representation of a preferred embodiment of the labelled platform conjugate.

The labelled moieties described above can be conveniently prepared from precursor molecules these form further aspects of the invention.

It should be noted that for reasons of brevity, disclosure of the various features or preferred embodiments of the invention are not repeated for each individual aspect. However all preferred or example embodiments, or other features of the invention, described with respect to any aspect will be understood to be disclosed mutatis mutandis in respect of all other relevant aspects of the invention also.

Unlabelled platform conjugates for label attachment

As discussed above the positron emitters are attached to the labelling platform at positron emitter attachment sites. These are preferably used to provide the labelled moieties described above in a quick and efficient manner.

Accordingly, another aspect of the present invention provides a labelling platform conjugate comprising:

(i) a labelling platform, and

(ii) a target binding moiety, wherein the labelling platform has a plurality of positron emitter attachment sites attached thereto.

As noted and described above, by "positron emitter attachment site" is meant a site at which a positron emitter can be conveniently attached, preferably in a single reaction step.

Preferably the labelling platform conjugate is water-soluble.

Preferably the labelling platform is as described above. Accordingly, the labelled platform may be, for example, a nanoparticle or a dendrimer.

Preferably the target binding moiety is as described above.

In some preferable embodiments, the positron emitter attachment site is a ¹⁸ F attachment site. By " ¹⁸ F attachment site" is meant a site at which it is possible to attach an ¹⁸ F atom, or an ¹⁸ F ^" anion.

Preferably, the positron emitter attachment site comprises a replaceable moiety, which is replaced by the positron emitter when it attaches to the positron emitter attachment site. In some embodiments, the replaceable moiety may be an isotope which is replaced with a positron emitter by isotopic substitution, for example ¹⁹ F. The replaceable moiety may be replaced by nucleophilic substitution. Examples of replaceable moieties include halide (e.g. Cl ^" , Br ^" , I ^" ), alkoxide (e.g. methoxy, ethoxy, propoxy, butoxy and alkoxides derived from dialcohols such as pinacolate), hydroxy, amine and sulfonate (e.g. mesylate, triflate and tosylate). Others include epoxide (for addition reaction/ring-opening reaction) or an α, β-unsaturated carbonyl group e.g. a ketone, an ester or an amide (addition reaction).

Preferably the replaceable moiety is attached to a B atom, a Si atom or a C atom.

In embodiments wherein the replaceable moiety is attached to a B atom, preferably it is alkoxide, more preferably pinacolate. In embodiments wherein the replaceable moiety is attached to a B atom preferably the positron emitter attachment site comprises boroaryl. Recently a boroaryl method for ¹⁸ F ^" labelling has been described ²³ .

In embodiments wherein the replaceable moiety is attached to a Si atom, preferably it is ¹⁹ F or alkoxide. In embodiments wherein the replaceable moiety is attached to a C atom, preferably it is a sulfonate, more preferably mesylate or triflate.

In embodiments wherein each positron emitter attachment site is able to attach to more than one positron emitter, it preferably comprises more than one replaceable moiety. In these embodiments, preferably each replaceable moiety is attached to the same atom.

Preferably the positron emitter attachment site comprises a moiety having one of the following structures:

tBu I

Sk R ^" ₁₉ ^tBu

R

R ¹

N R ²

(Y = ¹⁹ F or other halide, or OMs)

wherein R is a moiety (e.g. alkyl, aryl, or amido) which may serve as a linker for attaching the positron emitter attachment site to the labelling platform. Preferably this moiety is as described above. OMs is mesylate, OTf is triflate, Me is methyl and tBu is tertiary butyl.

The labelling platform conjugates of the present aspect may be used to prepare the labelled moieties of the first aspect by attaching positron emitters to the positron emitter attachment sites, for instance by use of the processes described below. Preferably the positron emitters are attached by replacement of the replaceable moieties as described above. More preferably the positron emitters are attached to the positron emitter attachment sites by isotopic substitution or nucleophilic substitution of the replaceable moieties.

Thus a process including the step of attaching positron emitters to the positron emitter attachment sites to the unlabelled platform conjugates of the present aspect forms one aspect of the invention.

In another embodiment described below, the labelled moieties of the first aspect may be synthesised by methods including the step of attaching the target binding moiety to the labelling platform already having a plurality of labelled positron emitter attachment sites attached thereto, via a target binding moiety attachment site.

Labelled platform conjugates for target-binding moiety attachment

Accordingly, a further aspect of the invention provides a labelled platform conjugate, comprising:

(i) a labelling platform, and

(ii) a target binding moiety attachment site, wherein the labelling platform has at least one positron emitter attached thereto.

Preferably the labelling platform has a plurality of positron emitters attached thereto. These will be attached to the labelling platform at positron emitter attachment sites. Preferably the labelling platform and positron emitter attachment sites are as described above.

By "target binding moiety attachment site" is meant a site to which a target binding moiety (preferably as described above) may be attached.

The target binding moiety attachment site may be any moiety capable of attaching to a target binding moiety. The target binding moiety attachment site may comprise a moiety selected from the group consisting of α-β-unsaturated carbonyl (e.g. succinimide), amine,

O-substituted hydroxylamine, acyl halide, ester, amide, ketone, ether, amine and aldehyde.

The target binding moiety attachment site may comprise any protecting group know in the art, such as Boc or Fmoc. Others include the benzyloxycarbonyl group (Z group), benzyl group, silyl group, trifluoroacetyl group or sulfonyl group.

Preferably this protecting group is removable. In protection techniques, a protecting group is attached to prevent the attachment site from reacting with other chemicals. The protection group is then removed to allow attachment of the target binding moiety. The labelling platform conjugate may have more than one target binding moiety attachment site. It may have 2, 3, 4 or 5, for example.

Preferably, the labelling platform, positron emitters, labelled positron emitter attachment sites and target binding moiety attachment sites are as described above.

The labelled platform conjugates of the present aspect may be used to prepare the labelled moieties of the first aspect by attaching target binding moieties thereto, for instance by use of the processes described below and a process including the step of attaching the target binding moiety to the labelled platform conjugates of the present aspect forms one aspect of the invention.

Processes for producing conjugates

According to another aspect of the invention there is provided a process for making a labelling platform conjugate comprising the steps of i) preparing or providing a labelling platform; ii) attaching a plurality of positron emitter attachment sites to the labelling platform; iii) attaching a target binding moiety attachment site to the labelling platform; and iv) attaching a target binding moiety to the target binding moiety attachment site.

Preferably, the labelling platform, positron emitter attachment sites, target binding moiety attachment site and target binding moiety are as described above.

Steps i) to iv) may be performed in any appropriate order. For example, step iii) and optionally step iv) may be performed before step ii). In this case, the target binding moiety attachment site (and optionally the target binding moiety) are attached to the labelling platform prior to the attachment of the plurality of positron emitter attachment sites.

Step iii) may be omitted if the target binding moiety attachment site is integral to the labelling platform. This may be the case, for example, when the labelling platform is a dend rimer.

Preparation of nanoparticles

In embodiments wherein the labelling platform is a nanoparticle, step i) may comprise forming the nanoparticle by a hydrothermal method ²⁶ . The nanoparticle may be coated using fatty acids. In such embodiments, the nanoparticle may be formed by precipitation from a mixture containing the fatty acids. The fatty acids may be unsaturated, preferably terminally unsaturated. In such embodiments the process for making the labelling platform may further comprise the step of converting the terminal double bond into an aldehyde to facilitate attachment of positron emitter attachment sites and target binding moieties. This may be done by treatment with ozone (O ₃ ). The aldehyde may further be reacted to introduce a further functional group, for example an amine by reaction with 1 ,2- bis(2-aminoethoxy)ethane.

Preparation of dendήmers

In embodiments wherein the labelling platform is a dendrimer, step i) may comprise forming a dendrimer by an iterative method. Preferably the dendrimer is a poly(amidoamine) (PAMAM) dendrimer. These may be formed by reaction of diaminopolyethylene glycol (preferably monoprotected with Boc) which forms the core of the dendrimer. This core may be sequentially reacted with extension reactants such as methyl acrylate and ethylene diamine to form the first generation dendrimer, then second, third and fourth etc generation dendrimers (see below).

Preferably the addition of extension reactants such as methyl acrylate and ethylene diamine is repeated six times to attain a sixth generation dendrimer (but may be repeated

for example five times to form the fifth generation dendrimer, or a suitable number of times to form the second, third, fourth, seventh or eighth generation dendrimer).

Addition of positron emitter attachment site

In step ii), it may be desirable to provide reactants for forming a positron emitter attachment site in sub-stoichiometric amounts. This is to allow there to be free reaction sites (e.g. amine groups) for attachment of the target binding moieties (or target binding moiety attachment sites).

Addition of target binding moieties

In some embodiments, labelling platforms conjugated to more than one target binding moiety may be below renal clearance limit, and it may be desirable to produce multivalent labelling platforms. The mean number of target binding moieties per labelling platform can be adjusted by varying the target binding moiety attachment site to labelling platform ratio during step iii) or the target binding moiety to labelling platform ratio during step iv).

General preparative methods

The process for making a labelling platform conjugate may comprise protection or deprotection steps. Protection techniques are well known in the art. In protection techniques, a protecting group is attached to prevent the attachment site from reacting with other chemicals. Any suitable protecting group may be used, for example Boc or Fmoc.

For example, the amino groups of the dendrimer may be protected (in embodiments wherein the labelling platform is a dendrimer). The target binding moiety attachment site may be protected, as may the positron emitter attachment site. Indeed, any moiety may be protected to prevent it from reacting in an undesirable manner. Where a moiety is protected, preferably the process comprises a deprotection wherein the protecting group is removed from the moiety is was protecting.

The method of making a labelling platform conjugate may further comprise a purification step, performed after step iv) to remove any un-conjugated target binding moiety. The purification step may comprise filtration. In some embodiments, the conjugates will be at

least 35kDa (monovalent conjugate) in size and unconjugated Affibodies 7kDa, so filtration through a Centricon filter will remove non-conjugated Affibody which would otherwise compete for receptor binding.

Preferably the process for making a labelling platform conjugate can be carried out at one location. Preferably the process can be carried out without the need for specialist equipment. Preferably the process can be carried out without the need for specialist practitioners.

Addition of positron emitters

According to another aspect of the invention there is provided a process for making a labelled platform of the invention, comprising the process above plus: v) attaching at least one positron emitter to a positron emitter attachment site.

In other embodiments, two or more positron emitters may be added.

Steps i) to v) may be performed in any appropriate order. For example, step iii) and optionally step iv) may be performed before step ii). In this case, the target binding moiety attachment site (and optionally the target binding moiety) are attached to the labelling platform prior to the attachment of the plurality of positron emitter attachment sites. Step v) must be performed after step ii), but may be performed before step iv) and optionally step iii). In this case, the positron emitters are attached to the positron emitter attachment sites before the attachment of the target binding moiety and optionally before the attachment of the target binding moiety attachment site.

Step iii) may be omitted if the target binding moiety attachment site is integral to the labelling platform. This may be the case, for example, when the labelling platform is a dendrimer.

It should be noted that generally speaking the platform substrate will be present at large excess over the ¹⁸ F - for example more than 10x, 10Ox or typically 100Ox excess. Thus only a small proportion of the substrate will be labelled. However due to the plurality of positron emitter attachment sites, this is nevertheless efficient compared to conventional methods, and can thus yield a product with a relatively high specific activity.

The method of making a labelling platform conjugate may further comprise a purification step. This may be performed after step iv) or v) to remove any un-conjugated target binding moiety, as described above.

Preferably the nature of the positron emitter attachment site allows attachment of positron emitters at a late or final stage of synthesis. In embodiments wherein the positron emitter is ¹⁸ F, the positron emitters may be attached to the positron emitter attachment sites by administration of an ¹⁸ F ^' containing composition. Preferably the ¹⁸ F ^" containing composition comprises K ¹⁸ F optionally with Kryptofix ®. Alternatively the ¹⁸ F ^" containing composition ¹⁸ FV[ ¹⁸ O]H ₂ O. By Kryptofix ® is meant 4,7,13,16,21,24-Hexaoxa-1 ,10- diazabicyclo[8.8.8]-hexacosane. These methods are particularly preferable when the positron emitter attachment site comprises a boron atom, more preferably a boroaryl group, or when the replaceable moiety is mesylate or triflate. Methods comprising fluorination as described above may further comprise a purification step comprising passage through an ion exchange cartridge and\or size exclusion chromatography. In particular the latter may be preferred where the platform is a dendrimer.

Alternatively, the positron emitters may be attached by isotopic substitution, for example using di-(tert-butyl)fluorosilylaryl surface groups ²⁴ . According to Schirrmacher et al ²⁴ this can be performed in only 15 minutes in acetonitrile (room temperature) or in 30 minutes in aqueous medium (9O ⁰ C), eliminating the need for a solvent removal step.

¹⁸ F ^' is often produced in cyclotrons in hospitals. The simple methods of fluorination disclosed herein may allow for the attachment of the positron emitters on site in hospitals, using cyclotron-produced ¹⁸ F ^' .

In some preferable embodiments, the target binding moiety is biotin. Biotin is a resilient molecule that can be exposed to more extreme conditions than can some proteins. This has certain advantages, for example allowing the attachment of a positron emitter, e.g. ¹⁸ F as described above to be carried out in the presence of organic solvents and/or at relatively high temperatures. Consequently this can be the final step of a synthesis. In embodiments wherein the position emitter is ¹⁸ F, purification may require only passage though an ion exchange cartridge (which can potentially be performed in a few seconds) or gel-filtration column (or other size exclusion chromatography) to remove unattached ¹⁸ F.

Compared with current protein and peptide ¹⁸ F ^" radio-labelling techniques the preparation of the labelling unit is simple, not requiring a multi-step synthesis of ¹⁸ F-prosthetic groups currently used in the ¹⁸ F-labelling of proteins. Preferably the process for making a labelled platform conjugate can be carried out at one location, for example a hospital radiopharmacy. Preferably the process can be carried out without the need for specialist equipment (such as sophisticated rigs). Preferably the process can be carried out without the need for specialist practitioners.

Additionally, the specific activity of the labelled platform conjugate is improved compared to conventional labels due to the presence of multiple positron emitter attachment sites per labelling platform conjugate that can potentially be fluorinated (e.g. with ¹⁸ F ^' ), reducing the substrate excess required.

Use of labelled moieties

The labelled moieties of the invention may be used in methods of binding targets, wherein the method comprises the step of contacting the target with the labelled moiety. Preferably this is in vivo in a living subject such as a human or animal patient. Less preferably the subject may be a biological sample. The subject will be one which it is suspected may contains targets to which the target binding moiety may bind (or may be control which it is believed does not contains targets to which the target binding moiety may bind).

Preferably the labelled moiety is then observed or detected (for example using PET). Optionally the result of the observation or detection is used in a method of diagnosis or prognosis for a pathology associated with the presence and\or location and\or amount of the target so observed or detected. Thus the invention also provides an imaging method, comprising administering a labelled platform conjugate to a subject and scanning using PET to generate an image, and the use of the same in diagnosis or prognosis of a pathology - for example disease or disorder.

Targets

In some preferable embodiments the targets are associated with cancer. In some preferable embodiments the target is an antigen e.g. a cell surface receptor. Preferably the receptor is tumour specific, or over-expressed in tumours e.g. in breast cancer.

The selected biomarker can, for example, be a general diagnostic or prognostic marker useful for multiple types of cancer, such as CA 125, CEA or LDH, or can be a cancer- specific diagnostic or prognostic marker, such as a colon cancer marker (for example, sialosyl-TnCEA, CA19-9, or LASA), breast cancer marker (for example, CA 15-2. Her- 2/neu and CA 27.29), ovarian cancer marker (for example, CA72-4), lung cancer (for example, neuron-specific enolase (NSE) and tissue polypeptide antigen (TPA)), prostate cancer (for example, PSA, prostate-specific membrane antigen and prostatic acid ' phosphatase), melanoma (for example, S-100 and TA-90), as well as other biomarkers specific for other types of cancer.

The target binding moieties of the labelled moieties will be dictated by the nature of the target. For example in some preferable embodiments the target binding moiety is an anti- HER-2 Affibody ® (molecular weight is 7 kDa). In these embodiments, the present invention finds use in an improved method for the identification of patients who could benefit from treatment with Herceptin. Metastatic expression of HER-2 mirrors that of the primary ²¹ so the imaging agent would also detect metastasis. It may also be utilised in serial PET scans to detect response to anti-HER-2 treatment.

In other preferable embodiments the target may be an affinity molecule useful in a pre- targeting method (see below).

PET scanning

Preferably the scanning is performed between 0 and 10 hours after administration of the labelled platform conjugate, more preferably between 30 minutes and 5 hours, between 1 and 5 hours or between 1 and 3 hours, most preferably between 1 and 2 hours after administration. This is to allow the binding of the target binding moiety to the target, and to allow any unbound labelled platform conjugate to clear from the blood.

The imaging method may be repeated at intervals, for example to detect response to treatment. For example, imaging methods involving labelled platform conjugates wherein the target binding moiety is the anti-HER " Affibody ® may be repeated to detect response to treatment with Herceptin ®.

Pre-targeting methods

The imaging method may be a pre-targeting imaging method. In pre-targeting methods the target may be pre-targeted with any target binding moiety as described above, but usually with a protein, preferably an antibody, conjugated to an affinity molecule such as streptavidin. Several hours or days later, the unbound target binding moiety conjugated to an affinity molecule will have cleared from the blood (this may be assisted by addition of a clearing molecule). Thus, a small target binding moiety, such as biotin, which selectively binds to the affinity molecule already administered may be used to deliver the labelling platform conjugate to the target. Similar pre-targeting methods are known in the art, for example they have been carried out with ⁶⁸ Ga labelling ²² . Thus in some embodiments it is preferable that the target binding moiety is an affinity molecule suitable for use in such pre-targeting methods, more preferably biotin. Although streptavidin/biotin is the most commonly used affinity pair for pretargeted applications, other possibilities include antihapten antibody/hapten, and oligonucleotides/complementary oligonucleotide (cited in: McBride WJ et al Bispecific Antibody Pretargeting PET (ImmunoPET) with an ¹²⁴ l-Labeled Hapten-Peptide, Journal of Nuclear Medicine Vol. 47 1678-1688).

Accordingly, the imaging method may comprise a step of administering to a human or animal subject a target binding moiety conjugated to an affinity molecule for pre-targeting. The method may further comprise addition of a clearing molecule to clear the target binding moiety conjugated to an affinity molecule from the blood of the subject. The methods of the invention as described above are then performed, wherein the affinity molecule is targeted by the labelled moiety of the invention e.g. which has as its target binding moiety one member of a specific binding pair which binds the affinity molecule.

Such pre-targeting methods may improve image quality when compared with the administration of directly labelled antibodies ²⁵ .

The target binding moiety conjugated to an affinity molecule for pre-targeting may comprise a dendrimer. This dendrimer may be conjugated to an affinity molecule which is one member of a binding pair such as streptavidin and biotin, preferably streptavidin. The dendrimer may further be conjugated to multiple target binding moieties, for example multiple HER-2 Affibodies ®.

Other aspects and uses

A further aspect of the present invention is the use of the labelled platform conjugate of the present invention in any of the above disclosed methods e.g. an imaging method of the present invention.

A further aspect of the invention is the use of a labelling platform conjugate or labelled platform conjugate of the present invention in the manufacture of a medicament. Preferably the medicament is for a method as described above e.g. a medical imaging method employing Positron Emission Tomography. Preferably the medical imaging method is for the diagnosis or visualisation of cancer. Most preferably the medical imaging method is an imaging method as described above.

Kits

Kits of the present invention may be provided e.g. for diagnostic or prognostic purposes.

Such kits may comprise two or all of:

(1) a labelling platform e.g. nanoparticle

(2) a target binding moiety e.g. an affinity reagent such as an Affibody.

(3) a positron emitter attachment site moiety

Kits may alternatively provide:

(1) an unlabelled platform conjugates for label attachment, or

(2) a labelled platform conjugates for target-binding moiety attachment, in each case as described above.

Kits may additionally provide:

(1) instructions for preparing labelled platform conjugates of the invention e.g. in accordance with the processes described herein,

(2) instructions for using labelled platform conjugates of the invention e.g. in accordance with the methods described herein,

(3) a label or insert indicating regulatory approval for diagnostic, prognostic or therapeutic use or any suitable combination thereof.

AII preferred or example embodiments, or other features of the invention, described with respect to any aspect will be understood to be disclosed mutatis mutandis in respect of all other relevant aspects of the invention also.

Any sub-titles herein are included for convenience only, and are not to be construed as limiting the disclosure in any way.

The invention will now be further described with reference to the following non-limiting Figures and Examples. Other embodiments of the invention will occur to those skilled in the art in the light of these.

The disclosure of all references cited herein, inasmuch as it may be used by those skilled in the art to carry out the invention, is hereby specifically incorporated herein by cross- reference.

Figures

Fig 1 is a reaction scheme showing the synthesis of an ¹⁸ F-labelled TiO ₂ nanoparticle- Affibody conjugate as described in Example 2 below.

Fig 2 shows a general structure of a dendrimer-biotin conjugate.

Fig 3 is a reaction scheme showing the synthesis of a functionalised Generation 6 dendrimer.

Fig 4 is a reaction scheme showing the synthesis of a radiolabeled of the dendrimer- biotin conjugate.

Fig 5 shows "Sample 2" from Example 14 (a radiofluorinated boroarylated dendrimer- biotin conjugate) run on TLC using acetonitrile 95%:5% H ₂ O mobile phase.

Examples

Example 1- Synthesis and Characterisation of TiQ? Nanoparticles

Synthesis: TiO ₂ nanoparticles are coated using a mixture of stearic acid (C ₁₈ H ₃₆ O ₂ ) and monounsaturated 21-docosenoic acid (C ₂₂ H ₄₂ O ₂ ) as described below. The use of fatty acids allows the formation of nanoparticles of diameter <5 nm (this means that the overall size when combined e.g. with an anti HER-2 Affibody ® is <15nm. This is preferred for rapid renal clearance of non-receptor-bound fragments). The terminal double bonds of the unsaturated fatty acid are used to introduce appropriate functional groups for conjugation to Affibodies and radio-labelling.

Coated TiO ₂ nanoparticles are prepared by a hydrothermal method ²⁶ by dissolving tert- butylamine in water and transferring the solution to a stainless steel vessel, lined with PTFE, (inner diameter = 26mm and length of = 95 mm). Titanium (IV) n-propoxide and a mixture of stearic acid (C ₁₈ H ₃₆ O ₂ ) and monounsaturated 21-docosenoic acid (C ₂₂ H ₄₂ O ₂ ) are dissolved in toluene and the solution then transferred to the stainless steel autoclave. The autoclave is sealed and maintained at 180°C for 12 hours then cooled to RT. The nanocrystals are precipitated with methanol and isolated by centrifugation and decantation. Under these conditions surface coated TiO ₂ nanoparticles, for example of around 5 nm or less, can be formed which consist solely of the anatase form of TiO ₂ . Nanoparticle size is controlled by the volume of terf-butylamine added ²⁷ .

Characterisation: X-ray diffraction using a Bruker D8 Advance diffractometer is employed to check phase composition, particle size and crystalinity of the TiO ₂ nanoparticles. Transmission electron microscopy is also used in order to determine the size, dispersibility and crystalinity of the surface coated nanoparticles. X-ray photoelectron spectroscopy (XPS) is employed to confirm that the surface coated nanoparticles consist of a TiO ₂ core and a mixed stearic acid (C ₁₈ H ₃₆ O ₂ ) and monounsaturated 21-docosenoic acid (C ₂₂ H ₄₂ O ₂ ) coating; Furthermore the technique of Raman scattering is used to check that the desired surface coated TiO ₂ nanoparticles have formed. Recent results have shown that a blue shift in the Raman scattering is observed upon coating TiO ₂ nanoparticles with surface active molecules such as dodecylbenzenesulfonic acid or stearic acid ²⁸ i.e. the Raman peaks move to higher wavenumber.

Example 2 - Nanoparticle functionalisation and conjugation to Affibody

Coated titanium oxide nanoparticle 1 (see Fig 1), synthesised for example as described above, is treated with ozone to oxidise the terminal double bonds of the coating into aldehydes. Reaction with 1 ,2-bis(2-aminoethoxy)ethane followed by reduction with

sodium borohydride then permits to extend the chains of the coating and introduce free amino groups at the periphery of the nanoparticles. The introduction of 1 ,2-bis(2- aminoethoxy)ethane enhances the nanoparticle water solubility and help inhibit coating by plasma components (opsonisation) ²⁹ . Nearly all of these free amino groups are coupled to boroaryl carboxylic acid 4 using substoichiometric amounts of reagent. The remaining amino group(s) are reacted with the succinimidyl 4-(N- maleimidomethyl)cyclohexan-1-carboxylate (SMCC) linker to give nanoparticle 6, which is subsequently conjugated to anti-HER2 Affibodies to provide conjugate system 7. ³⁰ At this stage, unconjugated Affibodies are removed by filtration through a Centricon filter (using either a 10kDa or 30 kDa cut off filter - dependent on the filtration behaviour of the conjugates) and stored. Finally, ¹⁸ F-radiolabelling is achieved by treatment of the boronate groups with potassium 18-fluoride or other suitable source. The fluorination reaction takes 1h at pH 4.5 and room temperature but increasing the temperature to 37 ⁰ C and working at higher concentration can speed it up. Each step of the synthetic sequence is assessed using IR, Raman and NMR spectroscopy. The ¹⁸ F-nanoparticle-affibody conjugates are ready to use for medical applications after rapid purification by passage through an ion exchange cartridge.

For comparison the nanoparticle prior may be fluorinated prior to Affibody conjugation, and this may result in higher radiolabel yields. The nanoparticles are then rapidly passed through an ion exchange cartridge (30s), conjugated to the Affibody via the maleimide (free) group of end of the SMCC linker (30 minutes at room temperature, www.Piercenet.com/objects/view.cfm?type=file&ID=0581 , faster at 37 ⁰ C), filtered onto a Centricon filter (5-10min) to remove unconjugated Affibody.

This involves more steps but these are quick and readily carried out in a hospital radiopharmacy which generally have centrifuges for blood labelling procedures.

Example 3 - Comparison by labelling of Affibodies ® with ^99m Tc

The anti HER2 Affibody is labelled with ^99m Tc via the C terminal cysteine either directly or after conjugation with the linker HYNIC ³¹ . The cell binding of ^99m Tc-labelled anti HER-2 Affibody is compared with that of the ¹⁸ F-nanoparticle conjugates.

Example 4 - Stability of the ¹⁸ F-nanoparticle and protein conjugates

To determine the stability of the fluorinated nanoparticles, they are placed in dialysis units (Dispo-Biodialyzers, Sigma, Poole UK) with a molecular weight cut-off of IkDa and dialysed against serum and the serum periodically sampled and counted for ¹⁸ F. These techniques are routinely used in our laboratories. ³² Further verification of the intactness of the tracer will be achieved using size exclusion High Performance Liquid Chromatography (HPLC).

Example 5 - Immunoreactivitv by competition with unlabelled Affibodv and herceptin

To verify that the labelled Affibodies have retained their immunoreactivity they are incubated with excess antigen and submitted to size exclusion HPLC to ensure antigen/antibody complex formation has occurred.

Example 6 - Cell binding

Receptor specific binding of the anti-HER2- ¹⁸ F-nanoparticle is determined by comparing binding by breast tumour cell lines that express HER2 (SK-OV-3 (intensity 3+) and MDA- MB-453 (intensity 2+)) with HER2 negative cells (MDA-MB-468.) ³³ Kinetic cell binding studies are carried out using increasing concentrations of conjugated Affibody in the presence and absence of blocking levels of non-conjugated Affibody and also of herceptin. The binding of the Affibody ¹⁸ F-nanoparticle conjugates is compared with the binding of the ^99m Tc-labeled Affibody.

Example 7 - In Vivo

The in-vivo specificity of the conjugate for the HER-2 receptor is investigated by administration of the conjugate, with and without excess non-conjugated anti HER-2 Affibody or Herceptin, to nude mice (6 per group) bearing tumours established from either SK-OV-3 (HER-2 positive) or MDA-MB-468 (HER-2 receptor negative). At appropriate times after injection the mice are killed and tracer bio-distribution determined. Administration to kill times of 1 ,2 and 4h (as W ₂ of ¹⁸ F is 110 min 4h is longest practical time point) are also investigated.

Example 8 - Synthesis of the activated PAMAM dendrimers

The synthesis of PAMAM dendrimers is well documented ³⁴ . Monoprotected diamine 2 is readily synthesised from commercially available 2-(2-aminoethoxy)ethanol and 1,2-bis(2- chloroethoxy)ethane. Then, it is sequentially reacted with methyl acrylate and ethylenediamine to form a GO-dendrimer (generation 0 - dendrimer) possessing two terminal amino groups (PAMAM-(NH ₂ ) ₂ ). Dendrimers of higher generations are simply obtained by reiteration of these steps until the desired dendrimers' size is attained ³⁵ . For example, fourth generation PAMAM-(NH ₂ )3 ₂ dendrimer would roughly measure 3.7 nm in diameter and possess 32 amino groups at its periphery (Fig 3). Sixth generation dendrimer 4, possessing 128 amino surface groups is prepared and coupled to Fmoc protected (aminooxy)acetic acid to give functionalised dendrimer 5.

Example 9 - Conjugation to targeting units and fluorination

The dendrimer-biotin conjugate is readily prepared by coupling dendrimer 5 (G6) to biotin (sulfo-NHS-LC-biotin, Pierce Chemical Group, Rockford, IL) after deprotection of the amino group of the polyethylene glycol linker (Fig 4).

Finally, radio-labelled dendrimer-biotin conjugate 7 is obtained by coupling conjugate 6 with p-(di-tert-butylfluorosilyl) benzaldehyde and radio-labeling using ¹⁸ F7Kryptofix ®/K ⁺ complex in acetonitrile at room temperature for 15 minutes ³⁶ . These conditions enabled Schirrmacher et al to prepare radio-labeled conjugates in high yields (> 90%). Alternatively, the same authors successfully radio-labeled their di-(tert-butyl)fluoroaryl derivatives by treatment with the commercially available ¹⁸ FV[ ¹⁸ O]H ₂ O solution at 90 ⁰ C for 30 minutes.

Alternatively, classical approaches using mesylate ³⁷ and/or triflate ³⁸ derivatives, which are readily prepared from dendrimer 4 (Fig 4), may be employed.

Chemical integrity of all the dendrimers is assessed by IR, HPLC (size exclusion) and mass spectrometry. Radiochemical purity of the conjugates is checked by instant thin layer chromatography.

Example 10 - Stability of the ¹⁸ F-dendrimer

To determine the stability of the fluorinated dendrimers, they are placed in dialysis units (Dispo-Biodialyzers, Sigma, Poole UK) with a cut-off of IkDa Mwt and dialysed against

human serum and the serum periodically sampled and counted for ¹⁸ F. These techniques are routinely used in our laboratories ³⁹ . Further verification of the intactness of the tracer is achieved using size exclusion High Performance Liquid Chromatography (HPLC).

Example 11 - Immunoreactivitv by competition with unlabelled antibody fragments

To verify that the labelled antibody fragments have retained their immunoreactivity they are incubated with excess antigen and submitted to size exclusion HPLC to ensure antigen/antibody complex formation occurred.

Example 12 - Cell binding

In-vitro: The receptor specific uptake of ¹⁸ F by cells expressing the glycoprotein antigen target of Nr-Lu-10 incubated with ¹⁸ F-dendrimer-biotin is determined by pre-targeting with streptavidin-Nr-Lu-10 fragment antibody (fab) in the presence and absence of competing Nr-Lu-10. Competition assays are also carried out in the presence of non fluorinated biotin-dendrimer conjugates. Similar experiments are carried out with HER-2 pre-targeting receptors.

In-vivo: Nude mice bearing Nr-Lu-10 and MDA-MB-453, fed on a biotin-free diet, are injected with the Nr-Lu-10 fab- or HER-2-straptavidin conjugates respectively in the presence and absence of competing non-conjugated fragment antibody. 18F-dendrimer~ biotin is administered 2 or 5 days after pre-targeting with the streptavidin antibody fragment conjugates. Some experiments involve inclusion of a clearance step between pre-targeting and administration of the ¹⁸ F-dendrimer-biotin. The time intervals between administration of ¹⁸ F-dendrimer-biotin and kill of 1, 2 and 4h (as ¹⁸ F has a t1/2 of 110min 4h is the longest practical time point) may also be investigated.

Example 13 - Dendrimers in pre-targeting

The performance of the labelled dendrimer is examined using a well established model system for pre-targeting): The NR-LU10 - streptavidin conjugate (NrLuIO-SA) will be prepared in water according to the method described by Axworthy et al ⁴⁰ using the activated succinimidyl 4-(N-maleimido-methyl)cyclohexane-1-carboxylate (SMCC) or the water soluble sulfo-SMCC. The targeting NrLuIO-SA conjugate is incubated with LS180 human colon adenocarcinoma cells or injected into mice bearing LS180 xenografts. After

72h, unbound antibody is cleared from the blood, by injection of a clearance molecule consisting of biotin-galactose-human serum albumin (prepared as described ⁴⁰ ) which due to the presence of specific receptors in the liver is rapidly removed from circulation. Streptavidin conjugated to the bound antibody will be localised by addition of the ¹⁸ F ^" dendrimer-biotin conjugate, exploiting the very high affinity of biotin for streptavidin ⁴⁰ . The biodistribution of ¹⁸ F-dendrimer-biotin in animals not pre-targeted with the streptavidin- NrLUIO conjugate is also determined.

The labelled dendrimer is also tested by pre-targeting HER-2 expressing MDA-MB-453 or SKBr3 tumour cells and xenografts grown in nude mice with Streptavidin-HER-2- affibodies. Pre-targeting is compared with the performance of directly labelled HER-2 receptors.

Example 14 - Preparation of radiofluorinated boroarylated dendrimer-biotin conjugate (BDB)

Preparation of dendrimers

Methyl acrylate (47.8 mg, 50 μl_, 0.555 mmol) was added to a solution of O-(2- aminoethyl)-O'-[2-(Boc-amino)ethyl]hexaethylene glycol (96.3 mg, 0.206 mmol) in anhydrous methanol (500 μl_) and the reaction mixture was stirred under Ar for 36 hours. The solvent and excess methyl acrylate were removed under reduced pressure to leave the desired product as a viscous oil (131 mg, 99 %); δ ^" _H (250 MHz; CDCI ₃ ) 1.35 (9 H, s), 2.38 (4 H, t, J 7.0), 2.59 (2 H, t, .76.2), 2.75 (4 H, t, J 7.2), 3.20-3.23 (2 H, m), 3.43-3.46 (4 H, m), 3.50-3.56 (24 H, m), 3.57 (6 H, s), 5.12 (1 H, bs); δ ^" _c (62.5 MHz; CDCI ₃ ) 28.3, 32.3, 40.2, 49.7, 51.4, 53.0, 69.3, 70.1 , 70.3, 70.4, 70.7, 70.9, 79.0 155.9, 172.8.

A solution of ethylenediamine (70.7 mg, 100 μl_, 1.18 mmol) in anhydrous methanol (500 μL) was added to the growing dendron (113 mg, 0.176 mmol) and the reaction mixture was stirred under Ar for 5 days. The solvent and excess ethylenediamine were removed under reduced pressure at 40 ⁰ C to leave the desired product as a viscous oil (110 mg, 89 %); u _max (neat, film)/cnr ¹ 3551-3140, 2983, 2872, 1707, 1654, 1545, 1459, 1365, 1252, 1107; δ ^~ _H (250 MHZ; CDCI ₃ ) 1.40 (9 H, s), 1.94 (4 H, bs), 2.35 (4 H, t, J 6.1), 2.60-2.81 (10 H, m), 3.23-3.32 (6 H, m), 3.46-3.61 (28 H, m), 5.08 (1 H, bs), 7.58 (2 H, bs); ό ^~ _c (62.5 MHz; CDCI ₃ ) 28.4, 34.3, 40.3, 41.4, 41.8, 51.1, 53.8, 69.0, 69.1 , 70.1 , 70.4, 79.0, 155.9, 172.8.

This sequence of reactions was continued in order to produce the corresponding dendron with 16 terminal amines. The yields are shown below in Table 2:

Table 2: Dendron Synthesis

Data for the 16-NH ₂ dendron; ό ^~ _H (250 MHz; CD ₃ OD) 1.44 (9 H, s), 2.34-2.43 (60 H, m), 2.55-2.63 (30 H, m), 2.70-2.90 (90 H, m), 3.19-3.42 (74 H, m, also contains some CH ₃ OD from the CD ₃ OD) 3.50-3.68 (28 H, m); <5 _C (62.5 MHz; CD ₃ OD) 29.1, 34.9, 38.7, 42.0, 42.6, 51.3, 53.6, 70.3, 71.5, 80.3, 158.7, 175.0, 175.6.

Preparation ofboroaryl groups for functionalising dendrimer

Pyridine (158 mg, 162 μl_, 2.00 mmol) was added to a solution of 4-aminophenylboronic acid pinacol ester (399 mg, 1.82 mmol) in DCM (8.5 ml_) and after cooling to 0 ⁰ C, a solution of acryloyl chloride (182 mg, 163 μl_, 2.01 mmol) in DCM (4 mL) was added. The mixture was slowly warmed to room temperature and stirred under Ar for 15 hours. The reaction was quenched with sat. aq. NH ₄ CI solution (12.5 mL), then the organic layer was separated and the aqueous layer extracted with DCM (2 * 12.5 mL). The combined organic portions were dried (MgSO ₄ ), filtered and evaporated to leave a white solid. Purification was effected by flash column chromatography (Petroleum ether 40-60: Ethyl acetate, 4:1) to yield the product as a white solid (468 mg, 94 %), m.p. 157.5-158.5 ⁰ C; ό ^~ _H (250 MHZ; CD ₃ OD) 1.33 (12 H, s), 5.75 (1 H, d, J 10.1 ), 6.24 (1 H, dd, J 16.5, 10.1 ), 6.43 (1 H, d, J 16.5), 7.58-7.61 (3 H, m), 7.77 (2 H, d, J 7.9); ό ^~ _c (62.5 MHz; CD ₃ OD) 24.8, 83.8, 118.8, 128.1, 131.0, 135.8, 140.4, 163.6.

Addition of boroaryl groups to amine end groups of the dendrimer

The 16-NH ₂ dendron (34 mg, 8.73 μmol) and /V-[4-(4,4,5,5-tetramethyl- [1,3,2]dioxaborolan-2-yl)-phenyl]-acrylamide (77 mg, 0.282 mmol) were dissolved in anhydrous toluene (5 ml_) and stirred at 100 ⁰ C for 6 hours. The solvent was evaporated to leave the desired product as a viscous oil (110 mg, 100 %). ¹ H-NMR confirmed the presence of product.

Conjugation of boroarylated dendrimer with biotin

The 32-boroaryl-containing dendron (110 mg, 8.73 μmol) was dissolved in trifluoroacetic acid (7.68 g, 5 ml_, 67.3 mmol) and the reaction mixture stirred for 10 minutes. The solvent was then evaporated and the residue dissolved in DCM (5 ml_). (+)- Biotin λ/-hydroxysuccinimide ester (3.0 mg, 8.79 μmol) and triethylamine (87.8 mg, 121 μl_, 0.868 mmol) were then added and the reaction mixture stirred for 15 hours. Evaporation of the solvent yielded the desired product as a viscous oil. ¹ H-NMR demonstrated the presence of the product.

Radiofluorination of boroarylated dendrimer-biotin conjugate (BDB)

6μl of DBD (4OmM) was added to 28μl of Ethanol and mixed. 5μl of 18F-fluoride (2MBq) was added then 3μl of KHF2 (0.8M solution) and mixed and incubated for 30min at 37 ⁰ C. Sampling 1: 2μl was applied to a silica gel thin layer chromatography plate and 2μl diluted in 200μl phosphate buffered saline and centrifuged on a 5000 Mwt cutoff filter. The reaction left for another 30min at 37 ⁰ C. Sampling 2; 2μl was applied to a silica gel thin layer chromatography plate and 2μl diluted in 200μl phosphate buffered saline and centrifuged on a 5000 Mwt cutoff filter.

Thin layer chromatography

Using silica gel on aluminium plates cut 8cm by 2cm. 2μl samples were applied 1cm up the plate, dried and the plate placed in a chromatography jar containing 95% acetonitrile and 5% water to a depth of 5mm. The TLC was run until the solvent front within 5mm of the top of the plate. They were then dried and movement of activity measured on a GITA star flatbed TLC reader (Raytest, Germany). 18F-fluoride remains at the origin.

Figure 5 shows sample 2 with 18% having migrated with the solvent with an RF of 0.7. Seven % of sample 1 migrated from the origin.

Size-exclusion filtration on 5000 mwt cutoff filters

The boroarylated dendrimers have a molecular weight of about 12,000 g so will be retained by the 5000 mwt cut off filters. Thus activity retained by the filter as a percentage of total activity will be a measure of labelling efficiency. About 6% of activity was retained by the filter after 30min (sample 1) but by 60min 25% was retained (sample 2).

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