This application claims priority from Australian provisional patent application no. 2021903919, filed on 3 December 2021, the entire contents of which are incorporated herein by this reference.

Field

This disclosure relates to metal-organic framework (MOF) biocomposites, in particular biocomposites comprising partially embedded biomolecules such as antibodies. The disclosure also relates to methods of producing the MOF biocomposites, and uses of the MOF biocomposites in applications such as imaging, sensing and detection, diagnosis and therapy.

Background

Antibodies (Abs), especially monoclonal antibodies (mAbs), are cornerstone biopharmaceuticals that are involved in the treatment of a wide range of conditions, including chronic inflammatory, infectious diseases, cardiovascular diseases, and cancer ^[1,2] providing precision medicine not possible with other drugs. ^[3,4] The efficacy of Abs can be attributed to their capacity to specifically bind molecular components with excellent targeting selectivity. ^[2,4,5] Furthermore, many of their properties such as antigen-binding specificity, affinity, and cellular internalization can be fine-tuned to improve their clinical utility. ^[1] To expand their utility, Abs can be combined with functional nanomaterials such as quantum dots (QDs), gold or iron oxide nanoparticles for imaging, sensing, and targeted drug delivery applications^.

However, when a nanoparticle is decorated with antibodies, the orientation of the protein is typically lost and binding sites are incompletely available for target recognition ^[7,8] . A precise and homogeneous Ab orientation would greatly reduce nonspecific tissue interactions and off-target effects in vivo. ^[7,9] However, precise Ab orientation on particles is made cumbersome by employing involved chemical protocols (e.g. protein fusion methods) or is limited to specific materials (e.g. gold). ^[8]

Recently, metal-organic frameworks (MOFs) ^[10] as nanocrystals have shown promise in biotechnology and biomedical applications. ^[11-15] MOFs have been combined with Abs, either via encapsulation or site-specific conjugation, for bio-banking and immunosensing applications. ^{[11-13-16-22]} However, to the best of the inventors' knowledge, there are no literature reports on the controlled self-assembly of MOFs on selected regions of Abs.

It would be desirable to provide new facile and versatile Ab-directed chemistries which facilitate the use of Ab-targeted particles in modern medicine. ^[8]

Summary of the Invention

In one aspect, there is provided a metal-organic framework (MOF) biocomposite comprising a partially embedded biomolecule which is an antibody, wherein the MOF comprises Zn ²⁺ ions and an azole-containing ligand, and wherein the antibody is oriented such that the antigen-binding regions of the antibody are not embedded in the MOF and are available for binding.

In another aspect, there is provided a metal-organic framework (MOF) biocomposite comprising a partially embedded biomolecule, wherein the biomolecule comprises an antibody F _c region amino acid sequence or variant thereof, the MOF comprises Zn ²⁺ ions and an azole- containing ligand, and wherein at least part of the F _c region is embedded in the MOF. In some embodiments, the biomolecule comprises an antibody F _c region amino acid sequence. In some embodiments, the biomolecule is an antibody. In some embodiments, the antibody is oriented such that the antigen-binding regions of the antibody are not embedded in the MOF and are available for binding.

Advantageously, it has been found that the crystallisation of a MOF can be triggered by the fragment crystallisable (F _c ) region of various antibodies, with the selective growth yielding biocomposites with oriented antibodies on the MOF nanocrystals (MOF* Ab). The F _c regions of the antibodies are partially inserted within the MOF surface and the antigen-binding regions protrude from the MOF surface towards the target. This ordered configuration imparts antibody- antigen recognition properties to the biocomposite and shows preserved target binding comparable to the parental antibodies.

In some embodiments, only the F _c region of the antibody is embedded within the MOF.

In some embodiments, the biomolecule is bonded to the MOF by non-covalent interactions.

In some embodiments, the antibody is an antibody in a class selected from the group consisting of IgG, IgE and IgD. In some embodiments, the antibody is an IgG antibody. In some embodiments, the IgG antibody is type 1, type 2, type 3, or type 4. In some embodiments, the antibody is an anti-HER2, an anti-P-selectin, anti-fibrin, anti- PSMA or human IgG antibody. In some embodiments, the antibody is trastuzumab.

In some embodiments, the antibody is human, humanised or chimeric.

In some embodiments, the F _c region has a net negative charge.

In some embodiments, the azole-containing ligand is an imidazole-containing ligand.

In some embodiments, the azole-containing ligand is 2-methylimidazole.

In some embodiments, the MOF comprises carbonate ions.

In some embodiments, the MOF is a crystalline material with orthorhombic unit cell.

In some embodiments, the MOF is ZIF-C.

In some embodiments, the MOF biocomposite comprises a therapeutic, imaging, diagnostic, signalling or sensing agent.

In some embodiments, the MOF biocomposite comprises a fluorescent protein, a quantum dot or a dye.

In some embodiments, the MOF biocomposite comprises an additional metal ion . In some embodiments, the MOF comprises Cu ions.

In some embodiments, the MOF biocomposite comprises a radionuclide. In some embodiments, the radionuclide is ⁶⁴ Cu or ⁶⁷ Cu.

In some embodiments, the MOF biocomposite comprises magnetic particles.

In some embodiments, MOF biocomposite comprises a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic agent. In some embodiments, the therapeutic agent is doxorubicin.

In some embodiments, the MOF comprises a nucleic acid.

In another aspect, there is provided a method of producing a MOF biocomposite as defined herein, the method comprising: contacting a biomolecule comprising an antibody F _c region amino acid sequence or variant thereof, with an azole-containing ligand, and with Zn ²⁺ ions, in aqueous solution, thereby producing the MOF biocomposite.

In some embodiments, the aqueous solution contains a buffer. In some embodiments, the buffer is a zwitterionic buffer. In some embodiments, the buffer is selected from the group consisting of MOPS, CAPS, HEPES, MES and TRIS.

In some embodiments, the contacting step is carried out a temperature in the range of from 10 °C to 50 °C. In some embodiments, the contacting step is carried out for a time period of up to 24 hours.

In some embodiments, the contacting step is carried out in the presence of carbonate ions.

In some embodiments, the contacting step is carried out under ambient atmosphere, and carbon dioxide present in the atmosphere provides a source of carbonate in the aqueous solution.

In some embodiments, the Zn ²⁺ ions are provided in the form of zinc acetate dihydrate. In some embodiments, the contacting step is carried out using zinc acetate dihydrate at a concentration in the range of from 10 to 50 mM.

In some embodiments, the azole-containing ligand is 2-methylimidazole. In some embodiments, the contacting step is carried out using 2-methylimidazole at a concentration in the range of from 40 to 400 mM.

In some embodiments, the contacting step is carried out using an amount of biomolecule in the range of from 25 μg to 75 μg per 100μL of aqueous solution.

In some embodiments, the method comprises the steps of: a. contacting the biomolecule with an aqueous solution containing azole- containing ligand; and b. contacting the solution of a. with an aqueous solution containing Zn ²⁺ ions.

In another aspect, there is provided a metal-organic framework (MOF) biocomposite when produced according to a method as defined herein.

In another aspect, there is provided a pharmaceutical composition comprising a metal organic framework (MOF) biocomposite as defined herein and a pharmaceutically acceptable excipient.

In another aspect, there is provided a method of treating and/or preventing a disease or disorder in a subject, comprising administering a metal-organic framework (MOF) biocomposite or a pharmaceutical composition as defined herein, to the subject.

In another aspect, there is provided use of a metal-organic framework (MOF) biocomposite as defined herein in the manufacture of a medicament for diagnosing, treating and/or preventing a disease or disorder.

In another aspect, there is provided a metal-organic framework (MOF) biocomposite or a pharmaceutical composition as defined herein for use in the diagnosis, treatment and/or prevention of a disease or disorder. In some embodiments, the biomolecule is an anti-HER-2 antibody, such as trastuzumab, and the disease or disorder is a cancer, for example a cancer selected from the group consisting of breast cancer, metastatic gastric adenocarcinoma and gastroesophageal junction adenocarcinoma.

In some embodiments, the biomolecule is an anti-PSMA antibody, and the disease of disorder is a cancer, for example prostate cancer.

In some embodiments, the biomolecule is an anti-fibrin antibody (e.g. 59D8), and the disease or disorder is multiple sclerosis, a neurodegenerative disease or disorder, or a cardiovascular disease or disorder.

In some embodiments, the biomolecule is an anti-P-selectin antibody (e.g. VH10), and the disease or disorder is an inflammatory disease or disorder, or a cardiovascular disease or disorder.

In another aspect, there is provided a method of imaging or biosensing a subject, comprising administering to a subject a MOF biocomposite as defined herein which comprises an imaging, diagnostic, signalling or sensing agent, and carrying out imaging or biosensing of the subject.

In another aspect, there is provided a method of diagnosing whether a subject has a disease or disorder, comprising administering to a subject a MOF biocomposite as defined herein which comprises an imaging, diagnostic, signalling or sensing agent, carrying out imaging or biosensing of the subject, and determining whether the subject has a disease or disorder based on the imaging results.

In another aspect, there is provided a method of diagnosing whether a subject has a disease or disorder, comprising contacting a biological sample isolated from a subject with a MOF biocomposite as defined herein which comprises an imaging, diagnostic, signalling or sensing agent, carrying out imaging or biosensing of the biological sample, and determining whether the subject has a disease or disorder based on the imaging results.

In another aspect, there is provided a method of diagnosis and therapy of a disease or disorder in a subject, comprising carrying out a method of diagnosis as defined above and, if the subject is determined to have a disease or disorder susceptible to treatment with a therapy, administering the therapy to the subject.

In some embodiments, the imaging, diagnostic, signalling or sensing agent is a radionuclide.

In some embodiments, the disease or disorder is cancer. In some embodiments, the therapy is a MOF biocomposite as defined herein which comprises a therapeutic agent. In some embodiments, the therapeutic agent is a radionuclide or a cytotoxic agent.

In another aspect, there is provided a method of determining the presence and/or concentration of a target molecule or analyte in a sample, comprising: contacting a sample with a metal-organic framework (MOF) biocomposite as defined herein which comprises a sensing or signalling agent, wherein the biomolecule is an antibody having an antigen-binding region which binds to a target molecule or analyte; and if the sample comprises the target molecule or analyte, detecting the presence and/or concentration of the target molecule or analyte in the sample through binding of the target molecule or analyte to the antibody so as to generate a signal from the sensing or signalling agent.

Brief Description of the Drawings

Figure 1 shows a schematic representation of the spatially controlled crystallisation of a Zn-based ZIF-C*Ab. (a) Antibody and MOF precursors (Zn ²⁺ ions (Zn ²⁺ ), azole containing ligand (2-methylimidazole - 2-mIM), carbonate source (CO ₃ ^2- )); (b) Nucleation of ZIF-C around the Fc region (inset shows ZIF-C crystal structure); (c-e) growth of plate-like nanocrystals where antibodies are only partially embedded in the MOF (here named as ZIF- C*Ab); (f) agglomeration of ZIF-C*Ab nanocrystals; (g) proposed targeting application for ZIF-C*Ab.

Figure 2 shows the XRD (X-ray powder diffraction) patterns, FTIR (Fourier transform infrared) vibrational modes, Time -resolved SAXS (Small Angle X-ray Scattering) patterns, SEM (scanning electron microscope) image, AFM (atomic force microscope) image, and TEM (transmission electron microscope) image of a ZIF-C*Ab. (a) XRD patterns of ZIF-C*α-HER2 and calculated ZIF-C. (b) FTIR spectra of α-HER2 and ZIF-C*α-HER2. (c) Time-resolved SAXS patterns of the nucleation and growth of ZIF-C*α-HER2. (d) SEM image of ZIF-C*α- HER2. (e) AFM amplitude image of pyrolyzed ZIF-C*α-HER2; the inset shows a 3D topography of a 100x100 nm ² region with cavities, (f) TEM image of pyrolyzed ZIF-C*α- HER2.

Figure 3 shows characterisation of ZIF-C*α-fHER2 and QD@ZIF-C*α-fHER2. (a) Photograph of a ZIF-C*α-fHER2 solution under UV light, (b) Schematic illustrations of ZIF- C*α-fHER2 with Ab Fc region and Ab antigen binding site and (c-d) binding of ZIF-C *α-

RECTIFIED SHEET (RULE 91) fHER2 to its target cell, (e) 2D and 3D confocal microscopy images of ZIF-C*fAb internalization. SKOV-3 (HER2-R ⁺ ) cells post 30 min incubation at 37 °C with either ZIF- C*fHER2 (left) or ZIF-C*fIgG (right) presented in either 2D (top) or 3D (bottom). Cell nuclei were stained with 4’,6-diamidino-2-phenylindole and cell membranes stained with Wheat Germ Agglutinin, Alexa FluorTM 647 in the 2D data set. Scale bar 5 pm. (f) Statistical analysis of the immunofluorescence assay (e), wherein the mean of the ZIF-C*fAb as positive signal in the cell was measured, (g) Photograph of a QD@ZIF-C*α-fHER2 solution under UV light, (h, i) Schematic illustrations of QD@ ZIF-C *α-fHER2 (h) and binding of QD@ ZIF-C *α-fHER2 to its target cell (i). (j) TEM image of QD@ZIF-C*α-fHER2. Co-localization of Alexa-488 fluorochrome and QD 624 nm. (k) Representative dot plots of the ZIF-C*fAb binding efficiency to different cell lines. ZIF-C*fHER2 and QD@ZIF-C*fHER2 to SKOV-3 are shown. ZIF-C*fhIgG was used as a control for binding to SKOV-3 cells and ZIF-C*α-fHER2 was used as a control for binding to MDA-MB-231 cells. The displayed values represent fluorescence intensity in percentage. (1) Flow cytometry analysis based on the co-localization of QD 625 nm and A1488 fluorescence positive signal. Statistical analysis was performed using two-way ANOVA followed by a multiple comparison test (Tukey test) (± SEM) (n = 3); with **p ≤ 0.05, ****p ≤ 0.001 considered as statistically significant.

Figure 4 shows ZIF-C* Growth Kinetics Model. A 96-well plate was coated with or without 2-methylimidazole (2-mIM) and 50 μg of A) BSA, B) 59D8, or C) VH10. Zinc ions (Zn ²⁺ ) was injected after 60 seconds (up arrow), and absorbance as optical density (OD) at 595 nm was measured every 60 seconds for a length of 30 minutes (31 cycles) at 37°C. D) pH sensitivity (anti-HER2/anti-PSMA) was tested via kinetic growth by adding 1% HC1 to change the pH to 5 (solid down arrow) and then to pH 2 (hollow down arrow). Each dot represents the cycles mean OD + SEM (n=3). A repeated unpaired T-test was performed at each cycle point (ns = not significant, P>0.05; *** P<0.001; **** P<0.0001).

Figure 5 shows XRD patterns of ZIF-C* α-HER2, ZIF-C*hIgG, and ZIF-C.

Figure 6 shows FTIR spectra of α-HER2 (d), hlgG (e), ZIF-C*α-HER2 (a), ZIF- C*hIgG (b), and pure ZIF-C (c) and schematic representations of orientation of solutions within MOF. a) The spectrum of ZIF-C, Zn ₂ (2-mIM) ₂ (CO ₃ ), and the related ZIF-C*Ab shows bands in the 700-850 and 1300-1600 cm' ¹ regions that can be assigned to bending (828 cm' ¹ ) and asymmetric stretching modes (1575 and 1375 cm' ¹ ) of CO ₃ ² ’. The Zn-N stretching mode, typically at 420 cm ^-1 for sodalite (sod) or diamondoid (dia) ZIF-8 topologies, Zn(2-mIM)2, for ZIF-C is blue-shifted (e.g. centred at 425 cm ^-1 ) because of the coordination of CO ₃ ² ’ with Zn cations and the related change in coordination environment, bi) coordination mode of Zn with CO ₃ ^2- circled; bi) crystalline framework of ZIF-C viewed along the c axis; b ₃ ) crystalline framework of ZIF-C viewed along the a axis.

Figure 7 shows Antibody Immobilisation Efficacy and Stability of ZIF-C*Ab. A) Protein concentration of the supernatant for produced ZIF-C produced in the presence of BSA, ZIF-C*VH10, ZIF-C*59D8, or ZIF-C*hIgG was measured using a BCA assay and immobilisation efficacy was calculated. Bar graph of the mean + SEM (n=3) and a one-way ANOVA test was performed (ns = not significant, P>0.05). B) ZIF-C* VH10 and ZIFC-C*hIgG were produced and immobilisation efficacy measured for the supernatant at production and after the nanoparticles were stored at 4°C for 4 weeks. Bar graph of the mean + SEM (n=2) and a one-way ANOVA test was performed (ns = not significant, P>0.05).

Figure 8 shows SEM images. Low- and high-magnification SEM images of (a, b) ZIF- C*α-HER2 and (c, d) ZIF-C*hIgG are shown.

Figure 9 shows TEM images. Low- and high- magnification of ZIF-C*α-HER2 are shown.

Figure 10 shows schematic of the stopped-flow setup used for the SAXS measurements.

Figure 11 shows time-resolved SAXS patterns, (a) Plots of growth of ZIF-C*α-HER2. The data were successfully fitted using a sphere (DAB) model (time < 3.7 s) and a plate model (time > 3.7 s). (b) invariant Q, (c) mean particle size (R or T depending on the particle growth model), and (d) mean inter-particle distance (<d>) were calculated. Lateral dimension of the plates cannot be quantified as they are larger than the 100 nm resolution limit.

Figure 12 shows SAXS pattern of the dry powder of ZIF-C*α-HER2 and fitted data. Cavities with an average size of 3 nm and an average distance of 13.4 nm between the cavities were measured. These cavities correspond to the Fc fragments of the Ab that are embedded in the MOF particles.

Figure 13 shows time-resolved SAXS patterns, (a) Plots of growth of bare MOF, i.e. no Ab). The data were successfully fitted with the spheres ScS model, (b) invariant Q and (c) mean sphere radius (R, and parameter σ of the Shultz model) were calculated.

Figure 14 shows representations of the different Ab components that were investigated using time-resolved SAXS.

Figure 15 shows time-resolved SAXS patterns, (a) Plots of growth of ZIF-C grown in the presence of Fc (ZIF-C/Fc composite). The data were successfully fitted using the plate model, (b) invariant Q, (c) mean particle size (T, calculated for time > 3.7 s), and (d) mean inter-particle distance (<d>) were calculated . The calculated average thickness of the plates was 16 ± 1 nm. The calculated mean inter-particle distance was calculated after 5 s.

Figure 16 shows time-resolved SAXS patterns, (a) Plots of growth of ZIF-C grown in the presence of Fab (ZIF-C/Fab composite). The data were successfully fitted with the spheres ScS model, (b) invariant Q and (c) mean sphere radius (R, and parameter σ of the Shultz model) were calculated.

Figure 17 shows time-resolved SAXS patterns, (a) Plots of growth of ZIF-C grown in the presence of F(ab')2 (ZIF-C/F(ab')2 composite). The data were successfully fitted using the spheres ScS model combined with the structure factor SSHS(q) (time < 6.2 s) and the rod model (time > 6.2 s). (b) invariant Q and (c) mean particle size (R (spheres) or Rc (rods)) were calculated.

Figure 18 shows time-resolved SAXS patterns, (a) Plots of growth of ZIF-C grown in the presence of SGP (ZIF-C/SGP composite). The data were successfully fitted using the hierarchical spheres ScS model, (b) invariant Q and (c) mean sphere radius (R, and parameter σ of the Shultz model) were calculated.

Figure 19 shows time-resolved SAXS patterns, (a) Plots of growth of ZIF-C grown in the presence of SG (ZIF-C/SG composite). The data were successfully fitted using the hierarchical spheres ScS model, (b) invariant Q and (c) mean sphere radius (R, and parameter σ of the Shultz model) were calculated.

Figure 20 shows XRD patterns before and after pyrolysis at 325 °C. (a) ZIF-C*α-HER2 and (b) ZIF-C*hIgG.

Figure 21 shows SEM images of ZIF-C*α-HER2 after pyrolysis at 325 °C. (a) low magnification and (b) high magnification.

Figure 22 shows AFM images of pyrolysed ZIF-C* α-HER2 at 325 °C. (a, d) topography, (b, e) amplitude, and (c, f) phase contrast images show a plate-like morphology, (d-f) high magnification of the edge of the structure.

Figure 23 shows AFM analysis of pyrolyzed ZIF-C*α-HER2 at 325 °C. (a) amplitude and (b) phase images of the edge of the layered structure, (c) Typical line profile obtained from the phase image, (d) Histogram of the layer thickness populated from several line profiles obtained from different sample regions. The plate thickness typically ranged from 3 to 5 nm.

Figure 24 shows AFM analysis of pyrolyzed ZIF-C*α-HER2 at 325 °C. (a) topography and (b) amplitude images, (c) Typical line profile obtained from topography analysis in (a) showing the cavities on the surface, (d) Histogram of the cavity diameter values obtained from different line profiles obtained from different sample regions and a corresponding Gaussian distribution fit. The calculated cavity diameter value was 11 ± 3 nm (centre and standard deviation value).

Figure 25 shows cellular internalization of ZIF-C*α-fHER2. Representative confocal fluorescence microscopy Z stack images of SKOV-3 cells after 30 min incubation at 37 °C with either ZIF-C*fHER2 (top row) or ZIF-C*fIgG (bottom row). Z stack images were acquired from the bottom to the top of the cells Nuclei are stained with 4',6-diamidino-2-phenylindole and cell membrane with Wheat Germ Agglutinin, Alexa Fluor™ 647. Scale bar, 5 pm. Slice numbers (1-33) indicate the image position within Z stack of the cell.

Figure 26 shows 3D confocal microscopy images of ZIF-C*fAb internalization (fAb = Alexa-Fluor 488 pre-labelled α-HER2 or hlgG). SKOV-3 (HER2-R+) or MDA-MB-231 (HER2-R-) cells were incubated with either ZIF-C* α-fHER2 or ZIF-C*fhIgG and DAPI DNA stain.

Figure 27 shows FTIR spectra and XRD patterns of QD only and QD@ZIF-C*α- HER2, QD@ZIF-C*hIgG. (a) FTIR spectra and (b) XRD patterns.

Figure 28 shows TEM images of QD@ZIF-C*α-HER2.

Figure 29 shows SEM image and a representative EDX spectrum collected from a 0.5 x 0.5 pm ² region of QD@ZIF-C*α-HER2.

Figure 30 shows Flow Cytometric Analysis of Fluorescent Labelled ZIF-C*VH10- AF488. A) hypothetical depiction of fluorescent labelled nanoparticle (ZIF-C*VH10-AF488).

B) Images of ZIF-C*VH10-AF488 particles illuminated with daylight (left) and ultraviolet light (middle), and confocal microscopy image (right). Human Platelet Rich Plasma was activated with ADP (lines) and compared to non-activated platelets (dots) by flow cytometry.

C) Histograms of ZIF-C*VH10-AF488 and ZIF-C*hIgG-AF488 (2.5 μg/mL) incubated on platelets (n=2). D) Column graph showing mean percentage ± SEM of platelets with ZIF- C*VH10-AF488 or h!gG-AF48 signal (n=2) with a one-way ANOVA performed followed by Tukey’s multiple comparison post hoc test (ns = not significant, P>0.05; * P<0.05; ** P<0.01).

Figure 31 shows Illustration and Image of Dual Labelled QD@ZIF-C*VH10-AF488. A) Hypothetical depiction of ZIF-C biocomposite constructed with AF488 labelled VH10, then encapsulated with quantum dots for dual labelling and B) images of QD@ZIF-C*VH10 - AF488 particles illuminated with daylight (left) and ultraviolet light (right). C) Confocal microscopy images showing co-localisation of AF488 labelled VH10 and quantum dots. Figure 32 shows Illustration and Images of Iron Oxide Encapsulated ZIF-C* VH10. A) Illustration depicting IONP encapsulated ZIF-C biocomposite produced with VH10 - AF488.

B) Photograph IONP Cy5@VH10- AF488 (left) and collection of particles by applying a magnetic field (right). C) Confocal microscopy image showing co-localisation of AF488 labelled VH10 and Cy5 labelled IONP.

Figure 33 shows A) schematic of bispecific MOFs. Dot plot of HER2+cells (B) and PBMC (C) incubated with bispecific MOF.

Figure 34 shows A) Schematic layout of the Microfluidic channels with a neodymium magnetic disc. B) Representative fluorescence images sequence showing the MP/ZIF- C*fPSMA or MP@ZIF-C*fPSMA aggregation occurring under the magnetic area post 60 sec.

C) Statistic analysis of the MP/ZIF-C*fPSMA or MP@ZIF-C*fPSMA accumulation in the magnetic area post 60 sec in comparison to the control. D) Statistic analysis in the magnetic area over a time period of 60 sec in comparison to the control. E) Representative fluorescence images of the upstream area and magnetic area indicate the C4.2488 accumulation after pre- incubation of magnetic MPQD@ZIF-C*PSMA or control MPQD@ZIF-C*hIgG post 60 sec of flow. F) Statistic analysis of the C4.2488 accumulation in the magnetic area at the end point 60 sec in comparison to the controls.

Figure 35 shows targeting in vivo in tumour-bearing mice using an in vivo imaging system (IVIS). A) Xenograft mouse model was performed by inoculation of 2x10 ⁶ HER+ cells s.c. into the right flank of 6 week-old immunodeficient BALB/c-Foxnlnu mice. ZIF- C*fHER2 and ZIF-C*fC (control) were pre-labelled with Cy5-NHS Dye, injected i.v. into the animal and biodistribution performed 4 h post injection. HER2 ⁺ xenograft demonstrates the targeting property in vivo of Ab-NP compared to non-binding antibody control B) Statistical analysis of xenograft animal confirms a high binding to its target organ compared to the muscle in vivo. Two-way ANOVA (n=3-5).

Figure 36 shows toxicity study. The viability of untreated SKOV-3 cells or SKOV-3 cells incubated with 2.5ug DOX@ZIF-C*HER2 or free drug were analysed 48 h post incubation; n=3; ANOVA Tukey post hoc, with p < 0.05 considered statistically significant.

Figure 37 shows protein visualisation of ZIF-C biocomposite supernatants. Coomassie brilliant blue stained SDS-PAGE of supernatants collected from ZIF-C produced in the presence of BSA (left) and VH10, XIIF9, hlgG, 59D8, or without protein (right). Alongside each ZIF-C biocomposite, a neat sample of the equivalent mAb was run. "Standard" corresponds to BioRad Precision Plus Dual Colour Standard. Detailed Description

Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., chemistry, biology, and the like).

The present disclosure refers to the entire contents of certain documents being incorporated herein by reference. In the event of any inconsistent teaching between the teaching of the present disclosure and the contents of those documents, the teaching of the present disclosure takes precedence.

It is to be understood that if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art in Australia or any other country

As used herein, the term "and/or", e.g., "X and/or Y" shall be understood to mean either "X and Y" or "X or Y" and shall be taken to provide explicit support for both meanings or for either meaning.

As used herein, the term about, unless stated to the contrary, refers to +/- 10%, of the designated value.

As used herein, the terms "a", "an" and "the" include both singular and plural aspects, unless the context clearly indicates otherwise.

Unless otherwise indicated, terms such as "first," "second," etc. are used herein merely as labels, and are not intended to impose ordinal, positional, or hierarchical requirements on the items to which these terms refer. Moreover, reference to a "second" item does not require or preclude the existence of lower-numbered item (e.g., a "first" item) and/or a higher- numbered item (e.g., a "third" item).

As used herein, the phrase "at least one of", when used with a list of items, means different combinations of one or more of the listed items may be used and only one of the items in the list may be needed. The item may be a particular object, thing, or category. In other words, "at least one of’ means any combination of items or number of items may be used from the list, but not all of the items in the list may be required. For example, "at least one of item A, item B, and item C" may mean item A; item A and item B; item B; item A, item B, and item C; or item B and item C. In some cases, "at least one of item A, item B, and item C" may mean, for example and without limitation, two of item A, one of item B, and ten of item C; four of item B and seven of item C; or some other suitable combination. As used herein, the word "comprise" and other forms of the word, such as "comprising" and "comprises," means including but not limited to, and is not intended to exclude, for example, other additives, components, integers, or steps.

As used herein, the term "subject" refers to any organism that is susceptible to a disease or condition. For example, the subject can be an animal, a mammal, a primate, a livestock animal (e.g., sheep, cow, horse, pig), a companion animal (e.g., dog, cat), or a laboratory animal (e.g., mouse, rabbit, rat, guinea pig, hamster). In one embodiment, the subject is a mammal. In one embodiment, the subject is human. In one embodiment, the subject is a non-human animal.

As used herein, the term "treating" includes alleviation of symptoms associated with a specific disorder or condition.

As used herein, the term "prevention" includes prophylaxis of the specific disorder or condition.

The term "therapeutically effective amount", as used herein, refers to a therapy being administered in an amount sufficient to alleviate or prevent to some extent one or more of the symptoms of the disorder or condition being treated. The result can be the reduction and/or alleviation of the signs, symptoms, or causes of a disease or condition, or any other desired alteration of a biological system. The term, an "effective amount", as used herein, refers to an amount of a therapy effective to achieve a desired pharmacologic effect or therapeutic improvement without undue adverse side effects. The term "therapeutically effective amount" includes, for example, a prophylactically effective amount. It is understood that "an effective amount" or "a therapeutically effective amount" can vary from subject to subject, due to variation in metabolism of the compound and any of age, weight, general condition of the subject, the condition being treated, the severity of the condition being treated, and the judgment of the prescribing physician.

The term "diagnosis", as used herein, may for example include a process of administering a biocomposite of the disclosure to a subject having or suspected of having a condition, disease or disorder, and subsequently using a technique to provide information on the level of radioactivity in various parts of the body, for example imaging a part or parts of the subject’ s body, in order to enable a decision to be made regarding the existence of a disease, disorder or condition and/or regarding the status, staging and/or extent of the disease, disorder or condition. In some embodiments, the term "diagnosis" may include the act of identifying and/or classifying the status, staging or extent of a disease, disorder or condition from signs or symptoms. Metal-organic framework (MOF) biocomposites

There is provided a metal-organic framework (MOF) biocomposite comprising a partially embedded biomolecule which is an antibody, wherein the MOF comprises Zn ²⁺ ions and an azole-containing ligand, and wherein the antibody is oriented such that the antigen- binding regions of the antibody are not embedded in the MOF and are available for binding. There is also provided a metal-organic framework (MOF) biocomposite comprising a partially embedded biomolecule, wherein the biomolecule comprises an antibody F _c region amino acid sequence or variant thereof, the MOF comprises Zn ²⁺ ions and an azole-containing ligand, and wherein at least part of the F _c region is embedded in the MOF.

As discussed above, it has been found that the crystallisation of a MOF can be triggered by the fragment crystallisable (F _c ) region of various antibodies, with the selective growth yielding biocomposites with oriented antibodies on the MOF nanocrystals (MOF* Ab). The F _c regions of the antibodies are at least partially inserted within the MOF surface and the antigen- binding regions protrude from the MOF surface towards the target. This ordered configuration imparts antibody-antigen recognition properties to the biocomposite and shows preserved target binding when compared to the parental antibodies. The biocomposites find use in applications such as imaging, diagnostics, biosensing, therapy, and detection of biological target molecules in samples.

MOF biocomposites described herein may for example be referred to using the following nomenclature MOF*Bio, wherein MOF is the metal-organic framework and Bio is the biomolecule. The notation * indicates that the group Bio is partially embedded in the MOF. For example, ZIF-C*α-HER2 refers to a MOF biocomposite in which the metal-organic framework is ZIF-C and the biomolecule is an anti-HER2 antibody, with the antibody being partially embedded in the MOF.

The notation AA@MOF refers to a composite in which AA is an additional agent as defined herein, MOF is the metal-organic framework, and @ indicates that the AA is encapsulated into the MOF.

MOF biocomposites described herein may for example be referred to using the following nomenclature AA@MOF*Bio, wherein MOF is the metal-organic framework, Bio is the biomolecule, and AA is an additional agent as defined herein. For example, DOX@ZIF- C*Ab refers to a MOF biocomposite in which the metal-organic framework is ZIF-C, the biomolecule is an antibody, and the additional agent is doxorubicin, with the antibody being partially embedded in the MOF, and the doxorubicin being encapsulated into the MOF.

Metal-Organic Framework

The MOF biocomposites comprise a metal-organic framework (MOF). Metal-organic frameworks are a class of compounds containing metal ions coordinated to organic ligands, which can form two or three dimensional structures. A metal-organic framework, abbreviated to MOF, is a coordination network with organic ligands containing potential voids. A coordination network may be defined as a coordination compound extending, through repeating coordination entities, in 1 dimension, but with cross-links between two or more individual chains, loops, or spiro-links, or a coordination compound extending through repeating coordination entities in 2 or 3 dimensions. See for example, Pure Appl. Chem., Vol. 85, No. 8, pp. 1715-1724, 2013.

The MOF of the present disclosure comprises Zn ²⁺ ions and an azole-containing ligand. Any suitable source of Zn ²⁺ ions may be used, such as for example a zinc salt. In some embodiments, the Zn ²⁺ ions are provided in the form of zinc acetate. In some embodiments, the Zn ²⁺ ions are provided in the form of zinc acetate dihydrate.

The azole-containing ligand is a ligand which is capable of forming a MOF with zinc ions and which contains an azole group, such as an imidazole or a triazole. In some embodiments, the azole group is one which contains an acidic proton. In some embodiments, the azole-containing ligand is an imidazole-containing ligand. In some other embodiments, the azole-containing ligand is a triazole-containing ligand. In some embodiments, the azole- containing ligand is an imidazole-containing ligand containing an acidic proton.

The azole-containing group may be substituted or unsubstituted. In some embodiments, the azole-containing ligand is substituted with one or more Ci-6 alkyl groups, e.g. one or more methyl groups.

In some embodiments, the azole-containing ligand is 2-methylimidazole.

In some embodiments, the molar ratio of azole-containing ligand to Zn ²⁺ ions is in the range of from 16:1 to 2:1, or from 8:1 to 2:1, or from 6:1 to 2:1, or from 5:1 to 3:1, or about 4:1.

In some embodiments, the MOF comprises carbonate ions. Any suitable source of carbonate ions may be used. For example, in some embodiments, production of the MOF biocomposite is carried out under ambient atmosphere, and carbon dioxide present in the atmosphere provides a source of carbonate. In some other embodiments, a carbonate or bicarbonate (e.g. metal bicarbonate or carbonate) may be added.

In some embodiments, the MOF is a zeolitic imidazolate framework (ZIF). ZIFs are a class of MOF which are comprised of imidazolate linkers and metal ions (e.g. zinc ions), with structures similar to conventional zeolites. ZIFs have good thermal and chemical stability, including stability in biological systems. ZIFs can also demonstrate pH-dependent stability properties, such that in neutral media they can demonstrate good stability, whilst on exposure to acidic conditions they can degrade.

The MOF present in the MOF biocomposites may for example be crystalline. In some embodiments, the MOF has an orthorhombic crystal structure.

In some embodiments, the MOF is ZIF-C. ZIF-C is a previously reported MOF having a zeolitic imidazolate framework, see e.g. Poddar et al, Chem. Commun., 2020, 56, 15406- 15409; Pyreddy et al. ZIF-C as non-viral delivery system for CRISPR/Cas9 mediated hTERT knockdown in cancer cells . ChemRxiv. Cambridge: Cambridge Open Engage; 2021; Carraro et al, Chem. Sci, 2020, 11, 3397-3404; Basnayake et al., Inorg. Chem. 2015, 54, 4, 1816— 1821.

Biomolecules

The MOF biocomposite comprises a biomolecule which is either an antibody or which is a biomolecule that comprises an antibody F _c region amino acid sequence or variant thereof. Bioactivity can be strongly associated with special configuration. As discussed above, the F _c region is at least partially embedded in the MOF, allowing anchoring of the biomolecule to the MOF structure, but allowing exposure of other regions or parts of the biomolecule structure to the surrounding environment, such that it is available for interaction (e.g. binding) to other components, for example allowing antigen-binding regions of antibodies to retain their ability to bind antigens and target molecules.

In some embodiments, the biomolecule is an antibody. The term "antibody" should be understood to mean an immunoglobulin molecule that recognises and specifically binds to a target antigen, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combination thereof through at least one antigen recognition site within the variable region of the immunoglobulin molecule. The term "antibody" encompasses polyclonal antibodies, monoclonal antibodies, multispecific antibodies such as bispecific antibodies, chimeric antibodies, humanised antibodies, human antibodies. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a polyclonal antibody.

In some embodiments, the antibody is an antibody in a class selected from the group consisting of IgG, IgE and IgD. In some embodiments, the antibody is an IgG antibody.

In some embodiments, the IgG antibody is type 1, type 2, type 3, or type 4.

In some embodiments, the antibody is human, humanised or chimeric.

In some embodiments, the antibody is an anti-HER2, an anti-P-selectin, anti-fibrin, anti-PSMA or human IgG antibody.

In some embodiments, the antibody is selected from the group comprising adalimumab, pembrolizumab, nivolumab, bevacizumab, rituximab, ustekinumab, trastuzumab, infliximab, denosumab, eculizumab, ranibizumab, ocrelizumab, secukinumab, pertuzumab, omalizumab, daratumumab, vedolizumab, dupilumab and tocilizumab. In some embodiments, the antibody is trastuzumab.

Sequence information for selected antibodies is provided below:

SEQ ID NO: 1 - Human IgGl F _c region

In IgG, IgD and IgA antibodies, the F _c region is composed of two identical protein sequences. Whilst the other part of an antibody (the F _ab region) contains variable sequence, the F _c region is typically highly conserved and may be identical for antibodies in the same class, or may be highly similar (e.g. varying by only one or a few amino acid residues, e.g. 1, 2, 3, 4, 5 amino acid residues). Percent sequence identity for the Fc region of trastuzumab with other antibodies is provided in the table below:

In some embodiments, the biomolecule comprises an antibody F _c (fragment crystallisable) region amino acid sequence or variant thereof.

In some embodiments, the biomolecule comprises the entire amino acid sequence of an antibody F _c region.

In some embodiments, the biomolecule comprises an Fc region which is identical to the Fc region of human IgGl, adalimumab, pembrolizumab, nivolumab, bevacizumab, rituximab, ustekinumab, trastuzumab, infliximab, denosumab, eculizumab, ranibizumab, ocrelizumab, secukinumab, pertuzumab, omalizumab, daratumumab, vedolizumab, dupilumab or tocilizumab.

In some embodiments, the biomolecule comprises an Fc region which is identical to the Fc region of trastuzumab or human IgGl.

In some embodiments, the biomolecule comprises a variant of an antibody F _c (fragment crystallisable) region amino acid sequence. A variant of an antibody F _c region amino acid sequence is an amino acid sequence which retains the functional properties of an antibody F _c region amino acid sequence. In some embodiments, a variant is an amino acid sequence which enables crystallisation of zinc(II) and azole-containing ligand to form a MOF biocomposite in which the variant amino acid sequence is embedded within the MOF.

In some embodiments, a variant of an antibody F _c region amino acid sequence is a truncated form of an antibody F _c region amino acid sequence, for example having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the amino acid residues of an antibody F _c region amino acid sequence.

In some embodiments, a variant of an antibody F _c region amino acid sequence is a truncated form of an antibody F _c region amino acid sequence, for example having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the amino acid residues of the F _c region amino acid sequence of human IgGl, adalimumab, pembrolizumab, nivolumab, bevacizumab, rituximab, ustekinumab, trastuzumab, infliximab, denosumab, eculizumab, ranibizumab, ocrelizumab, secukinumab, pertuzumab, omalizumab, daratumumab, vedolizumab, dupilumab or tocilizumab.

In some embodiments, a variant of an antibody F _c region amino acid sequence is a truncated form of an antibody F _c region amino acid sequence, for example having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the amino acid residues of the F _c region amino acid sequence of trastuzumab or human IgGl.

In some embodiments, a variant of an antibody F _c region amino acid sequence is an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to an antibody F _c region amino acid sequence.

In some embodiments, a variant of an antibody F _c amino acid sequence is an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the F _c region amino acid sequence of human IgGl, adalimumab, pembrolizumab, nivolumab, bevacizumab, rituximab, ustekinumab, trastuzumab, infliximab, denosumab, eculizumab, ranibizumab, ocrelizumab, secukinumab, pertuzumab, omalizumab, daratumumab, vedolizumab, dupilumab or tocilizumab.

In some embodiments, a variant of an antibody F _c amino acid sequence is an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the F _c region amino acid sequence of trastuzumab or human IgGl.

Percentage of sequence identity may be determined based on assessing the number of identical amino acid residues over a defined length in a given alignment. Amino acid sequences can for example be modified by substitution of amino acid residues, addition of amino acid residues, and/or deletion of amino acid residues.

In some embodiments, percentage sequence identity is determined based on the BLAST (Basic Local Alignment Search Tool) sequence comparison algorithm (Atlschul et al, J. Mol. Biol., 1990, 215(3), 403-410).

For example, a sequence which has 90% of the full length Fc region sequence of human IgGl but which is truncated, i.e. has the end 10% of the Fc region sequence deleted, is determined to have 90% sequence identity to the Fc region of human IgGl. As another example, a sequence which has an amino acid sequence of the Fc region of human IgGl except that it has 10% of the amino acid residues present in the human IgGl sequence substituted for other amino acids has 90% sequence identity to the Fc region sequence of human IgGl.

The term conservative substitution refers to the replacement of an amino acid residue by another, biologically similar residue in a polypeptide. Typical but not limiting conservative substitutions are the replacements, for one another, among the aliphatic amino acids Ala, Vai, Leu and He: interchange of hydroxyl-containing residues Ser and Thr, interchange of the acidic residues Asp and Glu, interchange between the amide- containing residues Asn and Gin, interchange of the basic residues Lys and Arg, interchange of the aromatic residues Pile and Tyr, and interchange of the small-sized amino acids Ala, Ser, Thr, Met and Gly. Additional conservative substitutions include the replacement of an amino acid by another of similar spatial or steric configuration, for example the interchange of Asn for Asp, or Gin for Glu.

In some embodiments, a variant of an antibody F _c region amino acid sequence is a truncated form of an antibody F _c region amino acid sequence, for example having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the amino acid residues of an antibody F _c region amino acid sequence, wherein any substitution is a conservative substitution.

In some embodiments, a variant of an antibody F _c region amino acid sequence is a truncated form of an antibody F _c region amino acid sequence, for example having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the amino acid residues of the antibody F _c region amino acid sequence of human IgGl, adalimumab, pembrolizumab, nivolumab, bevacizumab, rituximab, ustekinumab, trastuzumab, infliximab, denosumab, eculizumab, ranibizumab, ocrelizumab, secukinumab, pertuzumab, omalizumab, daratumumab, vedolizumab, dupilumab or tocilizumab, wherein any substitution is a conservative substitution.

In some embodiments, a variant of an antibody F _c region amino acid sequence is a truncated form of an antibody F _c region amino acid sequence, for example having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the amino acid residues of the antibody F _c region amino acid sequence of human IgGl or trastuzumab, wherein any substitution is a conservative substitution.

In some embodiments, a variant of an antibody F _c region amino acid sequence is a truncated form of an antibody F _c region amino acid sequence, for example having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the amino acid residues of an antibody F _c region amino acid sequence, wherein any variation in sequence is a conservative substitution.

In some embodiments, a variant of an antibody F _c region amino acid sequence is a truncated form of an antibody F _c region amino acid sequence, for example having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the amino acid residues of the antibody F _c region amino acid sequence of human IgGl, adalimumab, pembrolizumab, nivolumab, bevacizumab, rituximab, ustekinumab, trastuzumab, infliximab, denosumab, eculizumab, ranibizumab, ocrelizumab, secukinumab, pertuzumab, omalizumab, daratumumab, vedolizumab, dupilumab or tocilizumab, wherein any variation in sequence is a conservative substitution.

In some embodiments, a variant of an antibody F _c region amino acid sequence is a truncated form of an antibody F _c region amino acid sequence, for example having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the amino acid residues of the antibody F _c region amino acid sequence of human IgGl or trastuzumab, wherein any variation in sequence is a conservative substitution.

In some embodiments, the biomolecule is a fusion protein, e.g. of an antibody F _c region, and of a ligand binding domain of a receptor.

Biomolecules such as antibodies may be produced by techniques known in the art. Antibodies may be obtained, for example, where immunisation of an animal is necessary, by administering polypeptides to an animal, preferably a non-human animal, using well-known and routine protocols, see for example Handbook of Experimental Immunology, D. M. Weir (ed.), Vol 4, Blackwell Scientific Publishers, Oxford, England, 1986. Many warm- blooded animals, such as rabbits, mice, rats, sheep, cows, camels or pigs may be immunized. However, mice, rabbits, pigs and rats are generally most suitable.

Monoclonal antibodies may be prepared by any method known in the art such as the hybridoma technique (Kohler & Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today, 4:72) and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, pp77-96, Alan R Liss, Inc.). Antibodies may also be generated using single lymphocyte antibody methods by cloning and expressing immunoglobulin variable region cDNAs generated from single lymphocytes selected for the production of specific antibodies by, for example, the methods described by Babcook, J. et al., 1996, Proc. Natl. Acad. Sci. USA 93:7843-7848.

Antibodies may for example be produced by cultivating a host cell comprising a nucleic acid or vector encoding an antibody; recovering the antibody from the host cell or the cultivation medium; and purifying the antibody. A host cell refers to cells into which an exogenous nucleic acid or vector has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

The term cell includes cells which are used for the expression of nucleic acids. In one embodiment the host cell is a CHO cell, or a BHK cell, or a NSO cell, or a SP2/0 cell, or a HEK 293 cell, or a HEK 293 EBNA cell, or a PER.C6® cell, or a COS cells. As used herein, the expression "cell" includes the subject cell and its progeny.

Various well-established methods may be used for protein recovery and purification. Recovery methods may include, but are not limited to, affinity chromatography with microbial proteins (e.g. protein A or protein G affinity chromatography), affinity chromatography with a recombinant protein as ligand (e.g. single chain Fv as ligand, e.g. Kappa select), or ion exchange chromatography (e.g. cation exchange (carboxymethyl resins), anion exchange (amino ethyl resins) and mixed-mode exchange). Recovery methods may be combined independently in different embodiments as reported herein.

The metal-organic framework (MOF) biocomposite comprises a partially embedded biomolecule. The term ‘partially embedded’ means that part of the biomolecule (e.g. antibody) is embedded in the MOF, but another part of the molecule is not embedded in the MOF and is instead exposed to the surrounding environment.

At least part of the F _c region of the biomolecule (e.g. antibody) is embedded in the MOF. In other words, amino acid sequence from the Fc region penetrates the surface of the MOF and is situated within its interior.

In some embodiments, the biomolecule is bonded to the MOF by non-covalent interactions, i.e. there are no covalent bonds between the biomolecule (in particular, the Fc region of the biomolecule) and the MOF. For example, the biomolecule may be bonded to the MOF by ionic interactions, hydrogen bonding interactions, Van der Waals interactions, TC-TT interactions, hydrophobicity, or a combination thereof.

In some embodiments, only the F _c region or a part thereof is embedded within the MOF.

In some embodiments, at least 50%, at least 75%, at least 90%, or at least 95% of the F _c region is embedded in the MOF. In some embodiments, the entire F _c region is embedded within the MOF,

In some embodiments, at least 50%, or at least 75%, or at least 90%, or at least 95% of the biomolecules present in the MOF biocomposite are partially embedded in the MOF and have at least part of the F _c region embedded in the MOF. In some embodiments, all or substantially all of the biomolecules present in the MOF biocomposite are partially embedded in the MOF and have at least part of the F _c region embedded in the MOF.

Where the biomolecule is an antibody, in some embodiments, the antibody is oriented such that the antigen-binding regions of the antibody are not embedded in the MOF and are available for binding (i.e. the CDRs are not partially embedded in the MOF biocomposite).

In some embodiments, the biomolecule is an antibody and the antibody when present in the MOF biocomposite retains at least 1%, at least 2.5%, at least 5%, 10%, at least 25%, at least 50%, at least 75%, at least 90% of the binding affinity for its target antigen compared with the same antibody when free (i.e. when not partially embedded in the MOF biocomposite).

Determination of biomolecule embedding in the MOF biocomposite can be carried out using any appropriate technique. For example, determination of the binding affinity of the MOF biocomposite for its target antigen may be carried out, and compared to binding affinity data for a reference free antibody. As a further example, pyrolysis of the MOF biocomposite can be carried out, followed by TEM analysis of the pyrolyzed biocomposite. As a further example, pyrolysis of the MOF biocomposite can be carried out, followed by AFM analysis of the pyrolyzed biocomposite.

In some embodiments, the MOF-biocomposite is stable to loss of biomolecule in aqueous solution at pH 7.4 (e.g. in water adjusted to pH 7.4) such that at least 50%, at least 75%, at least 90%, or at least 95% of the biomolecule remains partially embedded in the MOF when contacted with the aqueous solution for a period of at least 6 hours. In some embodiments, the MOF-biocomposite is stable to loss of biomolecule in aqueous solution at pH 7.4 (e.g. in water adjusted to pH 7.4) such that at least 50%, at least 75%, at least 90%, or at least 95% of the biomolecule remains partially embedded in the MOF when contacted with the aqueous solution for a period of at least 12 hours. In some embodiments, the MOF-biocomposite is stable to loss of biomolecule in aqueous solution at pH 7.4 (e.g. in water adjusted to pH 7.4) such that at least 50%, at least 75%, at least 90%, or at least 95% of the biomolecule remains partially embedded in the MOF when contacted with the aqueous solution for a period of at least 24 hours.

Additional Agents

The MOF biocomposite may, in some embodiments, contain an additional agent which is useful for imaging, diagnosis, biosensing and/or therapy. Such an agent may for example be incorporated within the biocomposite.

In some embodiments, the MOF biocomposite comprises a therapeutic, imaging, diagnostic, signalling or sensing agent.

In some embodiments, the MOF biocomposite comprises a fluorescent protein or a dye.

In some embodiments, the MOF biocomposite comprises a fluorescent protein. The fluorescent protein may for example be a blue, cyan, green, yellow, orange, red, or near infra- red fluorescent protein. In some embodiments, the fluorescent protein is EBFP, ECFP, EGFP, EYFP, mOrange2, mCherry, or NirFP.

In some embodiments, the MOF biocomposite comprises a dye. In some embodiments the dye is a ultra violet blue, cyan, green, yellow, orange, red, far-red or near infra-red emitting dye. In some embodiments, the dye is an Alexa Fluor (AF) dye. In some embodiments, the AF dye is AF350, AF405, AF488, AF532, AF555, AF594, AF647, AF750.In some embodiments, the dye is coumarin, Cy2, fluorescein (FITC), Cy3, rhodamine (TRITC), Texas Red, Cy5, or Cy7.

In some embodiments where the MOF biocomposite comprises a fluorescent protein or dye, the fluorescent protein or dye component may be conjugated to another component, for example it may be conjugated to the biomolecule.

In some embodiments, the MOF biocomposite comprises a quantum dot (QD). Quantum dots are semiconductor particles of small size, typically of the nanometer order. When subjected to UV light, an excited electron is produced, which on dropping back to the valence band results in emission of light. Quantum dots can have high extinction coefficients, and find use in, for example, imaging applications.

In some embodiments, where the MOF biocomposite comprises quantum dots (QD), the QD has a mean diameter in the range of from Inm to lOOnm. In some embodiments, the QD is a Core-type QD, Core-shell QD, or Alloyed QD. In some embodiments, the materials used is a chalcogenide (e.g., selenides, sulphides, or tellurides), cadmium, lead, or zinc. In some embodiments, the QD is a PbS, an InP, a CdTe, a CdSe, a CdS, a ZnSe, or a ZnS QD.

In some embodiments, the fluorescent protein, QD, or dye is a non-fluorescent version, i.e., a quencher.

In some embodiments, the MOF biocomposite comprises an additional metal ion. In some embodiments, the MOF comprises Cu ions.

In some embodiments, the MOF biocomposite comprises a radionuclide. MOF biocomposites find use in medical imaging and therapeutic applications. For example, the biocomposite may contain and antibody which binds to an antigen present on tumor cells, and further contain a radionuclide useful for imaging or which can be used to destroy tumor cells.

In some embodiments, the radionuclide is selected from the group consisting of ⁶⁴ Cu, ⁶⁷ Cu, ⁹⁹ Mo, ⁸² Rb, ⁸² Sr, ²⁰¹ T1, ⁶⁷ Ga, ^81m Kr, ^99m Tc, ¹¹¹ ln, ¹³³ Xn, ¹⁷⁷ Lu, ²²⁵ Ac, ²¹¹ As, ²¹² Bi, ²¹³ Bi, ²¹² Pb, ⁹⁰ Y, ¹⁸⁶ Re, ⁶⁸ Ga or ⁸⁹ Zr. In some embodiments, the radionuclide is a copper radionuclide. In some embodiments, the radionuclide is ⁶⁴ Cu or ⁶⁷ Cu. In some embodiments, the radionuclide is ⁶⁷ Cu, ¹⁷⁷ LU, ²²⁵ AC, ²¹¹ AS, ²¹² Bi, ²¹³ Bi, ²¹² Pb, ⁹⁰ Y or ¹⁸⁶ Re. In some embodiments, the radionuclide is ⁶⁴ Cu, ⁶⁸ Ga or ⁸⁹ Zr.

In some embodiments, the MOF biocomposite comprises magnetic particles. In some embodiments, the magnetic particles are iron oxide nanoparticles (IONP), IONP shell magnetic particles, gadolinium, manganese, or iron platinum nanoparticles. In some embodiments, where IONP shell magnetic particles are used, the shell is a silica shell. In some embodiments, the magnetic particle is IONP.

In some embodiments the IONP have a mean diameter in the range of from 10 nm to about 75 nm, from 15 nm to about 75 nm, from 20 nm to about 75 nm, from 25 nm to about 75 nm, from 30 nm to about 75 nm, from 35 nm to about 75 nm, from 40 nm to about 75 nm, from 45 nm to about 75 nm, 10 nm to about 70 nm, from 15 nm to about 70 nm, from 20 nm to about 70 nm, from 25 nm to about 70 nm, from 30 nm to about 70 nm, from 35 nm to about 70 nm, from 40 nm to about 70 nm, from 45 nm to about 70 nm, 10 nm to about 65 nm, from 15 nm to about 65 nm, from 20 nm to about 65 nm, from 25 nm to about 65 nm, from 30 nm to about 65 nm, from 35 nm to about 65 nm, from 40 nm to about 65 nm, from 45 nm to about 65 nm, 10 nm to about 60 nm, from 15 nm to about 60 nm, from 20 nm to about 60 nm, from 25 nm to about 60 nm, from 30 nm to about 60 nm, from 35 nm to about 60 nm, from 40 nm to about 60 nm, from 45 nm to about 60 nm, 10 nm to about 55 nm, from 15 nm to about 55 nm, from 20 nm to about 55 nm, from 25 nm to about 55 nm, from 30 nm to about 55 nm, from 35 nm to about 55 nm, from 40 nm to about 55 nm, from 45 nm to about 55, 10 nm to about 50 nm, from 15 nm to about 50 nm, from 20 nm to about 50 nm, from 25 nm to about 50 nm, from 30 nm to about 50 nm, from 35 nm to about 50 nm, from 40 nm to about 50 nm, from 45 nm to about 50 nm.

In some embodiments, the magnetic particles are hydrodynamic monodisperse particles.

In some embodiments, the MOF biocomposite contains IONP which have a mean diameter in the range of from 40 to 50 nm, and which are hydrodynamic monodisperse particle.

In some embodiments, particles are used which have a mean core size in the range of from 5 to 25 nm. In some embodiments, the hydrodynamic monodisperse particles have a mean core size of about 15 nm.

In some embodiments, the particles contain iron in the range of from 1.5 to 3.5 mg of Fe.ml ¹ . In some embodiments the IONP particle has 2.5 mg Fe.ml ¹ .

In some embodiments, the IONP particles are coated. For example they may be coated in polyDMSO polymer.

In some embodiments, the IONP particles are fluorescently labelled. In some embodiments the fluorescent label is any one of the dyes or a quencher as outlined above.

In some embodiments, the IONP particle is coated in polyDMSO polymer and fluorescently labelled with Cy5 through a ‘click’ reaction. In some embodiments, the MOF biocomposite comprises a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic agent. In some embodiments, the cytotoxic agent is an anti-cancer drug. In some embodiments, the cytotoxic agent is selected from the group consisting of aflibercept, amsacrine, azacitidine, azathioprine, bendamustine, bleomycin, bortezomib, busulfan, cabazitaxel, capecitabine, carboplatin, carfilzomib, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin (actinomycin D), daunorubicin, docetaxel, doxorubicin, doxorubicin liposomal, epirubicin, eribulin, etoposide, etoposide phosphate, fludarabine, fluorouracil, fotemustine, ganciclovir, gemcitabine, hydroxyurea, idarubicin, ifosfamide, inotuzumab ozogamicin, irinotecan, ixazomib, lomustine, melphalan, mercaptopurine, methotrexate, mitomycin, mitozantrone, nab-paclitaxel, oxaliplatin, paclitaxel, pemetrexed, polatuzumab vedotin, pralatrexate, procarbazine, raltitrexed, romidepsin, temozolomide, teniposide, thiotepa, tioguanine, topotecan, trabectedin, trastuzumab emtansine, trifluridine/tipiracil, valganciclovir, vinblastine, vincristine, vindesine, vinflunine, vinorelbine and vismodegib. In some embodiments, the therapeutic agent is doxorubicin.

In some embodiments, the MOF biocomposite comprises a nucleic acid. In some embodiments, the nucleic acid is an antisense, siRNA, miRNA, aptamer, decoy, ribozyme, or CpG-oligodeoxynucelotide nucleic acid.

As discussed above, the MOF biocomposites containing zinc ions and azole-containing ligand are typically stable at neutral pH, but may be unstable on exposure to acidic pH. Such pH dependent properties can lead to selective release of the contents of the MOF biocomposite when exposed to an acidic environment, e.g. to selective release of therapeutic agents (e.g. cytotoxic agents and/or radionuclides). For example, tumors can have an acidic microenvironment. Accordingly, MOF biocomposites comprising an antibody which selectively binds an antigen present on a tumor cell surface thus enabling localisation to a tumor environment, and comprising a therapeutic agent (e.g. cytotoxic agent, radionuclide) within the MOF that can be selectively released in the vicinity of the tumor due to pH-dependent stability properties, may be particularly well suited for cancer therapy.

Pharmaceutical compositions

In some embodiments, the biocomposite may be used as part of a composition, containing additional components. Accordingly, there is also provided a pharmaceutical composition comprising a metal organic framework (MOF) biocomposite as defined herein and a pharmaceutically acceptable excipient.

The biocomposites of the present disclosure may for example be provided together with one or more pharmaceutically acceptable carriers, and optionally any other therapeutic ingredients, stabilisers, or the like.

The excipient(s) and/or carrier(s) must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the composition and not unduly deleterious to the recipient thereof.

Exemplary pharmaceutical excipients and/or additives suitable for use in the compositions according to the present disclosure are listed in "Remington: The Science & Practice of Pharmacy", 19.sup.th ed., Williams & Williams, (1995), and in the "Physician's Desk Reference", 52. sup. nd ed., Medical Economics, Montvale, N.J. (1998), and in "Handbook of Pharmaceutical Excipients", Third Ed., Ed. A. H. Kibbe, Pharmaceutical Press, 2000.

For example, the biocomposite may be formulated together with one or more excipients such as a suspending solvent (e.g. water), a buffer, pH adjusting agent (such as HC1 or NaOH) and/or a preservative.

The biocomposites of the present disclosure may be formulated in compositions including those suitable for administration by any suitable route, including for example by parenteral (including intraperitoneal, intravenous, subcutaneous, or intramuscular injection) or oral administration.

The compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy.

The composition may be a solid composition, a semisolid or liquid composition. For example, when a liquid composition is used, it may for example be in the form of an aqueous suspension containing the biocomposite. Where the composition is a solid composition, it may for example be in tablet, capsule, caplet, powder or granule form.

In some preferred embodiments, the composition is formulated for parenteral delivery. For example, the composition may be in the form of an aqueous suspension which is suitable for injection. As a further example, the composition may be a sterile, dry composition that is suitable for reconstitution in an aqueous vehicle (e.g. via suspension) prior to injection.

As discussed below, the biocomposites of the present disclosure may for example be used in combination with an additional pharmaceutically active agent. In some embodiments, the biocomposite is provided in combination with a further active, which may for example be present in the same composition or dosage form, or alternatively may be provided in different dosage forms.

Methods of producing MOF biocomposites

There is also provided a method of producing a MOF biocomposite as defined herein, the method comprising: contacting a biomolecule comprising an antibody F _c region amino acid sequence or variant thereof, with an azole-containing ligand, and with Zn ²⁺ ions, in aqueous solution, thereby producing the MOF biocomposite.

The method involves contacting the biomolecule (e.g. an antibody) with the azole- containing ligand and with Zn ²⁺ ions in aqueous solution. As discussed above, the F _c region of the biomolecule can trigger crystallisation of a MOF framework with the F _c region of the biomolecule becoming embedded within the MOF.

For example, aqueous solutions of each of the azole-containing ligand and a zinc(II) salt may be prepared, the biomolecule (e.g. antibody) added to one or other of the solutions, and then the solutions combined. In some embodiments, the biomolecule is added to the solution of azole-containing ligand, and then that solution is combined with the zinc-containing solution.

Accordingly, in some embodiments the method comprises the steps of: a. contacting the biomolecule with an aqueous solution containing azole- containing ligand; and b. contacting the solution of a. with an aqueous solution containing Zn ²⁺ ions.

The aqueous solution in which the MOF biocomposite is produced is typically water.

In some embodiments, the aqueous solution in which the MOF biocomposite is produced contains a buffer. For example, where separate solutions of antibody and zinc salt are prepared, one or both solutions may be prepared containing a buffer. In some embodiments, the antibody-containing solution contains a buffer.

Any suitable buffer may be used. In some embodiments, the buffer is a zwitterionic buffer. In some embodiments, the buffer is selected from the group consisting of MOPS, CAPS, HEPES, MES and TRIS. In some embodiments, the buffer is MOPS.

The step of contacting the biomolecule with the azole-containing ligand and with Zn ²⁺ ions in aqueous solution to produce the MOF biocomposite may be carried out at any temperature suitable for production of the MOF and which does not lead to significant denaturing of the biomolecule.

In some embodiments, the contacting step is carried out a temperature in the range of from 10 °C to 50 °C, or in the range of from 10 °C to 40 °C, or in the range of from 10 °C to 37 °C, or in the range of from 20 °C to 40 °C, or in the range of from 20 °C to 37 °C, or in the range of from 25 °C to 37 °C, or in the range of from 30 °C to 37 °C, or at a temperature of about 37 °C.

The step of contacting the biomolecule with the azole-containing ligand and with Zn ²⁺ ions in aqueous solution may be carried out for any period of time suitable to produce the MOF biocomposite. For example, in some embodiments, the contacting step is carried out for a time period of up to 24 hours, or up to 12 hours, or up to 6 hours, or up to 3 hours, or up to 2 hours, or up to 1 hour, or up to 30 minutes, or up to 15 minutes. In some embodiments, the contacting step is carried out for a time period in the range of from 5 minutes to 24 hours, or from 5 minutes to 12 hours, or from 5 minutes to 6 hours, or from 5 minutes to 3 hours, or from 5 minutes to 2 hours, or from 5 minutes to 1 hour, or from 5 minutes to 30 minutes, or from 5 minutes to 15 minutes.

In some preferred embodiments, the contacting step is carried out in the presence of carbonate ions. Any suitable source of carbonate ions may be used. For example, in some embodiments, the contacting step is carried out under ambient atmosphere, and carbon dioxide present in the atmosphere provides a source of carbonate in the aqueous solution. In some other embodiments, a carbonate or bicarbonate (e.g. metal bicarbonate or carbonate) may be added.

Any suitable source of Zn ²⁺ ions may be used, such as for example a zinc salt. In some embodiments, the Zn ²⁺ ions are provided in the form of zinc acetate. In some embodiments, the Zn ²⁺ ions are provided in the form of zinc acetate dihydrate.

In some embodiments, the contacting step is carried out using Zn ²⁺ ions at a concentration in the range of from 10 to 50 mM. In some embodiments, the contacting step is carried out using Zn ²⁺ ions at a concentration in the range of from 10 to 30 mM. In some embodiments, the contacting step is carried out using Zn ²⁺ ions at a concentration in the range of about 20 mM.

In some embodiments, the contacting step is carried out using zinc acetate dihydrate at a concentration in the range of from 10 to 50 mM. In some embodiments, the contacting step is carried out using zinc acetate dihydrate at a concentration in the range of from 10 to 30 mM. In some embodiments, the contacting step is carried out using zinc acetate dihydrate at a concentration in the range of about 20 mM.

The azole-containing ligand is a ligand which is capable of forming a MOF with zinc ions and which contains an azole group, such as an imidazole or a triazole. In some embodiments, the azole group is one which contains an acidic proton. In some embodiments, the azole-containing ligand is an imidazole-containing ligand. In some other embodiments, the azole-containing ligand is a triazole-containing ligand. In some embodiments, the azole- containing ligand is an imidazole-containing ligand containing an acidic proton.

The azole-containing group may be substituted or unsubstituted. In some embodiments, the azole-containing ligand is substituted with one or more Ci-6 alkyl groups, e.g. one or more methyl groups.

In some embodiments, the azole-containing ligand is 2-methylimidazole.

In some embodiments, the contacting step is carried out using azole-containing ligand at a concentration in the range of from 40 to 400 mM. In some embodiments, the contacting step is carried out using azole-containing ligand at a concentration in the range of from 40 to 200 mM. In some embodiments, the contacting step is carried out using azole-containing ligand at a concentration in the range of from 60 to 100 mM. In some embodiments, the contacting step is carried out using azole-containing ligand at a concentration of about 80 mM.

In some embodiments, the contacting step is carried out using 2-methylimidazole at a concentration in the range of from 40 to 400 mM. In some embodiments, the contacting step is carried out using 2-methylimidazole at a concentration in the range of from 40 to 200 mM. In some embodiments, the contacting step is carried out using 2-methylimidazole at a concentration in the range of from 60 to 100 mM. In some embodiments, the contacting step is carried out using 2-methylimidazole at a concentration of about 80 mM.

In some embodiments, the molar ratio of azole-containing ligand to Zn ²⁺ ions is in the range of from 16:1, or from 8:1, or from 6:1 to 2:1, or from 5:1 to 3:1, or about 4:1.

In some embodiments, the contacting step is carried out using an amount of biomolecule in the range of from 25 μg to 75 μg per lOOμL of aqueous solution, (i.e. from 250 to 750 mg/mL). In some embodiments, the contacting step is carried out using an amount of biomolecule in the range of from 40 μg to 60 μg per lOOμL of aqueous solution (i.e. from 400 to 600 mg/mL). In some embodiments, the contacting step is carried out using an amount of biomolecule of about 50 μg per lOOμL of aqueous solution (i.e. about 500 mg/mL). In some embodiments, where the MOF biocomposite comprises an additional agent, the step of contacting a biomolecule comprising an F _c region of an antibody, with an azole- containing ligand, and with Zn ²⁺ ions, in aqueous solution, is carried out in the presence of the additional agent (e.g., in the presence of quantum dots, additional metal ions, a radionuclide, magnetic particles, or a therapeutic agent such as a cytotoxic agent, or a nucleic acid).

For example, the additional agent can be added to an aqueous solution containing biomolecule and azole-containing ligand, and then combined with an aqueous solution containing zinc ions.

Once produced, the MOF biocomposite may be recovered, separated from other constituents of the reaction mixture and/or purified by any appropriate technique. For example, the MOF biocomposite may undergo one or more filtration, decanting or centrifugation steps. The MOF biocomposite may undergo one or more washing steps, for example it may be washed with aqueous buffer solution. The MOF biocomposite may undergo a drying step.

There is also provided a metal-organic framework (MOF) biocomposite when produced according to a method as defined herein.

Methods of imaging, diagnosis, and prevention and/or treatment of diseases and disorders

The MOF biocomposites of the present disclosure find use in imaging, diagnostic and therapeutic methods.

Accordingly, there is provided a method of treating and/or preventing a disease or disorder in a subject, comprising administering a metal-organic framework (MOF) biocomposite or a pharmaceutical composition as defined herein, to the subject.

There is also provided use of a metal-organic framework (MOF) biocomposite as defined herein in the manufacture of a medicament for diagnosing, treating and/or preventing a disease or disorder.

There is also provided a metal-organic framework (MOF) biocomposite or a pharmaceutical composition as defined herein for use in the diagnosis, treatment and/or prevention of a disease or disorder.

In some embodiments, the disease or disorder is a cancer, an inflammatory disease or disorder, a cardiovascular disease or disorder, multiple sclerosis, or a neurodegenerative disease or disorder. In some embodiments, the cancer is selected from the group consisting of breast cancer, metastatic gastric adenocarcinoma, gastroesophageal junction adenocarcinoma, and prostate cancer. The disease or disorder may depend on the biomolecule present in the biocomposite, for example where the biomolecule is an antibody, the disease or disorder may be one for which the antibody is indicated as a therapy for treating. For example, in some embodiments the biomolecule is an anti-HER-2, an anti-PSMA, an anti-fibrin, or an anti-P-selectin antibody, and the disease or disorder is a cancer, an inflammatory disease or disorder, a cardiovascular disease or disorder, multiple sclerosis, or a neurodegenerative disease or disorder. In some embodiments, the cancer is selected from the group consisting of breast cancer, metastatic gastric adenocarcinoma, gastroesophageal junction adenocarcinoma, and prostate cancer.

In some embodiments, the biomolecule is an anti-HER-2 antibody, such as trastuzumab, and the disease or disorder is a cancer, for example a cancer selected from the group consisting of breast cancer, metastatic gastric adenocarcinoma and gastroesophageal junction adenocarcinoma. In some embodiments, the cancer is characterised by an abnormal expression, or overexpression, of HER2.

In some embodiments, the biomolecule is an anti-PSMA antibody, and the disease of disorder is a cancer, for example prostate cancer. In some embodiments, the cancer is characterised by an abnormal expression, or overexpression, of PSMA.

In some embodiments, the biomolecule is an anti-fibrin antibody (e.g. 59D8), and the disease or disorder is multiple sclerosis, a neurodegenerative disease or disorder, or a cardiovascular disease or disorder.

In some embodiments, the biomolecule is an anti-P-selectin antibody (e.g. VH10), and the disease or disorder is an inflammatory disease or disorder, or a cardiovascular disease or disorder.

A therapeutically effective amount of the MOF biocomposite is used in the therapeutic methods and uses. It will be appreciated that the term "therapeutically effective amount" refers to a MOF biocomposite, or composition comprising the MOF biocomposite, being administered in an amount sufficient to alleviate or prevent to some extent one or more of the symptoms of the disorder or condition being treated.

The MOF biocomposite may be administered by any suitable route, including for example parenterally (e.g. intravenously) or orally.

In some embodiments, the MOF biocomposite comprises an antibody which binds to a specific target preferentially present on or in tissue or cells associated with a disease or disorder, and comprises an additional agent such as a radionuclide or cytotoxic agent. For example, the MOF biocomposite may comprise an antibody which selectively binds a receptor or protein present on the surface of tumour cells (and thus concentrates in the vicinity of a tumour), and may also contain a radionuclide or cytotoxic agent.

Where the MOF biocomposite is used for treatment purposes, it may for example contain a radionuclide such as ⁶⁴ Cu, ⁶⁷ Cu, ¹⁷⁷ Lu, ²²⁵ Ac, ²¹¹ As, ²¹² Bi, ²¹³ Bi, ²¹² Pb, ⁹⁰ Y or ¹⁸⁶ Re.

When used for treatment purposes, the MOF-biocomposite will typically be administered in an amount sufficient to deliver a therapeutically effective dose of radioactivity to the target (e.g. tumour), whilst at the same time avoiding unacceptable exposure of other parts of the body (e.g. other organs) to radiation. The precise dosage may be dependent on the nature of the radionuclide (e.g. alpha emitter, beta emitter), and the condition to be treated.

Where the MOF biocomposite is used for treatment purposes, it may for example contain a cytotoxic agent such as cis-platin, capecitabine, doxorubicin, cyclophosphamide, methotrexate, 5 -fluorouracil, vinblastine, vinorelbine, bleomycin, etoposide, docetaxel or cabazitaxel. In such cases, the MF-biocomposite will typically be administered in an amount sufficient to deliver a therapeutically effective dose of the cytotoxic agent to the target (e.g. tumour).

The MOF biocomposites described herein also find use as diagnostic, biosensing and/or imaging agents.

Accordingly, there is provided a method of imaging or biosensing a subject, comprising administering to a subject a MOF biocomposite as defined herein which comprises an imaging, diagnostic, signalling or sensing agent within the MOF, and carrying out imaging or biosensing of the subject.

In another aspect, there is provided a method of diagnosing whether a subject has a disease or disorder, comprising administering to a subject a MOF biocomposite as defined herein which comprises an imaging, diagnostic, signalling or sensing agent within the MOF, carrying out imaging or biosensing of the subject, and determining whether the subject has a disease or disorder based on the imaging results.

In another aspect, there is provided a method of diagnosing whether a subject has a disease or disorder, comprising contacting a biological sample isolated from a subject with a MOF biocomposite as defined herein which comprises an imaging, diagnostic, signalling or sensing agent within the MOF, carrying out imaging or biosensing of the biological sample, and determining whether the subject has a disease or disorder based on the imaging results.

In another aspect, there is provided a method of diagnosis and therapy of a disease or disorder in a subject, comprising carrying out a method of diagnosis as defined above and, if the subject is determined to have a disease or disorder susceptible to treatment with a therapy, administering the therapy to the subject.

In some embodiments, the imaging, diagnostic, signalling or sensing agent is a fluorescent protein, a quantum dot or a dye. In some embodiments, the imaging, diagnostic, signalling or sensing agent is a radionuclide. For example, where the MOF biocomposite is used for imaging or diagnostic purposes, it may contain a radionuclide such as for example ⁶⁴ Cu, ⁶⁸ Ga or ⁸⁹ Zr.

In the above methods and uses, any suitable means for administering an amount of MOF biocomposite sufficient for the imaging or diagnostic use may be utilised. For example, the conjugate or composition may administered intravenously to the subject.

Typically, when used for imaging and diagnostic purposes, the conjugate is administered and then the subject, or relevant part of the subject, is imaged after a suitable period of time. Suitable techniques for imaging radionuclide-containing samples, and for analysing the results, are known to the person skilled in the art, and may be used in the above methods and uses. In some embodiments, PET imaging is used. In some embodiments, PET- MRI, SPECT, SPECT-CT, CT, scintography or PET-CT imaging is used.

In some embodiments, the disease or disorder is cancer. The cancer may for example be any of the cancers discussed above in relation to therapeutic applications of the conjugates. For example, in some embodiments, the cancer is a solid tumour.

In some embodiments, the disease or disorder is a cancer selected from the group consisting of breast cancer, metastatic gastric adenocarcinoma, gastroesophageal junction adenocarcinoma and prostate cancer. In some embodiments, the cancer is characterised by an abnormal expression, or overexpression, of HER2. In some embodiments, the cancer is characterised by an abnormal expression, or overexpression, of PSMA.

In methods involving administration of a therapy, in some embodiments the therapy is a MOF biocomposite as defined herein which comprises a therapeutic agent within the MOF. In some embodiments, the therapeutic agent is a radionuclide or a cytotoxic agent, for example a radionuclide or cytotoxic agent as discussed above. In some other embodiments, the therapy administered may be other than a MOF biocomposite, for example it may be an antibody therapy or cytotoxic agent. Combinations

In some embodiments the MOF biocomposite is administered in combination with one or more further pharmaceutically active agents, for example one or more anti-cancer agents/drugs. The MOF biocomposite and the one or more further pharmaceutically active agents may be administered simultaneously, subsequently or separately. For example, they may be administered as part of the same composition, or by administration of separate compositions.

The one or more further pharmaceutically active agents may for example be anti-cancer therapeutic agents, such as cytotoxic agents or antibody therapies, e.g. cis-platin, capecitabine, doxorubicin, cyclophosphamide, methotrexate, 5 -fluorouracil, vinblastine, vinorelbine, bleomycin, etoposide, docetaxel or cabazitaxel.

The one or more further pharmaceutically active agents may for example be a therapeutic agent for the treatment of cardiovascular diseases and disorders, such as a statin (e.g. atorvastatin, rosuvastatin), an anticoagulant (warfarin, heparin), an antithrombotic agent, or a blood-pressure lowering agent (e.g. amlodipine, verapamil).

The one or more further pharmaceutically active agents may for example be therapeutic agents for the treatment of inflammatory diseases and disorders, such as steroids, aspirin, ibuprofen, and diclofenac.

The one or more further pharmaceutically active agents may for example be therapeutic agents for the treatment of multiple sclerosis, such as fingolimod or dimethyl fumarate.

Detection of target molecules

In another aspect, there is provided, a method of determining the presence and/or concentration of a target molecule or analyte in a sample, comprising: contacting a sample with a metal-organic framework (MOF) biocomposite as defined herein which comprises a sensing or signalling agent within the MOF, wherein the biomolecule is an antibody having an antigen-binding region which binds to a target molecule or analyte; and if the sample contains the target molecule or analyte, detecting the presence and/or concentration of the target molecule or analyte in the sample through binding of the target molecule or analyte to the antibody so as to generate a signal from the sensing or signalling agent.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the above-described embodiments and below-described examples, without departing from the broad general scope of the present disclosure. The present embodiments and examples are, therefore, to be considered in all respects as illustrative and not restrictive.

Examples

The present disclosure is further illustrated by the following non-limiting examples.

Materials and Methods

Biomolecules

Biomolecules used herein include monoclonal antibodies anti-HER2 (α-HER2) (Trastuzumab, Roche, Switzerland), α-P-selectin (VH10), α-Fibrin (59D8) (prepared internally), and α-PSMA (3A12) (prepared internally, see J. Nucl. Med., 2009, Jun; 50(6):958), Eluman IgG (hlgG) (Sigma), Purified fragments of hlgG (Bethyl Laboratories Inc., USA), including Fab (P80-215), F(ab')2 (P8O-13O), and fragment crystallizable (Fc) (P80-204), Kadcyla (Roche Genentech), Sialylglycan (SG, α-2,6) (Wako Chemical GmbH, Germany), Sialylglycopeptide (SGP, α-2,6) (PeptaNova GmbH, Germany), and Bovine Serum Albumin (BSA) (Sigma- Aldrich, A7906-100G Lot # SLBM9695V).

The sequence of the VL α-Fibrin (59D8) antibody (SEQ ID NO: 42):

The sequence of the VH α-Fibrin (59D8) antibody (SEQ ID NO: 43):

X-ray diffraction (XRD)

XRD patterns were acquired using a SmartLab II Rigaku X-ray diffractometer equipped with a Cu anode (λ = 1.5406 A) and operating at 9 kW. Prior to analysis, the biocomposites were washed three times by centrifugation (30 s, 1000g) with deionized water, the supernatant was discarded, and the products were dried at ambient pressure and temperature overnight.

Scanning electron microscopy (SEM) SEM images were captured using a Tescan VEGA 3 scanning electron microscope equipped with a tungsten source filament operating at 20 kV. Prior to analysis, the biocomposites were washed three times by centrifugation (30 s, 1000g) with deionized water, the supernatant was discarded, and the products were dried at ambient pressure and temperature overnight. The powder samples were then sputter-coated with gold. Energy-dispersive X-ray spectroscopy (EDX) analysis were conducted using the same instrument.

Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy

FTIR spectra were recorded on a Bruker ALPHA spectrometer fitted with an ATR accessory with a diamond window in the range of 500-4000 cm ^-1 . Prior to analysis, the biocomposites were washed three times by centrifugation (30 seconds, 1000g) with deionized water, the supernatant was discarded, and the products were dried at ambient pressure and temperature overnight.

Transmission electron microscopy (TEM)

TEM images were captured on a JEM- 1400 flash electron microscope operating at 100 kV. Samples were dispersed in deionized water by thorough vortex stirring and sonication. An aliquot (20 μL) of the dispersion was drop-cast on an ultrathin (-3 nm) continuous carbon film- coated holey carbon on a copper grid (Latech) and dried overnight

Atomic force microscopy (AFM)

AFM measurements were performed using an Asylum Research Cypher ES atomic force microscope (Oxford Instruments) in tapping mode using blueDrive photothermal excitation. The images were acquired in air, at room temperature and at a scanning rate of 2 Hz, using Oxford Instruments HQ75 silicon probes.

Time-resolved small-angle X-ray scattering (SAXS)

Time-resolved SAXS data were collected on the SAXS beamline at the ELETTRA synchrotron light source. ^[2] The experiments were conducted at a photon energy of 8 keV, covering the range of momentum transfer q (where q = 4n sin(0/2)/X) between 0.07 and 5 nm-1 at the scattering angle 0 and wavelength λ The nucleation and growth kinetics of the MOFs were monitored using a commercial stopped-flow apparatus SFM-4 (Bio-Logic, France) modified for synchrotron radiation SAXS investigations. Two independently stepper-motor- driven syringes were filled respectively with the Zn2+ solution and 2-mIM solution (or a mixture of 2-mIM and α-HER2 or α-HER2 fragments). Fixed volumes of both solutions were injected at a flow rate of several milliliters per second first into a mixer and subsequently into a quartz capillary (1.5 mm diameter) placed in the X-ray beam (the mixing/injection process lasts a few milliseconds). The start of the mixing sequence is triggered from the X-ray data- acquisition system, which captured images with a time resolution of 100 ms (detector: Pilatus3 IM, Dectris Ltd., Switzerland; sample-to-detector distance: 1260 mm, as determined with a silver behenate calibration sample). The volume ratio between the two solutions was set accordingly to maintain the conditions used for the syntheses in all batches. All the experiments were performed at room temperature. The SAXS pattern of Zn2+ in buffer solution was measured to assess and subtract the instrument background signal from the data. The resulting two-dimensional images were azimuthally integrated to obtain a one-dimensional pattern of normalized intensity (corrected for transmission and intensity fluctuations) versus scattering vector q.

SAXS Data evaluation

Data analysis was carried out with the software package Igor Pro (IGOR Pro 7.0.8.1, Wavemetrics, USA). The Porod-invariant was estimated as follows: within the q-region, in which the larger variations were observed, i.e. q ₁ = 0.087 and q ₂ = 0.406 nm ^-1 . An increasing trend of this quantity describes the formation of agglomerates, whereas a plateau points toward stable conditions within the dimensional window defined by the extremes of the q-integration.

Various models were applied to interpret the scattering of the samples. Generally, for hierarchical structures, the SAXS signal can be expressed as: in which the residual background given by the incoherent contributions of the solvents is defined by a constant B together with an (optional) power law (constant c and exponent p), which describes diffuse scattering from aggregates larger than the resolution limit of the experiment. The scattering of the aggregates formed during the synthesis is given by the product of a form factor P(q) and a structure factor S(q). The term P(q) takes into account the shape and size of the particles, whereas the term S(q) defines particle correlation/ordering. The scaling factor A represents the contribution of contrast and concentration of the particles. Here, for the structure factor S(q), two models were used: a sticky hard sphere (SHS) model [4,5] and a simplified model consisting of a single peak due to near-range order. [6] The first model SSHS(q) was used in most cases, in which particles grow into a spherical shape. Furthermore, the minimum inter-particle distance DSHS, the depth ε, and the relative extension λ of the attractive potential well were determined from the structure factor.

For the second simplified model, which was used to quantify a disordered near-range particle correlation, the structure factor Sord(q) was applied and described as: where K(x,s) is the Voigt function for the peak shape and the Voigt peak parameters αV, wV, qV, and sV are the peak amplitude, peak position, half- width at half-maximum, and peak shape factor, respectively. The average inter-particle distance (d) can be calculated from the peak position (d) — 2π/q _v .

As for the form factor P(q), different models were used, as detailed below:

• A simplified model for spheres with a large size distribution, which was approximated with the Debye-Anderson-Brumberger model[7] (DAB model), where ζ, is the correlation parameter. By comparison with the Guinier approximation for spherical particles, ζ can be related to a spherical particle with radius R where

• A simple empirical rod model similar to the DAB model with the adjustment of the asymptotic behaviour for q « IP; and q » IP; (rod model), expressed as where ζ is the correlation parameter and ε is related to the surface- to-volume ratio. By comparison with the Guinier approximation for quasi infinite rods, ζ can be related to a rod with cross-sectional radius expressed as R _c = 2ξ.

• A simplified model for quasi infinite large plates with a thickness T (plate model), which is based on the Cauchy integral[7] and the asymptotic behaviour of the scattering for the q-range q « IP; for plates with lateral size larger than thickness, expressed as where ζ is the thickness correlation parameter. By comparison with the Guinier approximation for quasi infinite plates, ζ can be related to a plate with thickness T where

• Polydisperse spherical particles with Schulz distribution for their sizes (spheres ScS model); the form factor describes a set of spheres following Schulz distribution for the number size distribution, [8, 9] P(q) = |F _scs (q,R ,σ ) l ² where FScS is the scattering amplitude of the spheres following the Shulz distribution, R is the average particle radius and σ is the standard deviation.

Statistical analysis

All quantitative data are reported as means ± standard deviations. Statistical analysis was performed using ANOVA followed by a multiple comparison test with p < 0.05 considered as statistically significant (Prism 7, GraphPad).

MOF and MOF biocomposite preparation

MOF (ZIF-C)

33.5 μL of an aqueous solution of 240 mM 2-methylimidazole (2-mIM) in MOPS buffer (MOPS 0.1 M prepared in the pH range 7.0-8.0) was added to 66.5 μL of an aqueous solution of 30 mM zinc acetate dihydrate (Zn ²⁺ solution) in MOPS buffer (MOPS 0.1 M prepared in the pH range 7.0-8.0) . These aqueous solutions were incubated together at 37 °C for 20 minutes under ambient atmosphere. Following incubation, the MOF composite (ZIF-C) was washed twice with MOPS buffer at pH 7.1, centrifuged for 30 seconds at 1000g and resuspended in 200 μL MOPS buffer.

MOF* biocomposites

50 μg of an antibody was added to 33.5 μL of 240 mM 2-methylimidazole (2-mIM) in MOPS buffer (MOPS 0.1 M prepared in the pH range 7.0-8.0). The antibody-mlM (Ab-2-mIM) solution was added to 66.5 μL of 30 mM zinc acetate dihydrate (Zn ²⁺ solution) in MOPS buffer (MOPS 0.1 M prepared in the pH range 7.0-8.0). These aqueous solutions were incubated together at 37 °C for 20 minutes under ambient atmosphere. Following incubation, the MOF biocomposite (ZIF-C* Ab) was washed twice with MOPS buffer at pH 7.1, centrifuged for 30 seconds at 1000g and resuspended in 200 μL MOPS buffer to achieve a final antibody concentration of 500 μg.rnL ^-1 .

Using the above method, a variety of MOF* biocomposites were produced, including ZIF-C*α-HER2, ZIF-C*VH10, ZIF-C*59D8, ZIF-C*3A12, ZIF-C*Kadcyla, and ZIF-C*hIgG (see e.g. Figure 1).

MOF* biocomposite with additional agent A MOF* biocomposite labelled with an additional agent was produced when an additional agents was added to the solution of 2-mIM and antibody and then incubated with the Zn ²⁺ solution. Examples of the resulting multi-labelled MOF* biocomposite include QD@ZIF- C*fAb biocomposite, QD/IONP Cy5@ZIF-C*Ab, QD/IONP Cy5@ZIF-C*fAb, QD@ZIF- C*Abi/Ab2 biocomposite, and ^x Cu@ZIF-C*Ab.

ZIF-C* and fluorescently-labelled antibody

Alexa Fluor 488 (AF488) dye (Invitrogen™, Germany) was used to fluorescently label antibodies using an N-hydroxy succinimide ester-activated following the manufacturer’s protocol. 1 molar equivalent of the antibody was incubated with 10 molar equivalent AF488 at pH 7.5 and 4 °C for 3 hours. The fluorescently labelled antibody (fAb) was purified using a 10k centrifugation column (Amicon, USA). 50 μg of a fAb was added to 33.5 μL of 240 mM 2 -methylimidazole (2-mIM) in MOPS buffer (MOPS 0.1 M prepared in the pH range 7.0-8.0) . This fAb-2-mIM solution was added to 66.5 μL of 30 mM zinc acetate dihydrate (Zn ²⁺ solution) in MOPS buffer (MOPS 0.1 M prepared in the pH range 7.0-8.0). These aqueous solutions were incubated together at 37 °C for 20 minutes under ambient atmosphere (see e.g. Figure 3), resulting in a fluorescent MOF* biocomposite, ZIF-C*fAB.

Quantum dots encapsulated into ZIF-C*Ab

1 μL of an 8 pM QD suspension (625 nm - carboxyl QDs) was added to a solution containing Ab and 2-mIM. The resulting solution was added to 66.5 μL of 30 mM zinc acetate dihydrate (Zn ²⁺ solution) in MOPS buffer (MOPS 0.1 M prepared in the pH range 7.0-8.0) . These aqueous solutions were incubated together at 37 °C for 20 minutes under ambient atmosphere (see e.g. Figure 31) resulting in a MOF* biocomposite, QD@ZIF-C*Ab.

Magnetic particles encapsulated into ZIF-C*Ab

Iron Oxide NanoParticles (IONP), an MRI contrast agent, with properties of -40-50 nm hydrodynamic monodisperse particles with a -15 nm core size and 2.5 mg of Fe per mL. The IONP were coated in polyDMSO polymer and fluorescently labelled with Cy5 through a "click" reaction (IONP Cy5). 12.5 μg of IONP Cy5 was added to a solution containing Ab and 2mlm. The resulting solution was added to 66.5 μL of 30 mM zinc acetate dihydrate (Zn ²⁺ solution) in MOPS buffer (MOPS 0.1 M prepared in the pH range 7.0-8.0). These aqueous solutions were incubated together at 37 °C for 20 minutes under ambient atmosphere (see e.g. Figure 32), resulting in a magnetic MOF* biocomposite, IONP Cy5@ZIF-C*Ab.

Chemotherapeutic agents encapsulated into ZIF-C*Ab

2 μg of Doxorubicin was incubated with 50 μg of α-HER2 antibody, 33.5 pl of 240 nM 2-mIM, 5 pl of MP and 66.5 pl 30 nM of Zn ²⁺ at 37°C for 20 min. Post incubation chemotherapeutic MOF biocomposites were washed twice with MOPS buffer pH 7.1 in a final volume of 200 pl and finally resuspended in buffer with a final concentration of 500 μg/ml (see e.g. Figure 36), resulting in a chemotherapeutic MOF biocomposite, DOX@ZIF-C*Ab.

Bi-specific ZIF-C*Abi/Ab2

25 μg of each of the monoclonal antibodies α-HER2-Cy5 and α-CD3-488 were incubated with 1 ul of QD, 33.5 pl of 240 nM 2-mIM, and 66.5 pl 30 nM of Zn ²⁺ at 37°C for 20 min. Post incubation MOFs were wash twice with MOPS buffer pH 7.1 in a final volume of 200 pl and finally resuspended in buffer with a final concentration of 500 μg/ml, resulting in ZIF- C*Abi/Ab2. Binding was tested via FACS (see e.g. Figure 33).

Radioactive element encapsulated into ZIF-C*Ab

Different concentrations of radioactive ⁶⁴ Cu (or ⁶⁷ Cu) (e.g.10 MBq of ^x Cu) was added to a solution of Ab and 2-mIM (50 μg of monoclonal antibody α-HER2 with 33.5 pl of 240 nM 2-mIM). The solution of radioactive Cu, Ab and 2-mIM was added to 66.5 μL of 30 mM zinc acetate dihydrate (Zn ²⁺ solution) in MOPS buffer (MOPS 0.1 M prepared in the pH range 7.0- 8.0) . These aqueous solutions were incubated together at 37 °C for 20 minutes under ambient atmosphere Post incubation, MOFs were centrifuged, resulting in a radioactive MOF biocomposite, ^x Cu@ZIF-C*Ab.

In the preparation of all MOF biocomposites with additional agents, following incubation the MOF biocomposite was washed twice with MOPS buffer at pH 7.1, centrifuged for 30 seconds at 1000g and resuspended in 200 μL MOPS buffer to achieve a final antibody concentration of 500 μg mL-1.

MOF results and analysis techniques

MOF biocomposite growth kinetics Evaluation of the ZIF-C biocomposite growth kinetics were analysed by monitoring changes in the optical density of the ZIF-C*Ab or ZIF-C solution, performed using a FEUOstar OPTIMA microplate reader (BMG Labtech, Germany) equipped with two built-in reagent injectors (see e.g. Figure 4). The injection pumps were filled with the following buffers: 240 mM 2-mIM (pump 1) and 30 mM Zn ²⁺ (pump 2); 31 cycles and 60 s per cycle were applied. The antibody samples in 10 mM MOPS buffer pH 7.1 were placed in a 96-well plate and 240 mM 2-mIM (33.5 μL) was added in cycle 1, followed by the addition of 30 mM of Zn ²⁺ (66.5 μL) in cycle 3. The increase in the optical density was measured and correlated with MOF growth.

All curves exhibit exponential increase in absorbance followed by eventual stabilisation and plateau by 30 minutes. However, this relationship is only applicable when all three components of the ZIF-C biocomposite are present. Control samples of Zn ²⁺ with either 2-mIM or the antibody alone was not sufficient for nanoparticle formation, evident by the constant curve. No statistically significant difference in absorbance was found at cycle 1 (minute 0) between samples with just the antibody and samples with antibody and 2-mIM (p=0.82 for 59D8, p=0.69 for VH10, and p=0.24 for BSA). However, rapidly following the introduction of Zn ²⁺ , the two groups diverge, and statistical significance is present from cycle 2 onwards. Formation of ZIF-C*59D8 begins to stabilise at minute 26 with an OD of 1.31 and continues to slightly increase reaching a maximum of 1.35 OD compared to a constant 0.2 OD for the antibody control group (p<0.0001 from cycle 2 - cycle 31). ZIF-C*VH10 formation stabilises at 20 minutes with an OD of 1.20 and a peak at 1.29 OD compared to a maximum of 0.3 OD for the antibody control group (p<0.001 from cycle 2 - cycle 31). To display that the formation of ZIF-C biocomposite is unique to immunoglobulins and not any other reagent, MOPS was used in place of an antibody. This resulted in unsuccessful ZIF-C biocomposite formation as shown by the linear curve as well as visually the lack of crystallisation.

Additionally, 1% HC1 was added to reduce the pH of the solution in each well down to approximately pH5 and optical density measured. A second addition of 1% HC1 to lower the pH down to approximately pH 2 was performed and optical density measured. A reduction in optical density is indication of dissociation of ZIF-C*Ab biocomposite back to original substrates.

MOF biocomposite immobilisation efficiency and stability Following the production of a ZIF-C*Ab, the amount of antibody within the MOF biocomposite was quantified by performing a BCA assay of each sample supernatant. Comparing the concentration of protein in the supernatant to a neat sample of the mAb in the same volume could be used to determine the immobilisation efficacy (IE%). ZIF-C*59D8 had a mean IE (±SEM) of 91% (±2.5) for the antibody immobilised, while ZIF-C*VH10 had an IE of 87% (±1.5). The immobilisation of BSA and hlgG was equally successful with an IE of 84% (±3.8) and 82% (±3.3) respectively, and no statistically significant difference between all four groups.

Long term stability of ZIF-C*VH10 and hlgG was assessed by comparing the immobilisation efficacy of the construct at production and 4 weeks after storage at 4°C. The results showed no significant difference in the immobilisation efficiency (IE%) between the time points for ZIF-C* VH10 (84.87% ±1.327 versus 85.73% ±2.067) andZIF-C* hlgG (88.14 ±0.885 versus 87.01 ±0.395) elucidating complete stability (see e.g. Figure 7).

MOF biocomposite validation

To validate the incorporation of the antibodies within the nanoparticles, the supernatants were analysed using gel electrophoresis in comparison to a neat sample of the equivalent mAb. The presence of protein in the gel represents antibodies that had unsuccessfully immobilised into the nanoparticle. Neat immunoglobulins when run on a gel electrophoresis separate into 2 distinct bands, a heavy chain (~50 kD) and a light chain band (~25 kD), due to the denaturing of the disulfide linker between the chains. The gel showed no distinct protein presence in the supernatant of ZIF-C*Ab (see e.g. Figure 37).

Cell/animal culture

HER2-receptor-positive (HER2-R+) cell line SKOV-3 and HER2-receptor-negative (HER2-R-) cell line MDA-MB-231 were grown in RPMI 1640 medium with 10% fetal bovine serum (Sigma, USA) and 1% penicillin (10,000 units. mL ^-1 )/streptomycin (10,000 μg.rnL ^-1 ) at 37 °C in a humidified atmosphere of 5% CO ₂ . Subcultures were prepared every 2-3 days to maintain exponential growth.

PSMA-positive cells were grown in RPMI 1640 medium with 10% fetal bovine serum (Sigma, USA) and 1% penicillin (10,000 units.mL ^-1 )/streptomycin (10,000 μg.rnL ^-1 ) at 37 °C in a humidified atmosphere of 5% CO ₂ . Subcultures were prepared every 2-3 days to maintain exponential growth. BALB/c-Foxnlnu mice were obtained from Walter and Eliza Hall Institute, Victoria, Australia (see e.g. Figure 35).

MOF* biocomposite binding studies

To evaluate the binding capability of the QD@ZIF-C*Ab via flow cytometry (CytoFlex & Canto, Beckman Coulter, USA), the QD MOF* biocomposites were prepared as above.

HER-R+ cells were freshly harvested from the tissue culture flasks and a single-cell suspension prepared in phosphate-buffered saline with 3% Fetal Calf Serum and 0.5 mM ethylenediaminetetraacetic acid. Specifically, 2xl0 ⁵ cells were incubated with 2.5 μg.mL ^-1 of the QD@ ZIF-C*Ab at room temperature and after washing, the relative fluorescence of the stained cells was measured using either BD FACSCantoTM II or CytoFLEX flow cytometer and analysed using BD FACSDiva (Becton Dickinson, USA) or CytExpert (Beckman Coulter, USA) software. Post-data acquisition analysis was performed using FlowJo (Version 10, FlowJo LLC, USA) (see e.g. Figure 3).

Confocal microscopy

To assess cell internalization, 1.25xl0 ⁴ HER-R+ cells per well were treated with 2.5 μg.mL ^-1 of either ZIF-C*α-fHER2 or ZIF-C*fhIgG and nuclear stain 4',6-diamidino-2- phenylindole at 1 μg. mF ¹ for 20 min at 37 °C. After incubation, the cells were washed several times and transferred to a CEELview™ cell culture slide (Greiner AG, Austria). One cohort was additionally stained with 4 μg. mL ^-1 cell membrane dye Wheat germ agglutinin, Alexa FluorTM 647 conjugate (Invitrogen/Thermo Fisher, USA). Confocal images and z-stacks were acquired using a Nikon Air confocal microscope with a 40x objective and a 4x scanner zoom. Lasers (405 and 488 nm) were used for excitation, and emission was detected using 450/50 nm and 535/50 nm filters. Quantification of the cell binding and internalization of the ZIF-C*fAb biocomposites was performed using Fiji software ^[1] . Region of interests (RO Is) were drawn around the outline of the bright-field cells in focus and analyzed through the stacks to ensure that the ROIs covered cells throughout the stack. An ROI was then added to the ROI manager and applied to 500-550 nm channel images. To simultaneously measure the AF488 signal in all ROIs on every slice, the multi-measure function was used to measure the integrated density, where measurement was limited to the threshold value. Data are expressed as integrated density per cell (see e.g. Figure 25). IONP Encapsulated ZIF-C*VH10

To determine the amount of IONP that had been loaded into the ZIF-C*Ab, a standard curve of IONP Cy5 standards was generated. The standard curve was then used to interpolate the amount of IONP present both in the ZIF-C grown in the presence of IONP Cy5 and BSA produced and the supernatant of each sample. Due to the limited sensitivity of the IVIS (in vivo imaging system - Fluorescence Imaging System for In Vivo scan of mice, some signals were generated below the ROI of the blank sample and were thus quantified mathematically by extrapolating the curve. The quantity of IONP in each sample was compared to the amount expected to be present based on the amount added when producing the ZIF-C* biocomposite (see e.g. Figure 32).

Samples loaded with 1.75 μg, 3.125 μg, and 6.25 μg of IONP all fell beneath the detection limit. Samples loaded with 25 μg of IONP appeared to only contain 45% of the expected amount. Finally, when 12.5 μg of IONP was used, 74.6% of the added IONP was loaded into the particle and the remaining IONP present in the supernatant. Based on this, 12.5 μg of 2.5 mg/mL IONP was determined to be the optimal maximum amount that could be loaded within the ZIF-C biocomposite.

Microfluidic channel fabrication and determination of presence of magnetic nanoparticles within biocomposites

Poly dimethyl siloxane (PDMS) chips containing microfluidic channels were fabricated via soft-lithography method using a SU-8 master mold (pre-generated via standard photolithography) on a silicon wafer as per previously reported protocols. Briefly, liquid PDMS prepolymer and curing agent (Sylgard 184) were mixed in 10: 1 (v/v), degassed and poured onto the master mold, following by baking at 100 °C for 1 hour. A nickel coated neodymium magnetic disc (1/8" x 1/16") with a pull force of 0.8 pounds (Apex Magnets) was incorporated within the PDMS chips on top of one of the micro fluidic channels. The newly designed chips contained 52 pm high, 300 pm wide and 17 mm long microfluidic channels. The fabricated chips were hydrophobically sealed with thin glass cover slips. Later, the channels were blocked with 1% BSA (w/v) via static incubation at RT for at least 20 minutes, followed by lOmM MOPS buffer pH 7.1 rinse prior further use.

Magnetic (IONP Cy5@ZIF-C* VH10 - AF488) and non-magnetic (ZIF-C* VH10 - AF488) nanoparticles were produced and perfused into the channels. The co-localisation of the AF488 labelled VH10 and the Cy5 labelled IONP was evident demonstrating the intact nanoparticle structure. The results also illustrated the strong magnetism of the IONP Cy5 @ZIF- C* VH10 - AF488 rapidly accumulating at the magnet compared to the non-magnetic nanoparticles showing no binding. The magnetic particles reached a maximum of 9.39% (±3.008) area aggregation at the magnetic zone after 1 minute compared to 0.507% (+0.02, P=0.01) aggregation of the non-magnet nanoparticle (see e.g. Figure 34). The results also demonstrated the pH sensitivity of ZIF-C* with the disappearance of the VH10 AF488 signal and retained IONP Cy5 signal following the perfusion of HC1 (pH 5) into the channel.

Superparamagnetic MOF biocomposite

To test the magnetic function of the nanoparticles, 50 μg of AF488-labelled monoclonal antibody α-PSMA488 were incubated with 33.5 pl of 240 nM 2-mIM, either with or without 5 pl of magnetic particles of iron oxide and 66.5 pl 30 nM of Zn ²⁺ at 37°C for 20 min. Post incubation supermagnetic MOF biocomposites were washed twice with MOPS buffer pH 7.1 in a final volume of 200 pl and finally resuspended in buffer with a final concentration of 500 μg/ml. Microfluidic channels under the magnet were perfused with either non-magnetic ZIF- C*fPSMA or magnetic MP@ZIF-C*fPSMA (MP = magnetic particles) at a concentration of 25 μg/ml in MOPS buffer pH 7.1 for 2 min at RT. The volumetric input flow rate of the nanoparticles was 1.87 pl/min corresponding to low venous flow rate. Aggregation of Alexa- 488 functionalised magnetic -/ control particles around the magnetic -t the upstream zone in these microfluidic channels were recorded via sCMOS Andor Zyla camera for 2 min at 500 ms frame intervals using a 4x objective lens on Nikon Ti-E inverted fluorescence microscope (Nikon Inc., Japan). Aggregate area coverage was analysed using FIJI image analysis software (see Figure 34D).

To test the targeting function of the nanoparticles, 50 μg of monoclonal antibody α- PSMA or IgG as control mAb were incubated with 33.5 pl of 240 nM 2-mIM, 5 pl of MP and 66.5 pl 30 nM of Zn2+ at 37°C for 20 min. Post incubation supermagnetic MOF biocomposites were wash twice with MOPS buffer pH 7.1 in a final volume of 200 pl and finally resuspended in buffer with a final concentration of 500 μg/ml. PSMA-positive cells were incubated with CellTracker™ Green CMFDA Dye (Invitrogen)(Ex/Em:495/519nm) in a final concentration of 2 pM for 10 min at 37°C and 5% CO2. Nanoparticles were made with the binding mAb 3A12 as well as control IgG, modified with QDs 625nm (Qd)(Ex/Em:405/625) and MPIOs, pre-incubated with C4.2 for 30 min on ice and washed with FACS buffer three times. Microfluidic channels under the magnet were perfused with either magnetic QD/MP@ZIF- C*IgG or magnetic QD/MP@ZIF-C*fPSMA at a concentration of 25 μg/ml in MOPS buffer pH 7.1 for 2 min at RT (see Figures 34 E, F).

Aggregation of Alexa-488 coloured cells around the magnetic -/ the upstream zone in these microfluidic channels were recorded via sCMOS Andor Zyla camera for 2 min at 500 ms frame intervals using a 4x objective lens on Nikon Ti-E inverted fluorescence microscope (Nikon Inc., Japan). Aggregate area coverage was analysed using FIJI image analysis software (see e.g. Figure 34).

QD Encapsulated ZIF-C*VH10

To ensure that the signal generated was not from free fVHlO, dual labelling was performed. ZIF-C biocomposites were produced with the fVHlO and QD were added to the solution during production. A positive co-signal of both QD and AF488 would correspond to an intact nanoparticle binding to platelets, compared to only AF488 expression implying a functional antibody with dismantled nanoparticle. Successful encapsulation of the QD was achieved as the ZIF-C biocomposite illuminated under ultraviolet (UV) light following washing of the nanoparticles, which would have removed excess QD. Further, confocal microscopy validated the co-localisation of the labelled antibody on the ZIF-C biocomposite surface and the QD encapsulated within (see e.g. Figure 31).

In vitro potency of DOX@ZIF-C*HER2.

To address in vitro potency, cell viability assays was carried out. 1.5xl0 ⁴ SKOV-3 cells were incubated with either 2.5ug DOX@ZIF-C*HER2 or with 2.5 ug free Dox for 20 min before washed two time with MOPS, free media added and incubated for 24 hrs. After incubation, lOpl of WST-1 reagent (Roche) were added and further incubated for 90 min at 37C, 5%CO ₂ . A stopping reagent (12 μL of 10% Triton X100 + 1% SDS for final concentration of 1% Triton X100Z 0.1% SDS) were added and incubated for 5 min at room temperature. Afterwards centrifuged in a microplate for 5 mins at 1200 RPM, taken 50 ul of supernatant and measured in microplate reader at ref 450 nm as endpoint kinetic. Untreated cells were used as 100%.

A cytotoxicity study showed a clear reduction in cell viability in those cells treated compared to untreated cells or free drug 48 hours post incubation (see e.g. Figure 36). The WST-1 assay is a variability test and the protocol is based on the cleavage of the tetrazolium salt WST-1 to formazan by cellular mitochondrial dehydrogenases. The larger the number of viable cells, the higher the activity of the mitochondrial dehydrogenases, and in turn the greater the amount of formazan dye formed which is detectable via a change of colour in the microplate reader.

Bispecific MOFs with HER2 and CD 3

25 μg of each of the monoclonal antibodies α-HER2-Cy5 and α-CD3-488 were incubated with 1 ul of QD, 33.5 pl of 240 nM 2-mIM, and 66.5 μl 30 nM of Zn2+ at 37°C for 20 min. Post incubation MOFs were wash twice with MOPS buffer pH 7.1 in a final volume of 200 pl and finally resuspended in buffer with a final concentration of 500 μg/ml. Binding were tested via FACS (see e.g. Figure 33).

Radioactive MOFs

The radioactivity of MOF biocomposites labelled with ⁶⁴ Cu, produced as described above, was determined. Post incubation, MOFs were centrifuged and radiation of the MOFs as well as supernatant measured.

Table: Radioactive MOFs α-HER2

Flow Cytometric Analysis ofZIF-C* VH10

The produced MOF biocomposite ZIF-C*VH10 was subsequently investigated for its antibody binding function and compared to the interaction of the non-binding control ZIF- C*hIgG using flow cytometry Human PRP was either left un-treated or stimulated with ADP to activate platelets. Platelet activation was confirmed using the commercial PE α-human CD62P (P-selectin) antibody.

Prior to testing ZIF-C*VH10 functionality, the binding of non-labelled VH10 antibody was assessed. Platelets were incubated with VH10 followed by the addition of a secondary AF488 α-mouse antibody. Percentage of platelets the construct was binding to was based on gating the platelets with positive AF488 signal on an unstained sample. The secondary antibody alone exhibited no binding. Figure 5 shows that VH10 binds to approximately 60.93% (±3.894) of the activated platelets. The binding was also determined to be approximately 3 -fold greater than to non-activated platelets (25.03% ±3.894, P=0.003). There was no difference in the binding of ability when 2.5 μg/mL or 5 μg/mL of VH10 was used (60.93 ±3.894 and 56.13 ±10.02 respectively, P=0.99) and so the lower concentration was used for all further investigations.

Following the confirmation of VH10 binding, ZIF-C biocomposites were produced with the AF488 labelled VH10 based on an IE of 85% mAb. Production of ZIF-C biocomposites with the fluorescent labelled antibodies (hlgG - AF488 and VH10 - AF488) did not appear to affect nanoparticle formation (Figure 6A-B). 2.5 μg/mL of the nanoparticles were incubated with PRP and the construct showed binding to 13.2% (±1.7) of activated platelets compared to 2.020% to non-activated platelets (±1.390, P=0.007 ).

Quadrant gating of a population of activated and non-activated platelets was performed on an unstained sample. The results indicated a 24% (±6.047) double -positive signal generated from QD@ZIF-C*VH10 - AF488 binding to activated platelets compared to only 9.303% (±3.026) on non-activated platelets (P=0.04) (Figure 8). Furthermore, significant differences were present between the binding of the QD @ ZIF-C* VH 10 - AF488 and the non-binding control (4.035% ±0.443, P=0.06) (see e.g. Figure 30).

MOF biocomposite pyrolysis

To further investigate the immobilization geometry of the Ab in the MOF lamellae, ZIF- C*α-HER2 was pyrolyzed at 325 °C following a protocol previously used for the localization of proteins in ZIF biocomposites ^[30] .

The crystallinity of ZIF-C*α-HER2 and the overall morphology of the MOF* Ab biocomposite were preserved after pyrolysis (see e.g. Figures 20 and 21). TEM analysis revealed the presence of pores with a diameter of 10-15 nm (see e.g. Figure 2f). AFM confirmed that pyrolyzed ZIF-C*α-HER2 were constituted of plates with a typical thickness of 3-5 nm (see e.g. Figure 22 and 23). AFM analysis of the surface of the pyrolyzed ZIF-C crystals also revealed the presence of cavities with a width of 11 ± 3 nm (see e.g. Figure 2e, 22, and 23), consistent with the TEM results. These cavities were formed upon thermal decomposition of the Fc regions in ZIF-C, while the thickness of the plates (3-5 nm) suggests that the F(ab')2 fraction is exposed from the plate with perpendicular orientation. Thus, these structural investigations support that preferential nucleation and growth of ZIF-C composite can occur on the Fc region.

X-ray diffraction (XRD) studies

XRD analysis was used to identify the crystalline phases present in a material and thereby reveal chemical composition information. Solid MOF biocomposites were collected by centrifugation, washed with water, and examined via X-ray diffraction (XRD) (see e.g. Figure 2a, 5, and 20).

Scanning electron microscopy (SEM)

The morphology of the MOF biocomposites ZIF-C*α-HER2 and ZIF-C*hIgG was examined by scanning electron microscopy (SEM).

SEM images showed the presence of two populations of particles: micrometer- sized lamellar particles and clusters of smaller particles. ZIF-C*α-HER2 was selected as a model system for an in-depth investigation of the biocomposite morphology (see e.g. Figures 2d and 8).

Attenuated total reflectance-Fourier transform infrared (ATR-FTIR ) spectroscopy

The ZIF-C*Ab were examined by Fourier transform infrared (FTIR) spectroscopy (see e.g. Figures 2b and 6).

The spectra featured vibrational modes assigned to ZIF-C ^[23 ’ ²⁴ ’ ²⁵ ’ ^26] (e.g., Zn-N stretching mode at 424 cm ^-1 , CO ₃ ^2- bending mode at 828 cm ^-1 , asymmetric stretching modes of CO ₃ ^2- at 1575 and 1375 cm ¹ ) and to the peptide backbone of the antibodies, amide I (1600-1710 cm-1) and amide II (1480-1595 cm-1) bands ^[27] , thus confirming the successful integration of the Abs within ZIF-C.

The spectrum of ZIF-C, and the related ZIF-C* Ab shows bands in the 700-850 and 1300- 1600 cm' ¹ regions that can be assigned to bending (828 cm ^-1 ) and asymmetric stretching modes (1575 and 1375 cm ^-1 ) of CO ₃ ² '. The Zn-N stretching mode, typically at 420 cm ^-1 for sod or dia ZIF-8, Zn(2-mIM) ₂ , for ZIF-C is centred at 425 cm' ¹ because of the coordination of CO ₃ ² ’ with Zn and the related change in coordination environment (see e.g. Figure 6).

Transmission electron microscopy (TEM) TEM analysis confirmed the lamellar structure of ZIF-C*α-HER2, constituting of clusters of plates (0.1-6 pm) made of lower and higher contrast lamellae inclusion (plate perpendicular and parallel to the electron beam, respectively). The estimated thickness of the lamellae was 2-3 nm with an average lateral dimension of 25 nm (see e.g. Figure 9).

TEM analysis revealed the presence of pores with a diameter of 10-15 nm (see e.g. Figure 2f). TEM analysis also revealed colocalization of AF488 and QD624 in samples of QD@ZIF- C*α-fHER2 (see e.g. Figure 3j).

Atomic force microscopy (AFM)

AFM was used to confirm that pyrolyzed ZIF-C* α-HER2 was constituted of plates with a typical thickness of 3-5 nm (see e.g. Figures 22 and 23). AFM analysis of the surface of the pyrolyzed ZIF-C*Ab crystals also revealed the presence of cavities with a width of 11 ± 3 nm (Figures 2e and 22-24), consistent with the TEM results. These cavities were formed upon thermal decomposition of the F _c regions in ZIF-C, while the thickness of the plates (3-5 nm) suggests that the F(ab') ² fraction is exposed from the plate with perpendicular orientation. Thus, these structural investigations support that preferential nucleation and growth of ZIF-C* Ab occurs on the F _c region.

Time-resolved small-angle X-ray scattering (SAXS)

SAXS experiments were used to study the formation of the ZIF-C* α-HER2 using a stopped-flow cell setup (see e.g. Figure 10) ^[28] , in the presence and absence of the Ab in the MOF precursor solution.

The formation of the biocomposite occurred over a time scale of seconds, as seen in the time-resolved SAXS patterns and calculated Porod invariant (see e.g. Figures 2c and 11). Modelling of the time-resolved SAXS curves revealed that near- spherical particles of about 10 nm rapidly form, followed by a transition to plate-like structures after 3.6 seconds upon mixing of the reagents (see e.g. Figure la-f). As the reaction proceeds, the plate-like structures aggregate to form layered superstructures in solution (see e.g. Figure If, g). The modelling results confirm the topological information obtained from the microscopy analysis. Fitting of the time-resolved SAXS pattern of the dry particles revealed the presence of cavities with an average size of 3 nm on the particles (see e.g. Figure 12). The size of the cavities of the MOF* biocomposites is comparable with the size of the F _c region. ^[29] Conversely, in the absence of an Ab, a slow three-dimensional (3D) growth of spherical MOF particles of < 100 nm was obtained (see e.g. Figure 13). From these in situ synchrotron time-resolved SAXS data, it was concluded that an antibody triggers the formation of the MOF biocomposite, e.g., ZIF-C*α-HER2 biocomposite formed via biomimetic mineralization. More importantly, the data suggest that the F _c region of the Ab is embedded in ZIF-C.

The influence of the different constituent fractions of the antibody (i.e., F _c , Fab, F(ab') ² , sialylglycopeptide (SGP), sialylglycan (SG), see e.g. Figure 14) on the MOF formation was examined by in situ time-resolved SAXS analysis. Rapid formation of MOF-containing particles with the same plate-like morphology as that obtained with α-HER2 as a whole was only observed in the presence of the F _c component (see e.g. Figure 15). In the presence of the other components, particle formation was comparable with that obtained in the absence of an antibody (Table 1, see e.g. Figures 16-19). These results confirmed that the F _c region of an antibody is the only responsible part for the formation of ZIF-C with a plate-like structure.

The table below summarizes the samples analysed and the corresponding model used for data fitting.

Table: Summary of SAXS models applied for the samples and reference to the corresponding figures References

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