Modified proteins

Abstract

The present invention provides an antigen binding protein comprising at least one disulfide bond and a mutation or modification of at least one residue to introduce a side chain capable of acting as a ligand for a metal are described. The present invention further provides an antigen binding protein comprising a helix bundle with at least three helices comprising mutation or modification of at least three residues to introduce a side chain capable of acting as a ligand for a metal. Metal coordinated antigen binding proteins are also described. The metal coordinated antigen binding proteins described can be used in therapeutic, diagnostic or theranostic applications.

Description

Modified proteins Cross-reference to related applications

[0001] This application claims the benefit of priority from Australian provisional application no. 2024903072, filed on 24 September 2024, and Australia provisional application no. 2025902405, filed on 12 June 2025, the contents of which are incorporated herein by reference in their entirety. Field of the invention

[0002] The present disclosure relates to a metal coordinated antigen binding protein, methods of making antigen binding proteins and metal coordinated antigen binding proteins, and their use in therapeutic and / or diagnostic (together theranostic) applications. Background of the invention

[0003] The integration of antigen binding proteins (such as monoclonal antibodies) with metals is crucial for therapeutic, diagnostic and theranostic applications. In theranostics, while the antigen binding protein (e.g. antibody) typically targets a specific biomolecule, such as a cancer cell receptor, the metal serves as a probe for imaging purposes or selectively destroys target cells through radiation.

[0004] For example, the monoclonal antibody trastuzumab, which binds to the human epidermal growth factor receptor 2 (HER2), can be labelled with the radioactive isotopes68Ga or111In for imaging purposes using positron emission tomography (PET) or single- photon emission computed tomography (SPECT), respectively. Combination of trastuzumab with the radioactive isotope213Bi allows the selective destruction of HER2- positive cells using targeted-alpha therapy (TAT).

[0005] While antibodies continue to dominate biopharmaceuticals, their large size (~150 kDa) limits tumor tissue penetration, and their glycosylation necessitates production in mammalian cells. Alternatively, single-domain antibodies (sdAb), known as nanobodies, are an emerging class of antigen-binding fragments originating from camelids and cartilaginous fishes. While they exhibit affinity and selectivity similar to conventional antibodies, their much smaller size (12–15 kDa) allows for better tissue penetration and they can be recombinantly expressed from bacterial culture.

[0006] Another class of antigen-binding molecules that have gathered interest are affibodies. Affibodies are small, engineered proteins based on a three-helix bundle structure. They combine high thermal stability with strong binding affinity and specificity. Their compact size (6–7 kDa) also allows for excellent tissue penetration, making affibodies potential tools for molecular imaging and targeted cancer therapy.

[0007] The conventional strategy for conjugating antigen binding proteins, such as antibodies, nanobodies or affibodies, to metals uses bifunctional linkers. One part of these linkers reacts with lysine or cysteine residues in the protein, while the other part contains a metal chelator. Where residue modification of an antibody, nanobody or affibody is required, they are typically randomly modified, resulting in heterogeneous conjugates and partial inactivation by altering the antigen-binding site.

[0008] In addition to challenges with harsh, incomplete and lengthy tagging processes, conventional chelators are large and highly charged, impacting on the properties of the conjugate. This is particularly concerning for some antigen binding proteins (e.g. nanobodies and affibodies) where the bifunctional linker significantly changes the overall mass and charge of the conjugate.

[0009] There exists a need for new and / or improved metal bound antigen binding proteins. Further, there also exists a need for new and / or improved processes for producing such metal bound antigen binding proteins.

[0010] Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and / or combined with other pieces of prior art by a skilled person in the art. Summary of the invention

[0011] In a first aspect, the present disclosure provides an antigen binding protein comprising an immunoglobulin domain comprising at least one disulfide bond, wherein the antigen binding protein comprises a mutation or modification of at least one residue that introduces a side chain capable of acting as a ligand for a metal.

[0012] In some embodiments, the metal is a radionuclide.

[0013] In some embodiments, the radionuclide is an alpha particle emitter. In some alternative embodiments, the radionuclide is a beta particle emitter. In some alternative embodiments, the radionuclide is a positron emitter. In some alternative embodiments, the radionuclide is a gamma emitter. In some alternative embodiments, the radionuclide is an Auger electron emitter.

[0014] In some embodiments, the radionuclide is an isotope of Al, Ga, Cu, Mn, Sc, Sr, Co, Zr, Rb, Tc, Y, In, Lu, Sm, Ac, At, Pb, Bi, Fr, K, Ti, Nb, Ta, Re, Fe, Rh, Ni, Pt, Au, Tl, Ge, Sn, As, Po, Ir, La, Pr, Pm, Tb, Dy, Ho, Tm, Yb, Lu, Th, Er, Cr, Cs or Ra. Preferably the radionuclide is an isotope of Ga, In, Bi, or Pb. Preferably the radionuclide is an isotope of Ga, In or Bi.

[0015] In some embodiments, the radionuclide is26Al,38K,66Ga,67Ga,68Ga,68Ge,61Cu,62Cu,64Cu,67Cu,51Mn,52Mn,44Sc,47Sc,45Ti,52Fe,57Ni,83Sr,89Sr,90Sr,51Cr,55Co,60Co, ,72As,89Zr,81Rb,82mRb,94mTc,99mTc,86Y,90Y,90Nb,103Pd,111In,110mIn,114mIn,140Pr,117mSn,131Cs,149Tb,152Tb,155Tb,161Tb,153Sm,166Ho,169Yb,169Er,177Lu,186Re,188Re,191mIr,192Ir,193mPt,195mPt,201Tl,225Ac,212Pb,211At,211Bi,212Bi,213Bi,211Fr,211Po,213Po,223Ra,224Ra, and227Th.

[0016] In some embodiments, the radionuclide is26Al,38K,66Ga,67Ga,68Ga,68Ge,61Cu,62Cu,64Cu,67Cu,51Mn,52Mn,44Sc,47Sc,45Ti,52Fe,57Ni,83Sr,89Sr,90Sr,51Cr,55Co,60Co, ,72As,89Zr,81Rb,82mRb,94mTc,99mTc,86Y,90Y,90Nb,103Pd,111In,110mIn,114mIn,140Pr,117mSn,131Cs,149Tb,152Tb,155Tb,161Tb,153Sm,166Ho,169Yb,169Er,177Lu,186Re,188Re,191mIr,192Ir,193mPt,195mPt,201Tl,225Ac,203Pb,212Pb,211At,211Bi,212Bi,213Bi,211Fr,211Po,213Po,223Ra,224Ra, and227Th.

[0017] In some alternative embodiments, the metal is a non-radioactive isotope.

[0018] In some embodiments, the non-radioactive isotope is an isotope of Al, Ga, Cu, Mn, Sc, Sr, Co, Zr, Rb, Tc, Y, In, Lu, Sm, Ac, At, Pb, Bi, Fr, K, Ti, Nb, Ta, Re, Fe, Rh, Ni, Pt, Au, Tl, Ge, Sn, As, Po, Ir, La, Pr, Pm, Tb, Dy, Ho, Tm, Yb, Lu, Th, Er, Cr, Cs or Ra. Preferably, the non-radioactive isotope is an isotope of Bi, Ga, In, As, Sb, Pb, Cu, Gd or Ba. In some embodiments, the side chain capable of acting as a ligand for a metal comprises a monodentate ligand.

[0019] In some embodiments, the side chain capable of acting as a ligand for a metal comprises a sulphur or selenium atom.

[0020] In some embodiments, the mutation of at least one residue introduces a cysteine or selenocysteine residue.

[0021] In some embodiments, the mutation of at least one residue introduces a cysteine residue.

[0022] In some embodiments, the alpha carbon atom of the at least one residue, or the alpha carbon atom of each of the two residues, is located within 10 Å, or within 9 Å, or within 8 Å, or within 7 Å, or within 6 Å, or within 5 Å or within 4 Å, or within 3 Å of the alpha carbon atom of one or both of the cysteines that form the at least one disulfide bond. In some alternative embodiments, the alpha carbon atom of the at least one residue, or the alpha carbon atom of each of the two residues, is located between about 3 Å to about 10 Å, or between about 3 Å to about 9 Å, or between about 4 Å to about 8 Å of the alpha carbon atom of one or both of the cysteines that form the at least one disulfide bond.

[0023] In some embodiments, the residue is located in a framework region of the immunoglobulin domain.

[0024] In some embodiments, the residue is located in framework region 1 of the immunoglobulin domain.

[0025] In some embodiments, residue is located in a framework region of a heavy chain variable domain.

[0026] In some embodiments, the residue is located at any one or more residues, or residues equivalent to, 4 or 6 in SEQ ID NOs: 1 to 10.

[0027] In some embodiments, the at least one residue that is mutated or modified is a hydrophobic residue.

[0028] In some embodiments, the hydrophobic residue is leucine or isoleucine.

[0029] In alternative embodiments, the at least one residue that is mutated or modified is a hydrophilic residue.

[0030] In some embodiments, the hydrophilic residue is glutamate or aspartate.

[0031] In some embodiments, the at least one disulfide bond is formed between native cysteine residues.

[0032] In some alternative embodiments, the at least one disulfide bond is formed between non-native cysteine residues.

[0033] In some embodiments, the non-native cysteine residues have been introduced by mutation or modification.

[0034] In some embodiments, the antigen binding protein comprises mutation or modification of two residues that introduces a side chain capable of acting as a ligand for a metal, wherein at least one residue is hydrophobic. In some embodiments, where the antigen binding protein comprises mutation or modification of two residues that introduces a side chain capable of acting as a ligand for a metal, one residue may be hydrophobic and the other non-hydrophobic (e.g. an amino acid with a polar uncharged side chain or an amino acid or an amino acid with an electrically charged side chain). In one embodiment, one residue is leucine and the other residue is glutamic acid.

[0035] In some embodiments, each of the two residues are hydrophobic or each of the two residues are hydrophilic.

[0036] In some embodiments, one of the two residues is hydrophobic and the other is hydrophilic, for example one residue is leucine and the other is glutamate.

[0037] In some embodiments, where the two residues are both hydrophobic or are both hydrophilic, each of the two residues are the same type of amino acid, for example both residues are leucine.

[0038] In some alternative embodiments, where the two residues both hydrophobic or are both hydrophilic, the two residues are different types of amino acid, for example one residue is leucine and the other is isoleucine.

[0039] In some embodiments, the two residues are located at any one or more residues, or residues equivalent to, 4 or 6 in SEQ ID NOs: 1 to 10.

[0040] In some embodiments, the radionuclide is coordinated by 3 residues of the antigen binding protein. Preferably, the metal is a radionuclide of Bi, As, Sb, Cr, Cu, Fe, Co or Mn.

[0041] In some embodiments, the radionuclide is coordinated by 4 residues of the antigen binding protein. In some embodiments, the radionuclide is a radionuclide of Al,Ge, Ga, Ti, Fe, Zr, Cu, Mn, Sc, Sr, Co, Rh, Ni, Pt, Tc, Re, In, Tl, Sn, Pd or Pb Preferably, the metal is a radionuclide of Al, Ge, Ga, Ti, Fe, Zr, Cu, Mn, Sc, Sr, Co, Rh, Ni, Pt, Tc, Re, In, Tl, Sn or Pb.

[0042] In some embodiments, the antigen binding protein is capable of binding more than one metal or radionuclide. Preferably, the antigen binding protein is capable of binding two metals or radionuclides. Preferably, the metal or radionuclide is in the 2+ oxidation state. Preferably, the metal or radionuclide is Pb, Fe, Cu, Mn, Sr, Co, Ni, Pt, Rh, Re, Pd or Sn. Optionally, the metal is Pb or a radionuclide of Pb.

[0043] In any aspect or embodiment, the ligand is not a chelator or chelating ligand. Preferably, the ligand is a monodentate ligand such that it bonds to the metal through a single atom or group as part of the side chain.

[0044] In some embodiments, the antigen binding protein binds to an antigen expressed on a cancer cell.

[0045] In some embodiments, the cancer cell is from a solid tumour.

[0046] In some alternative embodiments, the cancer cell is from a liquid or haematological tumour.

[0047] In some embodiments, the antigen is a tumour specific antigen.

[0048] In some embodiments, the antigen is a tumour associated antigen.

[0049] In some embodiments, the cancer cell is a blastoma (including medulloblastoma and retinoblastoma), sarcoma (including liposarcoma and synovial cell sarcoma), neuroendocrine tumours (including carcinoid tumours, gastrinoma, and islet cell cancer), mesothelioma, schwannoma (including acoustic neuroma), meningioma, adenocarcinoma, melanoma, leukaemia or lymphoid malignancies, lung cancer including small-cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer (including metastatic breast cancer), colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, analcarcinoma, penile carcinoma, testicular cancer, oesophageal cancer, tumours of the biliary tract, or head and neck cancer.

[0050] In some embodiments, the antigen is mesothelin (MSLN), prostate specific membrane antigen (PSMA), prostate stem cell antigen (PCSA), carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD123, CD133, CD138, epithelial glycoprotein (EGP 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), folate-binding protein (FBP), foetal acetylcholine receptor (AChR), folate receptor-α and β (FRα and β), Ganglioside G2 (GD2), Ganglioside G3 (GD3), human Epidermal Growth Factor Receptor 2 (HER-2 / ERB2), HER3, Epidermal Growth Factor Receptor vIII (EGFRvIII), ERB3, ERB4, human telomerase reverse transcriptase (hTERT), Interleukin-13 receptor subunit alpha-2 (IL-13Rα2), κ-light chain, kinase insert domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), L1 cell adhesion molecule (L1CAM), melanoma-associated antigen 1 (melanoma antigen family A1, MAGE-A1), Mucin 16 (Muc-16), Mucin 1 (Muc-1), NKG2D ligands, cancer-testis antigen NY-ESO-1, oncofoetal antigen (h5T4), tumour-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF- R2), Wilms’ tumour protein (WT-1), type 1 tyrosine-protein kinase transmembrane receptor (ROR1), B7-H3 (CD276), B7-H6 (Nkp30), Chondroitin sulfate proteoglycan-4 (CSPG4), DNAX Accessory Molecule (DNAM-1), Ephrin type A Receptor 2 (EpHA2), Fibroblast Associated Protein (FAP), Gpl00 / HLA-A2, Glypican 3 (GPC3), HA-1H, HERK-V, IL-11Ra, Latent Membrane Protein 1 (LMP1), Neural cell-adhesion molecule (N-CAM / CD56), or Trail Receptor (TRAIL R).

[0051] In one embodiment, the antigen binding protein comprises, consists essentially of or consist of one of more amino acid sequences of any one of SEQ ID NOs: 1 to 10, or a sequence at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to a sequence set forth in SEQ ID NOs: 1 to 10. Typically, the amino acid sequence does not include the HIS tag as shown in any one or more of SEQ ID NOs: 1 to 10.

[0052] In some embodiments, the protein is: i. a diabody;ii. a triabody; iii. a tetrabody; iv. a nanobody; v. a minibody; vi. a Fab; vii. a F(ab’)2; viii. a Fv; ix. a scFv; x. a bispecific antibody or other form of multispecific antibody (including a BiTE); or one of (i) to (x) linked to a constant region of an antibody, Fc or a heavy chain constant domain (CH) 2 and / or CH3.

[0053] In some embodiments, the antigen binding protein has been exposed to conditions that reduce the at least one disulfide bond.

[0054] In a further aspect, the present disclosure provides a metal coordinated antigen binding protein, wherein the antigen binding protein according to the present disclosure further comprises a metal coordinated by (a) reduced residues that when non-reduced form the disulphide bond, and (b) at least one mutated or modified residue that contains a side chain capable of acting as a ligand for the metal. In some embodiment, two metal atoms are coordinated. In some embodiments, two metal atoms are coordinated to the same site. In some alternative embodiments, two metal atoms are coordinated to different sites.

[0055] In a further aspect, the present disclosure provides a method of producing an antigen binding protein according to the present disclosure, the method comprising mutating or modifying at least one residue of an immunoglobulin domain comprising at least one disulfide bond to introduce a side chain capable of acting as a ligand for a metal.

[0056] In some embodiments of the method, the method comprises mutating or modifying two residues of an immunoglobulin domain comprising at least one disulfide bond to introduce two side chains capable of acting as a ligand for a metal.

[0057] In a further aspect, the present disclosure provides a method of producing a metal coordinated antigen binding protein according to the present disclosure, the method comprising: (a) contacting an antigen binding protein according to the present disclosure with a reducing agent such that at least one disulfide bond of the antigen binding protein is reduced to form a reduced antigen binding protein; and (b) contacting the reduced antigen binding protein with a metal source such that the reduced disulfide bond and at least one mutated or modified residue containing a side chain capable of acting as a ligand coordinate to the metal, thereby forming a metal coordinated antigen binding protein.

[0058] In some embodiments of the method of producing a metal coordinated antigen binding protein, the reducing agent is tris(2-carboxyethyl)phosphine (TCEP), β- mercaptoethanol (BME) or dithiothreitol (DTT). Preferably, the reducing agent is tris(2- carboxyethyl)phosphine (TCEP).

[0059] In some embodiments of the method of producing a metal coordinated antigen binding protein, step (a) is performed between about 15 °C below and about 15 °C above the denaturation midpoint temperature of the antigen binding protein. Preferably, between about 10 °C below and about 10 °C above the denaturation midpoint temperature of the antigen binding protein. More preferably, between about 8 to about 10 °C above or below the denaturation midpoint temperature of the antigen binding protein.

[0060] In some embodiments of the method of producing a metal coordinated antigen binding protein, step (a) further comprises a chemical denaturation agent. In some embodiments, the chemical denaturation agent is guanidinium hydrochloride or urea. Preferably, the chemical denaturation agent is guanidinium hydrochloride.

[0061] In some embodiments of the method of producing a metal coordinated antigen binding protein, the method is performed between pH 2 and pH 8. Preferably between pH 3 and pH 7.

[0062] In some embodiments of the method of producing a metal coordinated antigen binding protein, the concentration of the reducing agent is between about 1 mM to about 50 mM.

[0063] In some embodiments of the method of producing a metal coordinated antigen binding protein, the antigen binding protein and the metal source have a molar ratio of at least 1:1. Preferably, between 1:1 to 1:10, more preferably between 1:1 to 1:5.

[0064] In another aspect, the present disclosure provides an antigen binding protein comprising a helix bundle, the helix bundle comprising at least three helices, wherein the antigen binding protein comprises a mutation or modification of at least three residues, each mutation or modification introducing a side chain capable of acting as a ligand for a metal.

[0065] In some embodiments, the metal is a radionuclide.

[0066] In some embodiments, the radionuclide is an alpha particle emitter. In some alternative embodiments, the radionuclide is a beta particle emitter. In some alternative embodiments, the radionuclide is a positron emitter. In some alternative embodiments, the radionuclide is a gamma emitter. In some alternative embodiments, the radionuclide is an Auger electron emitter.

[0067] In some embodiments, the radionuclide is an isotope of Al, Ga, Cu, Mn, Sc, Sr, Co, Zr, Rb, Tc, Y, In, Lu, Sm, Ac, At, Pb, Bi, Fr, K, Ti, Nb, Ta, Re, Fe, Rh, Ni, Pt, Au, Tl, Ge, Sn, As, Po, Ir, La, Pr, Pm, Tb, Dy, Ho, Tm, Yb, Lu, Th, Er, Cr, Cs or Ra. Preferably the radionuclide is an isotope of Ga, In, Bi or Pb.

[0068] In some embodiments, the radionuclide is26Al,38K,66Ga,67Ga,68Ga,68Ge,61Cu,62Cu,64Cu,67Cu,51Mn,52Mn,44Sc,47Sc,45Ti,52Fe,57Ni,83Sr,89Sr,90Sr,51Cr,55Co,60Co, ,72As,89Zr,81Rb,82mRb,94mTc,99mTc,86Y,90Y,90Nb,103Pd,111In,110mIn,114mIn,140Pr,117mSn,131Cs,149Tb,152Tb,155Tb,161Tb,153Sm,166Ho,169Yb,169Er,177Lu,186Re,188Re,191mIr,192Ir,193mPt,195mPt,201Tl,225Ac,203Pb,212Pb,211At,211Bi,212Bi,213Bi,211Fr,211Po,213Po,223Ra,224Ra, and227Th.

[0069] In some alternative embodiments, the metal is a non-radioactive isotope.

[0070] In some embodiments, the non-radioactive isotope is an isotope of Al, Ga, Cu, Mn, Sc, Sr, Co, Zr, Rb, Tc, Y, In, Lu, Sm, Ac, At, Pb, Bi, Fr, K, Ti, Nb, Ta, Re, Fe, Rh, Ni,Pt, Au, Tl, Ge, Sn, As, Po, Ir, La, Pr, Pm, Tb, Dy, Ho, Tm, Yb, Lu, Th, Er, Cr, Cs or Ra. Preferably, the non-radioactive isotope is an isotope of Bi, Ga, In, As, Sb, Pb, Cu, Gd or Ba.

[0071] In some embodiments, the side chain capable of acting as a ligand for a metal comprises a monodentate ligand.

[0072] In some embodiments, the side chain capable of acting as a ligand for a metal comprises a sulphur or selenium atom.

[0073] In some embodiments, the mutation or modification introduces at least three cysteine residues, at least three selenocysteine residues, or a combination thereof.

[0074] In some embodiments, the mutation introduces at least two selenocysteine residues and at least one cysteine residue. In alternative embodiments, the mutation introduces at least two cysteine residues and at least one selenocysteine residue.

[0075] In some embodiments, the mutation introduces three cysteine residues.

[0076] In some embodiments, the alpha carbon atom of one of the at least three residues is located within 13 Å, or within 12 Å, or within 11 Å, or within 10 Å, or within 9 Å, or within 8 Å, or within 7 Å, or within 6 Å, or within 5 Å or within 4 Å, or within 3 Å of the alpha carbon atoms of the remaining modified or mutated residues. In some alternative embodiments, the alpha carbon atom of one of the at least three residues, is located between about 3 Å to about 13 Å, or between about 3 Å to about 10 Å, or between about 4 Å to about 10 Å, or between about 5 Å to about 10 Å of the alpha carbon atoms of the remaining modified or mutated residues.

[0077] In some embodiments, the at least one, two or all of the mutations or modifications are in at least one of the helices of the helix bundle. In one embodiment, each of the at least three mutations or modifications is in a different helix of the helix bundle.

[0078] In some embodiments, the three residues are located at, or residues equivalent to, 12, 34 and 41 in SEQ ID NOs: 11 to 14 (wherein the numbering does not include the HIS tag, or residues LQ following the HIS tag i.e. position 1 is alanine or valine). For example, equivalent to A12, L34, and S41.

[0079] In some embodiments, the at least three residues that are mutated or modified comprise a hydrophobic residue.

[0080] In some embodiments, the hydrophobic residue is leucine or alanine.

[0081] In alternative embodiments, the at least three residues that are mutated or modified comprise a hydrophilic residue.

[0082] In some embodiments, the hydrophilic residue is serine.

[0083] In some embodiments, the antigen binding protein comprises mutation or modification of three residues that introduce a side chain capable of acting as a ligand for a metal, wherein at least one residue is hydrophobic. In some embodiments, where the antigen binding protein comprises mutation or modification of three residues that introduce a side chain capable of acting as a ligand for a metal, one residue may be hydrophobic and the other non-hydrophobic (e.g. an amino acid with a polar uncharged side chain or an amino acid or an amino acid with an electrically charged side chain). In one embodiment, one residue is leucine, one residue is alanine, and the other residue is serine.

[0084] In some embodiments, each of the three residues are hydrophobic or each of the three residues are hydrophilic.

[0085] In some embodiments, two of the three residues are hydrophobic, and the other one is hydrophilic, for example the hydrophobic residues are leucine or alanine, and the hydrophilic residue is serine. In some alternative embodiments, two of the three residues are hydrophilic, and the other one is hydrophobic.

[0086] In some embodiments, where two of the three residues are both hydrophobic or are both hydrophilic, each of the two residues are the same type of amino acid, for example both residues are leucine or alanine.

[0087] In some alternative embodiments, where two of the three residues are both hydrophobic or are both hydrophilic, the two residues are different types of amino acid, for example one residue is leucine and the other is alanine.

[0088] In some embodiments, the metal is coordinated by at least 2 residues of the antigen binding protein. Preferably, the metal is coordinated by 3 residues of the antigenbinding protein. Preferably, the metal is a radionuclide of Bi, Ga, In, Pb, As, Sb, Cr, Cu, Fe, Co, or Mn. More preferably, the metal is a radionuclide of Bi, Ga, In, Pb.

[0089] In some embodiments, the metal is coordinated by 4 residues of the antigen binding protein. Preferably, the metal is a radionuclide of Al, Ge, Ga, Ti, Fe, Zr, Cu, Mn, Sc, Sr, Co, Rh, Ni, Pt, Tc, Re, In, Tl, Sn or Pb.

[0090] In any aspect or embodiment, the ligand is not a chelator or chelating ligand. Preferably, the ligand is a monodentate ligand such that it bonds to the metal through a single atom or group as part of the side chain.

[0091] In some embodiments, the antigen binding protein binds to an antigen expressed on a cancer cell.

[0092] In some embodiments, the cancer cell is from a solid tumour.

[0093] In some alternative embodiments, the cancer cell is from a liquid or haematological tumour.

[0094] In some embodiments, the antigen is a tumour specific antigen.

[0095] In some embodiments, the antigen is a tumour associated antigen.

[0096] In some embodiments, the cancer cell is a blastoma (including medulloblastoma and retinoblastoma), sarcoma (including liposarcoma and synovial cell sarcoma), neuroendocrine tumours (including carcinoid tumours, gastrinoma, and islet cell cancer), mesothelioma, schwannoma (including acoustic neuroma), meningioma, adenocarcinoma, melanoma, leukaemia or lymphoid malignancies, lung cancer including small-cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer (including metastatic breast cancer), colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, testicular cancer, oesophageal cancer, tumours of the biliary tract, or head and neck cancer.

[0097] In some embodiments, the antigen is mesothelin (MSLN), prostate specific membrane antigen (PSMA), prostate stem cell antigen (PCSA), carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD123, CD133, CD138, epithelial glycoprotein (EGP 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), folate-binding protein (FBP), foetal acetylcholine receptor (AChR), folate receptor-α and β (FRα and β), Ganglioside G2 (GD2), Ganglioside G3 (GD3), human Epidermal Growth Factor Receptor 2 (HER-2 / ERB2), HER3, Epidermal Growth Factor Receptor vIII (EGFRvIII), ERB3, ERB4, human telomerase reverse transcriptase (hTERT), Interleukin-13 receptor subunit alpha-2 (IL-13Rα2), κ-light chain, kinase insert domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), L1 cell adhesion molecule (L1CAM), melanoma-associated antigen 1 (melanoma antigen family A1, MAGE-A1), Mucin 16 (Muc-16), Mucin 1 (Muc-1), NKG2D ligands, cancer-testis antigen NY-ESO-1, oncofoetal antigen (h5T4), tumour-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF- R2), Wilms’ tumour protein (WT-1), type 1 tyrosine-protein kinase transmembrane receptor (ROR1), B7-H3 (CD276), B7-H6 (Nkp30), Chondroitin sulfate proteoglycan-4 (CSPG4), DNAX Accessory Molecule (DNAM-1), Ephrin type A Receptor 2 (EpHA2), Fibroblast Associated Protein (FAP), Gpl00 / HLA-A2, Glypican 3 (GPC3), HA-1H, HERK-V, IL-11Ra, Latent Membrane Protein 1 (LMP1), Neural cell-adhesion molecule (N-CAM / CD56), or Trail Receptor (TRAIL R).

[0098] In one embodiment, the antigen binding protein comprises, consists essentially of or consist of one of more amino acid sequences of any one of SEQ ID NOs: 11 to 14, or a sequence at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to a sequence set forth in SEQ ID NOs: 11 to 14. Typically, the amino acid sequence does not include the HIS tag or any amino acids as shown bold and underlined at the N-terminus in any one or more of SEQ ID NOs: 11 to 14.

[0099] In some embodiments, the protein is an affibody.

[0100] In some embodiments, the protein has a molecular weight from about 4 to about 10 kDa. Preferably from about 5 and to about 8 kDa. More preferably from about 6 to about 7 kDa.

[0101] In a further aspect, the present disclosure provides a metal coordinated antigen binding protein, wherein the antigen binding protein according to the present disclosure further comprises a metal coordinated by at least three mutated or modified residues that contain a side chain capable of acting as a ligand for the metal.

[0102] In a further aspect, the present disclosure provides a method of producing an antigen binding protein according to the present disclosure, the method comprising mutating or modifying at least two residues of a helix bundle comprising at least three helices, each mutation or modification to introduce a side chain capable of acting as a ligand for a metal.

[0103] In some embodiments of the method, the method comprises mutating or modifying at least three residues.

[0104] In some embodiments of the method, the method comprises mutating or modifying at least one residue in at least each of the three helices, or mutating or modifying at least two or three residues in the same helix.

[0105] In a further aspect, the present disclosure provides a method of producing a metal coordinated antigen binding protein according to the present disclosure, the method comprising contacting the antigen binding protein with a metal source such that the at least two mutated or modified residues containing a side chain capable of acting as a ligand coordinate to the metal, thereby forming a metal coordinated antigen binding protein.

[0106] In some embodiments, the method comprises contacting the antigen binding protein with a metal source such that the at least three mutated or modified residues containing a side chain capable of acting as a ligand coordinate to the metal, thereby forming a metal coordinated antigen binding protein.

[0107] In some embodiments of the method of producing a metal coordinated antigen binding protein, the contacting is performed between about 15 °C below and about 15 °C above the denaturation midpoint temperature of the antigen binding protein. Preferably, between about 10 °C below and about 10 °C above the denaturation midpoint temperature of the antigen binding protein. More preferably, between about 8 to about 10 °C above or below the denaturation midpoint temperature of the antigen binding protein.

[0108] In some alternative embodiments of the method of producing a metal coordinated antigen binding protein, the contacting is performed between about 0 °C and about 40 °C. Preferably, between about 15 °C and about 40 °C. More preferably, between about 15 °C and about 30 °C.

[0109] In some embodiments of the method of producing a metal coordinated antigen binding protein, the method is performed between pH 2 and pH 8. Preferably between pH 3 and pH 7.

[0110] In some alternative embodiments of the method of producing a metal coordinated antigen binding protein, the method is performed from pH 4 to pH 10, or between pH 4 and pH 10. Preferably from pH 5 to pH 9, or between pH 5 and pH 9. More preferably from pH 6 to pH 8, or between pH 6 and pH 8.

[0111] In some embodiments of the method of producing a metal coordinated antigen binding protein, the antigen binding protein and the metal source have a molar ratio of at least 1:1. Preferably, between 1:1 to 1:10, more preferably between 1:1 to 1:5.

[0112] In a further aspect, the present disclosure provides a method of making the antigen binding protein of the present disclosure, comprising cell culture expression, cell- free protein synthesis (using ribosomes outside cells) or solid-phase peptide synthesis.

[0113] In a further aspect, the present disclosure provides a nucleic acid comprising a nucleotide sequence encoding an antigen binding protein of the present disclosure.

[0114] In a further aspect, the present disclosure provides a vector comprising the nucleic acid of the present disclosure.

[0115] In some embodiments of the vector, the vector is an expression vector.

[0116] In a further aspect, the present disclosure provides a cell comprising a nucleic acid of the present disclosure or a vector of the present disclosure.

[0117] In a further aspect, the present disclosure provides a composition comprising an antigen binding protein of the present disclosure, or a metal coordinate antigen binding protein of the present disclosure.

[0118] In a further aspect, the present disclosure provides a pharmaceutical comprising an antigen binding protein of the present disclosure, or a metal coordinated antigenbinding protein of the present disclosure, and a pharmaceutically acceptable diluent, carrier or excipient.

[0119] In a further aspect, the present disclosure provides a method of treating cancer in an individual in need thereof, the method comprising administering a metal coordinated antigen binding protein of the present disclosure to the individual, thereby treating cancer in the individual.

[0120] In a further aspect, the present disclosure provides the use of a metal coordinated antigen binding protein of the present disclosure in the manufacture of a medicament for treating cancer in an individual in need thereof.

[0121] In a further aspect, the present disclosure provides a metal coordinated antigen binding protein of the present disclosure for use in the treatment of cancer.

[0122] In some embodiments of the method, use or metal coordinated antigen of the present disclosure, the cancer is a blastoma (including medulloblastoma and retinoblastoma), sarcoma (including liposarcoma and synovial cell sarcoma), neuroendocrine tumours (including carcinoid tumours, gastrinoma, and islet cell cancer), mesothelioma, schwannoma (including acoustic neuroma), meningioma, adenocarcinoma, melanoma, leukaemia or lymphoid malignancies, lung cancer including small-cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer (including metastatic breast cancer), colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, testicular cancer, oesophageal cancer, tumours of the biliary tract, or head and neck cancer.

[0123] In a further aspect, the present disclosure provides a method of diagnosing, monitoring or prognosing a cancer in an individual in need thereof, the method comprising: (a) administering a metal coordinated antigen binding protein of the present disclosure to the individual, wherein the metal is a radionuclide; and(b) detecting the radionuclide in the individual; whereby detection of the radionuclide enables an image of the cancer (if present) to be created to thereby diagnose, monitor or prognose the cancer.

[0124] In a further aspect, the present invention provides a method for in vivo imaging or detection of a cancer in a subject, wherein the method comprises: - administering to a subject in need thereof, a metal coordinated antigen binding protein of the present disclosure, wherein the metal is a radionuclide, - detecting the radionuclide in the individual.

[0125] In these aspects, the detection of the radionuclide, may be compared to a background or standard level to determine the presence of the cancer, thereby imaging or detecting the cancer in the subject.

[0126] In a further aspect, the present disclosure provides a theranostic method comprising: (1) administering a diagnostically-effective amount a metal coordinated antigen binding protein of the present disclosure to a patient or subject, and (2) administering a therapeutically-effective amount of a metal coordinated antigen binding protein of the present disclosure to the patient or subject in need thereof.

[0127] In some embodiments of the theranostic method, the metal coordinated antigen binding protein in step 1 and step 2 is the same.

[0128] In another aspect, the present disclosure also provides for a metal coordinated antigen binding protein of the present disclosure, or a composition comprising the same, as described herein, for use in a method of: - in vivo imaging or detection of a cancer; - diagnosis of a cancer; - producing an image of a cancer; - classifying a cancer as sensitive to treatment;- classifying or selecting a patient for eligibility for cancer therapy; - treating a cancer patient or cohort of cancer patients; wherein the methods are as described herein.

[0129] In another aspect, the present disclosure also provides for a use of a metal coordinated antigen binding protein of the present disclosure, in the manufacture of a composition or medicament for: - in vivo imaging or detection of a cancer; - diagnosis of a cancer; - producing an image of a cancer; - classifying a cancer as sensitive to treatment with an inhibitor; - classifying or selecting a patient for eligibility for cancer therapy with an inhibitor; - treating a cancer patient or cohort of cancer patients with an inhibitor; wherein the methods are as described herein.

[0130] In any aspect or embodiment, the antigen binding protein is recombinant, synthetic, isolated, purified, or substantially purified.

[0131] As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps.

[0132] Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings. Brief description of the drawings

[0133] Figure 1. (A) Scheme showing the conventional strategy to label nanobodies with bifunctional linkers. (B) Schematic showing a metal-binding site directly inside the nanobody. (C) Diagram of the structure of a wildtype nanobody (anti-SARS-CoV-2 spike nanobody mNb6; pdb: 7KKJ). The conserved disulfide bond (C22, C96) and the threecomplementarity-determining regions (CDRs) are indicated. (D) Diagram showing that a single mutation, L4C, generates a triple cysteine motif. (E) Diagram showing that an additional mutation, E6C, generates a quadruple cysteine motif. A single E6C mutations generates an alternative triple cysteine motif. (F) Reaction scheme showing bismuth(III) binding to the primed triple cysteine motif. (G) Reaction scheme showing indium(III) and gallium(III) binding to the primed quadruple cysteine motif.

[0134] Figure 2. (A–C) Spectra showing native mass spectrometry (MS) performed in 100 mM ammonium acetate pH 7.0 for mNb6-3C, mNb6-3C* and mNb6-4C before and after modification with bismuth (Bi), gallium (Ga) or indium (In). Nanobodies were reduced with 25 mM TCEP for 15 minutes at 50 °C (mNb6-3C and mNb6-3C*) or 60 °C (mNb6- 4C) prior addition of the trivalent metal. (D) Diagram showing optimized conditions for uptake of Ga, In and Bi by nanobodies containing two, three or four cysteine residues after reduction with 25 mM TCEP for 15 minutes at 55 °C (mNb6), 50 °C (mNb6-3C and mNb6-3C*) and 60 °C (mNb6-4C). Uptake determined by native MS is indicated in %. (E) SDS-PAGE of mNb6 nanobody constructs after periplasmic expression from E. coli and His-tag affinity purification. (F) Graph showing the time-dependent reduction and modification of mNb6-3C with 25 mM TCEP and 5 equiv. Bi (gastrodenol) at room temperature (RT) or 50 °C. The metal was either co-incubated or added after the reduction period as indicated. Bismuth uptake was monitored by native MS. (G) Graph showing the bismuth uptake of mNb6-3C monitored by native MS after incubation for 15 min at 50 °C with 5 equiv. gastrodenol depending on TCEP concentration and pH.

[0135] Figure 3: 800 MHz [15N,1H]-HSQC NMR spectrum of a 150 mM solution of15N- mNb6-3C in 20 mM MES pH 7.5, 150 mM NaCl, 10% D2O. Intact MS spectrum of the protein is shown in the inset.

[0136] Figure 4: 800 MHz [15N,1H]-HSQC NMR spectrum of a 150 mM solution of15N- mNb6-3C-Bi in 20 mM MES pH 7.5, 150 mM NaCl, 10 mM TCEP, 10% D2O. Native MS spectrum of the protein is shown in the inset.

[0137] Figure 5: Thermal denaturation curves determined by circular dichroism (CD) spectroscopy in 20 mM phosphate buffer pH 7.4 for mNb6 (A) (Tm = 65 °C), mNb6-3C-Bi (B) (Tm = 65 °C), and mNb6-3C*-Bi (C) (Tm = 62 °C).

[0138] Figure 6: Thermal denaturation curves determined by circular dichroism (CD) spectroscopy in 20 mM phosphate buffer pH 7.4 for mNb6-4C (A) (Tm = 65 °C), mNb6- 4C-Ga (B) (Tm = 56 °C), and mNb6-4C-In (C) (Tm = 51 °C).

[0139] Figure 7. Intact MS of mNb6 (A), mNb6-3C (B), and mNb6-4C (C) after time- dependent reduction and modification with 25 mM TCEP and 5 equiv. gastrodenol at room temperature (RT). The m / z ratios indicate the presence of an intact disulfide bond even after TCEP treatment. Species of higher m / z ratio that correspond to a partially cleaved N-terminal leader sequence are indicated with an asterisk (*).

[0140] Figure 8. Native MS of mNb6-3C after treatment with 1 M (A) and 6 M (B) guanidinium hydrochloride at room temperature for 20 minutes in the presence of 25 mM TCEP (pH 3), followed by addition of 5 equiv. BiBr3.

[0141] Figure 9. Graph showing the uptake of Bi(III) by mNb6-3C from BiBr3 (DMSO stock) after reduction with 25 mM TCEP at pH 3 and 50 °C heat shock over different durations up to 60 minutes.

[0142] Figure 10. Optimization of Bi(III) uptake by mNb6-3C using BiBr3. Native mass spectra of samples in triplicate after 1 min (A to C), 5 min (D to F), 10 min (G to I), 15 min (J to L), 30 min (M to O), 45 min (P to R), and 60 min (S to U) incubation of the protein at 50 °C in 25 mM TCEP at pH 3.0, followed by addition of BiBr3 (DMSO stock).

[0143] Figure 11. Circular dichroism (CD) spectra of mNb6 (A), mNb6-3C (B), mNb6- 3C-Bi (C), mNb6-3C* (D), and mNb6-3C*-Bi (E) measured at 15 ^^M in 20 mM phosphate buffer, pH 7.5.

[0144] Figure 12. (A) Graph showing the retention of Bi(III), In(III) and Ga(III) in engineered mNb6 variants after two weeks of storage at 4 °C in 100 mM ammonium acetate, pH 7.0. (B) Graph showing the uptake of Bi(III), In(III) and Ga(III) by engineered mNb6 variants two weeks after reduction and storage at 4 °C in 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 25 mM TCEP. (C, D) Graphs showing the retention of Bi(III), In(III) and Ga(III) bound to engineered mNb6 variants (50 mM for C, 25 mM for D) in presence of glutathione (C) and apo-transferrin (D) after 1 h incubation at 25 °C in 100 mM ammonium acetate, pH 7.0. (E–G) Original and deconvoluted high-resolution native MS spectra of mNb6-3C-Bi, mNb6-4C-In and mNb6-4C-Ga after 18 h dialysis at 4 °C in 20 mM HEPES,pH 7.8, 150 mM NaCl. Calculated m / z ratios for the indicated species are given in brackets.

[0145] Figure 13. Uptake of In(III), Ga(III) and Bi(III) by mNb6 determined using native MS after incubation at specified temperature and time in 25 mM TCEP at pH 3. (A) In(III), 50 °C, 15 min, (B) In(III), 55 °C, 15 min, (C) In(III), 60 °C, 15 min, (D) In(III), 50 °C, 90 min, (E) In(III), 55 °C, 90 min, (F) In(III), 60 °C, 90 min, (G) Ga(III), 50 °C, 15 min, (H) Ga(III), 55 °C, 15 min, (I) Ga(III), 60 °C, 15 min, (J) Ga(III), 50 °C, 90 min, (K) Ga(III), 55 °C, 90 min, (L) Ga(III), 60 °C, 90 min, (M) Bi(III), 50 °C, 15 min, (N) Bi(III), 55 °C, 15 min, (O) Bi(III), 60 °C, 15 min, (P) Bi(III), 50 °C, 90 min, (Q) Bi(III), 55 °C, 90 min, (R) Bi(III), 90 °C 90 min.

[0146] Figure 14. Heatmap of In(III), Ga(III) and Bi(III) uptake by mNb6 following incubation at indicated temperatures for 15 (A) and 90 minutes (B). Uptake determined by native MS is indicated in %. Conditions where excessive protein precipitation was observed are indicated by an asterisk (*).

[0147] Figure 15. Uptake of In(III), Ga(III) and Bi(III) by mNb6-3C determined using native MS after incubation at specified temperature and time in 25 mM TCEP at pH 3. (A) In(III), 50 °C, 15 min, (B) In(III), 55 °C, 15 min, (C) In(III), 60 °C, 15 min, (D) Ga(III), 50 °C, 15 min, (E) Ga(III), 55 °C, 15 min, (F) Ga(III), 60 °C, 15 min, (G) Bi 50(III), °C, 15 min, (H) Bi(III), 55 °C, 15 min, (I) Bi(III), 60 °C, 15 min, (J) Bi(III), 50 °C, 90 min, (K) Bi(III), 55 °C, 90 min, (L) Bi(III), 60°C, 90 min. The asterisks (*) indicate unknown protein impurities.

[0148] Figure 16. Heatmap of In(III), Ga(III) and Bi(III) uptake by mNb6-3C following incubation at indicated temperatures for 15 (Left) and 90 minutes (Right). Uptake determined by native MS is indicated in %. Conditions where excessive protein precipitation was observed are indicated by an asterisk (*).

[0149] Figure 17. Uptake of In(III), Ga(III) and Bi(III) by mNb6-4C determined using native MS after incubation at specified temperature and time in 25 mM TCEP at pH 3. (A) In(III), 50 °C, 15 min, (B) In(III), 55 °C, 15 min, (C) In(III), 60 °C, 15 min, (D) In(III), 50 °C, 90 min, (E) In(III), 55 °C, 90 min, (F) In(III), 60 °C, 90 min, (G) Ga(III), 50 °C, 15 min, (H) Ga(III), 55 °C, 15 min, (I) Ga(III), 60 °C, 15 min, (J) Ga(III), 50 °C, 90 min, (K) Ga(III), 55 °C, 90 min, (L) Ga(III), 60 °C, 90 min, (M) Bi(III), 50 °C, 15 min, (N) Bi(III),55 °C, 15 min, (O) Bi(III), 60 °C, 15 min, (P) Bi(III), 50 °C, 90 min, (Q) Bi(III), 55 °C, 90 min, (R) Bi(III), 90 °C 90 min.

[0150] Figure 18. Heatmap of In(III), Ga(III) and Bi(III) uptake by mNb6-4C following incubation at indicated temperatures for 15 (A) and 90 minutes (B). Uptake determined by native MS is indicated in %.

[0151] Figure 19. Native MS of mNb6-3C* after incubation at 50 °C in 25 mM TCEP, pH 3.0 for 15 minutes, followed by addition of In(III) (A) and Ga(III) (B).

[0152] Figure 20. Representative native MS showing quantitative Bi(III) uptake by mNb6-3C after incubation with 1 mM TCEP, pH 6.5 (A), 5 mM TCEP, pH 4 (B), 10 mM TCEP, pH 3.5 (C) and 25 mM TCEP, pH 3 (D) for 15 minutes at 50 °C.

[0153] Figure 21. Graph showing the stability of mNb6-3C in complex with Bi(III), In(III) or Ga(III) at 4 °C over a duration of 14 days. Ratio of bound metal was determined by native MS in triplicate.

[0154] Figure 22. Native MS for Bi(III) bound mNb6-3C in triplicate stored at 4 °C for 0 days (A-C), 1 day (D-F), 2 days (G-I), 7 days (J-L) and 14 days (M-O).

[0155] Figure 23. Native MS for In(III) bound mNb6-3C in triplicate stored at 4 °C for 0 days (A-C), 1 day (D-F), 2 days (G-I), 7 days (J-L) and 14 days (M-O).

[0156] Figure 24. Native MS for Ga(III) bound mNb6-3C in triplicate stored at 4 °C for 0 days (A-C), 1 day (D-F), 2 days (G-I), 7 days (J-L) and 14 days (M-O).

[0157] Figure 25. Graph showing the stability of mNb6-4C in complex with Bi (III), In(III) or Ga(III) at 4 °C over a duration of 14 days. Ratio of bound metal was determined by native MS in triplicate.

[0158] Figure 26. Native MS for Bi(III) bound mNb6-4C in triplicate stored at 4 °C for 0 days (A-C), 1 day (D-F), 2 days (G-I), 7 days (J-L) and 14 days (M-O).

[0159] Figure 27. Native MS for In(III) bound mNb6-4C in triplicate stored at 4 °C for 0 days (A-C), 1 day (D-F), 2 days (G-I), 7 days (J-L) and 14 days (M-O).

[0160] Figure 28. Native MS for Ga(III) bound mNb6-4C in triplicate stored at 4 °C for 0 days (A-C), 1 day (D-F), 2 days (G-I), 7 days (J-L) and 14 days (M-O).

[0161] Figure 29. Native MS (triplicate) for ‘primed’ (pre-reduced) mNb6-3C at different time points after reduction and storage at 4 °C before addition of Bi(III). Day 0 (A to C), day 1 (D to F), day 2 (G to I), day 3 (J to L), day 7 (M to O), day 14 (P to R).

[0162] Figure 30. Native MS (triplicate) for ‘primed’ (pre-reduced) mNb6-4C at different time points after reduction and storage at 4 °C prior to addition of In(III), Ga(III) or Bi(III). Day 0, In(III) (A to C), day 14, In(III) (D to F), day 0, Ga(III) (G to I), day 14, Ga(III) (J to L), day 0, Bi(III) (M to O), day 14, Bi(III) (P to R).

[0163] Figure 31. SPR sensograms (duplicate) showing the binding response of the SARS-CoV-2 receptor binding domain (RBD) to mNb6-3C-Bi (A), mNb6-4C-In (B), and mNb6-4C-Ga (C) immobilised on a CM5 chip in single kinetics mode. Rounded concentrations (in nM) are indicated above the response peaks.

[0164] Figure 32. SDS PAGE of cross-linked metal nanobody-RBD complexes. Metal- bound mNb6 constructs were incubated 1:1 with the SARS-CoV-2 spike RBD for 60 minutes at 4 °C and subsequently crosslinked by incubating with 100 equivalents of disuccinimidyl glutarate (DSG) at 4 °C for 60 minutes. The samples were then loaded and run on an SDS PAGE following usual procedures. The native MS spectra confirm full metal coordination prior to incubation. (a) indicates mNb6:RBD complex, (b) RBD and (c) metal-bound mNb6 construct.

[0165] Figure 33. (A–C) Binding curves and dissociation constants of the receptor binding domain (RBD) of SARS-CoV-2 and mNb6-3C-Bi (A), mNb6-4C-In (B) and mNb6- 4C-Ga (C) determined by surface plasmon resonance (SPR). (D) Native PAGE of Lam2- Bi nanobody and mCherry (mCh). Excised gel parts are indicated by boxes and their bismuth content, determined by ICP-MS, is indicated as parts per billion (ppb). Two bands for the complex are a result of mNb6-3C being expressed with (a) and without (b) part of its N-terminal leader sequence. (E) Isothermal titration calorimetry (ITC) of Lam2-Bi and mCh with indicated dissociation constant (KD). (F) Superimposed native MS spectra of Lam2-3C and Lam2-3C-Bi. Species of higher m / z ratio that correspond to a partially cleaved N-terminal leader sequence are indicated with an asterisk (*). (G, H) Superimposed intact MS spectra of 2Rs15d-2A-3C (G) and 2Rs15d-5C (H) in presence and absence of Bi(III).

[0166] Figure 34: Thermal denaturation curves determined by CD spectroscopy in 20 mM phosphate buffer pH 7.4 for Lam2 (A) (Tm = 70 °C) and Lam2-3C-Bi (B) (Tm = 66 °C).

[0167] Figure 35: Thermal denaturation curves determined by CD spectroscopy in 20 mM phosphate buffer pH 7.4 for 2Rs15d-2A (A) (Tm = 61 °C), 2Rs15d-2A-3C-Bi (B) (Tm = 58 °C), and 2Rs15d-5C-Bi (C) (Tm = 61 °C).

[0168] Figure 36. SDS-PAGE and Intact protein MS confirming successful periplasmic expression of Lam2 (A) and Lam2-3C (B) in E. coli, Species of higher m / z ratio that correspond to a partially cleaved N-terminal leader sequence are indicated with an asterisk (*). SDS-PAGE marker proteins and their masses in kDa are shown.

[0169] Figure 37. Circular dichroism (CD) spectra of Lam2 (A) and Lam2-3C-Bi (B) measured at 15 ^^M in 20 mM phosphate buffer, pH 7.5.

[0170] Figure 38. Uptake of Bi(III) by Lam2-3C in 25 mM TCEP at pH 3.0 following co- incubation with Bi(III) at 58 °C over varying durations.

[0171] Figure 39. Optimization of Bi(III) uptake by Lam2-3C. Native MS of samples in 25 mM TCEP, pH 3.0 after 0 min (A), 5 min (B), 10 min (C), 15 min (D), 30 min (E), 60 min (F), and 120 min (G) co-incubation at 58 °C with Bi(III).

[0172] Figure 40. Isothermal titration calorimetry (ITC) of Lam2 and mCherry with indicated dissociation constant (KD).

[0173] Figure 41. Circular dichroism (CD) spectra of 2Rs15d-5C (A), 2Rs15d-5C-Bi (B), 2Rs15d-2A-3C (C), and 2Rs15d-2A-3C-Bi (D) measured at 15 ^^M in 20 mM phosphate buffer, pH 7.5.

[0174] Figure 42. SDS-PAGE and intact protein MS confirming successful periplasmic expression of 2Rs15d-2C (A), 2Rs15d-2A-3C (B) and 2Rs15d-5C (C) in E. coli.

[0175] Figure 43: Native mass spectrometry (MS) of mNb6-3C (A) and mNb6-4C (C, F) in 100 mM ammonium acetate pH 7.0 before modification with Pb(II). Nanobodies were reduced with 25 mM TCEP for 5 min at 50 °C (B) or 5 min (D), 15 min (E) and 60 min at 60 °C (G) prior to addition of lead(II) nitrate [Pb(NO3)2].

[0176] Figure 44: Circular dichroism (CD) spectra of mNb6-3C-Pb (A) and mNb6-4C- 2Pb (B) in 20 mM phosphate buffer, pH 7.5. Thermal denaturation curves determined byCD in 20 mM phosphate buffer, pH 7.5 of mNb6-3C-Pb (C, Tm = 49 °C) and mNb6-4C- 2Pb (D, Tm = 53 °C). Binding curves and dissociation constants of the receptor binding domain (RBD) of SARS-CoV-2 and mNb6-3C-Pb (E), mNb6-4C-2Pb (F) determined by surface plasmon resonance (SPR).

[0177] Figure 45. Expression of affibodies and optimisation of metal uptake reactions. (A) SDS-PAGE of TNF-^^ protein and ZTNF-alpha1 affibody constructs after recombinant expression from E. coli and His6-tag affinity purification (WT: wildtype, 3C: A12C / L34C / S41C mutant). (B) SDS-PAGE of ZHER2:2891 affibody constructs after recombinant expression from E. coli and His6-tag affinity purification (WT: wildtype, 3C: A12C / L34C / S41C mutant). (C) Uptake of Bi(III) by ZHER2:2891-3C after treatment with 50 mM TCEP and gastrodenol (5 eq.) at different pH, temperature and time points as indicated. Bi(III) uptake was monitored by native mass spectrometry (MS) (n=1). The conditions of pH 7.5 and 25 °C were used for all subsequent experiments.

[0178] Figure 46: Native MS of the ZHER2:2891 wildtype (with His6-tag) in 100 mM ammonium acetate pH 7.0 indicate no uptake of Bi(III) (A,B), In(III) (C, D), Ga(III) (E, F) or Pb(II) (G,H) after exposure to gastrodenol (5 eq.), indium(III) chloride (5 eq.), gallium(III) nitrate hydrate (5 eq.) or lead(II) nitrate (5 eq) under the optimised conditions.

[0179] Figure 47: Native MS of the ZTNF-alpha1 wildtype (with His6-tag) in 100 mM ammonium acetate pH 7.0 indicate no uptake of Bi(III) (A,B), In(III) (C, D), Ga(III) (E, F) or Pb(II) (G,H) after exposure to gastrodenol (5 eq.), indium(III) chloride (5 eq.), gallium(III) nitrate hydrate (5 eq.) or lead(II) nitrate (5 eq) under the optimised conditions.

[0180] Figure 48. Affibody design and metal binding. (A) Visualisation of the structure of a wild type affibody according to one embodiment (ZHER2:2891) with exemplary residues involved in target binding highlighted. (B) Visualisation of the structure of a modified affibody according to one embodiment, with mutations at A12C, L34C and S41C to create a triple cysteine motif, avoiding residues essential for target binding. (C) Scheme showing Bi(III), In(III), Ga(III) and Pb(II) binding to the primed triple cysteine motif at pH 7.5, 25 °C.

[0181] Figure 49. Retention of Bi(III), In (III), Ga(III) and Pb(II) in engineered ZHER2:2891- 3C after seven days of storage at 4 °C in 100 mM ammonium acetate, pH 7.0 (n = 3 ^ SEM).

[0182] Figure 50: Native MS of Bi(III)-bound ZHER2:2891-3C in triplicate stored in 100 mM ammonium acetate pH 7 at 4 °C for 0 days (A-C), 1 day (D-F), and 7 days (G-I).

[0183] Figure 51: Native MS of In(III)-bound ZHER2:2891-3C in triplicate stored in 100 mM ammonium acetate pH 7 at 4 °C for 0 days (A-C), 1 day (D-F), and 7 days (G-I).

[0184] Figure 52: Native MS of Ga(III)-bound ZHER2:2891-3C in triplicate stored in 100 mM ammonium acetate pH 7 at 4 °C for 0 days (A-C), 1 day (D-F), and 7 days (G-I).

[0185] Figure 53: Native MS of Pb(II)-bound ZHER2:2891-3C in triplicate stored in 100 mM ammonium acetate pH 7 at 4 °C for 0 days (A-C), 1 day (D-F), and 7 days (G-I).

[0186] Figure 54: Metal retention in Bi(III)-bound ZHER2:2891-3C in the presence of increasing equivalents of EDTA (A) and GSH (B), Ga(III)-bound ZHER2:2891-3C in the presence of increasing equivalents of EDTA (C) and GSH (D), In(III)-bound ZHER2:2891-3C in the presence of increasing equivalents of EDTA (E) and GSH (F), and Pb(II)-bound ZHER2:2891-3C in the presence of increasing equivalents of EDTA (G) and GSH (H) incubated in 100 mM ammonium acetate pH 7, at 25°C for 1 hr (n = 2, ^ SEM).

[0187] Figure 55: Native MS of Bi(III)-bound ZHER2:2891-3C in duplicate after incubation for 1 hour at 25 °C in the presence of 0 eq (A, B), 1 eq (C, D), 10 eq (E,F), 20 eq (G, H), 50 eq (I, J) or 100 eq (K, L) EDTA in 100 mM ammonium acetate pH 7.

[0188] Figure 56: Native MS of Bi(III)-bound ZHER2:2891-3C in duplicate after incubation for 1 hour at 25 °C in the presence of 0 eq (A, B), 1 eq (C, D), 10 eq (E,F), 20 eq (G, H), 50 eq (I, J) or 100 eq (K, L) GSH in 100 mM ammonium acetate pH 7.

[0189] Figure 57: Native MS of Ga(III)-bound ZHER2:2891-3C in duplicate after incubation for 1 hour at 25 °C in the presence of 0 eq (A, B), 1 eq (C, D), 10 eq (E,F), 20 eq (G, H), 50 eq (I, J) or 100 eq (K, L) EDTA in 100 mM ammonium acetate pH 7.

[0190] Figure 58: Native MS of Ga(III)-bound ZHER2:2891-3C in duplicate after incubation for 1 hour at 25 °C in the presence of 0 eq (A, B), 1 eq (C, D), 10 eq (E,F), 20 eq (G, H), 50 eq (I, J) or 100 eq (K, L) GSH in 100 mM ammonium acetate pH 7.

[0191] Figure 59: Native MS of In(III)-bound ZHER2:2891-3C in duplicate after incubation for 1 hour at 25 °C in the presence of 0 eq (A, B), 1 eq (C, D), 10 eq (E,F), 20 eq (G, H), 50 eq (I, J) or 100 eq (K, L) EDTA in 100 mM ammonium acetate pH 7.

[0192] Figure 60: Native MS of In(III)-bound ZHER2:2891-3C in duplicate after incubation for 1 hour at 25 °C in the presence of 0 eq (A, B), 1 eq (C, D), 10 eq (E,F), 20 eq (G, H), 50 eq (I, J) or 100 eq (K, L) GSH in 100 mM ammonium acetate pH 7.

[0193] Figure 61: Native MS of Pb(II)-bound ZHER2:2891-3C in duplicate after incubation for 1 hour at 25 °C in the presence of 0 eq (A, B), 1 eq (C, D), 10 eq (E,F), 20 eq (G, H), 50 eq (I, J) or 100 eq (K, L) EDTA in 100 mM ammonium acetate pH 7. EDTA- ZHER2:2891- 3C adducts indicated by an asterisk (*).

[0194] Figure 62: Native MS of Pb(II)-bound ZHER2:2891-3C in duplicate after incubation for 1 hour at 25 °C in the presence of 0 eq (A, B), 1 eq (C, D), 10 eq (E,F), 20 eq (G, H), 50 eq (I, J) or 100 eq (K, L) GSH in 100 mM ammonium acetate pH 7.

[0195] Figure 63. Stability of metal-bound affibodies. (A) Retention of Bi(III) in engineered ZHER2:2891-3C (50 ^M) in the presence of glutathione (GSH) and EDTA after 1 h incubation in 100 mM ammonium acetate, pH 7.0 at 25 °C (n = 3 ^ SEM). (B) Retention of Bi(III) in engineered ZHER2:2891-3C (50 ^M) in the presence of 100 eq. of glutathione and EDTA after 1 h incubation in 100 mM ammonium acetate, pH 7.0 at 25 °C and subsequent storage at 4 °C for up to 14 days (n = 2 ^ SEM).

[0196] Figure 64: Native MS of Bi(III)-bound ZHER2:2891-3C in duplicate after incubation for 1 hour at 25 °C in the presence of 100 eq EDTA in 100 mM ammonium acetate pH 7 then subsequent storage at 4 °C for 1 day (A, B), 5 days (C, D), 8 days (E,F) and 14 days (G, H).

[0197] Figure 65: Native MS of Bi(III)-bound ZHER2:2891-3C in duplicate after incubation for 1 hour at 25 °C in the presence of 100 eq GSH in 100 mM ammonium acetate pH 7 then subsequent storage at 4 °C for 1 day (A, B), 5 days (C, D), 8 days (E,F) and 14 days (G, H).

[0198] Figure 66: Native MS of Bi(III)-bound ZHER2:2891-3C after incubation for 1 hour at 25 °C in the presence of 100 eq EDTA in 100 mM ammonium acetate pH 7. Collected with an orthogonal MS source (Synapt Native MS as described in the methods and materials).

[0199] Figure 67: Metal retention in Bi(III)-bound ZHER2:2891-3C in the presence of increasing equivalents of DTPA incubated in 100 mM ammonium acetate pH 7, at 25°C for 1 hr (n = 2, ^ SEM).

[0200] Figure 68: Native MS of Bi(III)-bound ZHER2:2891-3C in duplicate after incubation for 1 hour at 25°C in the presence of 0 eq (A, B), 1 eq (C, D), 10 eq (E,F) , 20 eq (G, H), 50 eq (I, J) or 100 eq (K, L) DTPA in 100 mM ammonium acetate pH 7.

[0201] Figure 69: Uptake of Bi(III) (A), In(III) (B), Ga(III) (C) and Pb(II) (D) by ZHER2:2891- 3C in triplicate determined by native MS after incubation at 25 °C in 20 mM Tris, pH 7.5, 150 mM NaCl, 50 mM TCEP across various times (n = 3, ± SEM).

[0202] Figure 70: Uptake of Bi(III) by ZHER2:2891-3C in triplicate determined by native MS after incubation at 25 °C in 20 mM Tris, pH 7.5, 150 mM NaCl, 50 mM TCEP for 5 min (A-C), 15 min (D-F), 30 min (G-I) and 60 min (J-L).

[0203] Figure 71: Uptake of In(III) by ZHER2:2891-3C in triplicate determined by native MS after incubation at 25 °C in 20 mM Tris, pH 7.5, 150 mM NaCl, 50 mM TCEP for 5 min (A-C), 15 min (D-F), 30 min (G-I) and 60 min (J-L).

[0204] Figure 72: Uptake of Ga(III) by ZHER2:2891-3C in triplicate determined by native MS after incubation at 25 °C in 20 mM Tris, pH 7.5, 150 mM NaCl, 50 mM TCEP for 5 min (A-C), 15 min (D-F), 30 min (G-I) and 60 min (J-L).

[0205] Figure 73: Uptake of Pb(II) by ZHER2:2891-3C in triplicate determined by native MS after incubation at 25 °C in 20 mM Tris, pH 7.5, 150 mM NaCl, 50 mM TCEP for 5 min (A-C), 15 min (D-F), 30 min (G-I) and 60 min (J-L).

[0206] Figure 74: Uptake of Bi(III) (A), In(III) (B), Ga(III) (C) and Pb(II) (D) by ZTNF-alpha1- 3C in triplicate determined by native MS after incubation at 25 °C in 20 mM Tris, pH 7.5, 150 mM NaCl, 50 mM TCEP across various times (n = 3, ± SEM).

[0207] Figure 75: Uptake of Bi(III) by ZTNF-alpha1-3C in triplicate determined by native MS after incubation at 25 °C in 20 mM Tris, pH 7.5, 150 mM NaCl, 50 mM TCEP for 5 min (A-C), 15 min (D-F), 30 min (G-I) and 60 min (J-L).

[0208] Figure 76: Uptake of In(III) by ZTNF-alpha1-3C in triplicate determined by native MS after incubation at 25 °C in 20 mM Tris, pH 7.5, 150 mM NaCl, 50 mM TCEP for 5 min (A-C), 15 min (D-F), 30 min (G-I) and 60 min (J-L).

[0209] Figure 77: Uptake of Ga(III) by ZTNF-alpha1-3C in triplicate determined by native MS after incubation at 25 °C in 20 mM Tris, pH 7.5, 150 mM NaCl, 50 mM TCEP for 5 min (A-C), 15 min (D-F), 30 min (G-I) and 60 min (J-L).

[0210] Figure 78: Uptake of Pb(II) by ZTNF-alpha1-3C in triplicate determined by native MS after incubation at 25 °C in 20 mM Tris, pH 7.5, 150 mM NaCl, 50 mM TCEP for 5 min (A-C), 15 min (D-F), 30 min (G-I) and 60 min (J-L). Minor impurities indicated by an asterisk (*).

[0211] Figure 79: Native MS in 100 mM ammonium acetate pH 7.0 of ZHER2:2891-3C before (A) and after modification with Bi(III) (B), In(III) (C), Ga(III) (D) and Pb(II) (E). Affibodies were reduced in 50 mM TCEP, pH 7.5 for 60 min at 25 °C prior to the addition of metal reagent (5 eq.).

[0212] Figure 80: Native MS in 100 mM ammonium acetate pH 7.0 of ZTNF-alpha1-3C before (A) and after modification with Bi(III) (B), In(III) (C), Ga(III) (D) and Pb(II) (E). Affibodies were reduced in 50 mM TCEP, pH 7.5 for 60 min at 25 °C prior to the addition of metal reagent (5 eq.).

[0213] Figure 81: Circular dichroism (CD) spectra of ZHER2:2891-3C compared to its wildtype (A) before (B) and after modification with Bi(III) (C), In(III) (D), Ga(III) (E) or Pb(II) (F) in 20 mM phosphate buffer, pH 7.5 (n = 2, average plotted)

[0214] Figure 82: Circular dichroism (CD) spectra of ZTNF-alpha1-3C compared to its wildtype (A) before (B) and after modification with Bi(III) (C), In(III) (D), Ga(III) (E) or Pb(II) (F) in 20 mM phosphate buffer, pH 7.5 (n = 2, average plotted).

[0215] Figure 83: Thermal denaturation curves determined by CD in 20 mM phosphate buffer, pH 7,5 of ZHER2:2891 (A, Tm = 74 °C), ZHER2:2891-3C (B, Tm= 71 °C), ZHER2:2891-3C-Bi (C, Tm= 69 °C), ZHER2:2891-3C-In (D, Tm = 63 °C), ZHER2:2891-3C-Ga (E, Tm = 71 °C), and ZHER2:2891-3C-Pb (F, Tm = 65 °C) (n = 2, average plotted).

[0216] Figure 84: Thermal denaturation curves determined by CD in 20 mM phosphate buffer, pH 7,5 of ZTNF-alpha1 (A, Tm= 56 °C), ZTNF-alpha1-3C (B, Tm = 59 °C), ZTNF-alpha1-3C-Bi(C, Tm = 61 °C), ZTNF-alpha1-3C-In (D, Tm= 58 °C), ZTNF-alpha1-3C-Ga (E, Tm= 59 °C), and ZTNF-alpha1-3C-Pb (F, Tm= 62 °C) (n = 2, average plotted).

[0217] Figure 85: Native MS of Bi(III)-bound ZHER2:2891-3C in after incubation for 10 minutes at 100 °C in the presence of 0 eq (A) or 100 eq (B, C) EDTA in 100 mM ammonium acetate pH 7.

[0218] Figure 86: Model of ZHER2:2891-3C with three cysteine residues bound to one Bi(III) atom in trigonal pyramidal geometry. (A) Cystine side chains shown as sticks. (B) All side chains shown as sticks. Detailed description of the embodiments

[0219] The present disclosure describes a strategy to coordinate a radionuclide metal to an antigen binding protein (such as an antibody, nanobody or affibody), thereby abolishing the need for a traditional bifunctional linker. This surprisingly provides highly stable metal-complexes that bind the metal with residues of the antigen binding protein, preventing the metal dissociating from said protein. This strategy provides compounds that could have a broad range of applications across targeted radiotherapy, diagnostics and chemical biology.

[0220] An advantage of the present invention is the ability to provide metal coordinated antigen binding proteins without the need of bifunctional linkers. Therefore, the invention avoids the resulting randomly modified antigen binding proteins, resulting in heterogeneous conjugates and partial inactivation by altering the antigen-binding site.

[0221] In addition to challenges with harsh, incomplete and lengthy tagging processes, conventional chelators are large and highly charged, impacting on the properties of the conjugate. This is particularly concerning for some antigen binding proteins (e.g. nanobodies or affibodies) where the bifunctional linker significantly changes the overall mass and charge of the conjugate. Again, the present invention avoids or minimises these challenges and complications. General definitions

[0222] It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentionedor evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

[0223] Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that the intention is not to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the present invention as defined by the claims.

[0224] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described. It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

[0225] All of the patents and publications referred to herein are incorporated by reference in their entirety.

[0226] For purposes of interpreting this specification, terms used in the singular will also include the plural and vice versa.

[0227] The general chemical terms used in the formulae herein have their usual meaning.

[0228] As used herein, the term "and / or" means "and", or "or", or both.

[0229] The term "(s)" following a noun contemplates the singular and plural form, or both.

[0230] It is intended that reference to a range of numbers disclosed herein (for example, 1 to 10) also incorporates reference to all rational numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9, and 10) and also any range of rational numbers within that range (for example, 2 to 8, 1.5 to 5.5, and 3.1 to 4.7) and, therefore, all sub-ranges of all ranges expressly disclosed herein are hereby expressly disclosed. These are only examples of what is specifically intended and all possible combinationsof numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application in a similar manner.

[0231] Various features of the invention are described with reference to a certain value, or range of values. These values are intended to relate to the results of the various appropriate measurement techniques, and therefore should be interpreted as including a margin of error inherent in any particular measurement technique. Some of the values referred to herein are denoted by the term “about” to at least in part account for this variability. The term “about”, when used to describe a value, may mean an amount within ±10%, ±5%, ±1% or ±0.1% of that value. Selected Definitions

[0232] The term "isolated protein" or "isolated polypeptide" is a protein or polypeptide that by virtue of its origin or source of derivation is not associated with naturally- associated components that accompany it in its native state; is substantially free of other proteins from the same source. A protein may be rendered substantially free of naturally associated components or substantially purified by isolation, using protein purification techniques known in the art. By “substantially purified” is meant the protein is substantially free of contaminating agents, e.g., at least about 70% or 75% or 80% or 85% or 90% or 95% or 96% or 97% or 98% or 99% free of contaminating agents.

[0233] The term “recombinant” shall be understood to mean the product of artificial genetic recombination. Accordingly, in the context of a recombinant protein comprising an antibody antigen binding domain, this term does not encompass an antibody naturally- occurring within a subject’s body that is the product of natural recombination that occurs during B cell maturation. However, if such an antibody is isolated, it is to be considered an isolated protein comprising an antibody antigen binding domain. Similarly, if nucleic acid encoding the protein is isolated and expressed using recombinant means, the resulting protein is a recombinant protein comprising an antibody antigen binding domain. A recombinant protein also encompasses a protein expressed by artificial recombinant means when it is within a cell, tissue or subject, e.g., in which it is expressed.

[0234] The term “protein” shall be taken to include a single polypeptide chain, i.e., a series of contiguous amino acids linked by peptide bonds or a series of polypeptide chains covalently or non-covalently linked to one another (i.e., a polypeptide complex). Forexample, the series of polypeptide chains can be covalently linked using a suitable chemical or a disulphide bond. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, Van der Waals forces, and hydrophobic interactions.

[0235] The term “polypeptide” or “polypeptide chain” will be understood from the foregoing paragraph to mean a series of contiguous amino acids linked by peptide bonds.

[0236] As used herein, the term “antigen binding protein” shall be taken to include a region of an antibody that is capable of specifically binding to an antigen, i.e., a VH or a VL or an Fv comprising both a VH and a VL. The antigen binding protein need not be in the context of an entire antibody, e.g., it can be in isolation (e.g., a domain antibody) or in another form, e.g., as described herein, such as a scFv.

[0237] For the purposes for the present disclosure, the term “antibody” includes a protein capable of specifically binding to one or a few closely related antigens by virtue of an antigen binding domain contained within a Fv. This term includes four chain antibodies (e.g., two light chains and two heavy chains), recombinant or modified antibodies (e.g., chimeric antibodies, humanized antibodies, human antibodies, CDR-grafted antibodies, primatized antibodies, de-immunized antibodies, synhumanized antibodies, half- antibodies, bispecific antibodies). An antibody generally comprises constant domains, which can be arranged into a constant region or constant fragment or fragment crystallizable (Fc). Exemplary forms of antibodies comprise a four-chain structure as their basic unit. Full-length antibodies comprise two heavy chains (~50 to 70 kD) covalently linked and two light chains (~23 kDa each). A light chain generally comprises a variable region (if present) and a constant domain and in mammals is either a κ light chain or a λ light chain. A heavy chain generally comprises a variable region and one or two constant domain(s) linked by a hinge region to additional constant domain(s). Heavy chains of mammals are of one of the following types α, δ, ε, γ, or μ. Each light chain is also covalently linked to one of the heavy chains. For example, the two heavy chains and the heavy and light chains are held together by inter-chain disulfide bonds and by non- covalent interactions. The number of inter-chain disulfide bonds can vary among different types of antibodies. Each chain has an N-terminal variable region (VH or VL wherein each are ~110 amino acids in length) and one or more constant domains at the C- terminus. The constant domain of the light chain (CL which is ~110 amino acids in length) is aligned with and disulfide bonded to the first constant domain of the heavy chain (CH1 which is 330 to 440 amino acids in length). The light chain variable region is aligned with thevariable region of the heavy chain. The antibody heavy chain can comprise 2 or more additional CH domains (such as, CH2, CH3 and the like) and can comprise a hinge region between the CH1 and CH2 constant domains. Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass. In one example, the antibody is a murine (mouse or rat) antibody or a primate (such as, human) antibody. In one example the antibody heavy chain is missing a C- terminal lysine residue. In one example, the antibody is humanized, synhumanized, chimeric, CDR-grafted or deimmunized.

[0238] The terms "full-length antibody", "intact antibody" or "whole antibody" are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antigen binding fragment of an antibody. Specifically, whole antibodies include those with heavy and light chains including an Fc region. The constant domains may be wild-type sequence constant domains (e.g., human wild-type sequence constant domains) or amino acid sequence variants thereof.

[0239] As used herein, “variable region” refers to the portions of the light and / or heavy chains of an antibody as defined herein that is capable of specifically binding to an antigen and, includes amino acid sequences of complementarity determining regions (CDRs); i.e., CDR1, CDR2, and CDR3, and framework regions (FRs). For example, the variable region comprises three or four FRs (e.g., FR1, FR2, FR3 and optionally FR4) together with three CDRs. VH refers to the variable region of the heavy chain. VL refers to the variable region of the light chain.

[0240] As used herein, the term “subject” shall be taken to mean any animal including humans, for example a mammal. Exemplary subjects include but are not limited to humans and non-human primates. For example, the subject is a human.

[0241] "Antibodies" or "immunoglobulins" or "Igs" are gamma globulin proteins that are found in blood, or other bodily fluids of vertebrates that function in the immune system to bind antigen, hence identifying and / or neutralising foreign objects.

[0242] Antibodies are generally a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. Each L chain is linked to a H chain by one covalent disulfide bond. The two H chains are linked to each other by oneor more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges.

[0243] H and L chains define specific Ig domains. More particularly, each H chain has at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the α and γ chains and four CH domains for μ and ε isotypes. Each L chain has at the N-terminus, a variable domain (VL) followed by a constant domain (CL) at its other end. The VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (CH1).

[0244] Antibodies can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated α, δ, ε, γ, and μ, respectively. The γ and α classes are further divided into subclasses on the basis of relatively minor differences in ¾ sequence and function, e.g., humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgAl, and IgA2. The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains.

[0245] The constant domain includes the Fc portion that comprises the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of antibodies such as ADCC are determined by sequences in the Fc region, which region is also the part recognised by Fc receptors (FcR) found on certain types of cells.

[0246] The pairing of a VHand VLtogether forms a "variable region" or "variable domain" including the amino -terminal domains of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as "VH." The variable domain of the light chain may be referred to as "VL." The V domain contains an "antigen binding site" that affects antigen binding and defines specificity of a particular antibody for its particular antigen. V regions span about 110 amino acid residues and consist of relatively invariant stretches called framework regions (FRs) (generally about 4) of 15-30 amino acids separated by shorter regions of extreme variability called "hypervariable regions" (generally about 3) that are each generally 9-12 amino acids long. The FRs largely adopt a β-sheet conformation and the hypervariable regions form loops connecting, and in some cases forming part of, the β-sheet structure.

[0247] "Hypervariable region" refers to the regions of an antibody variable domain that are hypervariable in sequence and / or form structurally defined loops. Generally, antibodies comprise six hypervariable regions; three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3).

[0248] As used herein, the term “complementarity determining regions” (syn. CDRs; i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of an antibody variable region the presence of which are major contributors to specific antigen binding. Each variable region domain (VH or VL) typically has three CDRs identified as CDR1, CDR2 and CDR3. The CDRs of VH are also referred to herein as CDR H1, CDR H2 and CDR H3, respectively, wherein CDR H1 corresponds to CDR 1 of VH, CDR H2 corresponds to CDR 2 of VH and CDR H3 corresponds to CDR 3 of VH. Likewise, the CDRs of VL are referred to herein as CDR L1, CDR L2 and CDR L3, respectively, wherein CDR L1 corresponds to CDR 1 of VL, CDR L2 corresponds to CDR 2 of VL and CDR L3 corresponds to CDR 3 of VL. In one example, the amino acid positions assigned to CDRs and FRs are defined according to Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991 (also referred to herein as “the Kabat numbering system”). In another example, the amino acid positions assigned to CDRs and FRs are defined according to the Enhanced Chothia Numbering Scheme (http: / / www.bioinfo.org.uk / mdex.html). The present invention is not limited to FRs and CDRs as defined by the Kabat numbering system, but includes all numbering systems, including the canonical numbering system or of Chothia and Lesk J. Mol. Biol.196: 901- 917, 1987; Chothia et al., Nature 342: 877-883, 1989; and / or Al-Lazikani et al., J. Mol. Biol.273: 927-948, 1997; the numbering system of Honnegher and Plükthun J. Mol. Biol. 309: 657-670, 2001; or the IMGT system discussed in Giudicelli et al., Nucleic Acids Res. 25: 206-211 1997. In one example, the CDRs are defined according to the Kabat numbering system. Optionally, heavy chain CDR2 according to the Kabat numbering system does not comprise the five C-terminal amino acids listed herein or any one or more of those amino acids are substituted with another naturally-occurring amino acid. In this regard, Padlan et al., FASEB J., 9: 133-139, 1995 established that the five C- terminal amino acids of heavy chain CDR2 are not generally involved in antigen binding.

[0249] "Framework" or "FR" residues are those variable domain residues other than the hypervariable region or CDR residues herein defined. The FRs of VH are also referred to herein as FR H1, FR H2, FR H3 and FR H4, respectively, wherein FR H1 corresponds toFR 1 of VH, FR H2 corresponds to FR 2 of VH, FR H3 corresponds to FR 3 of VH and FR H4 corresponds to FR 4 of VH. Likewise, the FRs of VL are referred to herein as FR L1, FR L2, FR L3 and FR L4, respectively, wherein FR L1 corresponds to FR 1 of VL, FR L2 corresponds to FR 2 of VL, FR L3 corresponds to FR 3 of VL and FR L4 corresponds to FR 4 of VL.

[0250] An "intact" or "whole" antibody is one that comprises an antigen-binding site as well as a CL and at least heavy chain constant domains, CH1, CH2 and CH3. The constant domains may be native sequence constant domains (e.g. human native sequence constant domains) or amino acid sequence variant thereof.

[0251] “Whole antibody related structures” include multimerized forms of whole antibody.

[0252] "Whole antibody fragments including a variable domain" include SD-mAb, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies, single-chain antibody molecules; minibodies; and multi-specific antibodies formed from antibody fragments.

[0253] The "Fab fragment" consists of an entire L chain along with the variable region domain of the H chain (VH), and the first constant domain of one heavy chain (CH1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen- binding site.

[0254] A "Fab' fragment" differs from Fab fragments by having additional few residues at the carboxy terminus of the CH1 domain including one or more cysteines from the antibody hinge region. Fab'- SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group.

[0255] A "F(ab')2 fragment" roughly corresponds to two disulphide linked Fab fragments having divalent antigen-binding activity and is still capable of cross-linking antigen.

[0256] An "Fv" is the minimum antibody fragment that contains a complete antigen- recognition and binding site. This fragment consists of a dimer of one heavy and one light chain variable region domain in tight, non-covalent association.

[0257] In a single-chain Fv (scFv) species, one heavy and one light chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a "dimeric" structure analogous to that in a two-chain Fv species.From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody.

[0258] "Single-chain Fv" also abbreviated as "sFv" or "scFv" are antibody fragments that comprise the VH and VL antibody domains connected to form a single polypeptide chain. Preferably, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

[0259] A "single variable domain" is half of an Fv (comprising only three CDRs specific for an antigen) that has the ability to recognise and bind antigen, although generally at a lower affinity than the entire binding site.

[0260] As meant herein, an "Fc region" is a dimer consisting of two polypeptide chains joined by one or more disulfide bonds, each chain comprising part or all of a hinge domain plus a CH2 and a CH3 domain. Each of the polypeptide chains is referred to as an "Fc polypeptide chain." To distinguish the two Fc polypeptide chains, one is referred to herein as an "A chain" and the other is referred to as a "B chain." More specifically, the Fc regions contemplated for use with the present invention are lgG Fc regions, which can be mammalian or human lgG1, lgG2, lgG3, or lgG4 Fc regions. Among human lgG1 Fc regions, at least two allelic types are known.

[0261] "Diabodies" refers to antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). The small antibody fragments are prepared by constructing sFv fragments (see preceding paragraph) with short linkers (about 5-10 residues) between the VH and VL domains such that interchain but not intra- chain pairing of the V domains is achieved, resulting in a bivalent fragment, i.e., a fragment having two antigen-binding sites.

[0262] Diabodies may be bivalent or bispecific. Bispecific diabodies are heterodimers of two "crossover" sFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains. Triabodies and tetrabodies are also generally known in the art.

[0263] An "isolated antibody" is one that has been identified and separated and / or recovered from a component of its pre-existing environment. Contaminant componentsare materials that would interfere with therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.

[0264] A "human antibody" refers to an antibody that possesses an amino acid sequence that corresponds to that of an antibody produced by a human. Human antibodies can be produced using various techniques known in the art, including phage -display libraries. Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled.

[0265] "Humanised' forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. For the most part, humanised antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired antibody specificity, affinity, and capability. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanised antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanised antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanised antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0266] "Monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site or determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesised uncontaminated by other antibodies. Monoclonal antibodies may be prepared by the hybridoma methodology. The "monoclonal antibodies" may also be isolated from phage antibody libraries using molecular engineering techniques.

[0267] "Binding affinity" generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high- affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present invention.

[0268] As used herein, the term “binds” in reference to the interaction of an antigen binding protein or an antigen binding domain thereof with an antigen means that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the antigen. For example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody binds to epitope "A", the presence of a molecule containing epitope “A” (or free, unlabelled “A”), in a reaction containing labelled “A” and the protein, will reduce the amount of labelled “A” bound to the antibody.

[0269] As used herein, the term “specifically binds” or “binds specifically” shall be taken to mean that an antigen binding protein of the invention reacts or associates more frequently, more rapidly, with greater duration and / or with greater affinity with a particular antigen or cell expressing same than it does with alternative antigens or cells.

[0270] As used herein, the term “does not detectably bind” shall be understood to mean that an antigen binding protein, e.g., an antibody, binds to a candidate antigen at a level less than 10%, or 8% or 6% or 5% above background. The background can be the level of binding signal detected in the absence of the protein and / or in the presence of a negative control protein (e.g., an isotype control antibody) and / or the level of binding detected in the presence of a negative control antigen. The level of binding is detected using biosensor analysis (e.g. Biacore) in which the antigen binding protein is immobilized and contacted with an antigen.

[0271] As used herein, the term “does not significantly bind” shall be understood to mean that the level of binding of an antigen binding protein of the invention to a polypeptide is not statistically significantly higher than background, e.g., the level of binding signal detected in the absence of the antigen binding protein and / or in the presence of a negative control protein (e.g., an isotype control antibody) and / or the level of binding detected in the presence of a negative control polypeptide. The level of binding is detected using biosensor analysis (e.g. Biacore) in which the antigen binding protein is immobilized and contacted with an antigen.

[0272] As used herein, the term "antigen" is intended to include substances that bind to or evoke the production of one or more antibodies and may comprise, but is not limited to, proteins, peptides, polypeptides, oligopeptides, lipids, carbohydrates, and combinations thereof, for example a glycosylated protein or a glycolipid. The term "antigen" as used herein refers to a molecular entity that may be expressed on a target cell and that can be recognised by means of the adaptive immune system including but not restricted to antibodies, or engineered molecules including but not restricted to scFvs or multimers thereof, Fab-fragments or multimers thereof, antibodies or multimers thereof, single chain antibodies or multimers thereof, or any other molecule that can execute binding to a structure with high affinity.

[0273] The phrase “pharmaceutically acceptable” indicates that the substance or composition must be compatible chemically and / or toxicologically, with the other ingredients comprising a formulation, and / or the mammal being treated therewith.

[0274] As used herein, the term “theranostic” refers to the ability of compounds / materials to be used for diagnosis as well as for therapy. The term "theranostic reagent" relates to any reagent which is both suitable for detection, diagnostic and / or the treatment of a disease or condition of a patient. The aim of theranostic compounds / materials is to overcome undesirable differences in biodistribution and selectivity, which can exist between distinct diagnostic and therapeutic agents.

[0275] “Ligand” as used herein refers to an atom or chemical group as part of a side chain capable of donating a pair of electrons to a central metal atom or ion to form a coordination complex with the metal.

[0276] In some embodiments, the ligand used in the present disclosure is not a chelator or chelating ligand. Preferably, the ligand is a monodentate ligand (ie it bonds to the metal through a single atom or group as part of the side chain.

[0277] A hydrophobic residue as used herein may be a non-polar amino acid with an aliphatic side chain or an aromatic side chain.

[0278] The words “treat” or “treatment” refer to therapeutic treatment wherein the object is to slow down (lessen) an undesired physiological change or disorder. For the purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment. Treatment may not necessarily result in the complete clearance of a disease or disorder but may reduce or minimise complications and side effects of infection and the progression of a disease or disorder.

[0279] The term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter in a cell. Antibodies

[0280] In one example, an antigen binding protein as described herein according to any example is an antibody. In an alternative example, an antigen binding protein as described herein according to any example is a nanobody. In another alternative example, an antigen binding protein as described herein is an affibody.

[0281] Methods for generating antibodies are known in the art and / or described in Harlow and Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988). Generally, in such methods an antigen or a region thereof (e.g., an extracellular region) or immunogenic fragment or epitope thereof or a cell expressing and displaying same (i.e., an immunogen), optionally formulated with any suitable or desired carrier, adjuvant, or pharmaceutically acceptable excipient, is administered to a non- human animal, for example, a mouse, chicken, rat, rabbit, guinea pig, dog, horse, cow, goat or pig. The immunogen may be administered intranasally, intramuscularly, subcutaneously, intravenously, intradermally, intraperitoneally, or by other known route.

[0282] The production of polyclonal antibodies may be monitored by sampling blood of the immunised animal at various points following immunisation. One or more further immunisations may be given, if required to achieve a desired antibody titre. The process of boosting and titreing is repeated until a suitable titre is achieved. When a desired level of immunogenicity is obtained, the immunised animal is bled and the serum isolated and stored, and / or the animal is used to generate monoclonal antibodies (mAbs).

[0283] Monoclonal antibodies are one exemplary form of antibody contemplated by the present invention. The term “monoclonal antibody" or “mAb” refers to a homogeneous antibody population capable of binding to the same antigen(s), for example, to the same epitope within the antigen. This term is not intended to be limited with regard to the source of the antibody or the manner in which it is made.

[0284] For the production of mAbs any one of a number of known techniques may be used, such as, for example, the procedure exemplified in US4196265 or Harlow and Lane (1988), supra.

[0285] For example, a suitable animal is immunised with an immunogen under conditions sufficient to stimulate antibody producing cells. Rodents such as rabbits, mice and rats are exemplary animals. Mice genetically-engineered to express human antibodies, for example, which do not express murine antibodies, can also be used to generate an antibody of the present invention (e.g., as described in WO2002 / 066630).

[0286] Following immunisation, somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the mAb generating protocol. These cells may be obtained from biopsies of spleens, tonsils or lymph nodes, or from a peripheral blood sample. The B cells from the immunised animal are then fused with cells of an immortal myeloma cell, generally derived from the same species as the animal that was immunised with the immunogen.

[0287] Hybrids are amplified by culture in a selective medium comprising an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary agents are aminopterin, methotrexate and azaserine.

[0288] The amplified hybridomas are subjected to a functional selection for antibody specificity and / or titre, such as, for example, by flow cytometry and / orimmunohistochemistry and / or immunoassay (e.g. radioimmunoassay, enzyme immunoassay, cytotoxicity assay, plaque assay, dot immunoassay, and the like).

[0289] Alternatively, ABL-MYC technology (NeoClone, Madison WI 53713, USA) is used to produce cell lines secreting MAbs (e.g., as described in Largaespada et al, J. Immunol. Methods.197: 85-95, 1996).

[0290] Antibodies can also be produced or isolated by screening a display library, e.g., a phage display library, e.g., as described in US6300064 and / or US5885793.

[0291] The antibody of the present invention may be a synthetic antibody. For example, the antibody is a chimeric antibody, a humanised antibody, a human antibody synhumanised antibody, primatised antibody or a de-immunised antibody.

[0292] Synthetic antibodies may be made by known methods such as solid-phase peptide synthesis (SPPS). Antibody binding domain-containing proteins Single domain antibodies

[0293] In some examples, a protein of the invention is or comprises a single-domain antibody (which is used interchangeably with the term “domain antibody”, “nanobody” or “sdAb”). A single-domain antibody is a single polypeptide chain comprising all or a portion of the heavy chain variable region of an antibody. In certain examples, a single-domain antibody is a human single-domain antibody. Diabodies, Triabodies, Tetrabodies

[0294] In some examples, a protein of the invention is or comprises a diabody, triabody, tetrabody or higher order protein complex such as those described in WO98 / 044001 and / or WO94 / 007921.

[0295] For example, a diabody is a protein comprising two associated polypeptide chains, each polypeptide chain comprising the structure VL-X-VH or VH-X-VL, wherein VL is an antibody light chain variable region, VH is an antibody heavy chain variable region, X is a linker comprising insufficient residues to permit the VH and VL in a single polypeptide chain to associate (or form an Fv) or is absent, and wherein the VH of one polypeptide chain binds to a VL of the other polypeptide chain to form an antigen binding domain, i.e.,to form a Fv molecule capable of specifically binding to one or more antigens. The VL and VH can be the same in each polypeptide chain or the VL and VH can be different in each polypeptide chain so as to form a bispecific diabody (i.e., comprising two Fvs having different specificity). Single Chain Fc (scFv)

[0296] The skilled artisan will be aware that scFvs comprise VH and VL regions in a single polypeptide chain and a polypeptide linker between the VH and VL which enables the scFv to form the desired structure for antigen binding (i.e., for the VH and VL of the single polypeptide chain to associate with one another to form a Fv). For example, the linker comprises in excess of 12 amino acid residues with (Gly4Ser)3 being one of the more favoured linkers for a scFv.

[0297] The present invention also contemplates a disulfide stabilised Fv (or diFv or dsFv), in which a single cysteine residue is introduced into a FR of VH and a FR of VL and the cysteine residues linked by a disulfide bond to yield a stable Fv.

[0298] Alternatively, or in addition, the present invention encompasses a dimeric scFv, i.e., a protein comprising two scFv molecules linked by a non-covalent or covalent linkage, e.g., by a leucine zipper domain (e.g., derived from Fos or Jun). Alternatively, two scFvs are linked by a peptide linker of sufficient length to permit both scFvs to form and to bind to an antigen, e.g., as described in US20060263367. Heavy Chain Antibodies

[0299] Heavy chain antibodies differ structurally from many other forms of antibodies, in so far as they comprise a heavy chain, but do not comprise a light chain. Accordingly, these antibodies are also referred to as “heavy chain only antibodies”. Heavy chain antibodies are found in, for example, camelids and cartilaginous fish (also called IgNAR).

[0300] The variable regions present in naturally occurring heavy chain antibodies are generally referred to as "VHH domains" in camelid antibodies and V-NAR in IgNAR, in order to distinguish them from the heavy chain variable regions that are present in conventional 4-chain antibodies (which are referred to as "VH domains") and from the light chain variable regions that are present in conventional 4-chain antibodies (which are referred to as "VL domains").

[0301] A general description of heavy chain antibodies from camelids and the variable regions thereof and methods for their production and / or isolation and / or use is found inter alia in the following references WO94 / 04678, WO97 / 49805 and WO 97 / 49805.

[0302] A general description of heavy chain antibodies from cartilaginous fish and the variable regions thereof and methods for their production and / or isolation and / or use is found inter alia in WO2005 / 118629. Mutations to proteins

[0303] The present invention also provides an antigen binding protein or a nucleic acid encoding same having a mutation or modification. In one example, an antigen binding protein or nucleic acid of the invention comprises sequence at least about 85% or 90% or 95% or 97% or 98% or 99% identical to a sequence disclosed herein.

[0304] Alternatively, or additionally, the antigen binding protein comprises a CDR (e.g., three CDRs) at least about 80% or 85% or 90% or 95% or 97% or 98% or 99% identical to CDR(s) of a VH or VL as described herein according to any example.

[0305] In another example, a nucleic acid of the invention comprises a sequence at least about 80% or 85% or 90% or 95% or 97% or 98% or 99% identical to a sequence encoding an antigen binding protein having a function as described herein according to any example. The present invention also encompasses nucleic acids encoding an antigen binding protein of the invention, which differs from a sequence exemplified herein as a result of degeneracy of the genetic code.

[0306] The % identity of a nucleic acid or polypeptide is determined by GAP (Needleman and Wunsch. Mol. Biol.48, 443-453, 1970) analysis (GCG program) with a gap creation penalty=5, and a gap extension penalty=0.3. The query sequence is at least 50 residues in length, and the GAP analysis aligns the two sequences over a region of at least 50 residues. For example, the query sequence is at least 100 residues in length and the GAP analysis aligns the two sequences over a region of at least 100 residues. For example, the two sequences are aligned over their entire length.

[0307] The present invention also contemplates a nucleic acid that hybridises under stringent hybridisation conditions to a nucleic acid encoding an antigen binding protein described herein. A “moderate stringency” is defined herein as being a hybridisationand / or washing carried out in 2 x SSC buffer, 0.1% (w / v) SDS at a temperature in the range 45°C to 65°C, or equivalent conditions. A “high stringency” is defined herein as being a hybridisation and / or wash carried out in 0.1 x SSC buffer, 0.1% (w / v) SDS, or lower salt concentration, and at a temperature of at least 65°C, or equivalent conditions. Reference herein to a particular level of stringency encompasses equivalent conditions using wash / hybridisation solutions other than SSC known to those skilled in the art. For example, methods for calculating the temperature at which the strands of a double stranded nucleic acid will dissociate (also known as melting temperature, or Tm) are known in the art. A temperature that is similar to (e.g., within 5°C or within 10°C) or equal to the Tm of a nucleic acid is considered to be high stringency. Medium stringency is to be considered to be within 10°C to 20°C or 10°C to 15°C of the calculated Tm of the nucleic acid.

[0308] The present invention also contemplates mutant forms of an antigen binding protein of the invention comprising one or more conservative amino acid substitutions compared to a sequence set forth herein. In some examples, the antigen binding protein comprises 10 or fewer, e.g., 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1 conservative amino acid substitutions. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain and / or hydropathicity and / or hydrophilicity.

[0309] Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine, leucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Hydropathic indices are described, for example in Kyte and Doolittle J. Mol. Biol., 157: 105-132, 1982 and hydrophilic indices are described in, e.g., US4554101.

[0310] The present invention also contemplates non-conservative amino acid changes. For example, of particular interest are substitutions of charged amino acids with another charged amino acid and with neutral or positively charged amino acids. In some examples, the antigen binding protein comprises 10 or fewer, e.g., 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1 non-conservative amino acid substitutions.

[0311] In one example, the mutation(s) occur within a FR of an antigen binding domain of an antigen binding protein of the invention. In another example, the mutation(s) occur within a CDR of an antigen binding protein of the invention.

[0312] Exemplary methods for producing mutant forms of an antigen binding protein include: • mutagenesis of DNA (Thie et al., Methods Mol. Biol.525: 309-322, 2009) or RNA (Kopsidas et al., Immunol. Lett.107:163-168, 2006; Kopsidas et al. BMC Biotechnology, 7: 18, 2007; and WO1999 / 058661); • introducing a nucleic acid encoding the polypeptide into a mutator cell, e.g., XL- 1Red, XL-mutS and XL-mutS-Kanr bacterial cells (Stratagene); • DNA shuffling, e.g., as disclosed in Stemmer, Nature 370: 389-91, 1994; and • site directed mutagenesis, e.g., as described in Dieffenbach (ed) and Dveksler (ed) (In: PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratories, NY, 1995).

[0313] Alternative methods for producing mutant forms of an antigen binding protein may include solid-phase peptide synthesis, where selected residues are replaced by alternative and / or modified residues.

[0314] Exemplary methods for determining biological activity of the mutant antigen binding proteins of the invention will be apparent to the skilled artisan and / or described herein, e.g., antigen binding. For example, methods for determining antigen binding, competitive inhibition of binding, affinity, association, dissociation and therapeutic efficacy are described herein.

[0315] In some embodiments, the alpha carbon atom of the at least one residue, or the alpha carbon atom of each of the two residues, is located within 10 Å, or within 9 Å, or within 8 Å, or within 7 Å, or within 6 Å, or within 5 Å or within 4 Å, or within 3 Å of the alpha carbon atom of one or both of the cysteines that form the at least one disulfide bond. In some alternative embodiments, the alpha carbon atom of the at least one residue, or the alpha carbon atom of each of the two residues, is located between about 3 Å to about 10 Å, or between about 3 Å to about 9 Å, or between about 4 Å to about 8 Å of the alpha carbon atom of one or both of the cysteines that form the at least one disulfide bond. Itwill be appreciated by the person skilled in the art that the distance between the alpha carbon atoms of the at least one residue, or at least two residues, and the alpha carbon of the one or both of the cysteines that form the at least one disulfide bond of the antigen binding protein may be selected depending on the size of metal to be coordinated. The location of the mutated or modified residue may be selected to achieve a particular distance between the alpha carbon atoms of the at least one residue, or at least two residues, and the alpha carbon of the one or both of the cysteines that form the at least one disulfide bond of the antigen binding protein.

[0316] In some embodiments, the alpha carbon atom of one of the at least three residues is located within 13 Å, or within 12 Å, or within 11 Å, or within 10 Å, or within 9 Å, or within 8 Å, or within 7 Å, or within 6 Å, or within 5 Å or within 4 Å, or within 3 Å of the alpha carbon atoms of the remaining modified or mutated residues. In some alternative embodiments, the alpha carbon atom of one of the at least three residues, is located between about 3 Å to about 13 Å, or between about 3 Å to about 10 Å, or between about 4 Å to about 10 Å, or between about 5 Å to about 10 Å of the alpha carbon atoms of the remaining modified or mutated residues.

[0317] Methods for determining the location of a residue for mutation or modification, after which, introduces a side chain capable of acting as a ligand for a metal are known in the art and described herein, including the Examples. Further, methods to determine whether the alpha carbon atom of the at least one residue, or the alpha carbon atom of each of the two residues, or the alpha carbon of any two residues, is located within 10 Å, or within 9 Å, or within 8 Å, or within 7 Å, or within 6 Å, or within 5 Å or within 4 Å, or within 3 Å of the alpha carbon atom of one or both of the cysteines that form the at least one disulfide bond, are also known in the art and described herein, including the Examples.

[0318] Methods for determining metal co-ordination of a mutated or modified antigen binding protein are also described herein, including the Examples. Protein production

[0319] In one example, an antigen binding protein described herein according to any example is produced by culturing a hybridoma under conditions sufficient to produce the protein, e.g., as described herein and / or as is known in the art. Recombinant expression

[0320] In another example an antigen binding protein described herein according to any example is recombinant.

[0321] The production of an antigen binding protein of the invention generally requires an expression vector containing a polynucleotide that encodes the antigen binding protein of the invention. A polynucleotide encoding an antigen binding protein of the invention may be obtained and sub cloned into a vector for the production of an antigen binding protein by recombinant DNA technology using techniques well-known in the art, including techniques described herein. Many different expression systems are contemplated including the use of mammalian cells including human cells for production and secretion of antigen binding proteins. Examples of cells include 293F, CHO and the NSO cell line.

[0322] Expression vectors containing protein coding sequences and appropriate transcriptional and translational control signals can be constructed using methods known in the art. These include in vitro recombinant DNA techniques, synthetic techniques and in vivo genetic recombination. In certain embodiments there is provided a replicable vector having a nucleic acid encoding an antigen binding protein operably linked to a promoter.

[0323] Cells transfected with an expression vector may be cultured by conventional techniques to produce an antigen binding protein. Thus, in certain embodiments, there is provided host cells or cell transfectants containing a polynucleotide encoding an antigen binding protein of the invention operably linked to a promoter. The promoter may be heterologous. A variety of host-expression vector systems may be utilized and in certain systems the transcription machinery of the vector system is particularly matched to the host cell. For example, mammalian cells such as Chinese hamster ovary cells (CHO) may be transfected with a vector including the major intermediate early gene promoter element from human cytomegalovirus. Additionally or alternatively, a host cell may be used that modulates the expression of inserted sequences, or modifies and processes the gene product as required, including various forms of post translational modification. Examples of mammalian host cells having particular post translation modification processes include CHO, VERY, BHK, HeIa, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NSO, CRL7O3O and HsS78Bst cells.

[0324] Depending upon the use intended for the protein molecule, a number of bacterial expression vectors may be advantageously selected. In one example, vectors that causethe expression of high levels of fusion protein products that are readily purified, such as the E. coli expression vector pUR278 may be used where a large quantity of an antigen binding protein is to be produced. The expression product may be produced in the form of a fusion protein with lacZ. Other bacterial vectors include pIN vectors and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione-S-transferase (GST). These fusion proteins are generally soluble and can easily be purified from lysed cells by adsorption and binding to glutathione-agarose affinity matrix followed by elution in the presence of free glutathione. A thrombin and / or factor Xa protease cleavage site may be provided in the expressed polypeptide so that the cloned target gene product can be released from the GST moiety.

[0325] Autographa californica nuclear polyhedrosis virus (AcNPV) may be used as a vector to express foreign genes in an insect system including Spodoptera frugiperda cells. The particular promoter used may depend on where the protein coding is inserted into the sequence. For example, the sequence may be cloned individually into the polyhedrin gene and placed under control of the polyhedrin promoter.

[0326] Virus based expression systems may be utilized with mammalian cells such as an adenovirus whereby the coding sequence of interest may be ligated to the adenoviral late promoter and tripartite leader sequence. In vitro or in vivo recombination may then be used to insert this chimeric gene into the adenoviral genome. Insertions into region E1 or E3 will result in a viable recombinant virus that is capable of expressing the antigen binding protein in infected host cells. Specific initiation signals including the ATG initiation codon and adjacent sequences may be required for efficient translation of inserted antigen binding protein coding sequences. Initiation and translational control signals and codons can be obtained from a variety of origins, both natural and synthetic. Transcription enhancer elements and transcription terminators may be used to enhance the efficiency of expression of a viral based system.

[0327] Where long-term, high-yield production of recombinant proteins is required, stable expression is preferred. Generally a selectable marker gene is used whereby following transfection, cells are grown for 1-2 days in an enriched media and then transferred to a medium containing a selective medium in which cells containing the corresponding selectable marker, for example, antibiotic resistance can be screened. The result is that cells that have stably integrated the plasmid into their chromosomes grow and form foci that in turn can be cloned and expanded into cell lines. The herpes simplexvirus thymidine kinase, hypoxanthineguanine phosphoribosyltransferase and adenine phosphoribosyltransferase genes are examples of genes that can be employed in tk-, hgprt- or aprT- cells, respectively, thereby providing appropriate selection systems. The following genes: dhfr, which confers resistance to methotrexate; gpt, which confers resistance to mycophenolic acid; neo, which confers resistance to the aminoglycoside G- 418; and hygro, which confers resistance to hygromycin are examples of genes that can be used in anti-metabolite selection systems.

[0328] An antigen binding protein of the invention may be purified by a recombinant expression system by known methods including ion exchange chromatography, affinity chromatography (especially affinity for the specific antigens Protein A or Protein G) and gel filtration column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Purification may be facilitated or assisted by providing the antigen binding protein in the form of a fusion protein.

[0329] Large quantities of the antigen binding proteins of the invention may be produced by a scalable process starting with a pilot expression system in a research laboratory that is scaled up to an analytical scale bioreactor (typically from 5L to about 50L bioreactors) or production scale bioreactors (for example, but not limited to 75L, 100L, 150L, 300L, or 500L). Desirable scalable processes include those wherein there are low to undetectable levels of aggregation as measured by HPSEC or rCGE, typically no more than 5% aggregation by weight of protein down to no more than 0.5% by weight aggregation of protein. Additionally or alternatively, undetectable levels of fragmentation measured in terms of the total peak area representing the intact antigen binding protein may be desired in a scalable process so that at least 80% and as much as 99.5% or higher of the total peak area represents intact antigen binding protein. In other embodiments, the scalable process of the invention produces antigen binding proteins at production efficiency of about from 10 mg / L to about 300 mg / L or higher.

[0330] Various techniques have been developed for the production of antibody fragments including proteolytic digestion of intact antibodies and recombinant expression in host cells. With regard to the latter, as described below, Fab, Fv and scFv antibody fragments can all be expressed in and secreted from E. coli, antibody fragments can be isolated from the antibody phage libraries and Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments. In another approach, F(ab')2 fragments are isolated directly from recombinant host cell culture.

[0331] In another embodiment there is provided a vector including a nucleic acid described above. The vector may, for example, be in the form of a plasmid, cosmid, viral particle, or phage. The appropriate nucleic acid sequence may be inserted into the vector by a variety of procedures. In general, DNA is inserted into an appropriate restriction endonuclease site(s) using techniques known in the art. Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan.

[0332] The antigen binding protein may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. In general, the signal sequence may be a component of the vector, or it may be a part of the antigen binding protein-encoding DNA that is inserted into the vector. The signal sequence may be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders. For yeast secretion the signal sequence may be, e.g., the yeast invertase leader, alpha factor leader, or acid phosphatase leader or the C. albicans glucoamylase leader. In mammalian cell expression, mammalian signal sequences may be used to direct secretion of the protein, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders.

[0333] Polynucleotide sequences encoding polypeptide components of the antigen binding protein of the invention can be obtained using standard recombinant techniques as described above. Polynucleotides can be synthesized using nucleotide synthesizer or PCR techniques. Once obtained, sequences encoding the polypeptides are inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in prokaryotic hosts. Many vectors that are available and known in the art can be used for the purpose of the present invention. Selection of an appropriate vector will depend mainly on the size of the nucleic acids to be inserted into the vector and the particular host cell to be transformed with the vector. Each vector contains various components, depending on its function (amplification or expression of heterologous polynucleotide, or both) and its compatibility with the particular host cell in which it resides.

[0334] In general, plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts. Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells, as well as marking sequences which are capable of providing phenotypic selection in transformed cells. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication from the plasmid pBR322, which contains genes encoding ampicillin (Amp) and tetracycline (Tet) resistance and thus provides easy means for identifying transformed cells, is suitable for most Gram-negative bacteria, the 2μm plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells. pBR322, its derivatives, or other microbial plasmids or bacteriophage may also contain, or be modified to contain, promoters which can be used by the microbial organism for expression of endogenous proteins.

[0335] In addition, phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as transforming vectors in connection with these hosts. For example, bacteriophage such as λGEM.TM.-11 may be utilized in making a recombinant vector which can be used to transform susceptible host cells such as E. coli LE392.

[0336] The expression vector of the invention may comprise two or more promoter- cistron (a cistron being segment of DNA that contains all the information for production of single polypeptide) pairs. A promoter is an untranslated regulatory sequence located upstream (5') to a cistron that modulates its expression. Prokaryotic promoters typically fall into two classes, inducible and constitutive. Inducible promoter is a promoter that initiates increased levels of transcription of the cistron under its control in response to changes in the culture condition, e.g. the presence or absence of a nutrient or a change in temperature.

[0337] A large number of promoters recognized by a variety of potential host cells are well known. The selected promoter can be operably linked to cistron DNA encoding the light or heavy chain by removing the promoter from the source DNA via restriction enzyme digestion and inserting the isolated promoter sequence into the vector of the invention. Both the native promoter sequence and many heterologous promoters may be used to direct amplification and / or expression of the target genes. In some embodiments, heterologous promoters are utilized, as they generally permit greater transcription andhigher yields of expressed target gene as compared to the native target polypeptide promoter.

[0338] Promoters recognized by a variety of potential host cells are well known. Promoters suitable for use with prokaryotic hosts include the PhoA promoter, the β- galactamase and lactose promoter systems, alkaline phosphatase, a tryptophan (trp) promoter system and hybrid promoters such as the tac or the trc promoter. Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding an antigen binding protein of the invention. However, other promoters that are functional in bacteria (such as other known bacterial or phage promoters) are suitable as well. Their nucleotide sequences have been published, thereby enabling a skilled person operably to ligate them to cistrons encoding the target light and heavy chains using linkers or adaptors to supply any required restriction sites.

[0339] In one aspect of the invention, each cistron within the recombinant vector comprises a secretion signal sequence component that directs translocation of the expressed polypeptides across a membrane. In general, the signal sequence may be a component of the vector, or it may be a part of the target polypeptide DNA that is inserted into the vector. The signal sequence selected for the purpose of this invention should be one that is recognized and processed (i.e. cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the signal sequences native to the heterologous polypeptides, the signal sequence is substituted by a prokaryotic signal sequence selected, for example, from the group consisting of the alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II (STII) leaders, LamB, PhoE, PeIB, OmpA and MBP. In one embodiment of the invention, the signal sequences used in both cistrons of the expression system are STII signal sequences or variants thereof.

[0340] In another aspect, the production of the immunoglobulins according to the invention can occur in the cytoplasm of the host cell, and therefore does not require the presence of secretion signal sequences within each cistron. In that regard, immunoglobulin light and heavy chains are expressed, folded and assembled to form functional immunoglobulins within the cytoplasm. Certain host strains (e.g., the E. coli trxB strains) provide cytoplasm conditions that are favourable for disulfide bond formation, thereby permitting proper folding and assembly of expressed protein subunits.

[0341] The present invention provides an expression system in which the quantitative ratio of expressed polypeptide components can be modulated in order to maximize the yield of secreted and properly assembled antigen binding proteins of the invention. Such modulation is accomplished at least in part by simultaneously modulating translational strengths for the polypeptide components.

[0342] In terms of expression in eukaryotic host cells, the vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0343] A vector for use in a eukaryotic host cell may also contain a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide of interest. The heterologous signal sequence selected preferably is one that is recognized and processed {i.e., cleaved by a signal peptidase) by the host cell. In mammalian cell expression, mammalian signal sequences as well as viral secretory leaders, for example, the herpes simplex gD signal, are available.

[0344] The DNA for such precursor region is ligated in reading frame to DNA encoding the antibody.

[0345] Generally, an origin of replication component is not needed for mammalian expression vectors. For example, the SV40 origin may typically be used only because it contains the early promoter.

[0346] Expression and cloning vectors will typically contain a selection gene, also termed a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.

[0347] One example of a selection scheme utilizes a drug to arrest growth of a host cell. Those cells that are successfully transformed with a heterologous gene produce a protein conferring drug resistance and thus survive the selection regimen. Examples of such dominant selection use the drugs neomycin, mycophenolic acid and hygromycin.

[0348] An example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the antigen binding protein- encoding nucleic acid, such as DHFR or thymidine kinase, metallothionein-I and -II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc. An appropriate host cell when wild-type DHFR is employed is the CHO cell line deficient in DHFR activity (e.g., ATCC CRL-9096), prepared and propagated. For example, cells transformed with the DHFR selection gene are first identified by culturing all of the transformants in a culture medium that contains methotrexate (Mtx), a competitive antagonist of DHFR. Alternatively, host cells (particularly wild-type hosts that contain endogenous DHFR) transformed or co- transformed with DNA sequences encoding an antibody, wild-type DHFR protein, and another selectable marker such as aminoglycoside 3 '-phosphotransferase (APH) can be selected by cell growth in medium containing a selection agent for the selectable marker such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418.

[0349] Expression and cloning vectors usually contain a promoter operably linked to the antigen binding protein encoding nucleic acid sequence to direct mRNA synthesis. Promoters recognized by a variety of potential host cells are well known.

[0350] Eukaryotic genes generally have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CNCAAT region where N may be any nucleotide. At the 3' end of most eukaryotic genes is an AATAAA sequence that may be the signal for addition of the poly A tail to the 3' end of the coding sequence. All of these sequences are suitably inserted into eukaryotic expression vectors.

[0351] Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase or other glycolytic enzymes including enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3 -phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.

[0352] Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions foralcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3- phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization.

[0353] Antigen binding protein transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems.

[0354] Transcription of a DNA encoding the antigen binding protein by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancer sequences include those known from mammalian genes (globin, elastase, albumin, α- fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0355] Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding an antigen binding protein.

[0356] In another embodiment there is provided a cell including a vector or nucleic acid described above. The nucleic acid molecule or vector may be present in the genetically modified host cell or host either as an independent molecule outside the genome, preferably as a molecule which is capable of replication, or it may be stably integrated into the genome of the host cell or host.

[0357] The host cell of the present invention may be any prokaryotic or eukaryotic cell.

[0358] Examples of prokaryotic cells are those generally used for cloning like E. coli or Bacillus subtilis. Furthermore, eukaryotic cells comprise, for example, fungal or animal cells.

[0359] Examples for suitable fungal cells are yeast cells, preferably those of the genus Saccharomyces and most preferably those of the species Saccharomyces cerevisiae.

[0360] Examples of animal cells are, for instance, insect cells, vertebrate cells, preferably mammalian cells, such as e.g. HEK293, NSO, CHO, MDCK, U2-OS, Hela, NIH3T3, MOLT-4, Jurkat, PC-12, PC-3, IMR, NT2N, Sk-n-sh, CaSki, C33A. These host cells, e.g. CHO-cells, may provide post- translational modifications to the antibody molecules of the invention, including leader peptide removal, folding and assembly of H (heavy) and L (light) chains, glycosylation of the molecule at correct sides and secretion of the functional molecule.

[0361] Further suitable cell lines known in the art are obtainable from cell line depositories, like the American Type Culture Collection (ATCC).

[0362] In another embodiment there is provided an animal including a cell described above. In certain embodiments, animals and tissues thereof containing a transgene are useful in producing the antigen binding proteins of the invention. The introduction of the nucleic acid molecules as transgenes into non-human hosts and their subsequent expression may be employed for the production of the antigen binding proteins, for example, the expression of such a transgene in the milk of the transgenic animal provide for means of obtaining the antigen binding proteins in quantitative amounts. Useful transgenes in this respect comprise the nucleic acid molecules of the invention, for example, coding sequences for the antigen binding proteins described herein, operatively linked to promoter and / or enhancer structures from a mammary gland specific gene, like casein or beta-lactoglobulin. The animal may be non-human mammals, most preferably mice, rats, sheep, calves, dogs, monkeys or apes. Assaying binding of antigen binding proteins

[0363] It will be apparent to the skilled artisan that antigen binding proteins of the present invention bind to an antigen. Methods for assessing binding to a protein are known in the art, e.g., as described in Scopes (In: Protein purification: principles and practice, Third Edition, Springer Verlag, 1994). Such a method generally involves immobilising theantigen binding protein and contacting it with labelled antigen. Following washing to remove non-specific bound protein, the amount of label and, as a consequence, bound antigen is detected. Of course, the antigen binding protein can be labelled and the antigen immobilised. Panning-type assays can also be used. Alternatively, or additionally, surface plasmon resonance assays can be used.

[0364] Optionally, the dissociation constant (Kd), association constant (Ka) and / or affinity constant (KD) of an immobilised antigen binding protein for its cognate antigen or an epitope thereof is determined. The "Kd" or "Ka" or “KD” for antigen is in one example measured by a radiolabelled or fluorescently-labelled antigen / ligand binding assay. In the case of a “Kd”, this assay equilibrates the antigen binding protein with a minimal concentration of labelled antigen or epitope thereof in the presence of a titration series of unlabelled antigen. Following washing to remove unbound antigen or epitope thereof, the amount of label is determined, which is indicative of the Kd of the protein.

[0365] According to another example the Kd, Ka or KD is measured by using surface plasmon resonance assays, e.g., using BIAcore surface plasmon resonance (BIAcore, Inc., Piscataway, NJ) with immobilised antigen or a region thereof or immobilised antigen binding protein. Affibodies

[0366] “Affibody” or “affibodies” as used herein refers to an antigen binding protein based on a three-helix bundle structure with a Z domain based on a region of a surface protein of Staphylococcal Protein A. Affibodies are small, engineered protein molecules that have been used in various applications such as imaging, diagnostics and therapeutics.

[0367] In some embodiments, the protein has a molecular weight from about 4 to about 10 kDa. Preferably from about 5 and to about 8 kDa. More preferably from about 6 to about 7 kDa. In some embodiments, affibodies according to the present disclosure have a molecular weight from about 6 to about 7 kDa. Radionuclides

[0368] “Radionuclide” or “radioisotope” as used herein refers to an unstable form of a chemical element that releases radiation as it breaks down and becomes more stable.Radionuclides may occur in nature or be made in a laboratory. Preferably, the radionuclides may be used in imaging tests or in treatment.

[0369] Examples of suitable radionuclides may include26Al,38K,66Ga,67Ga,68Ga,68Ge,61Cu,62Cu,64Cu,67Cu,51Mn,52Mn,44Sc,47Sc,45Ti,52Fe,57Ni,83Sr,89Sr,90Sr,51Cr,55Co,60Co, ,72As,89Zr,81Rb,82mRb,94mTc,99mTc,86Y,90Y,90Nb,103Pd,111In,110mIn,114mIn,,186Re,188Re,191mIr,192Ir,193mPt,195mPt,201Tl,225Ac,203Pb,212Pb,211At,211Bi,212Bi,213Bi,211Fr,211Po,213Po,223Ra,224Ra, and227Th. Other radionuclides known in the art are contemplated.

[0370] In some embodiments, the radionuclide may be an alpha particle emitter. In some alternative embodiments, the radionuclide may be a beta particle emitter. In some alternative embodiments, the radionuclide may be a positron emitter. In some alternative embodiments, the radionuclide may be a gamma emitter. In some alternative embodiments, the radionuclide is an Auger electron emitter.

[0371] The skilled person will be aware of the therapeutic or diagnostic potential of these radionuclides. As used herein the term “diagnostic radionuclide” or “diagnostic radio isotope” refer to a radionuclide useful in diagnostic methods, typically capable of use as a contrast agent for an imaging technique. As used herein the term “therapeutic radionuclide” refers to a radionuclide useful in therapy, and typically possessing post- administration cytotoxic activity.

[0372] The skilled addressee will also be able to determine which radionuclides may be used for therapy and which may be used for diagnosis. For example, complexes of213Bi are typically therapeutic agents, while complexes of68Ga and111In are typically diagnostic agents. Non-radioactive isotopes

[0373] “Non-radioactive isotope” or “cold isotope” as used herein refers to a stable form of a chemical element that does not release radiation. Preferably, non-radioactive isotopes may be used in imaging tests that do not require radiation therapy. For example, contrast agents in X-ray / CT / MRI or as mass spectrometry tags in single-cell mass cytometry.

[0374] In some embodiments, the non-radioactive isotope is an isotope of Al, Ga, Cu, Mn, Sc, Sr, Co, Zr, Rb, Tc, Y, In, Lu, Sm, Ac, At, Pb, Bi, Fr, K, Ti, Nb, Ta, Re, Fe, Rh, Ni, Pt, Au, Tl, Ge, Sn, As, Po, Ir, La, Pr, Pm, Tb, Dy, Ho, Tm, Yb, Lu, Th, Er, Cr, Cs or Ra. Examples of suitable non-radioactive isotopes include Bi, Ga, In, As, Sb, Pb, Cu, Gd or Ba. Other non-radioactive isotopes known in the art are contemplated.

[0375] The skilled addressee will also be able to determine which non-radioactive isotopes may be used for diagnosis. For example, complexes of Gd are typically used as MRI contrast agents, while complexes of Bi may be used in X-ray / CT contrast agents or as mass spectrometry tags in single-cell mass cytometry. Conditions to be treated

[0376] The present invention provides methods for identifying, detecting or imaging cancers in vivo and / or classifying such cancers for treatment. The antigen binding proteins, metal coordinated antigen binding proteins or compositions comprising the same, of the invention have particularly utility in the manufacture of medicaments (e.g., antibodies, antibody-drug conjugates).

[0377] Examples of cancers which may be treated according to the methods of the present invention include, pre-neoplastic and neoplastic diseases. Broad examples include breast tumours, colorectal tumours, adenocarcinomas, mesothelioma, bladder tumours, prostate tumours, germ cell tumour, hepatoma / cholongio, carcinoma, neuroendocrine tumours, pituitary neoplasm, small round cell tumour, squamous cell cancer, melanoma, atypical fibroxanthoma, seminomas, nonseminomas, stromal leydig cell tumours, Sertoli cell tumours, skin tumours, kidney tumours, testicular tumours, brain tumours, ovarian tumours, stomach tumours, oral tumours, bladder tumours, bone tumours, cervical tumours, esophageal tumours, laryngeal tumours, liver tumours, lung tumours, vaginal tumours and Wilm's tumour.

[0378] Examples of particular cancers include but are not limited to adenocarcinoma, adenoma, adenofibroma, adenolymphoma, adontoma, AIDS related cancers, acoustic neuroma, acute lymphocytic leukaemia, acute myeloid leukaemia, adenocystic carcinoma, adrenocortical cancer, agnogenic myeloid metaplasia, alopecia, alveolar soft- part sarcoma, ameloblastoma, angiokeratoma, angiolymphoid hyperplasia with eosinophilia, angioma sclerosing, angiomatosis, apudoma, anal cancer, angiosarcoma,aplastic anaemia, astrocytoma, ataxia-telangiectasia, basal cell carcinoma (skin), bladder cancer, bone cancers, bowel cancer, brain stem glioma, brain and CNS tumours, breast cancer, branchioma, CNS tumours, carcinoid tumours, cervical cancer, childhood brain tumours, childhood cancer, childhood leukaemia, childhood soft tissue sarcoma, chondrosarcoma, choriocarcinoma, chronic lymphocytic leukaemia, chronic myeloid leukaemia, colorectal cancers, cutaneous T-cell lymphoma, carcinoma (e.g. Walker, basal cell, basosquamous, Brown-Pearce, ductal, Ehrlich tumour, Krebs 2, Merkel cell, mucinous, non-small cell lung, oat cell, papillary, scirrhous, bronchiolar, bronchogenic, squamous cell, and transitional cell), carcinosarcoma, cervical dysplasia, cystosarcoma phyllodies, cementoma, chordoma, choristoma, chondrosarcoma, chondroblastoma, craniopharyngioma, cholangioma, cholesteatoma, cylindroma, cystadenocarcinoma, cystadenoma, dermatofibrosarcoma- protuberans, desmoplastic-small-round-cell- tumour, ductal carcinoma, dysgerminoam, endocrine cancers, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, extra-hepatic bile duct cancer, eye cancer, eye: melanoma, retinoblastoma, fallopian tube cancer, fanconi anaemia, fibroma, fibrosarcoma, gall bladder cancer, gastric cancer, gastrointestinal cancers, gastrointestinal-carcinoid-tumour, genitourinary cancers, germ cell tumours, gestationaltrophoblastic- disease, glioma, gynaecological cancers, giant cell tumours, ganglioneuroma, glioma, glomangioma, granulosa cell tumour, gynandroblastoma, haematological malignancies, hairy cell leukaemia, head and neck cancer, hepatocellular cancer, hereditary breast cancer, histiocytosis, Hodgkin's disease, human papillomavirus, hydatidiform mole, hypercalcemia, hypopharynx cancer, hamartoma, hemangioendothelioma, hemangioma, hemangiopericytoma, hemangiosarcoma, hemangiosarcoma, histiocytic disorders, histiocytosis malignant, histiocytoma, hepatoma, hidradenoma, hondrosarcoma, immunoproliferative small, opoma, ontraocular melanoma, islet cell cancer, Kaposi's sarcoma, kidney cancer, langerhan's cell-histiocytosis, laryngeal cancer, leiomyosarcoma, leukaemia, li-fraumeni syndrome, lip cancer, liposarcoma, liver cancer, lung cancer, lymphedema, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leigomyosarcoma, leukaemia (e.g. b-cell, mixed cell, null-cell, t-cell, t-cell chronic, htlv-ii-associated, lymphangiosarcoma, lymphocytic acute, lymphocytic chronic, mast-cell and myeloid), leukosarcoma, leydig cell tumour, liposarcoma, leiomyoma, leiomyosarcoma, lymphangioma, lymphangiocytoma, lymphagioma, lymphagiomyoma, lymphangiosarcoma, male breast cancer, malignant- rhabdoid-tumour-of-kidney, medulloblastoma, melanoma, Merkel cell cancer,mesothelioma, metastatic cancer, mouth cancer, multiple endocrine neoplasia, mycosis fungoides, myelodysplastic syndromes, myeloma, myeloproliferative disorders, malignant carcinoid syndrome carcinoid heart disease, medulloblastoma, meningioma, melanoma, mesenchymoma, mesonephroma, mesothelioma, myoblastoma, myoma, myosarcoma, myxoma, myxosarcoma, nasal cancer, nasopharyngeal cancer, nephroblastoma, neuroblastoma, neurofibromatosis, Nijmegen breakage syndrome, non- melanoma skin cancer, non-small-cell-lung-cancer-(nsclc), neurilemmoma, neuroblastoma, neuroepithelioma, neurofibromatosis, neurofibroma, neuroma, neoplasms (e.g. bone, breast, digestive system, colorectal, liver), ocular cancers, oesophageal cancer, oral cavity cancer, oropharynx cancer, osteosarcoma, ostomy ovarian cancer, pancreas cancer, paranasal cancer, parathyroid cancer, parotid gland cancer, penile cancer, peripheral- neuroectodermal-tumours, pituitary cancer, polycythemia vera, prostate cancer, osteoma, osteosarcoma, ovarian carcinoma, papilloma, paraganglioma, paraganglioma nonchromaffin, pinealoma, plasmacytoma, protooncogene, rare-cancers-and-associated- disorders, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, Rothmund-Thomson syndrome, reticuloendotheliosis, rhabdomyoma, salivary gland cancer, sarcoma, schwannoma, Sezary syndrome, skin cancer, small cell lung cancer (sclc), small intestine cancer, soft tissue sarcoma, spinal cord tumours, squamous-cell-carcinoma-(skin), stomach cancer, synovial sarcoma, sarcoma (e.g. Ewing's experimental, Kaposi's and mast-cell sarcomas), Sertoli cell tumour, synovioma, testicular cancer, thymus cancer, thyroid cancer, transitional-cell-cancer-(bladder), transitional-cell-cancer-(renal-pelvis- / -ureter), trophoblastic cancer, teratoma, theca cell tumour, thymoma, trophoblastic tumour, urethral cancer, urinary system cancer, uroplakins, uterine sarcoma, uterus cancer, vaginal cancer, vulva cancer, Waldenstrom' s-macroglobulinemia and Wilms' tumour.

[0379] By "therapeutically effective amount" is meant a dose that produces the effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques. As is known in the art and described above, adjustments for systemic versus localised delivery, age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art.

[0380] The objective or outcome of treatment may be to reduce the number of cancer cells; reduce the primary tumour size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumour metastasis; inhibit, to some extent, tumour growth; and / or relieve to some extent one or more of the symptoms associated with the disorder.

[0381] Efficacy of treatment can be measured by assessing the duration of survival, time to disease progression, the response rates (RR), duration of response, and / or quality of life.

[0382] The objective or outcome of treatment may be any one or more of the following: - to reduce the number of cancer cells; - reduce the primary tumour size; - inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; - inhibit (i.e., slow to some extent and preferably stop) tumour metastasis; - inhibit, to some extent, tumour growth; - relieve to some extent one or more of the symptoms associated with the disorder.

[0383] In one embodiment, subjects requiring treatment include those having a benign, pre-cancerous, non-metastatic tumour.

[0384] The cancer may be a solid or a “liquid” tumour. In other words, the cancer may be growth in a tissue (carcinoma, sarcoma, adenomas etc) or it may be a cancer present in bodily fluid such as in blood or bone marrow (e.g., lymphomas and leukaemias).

[0385] The antigen binding protein may bind, or specifically bind to, an antigen that is a tumour antigen. Tumour antigens or neo-antigens may be antigens that are presented by MHC I or MHC II molecules on the surface of tumour cells. These antigens may sometimes be presented by tumour cells and never by the normal cells. In this case, they are called tumour-specific antigens (TSAs) and, in general, result from a tumour-specific mutation. More common are antigens that are presented by tumour cells and normal cells, and they are called tumour-associated antigens (TAAs). A TAA is not unique to a tumourcell and instead is also expressed on a normal cell under conditions that fail to induce a state of immunologic tolerance to the antigen. The expression of the antigen on the tumour may occur under conditions that enable the immune system to respond to the antigen. TAAs may be antigens that are expressed on normal cells during foetal development when the immune system is immature and unable to respond or they may be antigens that are normally present at extremely low levels on normal cells but which are expressed at much higher levels on tumour cells. Cytotoxic T lymphocytes that recognise these antigens may be able to destroy the tumour cells before they proliferate or metastasise. Tumour antigens may also be on the surface of the tumour in the form of, for example, a mutated receptor, in which case they may be recognised by B cells.

[0386] Non-limiting examples of TSA or TAA antigens include the following: Differentiation antigens such as MART-1 / MelanA (MART-I), gp 100 (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumour-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumour-suppressor genes such as p53, Ras, HER-2 / neu; unique tumour antigens resulting from chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus antigens EBVA and the human papillomavirus (HPV) antigens E6 and E7. Other large, protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23Hl, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, 20 CD68\Pl, C0-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-C0-1, RCASl, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP, and TPS.

[0387] The antigen binding protein may bind, or specifically bind to, an antigen selected from the group consisting of any surface expressed antigens. Exemplary target antigens may comprise but are not limited to: CD33 (Siglec-3), CD123 (IL3RA), CD135 (FLT-3), CD44 (HCAM), CD44V6, CD47, CD184 (CXCR4), CLEC12A (CLL1), LeY, FRβ, MICA / B, CD305 (LAIR-1), CD366 (TIM-3), CD96 (TACTILE), CD133, CD56, CD29 (ITGB1), CD44 (HCAM), CD47 (IAP), CD66 (CEA), CD112 (Nectin2), CD117 (c-Kit), CD133, CD146 (MCAM), CD155 (PVR), CD171 (L1CAM), CD200 (OX-2), CD221 (IGF1), CD227(MUC1), CD243 (MRD1), CD246 (ALK), CD271 (LNGFR), CD19, CD20, GD2, and EGFR. Other target antigens include TCR, sugars, lipids, carbohydrates or any other molecule expressed on the surface of the target cell.

[0388] Exemplary antigen binding proteins are those described herein and known in the art, including, but not limited to, trastuzumab (for binding to Her2), alemtuzumab (for binding CD52), bevacizumab (for binding VEGF-A), brentuximab (for binding CD30, gemtuzumab (for binding CD33), ipilimumab (for binding VTLA-4), ibritumomab (for binding CD20), panitumumab (for binding EGFR), cetuximab (for binding EGFR), rituximab (for binding CD20), and fragments thereof. Methods of imaging, diagnosis, prognosis and monitoring

[0389] Also described is a method of imaging a cancer, comprising administering to a subject in need thereof a metal coordinated antigen binding protein of the present disclosure or, or a pharmaceutical comprising an antigen binding protein or a metal coordinated antigen binding protein of the present disclosure, and imaging the cancer.

[0390] Also described is a method of diagnosing, monitoring or prognosing a cancer in an individual in need thereof, the method comprising: (a) administering a metal coordinated antigen binding protein of the present disclosure to the individual, wherein the metal is a radionuclide; and (b) detecting the radionuclide; whereby detection of the radionuclide enables an image of the cancer (if present) to be created to thereby diagnose, monitor or prognose the cancer.

[0391] In these methods of diagnosing, monitoring or prognosing a cancer, the detecting step may comprise subjecting the subject to an imaging technique. The imaging technique may allow imaging of an area comprising a cancer to determine the presence or change in concentration of radionuclide.

[0392] The skilled person will be familiar with methods for selecting suitable diagnostic agents for use with the antigen binding proteins or metal coordinated antigen binding proteins of the invention, including radionuclides for use in radioimaging for diagnosingconditions described herein. Further, the skilled person will be familiar with imaging techniques for use in conjunction with the diagnostic reagents described herein.

[0393] In some embodiments, the diagnostic methods (including imaging methods) comprise subjecting the subject to positron emission tomography (PET) imaging, preferably immuno-PET imaging. PET imaging is a functional imaging technique applied in nuclear medicine, whereby a three-dimensional image (e.g. of functional processes) in the body is produced. The system detects pairs of gamma rays emitted indirectly by a positron-emitting radionuclide, which is introduced into the body in form of a pharmaceutical compound, for example, a metal coordinated antigen binding protein.

[0394] In some embodiments, the diagnostic method comprises subjecting the subject to single-photon emission computed tomography (SPECT), preferably wherein the radionuclide of diagnostic potential is gallium (68Ga) or indium (111In).

[0395] In some embodiments, the diagnostic method of the invention may be used in combination with another diagnostic method, such as magnetic resonance imaging (MRI), radiography, ultrasound, elastography, photoacoustic imaging, tomography (including computed tomography) and echocardiography; preferably magnetic resonance imaging (MRI) and tomography (including computed tomography). Kits

[0396] The present invention additionally comprises a kit comprising one or more of the following: (i) an antigen binding protein of the invention or expression construct(s) encoding same; (ii) a cell of the invention; (iii) a complex of the invention; or (iii) a pharmaceutical composition of the invention.

[0397] In the case of a kit for a use described herein, the kit can additionally comprise a pharmaceutically acceptable carrier.

[0398] Optionally a kit of the invention is packaged with instructions for use in a method described herein according to any example.

[0399] It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention. Examples

[0400] The present disclosure describes a strategy to attach a metal to an antigen binding protein (such as an antibody or nanobody), thereby abolishing the need for a traditional bifunctional linker. This surprisingly provides highly stable metal-complexes that bind the metal with residues of the antigen binding protein, preventing the metal dissociating from said protein. This strategy provides compounds that could have a broad range of applications across targeted radiotherapy, diagnostics and chemical biology.

[0401] The examples described herein include the coordination of multiple antibodies using multiple metals, highlighting the applicability of the described invention to various antigen binding proteins and various metals. Example 1 – Materials and methods Materials

[0402] All plasmids, with N-terminal periplasmic leader sequence and C-terminal His6 tag were obtained from Twist Bioscience, USA. Receptor binding domain (aa 319-541) of the SARS-CoV2 spike protein, His6 tagged (RP-87678) was purchased from Invitrogen, Thermo Fisher Scientific, Australia. Recombinant Human Her2 / ERBB2 protein (ECD, His Tag, 10004-H08H) was purchased from Sino Biological, China. Gastrodenol (bismuth tripotassium dicitrate), glutathione (~98% reduced), Tris, and IPTG were purchased from AK Scientific, USA. Indium(III) chloride, Gallium(III) nitrate hydrate, ammonium acetate (LC-MS grade), imidazole, and human apo-transferrin were purchased from Sigma Aldrich, Australia. TCEP.HCl was purchased from AmBeed, USA. HEPES (free acid) and MES (free acid) monohydrate were purchased from Astral Scientific, Australia.15N- labelled ammonium chloride (>99%) was purchased from Martek Isotopes LLC, USA. Kanamycin was purchased from AG Scientific, USA. Other chemicals and buffercompositions, such as sodium, potassium, magnesium salts, and EDTA were purchased from Sigma Aldrich, Australia. Bacterial media components were purchased from Gibco, Thermo Fisher Scientific, USA. Series S CM5 chip, amine coupling kit, and HisTrapTM(5 mL) column were purchased from Cytiva, USA. Pre-cast SDS-PAGE and native-PAGE gels (Novex Bis-Tris Plus Mini Protein Gels) were purchased from Invitrogen, Thermo Fisher Scientific, USA. Unstrained protein standard, broad range (10-200 KDa) was purchased from New England Biolabs, USA. Expression and purification of nanobodies

[0403] Nanobody (Nb) sequences with N-terminal periplasmic leader sequence (MKYLLPTAAAGLLLLAAQPAMA – SEQ ID NO:15) and C-terminal His6 tag were cloned into the pET-29b(+) expression vector (Twist Bioscience, USA) and transformed into BL21 (DE3) strain of E. coli. The expression protocol was followed as described by Schoof et al. with minor changes (Science, 370, 1473-1479 (2020)). Isolated single colonies were grown in Luria Broth (LB) overnight, transferred to Terrific Broth and grown at 37oC with gentle shaking until the OD reached 0.6-0.8. This was followed by induction of overexpression using 1 mM IPTG for 18-20 hours at 25oC with gentle shaking. The E. coli cells were harvested (centrifuged at 5000 x g, 30 minutes, 4oC) and resuspended in lysis buffer (200 mM Tris, pH 8.0, 500 mM sucrose, 0.5 mM EDTA) for 30 minutes on ice. This was followed by a 45-minute osmotic shock with a two-fold volume addition of cold water on ice.150 mM NaCl, 2 mM MgCl2and 40 mM imidazole were added to the lysate before centrifugation at 17,000 x g for 30 minutes at 4 °C to separate cell debris from the periplasmic fraction. The soluble fraction was then loaded onto a 5 mL HisTrapTMHP column (Cytiva) which had been equilibrated with binding buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 30 mM imidazole). The column was then washed with 5 column volumes of binding buffer. Bound proteins were then eluted using 5 column volumes of elution buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 500 mM imidazole). The nanobodies were desalted using desalting buffer (20 mM Tris, pH 7.5, 150 mM NaCl) and further concentrated using a 3 kDa MWCO centrifugal filter unit (Amicon®-Ultra 15). Nanobody solutions were aliquoted, and flash frozen in liquid nitrogen for long-term storage at -80oC. Expression of uniformly15N-labelled nanobody mNb6-3C

[0404] Cells were grown at 37oC until the OD reached 0.6-0.8 in Terrific Broth. Cells were centrifuged (5000 x g, 30 minutes, 4oC) and resuspended in15N-minimal media (50mM NaHPO4, 25 mM KH2PO4, 10 mM NaCl, pH 8.0) supplemented with 5 mM MgSO4, 0.2 mM CaCl2, 0.25% metal mix,15NH4Cl (1g / L) as the only nitrogen source, and 1% glucose. Cells were grown in15N-minimal media at 37oC for 30 minutes before induction using 1 mM IPTG and overexpression for 18-20 hours at 25oC. Cell lysis, extraction, and purification were performed as mentioned above. The labelled nanobody was buffer exchanged into NMR buffer (20 mM MES, pH 6.5, 150 mM NaCl, 10% D2O), flash frozen in liquid nitrogen and stored at -80oC. Expression and purification of mCherry

[0405] The mCherry sequence with C-terminal His6 tag was cloned into the pET-29b(+) expression vector (Twist Bioscience, USA) and transformed into the BL21 (DE3) strain of E. coli. The expression protocol was followed as described by Wang et al (Protein Sci. 30, 2298-2309 (2021)) with minor changes. Isolated single colonies were grown in 10 mL Luria Broth (LB) overnight, transferred to 1 L LB and grown at 37oC until an OD of 0.8 was reached. This was followed by induction of overexpression using 0.2 mM IPTG for 18 hours at 18oC. The E. coli cells were harvested (centrifuged at 5000 x g, 30 minutes, 4oC) and resuspended in binding buffer (100 mM Tris, pH 8, 5% glycerol, 150 mM NaCl, 20 mM imidazole). Cells were lysed by sonication (Omni Sonic Ruptor 400 Ultrasonic homogenizer) three times at 50% power for 30 seconds on ice, before centrifugation at 17,000 x g for 30 minutes at 4 °C to separate cell debris. The soluble fraction was then loaded onto a 5 mL HisTrapTMHP column (Cytiva) which had been equilibrated with binding buffer. The column was then washed with 5 column volumes of the binding buffer. Bound protein was eluted using 5 column volumes of elution buffer (100 mM Tris, pH 8, 5% glycerol, 150 mM NaCl, 300 mM imidazole). The protein was desalted using desalting buffer (20 mM Tris, pH 7.5, 150 mM NaCl) and further concentrated using a 3 kDa MWCO centrifugal filter unit (Amicon®-Ultra 15). The protein solution was aliquoted, and flash frozen in liquid nitrogen for long-term storage at -80oC.

[0406] All proteins were characterized by SDS-PAGE and Intact protein MS (positive ion mode). Protein mass spectrometry Intact protein MS

[0407] Intact protein analysis was performed on Orbitrap Elite and Orbitrap Fusion™ Tribrid™ mass spectrometers (Thermo Fisher Scientific, USA) connected to a Thermo Fisher Scientific UltiMate 3000 HPLC system equipped with ZORBAX 300SB-C3, 3.5 µm, 4.6 x 50 mm HPLC column (Agilent Technologies, USA).10-20 ^M protein samples were injected using a 500 ^L / min linear gradient of solvent A (0.1% (v / v) formic acid in water) and solvent B (0.1% (v / v) formic acid in acetonitrile), ramping solvent B from 5% solvent B at the start to 80% after 12 min. The ion source was H-ESI with a static spray voltage set to 3500 V in positive ion mode. The sheath, auxiliary and sweep gasses were set to 50 Arb, 10 Arb and 1 Arb, respectively. The ion transfer tube temperature was set to 325 °C and the vaporizer temperature was set to 350 °C. The resolution of the Orbitrap MS detector was set to 120000, and the scan range set to 500-2000 m / z. Protein mass was determined by deconvolution using the program Xcalibur 3.0.63 (Thermo Fisher Scientific, USA). Native MS with prior buffer exchange

[0408] Samples for native MS were buffer exchanged into 100 mM NH4OAc (pH 7) in a 3kDa MWCO centrifugal filter unit (Amicon-Ultra 0.5) prior to analysis. Native protein analysis was performed on an Orbitrap Fusion™ Tribrid™ mass spectrometer (Thermo Fisher Scientific, USA) connected to a Thermo Fisher Scientific UltiMate 3000 HPLC system.10-20 ^M protein samples were injected using a 5 minute 100 ^L / min isocratic elution mode in 100 mM NH4OAc (pH 7). The ion source was H-ESI with a static spray voltage set to 3000 V in positive ion mode. The sheath, auxiliary and sweep gasses were set to 25 Arb, 5 Arb and 0 Arb, respectively. The ion transfer tube temperature was set to 275 °C and the vaporizer temperature was set to 50 °C. The MS detector was type as an Orbitrap with the resolution set to 120000, and the scan range set to 500-4000 m / z. Native protein mass was determined by deconvolution using the program Xcalibur 3.0.63 (Thermo Fisher Scientific, USA). Native MS with online buffer exchange

[0409] Native protein analysis was performed on an Orbitrap Fusion™ Tribrid™ mass spectrometer (Thermo Fisher Scientific, USA) connected to a Thermo Fisher Scientific UltiMate 3000 HPLC system equipped with a NativePac OBE-1, 3 µm, 2.1 x 50 mm HPLC column (Thermo Scientific, Australia).10-50 ^M protein samples were injected using a 3 minute 100 ^L / min isocratic elution mode in 100 mM NH4OAc (pH 7). The divert valvewas set to send the flow to the source for the first 1.1 minutes before sending the flow to waste for the remainder of the run. The ion source was H-ESI with a time dependent spray voltage set to 3000 V in positive ion mode for the first 2 minutes before changing to 0 for the remainder of the run. The sheath, auxiliary and sweep gasses were set to 25 Arb, 5 Arb and 0 Arb, respectively. The ion transfer tube temperature was set to 275 °C and the vaporizer temperature was set to 50 °C. The MS detector was type as an Orbitrap with the resolution set to 120000, and the scan range set to 500-4000 m / z. Native protein mass was determined by deconvolution using the program Xcalibur 3.0.63 (Thermo Fisher Scientific, USA). Metal uptake reaction optimization

[0410] Initial attempts to incorporate metal into nanobodies involved incubating mNb6- 3C in 1M and 6M guanidinium hydrochloride (GdmCl) for 20 minutes at room temperature (RT), using 25 mM TCEP at pH 3. These conditions resulted in low metal uptake, and the removal of excess denaturant (GdmCl) to obtain functional protein proved challenging. Consequently, further optimizations were focused on applying simultaneous temperature and pH shock at different time points.

[0411] Initially, for Bi(III), In(III) and Ga(III) binding optimization, the nanobodies (50-100 ^M) were reduced either at room temperature (RT) or at a temperature range 8-10 °C below their respective denaturation midpoint (Tm) in presence of 1 (pH 6.5), 5 (pH 4.0), 10 (pH 3.5) or 25 (pH 3.0) mM TCEP for a maximum of 120 minutes. Reduced nanobodies were aliquoted at different time points, followed by the addition of 1-5 equivalents of the metal salts BiBr3 and gastrodenol (for bismuth), InCl3 hexahydrate (for indium), and Ga(NO3)3 hydrate (for gallium) to the reaction solution from their respective stocks, and immediately vortexed for 60 s. This protocol was effective for the mNb6 and 2Rs15d nanobody libraries. For the Lam2 nanobody subset, gastrodenol (for Bi(III) uptake) was co-incubated with the nanobodies for up to 120 minutes. Simultaneously, the nanobodies were subjected to pH and temperature shock to optimize metal uptake. Following the reaction, the solution was quickly centrifuged, and the supernatant was collected for further analysis. For quantification of metal uptake of mNb6-3C, mNb6-3C* and mNb6- 4C by native MS, the supernatant was buffer exchanged to 100 mM NH4OAc (pH 7). For native MS of Lam2-3C-Bi using online buffer exchange (OBE) column, the supernatant was directly used for data acquisition and analyses. Detailed optimization conditions for the metal-uptake reactions on the individual nanobodies are as mentioned in Table 1.Table 1. Optimization conditions for metal uptake.

[0412] Conditions which resulted in >98% metal uptake (Table 2), without denaturing the nanobodies were subsequently used for all other experiment and assays. Table 2. Optimal conditions for metal uptake.Evaluating the stability of metal-bound nanobodies

[0413] Nanobodies were reduced and saturated with either bismuth (III), indium (III) or gallium (III) as per the protocol described above to obtain >98% metal saturation, prior to buffer exchange into 100 mM NH4OAc (pH 7). Samples were then stored at 4 °C for up to 14 days and analyzed via native MS at different time points to determine the stability of the metal-bound nanobody. Assessing metal uptake after ‘priming’ (pre-reducing) nanobodies

[0414] Nanobodies were reduced as described above and stored at 4 °C for up to 14 days prior to the addition of either bismuth (III), indium (III) or gallium (III) as per the protocol described above to obtain >98% metal saturation. At different time points, metal- bound nanobodies were then buffer exchanged into 100 mM NH4OAc (pH 7) andanalyzed via native MS to determine the ability of the pre-reduced (‘primed’) nanobodies to bind Bi(III), In(III) or Ga(III). Dialysis to assess stability of metal nanobodies

[0415] In order to monitor any potential metal dissociation and to rule out the impact of excess metal salts and TCEP on the stability of the nanobodies, the metal-nanobody conjugates were dialysed (1000:1 v / v) overnight for 18 h at 4 °C in 20 mM HEPES, pH 7.8, 150 mM NaCl. mNb6-4C-In and mNb6-4C-Ga were co-dialysed in separate 3 kDa MWCO membranes within the same dialysis tank under the above conditions to identify metal cross-contamination. Dialysed nanobodies were then examined via native MS. Glutathione (GSH) competition assay

[0416] At first, reduction and Bi(III), In(III), Ga(III) uptake reactions were performed with the nanobodies (mNb6-3C, mNb6-3C* and mNb6-4C respectively) as per the optimized protocol to obtain >98% metal saturation. The metal-bound nanobody samples were buffer exchanged to 100mM NH4OAc (pH 7). A reduced glutathione (GSH) stock was prepared in 100 mM NH4OAc and adjusted to pH 7. Competition experiments were performed with 1, 10, 20, 50, and 100 eq. of GSH with respect to the concentrations of the metal-bound nanobodies (50 ^M) for 1 h at 25 °C. Native MS of the nanobodies was measured in 100 mM NH4OAc. Metal uptake in nanobodies (%) was plotted as a function of increasing equivalents of GSH in GraphPad Prism 10 (Dotmatics, USA). One-phase exponential decay was used as the fitting function. Competition with human apo-transferrin

[0417] Similar to the GSH competition assay protocol, Bi(III), In(III), Ga(III) uptake reactions were performed on the nanobodies (mNb6-3C, mNb6-3C* and mNb6-4C respectively) as per the optimized protocol to obtain >98% metal saturation. Human apo- transferrin from Sigma-Aldrich (T4382) was resuspended in 100 mM NH4OAc (pH 7) to prepare a stock concentration of 500 ^M. Competition experiments were performed at 1, 2, and 4 eq. of apo-transferrin with respect to the concentrations of the metal-bound nanobodies (25 ^M) for 1 h at 25 °C. Native MS of the nanobodies was measured in 100 mM NH4OAc. Metal uptake in nanobodies (%) was plotted as a function of increasing equivalents of apo-transferrin in GraphPad Prism 10 (Dotmatics, USA). One-phase exponential decay was used as the fitting function.Circular dichroism (CD) spectroscopy

[0418] Secondary structures of the nanobodies were assessed by circular dichroism using a Chirascan spectropolarimeter from Applied Photophysics equipped with a temperature control module using a 0.1 cm path-length cuvette. Each CD spectrum was averaged over 2 scans, and the baseline correction was done by subtraction of the spectrum with the appropriate blank solution. Individual nanobodies were diluted to 10- 20 ^M in 20 mM phosphate buffer, pH 7.4. Scans were carried out at 25oC over a range of 200-260 nm with a 0.5 nm step size and 1 nm bandwidth. Thermal denaturation experiments were monitored at 204 nm (mNb6, Lam2 nanobodies) or 222 nm (2Rs15d nanobodies) over a temperature range of 20-80oC (1oC / min heating rate). Temperature- dependent CD data were fitted to a two-state unfolding model using Boltzmann sigmoidal equation and plotted in GraphPad Prism 10 (Dotmatics, USA) to obtain the denaturation midpoint (Tm). NMR spectroscopy

[0419] All NMR spectra were recorded at 25oC using an 800 MHz Bruker Avance NMR spectrometer equipped with a cryoprobe. [15N,1H]-HSQC spectra were recorded in a 3 mm NMR tube (sample volume 180 ^L). The NMR spectrum of mNb6-3C was acquired in NMR buffer (20 mM MES, pH 6.5, 150 mM NaCl):D2O (9:1). For bismuth uptake, the protein sample was subjected to heating at 50oC in presence of 10 mM TCEP (pH 6.5) for 15 minutes.5 equiv. of gastrodenol (bismuth source) were added and the sample was immediately vortexed. After centrifugation, the sample concentration was 0.15 mM. The [15N,1H]-HSQC spectrum of mNb6-3C-Bi was acquired with same parameters as the apo- nanobody. Isothermal Titration Calorimetry (ITC)

[0420] Sample details for ITC experiments are shown in Table 3. mCherry, Lam2, and Lam2-3C-Bi were dialyzed overnight in 20 mM HEPES, pH 8, 150 mM NaCl (ITC buffer) in 10 MWCO and 3MWCO dialysis tubings respectively from SpectraPor^. Post-dialysis, proteins were concentrated using Amicon®-Ultra 15 centrifugal filter units with 10 MWCO (mCherry) and 3 MWCO (Nbs) to 700 ^M and 100 ^M respectively.Table 3. Sample details for ITC experiments.

[0421] Titrations were performed in a TA Instruments Benchtop Nano ITC. All stocks and dilutions were prepared using the buffer from the overnight dialysis. mCherry and its relevant nanobody samples were degassed prior to each ITC experiment. All titrations were performed as titrations of mCherry (protein) into nanobodies (ligands) at 25 °C with a stirring rate of 350 rpm. Initial and final baselines were generated over 120 s. The first injection of each titration was a 1 μL blank injection, followed by 22 injections of 2 μL each, with an injection interval of 300 s. The background heat was estimated as the average heat associated with each injection in a control titration of protein into buffer and subtracted from each titration. Results were analyzed in NITPIC for baseline detection, blank subtraction, and integration of baseline-subtracted power (Methods San Diego Calif 76, 87-98 (2015)). SEDPHAT was used to fit integrated heats to the single binding site model (A + B ←→ AB, hetero association) by global fitting with the Simplex algorithm (Methods San Diego Calif 76, 137-148 (2015)). Thermograms were plotted in GUSSI (Nat Protoc 11, 882-894 (2016)). Surface Plasmon Resonance (SPR)

[0422] SPR measurements were performed with 20 mM HEPES, pH 7.8, 150 mM NaCl, 0.05% Tween-20 as running buffer. For each experiment, 100–150 μg of metal nanobodies (>98% metal saturation), mNb6-3C-Bi, mNb6-4C-In and mNb6-4C-Ga, in 10 mM sodium acetate pH 4.8 buffer were freshly immobilised on a CM5, Series S sensor chip flow-cell (Cytiva, 29104988, USA) using EDC / NHS chemistry at 25 °C. The contact time for ligand binding was set to 1800 s with a flow rate of 5 ^l / min. The unbound sites were blocked using ethanolamine hydrochloride solution. An amine coupling kit (Cytiva, BR100050, USA) was used for the coupling step. For the experiments, the response unit (RU) after coupling ranged from 300-1000. The starting concentration of the analyte, SARS-CoV-2 receptor binding (RBD), was 25-50 nM which was diluted to 0.19 nM to evaluate its binding affinity to the nanobodies. All binding experiments were conducted at20 °C in single-cycle kinetics mode. SPR experiments were performed on a Biacore 8K (Cytiva, USA). The instrument was operated using Biacore Insight 5 software (Cytiva, USA). The data were plotted and analyzed in GraphPad Prism 10 (Dotmatics, USA) using the non-linear fit ‘one site – specific binding’. KDvalues of duplicate measurements are represented as the mean value ± standard deviation. Native Gel Electrophoresis

[0423] Lam2-3C-Bi:mCherry complex was created by incubating the metal-bound nanobody (>98% metal saturation) and mCherry together in 20 mM Tris, pH 7.5, 150 mM NaCl on ice for 60 minutes. The NativePAGE Novex Bis-Tris Gel system protocol was followed with minor changes.15 uL of nanobody:protein complexes were added to 5 uL of 4x loading dye (200 mM BisTris-Cl, 200 mM NaCl, 40% glycerol, 0.004% Ponceau S, pH 7.2). The gel was washed 3 times with running buffer (50 mM BisTris, 50 mM tricine, pH 6.8) and 20 uL of sample was loaded. The gel was run in running buffer at 4oC for 60 minutes at 150 V, then a further 45 minutes at 250 V (Thermo EC250-90 powerpack). The gel was viewed under UV light on a Biorad ChemiDock MP imaging system to observe fluorescent proteins then fixed in 40% methanol, 10% acetic acid for 30 minutes and stained with 0.02% Coomassie R-250 in 30% methanol and 10% acetic acid for 30 minutes before destaining in 8% acetic acid until desired background was obtained. The gel was then visualized with a Biorad ChemiDock MP imaging system. SDS-PAGE with chemical cross linking

[0424] mNb6-3C-Bi:SARS-CoV-2 receptor binding domain (RBD) complexes were created by incubating the metal-bound nanobody (>98% metal saturation) and SARS- CoV-2 RBD together in phosphate buffered saline (PBS) on ice for 60 minutes. Nanobody:protein complexes were crosslinked by incubating with 100 equivalents of disuccinimidyl glutarate on ice for 60 minutes (relative to nanobody:protein complex concentration). The samples were then prepared and run on SDS-PAGE following usual running and staining procedures. The gel was visualised with a Biorad ChemiDock MP imaging system. Inductively Coupled Plasma Mass Spectrometry (ICP-MS)

[0425] Relevant lanes of the native PAGE gel were excised with a scalpel and soaked in concentrated nitric acid for 10 minutes. The samples were then diluted to 10% nitricacid for analysis. A ThermoFisher iCap RQ ICP-MS was used ty yyyyyyy yysmuth abundance in the samples. A calibration curve (0.1 ppb, 1 ppb, 10 ppb, and 100 ppb) of Agilent Intelliquant standard with the isotope209Bi was run prior to the diluted gel samples, with calibration curve fits of better than 0.999 accepted. Sequence listing

[0426] Sequences of nanobody constructs disclosed in this application are shown in the below table. H6 tag and mutations are indicated.Example 2 - Designing metal binding motifs in nanobodies

[0427] The conventional strategy of labelling an antigen binding protein is shown in Figure 1A. The approach of directly attaching the metal to the antigen binding protein described by the present disclosure is shown in Figure 1B.

[0428] As gallium, indium and bismuth cover the major applications in imaging and radiotherapy with PET, SPECT and TAT, these metals were targeted. A common binding site using cysteine residues based on the thiophilic properties of these elements was identified. The disulfide bond between C22 and C96 in nanobodies (Figure 1C), which, while highly conserved, is not essential for stability and activity, nor close to binding epitopes. A third cysteine residue was introduced within 4-8 Å of the native disulfide bond in nanobodies to create a triple cysteine motif for bismuth recognition (Figure 1, D and F). Because In(III) and Ga(III) form negatively charged indates and gallates with cysteine- based ligands, a nanobody mutant with two additional cysteine residues adjacent to the native disulfide was designed, creating a tetrahedral quadruple cysteine motif for gallium and indium recognition (Figure 1, E and G). Example 3 - Optimizing disulfide reduction and metal uptake

[0429] As an initial model system, a previously reported nanobody was chosen, mNb6, raised against the receptor binding domain (RBD) of the spike protein of SARS-CoV-2. Periplasmic expression in E. coli of the wildtype, the L4C (mNb6-3C) and E6C (mNb6- 3C*) single mutants, and the L4C / E6C (mNb6-4C) double mutant was conducted (Figure 1, C to E, Figure 2E). The wildtype mNb6 and mutants mNb6-3C and mNb6-3C* contain one disulfide bond after expression, while mNb6-4C displayed two disulfides (Figure 2,A, B and C). To coordinate Bi(III) to mNb6-3C, reduction of the disulfide bond was attempted in large excess of the reducing agent TCEP at room temperature. Neither co- incubation with the water-soluble Bi(III) reagent gastrodenol (bismuth tripotassium dicitrate) nor its addition at a later point resulted in any significant formation of the mNb6- 3C-Bi conjugate (Figure 2F). As mass spectrometry (MS) confirmed the retention of the disulfide bond after TCEP treatment (Figure 7), it was concluded that the disulfide is inaccessible, and that partial unfolding would be required. Initial attempts to incorporate metal into nanobodies involved incubating mNb6-3C in 1M and 6M guanidinium hydrochloride (GdmCl) as a denaturing agent for 20 minutes at room temperature (RT), using 25 mM TCEP at pH 3. These conditions resulted in some metal uptake (Figure 8), but the removal of excess denaturant (GdmCl) to obtain functional protein proved challenging. In an alternative approach, informed by the denaturation temperature (Figure 5), short heating to 50 °C indeed resulted in the quantitatively bismuth-modified nanobodies mNb6-3C-Bi and mNb6-3C*-Bi, as confirmed by native MS (Figure 2, A, B and F).

[0430] While the 50 °C heat shock proved crucial for disulfide reduction, it resulted in the loss of up to 75% of soluble nanobody after 15 minutes at pH 7.5. Contrary, at pH 3 most of the nanobody remained soluble at this temperature, highlighting the extraordinary stability of nanobodies at pH extremes (Table.4). Table 4. Estimation of soluble mNb6-3C protein fraction after TCEP treatment at different pH.

[0431] Simultaneous increase of the TCEP concentration and decrease of pH resulted in fastest disulfide reduction and absence of protein precipitation during heat shock (Figure 2G). While treatment with BiBr3 is possible (Figure 9 and 10), quantitative modification was observed within only 3 minutes using water-soluble gastrodenol (Figure 2F). The thermal denaturation curves of mNb6-3C-Bi and mNb6-3C*-Bi were found to bealmost identical to the wildtype mNb6 (Figure 5), indicating that the bismuth-bridged cysteines mimic the native disulfide bond without impacting the overall stability.

[0432] Despite the likelihood of partial unfolding during the heat and pH shocks, circular dichroism (CD) spectroscopy indicated correctly folded nanobody after the procedure (Figure 11). To further confirm that the nanobody structure remains intact throughout the modification procedure, nuclear magnetic resonance (NMR) spectroscopy was conducted with uniformly15N-labelled mNb6-3C (Figure 3 and 4). Direct reduction in the NMR buffer at pH 6.5 for 15 minutes at 50 °C resulted in only 70% transformation into15N- mNb6-3C-Bi (Figure 3 and 4), confirming that fast and quantitative modification is best achieved at pH 3. Superimposition of NMR spectra reveal only minimal peak perturbations upon bismuth binding (Figure 3 and 4). Bound and unbound species are in slow exchange on the NMR time scale, indicating a kinetically stable complex.

[0433] Applying the heat-and-pH-shock procedure to the quadruple cysteine motif in mNb6-4C, it was possible to completely modify the nanobody with Ga(III) and In(III), using water-soluble Ga(NO3)3 and InCl3, respectively (Figure 2C). Gallium, as a hard Lewis acid, is more drawn toward oxygen and nitrogen ligands; hence, full complexation of Ga(III) in particular by mNb6-4C was surprising. High-resolution native MS confirmed that all four cysteine thiols are deprotonated, engaging in the formation of negatively charged indate [InSR4]–and gallate [GaSR4]–complexes (Figure 1G, Figure 12, F and G). Uptake of Bi, In and Ga by nanobodies comprising two (mNb6), three (mNb6-3C and mNb6-3C*) and four (mNb6-4C) cysteine residues was quantified and comprehensively compared after a 15-minute heat-and-pH-shock using native MS (Figure 2D, Figures 13 – 20). Only bismuth binds to the wildtype 2C motif, although not quantitatively unless fully denatured (Figure 14). The 3C motif quantitatively binds bismuth and indium, while gallium can only be fully complexed by the 4C motif. In summary and aligning with the expected chemistry, the data demonstrates that the 3C motif is optimal for bismuth binding, while the 4C motif appears optimal for indium and gallium. Example 4 - Stability of metal nanobodies

[0434] After identifying suitable binding motifs for bismuth, indium and gallium in the model nanobody mNb6, the stability of these metal-nanobody conjugates was studied (Figure 8). Following modification and buffer exchange, all three major conjugates, mNb6- 3C-Bi, mNb6-4C-In and mNb6-4C-Ga, remain fully intact if stored for two weeks at 4 °C(Figure 12A, Figures 21 – 28). The stability of metal bound mNb6-3C and mNb6-4C are also shown in Tables 5 and 6). Table 5. Stability of metal bound mNb6-3C after reduction, metal binding and storing at 4 °C (n=3).Table 6. Stability of metal bound mNb6-4C after reduction, metal binding and storing at 4 °C (n=3).

[0435] To provoke metal dissociation and fully eliminate the possibility that slight metal excess or reagents like TCEP could impact the stability assessment, the nanobody-metal conjugates were dialyzed (1000:1 v / v) for 18 h at 4 °C and it was observed that after this period >98% of the metal remained bound (Figure 12, E, F and G). Compounds mNb6- 4C-In and mNb6-4C-Ga were further co-incubated, which share the identical construct, during dialysis and only <1% cross-contamination of indium and gallium was identified after 18 h (Figure 12, F and G), demonstrating extremely slow metal dissociation sufficient for clinical applications.

[0436] As it can be beneficial for clinical set-ups dealing with radioactive metals with limited half-lives, such as213Bi (T1 / 2 = 46 min) used in TAT, to add the metal at a very late stage, it was explored if reduction and metal uptake can be divided into two distinct steps, separated by days if not weeks. After standard reduction procedure, the ‘primed’ nanobodies were kept at 4 °C and the metals added after different time points (Figures 29, 30). Even after two weeks of storage at 4 °C, >98% metal uptake was observed acrossall nanobodies and metals tested (Figure 12B), highlighting that primed nanobodies can be transported to the site where both the reactor and patient are present. Example 5 - Competition with endogenous metal binders

[0437] In order to further assess the complex stability of mNb6-3C-Bi, mNb6-4C-In and mNb6-4C-Ga, competition experiments with glutathione (Figure 12C) and human transferrin (Figure 12D) were performed. Glutathione (GSH) is the most abundant thiol in human cells (1–2 mM), playing a key role in maintaining the reducing environment, detoxification, and metal homeostasis. Though the primary function of transferrin is to bind and transport iron, it also binds various heavy metals to support detoxification. While the thiol in GSH can be considered a soft ligand, transferrin is rather a hard ligand with nitrogen and oxygen donors in its two binding sites comprised of tyrosines, histidine, arginine and aspartate.

[0438] The three nanobody-metal conjugates proved remarkably stable against GSH after 1 h of incubation (Figure 12C). At 5 mM GSH, which even exceeds most cellular levels, 80–90% of the nanobody molecules retained the metal, as determined by native MS. At a more realistic concentration of 50 ^M, which still exceeds typical plasma levels, >98% of nanobody-metal conjugates remained intact (Figure 12C). The complex stability constant logK between Bi(III) and three GSH ligands, [Bi(GS)3], has previously been determined by displacement of EDTA to be 30.1 at pH 7.3 (Chem. Eur. J.2, 701-708 (1996)). The fact that even 100 equivalents of GSH displace less than 10% Bi(III) from mNb6-3C-Bi (Figure 12C) demonstrates that the thermodynamic complex stability is within the range of conventional as well as contemporary Bi(III) complexing agents.

[0439] The transferrin competition experiments yielded a more distinct picture with much more pronounced differences between bismuth, indium and gallium (Figure 12D). All three elements are known to bind to human transferrin with similar stability constants, and a crystal structure for bismuth has been reported (N. Yang, H. Zhang, M. Wang, Q. Hao, H. Sun, Sci. Rep. 2012, 2, 999). To avoid any unwanted interference from iron, apo- transferrin was opted to be used, which has both metal-binding sites empty, unlike transferrin in healthy humans, which is typically 30% saturated. After 1 h incubation with 50 ^M apo-transferrin, mNb6-3C-Bi maintains >90% bismuth saturation. This concentration exceeds typical plasma levels of transferrin and relates to a 4:1 ratio of available metal-binding sites in transferrin and the nanobody. While the impact on bismuthbinding was marginal, transferrin had a much more pronounced effect on indium and gallium bound to mNb6-4C (Figure 12D). While at 50 ^M transferrin, mNb6-4C still showed 50% saturation with indium, it only displayed 25% gallium saturation. These observations align with the hard and soft acids and bases (HSAB) theory. Gallium is a harder Lewis acid than indium and the engineered nanobody is a softer ligand than transferrin. Overall, GSH and transferrin competition indicated the following ranking of inertness of the nanobody-metal conjugates: Bi >> In > Ga. The stability of mNb6-3C*-Bi after GSH competition and human apo-transferrin competition are shown in Table 7 and Table 8. Table 7. Stability of mNb6-3C*-Bi after GSH competition (n=3)Table 8. Stability of mNb6-3C*-Bi after Human apo-transferrin competition (n=3)Example 6 - Metal-nanobody conjugates remain fully functional

[0440] The engineered nanobodies were assessed to determine whether they still bind their target proteins after modification with the investigated main group metals. After confirming full modification by native MS, mNb6-3C-Bi, mNb6-4C-In and mNb6-4C-Ga were immobilized on a CM5 chip and surface plasmon resonance (SPR) experiments conducted by flushing the RBD of the SARS-CoV-2 spike protein over the chip surface (Figure 31 and Figure 33). These experiments confirmed fully functional nanobodies withdissociation constants (KD) between 17 and 34 nM (Figure 33, A, B and C), demonstrating tight binding of the bismuth, indium and gallium nanobodies to their target.

[0441] In order to further expand the scope and validate that the approach is broadly applicable to nanobodies raised against various targets, the nanobody Lam2 was selected, binding to the red fluorescent protein mCherry (mCh) frequently used in chemical biology. Following from the work with mNb6-3C, the same L4C mutation was introduced in Lam2-3C. Periplasmic expression resulted in species where the N-terminal leader sequence was either fully or only partially cleaved (Figure 36). Informed by the thermal denaturation curve (Figure 34), the heat-and-pH-shock method led to correctly folded (Figure 37) Lam2-3C-Bi with >98% bismuth saturation as confirmed by native MS (Figure 33F). Uptake of Bi(III) by Lam2-3C is shown in Figure 38 and Figure 39. Isothermal titration calorimetry (ITC) of Lam2-3C-Bi with mCherry confirmed fully functional modified nanobody with a KD of 46 nM (Figure 33E, Figure 40, and shown in Table 9). Table 9. Thermodynamic parameters for Lam2 and Lam2-3C-Bi binding to mCherry determined by isothermal titration calorimetry (ITC).

[0442] To demonstrate beyond any doubt that bismuth remains conjugated when the nanobody binds to its target protein, native PAGE with Lam2-3C-Bi and mCherry was conducted, followed by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) analysis of individual gel bands (Figure 33D) This combination of techniques confirmed not only the formation of the mCherry-Lam2-3C-Bi complex under native conditions, but also proved the presence of bismuth only in the complex and not in mCherry itself (Figure 33D) and shown in Table 10. Table 10. Expected and detected bismuth content in native PAGE samples by ICP-MS.

[0443] Finally, another therapeutically relevant nanobody was evaluated, 2Rs15d, that binds to the HER2 receptor overexpressed in some cancer cells. This nanobody contains an additional non-canonical disulfide bond between CDR 1 and CDR 3 (C32 and C98) that is typical for single-domain antibodies of camelid origin. While without impact on functionality, this optional disulfide bond can contribute to overall nanobody stability. Hence, an L4C mutant of 2Rs15d with both disulfide bonds retained was investigated, resulting in a total of five cysteine residues in 2Rs15d-5C for which correct folding (Figure 41) and thermal denaturation above 60 °C (Figure 35) was confirmed by CD spectroscopy. The usual heat-and-pH-shock and treatment with 5 equivalents of gastrodenol yielded 2Rs15d-5C-Bi with two bound bismuth atoms as the dominant species (Figure 33H). The fact that the extra disulfide bond is capable of binding Bi(III) aligns with the mNb6 experiments, where 76% bismuth saturation was observed for soluble protein (Figure 2D).

[0444] While 2Rs15d-5C demonstrates the opportunity for increasing the overall bismuth load per nanobody, it must be noted that the 2C-bismuth conjugate appears to be much more labile than the engineered 3C bismuth trap, as indicated by the presence of the single bismuth species in 2Rs15d-5C-Bi (Figure 33H). Therefore, a 2Rs15d-2A-3C mutant was additionally explored, in which the non-canonical disulfide bond was mutated to two alanine residues. The wildtype 2Rs15d-2A was also expressed lacking the extra cysteine residue and folding and denaturation parameters in comparison to 2Rs15d-2A- 3C (Figure 42, Figure 35) were confirmed. Like mNb6-3C and Lam-2-3C, 2Rs15d-3C could be fully saturated with one bismuth atom using the heat-and-pH-shock procedure (Figure 33G). Overall, these experiments showcase that the L4C mutation reliably generates a conserved binding motif for main group metal binding across a large variety of antigen binding proteins. Example 7 – Materials and methods for affibody studies Materials

[0445] All plasmids encoding an N-terminal His6-tag were obtained from Twist Bioscience, USA. Gastrodenol (bismuth tripotassium dicitrate), glutathione (~98% reduced), Tris and IPTG were purchased from AK Scientific, USA. Indium(III) chloridehexahydrate, gallium(III) nitrate hydrate, lead(II) nitrate, ammonium acetate (LC-MS grade) and imidazole were purchased from Sigma Aldrich, Australia. TCEP hydrochloride was purchased from AmBeed, USA. Kanamycin was purchased from AG Scientific, USA. Other chemicals and buffer compositions, such as sodium and potassium salts as well as EDTA and DTPA were purchased from Sigma Aldrich, Australia. LC-MS grade solvents were purchased from Fisher Scientific, Australia. Bacterial media components were purchased from Gibco, Thermo Fisher Scientific, USA. HisTrapTM(5 mL) columns were purchased from Cytiva, USA. Pre-cast SDS-PAGE and native-PAGE gels (Novex Bis- Tris Plus Mini Protein Gels) were purchased from Invitrogen, Thermo Fisher Scientific, USA. Unstained protein standard, broad range (10-200 KDa) was purchased from New England Biolabs, USA. Amicon®- Ultra centrifugal filters were purchased from Merck Millipore, USA. Protein mass spectrometry (MS)

[0446] Intact protein analysis was performed on Orbitrap Elite and Orbitrap Fusion™ Tribrid™ mass spectrometers (Thermo Fisher Scientific, USA) connected to a Thermo Fisher Scientific UltiMate 3000 HPLC system equipped with a ZORBAX 300SB-C3, 3.5 μm, 4.6 × 50 mm HPLC column (Agilent Technologies, USA).10–20 μM protein samples were injected using a 500 μL / min linear gradient of solvent A (0.1% (v / v) formic acid in water) and solvent B (0.1% (v / v) formic acid in acetonitrile), ramping solvent B from 5% at the start to 80% after 12 min. The ion source was H-ESI with a static spray voltage set to 3500 V in positive ion mode. The sheath, auxiliary and sweep gasses were set to 50 Arb, 10 Arb and 1 Arb, respectively. The ion transfer tube temperature was set to 325 °C and the vaporizer temperature was set to 350 °C. The resolution of the Orbitrap MS detector was set to 120000, and the scan range was set to 500–4000 m / z. Protein mass was determined by deconvolution using the program Xcalibur 3.0.63 (Thermo Fisher Scientific, USA).

[0447] Samples for native MS were buffer-exchanged into 100 mM NH4OAc (pH 7) in a 3 kDa MWCO centrifugal filter unit (Amicon®-Ultra 0.5) prior to analysis. Native protein analysis was performed on an Orbitrap Fusion™ Tribrid™ mass spectrometer (Thermo Fisher Scientific, USA) connected to a Thermo Fisher Scientific UltiMate 3000 HPLC system.10–20 μM protein samples were injected using a 5 minute 100 μL / min isocratic elution mode in 100 mM NH4OAc (pH 7). The ion source was H-ESI with a static spray voltage set to 3000 V in positive ion mode. The sheath, auxiliary and sweep gasses wereset to 25 Arb, 5 Arb and 0 Arb, respectively. The ion transfer tube temperature was set to 275 °C and the vaporizer temperature was set to 50 °C. The MS detector was an Orbitrap with the resolution set to 120000, and the scan range was set to 500–4000 m / z. Native protein mass was determined by deconvolution using the program Xcalibur 3.0.63 (Thermo Fisher Scientific, USA).

[0448] An orthogonal native MS method was performed on a Waters Synapt G2-Si HDMS qTOF mass spectrometer connected to a Waters Acquity UPLC I Class plus LC unit. 10–20 μM protein samples were injected using a 3 minute 100 μL / min isocratic elution mode in 100 mM NH4OAc (pH 7). The mass spectrometer was operated in positive, full MS, resolution and TOF modes. Capillary voltage and cone voltage were set to 1.5 kV and 30 V, respectively. The source temperature was set to 120 °C and the desolvation temperature was set to 350 °C. Leucine enkephalin was used as the Lockspray reference compound. Protein expression and purification

[0449] Affibody sequences with N-terminal His6-tag (ZHer2:2891 and ZHer2:2891-3C) were cloned into a pET-29b(+) expression vector (Twist Bioscience, USA) and transformed into E. coli BL21 (DE3) cells. The expression protocol is described as follows. Isolated single colonies were grown in Luria Broth (LB) overnight, transferred to 1 L LB and grown at 37 °C with gentle shaking until the OD reached 0.6–0.8. This was followed by induction of overexpression using 0.5 mM IPTG for 18–20 h at 25 °C. The E. coli cells were harvested (centrifuged at 5000 × g, 30 minutes, 4 °C) and resuspended in lysis buffer (20 mM tris- HCl, pH 8) for 30 minutes on ice. Cells were lysed by sonication (Omni Sonic Ruptor 400 Ultrasonic homogenizer) three times at 50% power for 30 seconds on ice, before centrifugation at 17,000 × g for 30 minutes to separate cell debris. The soluble fraction was then loaded onto a 5 mL HisTrapTMHP column (Cytiva) which had been equilibrated with binding buffer (20 mM tris-HCl, pH 8.0, 300 mM NaCl, 15 mM imidazole). The column was then washed with 5 column volumes of the binding buffer. Protein was eluted using 5 column volumes of elution buffer (20 mM tris-HCl, pH 8.0, 200 mM NaCl, 300 mM imidazole), and buffer exchanged then concentration in storage buffer (20 mM potassium phosphate, pH 6.0) using a 3 kDa MWCO centrifugal filter unit (Amicon®-Ultra 15). The protein solution was aliquoted, and flash frozen in liquid nitrogen for long term storage at -80 °C.

[0450] Affibody sequences with N-terminal His6-tag (ZTNF-alpha1 and ZTNF-alpha1-3C) were cloned into a pET-29b(+) expression vector (Twist Bioscience, USA) and transformed into E. coli BL21 (DE3) cells. The expression protocol was followed as described by Kronqvist et al. with minor changes (Protein Eng Des Sel 21, 247-255 (2008)). Isolated single colonies were grown in Luria Broth (LB) overnight, transferred to 1 L LB and grown at 37 °C with gentle shaking until the OD reached 0.8–1.0. This was followed by induction of overexpression using 1 mM IPTG for 4 h at 37 °C. The E. coli cells were harvested (centrifuged at 5000 × g, 30 minutes, 4 °C) and resuspended in binding buffer (PBS, pH 7.4, 30 mM imidazole) for 30 minutes on ice. Cells were lysed by sonication (Omni Sonic Ruptor 400 Ultrasonic homogenizer) three times at 50% power for 30 seconds on ice, before centrifugation at 17,000 × g for 30 minutes to separate cell debris. The soluble fraction was then loaded onto a 5 mL HisTrapTMHP column (Cytiva) which had been equilibrated with binding buffer. The column was then washed with 5 column volumes of the binding buffer. Protein was eluted using 5 column volumes of elution buffer (PBS, pH 7.4, 200 mM imidazole), followed by concentration using a 3 kDa MWCO centrifugal filter unit (Amicon®-Ultra 15). The protein solution was aliquoted, and flash frozen in liquid nitrogen for long term storage at -80 °C.

[0451] The TNFα protein sequence with an N-terminal His6-tag was cloned into a pET- 29b(+) expression vector (Twist Bioscience, USA) and transformed into E. coli BL21 (DE3) cells. The expression protocol was followed as described by Damough et al. with minor changes (Protein Expres Purif 179 (2021)). Isolated single colonies were grown in Luria Broth (LB) overnight, transferred to 1 L LB and grown at 37 °C with gentle shaking until the OD reached 0.6–0.8. This was followed by induction of overexpression using 0.5 mM IPTG for 18–20 h at 25 °C. The E. coli cells were harvested (centrifuged at 5000 × g, 30 minutes, 4 °C) and resuspended in binding buffer (50 mM NaH2PO4, pH 8.0, 300 mM NaCl, 10 mM imidazole) for 30 minutes on ice. Cells were lysed by sonication (Omni Sonic Ruptor 400 Ultrasonic homogenizer) three times at 50% power for 30 seconds on ice, before centrifugation at 17,000 × g for 30 minutes to separate cell debris. The soluble fraction was then loaded onto a 5 mL HisTrapTM HP column (Cytiva) which had been equilibrated with binding buffer. The column was then washed with 5 column volumes of the binding buffer. Protein was eluted using 5 column volumes of elution buffer (50 mM NaH2PO4, pH 8.0, 300 mM NaCl, 250 mM imidazole), followed by buffer exchange and concentration in storage buffer (20 mM phosphate buffer, pH 7.2, 100 mM NaCl) using a10 kDa MWCO centrifugal filter unit (Amicon®-Ultra 15). The protein solution was aliquoted, and flash frozen in liquid nitrogen for long term storage at -80 °C.

[0452] All proteins were characterized by SDS-PAGE and intact protein MS (positive ion mode). Computational Modelling of ZHER2:2891-3C

[0453] The amino acid sequence of ZHer2:2891 was used as input to generate a structural model using ColabFold v1.5.4:AlphaFold2. This structure was imported into BIOVIA Discovery Studio Visualiser 2025, where amino acids 12, 34 and 41 were mutated to cysteine residues. One bismuth(III) atom was introduced and connected to the three cysteines. The charges on the cysteine residues and bismuth atom were neturalised and the distance constraints based on known tris(L-cysteinato-S)-bismuth(III) monohydrate (CDC entry: CIYPIK) coordination geometry were applied. Geometry optimisation was then performed using the CHARMM force field. Circular dichroism (CD) spectroscopy

[0454] Secondary structures of the affibodies were assessed by circular dichroism using a Chirascan spectropolarimeter from Applied Photophysics equipped with a temperature control module using a 0.1 cm path-length cuvette. Each CD spectrum was averaged over 2 scans, and the baseline correction was done by subtraction of the spectrum with the appropriate blank solution. Individual affibodies were diluted in 20 mM phosphate buffer, pH 7.5. Scans were carried out at 25 °C over a range of 200–280 nm with a 1 nm step size and 1 nm bandwidth. Thermal denaturation experiments were monitored at 222 nm over a temperature range of 20–90 °C (1 °C / min heating rate). Temperature- dependent CD data were fitted to a two-state unfolding model (Boltzmann sigmoidal equation) and plotted in GraphPad Prism 10 (Dotmatics, USA) to obtain the denaturation midpoint (Tm). Sequence listing

[0455] Sequences of affibody constructs disclosed in this application are shown in the below table. H6 tag, leaders used for purification and mutations are indicated in bold and underlined.Example 8 – Affibody metal uptake reaction

[0456] Initial attempts to reduce and modify the affibodies by metal coordination were adapted from the previously established methods described for nanobodies in the present disclosure (Angew Chem Int Ed, e202419455 (2024)), whereby the protein is first reduced at a temperature 8 – 10 °C below their denaturation midpoint (Tm) under acidic conditions for up to 60 minutes prior to metal addition. With these methods proving successful, milder reduction conditions were evaluated, with metal coordination assessed at 25 °C and / or with a pH of 7.5 (Figure 45C). The metal binding was able to occur at 25 °C at pH 7.5, which is shown schematically in Figure 48.

[0457] In one embodiment, affibodies (100 µM in 10 mM Tris-HCl, 150 mM NaCl, pH 7.5) were reduced at 25 °C in the presence of 50 mM TCEP (pH 7.5) for a maximum duration of 60 minutes.5 equivalents of the metal salts gastrodenol (for bismuth), InCl3 hexahydrate, Ga(NO3)3 hydrate or Pb(NO3)2 were added to the reaction solution from their respective stocks, and immediately vortexed for 60 s. Following the reaction, the solution was quickly centrifuged, and the supernatant was collected for further analysis.For quantifying metal uptake by native MS, the supernatant was buffer exchanged to 100 mM NH4OAc (pH 7). Native MS spectra are shown in Figures 70-73 and 75-78.

[0458] Metal uptake of the engineered affibodies and their properties are shown in Figures 69-84. Example 9 – Stability of metal affibodies

[0459] Affibodies were reduced and saturated with either Bi(III), Ga(III) or Pb(II) as per the protocol in Example 8 to obtain >98% metal saturation, prior to buffer exchange into 100 mM NH4OAc (pH 7). Samples were then stored at 4 °C for up to 7 days and analysed via native MS at different time points to determine the stability of the metal-bound nanobody. The results of the stability testing are shown in Figures 49-53. Example 10 – Metal affibody competition with endogenous metal binders

[0460] In order to assess the complex stability of the metal bound affibodies, competition experiments with glutathione (GSH) (Figure 54B, D, F and H, Figures 56, 58, 60 and 62), ethylenediaminetetraacetic acid (EDTA) (Figure 54A, C, E and G, Figures 55, 57, 59 and 61) and diethylenetriamine pentaacetate (DTPA) (Figures 67 and 68) were conducted.

[0461] For experiments with glutathione, reduction followed by uptake reaction with Bi(III), In(III), Ga(III) and Pb(II) was performed with the ZHer2:2891-3C affibody as per the protocol in Example 8 to obtain >98% metal saturation. The metal-bound affibody samples were buffer-exchanged to 100 mM NH4OAc (pH 7). A reduced glutathione (GSH) stock was prepared in 100 mM NH4OAc and adjusted to pH 7. Competition experiments were performed with 1, 10, 20, 50 and 100 equiv. of GSH with respect to the concentrations of the metal-bound affibodies (50 μM) for 1 h at 25 °C. Native MS of the affibodies was performed in 100 mM NH4OAc. Metal bound affibodies (%) were plotted as a function of increasing equivalents of GSH in GraphPad Prism 10 (Dotmatics, USA). One-phase exponential decay was used as the fitting function. The 100 equiv. GSH samples were then stored at 4 °C for up to 14 days and the stability of the metal-bound affibody in the presence of GSH was monitored by native MS over time. Metal-bound affibodies (%) were plotted as a function of time (days) in GraphPad Prism 10 (Dotmatics, USA). One-phase exponential decay was used as the fitting function.

[0462] ZHER2:2891-3C shows high retention of Bi(III) when exposed to 100 equiv. of the strong metal chelators EDTA and DTPA (Figure 54A, Figure 67). The three-dimensional model of ZHER2:2891-3C-Bi (Figure 86) indicates that Bi(III) is likely inaccessible to the chelators due to its buried position within the affibody core (Figures 86). This is confirmed, as partial denaturation of the affibody-Bi(III) complex through heating for 10 minutes at 100 °C in presence of 100 equiv. EDTA resulted in a partial loss of the metal, indicating that the affibody is ‘opened’ and the metal becomes available to the chelator (Figure 85).

[0463] For experiments with EDTA and DTPA (for bismuth only), reduction followed by uptake reaction with Bi(III), In(III), Ga(III) and Pb(II) was performed with the ZHer2:2891-3C affibody as per the protocol in Example 8 to obtain >98% metal saturation. The metal- bound affibody samples were buffer-exchanged to 100 mM NH4OAc (pH 7). An EDTA / DTPA stock was prepared in 100 mM NH4OAc and adjusted to pH 7. Competition experiments were performed with 1, 10, 20, 50 and 100 equiv. of EDTA / DTPA with respect to the concentrations of the metal-bound affibodies (50 μM) for 1 h at 25 °C. Native MS of the affibodies was performed in 100 mM NH4OAc. Metal bound affibodies (%) were plotted as a function of increasing equivalents of EDTA / DTPA in GraphPad Prism 10 (Dotmatics, USA) (Figure 54). One-phase exponential decay was used as the fitting function. The 100 equiv. EDTA samples were then stored at 4 °C for up to 14 days and the stability of the metal-bound affibody in the presence of EDTA was monitored by native MS over time (Figure 63B). Metal-bound affibodies (%) were plotted as a function of time (days) in GraphPad Prism 10 (Dotmatics, USA). One-phase exponential decay was used as the fitting function. A second example of the 100 equiv. EDTA sample for Bi(III) was then also analysed with an orthogonal native MS method on a Waters Synapt G2-Si HDMS qTOF mass spectrometer (Figure 66). A third example was incubated for 10 minutes at 100 °C in the presence of 0 eq or 100 eq EDTA in 100 mM NH4OAc pH 7. Native MS of the affibodies was performed in 100 mM NH4OAc (Figure 85).

[0464] All experiments show that under these harsh conditions, the bismuth was able to remain bound to the affibody construct. Example 11 - Materials and methods for Pb nanobody studies

[0465] Expression and purification of nanobodies, Protein mass spectrometry (MS) (native MS), and circular dichroism (CD) spectroscopy were conducted in accordance with Example 1 (Figures 43-44).Conditions for Pb(II) uptake

[0466] For Pb(II) uptake, the nanobodies, mNb6-3C and mNb6-4C (50–100 µM), were reduced at a temperature range of 8–10 °C below their respective denaturation midpoint (Tm) in presence of 25 mM TCEP, pH 3.0 for a maximum of 60 minutes. Reduced nanobodies were aliquoted at different time points, followed by the addition of 5 equivalents of lead(II) nitrate [Pb(NO3)2] to the reaction solution from a 50 mM stock stock in milliQ water, and immediately vortexed for 60 s. Following the reaction, the solution was quickly centrifuged, and the supernatant was collected for further analysis. For quantifying lead(II) uptake by native MS, the supernatant was buffer-exchanged to 100 mM NH4OAc (pH 7). Lead(II) nitrate was obtained from commercial sources and used as received. Conditions for Pb(II)-uptake reactions on the individual nanobodies are as mentioned in Table 11. Table 11. Conditions tested for Pb(II) uptake.

[0467] Conditions which resulted in >98% Pb(II) uptake (Table 12), without denaturing the nanobodies were subsequently used for all other experiment and assays. Table 12. Conditions for Pb(II) uptake.Surface plasmon resonance (SPR)

[0468] SPR measurements were performed with 20 mM HEPES, pH 7.8, 150 mM NaCl, 0.05% Tween-20 as running buffer. For each experiment, 100–150 μg of metal nanobodies (>98% metal saturation confirmed by native MS), mNb6-3C-Pb and mNb6- 4C-2Pb, in 10 mM sodium acetate buffer, pH 4.8 were freshly immobilised on a CM5 Series S sensor chip flow-cell (Cytiva, 29104988, USA) using EDC / NHS chemistry at25 °C. The contact time for ligand binding was set to 1800 s with a flow rate of 5 mL / min. The unbound sites were blocked using ethanolamine hydrochloride solution. An amine coupling kit (Cytiva, BR100050, USA) was used for the coupling step. For the experiments, the response unit (RU) after coupling ranged from 300–1000. The initial concentration of the analyte, SARS-CoV-2 receptor binding domain (RBD) was 50 nM and was serially diluted down to 0.39 nM to evaluate its binding affinity to the nanobodies. All binding experiments were conducted at 20 °C in single-cycle kinetics mode. SPR experiments were performed on a Biacore 8K (Cytiva, USA). The instrument was operated using Biacore Insight 5 software (Cytiva, USA). The data were plotted and analysed in GraphPad Prism 10 (Dotmatics, USA) using the non-linear fit ‘one site – specific binding’ to obtain KD values (Figure 44E, F). Example 12 – Analysis for Pb nanobodies

[0469] After periplasmic expression of both nanobodies, mNb6-3C and mNb6-4C, protein mass spectrometry confirmed the presence of one and two intact disulfide bonds, respectively. Therefore, for lead(II) uptake, the heat-and-pH shock procedure as described in the pervious examples was undertaken, followed by addition of 5 equivalents of lead(II) nitrate [Pb(NO3)2].

[0470] For mNb6-3C, we observed >98% lead(II)-bound nanobody mNb6-3C-Pb within 5 minutes of heating at 50 °C, in presence of 25 mM TCEP (pH 3.0) (Figure 43A and B). A similar trend was observed in case of mNb6-4C, successfully converting to mNb6-4C- Pb after binding to a single lead(II) atom, within the first 5 minutes of a heat-and-pH shock at 60 °C (Figure 43C, D and E). Interestingly, increasing the duration of heat-and-pH shock of led to the uptake of two lead(II) atoms by the nanobody, resulting in 62% of mNb6-4C-2Pb species. After 60 minutes of heat-and-pH shock, >98% conversion to mNb6-4C-2Pb was observed (Figure 43F and G).

[0471] Without being bound by theory, it is possible that uptake of two lead(II) atom resulting in mNb6-4C-2Pb could potentially be supported by tetrel bonds where two Pb(II) are jointly coordinated to four sulphur atoms. This demonstrates that a single nanobody is capable of coordinating two lead(II) atoms to one engineered site, suggesting the potential for enhanced therapeutic payloads when applied to radioisotopes.

[0472] The impact of lead(II) uptake on the structural stability and functionality of both nanobodies was then assessed. Despite the possibility of partial unfolding during heat- and-pH shock, circular dichroism (CD) spectroscopy indicates retention of the tertiary structure post lead(II) uptake (Figure 44A and B). Thermal denaturation curves of lead(II)- nanobodies were identical to the native (mNb6) and apo-nanobodies (mNb6-3C and mNb6-4C) (Figure 44C and D). Surface plasmon resonance (SPR) experiments confirmed that lead(II)-bound nanobodies retained high target affinity towards the receptor binding domain (RBD) of SARS-CoV-2, with KD values of 17 nM for mNb6-3C- Pb and 23 nM for mNb6-4C-2Pb (Figure 44E and F). This demonstrated that their functionality remained fully intact after conjugation to lead(II).

[0473] Overall, these findings suggest that engineered nanobodies are capable of binding to lead(II), a key element of interest in radiotheranostics. A single Pb(II) atom binds to the triple cysteine motif in mNb6-3C within a few minutes, whereas two Pb(II) atoms bind to the quadruple cysteine motif in mNb6-4C.

Claims

CLAIMS 1. An antigen binding protein comprising an immunoglobulin domain comprising at least one disulfide bond, wherein the antigen binding protein comprises a mutation or modification of at least one residue that introduces a side chain capable of acting as a ligand for a metal.

2. The antigen binding protein according to claim 1, wherein the metal is a radionuclide.

3. The antigen binding protein according to claim 2, wherein the radionuclide is an isotope of Al Ga, Cu, Mn, Sc, Sr, Co, Zr, Rb, Tc, Y, In, Lu, Sm, Ac, At, Pb, Bi, Fr, K, Ti, Nb, Ta, Re, Fe, Rh, Ni, Pt, Au, Tl, Ge, Sn, As, Po, Ir, La, Pr, Pm, Tb, Dy, Ho, Tm, Yb, Lu, Th, Er, Cr, Cs or Ra.

4. The antigen binding protein according to claim 2, wherein the radionuclide is26Al,38K,66Ga,67Ga,68Ga,68Ge,61Cu,62Cu,64Cu,67Cu,51Mn,52Mn,44Sc,47Sc,45Ti,52Fe,57Ni,83Sr,89Sr,90Sr,51Cr,55Co,60Co, ,72As,89Zr,81Rb,82mRb,94mTc,99mTc,86Y,90Y,90Nb,103Pd,111In,110mIn,114mIn,140Pr,117mSn,131Cs,149Tb,152Tb,155Tb,161Tb,153Sm,166Ho,169Yb,169Er,177Lu,186Re,188Re,191mIr,192Ir,193mPt,195mPt,201Tl,225Ac,212Pb,211At,211Bi,212Bi,213Bi,211Fr,211Po,213Po,223Ra,224Ra, and227Th.

5. The antigen binding protein according to any one of claims 1 to 4, wherein the side chain capable of acting as a ligand for a metal comprises a monodentate ligand.

6. The antigen binding protein according to any one of claims 1 to 5, wherein the side chain capable of acting as a ligand for a metal comprises a sulphur or selenium atom.

7. The antigen binding protein according to any one of claims 1 to 6, wherein the mutation of at least one residue introduces a cysteine or selenocysteine residue.

8. The antigen binding protein according to any one of claims 1 to 7, wherein the mutation of at least one residue introduces a cysteine residue.

9. The antigen binding protein according to any one of claims 1 to 8, wherein the residue is a hydrophobic residue, preferably leucine or isoleucine.

10. The antigen binding protein according to any one of claims 1 to 8, wherein the residue is a hydrophilic residue, preferably glutamate or aspartate.

11. The antigen binding protein according to any one of claims 1 to 10, the alpha carbon atom of the at least one residue, or the alpha carbon atom of each of the two residues, is located within 10 Å, or within 9 Å, or within 8 Å, or within 7 Å, or within 6 Å, or within 5 Å or within 4 Å, or within 3 Å of the alpha carbon atom of one or both of the cysteines that form the at least one disulfide bond.

12. The antigen binding protein according to any one of claims 1 to 11, wherein the residue is located in a framework region of the immunoglobulin domain.

13. The antigen binding protein according to any one of claims 1 to 11, wherein the residue is located in framework region 1.

14. The antigen binding protein according to any one of claims 1 to 11, wherein the residue is located at any one or more residues, or residues equivalent to, 4 or 6 in SEQ ID NO: 1 to 10.

15. The antigen binding protein according to any one of claims 1 to 12, wherein the at least one disulfide bond is formed between native cysteine residues.

16. The antigen binding protein according to any one of claims 1 to 12, wherein the at least one disulfide bond is formed between non-native cysteine residues.

17. The antigen binding protein according to claim 16, wherein the non-native cysteine residues have been introduced by mutation or modification.

18. The antigen binding protein according to any one of claims 1 to 17, wherein the metal is a radionuclide of Bi, As, Sb, Cr, Cu, Fe, Co or Mn.

19. The antigen binding protein according to any one of claims 1 to 17, wherein the antigen binding protein comprises mutation or modification of two residues that introduces a side chain capable of acting as a ligand for a metal.

20. The antigen binding protein according to claim 19, wherein the two residues are the same type of amino acid.

21. The antigen binding protein according to claim 19, wherein the two residues are different types of amino acid.

22. The antigen binding protein according to any one of claims 19 to 21, wherein the two residues are located at any one or more residues, or residues equivalent to, 4 or 6 in SEQ ID NO: 1 to 10.

23. The antigen binding protein according to any one of claims 19 to 21, wherein the metal is a radionuclide of Al, Ge, Ga, Ti, Fe, Zr, Cu, Mn, Sc, Sr, Co, Rh, Ni, Pt, Tc, Re, In, Tl, Sn or Pb.

24. The antigen binding protein according to any one of claims 1 to 23, wherein the antigen binding protein binds to an antigen expressed on a cancer cell.

25. The antigen binding protein according to claim 24, wherein the cancer cell is from a solid tumour.

26. The antigen binding protein according to claim 24, wherein the cancer cell is from a liquid or haematological tumour.

27. The antigen binding protein according to claim 24, wherein the antigen is a tumour specific antigen.

28. The antigen binding protein according to claim 24, wherein the antigen is a tumour associated antigen.

29. The antigen binding protein according to claim 24, wherein the cancer cell is a blastoma (including medulloblastoma and retinoblastoma), sarcoma (including liposarcoma and synovial cell sarcoma), neuroendocrine tumours (including carcinoid tumours, gastrinoma, and islet cell cancer), mesothelioma, schwannoma (including acoustic neuroma), meningioma, adenocarcinoma, melanoma, leukaemia or lymphoid malignancies, lung cancer including small-cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer (including metastatic breast cancer), colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulvalcancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, testicular cancer, oesophageal cancer, tumours of the biliary tract, or head and neck cancer.

30. The antigen binding protein according to claim 24, wherein the antigen is mesothelin (MSLN), prostate specific membrane antigen (PSMA), prostate stem cell antigen (PCSA), carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD123, CD133, CD138, epithelial glycoprotein (EGP 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), folate-binding protein (FBP), foetal acetylcholine receptor (AChR), folate receptor-α and β (FRα and β), Ganglioside G2 (GD2), Ganglioside G3 (GD3), human Epidermal Growth Factor Receptor 2 (HER-2 / ERB2), HER3, Epidermal Growth Factor Receptor vIII (EGFRvIII), ERB3, ERB4, human telomerase reverse transcriptase (hTERT), Interleukin-13 receptor subunit alpha-2 (IL-13Rα2), κ-light chain, kinase insert domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), L1 cell adhesion molecule (L1CAM), melanoma-associated antigen 1 (melanoma antigen family A1, MAGE-A1), Mucin 16 (Muc-16), Mucin 1 (Muc- 1), NKG2D ligands, cancer-testis antigen NY-ESO-1, oncofoetal antigen (h5T4), tumour-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF- R2), Wilms’ tumour protein (WT-1), type 1 tyrosine-protein kinase transmembrane receptor (ROR1), B7-H3 (CD276), B7-H6 (Nkp30), Chondroitin sulfate proteoglycan-4 (CSPG4), DNAX Accessory Molecule (DNAM-1), Ephrin type A Receptor 2 (EpHA2), Fibroblast Associated Protein (FAP), Gpl00 / HLA-A2, Glypican 3 (GPC3), HA-1H, HERK-V, IL-11Ra, Latent Membrane Protein 1 (LMP1), Neural cell- adhesion molecule (N-CAM / CD56), or Trail Receptor (TRAIL R) 31. The antigen binding protein according to any one of claims 1 to 30, wherein the protein is: i. a diabody; ii. a triabody; iii. a tetrabody; iv. a nanobody; v. a minibody;vi. a Fab; vii. a F(ab’)2; viii. a Fv; ix. a scFv; x. a bispecific antibody or other form of multispecific antibody (including a BiTE); or one of (i) to (x) linked to a constant region of an antibody, Fc or a heavy chain constant domain (CH) 2 and / or CH3.

32. The antigen binding protein according to any one of claims 1 to 31, wherein the antigen binding protein has been exposed to conditions that reduce the at least one disulfide bond.

33. A metal coordinated antigen binding protein, wherein the antigen binding protein of any one of claims 1 to 32 further comprises a metal coordinated by (a) reduced residues that when non-reduced form the disulphide bond, and (b) at least one mutated or modified hydrophobic residue that contains a side chain capable of acting as a ligand for the metal.

34. A method of producing an antigen binding protein according to any one of claims 1 to 32, the method comprising mutating or modifying at least one residue of an immunoglobulin domain comprising at least one disulfide bond to introduce a side chain capable of acting as a ligand for a metal.

35. The method of claim 34, comprising mutating or modifying two residues of an immunoglobulin domain comprising at least one disulfide bond to introduce two sides chain capable of acting as a ligand for a metal.

36. A method of producing a metal coordinated antigen binding protein according to claim 33, the method comprising: (a) contacting an antigen binding protein according to any one of claims 1 to 32 with a reducing agent such that the at least one disulfide bond of the antigen binding protein is reduced to form a reduced antigen binding protein; and(b) contacting the reduced antigen binding protein with a metal source such that the reduced disulfide bond and at least one mutated or modified residue containing a side chain capable of acting as a ligand coordinate to the metal, thereby forming a metal coordinated antigen binding protein.

37. The method of claim 36, wherein the reducing agent is tris(2- carboxyethyl)phosphine (TCEP), β-mercaptoethanol (BME) or dithiothreitol (DTT).

38. The method of claim 36 or claim 37, wherein step (a) is performed between about 15 °below and about 15 °C above the denaturation midpoint temperature of the antigen binding protein.

39. The method of claim 36 or claim 37, wherein step (a) further comprises a chemical denaturation agent.

40. The method of any one of claims 36 to 39, wherein the method is performed between pH 2 and pH 8.

41. The method of any one of claims 36 to 40, wherein the concentration of the reducing agent is between about 1 mM to about 50 mM.

42. The method of any one of claims 36 to 41, wherein the antigen binding protein and the metal source have a molar ratio of at least 1:1, preferably between 1:1 to 1:

10.

43. An antigen binding protein comprising a helix bundle, the helix bundle comprising at least three helices, wherein the antigen binding protein comprises a mutation or modification of at least three residues, each mutation or modification introducing a side chain capable of acting as a ligand for a metal.

44. The antigen binding protein of claim 43, wherein the metal is a radionuclide.

45. The antigen binding protein of claim 44, wherein the radionuclide is an isotope of Al, Ga, Cu, Mn, Sc, Sr, Co, Zr, Rb, Tc, Y, In, Lu, Sm, Ac, At, Pb, Bi, Fr, K, Ti, Nb, Ta, Re, Fe, Rh, Ni, Pt, Au, Tl, Ge, Sn, As, Po, Ir, La, Pr, Pm, Tb, Dy, Ho, Tm, Yb, Lu, Th, Er, Cr, Cs or Ra.

46. The antigen binding protein according to claim 44, wherein the radionuclide is26Al,38K,66Ga,67Ga,68Ga,68Ge,61Cu,62Cu,64Cu,67Cu,51Mn,52Mn,44Sc,47Sc,45Ti,52Fe,57Ni,83Sr,89Sr,90Sr,51Cr,55Co,60Co, ,72As,89Zr,81Rb,82mRb,94mTc,99mTc,86Y,90Y,90Nb,103Pd,111In,110mIn,114mIn,140Pr,117mSn,131Cs,149Tb,152Tb,155Tb,161Tb,153Sm,166Ho,169Yb,169Er,177Lu,186Re,188Re,191mIr,192Ir,193mPt,195mPt,201Tl,225Ac,212Pb,211At,211Bi,212Bi,213Bi,211Fr,211Po,213Po,223Ra,224Ra, and227Th.

47. The antigen binding protein according to any one of claims 43 to 46, wherein the side chain capable of acting as a ligand for a metal comprises a monodentate ligand.

48. The antigen binding protein according to any one of claims 43 to 47, wherein the side chain capable of acting as a ligand for a metal comprises a sulphur or selenium atom.

49. The antigen binding protein according to any one of claims 43 to 48, wherein the mutation or modification introduces at least three cysteine residues, selenocysteine residues, or a combination thereof.

50. The antigen binding protein according to any one of claims 43 to 48, wherein the mutation introduces three cysteine residues.

51. The antigen binding protein according to any one of claims 43 to 50, wherein the residue is a hydrophobic residue, preferably leucine or alanine.

52. The antigen binding protein according to any one of claims 43 to 51, wherein the residue is a hydrophilic residue, preferably serine.

53. The antigen binding protein according to any one of claims 43 to 52, the alpha carbon atom of one of the at least three residues is located within 13 Å, or within 12 Å, or within 11 Å, or within 10 Å, or within 9 Å, or within 8 Å, or within 7 Å, or within 6 Å, or within 5 Å or within 4 Å, or within 3 Å of the alpha carbon atoms of the remaining modified or mutated residues.

54. The antigen binding protein according to any one of claims 43 to 53, wherein the residue is located at any one or more residues, or residues equivalent to, 12, 34 and 41 in SEQ ID NO: 11 to 14.

55. The antigen binding protein according to any one of claims 43 to 54, wherein the metal is coordinated by at least 2 residues of the antigen binding protein.

56. The antigen binding protein according to claims 43 to 54, wherein the metal is coordinated by 3 or 4 residues of the antigen binding protein.

57. The antigen binding protein according to any one of claims 43 to 56, wherein the antigen binding protein binds to an antigen expressed on a cancer cell.

58. The antigen binding protein according to claim 57, wherein the cancer cell is from a solid tumour.

59. The antigen binding protein according to claim 57, wherein the cancer cell is from a liquid or haematological tumour.

60. The antigen binding protein according to claim 57, wherein the antigen is a tumour specific antigen.

61. The antigen binding protein according to claim 57, wherein the antigen is a tumour associated antigen.

62. The antigen binding protein according to claim 57, wherein the cancer cell is a blastoma (including medulloblastoma and retinoblastoma), sarcoma (including liposarcoma and synovial cell sarcoma), neuroendocrine tumours (including carcinoid tumours, gastrinoma, and islet cell cancer), mesothelioma, schwannoma (including acoustic neuroma), meningioma, adenocarcinoma, melanoma, leukaemia or lymphoid malignancies, lung cancer including small-cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer (including metastatic breast cancer), colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, testicular cancer, oesophageal cancer, tumours of the biliary tract, or head and neck cancer.

63. The antigen binding protein according to claim 57, wherein the antigen is mesothelin (MSLN), prostate specific membrane antigen (PSMA), prostate stem cell antigen (PCSA), carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD123, CD133, CD138, epithelial glycoprotein (EGP 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), folate-binding protein (FBP), foetal acetylcholine receptor (AChR), folate receptor-α and β (FRα andβ), Ganglioside G2 (GD2), Ganglioside G3 (GD3), human Epidermal Growth Factor Receptor 2 (HER-2 / ERB2), HER3, Epidermal Growth Factor Receptor vIII (EGFRvIII), ERB3, ERB4, human telomerase reverse transcriptase (hTERT), Interleukin-13 receptor subunit alpha-2 (IL-13Rα2), κ-light chain, kinase insert domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), L1 cell adhesion molecule (L1CAM), melanoma-associated antigen 1 (melanoma antigen family A1, MAGE-A1), Mucin 16 (Muc-16), Mucin 1 (Muc- 1), NKG2D ligands, cancer-testis antigen NY-ESO-1, oncofoetal antigen (h5T4), tumour-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF- R2), Wilms’ tumour protein (WT-1), type 1 tyrosine-protein kinase transmembrane receptor (ROR1), B7-H3 (CD276), B7-H6 (Nkp30), Chondroitin sulfate proteoglycan-4 (CSPG4), DNAX Accessory Molecule (DNAM-1), Ephrin type A Receptor 2 (EpHA2), Fibroblast Associated Protein (FAP), Gpl00 / HLA-A2, Glypican 3 (GPC3), HA-1H, HERK-V, IL-11Ra, Latent Membrane Protein 1 (LMP1), Neural cell- adhesion molecule (N-CAM / CD56), or Trail Receptor (TRAIL R).

64. The antigen binding protein according to any one of claims 43 to 63, wherein the protein is an affibody.

65. The antigen binding protein according to claim 64, wherein the protein has a molecular weight from about 4 to about 10 kDa, or from about 5 and to about 8 kDa, or from about 6 to about 7 kDa.

66. A metal coordinated antigen binding protein, wherein the antigen binding protein of any one of claims 43 to 65 further comprises a metal coordinated by at least three mutated or modified residues that contain a side chain capable of acting as a ligand for the metal.

67. A method of producing an antigen binding protein according to any one of claims 43 to 65, the method comprising mutating or modifying at least two residues of a helix bundle comprising at least three helices, each mutation or modification to introduce a side chain capable of acting as a ligand for a metal.

68. A method of producing a metal coordinated antigen binding protein according to claim 66, the method comprising contacting the antigen binding protein of any one of claims 43 to 65 with a metal source such that the at least two mutated or modifiedresidues containing a side chain capable of acting as a ligand coordinate to the metal, thereby forming a metal coordinated antigen binding protein.

69. The method of claim 67 or claim 68, wherein the contacting is performed (a) between about 15 °C below and about 15 °C above the denaturation midpoint temperature of the antigen binding protein, or (b) between about 0 °C and about 40 °C.

70. The method of any one of claims 67 to 69, wherein the method is performed (a) between pH 2 and pH 8, or (b) between pH 4 and pH 10.

71. The method of any one of claims 67 to 69, wherein the antigen binding protein and the metal source have a molar ratio of at least 1:1, preferably between 1:1 to 1:

10.

72. A nucleic acid comprising a nucleotide sequence encoding an antigen binding protein of any one of claims 1 to 32 or 43 to 65.

73. A vector comprising the nucleic acid of claim 72.

74. The vector of claim 73, wherein the vector is an expression vector.

75. A cell comprising a nucleic acid of claim 72 or a vector of claim 73 or 74.

76. A composition comprising an antigen binding protein of any one of claims 1 to 32 or 43 to 65, or a metal coordinate antigen binding protein of claim 33 or 66.

77. A pharmaceutical comprising an antigen binding protein of any one of claims 1 to 32 or 43 to 65, or a metal coordinate antigen binding protein of claim 33 or 66, and a pharmaceutically acceptable diluent, carrier or excipient.

78. A method of treating cancer in an individual in need thereof, the method comprising administering a metal coordinated antigen binding protein of claim 33 or 66 to the individual, thereby treating cancer in the individual.

79. Use of a metal coordinated antigen binding protein of claim 33 or 66 in the manufacture of a medicament for treating cancer in an individual in need thereof.

80. A metal coordinated antigen binding protein of claim 33 or 66 for use in the treatment of cancer.

81. The method of claim 78, use of claim 79 or metal coordinated antigen of claim 80, wherein the cancer is a blastoma (including medulloblastoma and retinoblastoma), sarcoma (including liposarcoma and synovial cell sarcoma), neuroendocrine tumours (including carcinoid tumours, gastrinoma, and islet cell cancer), mesothelioma, schwannoma (including acoustic neuroma), meningioma, adenocarcinoma, melanoma, leukaemia or lymphoid malignancies, lung cancer including small-cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer (including metastatic breast cancer), colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, testicular cancer, oesophageal cancer, tumours of the biliary tract, or head and neck cancer.

82. A method of diagnosing, monitoring or prognosing a cancer in an individual in need thereof, the method comprising: (a) administering a metal coordinated antigen binding protein of claim 33 or 66 to the individual, wherein the metal is a radionuclide; and (b) detecting the radionuclide; whereby detection of the radionuclide enables an image of the cancer (if present) to be created to thereby diagnose, monitor or prognose the cancer.

83. A theranostic method comprising: (1) administering a diagnostically-effective amount a metal coordinated antigen binding protein of claim 33 or 66 to a patient or subject, and (2) administering a therapeutically-effective amount of a metal coordinated antigen binding protein of claim 33 or 66 to the patient or subject in need thereof.

84. The theranostic method of claim 83, wherein the metal coordinated antigen binding protein in step 1 and step 2 is the same.

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