WO2021136571A1

WO2021136571A1 - Potency Assay for Radiolabeled Antibodies

Info

Publication number: WO2021136571A1
Application number: PCT/DK2020/050405
Authority: WO
Inventors: Claus J. MØLLER SAN-PEDRO; Ahmed Mahiuddin; Sonia SEQUEIRA
Original assignee: Y-Mabs Therapeutics, Inc.
Priority date: 2019-12-30
Filing date: 2020-12-22
Publication date: 2021-07-08

Abstract

The present invention relates to a radioimmunoreactivity or immune reactive fraction assay to evaluate the potency of radioimmunotherapies or radiodiagnostics (e.g. radiotheranostics), radiolabeled with alpha, beta or positron emitting radionuclides. The invention further relates to a kit of parts adapted to carry out the inventive method and the production of the target antigens materials for the assay. More specifically, the present invention relates to a biotinylated antigen-streptavidin coated bead assay for the assessment of potency of B7H3, GD2, CD33 or TIM-3 targeting radiolabeled antibodies for the diagnosis or treatment of cancer. Furthermore, a potency assay for ¹⁷⁷Lu-DTPA-8H9 antibody (CAR 3), ¹³¹I-8H9 antibody or ¹³¹I-humanized 8H9 antibody.

Description

Potency Assay for Radiolabeled Antibodies

The present invention relates to a radioimmunoreactivity or immune reactive fraction assay to evaluate the potency of radioimmunotherapies or radiodiagnostics (e.g. radiotheranostics), radiolabeled with alpha, beta or positron emitting radionuclides. The invention further relates to a kit of parts adapted to carry out the inventive method and the production of the target antigens materials for the assay.

More specifically, the present invention relates to a biotinylated antigen-streptavidin coated bead assay for the assessment of potency of B7FI3, GD2, CD33 or TIM-3 targeting radiolabeled antibodies for the diagnosis or treatment of cancer. Furthermore, a potency assay for ¹⁷⁷Lu-DTPA-8FI9 antibody (CAR 3), ¹³¹I-8H9 antibody or ¹³¹l-humanized 8H 9 antibody.

Technical Background

IRF is the measured fraction of an antibody or engineered derivative to its cognate antigen/target upon compared to a reference standard. The IRF is used to evaluate the extent to which an antibody retains its binding to the target upon modifications such as bioconjugation and radiolabeling or as a measure of stability in defined conditions.

The use of cell-based assays to test the immune reactive fraction (IRF, aka target binding fraction orTBF) of radiopharmaceuticals is prone to inaccuracies and has limitations due to both intrinsic and extrinsic factors. Sharma et al. (Sharma et al. A rapid bead-based radioligand biding assay for the determination of target-binding fraction and quality control of radiopharmaceuticals, Nuclear Medicine and Biology 71, 2019) has reported a cell-free quantitative method of analysis to determine the IRF of radioligands that includes the incubation of magnetic beads functionalized with Ni-NTA (Nickel-Nitrilotriacetic acid; HisPurTM Ni-NTA magnetic beads; 88831: Thermo Scientific) or streptavidin (65601; DynaBeadsTM MyOneTM Streptavidin Tl; InvitrogenTM) with a histidine-tagged or biotinylated antigen of choice for 15 min, followed by the addition of the radioactive antigen-targeting agent for 30 min, washing of the beads and collection of supernatant, wash and beads for gamma counting. The assay included a positive and negative control, for specificity of radioligand binding to the cognate antigen in the presence of an excess of unlabeled ligand, and for non-specific binding (NSB) of the radioligand to beads lacking the target antigen, respectively. The IRF of 1311-omburtamab was measured using a his-tagged B7-H3 antigen bound to streptavidin beads. IRF's of greater than 90% at end of synthesis (EOS) were consistently achieved and an assessment of stability over time indicated a reduction in potency at 76h plausibly due to 1311-induced radiolysis of the sample in the chosen storage conditions. The specificity of radioligand binding to His-tagged B7-H3 protein was demonstrated by substituting 1311-Omburtamab with 89Zr-DFO-Pertuzumab, which yielded low NSB. The entire assay may be completed within 2 hours.

These encouraging results warrant further optimization and standardization of a bead-based assay for routine IRF testing of radiopharmaceuticals.

Summary of the invention

Immunoreactivity is an important quality control test to ensure that an immunodiagnostic (RID) or immunotherapy (RIT) retains the ability to bind to its target after radiolabeling. The immunoreactivity influences the absolute uptake of an antibody by the tumor and the target-to-background ratio. Deterioration of immunoreactivity is usually caused by conjugation of chelating agents, labeling procedures, specific activity, and by radiolysis during storage. The FDA requires a potency test for the release of clinical products however there is no standard protocol.

The most common radiopharmaceutical potency assay is the Lindmo cell-based assay (Lindmo, 1984) in which the fraction of radiolabeled antibody bound to antigen is extrapolated to conditions of "infinite antigen excess". Cell based assays such as this are cell batch and experimental condition dependent, and results are provided post-release given the length of the procedure (several hours), not allowing for the determination of immunoreactivity before the radiopharmaceutical is given to the patient. Cell based assays are also not commonly available to radiopharmacies.

The present invention relates to a robust, off the shelf assay that provides IRF results for radiolabeled antibodies in 60 minutes or under 30 minutes, reducing operator exposure time and reducing time from end-of-synthesis to patient administration. This is particularly important in the context of radiolabels with a short half-life. The invention only requires disposable materials readily available in radiopharmacies and does not require specialized equipment. Furthermore, the method may overcome inconsistencies and tediousness of cell- based IRF assays and provide a reproducible readout of stability and expiry for patient use.

A key part of the invention is the antigen pre-coated bead material, which obviates the need for an extra incubation step, reduces lot-to lot or operator variability, is stable for months and is ready for use. In particular, the invention relates to off the shelf biotinylated 2lg and 4lg 4 B7-FI3, CD33 or GD2 coated beads, produced in stable yeast cell lines, followed by complete biotinylation according to the invention and binding to streptavidin.

The invention relates to the specific method that allows for testing IRF in 30-60 min, including amounts of protein, buffers, number and volume of washes, controls.

Moreover, the invention relates to a potency assay for radioimmunotherapies or diagnostics labeled with a, b or g emitting radionuclides that include 177Lu-DTPA-omburtamab and 1311-humanized omburtamab.

Ultimately this invention relates to an IRF or potency kit of parts, that include the pre-coated biotinylated antigen beads and incubation/wash buffer.

Dynabeads™ MyOne™ Streptavidin T1 magnetic beads are uniform, 1.0 pm superpara- magnetic beads with a streptavidin monolayer covalently coupled to the hydrophilic bead surface. This layer ensures negligible streptavidin leakage while the lack of excess adsorbed streptavidin ensures batch consistency and reproducibility of results. A method for biotinylating a protein is described in US patent no.: 5,874,239. Biotinylation technology is also reported to be offered by the company Creative Biolabs using the trademark BtAP-Tag.

According to an aspect, the invention concerns an in vitro method for determining the efficacy of a radiolabeled antibody for administration to an individual, comprising the steps of: i. Providing a biotinylated target antigen by attaching biotin to a target antigen, preferably wherein the biotinylation is single and site specific; ii. Conjugating said target antigen to a solid phase support; iii. Radiolabeling an antibody with a radioactive entity, preferably after step ii.; iv. Adding said conjugated antibody to said solid phase support; and v. Measuring the binding of said antibody to said target antigen, preferably using a gamma counter. Step i. concerns providing a target antigen attached to biotin. Conjugating a target antigen to a solid phase support may be performed before step iii. The solid phase support including the target antigen may be frozen for storage and thawed before use. Performing the subsequent step of conjugating the antibody with a radioactive entity after step ii. allows for the time for completion of the rest of the method to be minimized. According to another aspect, the invention concerns an in vitro method for determining the immune reactive fraction (IRF) of a radiolabeled antibody, or antigen binding fragment thereof, wherein said antibody or antigen binding fragment binds to a target antigen, comprising the steps: i. Conjugating the target antigen to a solid phase support; ii. Radiolabeling an antibody with a radioactive entity after step i.; iii. Adding said antibody or antigen binding fragment to said solid phase support; and iv. Measuring the binding of said antibody or antigen binding fragment to said target antigen, preferably using a Geiger gamma counter. In step i, the beads are preferably pre-coated before use.

The target-binding fraction is the fraction of an antibody which retains its binding to the cognate target (TBF). This is also the case when the antibody has been undergoing modifications including bioconjugation and radiolabeling. This parameter may be referred to as "immunoreactivity" or "immunoreactive fraction" (IRF) According to another aspect, the invention concerns a polypeptide conjugate comprising a self-assembly disassembly (SADA) polypeptide, and at least a first binding domain that binds to a first target and is covalently linked to the SADA polypeptide, wherein said polypeptide conjugate is for use in a method according to the invention. According to another aspect, the invention concerns a reagent for use in a method according to the invention, comprising a solid phase support, a primary coating of antigen on the solid phase support, adapted to support a secondary coating of an antibody or antigen binding fragment thereof over said primary coating. According to another aspect, the invention concerns a vial comprising the reagent according to the invention.

According to another aspect, the invention concerns a method of preparing the antigen according to the invention, wherein said method comprises the steps: i. Reconstitution of said antigen in sterile deionized water. ii. Storing at 2 - 8 °C and/or -70 °C.

According to another aspect, the invention concerns a method of preparing a solid phase support according to the invention, wherein said method comprises the steps: i. Providing a magnetic bead ii. Incubation with the antigen according to the invention iii. Separation of antibody-coated beads with a magnet iv. Transfer of supernatant to a first vial v. Measurement of UV absorbance, preferably by nanodrop vi. Wash of said antigen-coated beads vii. Collecting the washes of step vi. in a second vial viii. Measurement of the UV absorbance in the second vial, preferably by nanodrop ix. Wash of said antigen coated beads x. Separation of the antigen-coated beads with a magnet xi. Resuspending the antigen-coated beads in PBS xii. Storing at - 80 °C.

Wherein said magnetic beads are conjugated to streptavidin, and wherein said antigen is biotinylated, and wherein said magnetic beads are conjugated to said antigen through binding of streptavidin to biotin.

PBS may be referred to as phosphate-buffered saline. According to another aspect, the invention concerns a method of treating cancer in an individual, wherein the method comprises use of an radiolabeled antibody or antigen binding fragment thereof, and wherein the potency and/or efficacy of said antibody is tested in a method according to the invention before use in the method of treatment. Potency may be defined as a measure of drug activity expressed in terms of the amount required to produce an effect of a given intensity.

According to another aspect, the invention concerns a kit of parts, wherein said kit comprises a vial, and wherein said vial comprises a solid phase support according to the invention, and wherein said kit further comprises an antigen according to the invention, and wherein said kit further comprises a wash buffer and/or a wash formulation buffer, and wherein said kit is adapted to be used in a method according to the invention.

According to another aspect, the invention concerns a kit of parts, wherein said kit comprises a magnetic bead surface coated with streptavidin conjugated to biotinylated antigen, and wherein said kit further comprises a wash buffer and/or a wash formulation buffer, and wherein said kit is adapted to be used in a method according to the invention.

Detailed Disclosure

In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided. Affinity: As is known in the art, "affinity" is a measure of the tightness with a particular ligand (e.g., an antibody) binds to its partner (e.g., an epitope). Affinities can be measured in difference ways.

Antibody: The term "antibody" is art-recognized terminology and is intended to include molecules or active fragments of molecules that bind to known antigens. Examples of active fragments of molecules that bind to known antigens include Fab and F(ab')2fragments. These active fragments can be derived from an antibody of the present invention by a number of techniques. For example, purified monoclonal antibodies can be cleaved with an enzyme, such as pepsin, and subjected to HPLC gel filtration. The appropriate fraction containing Fab fragments can then be collected and concentrated by membrane filtration and the like. The term "antibody" also includes bispecific and chimeric antibodies and other available formats.

Antibody fragment: An antibody fragment is a portion of an antibody such as F(ab')2, F(ab)2, Fab', Fab, Fv, sFv and the like. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody. For example, an 3F8 monoclonal antibody fragment binds with an epitope recognized by 3F8. The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex. For example, antibody fragments include isolated fragments consisting of the variable regions, such as the "Fv" fragments consisting of the variable regions of the heavy and light chains, recombinant single chain polypeptide molecules in which light and heavy variable regions are connected by a peptide linker ("scFv proteins"), and minimal recognition units consisting of the amino acid residues that mimic the hypervariable region.

Bispecific antibody: A bispecific antibody is an antibody that can bind simultaneously to two targets which are of different structure. Bispecific antibodies (bsAb) and bispecific antibody fragments (bsFab) have at least one arm that specifically binds to an antigen, for example, GD2 and at least one other arm that specifically binds to another antigen, for example a targetable conjugate that bears a therapeutic or diagnostic agent. A variety of bispecific fusion proteins can be produced using molecular engineering. In one form, the bispecific fusion protein is divalent, consisting of, for example, a scFv with a single binding site for one antigen and a Fab fragment with a single binding site for a second antigen. In another form, the bispecific fusion protein is tetravalent, consisting of, for example, an IgG with two binding sites for one antigen and two identical scFv for a second antigen.

Chimeric antibody: A chimeric antibody is a recombinant protein that contains the variable domains including the complementarity-determining regions (CDRs) of an antibody derived from one species, for example a rodent antibody, while the constant domains of the antibody molecule is derived from those of a human antibody. The constant domains of the chimeric antibody may also be derived from that of other species, such as a cat or dog.

Effective amount: As used herein, the term "effective amount" refers to an amount of a given compound, conjugate or composition that is necessary or sufficient to realize a desired biologic effect. An effective amount of a given compound, conjugate or composition in accordance with the methods of the present invention would be the amount that achieves this selected result, and such an amount can be determined as a matter of routine by a person skilled in the art, without the need for undue experimentation.

Humanized antibody: A humanized antibody is a recombinant protein in which the CDRs from an antibody from one species; e.g., a rodent antibody, is transferred from the heavy and light variable chains of the rodent antibody into human heavy and light variable domains. The constant domain of the antibody molecule is derived from those of a human antibody.

A human antibody may be an antibody obtained from transgenic mice that have been "engineered" to produce specific human antibodies in response to antigenic challenge. In this technique, elements of the human heavy and light chain locus are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy chain and light chain loci. The transgenic mice can synthesize human antibodies specific for human antigens, and the mice can be used to produce human antibody-secreting hybridomas.

Prevent: As used herein, the terms "prevent", "preventing" and "prevention" refer to the prevention of the recurrence or onset of one or more symptoms of a disorder in a subject as result of the administration of a prophylactic or therapeutic agent.

Radioactive isotope: Examples of radioactive isotopes that can be conjugated to antibodies for use diagnostically or therapeutically include, but are not limited to, ²¹¹At, ¹⁴C, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁶⁷Cu, ¹⁵²Eu, ⁶⁷Ga, ³H, ^mln, ⁵⁹Fe, ²¹²Pb, ¹⁷⁷Lu, ³²P, ²²³Ra, ²²⁴Ra, ¹⁸⁶Re, ¹⁸⁸Re, ⁷⁵Se, ³⁵S, ^99mTc, ²²⁷Th, ⁸⁹Zr, ⁹⁰Y, ¹²³l, ¹²⁴l, ¹²⁵l, ¹³¹l, ^94mTc, ⁶⁴Cu, ⁶⁸Ga, ⁶⁶Ga, ⁷⁶Br, ⁸⁶Y, ⁸²Rb, ^110mln, ¹³N, ⁿC, ¹⁸F and alpha-emitting particles. Non-limiting examples of alpha-emitting particles include

²⁰⁹Bi, ²¹¹Bi, ²¹²Bi, ²¹³Bi, ²¹⁰Po, ²¹¹Po, ²¹²Po, ²¹⁴Po, ²¹⁵Po, ²¹⁶Po, ²¹⁸Po, ²¹¹At, ²¹⁵At, ²¹⁷At, ²¹⁸At, ²¹⁸Rn, ²¹⁹Rn, ²²⁰Rn, ²²²Rn, ²²⁶Rn, ²²¹Fr, ²²³Ra, ²²⁴Ra, ²²⁶Ra, ²²⁵Ac, ²²⁷Ac, ²²⁷Th, ²²⁸Th, ²²⁹Th, ²³⁰Th, ²³²Th, ²³¹Pa, ²³³U, ²³⁴U, ²³⁵U, ²³⁶U, ²³⁸U, ²³⁷Np, ²³⁸Pu, ²³⁹Pu, ²⁴⁰Pu, ²⁴⁴Pu, ²⁴¹Am, ²⁴⁴Cm, ²⁴⁵Cm, ²⁴⁸Cm, ²⁴⁹Cf, and ²⁵²Cf.

Subject: By "subject" or "individual" or "animal" or "patient" or "mammal," is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans and other primates, domestic animals, farm animals, and zoo, sports, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and the like.

Treatment: As used herein, the terms "treatment, " "treat, " "treated" or "treating" refer to prophylaxis and/or therapy, particularly wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the progression of multiple sclerosis. 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. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

According to an embodiment, the invention concerns an in vitro method for determining the efficacy of a radiolabeled antibody for administration to an individual, comprising the steps of: i. Providing a biotinylated target antigen by attaching biotin to a target antigen, preferably wherein the biotinylation is single and site specific; ii. Conjugating said target antigen to a solid phase support; iii. Radiolabeling an antibody with a radioactive entity, preferably after step ii.; iv. Adding said conjugated antibody to said solid phase support; and v. Measuring the binding of said antibody to said target antigen, preferably using a gamma counter.

According to an embodiment, the invention concerns an in vitro method for determining the immune reactive fraction (IRF) of a radiolabeled antibody, or antigen binding fragment thereof, wherein said antibody or antigen binding fragment binds to a target antigen, comprising the steps: i. Conjugating the target antigen to a solid phase support; ii. Radiolabeling an antibody with a radioactive entity after step i.; iii. Adding said antibody or antigen binding fragment to said solid phase support; and iv. Measuring the binding of said antibody or antigen binding fragment to said target antigen, preferably using a Geiger gamma counter.

According to an embodiment, the invention concerns the method, wherein said steps are performed in said order.

According to an embodiment, the invention concerns the method, wherein a plurality of solid phase supports are prepared in step i. for subsequent use.

According to an embodiment, the invention concerns the method, wherein one or more solid phase supports are frozen after step i. and thawed before step ii.

According to an embodiment, the invention concerns the method, wherein said solid phase comprises a magnetic bead.

According to an embodiment, the invention concerns the method, wherein step ii. thorough iv. and/or step ii. through iv. is adapted to be performed in less than 120 minutes, 115 minutes, 110 minutes, 105 minutes, 100 minutes, 95 minutes, 90 minutes, 85 minutes, 80 minutes, 75 minutes, 70 minutes, 65 minutes, 60 minutes, 55 minutes, 50 minutes, 45 minutes, 40 minutes, 35 minutes, 30 minutes, 29 minutes, 28 minutes, 27 minutes, 26 minutes, 25 minutes, 24 minutes, 23 minutes, 22 minutes, 21 minutes, 20 minutes, 19 minutes, 18 minutes, 17 minutes, 16 minutes, 15 minutes, 14 minutes, 13 minutes, 12 minutes, 11 minutes or less than 10 minutes.

According to an embodiment, the invention concerns the method, wherein said method measure the immunoreactive fraction/target binding fraction of said antibody or antigen binding fragment thereof, and wherein said immunoreactive fraction/target binding fraction is above 60 %, 65 %, 70%, 75 %, 80 %, 85 %, 87 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % or above 99 %.

According to an embodiment, the invention concerns the method, wherein said measuring of the binding of said antibody or antigen binding fragment to the target antigen, is measured by UV absorbance.

According to an embodiment, the invention concerns the method, wherein said method comprises at least one step of incubation. According to an embodiment, the invention concerns the method, wherein said method comprises at least one step of isolating an antibody-coated bead using a magnet.

According to an embodiment, the invention concerns the method, wherein said antibody is for use in a method of treatment of a disease and/or for use in a prophylaxis of a disease.

According to an embodiment, the invention concerns the method, wherein said antibody is use in the diagnostics of a disease.

According to an embodiment, the invention concerns the method, wherein said disease is a cancer, tumor, metastasis or a neurodegenerative disease.

According to an embodiment, the invention concerns the method, wherein said cancer, said tumor and/or said metastasis is prostate cancer, a desmoplastic small round cell tumor, ovarian cancer, gastric cancer, pancreatic cancer, liver cancer, renal cancer, breast cancer, non-small cell lung cancer, melanoma, alveolar rhabdomyosarcoma, embryonal rhabdomyosarcoma, Ewing sarcoma, Wilms tumor, neuroblastoma, ganglioneuroblastoma, ganglioneuroma, medulloblastoma, high-grade glioma, diffuse intrinsic pontine glioma, embryonal tumors with multilayered rosettes, or a cancer expressing B7H3, GD2, CD33, TIM- 3, CD3 and/or CD22.

According to an embodiment, the invention concerns the method, wherein said target binding fraction or immune reactive fraction is determined prior to administration of said antibody. the present invention relates to a robust, off the shelf assay that provides IRF results for radiolabeled antibodies in 60 minutes or under 30 minutes, reducing operator exposure time and reducing time from end-of-synthesis to patient administration. This is particularly important in the context of radiolabels with a short half-life.

According to an embodiment, the invention concerns the method, wherein said magnetic beads are coated with streptavidin.

According to an embodiment, the invention concerns the method, wherein said antigen is selected among B7H3, GD2, CD33, TIM-3 and CD22.

According to an embodiment, the invention concerns the method, wherein said antigen is produced in yeast.

According to an embodiment, the invention concerns the method, wherein said antigen comprises the sequence according to SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 15 or SEQ ID No. 16. According to an embodiment, the invention concerns the method, wherein said antigen comprises the sequence according to SEQ ID No. 13 or SEQ ID No.14.

According to an embodiment, the invention concerns the method, wherein a single lysine residue in said antigen is labeled with biotin. According to an embodiment, the invention concerns the method, wherein a single lysine residue in sequence ID No. SEQ ID No. 13, 15 or 16 is labeled with biotin.

According to an embodiment, the invention concerns the method, wherein said antigen is biotinylated.

Biotinylation is the process of attaching biotin to proteins and other molecules. Biotinylation may not disturb the natural function of the molecule.

According to an embodiment, the invention concerns the method, wherein said biotinylation is above 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 % or about 100 %.

According to an embodiment, the invention concerns the method, wherein said antigen binding fragment is a single chain variable fragment (scFv).

According to an embodiment, the invention concerns the method, wherein said antibody or antigen binding fragment is a murine antibody or an antigen binding fragment thereof.

According to an embodiment, the invention concerns the method, wherein said antibody or antigen binding fragment is a chimeric antibody or an antigen binding fragment thereof. According to an embodiment, the invention concerns the method, wherein said antibody or antigen binding fragment is a humanized antibody or an antigen binding fragment thereof.

According to an embodiment, the invention concerns the method, wherein said radioactive isotope is selected among a PET label and a SPECT label.

According to an embodiment, the invention concerns the method, wherein said PET label is selected among 1241, 68Ga and 89Zr.

According to an embodiment, the invention concerns the method, wherein said SPECT label is selected among 1311, 177Lu, 99mTc, 64Cu and 89Zr.

According to an embodiment, the invention concerns the method, wherein said antibody or antigen binding fragment thereof is conjugated to a chelator compound. According to an embodiment, the invention concerns the method, wherein said chelator compound is bound to a radioactive isotope.

According to an embodiment, the invention concerns the method, wherein said radioactive isotope is selected among 1241, 1311, 68Ga and 177Lu or 99mTc, 64Cu and 89Zr.

According to an embodiment, the invention concerns the method, wherein said chelator compound is selected among DOTA, DTPA, NOTA and DFO and their derivatives.

According to an embodiment, the invention concerns the method, wherein said DOTA is a variant of DOTA, such as Benzyl-DOTA.

According to an embodiment, the invention concerns the method, wherein said DTPA is a variant of DTPA, such as CHX-A"-DTPA.

According to an embodiment, the invention concerns the method, wherein said antibody or antigen binding fragment is conjugated to a DTPA, and wherein said DTPA is bound to 177Lu.

According to an embodiment, the invention concerns the method, wherein said antibody or antigen binding fragment is bound to 1311.

According to an embodiment, the invention concerns the method, wherein said antibody or antigen binding fragment is bound to 225Ac or 211At, and wherein said antigen is CD33.

According to an embodiment, the invention concerns the method, wherein said antibody or antigen binding fragment is bound to 225Ac or 211At, and wherein said antigen is TIM-3.

According to an embodiment, the invention concerns the method, wherein said antibody or antigen binding fragment is bound to 225Ac or 211At, and wherein said antigen is GD2.

According to an embodiment, the invention concerns the method, wherein said antibody or antigen binding fragment comprises at least one sequence selected among a heavy chain variable region CDR1 according to SEQ ID No. 3, a heavy chain variable region CD2 according to SEQ ID No. 4, a heavy chain variable region CDR3 according to SEQ ID No. 5 a light chain variable region CDR1 according to SEQ ID No. 6, a light chain variable region CDR2 according to SEQ ID No. 7 and a light chain variable region CDR3 according to SEQ ID No. 8.

According to an embodiment, the invention concerns the method, wherein said antibody or antigen binding fragment comprises a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2. According to an embodiment, the invention concerns the method, wherein said antibody or antigen binding fragment thereof is humanized.

According to an embodiment, the invention concerns the method, wherein said antibody or antigen binding fragment comprises a heavy chain sequence according to SEQ ID No. 11 and a light chain sequence according to SEQ ID No. 12.

According to an embodiment, the invention concerns the method, wherein said radioactive isotope is an alpha, beta or positron emitting radionuclide.

According to an embodiment, the invention concerns the method, wherein said antibody or antigen binding fragment thereof is a bispecific and/or trispecific binding antibody.

According to an embodiment, the invention concerns the method, wherein said bispecific and/or trispecific binding antibody comprises a first antibody or antigen binding fragment thereof according to the invention, and a second antibody or antigen binding fragment for binding to a second antigen, and wherein said antibody is adapted for use in a method according to the invention.

According to an embodiment, the invention concerns the method, wherein said second antibody or antigen binding fragment thereof binds to derivatives of DOTA.

According to an embodiment, the invention concerns the method, wherein said antibody is linked to a self-assembly disassembly (SADA) polypeptide.

In certain embodiments, the antibody of antigen binding fragment thereof is linked to a self- assembly disassembly (SADA) polypeptide disclosed in International Patent Application Publication No. WO2018204873, all of which is incorporated by reference in its entirety.

According to an embodiment, the invention concerns the method, wherein said self- assembly disassembly (SADA) polypeptide has an amino acid sequence that shows at least 75% identity with that of a human homo-multimerizing polypeptide and being characterized by one or more multimerization dissociation constants (KD).

According to an embodiment, the invention concerns the method, wherein said method comprises use of a polypeptide conjugate, wherein said conjugate comprising the self- assembly disassembly (SADA) polypeptide according to the invention, and wherein said conjugate further comprises the bispecific antibody according to the invention, wherein said first antigen is B7H3 and wherein said second antigen is DOTA.

According to an embodiment, the invention concerns the method, wherein said polypeptide conjugate comprises the self-assembly disassembly (SADA) polypeptide according to the invention, and wherein said conjugate further comprises the bispecific antibody according to the invention, wherein said first antigen is B7H3 and wherein said second antigen is DOTA. According to an embodiment, the invention concerns a polypeptide conjugate comprising a self-assembly disassembly (SADA) polypeptide, and at least a first binding domain that binds to a first target and is covalently linked to the SADA polypeptide, wherein said polypeptide conjugate is for use in a method according to the invention.

According to an embodiment, the invention concerns the polypeptide conjugate according to the invention, wherein said self-assembly disassembly (SADA) polypeptide has an amino acid sequence that shows at least 75% identity with that of a human homo-multimerizing polypeptide and being characterized by one or more multimerization dissociation constants (KD); and wherein said conjugate is being constructed and arranged so that it adopts a first multimerization state and one or more higher-order multimerization states, wherein: the first multimerization state is less than about -70 kDa in size, at least one of the higher-order multimerization states is a homo-tetramer or higher-order homo multimer greater than 150 kDa in size, wherein the higher-order homo-multimerized conjugate is stable in aqueous solution when the conjugate is present at a concentration above the SADA polypeptide KD, and the conjugate transitions from the higher-order multimerization state(s) to the first multimerization state under physiological conditions when the concentration of the conjugate is below the SADA polypeptide KD.

According to an embodiment, the invention concerns a reagent for use in a method according to the invention, comprising a solid phase support, a primary coating of antigen on the solid phase support, adapted to support a secondary coating of an antibody or antigen binding fragment thereof over said primary coating.

According to an embodiment, the invention concerns the reagent, wherein said solid phase support comprises a magnetic bead.

According to an embodiment, the invention concerns the reagent, wherein said magnetic bead is conjugated to streptavidin.

According to an embodiment, the invention concerns the reagent, wherein said antigen is B7H3.

According to an embodiment, the invention concerns the reagent, wherein said antigen is biotinylated.

According to an embodiment, the invention concerns the reagent, wherein said antibody or antigen binding fragment is an antibody or antigen binding fragment according to the invention.

According to an embodiment, the invention concerns the reagent, wherein said reagent is adapted to withstand freezing.

According to an embodiment, the invention concerns a vial comprising the reagent according to the invention. According to an embodiment, the invention concerns the vial, wherein said vial is adapted to be frozen.

According to an embodiment, the invention concerns a method of preparing the antigen according to the invention, wherein said method comprises the steps: i. Reconstitution of said antigen in sterile deionized water. ii. Storing at 2 - 8 °C and/or -70 °C.

According to an embodiment, the invention concerns a method of preparing a solid phase support, wherein said method comprises the steps: i. Providing a magnetic bead ii. Incubation with the antigen according to the invention iii. Separation of antibody-coated beads with a magnet iv. Transfer of supernatant to a first vial v. Measurement of UV absorbance, preferably by nanodrop vi. Wash of said antigen-coated beads vii. Collecting the washes of step vi. in a second vial viii. Measurement of the UV absorbance in the second vial, preferably by nanodrop ix. Wash of said antigen coated beads x. Separation of the antigen-coated beads with a magnet xi. Resuspending the antigen-coated beads in PBS xii. Storing at - 80 °C.

PBS may be referred to as phosphate-buffered saline.

According to an embodiment, the invention concerns the method, wherein said method detects the clinical benefit of a radiolabeled antibody or antigen-binding fragment thereof in the treatment of cancer in an individual.

According to an embodiment, the invention concerns a method of treating cancer in an individual, wherein the method comprises use of an radiolabeled antibody or antigen binding fragment thereof, and wherein the potency and/or efficacy of said antibody is tested in a method according to the invention before use in the method of treatment.

According to an embodiment, the invention concerns the method, wherein the dosage to be used in the method of treatment is determined by said testing of said antibody or antigen binding fragment.

According to an embodiment, the invention concerns a kit of parts, wherein said kit comprises a vial, and wherein said vial comprises a solid phase support according to the invention, and wherein said kit further comprises an antigen according to the invention, and wherein said kit further comprises a wash buffer and/or a wash formulation buffer, and wherein said kit is adapted to be used in a method according to the invention.

According to an embodiment, the invention concerns a kit of parts, wherein said kit comprises a magnetic bead surface coated with streptavidin conjugated to biotinylated antigen, and wherein said kit further comprises a wash buffer and/or a wash formulation buffer, and wherein said kit is adapted to be used in a method according to the invention. According to an embodiment, the invention concerns the kit, wherein said kit further comprises an antibody or antigen binding fragment according to the invention.

According to an embodiment, the invention concerns the kit, wherein said kit further comprises a magnet.

According to an embodiment, the invention concerns the kit, wherein said antigen is B7H3, TIM-3, CD33 and/or GD2.

According to an embodiment, the invention concerns the kit, wherein said antigen is 70- 100 % biotinylated.

According to an embodiment, the invention concerns the kit, wherein said wash buffer and/or a wash formulation buffer is 1% FBS in PBS. FBS may be referred to as Fetal Bovine Serum.

All cited references are incorporated by reference. The accompanying Examples are provided to explain rather than limit the present invention. It will be clear to the person skilled in the art that aspects, embodiments, claims and any items of the present invention may be combined.

Unless otherwise mentioned, all percentages are in weight/weight. Unless otherwise mentioned, all measurements are conducted under standard conditions (ambient temperature and pressure). Unless otherwise mentioned, test conditions are according to European Pharmacopoeia 8.0.

Examples Example 1: Production of antigen conjugated streptavidin beads

Objective: To prepare antigen conjugated beads for use in a bead IRF assay. (This examples takes an hour).

Techniques: UV absorbance.

Materials: Biotinylated human B7-H3 / CD276 protein, His tag and Avi Tag (termed B7H3 throughout).

Biotin: Protein ratio is 0.7 (only 70% of the B7H3 is biotinylated).

White powder

Magnetic beads with a streptavidin monolayer covalently coupled to the hydrophilic bead surface.

Preparation of Antigen:

1) Take one 200 μg vial of B7H3.

2) Reconstitute the B7H3 (200 μg) with 1000 μL of sterile deionized water. Resulting concentration 200 μg/mL. 3) Create serial dilutions of the reconstituted B7H3, 1:2 to 1:10. a) 1:2. Take 1 μL of B7H3 and dilute with 1 μL of lx PBS. Total volume 2 μL b) 1:4. Take 1 μL of B7H3 and dilute with 3 μL of lx PBS. Total volume 4 μL c) 1:6. Take 1 μL of B7H3 and dilute with 5 μL of lx PBS. Total volume 6 μL d) 1:8. Take 1 μL of B7H3 and dilute with 7 μL of lx PBS. Total volume 8 μL e) 1:10. Take 1 μL of B7H3 and dilute with 9 μL of lx PBS. Total volume 10 μL f) Measure the UV absorbance in the order of lowest protein conc. to highest conc. and include 1 μL of B7H3 reconstituted solution in the samples to be tested.

4) Record the date on the vial and if using over the next few days store at 2-8°C.

Otherwise, store at -70 °C. B7H3 is stable for 3 months at -70°C. Preparation of Antigen conjugated beads:

1) Vortex the magnetic beads stock vial (highest setting for 1 min).

2) Transfer 1.4 mL (14.0 mg) of magnetic beads to a 15 mL tube.

3) Wash the magnetic beads 3 times: a) Add an equal volume (1.4 mL) of lxPBS and resuspend. b) Place the tube on a magnet for 2 min and discard the supernatant. c) Remove the tube from the magnet and resuspend the washed magnetic beads in 3 mL of lxPBS d) Repeat steps a-c twice, for a total of 3 washes. e) Resuspend in 2 mL lxPBS at the completion of the washes 4) Incubate the washed magnetic beads with the B7H3 antigen a) Thaw the vial of B7H3 (200 μg in 1 mL). i) Take a 1 μL sample for UV absorbance measurement by nanodrop. b) Once thawed, transfer the 2 mL of washed magnetic beads to the vial of B7H3. i) Total volume should be 3 mL c) Incubate the B7H3-bead mixture for 30 min at room temperature for 30 min using gentle rotation. d) Separate the antibody-coated beads with a magnet for 5 min or until the supernatant is clear. e) Carefully transfer supernatant to a separate vial. i) Take a sample (1 μL) of the supernatant and measure the UV absorbance via the nanodrop. ii) Remove tube from magnet and wash the coated beads with 3 mL of lxPBS. iii) Collect the supernatant from the washes in a separate vial for UV absorbance. f) Wash the antigen coated beads an additional 4 times as above. g) After the last wash, separate the antibody-coated beads with a magnet for 5 min, remove supernatant. h) Resuspend the beads in a total of 280 μL of PBS. i) Store beads at -80°C until use. j) Run the samples collected for UV absorbance in order of low protein concentration to high protein concentration (wash-supernatant, incubation- supernatant, B7H3).

5) Final antigen conjugated bead summary (assuming all biotinylated material binds to the beads): a) B7H3 concentration: 0.5 mg/ mL b) Bead concentration: 50 mg/mL c) B7H3/bead: 10 μg/mg

Example 2: Specific bead production batches Immunoreactivity Methods Antigen (B7H3) conjugated streptavidin beads were produced as described above. Specific bead production batches are described in Table 1. Table 1: B7H3-bead production summaries

Example 3: Immunoreactivity assay performed on the 177Lu-8H9 antibody derivatives Objective: To assess the immunoreactivity of Lutetium-177 labeled 8H9 antibody. Techniques: Radiochemistry, bead IRF assay.

Materials:

• 2 frozen vials of B7H3-beads (280 μL). o B7H3 cone. 0.5 mg/mL o Bead cone. 25 mg/mL o B7H3/bead 0.02 μg/μg

• Lutetium-177 labeled test article with the following specifications: o Radioconcentration: 20 μCi/mL o Specific Activity: 10-40 μCi/μg o Total volume of 177Lu-DOTA-mAb needed: at least 800 μL for triplicate assay or 525 μL for duplicate assay.

• 1.5 or 2 mL Eppendorf vials

• Gamma counting vials

Before the Experiment

1. Make 150 mL of 1% FBS in PBS (PBSF) on the day of experiment and store at RT.

2. Make vials of diluted IX beads as outlined in Table 2. Table 2: Dilutions needed for bead stock solutions

IRF assay. Perform once per Lutetium-mAb.

1) Assay to be performed in triplicates 2) Label the triplicate Eppendorf vials as indicated in the vial names in Table 2.

3) Label gamma counting tubes as "20μgB, 20μgUB" etc. and so forth in triplicate and 3 more labeled "REF" as per Table 2.

4) Wash each Eppendorf vial with 1 mL of PBSF.

5) Make the diluted bead solutions as outlined in Table 1. 6) According to Table 2, incubate the appropriate bead solution with 177Lu-mAb and PBSF.

Add bulk PBSF first, then smaller portion of PBSF, beads and lastly 177Lu-mAb.

7) Make 3 reference standards for each product (3 sets of three standards). a) Pipette 25 μL of 177Lu-mAb into three separate gamma counting tubes.

8) Incubate the vials at room temperature for 30 minutes on a 3D mixer. 9) At the end of the incubation, for all vials except the standards. a) Place each Eppendorf vial onto the magnetic rack or into the hole of the magnet for 2 min. b) After 2 minutes, draw off the supernatant and place in the corresponding gamma counting vial labeled "unbound". c) Add 365 μL of PBSF to each vial to wash the beads. d) Replace vial on the magnetic rack or in the hole of the magnet for another 2 minutes and draw off supernatant in the same way as described above. Place wash in the correlating gamma counting tube marked "unbound". e) Repeat the 365 μLPBSF wash a third time. f) After removing the final wash, add 500 μL of PBSF to each bead vial and gently mix by pipetting up and down ~6 times to re-suspend beads. Transfer the bead solution to the correlating gamma counting tube marked "bound". Add another 500 μL of PBSF to each vial to gently wash the vial for remaining beads. Transfer the solution to the Bound gamma counting tube. 10) Count all tubes in the gamma counter (separate tubes on rack as per requirements for

177Lu or local procedures) and make sure none of the readings are saturated.

11) Ensure that the three blank positions as background measurements and the three "standards" and are included in the gamma counter run. Table 3: Experiment setup. One set of vials for each radiolabeled 8H9 antibody.

Analysis:

For the analysis of the data please perform the following:

1) Perform the standard corrections (decay correction and background correction). 2) Normalize all values against the mean of the three reference standards. If the variance in the standards is > 10% of the mean, exclude the outlier standard in the reference mean.

3) Calculate the %Bound, %Unbound and %Lost for each sample (individual data).

4) Calculate the mean and STD of the % Bound, %Unbound and %Lost for each group (mean of 3 samples). a) If any individual sample shows > 20% (magnitude) Lost, exclude that sample from the calculation of the mean values.

5) Plot the %Bound, %Unbound and %Lost on a single plot against the mass of antigen in the vial. a) Plot with X-axis in linear and log scales. 6) Determine the IRF at the plateau values. Immunoreactivity Results

A summary of Immunoreactivity results for 1311 and 177Lu-DTPA-omburtamab (CAR 3) using the inventive method is included in Table 4. In summary, the immunoreactivity for 1311 and 177Lu-DTPA-omburtamab (CAR3) was in the range of 76-97%.

Table 4: Immunoreactivity results summary

Additional relevant sequences are provided below.

SEQ ID NO: 1: Murine 8H9 Heavy Chain

SEQ ID NO: 2: Murine 8H9 Light Chain

SEQ ID NO: 3: 8H9 Heavy Chain CDR-1

NYDIN SEQ ID NO: 4: 8H9 Heavy Chain CDR-2 WIFPGDGSTQY

SEQ ID NO: 5: 8H9 Heavy Chain CDR-3

QTTATWFAY

SEQ ID NO: 6: 8H9 Light Chain CDR-1 RASQSISDYLH

SEQ ID NO: 7: 8H9 Light Chain CDR-2 YASQSIS

SEQ ID NO: 8: 8H9 Light Chain CDR-3 QNGHSFPLT

SEQ ID NO: 9: 4lg-B7H3

SEQ ID NO: 10: 2lg-B7H3

SEQ ID NO: 11: hu8H9 Heavy Chain

SEQ ID NO: 12: hu8H9 Light Chain

SEQ ID NO: 13: Biotinylation - sequence 1

SEQ ID No: 14: Biotinylation - sequence 2

HHHHHHHHHH

SEQ ID No: 15: 2lg-B7H3 extra-cellular domain w tag

SEQ ID No: 16: 4lg-B7H3 extra-cellular domain w tag

Claims

1. An in vitro method for determining the efficacy of a radiolabeled antibody for administration to an individual, comprising the steps of: i. Providing a biotinylated target antigen by attaching biotin to a target antigen, preferably wherein the biotinylation is single and site specific; ii. Conjugating said target antigen to a solid phase support; iii. Radiolabeling an antibody with a radioactive entity, preferably after step ii.; iv. Adding said conjugated antibody to said solid phase support; and v. Measuring the binding of said antibody to said target antigen, preferably using a gamma counter.

2. An in vitro method for determining the immune reactive fraction (IRF) of a radiolabeled antibody, or antigen binding fragment thereof, wherein said antibody or antigen binding fragment binds to a target antigen, comprising the steps: i. Conjugating the target antigen to a solid phase support; ii. Radiolabeling an antibody with a radioactive entity after step i.; iii. Adding said antibody or antigen binding fragment to said solid phase support; and iv. Measuring the binding of said antibody or antigen binding fragment to said target antigen, preferably using a Geiger gamma counter.

3. The method according to any of the preceding claims, wherein said steps are performed in said order.

4. The method according to any of the preceding claims, wherein a plurality of solid phase supports are prepared in step i. for subsequent use.

5. The method according to any of the preceding claims, wherein one or more solid phase supports are frozen after step i. and thawed before step ii.

6. The method according to any of the preceding claims, wherein said solid phase comprises a magnetic bead.

7. The method according to any of the preceding claims, wherein step ii. thorough iv. and/or step ii. through iv. is adapted to be performed in less than 120 minutes, 115 minutes, 110 minutes, 105 minutes, 100 minutes, 95 minutes, 90 minutes, 85 minutes, 80 minutes, 75 minutes, 70 minutes, 65 minutes, 60 minutes, 55 minutes,

50 minutes, 45 minutes, 40 minutes, 35 minutes, 30 minutes, 29 minutes, 28 minutes, 27 minutes, 26 minutes, 25 minutes, 24 minutes, 23 minutes, 22 minutes,

21 minutes, 20 minutes, 19 minutes, 18 minutes, 17 minutes, 16 minutes, 15 minutes, 14 minutes, 13 minutes, 12 minutes, 11 minutes or less than 10 minutes.

8. The method according to any of the preceding claims, wherein said method measure the immunoreactive fraction/target binding fraction of said antibody or antigen binding fragment thereof, and wherein said immunoreactive fraction/target binding fraction is above 60 %, 65 %, 70%, 75 %, 80 %, 85 %, 87 %, 89 %, 90 %, 91 %, 92 %,

93 %, 94 %, 95 %, 96 %, 97 %, 98 % or above 99 %.

9. The method according to any of the preceding claims, wherein said measuring of the binding of said antibody or antigen binding fragment to the target antigen, is measured by UV absorbance.

10. The method according to any of the preceding claims, wherein said method comprises at least one step of incubation.

11. The method according to any of the preceding claims, wherein said method comprises at least one step of isolating an antibody-coated bead using a magnet.

12. The method according to any of the preceding claims, wherein said antibody is for use in a method of treatment of a disease and/or for use in a prophylaxis of a disease.

13. The method according to any of the preceding claims, wherein said antibody is use in the diagnostics of a disease.

14. The method according to any of the preceding claims, wherein said disease is a cancer, tumor or metastasis.

15. The method according to any of the preceding claims, wherein said cancer, said tumor and/or said metastasis is prostate cancer, a desmoplastic small round cell tumor, ovarian cancer, gastric cancer, pancreatic cancer, liver cancer, renal cancer, breast cancer, non-small cell lung cancer, melanoma, alveolar rhabdomyosarcoma, embryonal rhabdomyosarcoma, Ewing sarcoma, Wilms tumor, neuroblastoma, ganglioneuroblastoma, ganglioneuroma, medulloblastoma, high-grade glioma, diffuse intrinsic pontine glioma, embryonal tumors with multilayered rosettes, or a cancer expressing B7H3, GD2, CD33, TIM-3, CD3 and/or CD22.

16. The method according to any of the preceding claims, wherein said target binding fraction or immune reactive fraction is determined prior to administration of said antibody.

17. The method according to any of the preceding claims, wherein said magnetic beads are coated with streptavidin.

18. The method according to any of the preceding claims, wherein said antigen is selected among B7H3, GD2, CD33, TIM-3 and CD22.

19. The method according to any of the preceding claims, wherein said antigen is produced in yeast.

20. The method according to any of the preceding claims, wherein said antigen comprises the sequence according to SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 15 or SEQ ID No. 16.

21. The method according to any of the preceding claims, wherein said antigen comprises the sequence according to SEQ ID No. 13 or SEQ ID No. 14.

22. The method according to any of the preceding claims, wherein a single lysine residue in said antigen is labeled with biotin.

23. The method according to any of the preceding claims, wherein a single lysine residue in sequence ID No. 13, 15 or 16 is labeled with biotin.

24. The method according to any of the preceding claims, wherein said antigen is biotinylated.

25. The method according to any of the preceding claims, wherein said biotinylation is above 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 % or about 100 %.

26. The method according to any of the preceding claims, wherein said antigen binding fragment is a single chain variable fragment (scFv).

27. The method according to any of the preceding claims, wherein said antibody or antigen binding fragment is a murine antibody or an antigen binding fragment thereof.

28. The method according to any of the preceding claims, wherein said antibody or antigen binding fragment is a chimeric antibody or an antigen binding fragment thereof.

29. The method according to any of the preceding claims, wherein said antibody or antigen binding fragment is a humanized antibody or an antigen binding fragment thereof.

30. The method according to any of the preceding claims, wherein said radioactive isotope is selected among a PET label and a SPECT label.

31. The method according to any of the preceding claims, wherein said PET label is selected among 1241, 68Ga and 89Zr.

32. The method according to any of the preceding claims, wherein said SPECT label is selected among 1311, 177Lu, 99mTc, 64Cu and 89Zr.

33. The method according to any of the preceding claims, wherein said antibody or antigen binding fragment thereof is conjugated to a chelator compound.

34. The method according to any of the preceding claims, wherein said chelator compound is bound to a radioactive isotope.

35. The method according to any of the preceding claims, wherein said radioactive isotope is selected among 1241, 1311, 68Ga and 177Lu or 99mTc, 64Cu and 89Zr.

36. The method according to any of the preceding claims, wherein said chelator compound is selected among DOTA, DTPA, NOTA and DFO and their derivatives.

37. The method according to any of the preceding claims, wherein said DOTA is a variant of DOTA, such as Benzyl-DOTA.

38. The method according to any of the preceding claims, wherein said DTPA is a variant of DTPA, such as CHX-A"-DTPA.

39. The method according to any of the preceding claims, wherein said antibody or antigen binding fragment is conjugated to a DTPA, and wherein said DTPA is bound to 177Lu.

40. The method according to any of the preceding claims, wherein said antibody or antigen binding fragment is bound to 1311.

41. The method according to any of the preceding claims, wherein said antibody or antigen binding fragment is bound to 225Ac or 211At, and wherein said antigen is CD33.

42. The method according to any of the preceding claims, wherein said antibody or antigen binding fragment is bound to 225Ac or 211At, and wherein said antigen is TIM-3.

43. The method according to any of the preceding claims, wherein said antibody or antigen binding fragment is bound to 225Ac or 211At, and wherein said antigen is GD2.

44. The method according to any of the preceding claims, wherein said antibody or antigen binding fragment comprises at least one sequence selected among a heavy chain variable region CDR1 according to SEQ ID No. 3, a heavy chain variable region CD2 according to SEQ ID No. 4, a heavy chain variable region CDR3 according to SEQ ID No. 5 a light chain variable region CDR1 according to SEQ ID No. 6, a light chain variable region CDR2 according to SEQ ID No. 7 and a light chain variable region CDR3 according to SEQ ID No. 8.

45. The method according to any of the preceding claims, wherein said antibody or antigen binding fragment comprises a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2.

46. The method according to claim 44 - 45, wherein said antibody or antigen binding fragment thereof is humanized.

47. The method according to any of the preceding claims, wherein said antibody or antigen binding fragment comprises a heavy chain sequence according to SEQ ID No. 11 and a light chain sequence according to SEQ ID No. 12.

48. The method according to any of the preceding claims, wherein said radioactive isotope is an alpha, beta or positron emitting radionuclide.

49. The method according to any of the preceding claims, wherein said antibody or antigen binding fragment thereof is a bispecific and/or trispecific binding antibody.

50. The method according to any of the preceding claims, wherein said bispecific and/or trispecific binding antibody comprises a first antibody or antigen binding fragment thereof according to any of the preceding claims, and a second antibody or antigen binding fragment for binding to a second antigen, and wherein said antibody is adapted for use in a method according to any of the preceding claims.

51. The method according to any of the preceding claims, wherein said second antibody or antigen binding fragment thereof binds to derivatives of DOTA.

52. The method according to any of the preceding claims, wherein said antibody is linked to a self-assembly disassembly (SADA) polypeptide.

53. The method according to any of the preceding claims, wherein said self-assembly disassembly (SADA) polypeptide has an amino acid sequence that shows at least 75% identity with that of a human homo-multimerizing polypeptide and being characterized by one or more multimerization dissociation constants (KD).

54. The method according to any of the preceding claims, wherein said method comprises use of a polypeptide conjugate, wherein said conjugate comprising the self-assembly disassembly (SADA) polypeptide according to any of the preceding claim, and wherein said conjugate further comprises the bispecific antibody according to any of the preceding claim, wherein said first antigen is B7H3 and wherein said second antigen is DOTA.

55. The method according to any of the preceding claims, wherein said polypeptide conjugate comprises the self-assembly disassembly (SADA) polypeptide according to any of the preceding claims, and wherein said conjugate further comprises the bispecific antibody according to any of the preceding claims, wherein said first antigen is B7H3 and wherein said second antigen is DOTA.

56. A polypeptide conjugate comprising a self-assembly disassembly (SADA) polypeptide, and at least a first binding domain that binds to a first target and is covalently linked to the SADA polypeptide, wherein said polypeptide conjugate is for use in a method according to any of the preceding claims.

57. The polypeptide conjugate according to any of the preceding claims, wherein said self-assembly disassembly (SADA) polypeptide has an amino acid sequence that shows at least 75% identity with that of a human homo-multimerizing polypeptide and being characterized by one or more multimerization dissociation constants (KD); and wherein said conjugate is being constructed and arranged so that it adopts a first multimerization state and one or more higher-order multimerization states, wherein: the first multimerization state is less than about -70 kDa in size, at least one of the higher-order multimerization states is a homo-tetramer or higher-order homo multimer greater than 150 kDa in size, wherein the higher-order homo-multimerized conjugate is stable in aqueous solution when the conjugate is present at a concentration above the SADA polypeptide KD, and the conjugate transitions from the higher-order multimerization state(s) to the first multimerization state under physiological conditions when the concentration of the conjugate is below the SADA polypeptide KD.

58. A reagent for use in a method according to any of the preceding claims, comprising a solid phase support, a primary coating of antigen on the solid phase support, adapted to support a secondary coating of an antibody or antigen binding fragment thereof over said primary coating.

59. The reagent according to any of the preceding claims, wherein said solid phase support comprises a magnetic bead.

60. The reagent according to any of the preceding claims, wherein said magnetic bead is conjugated to streptavidin.

61. The reagent according to any of the preceding claims, wherein said antigen is B7H3.

62. The reagent according to any of the preceding claims, wherein said antigen is biotinylated.

63. The reagent according to any of the preceding claims, wherein said antibody or antigen binding fragment is an antibody or antigen binding fragment according to any of the preceding claims.

64. The reagent according to any of the preceding claims, wherein said reagent is adapted to withstand freezing.

65. A vial comprising the reagent according to any of the preceding claims.

66. The vial according to any of the preceding claims, wherein said vial is adapted to be frozen.

67. A method of preparing the antigen according to any of the preceding claims, wherein said method comprises the steps: i. Reconstitution of said antigen in sterile deionized water. ii. Storing at 2 - 8 °C and/or -70 °C.

68. A method of preparing a solid phase support according to any of the preceding claims, wherein said method comprises the steps: i. Providing a magnetic bead ii. Incubation with the antigen according to any of the preceding claims iii. Separation of antibody-coated beads with a magnet iv. Transfer of supernatant to a first vial v. Measurement of UV absorbance, preferably by nanodrop vi. Wash of said antigen-coated beads vii. Collecting the washes of step vi. in a second vial viii. Measurement of the UV absorbance in the second vial, preferably by nanodrop ix. Wash of said antigen coated beads x. Separation of the antigen-coated beads with a magnet xi. Resuspending the antigen-coated beads in PBS xii. Storing at - 80 °C;

69. The method according to any of the preceding claims, wherein said method detects the clinical benefit of a radiolabeled antibody or antigen-binding fragment thereof in the treatment of cancer or a neurodegenerative disease in an individual.

70. A method of treating cancer in an individual, wherein the method comprises use of an radiolabeled antibody or antigen binding fragment thereof, and wherein the potency and/or efficacy of said antibody is tested in a method according to any of the preceding claims before use in the method of treatment.

71. The method according to claim 70, wherein the dosage to be used in the method of treatment is determined by said testing of said antibody or antigen binding fragment.

72. A kit of parts, wherein said kit comprises a vial, and wherein said vial comprises a solid phase support according to any of the preceding claims, and wherein said kit further comprises an antigen according to any of the preceding claims, and wherein said kit further comprises a wash buffer and/or a wash formulation buffer, and wherein said kit is adapted to be used in a method according to any of the preceding claims.

73. A kit of parts, wherein said kit comprises a magnetic bead surface coated with streptavidin conjugated to biotinylated antigen, and wherein said kit further comprises a wash buffer and/or a wash formulation buffer, and wherein said kit is adapted to be used in a method according to any of the preceding claims.

74. The kit according to any of the preceding claims, wherein said kit further comprises an antibody or antigen binding fragment according to any of the preceding claims.

75. The kit according to any of the preceding claims, wherein said kit further comprises a magnet.

76. The kit according to any of the preceding claims, wherein said antigen is B7H3, TIM-3, CD33 and/or GD2.

77. The kit according to any of the preceding claims, wherein said antigen is 70-100 % biotinylated.

78. The kit according to any of the preceding claims, wherein said wash buffer and/or a wash formulation buffer is 1% FBS in PBS.