In vivo imaging of ALS biomarkers and methods thereof

Abstract

Provided are labeled antibody conjugates for use in methods of diagnosing ALS or monitoring ALS drug therapy and / or progression by immunoimaging in vivo detection and tracking of misfolded SOD1 in a subject, compositions and kits comprising the conjugates, and methods of using these conjugates for in vivo immunoimaging of superoxide dismutase (SOD1) that are used as markers of the efficacy of amyotrophic lateral sclerosis (ALS) or ALS drug treatment of a subject. Furthermore, SOD1 antibodies specific for misfolded and / or aggregated SOD1 are provided for use in the treatment of bone and / or joint related diseases.

Description

Technical Field

[0001] This invention relates to a method for in vivo immunoimaging of misfolded SOD1 (mSOD1). More specifically, the invention relates to antibodies and antibody conjugates, which can be used for the diagnosis and monitoring of the treatment / progression of diseases caused by or associated with misfolded and aggregated SOD1, particularly neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Furthermore, the invention relates to anti-SOD1 antibodies for targeting SOD1 in cartilage (e.g., the bones and joints of a subject). Background Technology

[0002] ALS is a rare neurodegenerative disease caused by the loss of motor neurons in the motor cortex, brainstem, and spinal cord. Currently, three out of every 100,000 people in the United States have ALS, and approximately 5,000 new cases are diagnosed each year. Most patients are between 40 and 70 years old (ALSA 2019). The death of motor neurons that enable movement, speech, swallowing, and breathing typically leads to a rapid decline in neurological function. This ineffective denervation of muscles results in muscle wasting and atrophy, eventually requiring assisted ventilation until death from respiratory failure, the most common cause of death in ALS (Tateno). et al. (2014).

[0003] Of all ALS cases, ~10% are hereditary (FALS), and the remainder are sporadic (SALS). Mutations in the SOD1 gene, leading to misfolding of its protein product, are the second most common genetic cause of ALS (Vu and Browser 2017). While the exact mechanism of mutant SOD1 toxicity remains unclear, most evidence suggests that pathological gain of function is associated with a tendency for the protein to misfold and aggregate (Li and Cashman 2014, Bosco). et al. 2010, Forsberg et al. 2011, Forsberg et al. 2010, Graffmo et al. (2013).

[0004] Preclinical disease modeling using transgenic mice (Tg) has generated data supporting the hypothesis that wild-type non-mutant SOD1 protein misfolds and may also generate a “toxic conformation” leading to ALS-like pathology. et al. 2016, Bidhendi et al.(2016). The detection of misfolded wild-type SOD1 in postmortem sporadic ALS samples has been used to support the hypothesis that misfolded wild-type SOD1 may contribute to the pathogenesis of sporadic ALS (Bosco). et al. 2010, Forsberg et al. 2011, Forsberg et al. 2010, Tokuda et al. 2019, Pare et al. 2018, Maier et al. (2018).

[0005] Preliminary data were generated using CSF from a cohort of SALS patients, indicating that 60-70% of SALS patients were also positive for misfolded SOD1. Overall, these results, along with IHC (immunohistochemical) data, suggest that the full penetrance of misfolded SOD1 expression in these patients was significantly higher than predicted by CSF testing. This implies the following assumption: while the CSF assay under development may be a useful tool supporting the diagnosis of ALS, it does not yet provide a suitable potential patient selection or diagnostic tool for identifying presymptomatic ALS. Furthermore, assays relying on detecting misfolded SOD1 in CSF may not be suitable for all patients, as factors may influence the presence of misfolded SOD1 in a patient's CSF relative to spinal cord tissue.

[0006] Currently, there are no approved diagnostic tools for ALS, leading to misdiagnosis and severe disease progression before a formal diagnosis. Therefore, ALS diagnosis is based on symptom analysis, including a complete patient history, and takes time, as regular neurological examinations are typically required to assess whether symptoms (such as muscle weakness, muscle atrophy, hyperreflexia, and spasticity) are worsening. Therefore, non-invasive imaging tests for early detection of ALS and monitoring disease progression will have significant diagnostic and prognostic value. To date, there is also no cure for ALS. The FDA has only approved two symptomatic treatments with minimal impact on disease progression; and successfully slowing disease progression requires early and accurate diagnosis to enable patients to access these standard care treatments.

[0007] Therefore, there is an urgent need for diagnostic tools, especially those that can be used in vivo, to detect misfolded SOD1 as an indicator of ALS or to monitor ALS progression or its treatment. Summary of the Invention

[0008] This invention generally relates to antibodies and equivalent binding molecules, which, combined with detectable label conjugates, allow for in vivo immunoimaging of misfolded and / or aggregated SOD1 in a subject. The labeled antibody conjugates are particularly useful in methods for diagnosing ALS and / or monitoring ALS treatment and / or progression in a subject. The method preferably includes detecting misfolded SOD1 by in vivo immunoimaging in a subject. The antibody and equivalent binding molecule specifically bind to misfolded and / or aggregated SOD1 and conjugate with a detection label. Preferably, the subject is a human.

[0009] This invention is based on a recombinant human monoclonal antibody (AP-101, Maier) that selectively targets mSOD1. et al. As described in SciTransl. Med. (2018):10 (470), it is coupled with a PET imaging agent to... 89 Characterized by the Zr-DFO-AP-101 radioactive tracer, it is used for PET imaging of mSOD1.

[0010] As shown in Example 1 and the preceding Materials and Methods section, by combining the AP-101 antibody with... p -SCN-Bn deferoxamine p -SCN-Bn deferoxamine (SCN-Bn-DFO) conjugate for use 89 Zr(t ½ =78.41h) marker was used to prepare DFO-AP-101. Longitudinal imaging studies were performed to determine the expression of mSOD1 in the transgene (Tg, [B6.Cg-Tg(SOD1)). The optimal mouse age for detecting mSOD1 aggregation in G93A)1Gur / J)]) mice and wild-type (Wt) mice and 89 Time following Zr-DFO-AP-101 administration. As a control, a subset of mice were co-injected with AP-101 (100 mg / kg) to assess target specificity. PET / CT data are expressed as percentage of injected dose per gram of tissue (%ID / g).

[0011] As shown in Example 4, 89 Zr-DFO-AP-101 binds to mSOD1 aggregates in the spinal cord of Tg mice, and imaging is optimal in 126-day-old Tg mice on day 10 post-administration. The number of aggregates detected on day 10 (18) is more significant than on day 7 (6). All spots were found between the T10 and L2 vertebrae. (The text abruptly ends here, likely due to an incomplete translation or source material.) 89Zr-DFO-AP-101 concentrations were significantly higher in Zr-DFO-AP-101 than in Wt mice (p=0.01). Co-injection of AP-101 significantly reduced the amount of detected mSOD1 aggregates, from 67% (6 / 9) to 15% (5 / 13). Alone use... 89 When Zr-DFO-AP-101 or in excess of AP-101 was present, the aggregate strength also decreased from 8.05±1.30 %ID / g to 5.73±0.28. AP-101 blockade significantly reduced the effects of AP-101 on the spinal cord (p=0.0002) and vertebrae (p<0.0001). 89 Intake of Zr-DFO-AP-101.

[0012] Furthermore, as shown in Example 7, in the context of these experiments, misSOD1 was also found in the bones / joints of SOD1 animals (PET signal, confirmed by IHC with other misSOD1 antibodies).

[0013] In short, with 89 The anti-SOD1 antibody conjugate labeled with Zr-DFO-AP-101, for example, showed unprecedented detection of mSOD1 aggregates in transgenic mice.

[0014] Furthermore, as shown in Example 10, the labeled antibody conjugate of the present invention is safe to use, and its clearance is primarily physical, with early excretion in the urine. Therefore, this tracer can be safely used in patients. Data to be obtained in ALS patients will validate its ability to measure in vivo mSOD1 levels for diagnostic and therapeutic monitoring. Thus, this labeled antibody conjugate has been found to be applicable to the early diagnosis and monitoring of therapeutic interventions for ALS. Furthermore, this construct, along with mSOD1 binding molecules (such as anti-SOD1 antibodies), can generally be used to target SOD1, i.e., misfolded and aggregated SOD1 in bone and / or joints, and may pave the way for potential treatments / improvements for “bone problems” in future ALS patients.

[0015] Therefore, the present invention generally relates to antibodies and equivalent SOD1-binding molecules for detecting misfolded and aggregated SOD1 in vivo, preferably for immunoimaging of misfolded and / or aggregated SOD1 in a subject. Imaging methods for detecting antibody-label conjugates in a subject are preferably selected from the group consisting of PET, SPECT, MRI, MPI, and optical imaging (including fluorescence imaging), or combinations thereof, or combinations thereof with CT (such as PET / CT, PET / MRI, PET / fluorescence, SPECT / CT imaging), or a combination thereof.

[0016] The labeling of the antibody conjugate or equivalent binding molecule is selected based on the imaging method intended for diagnosing ALS or monitoring ALS drug treatment and / or progression in a subject. If intended for PET and / or SPECT, the label is preferably a radionuclide or chemical compound (chelating agent or cofactor), wherein one or more atoms have been replaced by the radionuclide. If intended for PET only, a positron-emitting radionuclide is preferably selected. If intended for SPECT only, a gamma-ray-emitting radionuclide is preferably selected. If intended for PET or alternatively for SPECT, the antibody or equivalent binding molecule is preferably conjugated simultaneously with a cofactor and a chelating agent. In this way, a PET label (e.g., a positron-emitting radionuclide (e.g., F-18)) can be conjugated with a cofactor, and a SPECT label (e.g., a gamma-ray-emitting radionuclide (e.g., Ga-67)) can be conjugated with a chelating agent. In one embodiment, the antibody or equivalent binding molecule can be conjugated simultaneously with a cofactor (for labeling with PET labels exemplarily listed below) and a chelating agent (for binding with SPECT labels exemplarily listed below). Especially due to several technical advantages of PET over SPECT, PET or a combination of PET and CT is preferably used in the methods described herein according to the present invention. The advantages of PET over SPECT include higher photon penetration in PET, which can be combined with depth-dependent attenuation correction. In this way, the specificity of the imaging method is improved by reducing the number of false positives. Furthermore, due to the higher spatial and contrast resolution of PET, smaller defects can be detected by PET than by SPECT, thereby further improving sensitivity. Finally, PET allows for higher temporal resolution, thereby enabling dynamic imaging of tracer kinetics.

[0017] Preferably, the label to be included in the labeled antibody conjugate or labeled equivalent binding molecule is selected from: fluorine-18 (F-18), titanium-45 (Ti-45), manganese-52 (Mn-52), iron-52 (Fe-52), potassium-43 (K-43), scandium-43 (Sc-43), scandium-44 (Sc-44), cobalt-57 (Co-57), copper-60 (Cu-60), copper- 61 (Cu-61), Copper-62 (Cu-62), Copper-64 (Cu-64), Copper-67 (Cu-67), Gallium-67 (Ga-67), Gallium-68 (Ga-68), Bromine-76 (Br-76), Bromine-77 (Br-77), Krypton-81m (Kr-81m), Rubidium-81 (Rb-81), Yttrium-86 (Y-86), Strontium-87m (Sr- 87m), Zirconium-89 (Zr-89 or 89Zr), Technetium-99m (Tc-99m), Indium-111 (In-111), Indium-113m (In-113m), Antimony-117 (Sb-117), Iodine-123 (I-123), Iodine-125 (I-125), Cesium-127 (Cs-127), Cesium-129 (Cs-129), Iodine-131 (I-1 31) Iodine-132 (I-132), lutetium-177 (Lu-177), rhenium-186 (Re-186), mercury-197 (Hg-197), rhenium-188 (Re-188), lead-203 (Pg-203), bismuth-206 (Bi-206), actinium-225 (Ac-225), radium-225 (Ra-225), and any combination of two or more of these.

[0018] More preferably, the mark is selected from the group consisting of or including the following: (a) Used in PET, derived from: Fluorine-18 (F-18), Manganese-52 (Mn-52), Copper-64 (Cu-64), Gallium-68 (Ga-68), Antimony-117 (Sb-117), Scandium-43 (Sc-43), Scandium-44 (Sc-44), Titanium-45 (Ti-45), Bromine-76 (Br-76), Yttrium-86 (Y-86), Iodine-124 (I-124), and Zirconium-89 (Zr-89 or 89 Zr); (b) Used in SPECT, derived from: Indium-111 (In-111), Technetium-99m (Tc-99m), Gallium-67 (Ga-67), Iodine-123 (I-123), Iodine-131 (I-131), Lutetium-177 and Rhenium-186 (Re-186); (c) For use in MRI and MPI: magnetic, paramagnetic, or superparamagnetic ion complexes or particles, preferably containing gadolinium. 3+ (Gd 3+ ),manganese2+ (Mn 2+ );copper 2+ (Cu 2+ ); iron oxide (Fe3O4) contrast agent; and (d) Used for optical imaging, including fluorescence imaging, from: fluorescein, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, Texas Red® (Molecular Probes, Inc., Eugene, OR), AlexaFluor® (Molecular Probes, Inc., Eugene), AlexaFluor 350, AlexaFluor 405, AlexaFluor 430, AlexaFluor 488, AlexaFluor 500, AlexaFluor 532, AlexaFluor 546, AlexaFluor 568, AlexaFluor 594, AlexaFluor 610, AlexaFluor 633, AlexaFluor 647, AlexaFluor 660, AlexaFluor 680, AlexaFluor 700, AlexaFluor 750, BODIPY FL, BODIPY R6G, BODIPYTMR, BOPDIPY 530 / 550, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY 650 / 665, Rhodamine Green™-X (Molecular Probes, Inc.), Rhodamine Red™-X (Molecular Probes, Inc.), Rhodamine 6G, TMR, TAMRA™ (Applied Biosystems).

[0019] More preferably, the detection marker is a radionuclide. According to the invention, all radionuclides that can be detected in positron emission tomography (PET) and / or single-photon emission computed tomography (SPECT) can be used as part of the detection marker; however, preferably the physical half-life of the radionuclide should match its intended application, or more specifically, the biological half-life of the carrier (i.e., the antibody conjugated thereto).

[0020] Radionuclide tracers have energies of about 20 to about 4,000 keV. For SPECT, whether used alone or in combination with other imaging methods, radionuclides with energies of about 30 to about 300 keV are preferably used. Suitable radionuclide tracers include radionuclides that emit gamma rays (particularly suitable for SPECT), positrons (particularly suitable for PET), or a combination of gamma rays and positrons (suitable for PET or SPECT). In illustrative, non-limiting embodiments, the radionuclide tracers have suitable decay characteristics to optimize image resolution and quantitative accuracy and / or have suitable residual properties. For example, the radionuclide tracer may have a physical half-life (i.e., t½) that is time-compatible with the antibody to achieve the optimal specific-to-nonspecific binding ratio. Examples of radionuclide tracers suitable for PET imaging and suitable for inclusion in labeled antibody conjugates according to embodiments of the present disclosure are F-18 (t½ about 1.83 hours), Sc-44 (t½ about 3.97 hours), Ti-45 (t½ about 3 hours), Mn-52 (t½ about 5.6 days), Cu-64 (t½ about 12.7 hours), Ga-68 (t½ about 1.13 hours), Br-76 (t½ about 16.2 hours), Y-86 (t½ about 14.7 hours), Zr-89 (t½ about 78.4 hours), I-124 (t½ about 100.3 hours), or any combination of two or more thereof. Examples of radionuclide tracers suitable for SPECT imaging include Tc-99m (t½ about 6 h), In-111 (t½ about 2.80 days), I-123 (t½ about 13.2 hours), I-131 (t½ about 8 days), Lu-177 (t½ about 6.64 days), Re-186 (t½ about 3.7186 days), or any combination of two or more thereof.

[0021] Preferably, according to the invention, a radionuclide is used as part of the labeling, having a physical half-life between about 1 hour and about 10 days. The choice of labeling can also be based on whether the antibody or equivalent binding molecule (e.g., antibody fragment) needs to be labeled, preferably matching the biological half-life of the antibody or antibody fragment or another equivalent binding molecule. If the antibody is to be labeled, a half-life of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or about 10 days is preferred, more preferably about 3-10 days, and most preferably about 3-7 or 3-5 days. If the antibody fragment is to be labeled, a label with a shorter half-life than the antibody can be selected. Preferably, the half-life of the labeling of the antibody fragment is at least 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day, more preferably between about 1 hour and 5 days or between about 1 and 48 hours. Therefore, for labeling antibody fragments for PET, radionuclides with short physical half-lives are selected, preferably from the group consisting of Cu-64, F-18, Ti-45, Br-76, Y-86, Zr-89, or I-124, or any combination of two or more thereof. For labeling antibodies for PET, radionuclides with longer physical half-lives are preferred, preferably Zr-89 or I-124. For labeling antibodies or antibody fragments for SPECT, radionucleotides are preferably selected from the group consisting of Tc-99m, In-111, I-123, I-131, Lu-177, and Re-186, or any combination of two or more thereof.

[0022] In one embodiment, for PET, short-lived radionuclides (such as F-18 and Ga-68) are used to label the binding molecules for imaging on the same day after injection. Given their short half-lives, in one embodiment, antibody fragments with shorter biological half-lives are preferably used with these labels (such as single-domain antibodies, scFv, or biantibodies). However, in one embodiment, intact antibodies are also labeled with these radionucleotides. In one embodiment, longer-lived radionuclides (such as Cu-64, Zr-89, and I-124) are preferably used with intact antibodies or larger antibody fragments (such as microantibodies) to obtain optimal imaging contrast 24-48 hours or longer after injection.

[0023] If radionuclides are used below as radiotracers for detection labeling, then the labeling of the antibody conjugates of the present invention, as well as other potential entities (such as bifunctional chelators or cofactors), are also referred to as tracers. According to the present invention, bifunctional chelators are preferably used for labeling antibodies with radioactive metals (such as Ga-68, Zr-89, and Cu-64), and cofactors are preferably used for labeling with radioactive halides (such as F-18, I 123 / 124 / 131).

[0024] As shown in the embodiments, if a radionuclide is used as a label or part of a label according to the present invention, the preferred radionuclide is Zr-89. Therefore, in a preferred embodiment, the labeling of the antibody conjugate of the present invention is a radionuclide. 89 Zr.

[0025] Preferably, the detection label is conjugated to the antibody via a bifunctional chelating agent or prosthetic group. The bifunctional chelating agent or prosthetic group can be conjugated in any manner, resulting in covalent binding to the antibody. Preferably, the chelating agent or prosthetic group is randomly conjugated to an amino acid residue or a glycan region of the antibody. Preferably, the bifunctional chelating agent or prosthetic group is conjugated to a lysine or cysteine ​​residue of the antibody. Most preferably, the bifunctional chelating agent or prosthetic group is randomly conjugated to a lysine residue of the antibody.

[0026] According to the present invention, chelating agents, which have been shown to be useful for conjugating radionuclides, magnetic, paramagnetic, or supermagnetic ion complexes or particles with antibodies, can be commonly used. Most preferably, bifunctional chelating agents include… p -SCN-Bn deferoxamine (SCN-Bn-DFO).

[0027] A preferred embodiment of the labeled antibody conjugate of the present invention is antibody AP-101 or a fragment or biological derivative thereof. AP-101 is described in detail in international patent application published in WO 2012 / 080518 A1, wherein the antibody is disclosed as antibody NI-204.12G7. The VH and VL chain sequences of the antibody and their CDRs are described in WO 2012 / 080518 A1. Figure 1 B and Table A below. The constant region of the antibody is preferably derived from human IgG1. However, the human constant domain in the labeled antibody conjugate of the present invention may also be a different IgG allotype or a different allotype, for example, to avoid or reduce immunogenicity due to allogeneic immunization; see, for example, the review by Jefferis and Lefranc, MAbs 1 (2009), 332-338. The antibody AP-101 and the method of its production are further described in detail in the international patent application published in WO 2024 / 042250 A1. The entire disclosure of WO2012 / 080518 A1 and WO 2024 / 042250 A1, in particular the paragraphs expressly referenced herein, are incorporated herein by reference.

[0028] The antibody portion of the labeled antibody conjugate of the present invention can be glycosylated or deglycosylated. In a preferred embodiment, the antibody is glycosylated, preferably wherein the antibody is produced in CHO cells. CHO cells are the most widely used mammalian cells for producing recombinant monoclonal antibodies because they are capable of post-translational modification (PTM) of antibody molecules, a modification that also commonly occurs in humans. Different CHO daughter cells with improved quality have been established through mutagenesis genetic manipulation. These variants include CHO-K1, CHO-S, CHO-DXB11, and CHO-DG44. Therefore, in one embodiment, antibody AP-101 is produced in CHO cells, more preferably in CHO-K1 cells (ATCC No. CCL 61). Preferably, after production, the AP-101 antibody is purified from the cell culture medium for further use. In another embodiment, the antibody is deglycosylated. Preferably, the antibody is deglycosylated... N - Glycans are removed from antibodies. According to the invention, deglycosylation can be obtained by various methods. Fc-glycans can be deglycosylated, for example, by enzymatic treatment with an endoglycosidase used for complex N-glycans, or by a bacterial host in which glycosylation has not occurred (such as *Escherichia coli*). E. coli Antibodies are produced in the process.

[0029] As described in Example 7 on pages 104 and 105 of WO 2024 / 042250 A1, and particularly Table 18 on page 105, the major PTMs identified in antibody AP-101 are the HC-to-pyroglutamic acid modification of N-terminal glutamine, the loss of C-terminal lysine, and N-glycosylation. In this context, the N-glycosylation site is identified at position 303 (HC N303). Therefore, in one embodiment, the anti-SOD1 antibody used according to the invention has lost its C-terminal lysine, i.e., the antibody has undergone C-terminal lysine cleavage. Specifically, the C-terminal lysine of the antibody heavy chain (preferably each heavy chain of the antibody) is cleaved. Furthermore, or alternatively, the glutamine at the N-terminus is modified to pyroglutamic acid, i.e., the antibody has undergone N-terminal glutamine cyclization. Furthermore, or alternatively, the antibody is glycosylated, particularly N-glycosylated. More particularly, the antibody heavy chain is glycosylated, and even more particularly, the N303 of the heavy chain.

[0030] In a preferred embodiment, the anti-SOD1 antibody (preferably AP-101 antibody) used according to the invention lacks a C-terminal cysteine ​​residue, has modified glutamine at the N-terminus as pyroglutamic acid, and includes at least one N-glycosylation site, or, as in pharmaceutical formulations, preferably most antibody species present in the formulation have the aforementioned modifications.

[0031] The present invention also relates generally to the labeled antibody conjugates disclosed herein, wherein the labeled antibody conjugates are suitable for in vivo immunoimaging of misfolded SOD1 in subjects. The present invention also relates to a method for diagnosing ALS and monitoring ALS drug treatment and / or progression in subjects using the labeled antibody conjugates disclosed herein, wherein the method includes in vivo immunoimaging of the subjects. Preferably, imaging of the labeled antibody conjugates in subjects includes positron emission tomography (PET) imaging, single-photon emission computed tomography (SPECT) imaging, magnetic resonance imaging (MRI), magnetic particle imaging (MPI), or a combination of PET or SPECT with computed tomography (CT), MRI, or MPI.

[0032] In a further aspect, the present invention generally relates to a medicament for treating a patient who has been diagnosed with or is developing ALS, and / or for monitoring the efficacy of the medicament and / or the progression of ALS by the methods defined herein. Preferably, the medicament is selected from riluzole (Rilutek®, Tigultik, Exservan). TM ), Edaravone (Radicava®), Tofersen (BIIB067), AP-101, Dextromethorphan hydrobromide, and Quinidine sulfate Sulfate (Nuedexta®), Reldesemtiv (CY5031), Arimoclomol, Levosimendan, Fasudil, Pyrimethamine (Daraprim™), Rapamycin, Baclofen (Gablofen®, Kemstro®, Lioresal®), Diazepam (Diastat®, Valium®), Amitriptyline (Elavil®), Trihexyphenidyl, Scopoderm® (scopolamine patch), Glycopyrrolate (Robinul®), Ravulizumab (also known as BNJ441, ALXN1210, or Ultomiris®), and Eculizumab (also known as Soliris®).

[0033] Preferably, the labeled antibody conjugate is 89 Zr-DFO-AP-101, and includes (a) AP-101, as an antibody that specifically binds to misfolded SOD1; (b) p-SCN-Bn deferoxamine (SCN-Bn-DFO), as a bifunctional chelating agent, and (c) 89 Zr is a radioactive nuclide.

[0034] In a preferred embodiment, the labeled antibody conjugate of the present invention is glycosylated. In another preferred embodiment, the labeled antibody conjugate as defined above is further characterized in that the AP-101 antibody is deglycosylated. Such deglycosylated antibody is interchangeably referred to as deglycosylated antibody. 89 Zr-DFO-AP-101 or Degly- 89 Zr-DFO-AP-101. When labeled with Cu-64, this deglycosylated antibody is interchangeably referred to as deglycosylated. 64 Cu-NOTA-AP-101 or Degly- 64 Cu-NOTA-AP-101.

[0035] Furthermore, the present invention provides and relates to antibody conjugate intermediates, wherein the antibody portion of an antibody that specifically binds to misfolded SOD1 comprises a bifunctional chelating agent, preferably wherein the chelating agent is p -SCN-Bn deferoxamine (SCN-Bn-DFO). This antibody conjugate intermediate is particularly useful for preparing the labeled antibody conjugates of the present invention because it already includes a bifunctional chelating agent that conjugates with the antibody, and the label can be selected and conjugated according to the intended use. Specific labels that can be conjugated are listed above, depending on the imaging technology for which the conjugate is intended to be used, i.e., PET, SPECT, MRI or MPI, PET / CT, PET / MRI, SPECT / CT or SPECT / MRI.

[0036] The present invention also provides an imaging composition comprising an antibody or antibody conjugate as defined above, and further comprising a pharmaceutically acceptable excipient, preferably NaCl and gentianic acid, preferably at a pH of 4.5 to 8.0. Preferably, the imaging composition is a 10 mL solution in a sealed vial consisting of NaCl (0.9%) and gentianic acid (10 mg), containing about 10 mg of a labeled antibody conjugate and about 300 to 500 MBq, preferably wherein the amount of the labeled antibody conjugate is greater than 97% relative to the unlabeled antibody and / or wherein the apparent specific activity of the labeled antibody conjugate is about 0.656 GBq / mg, optionally wherein the injection solution may be diluted with NaCl prior to injection. When using deglycosylated... 89When preparing the corresponding imaging compositions using Zr-DFO-AP-101, the apparent specific activity of the labeled deglycosylated antibody conjugate was approximately 0.46 MBq / µg.

[0037] The present invention further provides diagnostic compositions or kits comprising labeled antibody conjugates or antibody conjugate intermediates as defined herein (i.e., possibly comprising deglycosylated forms or intermediate forms comprising chelating agents), optionally further comprising a detection marker, preferably a radionuclide, preferably wherein the radionuclide is Zr-89; or imaging compositions as defined above; and descriptions of methods optionally for diagnosing amyotrophic lateral sclerosis (ALS) or monitoring ALS drug treatment and / or progression in subjects, as defined above.

[0038] Furthermore, the present invention also relates to a method for in vivo immunoimaging of superoxide dismutase (SOD1), which can be used as a biomarker for amyotrophic lateral sclerosis (ALS) or a biomarker for the efficacy of ALS drug treatment in a subject, the method comprising: (a) Administration of the subject to the labeled antibody conjugate as defined above and below; and (b) Detection of the presence of labeled antibody conjugates in the subject's body by imaging. Imaging is preferably performed by methods selected from PET, SPECT, MRI and MPI, optical imaging including fluorescence imaging, or combinations thereof (such as PET / CT, SPECT / CT, PET / MRI and SPECT / MRI).

[0039] Preferably, a method for in vivo immunoimaging of superoxide dismutase (SOD1) is provided, wherein the method includes a radiographic imaging method of a subject after administration of a labeled antibody conjugate; and the preparation of a radiographic image of the subject for detecting the labeled antibody conjugate; wherein the radiographic image is used to diagnose the presence of misfolded and / or aggregated SOD1 in the subject.

[0040] Furthermore, the present invention provides a method for generating human images, preferably a method for generating human images of ALS patients, comprising administering a labeled antibody conjugate as defined herein to the human body and generating an image of at least a portion of the human body to which the labeled antibody conjugate has been distributed.

[0041] Preferably, a method for in vivo immunoimaging of superoxide dismutase (SOD1) or for generating human images is provided, wherein a labeled antibody conjugate is present in, or provided by, an imaging composition comprising, an antibody or antibody conjugate as defined above.

[0042] Preferably, one of the foregoing methods is provided, wherein the method includes contacting cells, tissues, or organs with a labeled antibody conjugate, wherein the labeled antibody conjugate is administered at a target dose in the range of 10-100 MBq; and preparing a radiographic image for detecting the labeled antibody conjugate. Particularly preferably, the labeled antibody conjugate is administered in such a manner that the target dose is in the range of about 40 MBq ± 10%.

[0043] Furthermore, in any of the foregoing methods, the labeled antibody conjugate is preferably administered at a target dose significantly lower than the therapeutic dose of the antibody. Preferably, the foregoing methods are provided in which the labeled antibody conjugate is administered at the target dose, wherein the amount of antibody protein is less than 2 mg. In these methods, the labeled antibody conjugate can be administered using different routes of administration; however, it is preferred that the labeled antibody conjugate be administered by intravenous injection. The final volume injected in these methods is preferably based on the content of HEPES or gentic acid, with specifications of ≤200 µg / injection and ≤5 mg / injection, respectively. In these methods, the labeled antibody conjugate can be administered in several doses; however, it is preferred to administer a single dose. The aforementioned immunoimaging or image generation methods can be performed shortly after administration of the labeled antibody conjugate. Preferably, these methods are performed at a time following administration of the labeled antibody conjugate, wherein this time is selected from the group consisting of about 1 hour, 2 hours, 3 hours, and 24 hours, and most preferably, the method is performed 2 hours after administration (in the preferred embodiment, intravenous injection).

[0044] Because AP-101 has a half-life of approximately 9.5 days in serum, and the labeled antibody conjugate is also very stable, as seen in the images obtained in animals 3 and 10 days after injection of the labeled antibody conjugate (see, for example, Figure 3 and 8 In a preferred embodiment, the imaging method described above is performed 1, 3, 7 and / or 10 days after administration (intravenous injection).

[0045] In addition, the aforementioned method is provided, wherein the imaging method is selected from the group consisting of planar imaging, positron emission tomography (PET), single-photon computed tomography (SPECT), and combinations thereof with computed tomography (CT).

[0046] Preferably, the method is provided in which regions of interest (ROIs) selected from a group of organs / tissues comprising the liver, kidneys, blood / heart (left ventricle), spleen, muscle, subcutaneous fat, red bone marrow, bone, joints, and cartilage are imaged. The most preferred method is provided in which ROIs are imaged, as further illustrated in the context of the experimental results presented herein, for example, in… Figure 1-4In 10, 13, 17 and 21, that is, in the spinal cord and in the bones of the vertebrae.

[0047] In a further embodiment, the aforementioned method is provided, wherein a labeled antibody conjugate is administered to predict the effectiveness of a therapeutic agent in treating diseases caused by and / or associated with the presence of misfolded and / or aggregated SOD1. Preferably, the therapeutic agent is selected from riluzole (Rilutek®, Tigultik, Exservan™), edaravone (Radicava®), tovsen (BIIB067), AP-101, dextromethorphan hydrobromide and quinidine sulfate (Nuedexta®), redisesentiel (CY5031), arellomo, levosimendan, fasudil, pyrimethamine (Daraprim™), rapamycin, baclofen (Gablofen®, Kemstro®, Lioresal®), diazepam (Diastat®, Valium®), amitriptyline (Elavil®), trihexyphenidyl, Scopoderm® (scopolamine patch), glycopyrronium bromide (Robinul®), piracetamab (also known as BNJ441, ALXN1210 or Ultomiris®), and eculizumab (also known as Soliris®).

[0048] Furthermore, the aforementioned method for in vivo immunoimaging and generating human images of superoxide dismutase (SOD1) is provided, wherein a labeled antibody conjugate is administered to diagnose ALS or monitor the effectiveness of therapeutic treatment for ALS. The method includes performing radiographic imaging on the subject after administration of the labeled antibody conjugate; and generating radiographic images of the subject for detecting the labeled antibody conjugate; wherein the radiographic images are used for diagnosing ALS and for assessing the effectiveness of therapeutic treatment for ALS, respectively.

[0049] In a further embodiment, the present invention relates to anti-SOD1 antibodies or equivalent SOD1-binding molecules, such as antibody fragments further defined below, for targeting misfolded and / or aggregated SOD1 in the bones and / or joints of a subject. In this document, bone and / or joint-related diseases are understood as diseases that impair the structure and / or function of the bones and / or joints of a subject. Preferably, the antibody or equivalent binding molecule is capable of binding to misfolded and / or aggregated SOD1. Preferably, the anti-SOD1 antibody or equivalent SOD1-binding molecule is antibody AP-101, its SOD1-binding fragment, or a labeled antibody conjugate as described herein. Preferably, the subject is a mammal, more preferably, the subject is a human. In a preferred embodiment, the disease is ALS. Furthermore, the invention also relates to... 89 Zr or 89Zr-DFO is used for immunoimaging of misfolded and / or aggregated SOD1 in subjects, preferably for use with the labeled antibody conjugates disclosed herein and / or for early diagnosis of ALS and / or for monitoring therapeutic interventions for ALS. Furthermore, in one embodiment, a pharmaceutical composition is provided comprising an anti-SOD1 antibody or equivalent binding molecule, a labeled antibody conjugate or equivalent SOD1 binding label molecule, and a pharmaceutically acceptable carrier for targeting misfolded and / or aggregated SOD1 in bone and / or joints.

[0050] In a further embodiment, the present invention also provides the use of the antibody or labeled antibody conjugate as defined above in the production of an imaging composition for use in one of the methods defined above.

[0051] In another embodiment, the present invention further provides a method for preparing the above-defined [material / material]. 89 A method for preparing a solution of a Zr-DFO-AP-101 antibody conjugate, the method comprising the following steps: (a) will 89 Zr chloride solution was combined with DFO-AP-101 to obtain a reaction solution; (b) The reaction solution is heated to a temperature of approximately 35ºC to approximately 42ºC, preferably approximately 37ºC, for a period of approximately 15 minutes to approximately 90 minutes, preferably approximately 45 minutes; and (e) the reaction solution containing [ 89 Zr]Zr-DFO-AP-101 is recovered into the recovery solution.

[0052] Preferably, the method further includes one or two of the following steps: (f) By adding salt water [ 89 The Zr-DFO-AP-101 recovery solution is adjusted to a final volume, preferably 10 mL; and / or (g) Filtration 89 Zr]Zr-DFO-AP-101 recovery solution.

[0053] In step (a), 89 The Zr-chloride solution is preferably adjusted to a pH of 6.5-8.0. Preferably, this is achieved by adding... 89 Sodium bicarbonate solution and HEPES buffer solution are added to the Zr-chloride solution for adjustment. In addition, recovery [ 89 The Zr-DFO-AP-101 process is preferably performed on a column comprising a gel filtration resin and includes the following steps: (c) Adjust the gel filter resin in the column with 10 mL of 0.9% saline solution, preferably at a rate of 1.5 mL / min; preferably, this step is repeated 3 times. The adjustment is preferably carried out during the warming of the reaction solution.

[0054] (d) Transfer the reaction solution from (b) and 4 mL of saline solution to the conditioning column from (c); (e) Recovery from the reaction solution [ 89 Zr]Zr-DFO-AP-101 is added to the solution recovered from the column.

[0055] Therefore, in a preferred embodiment, the above method includes the following steps: (a) Adjust the pH to 6.5-8.0. 89 Zr chloride solution was combined with DFO-AP-101 to obtain a reaction solution; (b) Warm the reaction solution to approximately 37ºC for approximately 45 minutes; (c) Adjust the gel filter resin in the column with 10 mL of 0.9% saline, preferably at a rate of 1.5 mL / min; preferably, this step is repeated 3 times; (d) Transfer the reaction solution from (b) and 4 mL of saline solution to the conditioning column from (c); (e) Recover from the column [ 89 Zr]Zr-DFO-AP-101 was used as the recovery solution; (f) By adding salt water [ 89 The Zr-DFO-AP-101 recovery solution is adjusted to a final volume, preferably 10 mL; and preferably... (g) Filtration 89 Zr]Zr-DFO-AP-101 solution.

[0056] Furthermore, the preparation method is provided, wherein in step (a) 89 Zr chloride solution is provided through the following steps: (a1) will 89 Zr-oxalate solution was fed onto a quaternary methyl ammonium (QMA) anion exchange column; (a2) A solution containing HCl was added to the QMA anion exchange column; and (a3) Collect the elution from the column 89 Zr chloride solution.

[0057] Preferably, the above-defined preparation method is provided, wherein the method further includes the following steps: (c) The filtered product obtained in step (g) is reacted by adding 0.9% saline and 10 mg gentianic acid. [89 The Zr]Zr-DFO-AP-101 solution was used to formulate the composition.

[0058] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While similar or equivalent methods and materials may be used in the practice or testing of this invention, exemplary methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. Materials, methods, and examples are for illustrative purposes only and are not intended to be limiting.

[0059] Other features and advantages of the invention will become apparent from the following detailed description and claims.

[0060] To avoid any doubt, it is necessary to emphasize that the use and meaning of expressions such as "in some embodiments," "in a certain embodiment," "in some aspects," "in certain aspects," "in some aspects," "in a further aspect," "in some cases," "in some cases," "in a further embodiment," and "in one embodiment" mean that when reading any embodiment described herein, one should consider combining each feature of these embodiments, and this disclosure must be treated in the same manner as if the combination of features of these embodiments were described in detail in one embodiment. The same applies to any combination of embodiments and features shown in the appended claims and embodiments, which are also intended to be combined with the features of the corresponding embodiments disclosed in the specification, wherein, for the sake of consistency and brevity only, embodiments are characterized by dependencies, and in practice, each combination of embodiments and features that can be explained by (multiple) dependencies must be considered as explicitly and unambiguously disclosed, and not as a choice among different options. Attached Figure Description

[0061] Figure 1Steps for mSOD1 aggregate identification and processing: (A) Visually identify spots on PET images and plot regions of interest (ROIs) including the entire spot. (B) Spots near the anterior vertebral bone where radiotracer spillage occurs are not retained for analysis. (C) In the same mouse, trace similar ROIs in the spinal cord region without mSOD1 to obtain an internal reference. (D) Obtain an external reference from the spinal cord of control mice by plotting discrete ROIs at levels corresponding to Tg mSOD1 spots or by tracing ROIs (outlines) including all control mouse spinal cords. (E) Report the amount of radiotracer in the mSOD1 spots in %ID / g and other observed parameters. (F) Overview table of mouse spots counted according to animal age. (G) Visualization of spot intensity compared to animal age. Older animals show more spots / animals.

[0062] Figure 2 Images from SOD1 G93A transgenic ALS mice: (A) Sagittal PET image showing the outlined region of interest (ROI) traced in the spinal cord and anterior vertebral region. (B) Axial (cross-sectional) PET image showing the ROI traced in the spinal cord and anterior vertebral region. (C) Axial CT image showing the ROI traced in the spinal cord.

[0063] Figure 3 IV administration to 4-month-old SOD1 G93A transgenic (Tg) mice 89 PET images taken on day 10 after Zr-DFO-AP-101 show the presence of radiotracers in the femoral head (top) and spleen (bottom). Arrows point to selected ROIs on the femoral head and spleen to quantify their radiotracer content. Signal intensity within the spinal cord increased over time from day 3 to day 10 (not shown).

[0064] Figure 3 (continued) PET images showed that on day 7, in the absence of [(A)Wt and (C)Tg] and in the presence of [(B)Wt and (D)Tg] co-injection of AP-101 (100 mg / kg), the bone (abdominal vertebrae, femoral head) and spleen were... 89 Zr-DFO-AP-101 uptake. AP-101 blocks and reduces the uptake of these tissues. 89 Intake of Zr-DFO-AP-101. 89 When Zr-DFO-AP-101 is present in the vertebrae, it can be strong enough to spill into the spinal cord, making the quantification of the amount of radiotracer in the mSOD-1 spot unreliable. To mitigate this effect, all images are corrected for spillover and recovery coefficients using well-developed mathematical models, such as those used by Émilie Gaudin. et al.The contents of Phys. Med. Biol. 66 (2021) 065019 are hereby incorporated in their entirety. Discovery 89 Zr-DFO-AP-101 uptake in bone and spleen is specific because it is blocked by co-injection of unlabeled AP-101. AP-101 binds not only to mSOD1 but also to Fc receptors; to reduce Fc binding, deglycosylation was synthesized and evaluated. 89 Zr-DFO AP-101 showed a large blocking effect in wild-type mice, indicating a significant contribution from misfolded SOD1 signaling.

[0065] Figure 3 (continued) In the scatter plot (EH), during injection 89 Zr-DFO- -miSOD1 and its blocking (100 mg / kg unlabeled) -miSOD1) and deglycosylation 89 Zr-DFO- Following -miSOD1, quantification of PET signal (%ID / g; individual mice with mean) was provided in the ROI of the spinal cord (E), vertebrae (F), femoral head (G), and spleen (H) of SOD1 G93A transgenic and wild-type mice. p<0.001, p<0.0001 (One-way ANOVA, Dunnett's multiple comparison test).

[0066] Figure 4 Using the recovery factor RC, based on the abdominal vertebrae... 89 The spillover and volumetric effects of Zr tracers correct for radioactivity measured within the spinal cord.

[0067] Figure 5 Partial volume effect (PVE) correction was performed using an ultramicro hotspot phantom. (A) Lateral view of a 0.3 mm slice obtained from the ultramicro hotspot phantom and the hotspot line profile at the location indicated by the yellow line. (B) Average activity of the hotspot (left y-axis) and recovery factor obtained by dividing the average activity of the hotspot by the maximum activity of the maximum hotspot (right y-axis) (Gaudin). et al. (2021).

[0068] Figure 6 nat Zr-DFO-AP-101 binds to A) denatured SOD1 and B) native SOD1. In a direct enzyme-linked immunosorbent assay (ELISA), unmodified α-miSOD1 (gray solid line with a solid circle) and DFO-labeled SOD1 are detected.nat Zr-DFO-AP-101 (black solid line with hollow triangle) and deglycosylation nat Zr-DFO-AP-101 (with a dotted line featuring hollow squares) binds with transgender SOD1(A) with high affinity, EC 50 The values ​​were 0.2, 0.3 and 2.4 nM, respectively, while showing only minimal binding to the physiological SOD1 dimer (B).

[0069] Figure 7 Characterization of Degly-DFO-AP-101. Characterization and comparison of binding properties of DFO-conjugated, deglycosylated, and unmodified AP-101. (A) Purified deglycosylated DFO-AP-101 (1), DFO- HPLC tracking of α-miSOD1(2) and unpurified deglycosylated DFO-AP-101(3) showed the presence of free polysaccharides (4) after incubation with ENDO-S2 for 2 h. (B) SDS-PAGE / Coomassie Brilliant Blue (2) of reduced AP-101(1) and deglycosylated α-miSOD1 after incubation with ENDO-S2 for 2 h.

[0070] Figure 8 Residual radioactive tracer content in isolated non-target organs of mice after perfusion. 89 Zr-DFO-AP-101 injection, followed by blockade with 100 mg / kg unlabeled α-miSOD1 (blocking), or degly 89 Residual radioactivity (expressed as a percentage of the injected dose per gram of tissue; %ID / g) in major non-target tissues of Zr-DFO-AP-101 (deglycosylated) SOD1 G93A transgenic (Tg; solid symbol) and wild-type (Wt; hollow symbol) mice 10 days post-injection was measured in whole blood (A) or after perfusion of the mouse spleen (B), liver (C), tibia (D), lung (E), and kidney (F). Mean %ID / g ± SEM; ns not significant. p<0.01, p<0.001, p<0.0001 (one-way ANOVA; Dunnett post-hoc test).

[0071] Figure 9 (A) prepared alone in PBS or (B) prepared in the presence of GA. 89 SDS-PAGE of Zr-DFO-AP-101. Monitoring under reducing conditions.

[0072] Figure 10This article describes the experimental groups / conditions: the distribution of transgenic (Tg or ALS) and wild-type (Wt or CTL) mice within different experimental groups. This study investigated the expression of misfolded SOD1 in Tg[B6.Cg-Tg(SOD1)]. The study was conducted on G93A)1Gur / J)] mice and control Wt[C56BL / 6J] mice. The aim was to select the optimal mouse age for PET imaging and 89 Time following Zr-DFO-AP-101 administration. For this study, mice older than 126 days were used, and imaging was performed on days 7 and 10 after administration of the radiotracer. The use of Zr-DFO-AP-101 was evaluated by blocking with unlabeled AP-101. 89 The feasibility of using Zr-DFO-AP-101 to detect accumulated mSOD1 in the spinal cord, to establish 89 Zr-DFO-AP-101 has specificity for mSOD1. Deglycosylation was also explored as an evaluation criterion. 89 The mode of Fc binding effect of Zr-DFO-AP-101. (A) Previous longitudinal studies aimed to select the optimal age and number of days after radiotracer administration for PET imaging. (B) Current study evaluates the use of... 89 Feasibility of using Zr-DFO-AP-101 for PET imaging of misfolded SOD1 (mSOD1).

[0073] Figure 11 Scatter plots and descriptive statistics were used to compare the body weights of 126-day-old Tg and Wt mice. An unpaired two-tailed Student's t-test was used to compare the body weights of Tg and Wt mice.

[0074] Figure 12 Scatter plots and descriptive statistics comparing the dose per gram of radiotracer administered to 126-day-old Tg and Wt mice. Unpaired two-tailed Student's t-test was used to compare the doses received by Tg and Wt mice.

[0075] Figure: Distribution of mSOD1 spots in the spinal cord of 13 mice. The left MIB PET / CT image shows the distribution using... 89 Spots detected by Zr-DFO-AP-101 and its deglycosylated analogues. The figure also includes false (+) spots from control (Wt) mice and blocking mice (Tg and Wt), which show valid spots. Data in the table are also included. Figure 21 Visualize in IJ.

[0076] Figure 14A bar graph was created comparing the frequency of mSOD1 spot detection (%), the mean intensity of detected mSOD1 spots, and the ratio of mSOD1 spots to an internal reference. Error bars represent the standard deviation (SDM) of the mean. The number at the top of the bar represents the number of mice in that group. Error bars indicate the standard deviation of the sample mean. A two-tailed Student's t-test was used to obtain the results. p "value.

[0077] Figure: Internal reference values ​​measured in the spinal cords of Tg mice receiving deglycosylated analogues were significantly higher than those receiving [other drugs]. 89 Internal reference values ​​were obtained from Zr-DFO-AP-101 Tg mice (two-tailed Student-t test). Higher background values ​​will help increase Degly- 89 %ID / g value of mSOD1 spots in Zr-DFO-AP-101 mice.

[0078] Figure: 16 Full-body maximum intensity projection PET / CT images 89 Biodistribution of Zr-AP-(A, D) in 4-month-old SOD1 G93A transgenic (Tg, A, B, C) and control wild-type (D, E, F) mice, and biodistribution of Zr-AP-(A, D) in the liver, spleen, spinal cord, abdominal vertebrae, and femoral head under blocking (B, E; 100 mg / kg unlabeled AP-101) and deglycosylation conditions (C, F). (G) positive regions are annotated.

[0079] Figure: 17 Comparisons 89 Zr-DFO-AP-101 and blocking conditions and Degly- 89 Scatter plots of spinal cord uptake (A), ventral vertebral bone uptake (B), and the ratio of spinal cord to ventral vertebral bone uptake (C) in Zr-DFO-AP-101 mice. %ID values ​​were obtained from contoured ROIs traced along the spinal cord. For each condition, a two-tailed Student's t-test was used to compare %ID / g values ​​measured in mice for Tg (orange; first scatter plot in each figure) and Wt (blue; second scatter plot in each figure), with p-values ​​given at the bottom of the figures. One-way ANOVA and Dunnett's multiple comparison test were used. 89 The %ID / g values ​​of Zr-DFO-AP-101 in Tg mice were compared with those in the blockade and deglycosylation groups to compare the effects of... 89 Zr-DFO-AP-101 values ​​obtained under blocking and deglycosylation conditions (dashed lines and "p" values).

[0080] Figure 18Correlation between %ID / g values ​​measured by PET imaging or by biodistribution. A significant correlation exists between %ID / g values ​​measured by PET imaging and biodistribution, except in the spinal cord where radiotracer levels in tissue are low.

[0081] Figure 19 Compilation of mSOD1 identification data on day 10 after administration of radiotracer and 89 The structure of Zr-DFO-AP-101 and its deglycosylated analogues.

[0082] Figure 20 In biological layer interferometry (BLI), 89 Zr-DFO-AP-101(B) binds to soluble misfolded SOD1 in a similar manner to unmodified AP-101(A). For chelator-modified AP-101, the absolute signal and K... D A slight decrease indicates that there are some subtle changes.

[0083] Figure 21 use 89 Zr-DFO-AP-101 was used to localize and quantify PET-positive misSOD1 aggregates, and immunohistochemistry of spinal cord sections was used to enable DFO-AP-101 to bind directly to misSOD1 aggregates. (A) Axial (left, circle) and sagittal (right) PET / CT images (arrows) of PET-positive spots identified in the spinal cord. (B) Rear view of CT images showing the vertebral and vertebral classification (V, left, T11-L6) of SOD1 G93A transgenic mice and the corresponding spinal cord regions used (Sc, right, L1-S1, based on Harrison, 2013). (C) Bar chart showing the results of using... 89 ImmunoPET was performed on Zr-DFO-AP-1011 (dark gray column) and its deglycosylated analogue (light gray column) to identify the number of misSOD1 aggregates on PET images at each vertebral level. (D)SOD1 G93A transgenic (Tg, dark gray) and wild-type (Wt, light gray) mice were used to treat the disease. 89 Zr-DFO-AP-101 89 Zr-DFO-AP-101 was co-injected with 100 mg / kg of AP-101 (blocking) and deglycosylation. 89 The Zr-DFO-AP-101 shows the percentage of effective spots. The total number of spots per imaging mouse is shown above each column. (E) Using 89Zr-DFO-AP-101, blocking or deglycosylated analogues, were used to quantify the radiotracer in misSOD1 aggregates (%ID / g) of transgenic mice (black) and Wt mice (hollow symbol). The mean ± SEM of all spots and the number of mice imaged are shown for each group. (For SOD1 G93A mice, ...) p<0.0001, one-way ANOVA, Dunnett's multiple comparison test). (F) In 89 Ratio of misSOD1 aggregate radiotracer content to internal background (internal reference ROI traced on areas without misSOD1 spots in the spinal cord of the same mouse) in SOD1 G93A transgenic mice (black symbols) and wild-type mice (hollow samples) under Zr-DFO-AP-101, blocking, and deglycosylation conditions. Mean ± SEM of all spots. (G) Axial histological sections through the spinal cord of SOD1 G93A transgenic mice, showing the effect of injection. 89 Following Zr-DFO-AP-101, immunohistochemistry with α-miSOD1 was used to pathologically label the sites of identified positive PET spots (see inset).

[0084] Figure 22 Using AP-101 and a commercially available misSOD1 antibody (B8H10 from MediMaps) in SOD1 G93A and SOD1G37R ( Figure 22 (continued) Misfolded SOD1 was detected in the bones of transgenic mice. Although no immune response was detected using allotype control antibodies (1 nM allotype control row) or in wild-type mice, misSOD1 aggregates were observed in the bone and cartilage tissue of the vertebrae and in the joint regions of two different SOD1 transgenic mice, G93A and G37R (exemplarily marked with black arrows).

[0085] Figure 23 89 Biodistribution of Zr-DFO-AP-101 in female (A) and male (B) C57BL6 mice at different time points. (Injection activity: 0.76–0.97 MBq).

[0086] Figure 24 HPLC chromatograms of DFO-AP-101 after purification (A; 2020-10-27) and 2 years later (B; 2022-09-14).

[0087] Figure 25 DFO-AP-101 (1-blue line, standard), purified 89 Zr-DFO-AP-101 (2-red line) and after 29 hours in saline. 89Size exclusion chromatography (SGC) analysis of Zr-DFO-AP-101 (3-black line) was performed. Analysis was conducted using a PRLP-S 10 µm, 1000A 4.6 × 250 mm column.

[0088] Figure 26 Five healthy volunteers aged 63±8 years ( Figure 26A There are 2 men in the middle. Figure 26B The percentage of the injection dose (for 3 women) was 41 ± 4 MBq. 89 Zr-DFO-AP-101. Whole-body PET / CT scans were performed two hours post-injection and on days 1, 3, 7, and 10 from a 45-minute scan. All PET scans were performed on a Biograph Vision 600 scanner (Siemens, Erlangen, Germany). Reconstruction was performed from list mode using an iterative algorithm with point spread function and time-of-flight modeling. The matrix size of the whole-body images was 440 × 440 voxels, with isotropic voxel sizes of 3.3 mm. 3 The five curves in the figure are shown below: Spleen – dark blue line (marked "a", the third line from top to bottom in both figures at time 0); Kidney – orange line (marked "b", the fourth line from top to bottom in both figures at time 0); Liver – gray line (marked "c", the top line in both figures at time 0); Heart – yellow line (marked "d", the second line from top to bottom in both figures at time 0); Bone – light blue line (marked "e", the lowest line in both figures at time 0).

[0089] Figure 27 shows a 62-year-old (yo) 79 kg woman who received an intravenous injection of 35 MBq. 89 Zr-DFO-AP-101. Whole-body PET / CT acquisitions were performed 2 hours post-injection (A) and on days 1 (B), 3 (C), 7 (D), and 10 (E). All PET scans were performed on a Biograph Vision 600 scanner (Siemens, Erlangen, Germany) with an axial field of view of 26 cm. Acquisitions were made from list mode, and reconstruction was performed using an iterative algorithm with point spread function and time-of-flight modeling. The matrix size of the whole-body images was 440 × 440 voxels, and the isotropic voxel size was 3.3 mm. 3 . Detailed Implementation

[0090] The present invention relates to embodiments as characterized in the claims, disclosed in the specification, and further illustrated in the following examples and drawings.

[0091] In particular, the present invention relates to antibodies and antibody conjugates, which can be used for the diagnosis and monitoring of the treatment / progression of diseases caused by or associated with misfolded SOD1, especially neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS).

[0092] This invention is based on the concept of using anti-SOD1 antibodies that selectively bind to misfolded and / or aggregated SOD1 to target such SOD1 species in human tissues of ALS patients, and visualizing target binding through detectable in vivo labeling. It is hoped that such labeled anti-SOD1 antibody conjugates can qualitatively and quantitatively determine the severity and progression of the disease.

[0093] There is a need for tracers that can facilitate ALS diagnosis, aid in prognosis, and measure drug pharmacodynamics to accelerate treatment development for ALS patients and identify patients early in the disease. Positron emission tomography (PET) imaging using appropriate radiotracers holds great potential for ALS because it will allow visualization of the central nervous system (CNS) pathology in individuals with the disease.

[0094] Recently, a recombinant human monoclonal antibody that selectively binds to human misfolded SOD1 (mSOD1) has been studied and named AP-101. This antibody was initially disclosed in international application WO 2012 / 080518 A1 and published in Maier. et al. Further characterization can be found in Sci. Transl. Med. 10 (2018)(470) doi: 10.1126 / scitranslmed.aah3924. In addition to its disease-modifying properties, recent experiments have shown that this anti-SOD1 antibody has high specificity for mSOD1.

[0095] AP-101 antibody has been shown to recognize mSOD1 in postmortem spinal cord tissue from familial and sporadic ALS patients, regardless of their SOD1 genotype (Maier). et al. (2018). To further confirm this hypothesis, the inventors developed a proprietary, sensitive assay for detecting mSOD1 in the biological fluids (particularly cerebrospinal fluid (CSF)) of ALS patients (an ELISA for detecting ALS). Based on preliminary data from a controlled collection dataset generated to support the assay's development, it was determined that mSOD1 is present in the CSF of most SOD1-mutant FALS patients tested. These results suggest that screening CSF in patients with known disease is useful for diagnosing ALS, but invasive assays may not yet have sufficient sensitivity to identify disease onset in all patients.

[0096] Direct imaging of the spine using positron emission tomography (PET) and single-photon emission computed tomography (SPECT), as well as magnetic resonance imaging (MRI), magnetic particle imaging (MPI), and optical imaging, may also serve as diagnostic tools to detect UMN pathology in ALS patients without upper motor neuron (UMN) symptoms, as a prognostic tracer that may help stratify patients for clinical trials, and as a pharmacodynamic tracer to measure the biological effects of drugs in the brain and spinal cord.

[0097] PET is a powerful imaging technique that can track the distribution of radiopharmaceuticals at picomolar concentrations. PET imaging is non-invasive, functional, and quantitative, and has proven to be a valuable technological platform for studying other neurodegenerative diseases. It also represents a powerful technology for efficacy validation studies in drug development. The introduction of sensitive PET imaging scanners for preclinical studies and PET-CT for preclinical and clinical applications, if a specific radiotracer is developed, can facilitate the early and definitive diagnosis of ALS. Similarly, SPECT, MRI, MPI, and optical imaging (including fluorescence imaging), or combinations thereof and / or combinations with PET (e.g., PET / CT, PET / MRI, PET / MPI), can be used to selectively visualize molecular targets that can be used with specific tracers, such as for detecting misfolded SOD1 and diagnosing ALS and / or monitoring disease progression / treatment.

[0098] To investigate this method, AP-101 antibody was combined with... p The labeled antibody conjugate DFO-AP-101 was prepared by randomly conjugating lysine residues with a chelating agent of -SCN-Bn deferoxamine (SCN-Bn-DFO), and then further processed using Streptococcus pyogenes (S. pyogenes). Streptococcus pyogenes Degly-DFO-AP-101 (a deglycosylated antibody chelator) was prepared by enzymatic treatment of DFO-AP-101 with an endoglucosidase, which specifically hydrolyzes the glycans at the Fc glycosylation sites of IgG (see the corresponding preparation section in Example 1 and Materials and Methods below). 89 Prior to Zr labeling, both antibodies were subjected to SDS-PAGE (see...). Figure 7 ) and SEC-HPLC were used for analysis, and given the intended use of PET for immunoimaging, it was selected 89 Zr was used as an exemplary radionuclide. The specific activity and amount of DFO chelating agent for each antibody were determined to fully characterize the PET tracer (see Example 1 and Table 1). A generation method was developed using a cartridge-based module. 89Automated process for Zr-DFO-AP-101 conjugates (see the corresponding preparation section in Example 1 and Materials and Methods below). Stability studies were conducted to establish the formulation and shelf life of the radiotracer (see the stability studies section in Example 2 and Materials and Methods). Transgenic (Tg, [B6.Cg-Tg(SOD1)) expressing misfolded SOD1 (leading to the formation of m-SOD1 aggregates) was used to identify the symptomatic and pre-symptomatic phases. The optimal mouse age for detecting misfolded SOD1 aggregation in G93A)1Gur / J)]) mice and 89 Longitudinal imaging studies were conducted to determine the optimal time following Zr-DFO-AP-101 administration. 3D regions of interest (ROIs) were plotted to include the overall mSOD1 spot, and the radiotracer content was expressed as a percentage of the injected dose per gram equivalent (%ID / g). 89 Zr-DFO-AP-101 and Degly- 89 Zr-DFO-AP-101 was used in a control imaging experiment in Wt mice. Administration was performed via the tail vein. 89 Zr-DFO-AP-101 or Degly- 89 After Zr-DFO-AP-101, mice were imaged using a small animal PET / CT scanner (see Examples 3-6, and for example, Figure 2 , 3 (13, 16). A group accepts 89 Mice were co-injected with unlabeled AP-101 to assess target specificity (see [link to relevant documentation]). Figure 3 (Continued 2) and Figure 10 , 13 (and 19). After the last imaging (day 10), the mice were euthanized, tissue samples were taken, and the radioactive tracer content was measured in a gamma counter.

[0099] The two radioactive tracers studied in this paper 89 Zr-DFO-AP-101 and Degly- 89 Zr-DFO-AP-101 can bind to mSOD1 aggregates in the spinal cord of Tg mice, and imaging is optimal in 126-day-old Tg mice and on days 7 and 10 after tracer administration. Figure 2-4 13). 89 Zr-DFO-AP-101 uptake is specific because it is blocked when co-injected with AP-101. Figure 3 (continued) as well as Figure 10 , 13 And 19). Degly- 89Zr-DFO-AP-101 showed improved uptake of mSOD1 aggregates, but this tracer also concentrated more in the anterior vertebrae of Tg mice, leading to radiation spillover into the spinal cord, resulting in similar aggregate-to-spinal-cord ratios for both radiotracers. Figure 3 , 4 Degly- was observed in the spinal cord region of Wt mice. 89 The low background of Zr-DFO-AP-101 results in better contrast between Wt and Tg mice. 89 Compared to Zr-DFO-AP-101, Degly- 89 Zr-DFO-AP-101 has low spleen and liver uptake (Example 4). Figure 17 and Figure 3 (Continued from 2)), but Tg mice showed higher bone uptake (vertebrae and femoral heads), indicating the presence of mSOD1 in the bones and / or bone marrow of Tg mice (Example 5, Figure 3 and Figure 22 ).

[0100] although 89 Zr-DFO-AP-101 and Degly- 89 Zr-DFO-AP-101 showed a similar mSOD1 to spinal cord ratio, but Degly- 89 Zr-DFO-AP-101 appears to have the greatest potential for clinical application. The rationale for this choice is that Degly- 89 Zr-DFO-AP-101 is more efficiently enriched in mSOD1 aggregates, resulting in high contrast between Wt and Tg mice.

[0101] Based on the results provided by the experiments described herein and summarized above, the present invention generally relates to methods for diagnosing amyotrophic lateral sclerosis (ALS) and / or monitoring ALS drug treatment and / or progression in subjects. The method preferably uses in vivo immunoimaging of misfolded SOD1 (mSOD1) in the subject. For this purpose, the labeled antibody conjugate preferably comprises an antibody that specifically binds to misfolded and / or aggregated SOD1, and a detection label conjugated to the antibody or antibody fragment. Preferably, the antibody (with or without labeling) specifically binds to misfolded and / or aggregated mammalian SOD1, such as mouse, rat, or human misfolded and / or aggregated SOD1, particularly preferably human misfolded and / or aggregated SOD1.

[0102] Preferably, the label is covalently bound to the antibody, either directly or indirectly. If the label is indirectly bound to the antibody, a bifunctional chelating agent or linker is preferably used, which is then covalently bound to both the label and the antibody.

[0103] As described herein, the labeled antibody conjugate, also interchangeably referred to herein as a "conjugate" (regardless of whether the label is directly bound to the antibody or indirectly bound according to preferred embodiments, e.g., whether the label is conjugated or complexed), is used in an in vivo immunoimaging method preferably including mSOD1, by which the conjugate allows determination of the presence of mSOD1 in a subject by making mSOD1 visible, and, if present, visualization of the distribution of mSOD1 in the subject's organs and / or tissues in addition to its presence. As mentioned above, the presence of mSOD1 in the spinal cord, particularly the presence of mSOD1 comprising aggregates in the spinal cord, is a marker of ALS in patients and transgenic (Tg) animal models. In this regard, any in vivo immunoimaging method known in the art can be used. However, preferably, the specific immunoimaging method is selected from PET, SPECT, MRI, MPI, and optical imaging (including fluorescence imaging), and combinations thereof or combinations with CT (such as PET / CT, SPECT / CT, and PET / MRI, SPECT / MRI), or groups thereof. Particularly preferably, the imaging or immunoimaging method includes PET or a combination of PET and CT, such as... Figure 2 , 3 As exemplarily shown in 21A.

[0104] Based on the imaging method intended for use in diagnosing ALS or monitoring ALS drug treatment and / or progression in subjects, a specific marker for the antibody conjugate is selected for labeling.

[0105] Preferably, the labeling in the labeled antibody conjugate or labeled antibody fragment conjugate includes a label selected from: fluorine-18 (F-18), titanium-45 (Ti-45), manganese-52 (Mn-52), iron-52 (Fe-52), potassium-43 (K-43), scandium-43 (Sc-43), scandium-44 (Sc-44), cobalt-57 (Co-57), copper-60 (Cu-60), and copper-61 (Cu-61). Copper-62 (Cu-62), Copper-64 (Cu-64), Copper-67 (Cu-67), Gallium-67 (Ga-67), Gallium-68 (Ga-68), Bromine-76 (Br-76), Bromine-77 (Br-77), Krypton-81m (Kr-81m), Rubidium-81 (Rb-81), Yttrium-86 (Y-86), Strontium-87m (Sr-87m), Zirconium-89 (Zr-89 or 89Zr), technetium-99m (Tc-99m), indium-111 (In-111), indium-113m (In-113m), antimony-117 (Sb-117), iodine-123 (I-123), iodine-125 (I-125), cesium-127 (Cs-127), cesium-129 (Cs-129), iodine-131 (I-131), iodine-132 (I-132), lutetium-177 (Lu-177), rhenium-186 (Re-186), mercury-197 (Hg-197), rhenium-188 (Re-188), lead-203 (Pg-203), bismuth-206 (Bi-206), actinium-225 (Ac-225), radium-225 (Ra-225), and any combination of two or more of these.

[0106] More preferably, the mark is selected from the group consisting of or including the following: (a) Used in PET, derived from: Fluorine-18 (F-18), Manganese-52 (Mn-52), Copper-64 (Cu-64), Gallium-68 (Ga-68), Antimony-117 (Sb-117), Scandium-43 (Sc-43), Scandium-44 (Sc-44), Titanium-45 (Ti-45), Bromine-76 (Br-76), Rubidium-82 (Rb-82), Yttrium-86 (Y-86), Iodine-124 (I-124), and Zirconium-89 (Zr-89 or 89 Zr); (b) Used in SPECT, derived from: Indium-111 (In-111), Technetium-99m (Tc-99m), Iodine-123 (I-123), Gallium-67 (Ga-67), Iodine-131 (I-131), Lutetium-177 and Rhenium-186 (Re-186); (c) For use in MRI and MPI: magnetic, paramagnetic, or superparamagnetic ion complexes or particles, preferably containing gadolinium. 3+ (Gd 3+ ),manganese 2+ (Mn 2+ );copper 2+ (Cu 2+ ); iron oxide (Fe3O4) contrast agent; and (d) Used for optical imaging, including fluorescence imaging, from: fluorescein, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, Texas Red® (Molecular Probes, Inc., Eugene, OR), AlexaFluor® (Molecular Probes, Inc., Eugene), AlexaFluor 350, AlexaFluor 405, AlexaFluor 430, AlexaFluor 488, AlexaFluor 500, AlexaFluor 532, AlexaFluor 546, AlexaFluor 568, AlexaFluor 594, AlexaFluor 610, AlexaFluor 633, AlexaFluor 647, AlexaFluor 660, AlexaFluor 680, AlexaFluor 700, AlexaFluor 750, BODIPY FL, BODIPY R6G, BODIPYTMR, BOPDIPY 530 / 550, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY 650 / 665, Rhodamine Green™-X (Molecular Probes, Inc.), Rhodamine Red™-X (Molecular Probes, Inc.), Rhodamine 6G, TMR, TAMRA™ (Applied Biosystems).

[0107] Preferably, an antibody-conjugate with a label according to the present invention is provided, wherein the detection label is a radionuclide. The label may include one or more radionuclides. More preferably, one or more radionuclides are selected from those listed in paragraph (a) or (b) above. Most preferably, the radionuclide is selected from those listed in paragraph (a) above. Particularly preferably, a label-antibody conjugate as defined herein is provided, wherein the radionuclide is Zr-89.

[0108] The presence and location of labeled antibody-conjugates or equivalent binding molecules in the subject are detected using a selected imaging method (preferably a real-time imaging method). Preferably, the imaging method is PET or a combination of PET and CT.

[0109] Since in vivo imaging methods such as PET require administering a labeled conjugate to the subject as a first step before performing the imaging method, less invasive screening methods are preferred. Preferably, in vitro methods are used in this regard, wherein markers indicating ALS are detected in bodily fluid samples obtained from the patient. A particularly preferred mSOD1 screening method is used, as described in another application of the inventors, WO 2021 / 185961 A1, the contents of which are incorporated herein by reference in their entirety.

[0110] Therefore, preferably, mSOD1 in the patient's body fluid samples is screened (pre-screening) before or as confirmation of the immunoimaging method according to the invention. The sample analyzed using the assay of the invention can be any body fluid suspected of containing pathological mSOD1, such as blood, CSF, or urine samples. In a preferred embodiment, the sample is whole blood lysate or CSF, preferably CSF.

[0111] The screening method employs an ELISA immunoassay, which involves contacting bodily fluids with two different anti-SOD1 antibodies. Specifically, pre-screening is performed using the methods described in Examples 1 and 2 on pages 23 to 25 of WO 2021 / 185961 A1 or in Example 3 on pages 26 to 28, which are explicitly incorporated herein by reference.

[0112] Preferably, the antibody detecting the specific binding of misfolded and / or aggregated SOD1 is conjugated via a bifunctional chelating agent or linker such as a prosthetic group. The bifunctional chelating agent or prosthetic group can be conjugated in any manner, resulting in covalent binding with the antibody. Preferably, the chelating agent or prosthetic group is randomly conjugated to amino acid residues or to the glycan region of the antibody. Preferably, the amino acid residues to which the chelating agent or prosthetic group is randomly conjugated are lysine or cysteine ​​residues. Most preferably, the bifunctional chelating agent or prosthetic group is randomly conjugated to one or more lysine residues of the antibody.

[0113] According to the present invention, a detection marker is directly or indirectly conjugated with an anti-SOD1 antibody to generate a labeled antibody conjugate.

[0114] According to the widely described embodiments, the direct conjugation of a detection marker to an anti-SOD1 (particularly anti-mSOD1) antibody, according to this disclosure, comprises the reaction of a functional group of the detection marker or a compound containing the detection marker with a corresponding functional group of the antibody or antibody fragment (such as a terminal amino group, a terminal carboxyl group, or a functional group of an amino acid side chain), thereby covalently binding the detection marker to the antibody or antibody fragment.

[0115] However, according to the present invention, it is preferable to indirectly conjugate the detection marker to the antibody or antibody fragment. Such indirect conjugation involves the reaction of the functional group of the linker, prosthetic group, or chelating agent with the corresponding functional group (such as the terminal amino group, terminal carboxyl group, or functional group of the amino acid side chain) of the antibody or antibody fragment, thereby covalently binding the prosthetic group or chelating agent to the antibody or antibody fragment and binding the detection marker, or a compound including the detection marker, to the linker or chelating agent.

[0116] Functional groups and exemplary conjugation reactions are known in the art, such as GT Hermanson, Bioconjugate Techniques, 2 nd The example is from Edition, Academic Press, 2008.

[0117] For indirect conjugation of a detection label to an antibody or antibody fragment, a suitable prosthetic group or chelating agent can be used, preferably a bifunctional chelating agent, wherein the prosthetic group or chelating agent binds to both the antibody or antibody fragment and the detection label. Preferably, the prosthetic group or chelating agent is bifunctional, thus allowing it to bind to both the antibody or antibody fragment and the detection label to produce a labeled antibody conjugate.

[0118] The cofactors, chelating agents, and methods of using for conjugating detection markers to antibodies or antibody fragments are well known in the art; see, for example, Shan S. Wong. et al. *Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation*, Second Edition, CRC Press, 2011. Preferred bifunctional chelating agents used according to the present invention are further listed below.

[0119] In one embodiment, the labeled antibody conjugate includes at least one detection marker conjugated to an antibody or antibody fragment, wherein the at least one detection marker includes a radionuclide. In another embodiment, the labeled antibody conjugate includes at least one detection marker, namely a fluorophore. In yet another embodiment, the at least one detection marker is a magnetic / paramagnetic or supermagnetic particle. In yet another embodiment, the labeled antibody conjugate includes at least one detection marker, a metal-containing particle. Specific particles include, but are not limited to, carbon, gold, or iron nanoparticles.

[0120] In this embodiment, there is no limitation on the number of labels included in the labeled antibody conjugate. In this embodiment, the labeled antibody conjugate includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more labels. When multiple labels are included, they may be the same or different.

[0121] According to the present invention, chelating agents, which have been shown to be effective for conjugating radionuclides, magnetic, paramagnetic, or supermagnetic ion complexes or particles with antibodies, can be commonly used. Preferably, the chelating agents according to the present invention for conjugating labels (preferably radionuclides) with antibodies comprise or are selected from the group consisting of: SCN-Bn DFO. SCN-Bn-HOPO, SCN-Bn-4HMSA, NOTA (2-[4,7-bis(carboxymethyl)-1,4,7-triazacyclo-1-yl]acetic acid) and its derivatives; DOTA (2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl]acetic acid) and its derivatives; methyl hydroxamates derived from triaza and tetraaza macrocycles (NOTHA2 and DOTHA2) and their derivatives; 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODAGA) and its derivatives; diethylenetriaminepentaacetic acid ester (DTPA) and its derivatives; 1,4,7,10-tetraazadodecane-1,4,7-triacetic acid ester (DO3A) and its derivatives; 3,6,9,15-tetraazabicyclo[9.3.1]pentadecan-1 (1 5), 11,13-triene-3,6,9-triacetic acid (PCTA) and its derivatives; 1,4,7,10-tetraazacyclotridecanetetraacetic acid (TRITA) and its derivatives; 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA) and its derivatives; 1,4,7,10-tetraazadodecanetetramethylacetate (DOTMA) and its derivatives; 1,4,7,10-tetraazadodecane-1,4,7-trimethylacetate (DO3MA) and its derivatives; N,N',N'',N'''-tetraphosphonomethyl-1,4,7,10-tetraazacyclododecane (DOTP) and its derivatives; 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetra(methylenemethylphosphonic acid) (DOTMP) and its derivatives; 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephenylphosphonic acid) (DOTPP) and its derivatives; N,N'-ethylenedi-L-cysteine ​​and its derivatives; N1,N1'-(butane-1,4-diyl)bis(N4-hydroxy-N1-(3-(4-(hydroxy(methyl)amino)-4-oxoylidenebutamido)propyl)-N4-methylsuccinamide (4HSM) and its derivatives; and p-SCN-Bn deferoxamine (SCN-Bn-DFO; also known as...) p -SCN-Bn deferoxamine).

[0122] In a preferred embodiment, if it is expected that the antibody will be labeled with Zr-89, it can typically be used. 89Zr-chelating agents, such as deferoxatin (DFC), HMSA, four 1-hydroxypyridin-2-one groups attached to linear tetraamine (HOPO), L1-4, L5, FSC derivatives, TAFC, FOXE, CP256, YM103, DFO-star (DFO ), oxoDFO and p -SCN-Bn deferoxamine.

[0123] Other chelating agents can be used in combination with other radioactive metals: NOTA or its derivatives; methyl hydroxamic acid esters derived from triaza and tetraaza macrocycles (NOTHA2 and DOTHA2); 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODAGA) or its derivatives; diethylenetriaminepentaacetic acid ester (DTPA) or its derivatives; 1,4,7,10-tetraazadodecanetetraacetic acid ester (DOTA) and its derivatives; 1,4,7,10-tetraazadodecane-1,4,7-triacetic acid ester (D03A) and its derivatives; 3,6,9,15-tetraazabicyclo[9.3.1]pentadecano-1(15),11,13-triene-3,6,9-triacetic acid (PCTA) or its derivatives; 1,4,7,10-tetraazacyclotetrazanetetraacetic acid (TRITA) and its derivatives. Biological compounds; 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA) and its derivatives; 1,4,7,10-tetraazadodecanetetramethylacetate (DOTMA) and its derivatives; 1,4,7,10-tetraazadodecane-1,4,7-trimethylacetate (DO3MA) and its derivatives; N,N',N'',N'''-tetraphosphonomethyl-1,4,7,10-tetraazacyclododecane (DOTP) and its derivatives; 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenemethylphosphonic acid) (DOTMP) and its derivatives; 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephenylphosphonic acid) (DOTPP) and its derivatives; or N,N'-ethylenedi-L-cysteine ​​or its derivatives.

[0124] The most preferred bifunctional chelating agents include p -SCN-Bn deferoxamine (SCN-Bn-DFO; also known as...) p -SCN-Bn deferoxamine).

[0125] According to the present invention, different cofactors can be used for 18F (or F-18) labeling. Preferred cofactors for this purpose include N-succinimide-4[18F]-fluorobenzoate ([18F]-SFB), 4-azidobenzoylmethyl-[18F]-fluoride ([18F]-APF), and 1-(3-(2-[18F]fluoropyridin-3-yloxy)propyl)pyrrole-2,5-dione ([18F]-FpyMe).

[0126] The antibody used in the method for diagnosing ALS or monitoring ALS pharmacological treatment and / or progression in the subjects specified herein may, in principle, be any anti-SOD1 antibody that selectively binds to misfolded and / or aggregated forms of SOD1, preferably preferentially or exclusively binding to the physiological form of SOD1.

[0127] In principle, the antibody that is part of the labeled antibody conjugate of the present invention can be any form that recognizes misfolded and / or aggregated SOD1, including, for example, chimeric antibodies, single-chain antibodies, Fab fragments, bispecific antibodies (biantibodies or microantibodies), fusion antibodies, monomers, or analogs of any of these. Corresponding methods for generating such variants are known to those skilled in the art and are described, for example, Harlow and Lane “Antibodies, A Laboratory Manual”, CSH Press, Cold Spring Harbor (1988) First edition; Second edition by Edward A. Greenfield, Dana-Farber Cancer Institute © 2014, ISBN 978-1-936113-81-1. For example, Fab and F(ab')2 fragments can be generated by recombinant synthesis or by using enzymes such as papain (to generate the Fab fragment) or pepsin (to generate the F(ab')2 fragment) to cleave immunoglobulin molecules via proteolytic cleavage. The F(ab')2 fragment contains a variable region, a light chain constant region, and a CH1 domain of the heavy chain.

[0128] In one embodiment, the antibody used according to the invention can therefore be provided in the form of a group selected from the group consisting of: single-chain Fv fragments (scFv), F(ab') fragments, F(ab) fragments and F(ab')2 fragments, Fd, Fv, single-chain antibodies, single-domain antibodies (sdAb, also known as VHH Ab or nanobody), and disulfide-linked Fv (sdFv) and / or chimeric mouse-human or humanized antibodies. Preferably, the anti-SOD1 antibody used comprises a human constant domain. As used herein, the term "equivalent binding molecule" is intended to refer to the form of the antibody alternative indicated in this and the preceding paragraphs.

[0129] Corresponding to WO 2012 / 080518 A1 and Maier et al., The antibody AP-101, characterized in Sci. Transl. Med. 10. (2018) doi: 10.1126 / scitranslmed.aah3924 (the disclosure of which is expressly incorporated herein by reference in its entirety), is particularly preferred and is a fully human IgG1m3 alloantibody having selective high affinity for binding misfolded SOD1 protein.

[0130] Therefore, in one embodiment, the labeled antibody used according to the invention, i.e., the labeled antibody particularly for the diagnostic method as defined above, is an anti-SOD1 antibody that recognizes an epitope comprising the amino acid sequence 73-GGPKDEERHVG-83 (SEQ ID NO: 11).

[0131] In a particularly preferred embodiment, the antibody used according to the invention is AP-101, derived from human antibody NI-204.12G7, and characterized in that its variable region, i.e., the binding domain, includes a structure having WO 2012 / 080518 A1. Figure 1 The variable heavy chain (V) of the amino acid sequence shown in B. H ) and variable light chains (V L The complementarity-determining regions (CDRs) of the antibodies, as shown in Table A below, are replicated or are equivalent to those of the antibodies. One or more of the CDRs may have amino acid sequences that are similar to those of WO 2012 / 080518 A1. Figure 1 CDRs B differ from those shown in Table A below, and in the cases of CDR2 and CDR3, differ by one, two, three, or more amino acids, and wherein the antibody exhibits substantially the same or identical immunological characteristics as the anti-SOD1 antibody NI-204.12G7 shown in the examples of WO 2012 / 080518 A1. The positions of the CDRs are as follows... Figure 1 As shown in B, and in WO 2012 / 080518 A1 Figure 1 The diagram is explained in the legend and is also shown in Table A below. The corresponding nucleotide sequences are shown in Table II on page 54 of WO 2012 / 080518 A1 and are listed in Table A below.

[0132] Table A: Amino acid and nucleotide sequences of the variable regions (i.e., heavy chain and κ / λ light chain) of the human antibody against AP-101. Frame regions (FR) and complementarity-determining regions (CDR) are indicated by underlining in the amino acid sequence. Heavy chain linker regions (JH) and light chain linker regions (JK) are also indicated. Due to cloning strategies, the amino acid sequences at the N-termini of the heavy and light chains may contain primer-induced changes in FR1, but this does not significantly affect the biological activity of the antibody. To provide consistent human antibodies, the nucleotide and amino acid sequences of the original clones were aligned and regulated against relevant human germline variable region sequences in databases; see, for example, Vbase (http: / / vbase.mrc-cpe.cam.ac.uk / ), hosted by the MRC Centre for Protein Engineering (Cambridge, UK). Amino acids that were considered to have deviated from the consensus germline sequence due to PCR primers and were therefore substituted in the amino acid sequence are shown in bold.

[0133]

[0134] In addition, or optionally, the frame area or the complete V H and / or V L The chain is related to Table A above (and WO 2012 / 080518 A1). Figure 1 The frame area shown in B) has 80% identity, and preferably is identical to that in Table A (and WO 2012 / 080518 A1) above. Figure 1 The frame region and V shown in B) H and / or V L The chains exhibit 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity. Furthermore, antibody NI-204.B was cloned and expressed according to the “Materials and Methods” section on pages 84-88 of WO 2012 / 080518 A1, and therefore the description of this method is incorporated herein by reference.

[0135] In a particularly preferred embodiment, the antibody is characterized by WO 2012 / 080518 A1. Figure 1 B and the CDR and / or V shown in Table A above H and / or V L chain.

[0136] Therefore, antibodies as defined herein preferably include (in addition to detection markers conjugated to the antibodies characterized herein): (i) A variable heavy chain (VH) including VH complementarity-determining regions (CDRs) 1, 2, and 3, and / or a variable light chain (VL) including VL CDRs 1, 2, and 3, wherein (a) VH-CDR1 comprises the amino acid sequence of SEQ ID NO: 3 or a variant thereof, wherein the variant comprises one or two amino acid substitutions. (b) VH-CDR2 comprises the amino acid sequence of SEQ ID NO: 4 or a variant thereof, wherein the variant comprises one or two amino acid substitutions. (c) VH-CDR3 comprises the amino acid sequence of SEQ ID NO: 5 or a variant thereof, wherein the variant comprises one or two amino acid substitutions. (d) VL-CDR1 comprises the amino acid sequence of SEQ ID NO: 8 or a variant thereof, wherein the variant comprises one or two amino acid substitutions. (e) VL-CDR2 comprises the amino acid sequence of SEQ ID NO: 9 or a variant thereof, wherein the variant comprises one or two amino acid substitutions, and (f) VL-CDR3 comprises the amino acid sequence of SEQ ID NO: 10 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; and / or (ii) VH chain and / or VL chain, wherein (a) The VH chain comprises the amino acid sequence shown in SEQ ID NO: 1 or 2, or a variant thereof, wherein the variant comprises one or more amino acid substitutions; and (b) The VL chain comprises the amino acid sequence shown in SEQ ID NO: 6 or 7 or a variant thereof, wherein the variant comprises one or more amino acid substitutions; Preferably, the amino acid sequences of the VH and VL chains have at least 90% identity with SEQ ID NO: 1 or 2 and 6 or 7, respectively.

[0137] In one embodiment, an antibody as defined herein is provided, wherein the VH chain comprises the amino acid sequence shown in SEQ ID NO: 1 or a variant thereof, wherein the variant comprises one or more amino acid substitutions; and the VL chain comprises the amino acid sequence shown in SEQ ID NO: 6 or a variant thereof, wherein the variant comprises one or more amino acid substitutions, preferably wherein the amino acid sequences of the VH and VL chains are at least 90% identical to those of SEQ ID NO: 1 and 6, respectively.

[0138] In one embodiment, an antibody as defined herein is provided, wherein the VH chain comprises the amino acid sequence shown in SEQ ID NO: 1 or a variant thereof, wherein the variant comprises one or more amino acid substitutions; and the VL chain comprises the amino acid sequence shown in SEQ ID NO: 7 or a variant thereof, wherein the variant comprises one or more amino acid substitutions, preferably wherein the amino acid sequences of the VH and VL chains are at least 90% identical to those of SEQ ID NO: 1 and 7, respectively.

[0139] In one embodiment, an antibody as defined herein is provided, wherein the VH chain comprises the amino acid sequence shown in SEQ ID NO: 2 or a variant thereof, wherein the variant comprises one or more amino acid substitutions; and the VL chain comprises the amino acid sequence shown in SEQ ID NO: 6 or a variant thereof, wherein the variant comprises one or more amino acid substitutions, preferably wherein the amino acid sequences of the VH and VL chains are at least 90% identical to those of SEQ ID NO: 2 and 6, respectively.

[0140] In one embodiment, an antibody as defined herein is provided, wherein the VH chain comprises the amino acid sequence shown in SEQ ID NO: 2 or a variant thereof, wherein the variant comprises one or more amino acid substitutions; and the VL chain comprises the amino acid sequence shown in SEQ ID NO: 7 or a variant thereof, wherein the variant comprises one or more amino acid substitutions, preferably wherein the amino acid sequences of the VH and VL chains are at least 90% identical to those of SEQ ID NO: 2 and 7, respectively.

[0141] Therefore, most preferably, the anti-SOD1 antibody used in the method for diagnosing amyotrophic lateral sclerosis (ALS) or monitoring ALS drug treatment and / or progression in subjects of the present invention is AP-101 or an equivalent binding molecule or an mSOD1 binding fragment derived from human antibody NI-204.12G7, such as WO 2012 / 080518 A1 and Maier et al., Characterized as described in Sci. Transl. Med. 10. (2018) doi: 10.1126 / scitranslmed.aah3924. Therefore, preferably, an anti-SOD1 antibody as defined herein is provided, wherein each heavy chain consists of 453 amino acid residues having SEQ ID NO: 14, and each light chain consists of 213 amino acid residues having SEQ ID: 15.

[0142] Furthermore, preferably, the antibodies used herein are produced in CHO-K1 host cells and purified from cell cultures.

[0143] The five main classes of immunoglobulins are IgG, IgM, IgA, IgD, and IgE. They are distinguished by the type of heavy chain found in their molecules. IgG molecules have a heavy chain called the γ-chain; IgM has a µ-chain; IgA has an α-chain; IgE has an ε-chain; and IgD has a δ-chain; see reviews, for example, Schroeder. et al. , J. Allergy Clin. Immunol. 125 (2010), S41‐S52. Furthermore, different subclasses exist, with IgA further divided into IgA1 and IgA2 subclasses, and IgG further divided into IgG1, IgG2, IgG3, and IgG4 subclasses. In addition, two types of light chains exist, namely kappa(κ) and lambda(λ).

[0144] In principle, the antibodies used according to the present invention can belong to any type of class and subclass, and can include any type of light chain, as long as the antibody binds to SOD1 in a misfolded form, preferably an aggregated form, and preferably as long as the binding specificity to SOD1 (as shown in the example for antibody NI-204.12G7 in WO 2012 / 080518 A1) remains unaffected. However, intact IgG antibodies are preferred, wherein the antibody includes constant structural domains. Thus, in one embodiment, the immunoglobulin heavy chain and / or light chain constant structural domains present in the antibody used according to the present invention belong to the IgG, IgM, IgA, IgD, or IgE type, preferably the IgG type. In one embodiment, the immunoglobulin heavy chain and / or light chain constant structural domains present in the antibody used according to the present invention belong to the IgA1, IgA1, IgG1, IgG2, IgG3, or IgG4 subclass, preferably the IgG1, IgG2, IgG3, or IgG4 subclass, and most preferably the IgG1 subclass.

[0145] The antibodies used as defined herein preferably include the human Ig constant region. Furthermore, the antibodies used as defined herein are preferably human IgG antibodies, more preferably human IgG1, and most preferably human IgG1m3 alloantibodies. Preferably, the antibodies used as defined herein include kappa (κ) or lambda (λ) light chains.

[0146] Recombinant expression of a complete human IgG1 antibody having a human or mouse constant structural domain can be performed substantially as described in the examples of WO 2012 / 080518 A1. Preferably, the antibody is a monoclonal antibody or derived from a monoclonal antibody.

[0147] In addition to the four subclasses mentioned above, human heavy and light chain genes also exhibit one or more extensive structural polymorphisms, which are tightly linked and inherited in haplotypes. Allotypes can be immunogenic and elicit antibody responses through alloimmunization. Therefore, allotype conversion is particularly significant for providing non-immunogenic antibodies. To date, extensive allotypes (polymorphisms) are known, but the focus has been on serologically defined allotypes. Allotypes of IgG proteins are defined by the expression of one or more unique epitopes identified by one or more unique serological reagents. Allotypes expressed on the constant region of the IgG heavy chain are labeled as Gm (genetic marker), along with a subclass (e.g., G1m) and an allotype number (or letter), such as G1m1 [or G1m(a)], G3m5 [or G3m(b1)]. Human immunoglobulin allotypes are listed in Table 1 of Jefferis and Lefrance, mAbs 1 (2009), 1–7 and Irani et al. , MolecularImmunology 67 (2015), 171-182 Figure 1 In section A, the contents of which are incorporated herein by reference. Therefore, in one embodiment, the antibody used according to the invention belongs to any, but is not limited to, the following allotypes: G1m1, G1m2, G1m3, G1m17, G2m23, G3m21, G3m28, G3m11, G3m5, G3m13, G3m14, G3m10, G3m15, G3m16, G3m6, G3m24, G3m26, G3m27, A2m1, A2m2, A2m3, Em1, Km1, Km2 and Km3, but preferably G1m2, G1m3 or G1m17, and most preferably G1m3.

[0148] As described above, antibody AP-101 is a fully human IgG1m3 allotype antibody and consists of two identical heavy chains of the IgG1 subtype and the IgG1m3 allotype. Furthermore, as described above, the original human antibody NI-204.12G7 belongs to the L-λ type; therefore, AP-101 consists of two identical λ subtype light chains. The sequences of the variable heavy chain (VH) and variable light chain (VL) of AP-101 are shown in SEQ ID NOs 1 and 2 and 6 and 7, respectively, and the sequences of the corresponding human constant regions are known in the art. For example, each allotype (such as the IgG1m3 allotype) has a unique amino acid sequence in its heavy chain constant region; see Jefferis and Lefrance (2009), ibid. Therefore, in one embodiment, the antibody used according to the invention is characterized by two heavy chains, each comprising the amino acid sequence shown in SEQ ID NO: 14, and two light chains, each comprising the amino acid sequence shown in SEQ ID NO: 15. In this preferred embodiment, each heavy chain consists of 453 amino acid residues, and each light chain consists of 213 amino acid residues. These four chains are preferably stabilized by twelve intrachain disulfide bonds and four interchain disulfide bonds. Each heavy chain preferably contains a single N-linked glycosylation site at Asn303. The N-linked glycosylation structure is preferably primarily a fucosylated complex biantennary glycan having 0 galactose residues (GOF) or 1 galactose residue (G1F).

[0149] Furthermore, but to a very small extent and preferably in negligible amounts, some antibody species used herein may have undergone other post-translational modifications (PTMs), such as partial cleavage, oxidation, deamidation, succinimide or pyroglutamate formation and isomerization. PTMs identified in AP-101 are shown in Example 7 on pages 104 and 105 of WO 2024 / 042250 A1, particularly in Table 18 on page 105, the disclosure of which is incorporated herein by reference.

[0150] In a preferred embodiment, the antibody used according to the invention has a heavy chain that does not include a C-terminal lysine. For example, in such an embodiment, the C-terminal lysine included in SEQ ID NO: 14 is absent.

[0151] Furthermore, or alternatively, the antibody used according to the invention has a heavy chain, wherein the glutamine at the N-terminus is replaced by pyroglutamic acid. This formation of pyroglutamic acid is also referred to as N-terminal cyclization.

[0152] Furthermore, or alternatively, the antibody used according to the invention has an N-glycosylated heavy chain, preferably wherein the N-linked glycosylation site is Asn303.

[0153] Most preferably, the antibody used according to the invention has a heavy chain that does not include a C-terminal lysine, i.e., the C-terminal lysine has undergone C-terminal lysine cleavage, wherein the glutamine at the N-terminus is replaced by pyroglutamic acid, i.e., the glutamine has undergone N-terminal glutamine cyclization and is N-glycosylated.

[0154] The amino acid sequences of the heavy and light chains are shown below: 1QVQLVQSGAE VKKPGASVTL SCKASGYTFT AYYIHWVRQA REQGLEWMGV 51INPSTGTTFY AQNFPDRVSV TRDTSTSTVF MELHNLKSED TAVYYCARAI 101SEHGSGSYSP YYWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGC 151 LVKDYFPEPV TVSWNSGALT SGVHTFPAVL QSSGLYSLSS VVTVPSSLG 201 TQTYICNVNH KPSNTKVDKR VEPKSCDKTH TCPPCPAPEL LGGPSVFLFP 251 PKPKDTLMIS RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE 301 QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR 351 EPQVYTLPPS REEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT 401 PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS 451 PGK (SEQ ID NO: 14, AP-101-HC, heavy chain amino acid sequence, wherein the amino acids in the constant region are underlined, and wherein the C-terminal lysine (K) is optional and / or the N-terminal glutamine (Q) undergoes intramolecular cyclization, resulting in the formation of pyroglutamic acid) 1SYELTQPPSV SVSLGQMAAI TCSGEALPKK YGYWYQQKPG QVPVLLIYRD 51VERPSGVPDR FSGSSSGTMV TLTISGVQAE DEADYYCLSA DSSGTWVFGG 101GTKLTVL GQP KAAPSVTLFP PSSEELQANK ATLVCLISDF YPGAVTVAWK 151 ADSSPVKAGV ETTTPSKQSN NKYAASSYLS LTPEQWKSHR SYSCQVTHEG 201 STVEKTVAPT ECS (SEQ ID NO: 15, AP-101-LC, light chain amino acid sequence, wherein amino acids in the constant region are underlined) Glycosylation, or the attachment of glycans, is a common post-translational modification that occurs during antibody production. et al. (2020)). As demonstrated in the experiments described in the following examples, deglycosylation increased the efficiency of the radiotracer in detecting mSOD1 aggregates from 67% to 100% (Example 3, Figure 14 However, it was found that both radiotracers (i.e., also glycosylated) could bind to spinal cord mSOD1 aggregates, Degly- 89 Zr-DFO-AP-101 yielded the highest uptake and detected number of mSOD1 aggregates. Figure 13 , 14 Deglycosylation significantly increases the uptake of radiotracers in the vertebrae and femoral heads (Example 5). Figure 3 G), indicating the presence of mSOD1 in bone or bone marrow and specific uptake in these tissues. Furthermore, in the case of Degly- 89 A lower spinal cord and vertebral background was observed in Wt mice using Zr-DFO-AP-101, leading to the use of this [based] 89 The contrast between Wt and Tg mice was better with the Zr AP-101 radiotracer (Example 4). Figure 17 and Figure 3 (Continued from 2)). For these reasons, both radioactive tracers are suitable; however, Degly- 89 Zr-DFO-AP-101 appears to be the most suitable radiotracer for clinical use. Therefore, the antibody, as specified herein, is provided in two forms: glycosylated or deglycosylated. Figure 19 (plan)).

[0155] Preferably, the antibody, as defined herein, is glycosylated. In another preferred embodiment, the antibody, as defined herein, is deglycosylated. Preferably, the antibody is removed from the antibody by deglycosylation. N - Polysaccharides (such as...) Figure 19 (As illustrated). According to the present invention, deglycosylation can be obtained by different methods. Fc-glycans can be deglycosylated, for example, by using them for complex... N Enzymatic treatment of polysaccharides with endoglucosidases can generate antibodies in bacterial hosts where glycosylation has not occurred, such as Escherichia coli.

[0156] As illustrated herein, labeled antibody-conjugates or labeled antibody fragments, as defined herein, allow for the detection of mSOD1 and mSOD1-aggregates using common in vivo immunoimaging methods (Examples 3-6 and, for example, Figure 2 , 316). Therefore, it is preferable to provide labeled antibodies or antibody fragments as specified herein, wherein the imaging includes PET imaging, SPECT imaging, CT imaging, MRI, MPI, optical imaging (including fluorescence imaging), or PET / CT and SPECT / CT.

[0157] Because immunoimaging methods (such as PET) are highly sensitive, early detection and quantification of mSOD1 and mSOD1 aggregates in subjects allow for early diagnosis of ALS, as well as close monitoring of disease progression and / or the efficacy of potential treatments. In the experiments described in the following examples, the preparation of labeled antibody conjugates is described, and the conjugates are validated as sensitive radiotracers for immunoimaging of misfolded SOD1 (mSOD1). By using conjugates in diagnostic methods, early diagnosis of ALS in subjects is possible, and treatment can be initiated earlier than with symptom-based diagnoses. Given the data provided herein, conjugates used as defined herein allow for detailed monitoring of disease status by visualizing mSOD1 and its aggregates (even in in vivo within spinal motor neurons) (see, e.g., Figure 3 , 13 (and 16), thereby allowing monitoring of ALS drug treatment and / or progression in subjects.

[0158] Therefore, the present invention also provides a medicament for treating patients who have been diagnosed with or are developing ALS, and / or for monitoring the efficacy of the medicament and / or the progression of ALS by the methods defined herein. Preferably, the medicament is selected from the group consisting of: riluzole (Rilutek®), edaravone (Radicava®), terbufos (BIIB067), dextromethorphan hydrobromide and quinidine sulfate (Nuedexta®), AP-101, remisente (CY5031), arellomo, levosimendan, fasudil, pyrimethamine (Daraprim). TM Rapamycin, baclofen (Gablofen®, Kemstro®, Lioresal®), diazepam (Diastat®, Valium®), amitriptyline (Elavil®), trihexyphenidyl, Scopoderm® (scopolamine patch), glycopyrronium bromide (Robinul®), piracetam (also known as BNJ441, ALXN1210, or Ultomiris®), and eculizumab (also known as Soliris®).

[0159] As described in the examples (see Example 1), due to experiments related to the present invention, in describing methods for diagnosing amyotrophic lateral sclerosis (ALS) or monitoring ALS drug treatment and / or progression in subjects, labeled antibody conjugates as defined herein are generated and provided, wherein the method includes in vivo immunoimaging of subjects, and the labeled antibody conjugates comprise an antibody that specifically binds to misfolded SOD1 and a detection marker conjugated to the antibody.

[0160] Therefore, in a further aspect, the present invention also provides labeled antibody conjugates in all preferred forms as defined above.

[0161] Preferably, the labeled antibody conjugate or labeled antibody fragment conjugate comprises: (i) A variable heavy chain (VH) including VH complementarity-determining regions (CDRs) 1, 2, and 3, and / or a variable light chain (VL) including VL CDRs 1, 2, and 3, wherein (a) VH-CDR1 comprises the amino acid sequence of SEQ ID NO: 3 or a variant thereof, wherein the variant comprises one or two amino acid substitutions. (b) VH-CDR2 comprises the amino acid sequence of SEQ ID NO: 4 or a variant thereof, wherein the variant comprises one or two amino acid substitutions. (c) VH-CDR3 comprises the amino acid sequence of SEQ ID NO: 5 or a variant thereof, wherein the variant comprises one or two amino acid substitutions. (d) VL-CDR1 comprises the amino acid sequence of SEQ ID NO: 8 or a variant thereof, wherein the variant comprises one or two amino acid substitutions. (e) VL-CDR2 comprises the amino acid sequence of SEQ ID NO: 9 or a variant thereof, wherein the variant comprises one or two amino acid substitutions, and (f) VL-CDR3 comprises the amino acid sequence of SEQ ID NO: 10 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; and / or (ii) VH chain and / or VL chain, wherein (a) The VH chain comprises the amino acid sequence shown in SEQ ID NO: 1 or 2, or a variant thereof, wherein the variant comprises one or more amino acid substitutions; and (b) The VL chain comprises the amino acid sequence shown in SEQ ID NO: 6 or 7 or a variant thereof, wherein the variant comprises one or more amino acid substitutions; Preferably, the amino acid sequences of the VH and VL chains have at least 90% identity with SEQ ID NO: 1 or 2 and 6 or 7, respectively.

[0162] More preferably, the labeled antibody conjugate or the labeled antibody fragment conjugate further comprises p-SCN-Bn deferoxamine (SCN-Bn-DFO) as a bifunctional chelating agent, and 89 Zr is used as a radionuclide tracer.

[0163] Particularly preferred, the labeled antibody conjugate is 89 Zr-DFO-AP-101, and includes AP-101 as an antibody that specifically binds to misfolded SOD1, p-SCN-Bn deferoxamine (SCN-Bn-DFO) as a bifunctional chelator / connector and Zr-89 as a radionuclide.

[0164] As discussed above, antibodies typically occur in glycosylated form. In a preferred embodiment, antibodies such as those disclosed herein are thus glycosylated. However, as illustrated in the examples and discussed above, deglycosylated tracers may be advantageous in immunoimaging because they enhance the detection of mSOD1 aggregates even in tissues where glycosylated antibody conjugates provide no signal or only a weak signal. Therefore, in another preferred embodiment, the labeled antibody conjugate is deglycosylated. More preferably, the labeled antibody conjugate in this embodiment is deglycosylated. 89 Zr-DFO-AP-101(Degly- 89 (Zr-DFO-AP-101). Exemplary preparation of this conjugate is described in the following examples, materials and methods section, "Preparation of Degly-DFO-AP-101".

[0165] In a further aspect, the present invention also provides an antibody that specifically binds to misfolded SOD1, the antibody comprising a bifunctional chelating agent, the chelating agent preferably being... p -SCN-Bn deferoxamine (SCN-Bn-DFO).

[0166] Antibodies that specifically bind to misfolded SOD1 preferably include: (i) A variable heavy chain (VH) including VH complementarity-determining regions (CDRs) 1, 2, and 3, and / or a variable light chain (VL) including VL CDRs 1, 2, and 3, wherein (a) VH-CDR1 comprises the amino acid sequence of SEQ ID NO: 3 or a variant thereof, wherein the variant comprises one or two amino acid substitutions. (b) VH-CDR2 comprises the amino acid sequence of SEQ ID NO: 4 or a variant thereof, wherein the variant comprises one or two amino acid substitutions. (c) VH-CDR3 comprises the amino acid sequence of SEQ ID NO: 5 or a variant thereof, wherein the variant comprises one or two amino acid substitutions. (d) VL-CDR1 comprises the amino acid sequence of SEQ ID NO: 8 or a variant thereof, wherein the variant comprises one or two amino acid substitutions. (e) VL-CDR2 comprises the amino acid sequence of SEQ ID NO: 9 or a variant thereof, wherein the variant comprises one or two amino acid substitutions, and (f) VL-CDR3 comprises the amino acid sequence of SEQ ID NO: 10 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; and / or (ii) VH chain and / or VL chain, wherein (a) The VH chain comprises the amino acid sequence shown in SEQ ID NO: 1 or 2, or a variant thereof, wherein the variant comprises one or more amino acid substitutions; and (b) The VL chain comprises the amino acid sequence shown in SEQ ID NO: 6 or 7 or a variant thereof, wherein the variant comprises one or more amino acid substitutions; Preferably, the amino acid sequences of the VH and VL chains have at least 90% identity with SEQ ID NO: 1 or 2 and 6 or 7, respectively.

[0167] As illustrated in the examples and described herein, most preferably, the antibody that specifically binds to misfolded and / or aggregated SOD1 is AP-101, i.e., an antibody comprising the following... (i) A variable heavy chain (VH) including VH complementarity-determining regions (CDRs) 1, 2, and 3, and a variable light chain (VL) including VL CDRs 1, 2, and 3, wherein (a) VH-CDR1 includes the amino acid sequence of SEQ ID NO: 3. (b) VH-CDR2 includes the amino acid sequence of SEQ ID NO: 4. (c) VH-CDR3 includes the amino acid sequence of SEQ ID NO: 5. (d) VL-CDR1 includes the amino acid sequence of SEQ ID NO: 8. (e)VL-CDR2 includes the amino acid sequence of SEQ ID NO: 9, and (f) VL-CDR3 includes the amino acid sequence of SEQ ID NO: 10; and (ii) VH chain and VL chain, where (a) The VH chain comprises the amino acid sequence shown in SEQ ID NO: 1 or 2; and (b) The VL chain includes the amino acid sequence shown in SEQ ID NO: 6 or 7.

[0168] The sequence of the heavy chain constant region is known in the art. For example, each isotype (and such as the IgG1m3 isotype) has a unique amino acid sequence in its heavy chain constant region; see Jefferis and Lefrance (2009), ibid. Therefore, in one embodiment, the antibody used according to the invention is characterized by two heavy chains, each comprising the amino acid sequence shown in SEQ ID NO: 14, and two light chains, each comprising the amino acid sequence shown in SEQ ID NO: 15. In this preferred embodiment, each heavy chain consists of 453 amino acid residues, and each light chain consists of 213 amino acid residues.

[0169] Preferably, the antibody (and therefore the labeled antibody-conjugate described herein) specifically binds to misfolded and / or aggregated mammalian SOD1, such as misfolded SOD1 from mice, rats, or humans, particularly preferably human misfolded SOD1. As explained in detail herein, the labeled antibody-conjugate of the present invention has been developed to provide new possibilities for imaging misfolded and / or aggregated mammalian SOD1 in animals, particularly for humans suffering from diseases associated with the occurrence of such misfolded and / or aggregated SOD1. Thus, in one embodiment, an imaging composition is provided, wherein the composition comprises a labeled antibody conjugate and further comprises NaCl at a pH of 4.5 to 8.0 (to make the solution more physiological) and gentianic acid (to avoid radiation degradation). Preferably, the imaging composition is provided in a sealed vial (to keep the composition sterile and pyrogen-free). The volume of the composition is preferably 10 mL, consisting of saline (0.9% NaCl) and 10 mg of gentianic acid. The vial preferably contains between about 300 and about 500 MBq. Preferably, the amount of the labeled antibody conjugate relative to the unlabeled antibody is greater than 97% and / or the apparent specific activity of the labeled antibody conjugate is about 0.66 GBq / mg, optionally wherein the injectable solution can be diluted with NaCl before injection. The final volume of the imaging composition for administration to animals or humans is preferably based on the content of HEPES or gentianic acid, preferably ≤200 µg / injection and ≤5 mg / injection, respectively.

[0170] Given that the labeled antibody-conjugates described herein have the ability to detect misfolded and / or aggregated SOD1 in a subject's body, as described herein and illustrated in the examples and figures, a further aspect of the invention relates to diagnostic compositions or kits comprising the imaging compositions as defined above, or the labeled antibody-conjugates or antibodies described herein, optionally further comprising a detection marker if the antibody is in the diagnostic composition or kit. Preferably, the antibody included in the diagnostic composition or kit is AP-101 or deglycosylated AP-101. Preferably, the AP-101 antibody comprises a bifunctional chelating agent, which is preferably... p -SCN-Bn deferoxamine (SCN-Bn-DFO).

[0171] The detection markers included in the diagnostic composition or kit are selected as described above for labeled antibody conjugates or labeled antibody fragment conjugates. This means that, depending on the intended use, the markers are preferably selected from the group consisting of or composed of the following: (a) Used in PET, derived from: Fluorine-18 (F-18), Manganese-52 (Mn-52), Copper-64 (Cu-64), Gallium-68 (Ga-68), Antimony-117 (Sb-117), Scandium-43 (Sc-43), Scandium-44 (Sc-44), Titanium-45 (Ti-45), Bromine-76 (Br-76), Yttrium-86 (Y-86), Iodine-124 (I-124), and Zirconium-89 (Zr-89 or 89 Zr); (b) Used in SPECT, derived from: Indium-111 (In-111), Technetium-99m (Tc-99m), Gallium-67 (Ga-67), Iodine-123 (I-123), Iodine-131 (I-131), Lutetium-177, and Rhenium-186 (Re-186). (c) For use in MRI and MPI: magnetic, paramagnetic, or superparamagnetic ion complexes or particles, preferably containing gadolinium. 3+ (Gd 3+ ),manganese 2+ (Mn 2+ );copper 2+ (Cu 2+ ); iron oxide (Fe3O4) contrast agent; and (d) Used for optical imaging, including fluorescence imaging, from: fluorescein, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, Texas Red® (Molecular Probes, Inc., Eugene, OR), AlexaFluor® (Molecular Probes, Inc., Eugene), AlexaFluor 350, AlexaFluor 405, AlexaFluor 430, AlexaFluor 488, AlexaFluor 500, AlexaFluor 532, AlexaFluor 546, AlexaFluor 568, AlexaFluor 594, AlexaFluor 610, AlexaFluor 633, AlexaFluor 647, AlexaFluor 660, AlexaFluor 680, AlexaFluor 700, AlexaFluor 750, BODIPY FL, BODIPY R6G, BODIPYTMR, BOPDIPY 530 / 550, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY 650 / 665, Rhodamine Green™-X (Molecular Probes, Inc.), Rhodamine Red™-X (Molecular Probes, Inc.), Rhodamine 6G, TMR, TAMRA™ (Applied Biosystems).

[0172] Preferably, the detection marker included in the diagnostic composition or kit is a radionuclide. More preferably, it is a radionuclide selected from or composed of the radionuclides included in items (a) to (c) above. Even more preferably, a diagnostic composition or kit is provided wherein the detection marker is a radionuclide selected from or composed of the following: fluorine-18 (F-18), copper-64 (Cu-64), gallium-67 (Ga-67), gallium-68 (Ga-68), antimony-117 (Sb-117), scandium-43 (Sc-43), scandium-44 (Sc-44), titanium-45 (Ti-45), indium-111 (In-111), lutetium-177 (Lu-177), and zirconium-89 (Zr-89 or 89 Zr). Particularly preferably, the detection marker is a radionuclide, wherein the radionuclide is 89 Zr.

[0173] Preferably, the labeled antibody conjugate or antibody included in the diagnostic composition or kit is 89 Zr-DFO-AP-101 or deglycosylated 89 Zr-DFO-AP-101(Degly- 89 Zr-DFO-AP-101).

[0174] The diagnostic composition or kit may optionally further include description of a method for diagnosing amyotrophic lateral sclerosis (ALS) or monitoring ALS drug treatment and / or progression in a subject, wherein the method includes in vivo immunoimaging of the subject, and the labeled antibody conjugate comprises an antibody that specifically binds to misfolded SOD1 and a detection label conjugated to the antibody, as defined above.

[0175] In a further related aspect, the present invention also relates to a method for in vivo immunoimaging of superoxide dismutase (SOD1), which can be used as a biomarker for amyotrophic lateral sclerosis (ALS) or as a biomarker for the efficacy of ALS drug treatment and / or ALS progression in a subject, the method comprising: (a) administering a subject with a labeled antibody conjugate as described herein; and (b) Detection of the presence of labeled antibody conjugates in the subject's body by imaging.

[0176] Preferably, the antibody used in this in vivo immunoimaging method is an antibody that specifically binds to misfolded SOD1, as described below, and more preferably, the subject is a human.

[0177] The antibodies specifically binding to misfolded SOD1, administered in the in vivo immunoimaging method, preferably include: (i) A variable heavy chain (VH) including VH complementarity-determining regions (CDRs) 1, 2, and 3, and / or a variable light chain (VL) including VL CDRs 1, 2, and 3, wherein (a) VH-CDR1 comprises the amino acid sequence of SEQ ID NO: 3 or a variant thereof, wherein the variant comprises one or two amino acid substitutions. (b) VH-CDR2 comprises the amino acid sequence of SEQ ID NO: 4 or a variant thereof, wherein the variant comprises one or two amino acid substitutions. (c) VH-CDR3 comprises the amino acid sequence of SEQ ID NO: 5 or a variant thereof, wherein the variant comprises one or two amino acid substitutions. (d) VL-CDR1 comprises the amino acid sequence of SEQ ID NO: 8 or a variant thereof, wherein the variant comprises one or two amino acid substitutions. (e) VL-CDR2 comprises the amino acid sequence of SEQ ID NO: 9 or a variant thereof, wherein the variant comprises one or two amino acid substitutions, and (f) VL-CDR3 comprises the amino acid sequence of SEQ ID NO: 10 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; and / or (ii) VH chain and / or VL chain, wherein (a) The VH chain comprises the amino acid sequence shown in SEQ ID NO: 1 or 2, or a variant thereof, wherein the variant comprises one or more amino acid substitutions; and (b) The VL chain comprises the amino acid sequence shown in SEQ ID NO: 6 or 7 or a variant thereof, wherein the variant comprises one or more amino acid substitutions; Preferably, the amino acid sequences of the VH and VL chains have at least 90% identity with SEQ ID NO: 1 or 2 and 6 or 7, respectively.

[0178] More preferably, the antibody that specifically binds to misfolded SOD1 and is administered in an in vivo immunoimaging method is AP-101.

[0179] Preferably, the antibody administered to the subject in the immunoimaging method comprises a bifunctional chelator, which is preferably... p -SCN-Bn deferoxamine (SCN-Bn-DFO).

[0180] Furthermore, the antibody is preferably labeled by a detection marker, wherein, depending on the intended use, the detection marker is selected from the group consisting of or composed of the following: (a) Used in PET, derived from: Fluorine-18 (F-18), Manganese-52 (Mn-52), Copper-64 (Cu-64), Gallium-68 (Ga-68), Antimony-117 (Sb-117), Scandium-43 (Sc-43), Scandium-44 (Sc-44), Titanium-45 (Ti-45), Bromine-76 (Br-76), Yttrium-86 (Y-86), Iodine-124 (I-124), and Zirconium-89 (Zr-89 or 89 Zr); (b) Used in SPECT, derived from: Indium-111 (In-111), Technetium-99m (Tc-99m), Gallium-67 (Ga-67), Iodine-123 (I-123), Iodine-131 (I-131), Lutetium-177, and Rhenium-186 (Re-186). (c) For use in MRI and MPI: magnetic, paramagnetic, or superparamagnetic ion complexes or particles, preferably containing gadolinium. 3+ (Gd 3+ ),manganese 2+ (Mn 2+ );copper 2+ (Cu 2+ ); iron oxide (Fe3O4) contrast agent; and (d) Used for optical imaging, including fluorescence imaging, from: fluorescein, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, Texas Red® (Molecular Probes, Inc., Eugene, OR), AlexaFluor® (Molecular Probes, Inc., Eugene), AlexaFluor 350, AlexaFluor 405, AlexaFluor 430, AlexaFluor 488, AlexaFluor 500, AlexaFluor 532, AlexaFluor 546, AlexaFluor 568, AlexaFluor 594, AlexaFluor 610, AlexaFluor 633, AlexaFluor 647, AlexaFluor 660, AlexaFluor 680, AlexaFluor 700, AlexaFluor 750, BODIPY FL, BODIPY R6G, BODIPYTMR, BOPDIPY 530 / 550, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY 650 / 665, Rhodamine Green™-X (Molecular Probes, Inc.), Rhodamine Red™-X (Molecular Probes, Inc.), Rhodamine 6G, TMR, TAMRA™ (Applied Biosystems).

[0181] The labeling is preferably covalently bound to the antibody via a bifunctional chelating agent or a prosthetic group, wherein, preferably, the bifunctional chelating agent or prosthetic group is randomly conjugated to one or more lysine or cysteine ​​residues of the antibody. More preferably, the bifunctional chelating agent or prosthetic group is randomly conjugated to one or more lysine residues.

[0182] Preferably, the detection marker is a radionuclide. More preferably, it is a radionuclide selected from or composed of the radionuclides included in or composed of items (a) to (c) above. Even more preferably, an immunoimaging method is provided, wherein the detection marker is a radionuclide selected from or composed of the following: fluorine-18 (F-18), copper-64 (Cu-64), gallium-67 (Ga-67), gallium-68 (Ga-68), antimony-117 (Sb-117), scandium-43 (Sc-43), scandium-44 (Sc-44), titanium-45 (Ti-45), indium-111 (In-111), lutetium-177 (Lu-177), and zirconium-89 (Zr-89 or 89 Zr). Particularly preferably, the detection marker is a radionuclide, wherein the radionuclide is 89 Zr.

[0183] Preferably, the labeled antibody conjugate or antibody used in the immunoimaging method is 89 Zr-DFO-AP-101 or deglycosylated 89 Zr-DFO-AP-101(Degly- 89 Zr-DFO-AP-101).

[0184] Preferably, the step of detecting the presence of labeled antibody conjugates in the subject's body by imaging includes PET imaging, SPECT imaging, MRI, MPI, optical imaging including fluorescence imaging, or PET and SPECT, preferably in combination with CT (i.e. PET / CT or SPECT / CT), and most preferably imaging includes PET.

[0185] Imaging methods In one or more embodiments, imaging methods are disclosed herein. In one embodiment, the imaging method includes: (a) administering a labeled antibody conjugate to a subject, wherein the labeled antibody conjugate comprises: an antibody that specifically recognizes and binds to mSOD1, and at least one detection label conjugated to the antibody, wherein the at least one detection label comprises at least one label, preferably a radionuclide, a fluorophore, a magnetic / paramagnetic / supermagnetic particle, or a combination thereof, or any two or more thereof; and (b) detecting the presence of the labeled conjugate in the subject's body by imaging. In one embodiment, the labeled antibody conjugate is as described herein with respect to antibody conjugates. In a further embodiment, the antibody and at least one detection label are as described herein with respect to antibody conjugates.

[0186] Preferably, the imaging method includes detecting the presence of a labeled conjugate in the subject's body via imaging. Preferably, the presence of the conjugate is detected in real time. Preferably, the presence of the labeled antibody conjugate is detected non-invasively and / or minimally invasively.

[0187] According to aspects of this disclosure, the antibody conjugate is an imaging agent that can be used to visualize mSOD1, such as in diagnostic procedures. Imaging can be performed by a number of procedures well known to those skilled in the art, such as PET, SPECT, Cerenkov imaging, photoacoustic imaging, ultrasound imaging, optical coherence tomography, optical imaging (including fluorescence imaging), magnetic resonance imaging, magnetic particle imaging, bioluminescence imaging, or any combination of two or more thereof.

[0188] In one implementation, the method includes administering a labeled antibody conjugate to a subject.

[0189] In one embodiment, the method includes administering an effective amount of a labeled antibody conjugate to a subject. The labeled antibody conjugate can be administered via any suitable route known to those skilled in the art. Preferably, administration of the labeled antibody conjugate is performed intravenously, intraarterially, intraperitoneally, intramuscularly, subcutaneously, intrapleurally, intrathecally, via a regional catheter, and / or directly into the lesion. If the labeled antibody conjugate is administered by injection, administration can be by continuous infusion, a single bolus, and / or multiple boluses. In some embodiments, the administration of the radiolabeled antibody conjugate is non-immunogenic to the subject. According to an embodiment, administering the labeled conjugate to a subject comprises administering a pharmaceutical composition comprising a labeled antibody conjugate and a pharmaceutically acceptable carrier.

[0190] Preferably, in relation to the diagnostic and treatment aspects described herein, the subject is a mammal. In some embodiments, the subject is a mammal selected from the following: humans, non-human primates, dogs, cats, rats, cattle, horses, goats, sheep, pigs, and rabbits. In a preferred embodiment, the subject is a rodent, including but not limited to rats, mice, or guinea pigs. Particularly preferred is a mouse or a human.

[0191] In a preferred embodiment, the labeled antibody conjugate administered to the subject comprises a radionuclide. Preferably, the labeled antibody conjugate administered to the subject comprises a labeled antibody conjugate wherein the label is solely a radionuclide (i.e., without fluorophores or magnetic / paramagnetic / supermagnetic particles). In some embodiments, the labeled antibody conjugate administered to the subject comprises a labeled antibody conjugate wherein the label is solely a fluorophore (i.e., without radionuclides or paramagnetic / supermagnetic particles). In some embodiments, the labeled antibody conjugate administered to the subject comprises a labeled antibody conjugate wherein the label is solely a paramagnetic / supermagnetic particle (i.e., without radionuclides or fluorophores).

[0192] In embodiments where the labeled antibody conjugate includes a fluorophore, the presence of the labeled antibody conjugate can be detected in vivo by optical imaging. Various in vivo optical imaging techniques are known to those skilled in the art. (See, for example, Ntziachristos, Annu. Rev. Biomed. Eng. 2006, 8:1-33; Troyan, SL) et al. ,Ann. Surg. Oneal. 16, 2943-2952 (2009); Luker, GD&Luker, KE, J. Nucl. Med. 49, 1-4 (2008); Tromberg, BJ et al. , Med. Phys. 35, 2443-2451 (2008), and their potential applicability in image-guided diagnostic and surgical approaches has been proposed in several preclinical studies; Kirsch, DG et al. , Nat. Med. 13, 992-997 (2007); von Burstin, J. et al. Int. J. Cancer 123, 2138-2147 (2008); and U.S. Patent No. 9,409,923.

[0193] In one embodiment, in vivo optical imaging is selected from confocal microscopy, planar imaging, fluorescence molecular tomography, whole-projection tomography, direct fluorescence tomography, two-photon in vivo imaging, or any combination of two or more thereof. In a further embodiment, planar imaging is selected from epi-illumination (i.e., photography) imaging, transmitted illumination imaging, tomography imaging, or any combination of two or more thereof.

[0194] In embodiments where the presence of labeled antibody conjugates is detected by in vivo optical imaging, the fluorophore can be selected such that it emits fluorescence in the visible or near-infrared region. In exemplary, non-limiting embodiments, the fluorophore emitting fluorescence in the visible or near-infrared region may be selected from fluorescein, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, TexasRed® (Molecular Probes, Inc., Eugene, OR), AlexaFluor® (Molecular Probes, Inc., Eugene), AlexaFluor 350, AlexaFluor 405, AlexaFluor 430, AlexaFluor 488, AlexaFluor 500, AlexaFluor 532, AlexaFluor 546, AlexaFluor 568, AlexaFluor 594, AlexaFluor 610, AlexaFluor 633, AlexaFluor 647, AlexaFluor 660, AlexaFluor 680, AlexaFluor 700, AlexaFluor 750, BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY 530 / 550, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY 650 / 665, Rhodamine Green™-X (Molecular Probes, Inc.), Rhodamine Red™-X (Molecular Probes, Inc.), Rhodamine 6G, TMR, TAMRA™ (Applied Biosystems), or any combination of two or more thereof.

[0195] In one embodiment, the presence of the labeled antibody conjugate is detected by in vivo photoacoustic imaging. In an illustrative, non-limiting embodiment, the photoacoustic dye included in the conjugate may be selected from methylene blue, Evan's blue, trypan blue, patent blue, indocyanine green, IRDye800CW, DiR, Cy7, Cy7.5, and porphyrin, or any combination of two or more thereof.

[0196] In embodiments where the labeled antibody conjugate comprises a combination of a radionuclide tracer and a fluorophore, the presence of the labeled antibody conjugate can be detected in vivo by a combination of PET or SPECT and optical imaging.

[0197] In embodiments where the labeled antibody conjugate comprises a combination of a radionuclide tracer and a fluorophore, the presence of the labeled antibody conjugate can be detected in vivo by a combination of PET or SPECT and photoacoustic imaging.

[0198] In implementation, the presence of labeled antibody conjugates can be detected in vivo by any combination of two or more of the following: PET, SPECT, CT, Cerenkov imaging, photoacoustic imaging, ultrasound imaging, optical coherence tomography, magnetic resonance imaging, magnetic particle imaging, optical imaging (including fluorescence imaging), and / or bioluminescence imaging.

[0199] As described in Example 7 and Figure 22 As shown, experiments conducted in the context of this application are the first to demonstrate the presence of misfolded and / or aggregated SOD1 in the bones and joints of transgenic SOD1G93A and G37R mice, which are commonly used as ALS models in this field. Impaired bone function and structure, such as affected osteoprogenitor formation, impaired osteoblast differentiation, increased osteoclast formation and bone resorption, have been observed in ALS patients and ALS model organisms, leading to decreased bone stability, manifested as osteopenia and fractures (Caplliure-Llopis at al. (2020), Zhu...). et al. (2015), Torres et al. ,2021 and Wang et al. (2018) confirmed the relationship between muscle and bone affected by progressive atrophy in ALS. The observations presented in this paper, together with the findings described in WO 2012 / 080518 A1, show that the application of SOD1-targeting antibodies leads to improvements in ALS symptoms, such as slowing progressive motor dysfunction, reducing weight loss in SOD1 transgenic animals and resulting in delayed disease onset, prolonged survival, and improved weight and motor performance. This strongly suggests that anti-SOD1 antibodies may also be used to treat and / or prevent bone and / or joint-related diseases, for example, by reducing and potentially preventing or delaying the onset of bone and / or joint deterioration in diseases (e.g., ALS) associated with the presence or accumulation of misfolded and / or aggregated SOD1 in bones and / or joints.

[0200] Therefore, in a further embodiment, the present invention provides an anti-SOD1 antibody or an antigen-binding fragment thereof for targeting misfolded and / or aggregated SOD1 in the bones and / or joints of a subject, preferably wherein the antibody is an AP-101 antibody or a miSOD1 binding fragment thereof, or wherein the anti-SOD1 antibody is a labeled antibody conjugate as described herein.

[0201] Particularly preferably, an anti-SOD1 antibody or its antigen-binding fragment is provided for targeting misfolded and / or aggregated SOD1 in subjects affected by or suffering from ALS. Furthermore, preferably, the subject is a mammal, most preferably a human.

[0202] Furthermore, the present invention relates to the use of the labeled antibody conjugates as defined herein in the production of imaging compositions for imaging methods as defined herein.

[0203] Furthermore, the present invention also provides a method for preparing a mixture comprising 89 The method for preparing the Zr-DFO-AP-101 antibody conjugate solution is described in detail in Example 9. The imaging solution thus prepared will be used in the clinical studies described in Example 8 and will be tested. Preferably, the method includes the following steps: (a) will 89 Zr-chloride solution is combined with DFO-AP-101 to obtain a reaction solution; preferably, 89 The Zr-chloride solution is adjusted to a pH of 6.5-8.0. Preferably, this adjustment is achieved by adding sodium bicarbonate solution and HEPES buffer solution to obtain the desired pH of 6.5-8.0.

[0204] (b) The reaction solution is heated to approximately 37ºC for approximately 45 minutes. Preferably, the heating of the solution is carried out in a reactor, i.e., in a controlled environment in which a uniform temperature can be obtained and maintained for the required period of time.

[0205] (e) Recycling 89 Zr-DFO-AP-101 is used as the recovery solution. Preferably, it includes... 89 The Zr-DFO-AP-101 solution is recovered into a container, such as a sealable vial.

[0206] The method preferably further includes the following steps (f) and / or (g): (f) By adding saline solution 89 The Zr-DFO-AP-101 recovery solution is adjusted to a final volume, preferably 10 mL. Preferably, 0.9% saline is used again in this step. (g) Filtration 89Zr-DFO-AP-101 solution.

[0207] In addition, recycling 89 The Zr-DFO-AP-101 process is preferably performed on a column comprising a gel filtration resin, and includes the following steps: (c) Adjust the gel filtration resin in the column with 10 mL of 0.9% saline, preferably at a flow rate of 1.5 mL / min; preferably, this step is repeated 3 times. In this step, a column filled with gel filtration resin is preferred, wherein the resin is preferably Sephadex G-25. In this step, a PD-10 column (GE Healthcare) is most preferably used.

[0208] (d) Transfer the reaction solution from (b) and 4 mL of saline solution to the conditioning column from (c). (e) Recovery from the reaction solution 89 Zr-DFO-AP-101 is added to the solution recovered from the column.

[0209] Therefore, in a preferred embodiment, the above method includes the following steps: (a) Adjust the pH to 6.5-8.0. 89 Zr chloride solution was combined with DFO-AP-101 to obtain a reaction solution; (b) The reaction solution is heated to approximately 37ºC for approximately 45 minutes; (c) Adjust the gel filtration resin in the column with 10 mL of 0.9% saline, preferably at a rate of 1.5 mL / min; wherein, preferably, this step is repeated 3 times; (d) Transfer the reaction solution from (b) and 4 mL of saline solution to the conditioning column from (c); and (e) Recover from the column 89 Zr-DFO-AP-101 was used as the recovery solution.

[0210] Preferably, the method further includes the steps (f) and / or (g). (f) By adding saline solution 89 The Zr-DFO-AP-101 recovery solution is adjusted to a final volume, preferably 10 mL; and (g) Filtration 89 Zr-DFO-AP-101 solution.

[0211] Preferably, the steps are performed in the indicated order.

[0212] In a preferred embodiment, a method is further provided, wherein step (a) of 89Zr chloride solution is provided by a method comprising the following steps: (a1) will 89 Zr-oxalate solution was fed onto a quaternary methyl ammonium (QMA) anion exchange column; (a2) A solution containing HCl was added to the QMA anion exchange column; and (a3) Collect the elution from the column 89 Zr chloride solution.

[0213] After performing steps (a1) to (a3), a mixture is prepared including 89 The method for preparing the solution of the Zr-DFO-AP-101 antibody conjugate continues with the steps defined above (a).

[0214] Get including 89 After preparing a solution of the Zr-DFO-AP-101 antibody conjugate, the solution can be further processed to obtain a composition that can be injected into a subject for imaging methods as defined herein. This composition has a shelf life of approximately 29 hours after synthesis. Therefore, it is preferable to provide a method for preparing a composition comprising... 89 A method for preparing a solution of a Zr-DFO-AP-101 antibody conjugate, the method further comprising the following steps: (h) The filtered product obtained in step (g) is reacted by adding 0.9% saline and 10 mg gentianic acid. 89 The Zr-DFO-AP-101 solution was used to prepare the composition. Preferably, the total volume of the prepared composition was 10 mL.

[0215] Therefore, in a preferred embodiment, the preparation includes... 89 The method for preparing the solution of the Zr-DFO-AP-101 antibody conjugate includes steps (a1)-(a3) and (a) to (h).

[0216] The resulting solution is preferably sterile and pyrogen-free, and can be used as a diagnostic agent for intravenous administration, with each injection preferably containing 5 mg or less. 89 Zr-DFO-AP-101. The shelf life of this solution is 29 hours after the end of synthesis (EOF). 89 The molecular weight of Zr-DFO-AP-101 is approximately 147,332-147,792 kDa. The method described above uses... 89 Zr isotopes and their corresponding salts can be obtained through a cyclotron accelerator. 89 Y(p,n) 89 Zr reaction preparation, as described in detail by Alnahwi et al. (2018), in which the obtained 89 Zr-oxalate is converted into a less toxic form using the above method.89 Zr chloride. As mentioned above, other sources can also be used in the above methods. 89 Zr isotopes, 89 Zr-oxalate and 89 Zr-chloride was used to label DFO-AP-101 and generate a product including 89 A solution of Zr-DFO-AP-101 is intended for use in anticipated clinical trials or is commonly used in imaging methods. (Depending on the use) 89 Zr-oxalate or 89 Zr chloride, as a starting material, can be incorporated into the specific steps of the above method as described above. For example, if 89 Since Zr chloride is already available for this method, steps (a1) to (a3) ​​can be skipped and the method can begin from step (a). When using the solution obtained by the above method or normally, according to the dosage of the tracer required for imaging,... 89 When Zr-DFO-AP-101 is used, no or minimal adverse effects are expected. This expectation is based on several factors. Animal studies have shown that... 89 Zr-DFO-AP-101 is stable in plasma and retains its affinity and specificity for misfolded and / or aggregated SOD1. It exhibits specific uptake in several animal regions of interest, including the spinal cord and vertebrae of ALS model mice. Over the past 10 years, 89 Data obtained in the Zr-DFO antibody field show that such antibodies are safe and well-tolerated by patients, leading to a large number of corresponding antibodies receiving FDA approval (e.g., trastuzumab, bevacizumab, cetuximab, and rituximab with corresponding labels; see, for example, Heskamp et al (2017), Stabin et al (1997), Laforest et al (2016), Dijkers et al (2010), Yoon et al (2020), Veldhuijzen van Zanten et al. (2018), Oosting et al. (2016), Even et al (2017), Makris et al (2015), (Jauw et al (2017) and Bruijnen et al (2016)). Clinical trials 1 and 2a testing AP-101, as described in Example 8, did not show any antibody-related adverse reactions. Due to the […] tested in the new clinical trials described in Example 8 89The Zr-DFO-AP-101 dose (planned dose of 1.5 mg per patient) is up to 1666 times lower than the doses tested in the AP-101 trial (100 mg, 500 mg, and 2500 mg doses), and therefore equivalent safety profiles can be expected. DFO chelators for conjugating radioisotopes to antibodies are known in the art, have been FDA approved, and data on their use in this field indicate relative safety and good patient tolerability. Therefore, in conclusion, it can be expected that… 89 Zr-DFO-AP-101 will also be proven to be relatively safe and well-tolerated by patients.

[0217] the term To avoid any ambiguity, it is necessary to emphasize expressions such as "in some embodiments," "in certain embodiments," "in some cases," "in some aspects," "in further embodiments," "in one embodiment," "in a further aspect," "in the first aspect," "in the second aspect," etc., and this means that when reading any embodiment described herein, one should take into account each feature of those embodiments, and this disclosure must be treated in the same way as if the combination of features of those embodiments would be described in detail in one embodiment. The same applies to any combination of embodiments and features shown in the appended claims and embodiments, which are also intended to be combined with features of the corresponding embodiments disclosed in the specification, wherein, for the sake of consistency and brevity only, embodiments are characterized by dependencies, and in practice, each combination of embodiments and features that can be interpreted due to (multiple) dependencies must be considered as explicitly and unambiguously disclosed, and not as a choice among different options.

[0218] The terms “radioactive nuclide,” “radioactive isotope,” “radioactive isotope,” or “radioactive isotope” are used interchangeably herein to refer to a nuclide having excess nuclear energy, making it unstable, and wherein the excess energy is emitted, for example, in the form of gamma rays and / or particles. Preferably, the emission occurs at least partially in the form of particles, such as beta particles (also known as beta rays or beta radiation), and most preferably in the form of positrons.

[0219] When referring to the antibodies or antibody peptides of the present invention, the terms "fragment," "variant," "derivative," and "analyte" include any peptide that retains at least some of the antigen-binding properties of the corresponding natural binding molecule, antibody, or peptide. As described above, the variable region allows the antibody to selectively recognize and specifically bind to epitopes on antigens. That is, the V of the antibody L Domain and V HA subset of structural domains, or complementarity-determining regions (CDRs), are combined to form variable regions that define three-dimensional antigen-binding sites. This quaternary antibody structure forms antigen-binding sites at the distal end of each arm of the Y-shape. More specifically, the antigen-binding site is formed by each V... H and V L The three CDRs on the chain define the binding fragment. Any antibody fragment containing sufficient structure to specifically bind to misfolded SOD1 is interchangeably referred to herein as a “binding fragment” or an “immune-specific fragment”. The antibody or antigen-binding fragments, variants or derivatives, and equivalent binding molecules of the present invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primate-derived, murine or chimeric antibodies, single-chain antibodies, epitope-binding fragments (e.g., Fab, Fab' and F(ab')2), Fd, Fvs, single-chain Fv (scFv), single-chain antibodies, single-domain antibodies, disulfide-linked Fv (sdFv), including V L or V H Fragments of the structural domain and fragments generated from Fab expression libraries. ScFv molecules are known in the art and described, for example, in U.S. Patent 5,892,019.

[0220] The terms “specific binding” and “specific recognition” are used interchangeably herein and generally refer to a binding molecule (e.g., an antibody or a fragment thereof) that binds to an epitope via its antigen-binding domain, and that such binding requires some complementarity between the antigen-binding domain and the epitope. By this definition, an antibody is said to be “specifically bound” to an epitope when it binds more readily to an epitope via its antigen-binding domain than to a random, unrelated epitope. The term “specific” is used herein to define the relative affinity of a particular antibody for a particular epitope. For example, antibody “A” may be considered to have higher specificity for a given epitope than antibody “B”, or antibody “A” may be said to have higher specificity for binding to epitope “C” than for binding to related epitope “D”. References to the binding of antibodies or fragments thereof to misfolded SOD1 as used herein have the same meaning as the terms “specific binding” or “specific recognition” as explained above. When referring to antibodies or fragments thereof as defined herein and their ability to bind to misfolded SOD1, the term “binding” is used interchangeably and includes the meaning of the terms “specific binding” or “specific recognition” as defined above.

[0221] The term "detection marker" refers to a chemical moiety that can be covalently attached to an antibody and serves to provide a detectable signal. Examples of such markers include fluorescent moieties, chemiluminescent moieties, bioluminescent moieties, nanoparticles, magnetic particles, metal-containing particles, and radioactive markers, such as radionuclides.

[0222] The term "bifunctional chelator" refers to a chemical moiety that attaches an antibody or antibody fragment to a label (e.g., a radionuclide). A bifunctional chelator comprises 1) a chelating agent moiety for binding the (radionuclide) label and 2) a reactive functional group. A bifunctional chelator functions by conjugating the label (e.g., a radionuclide) to the chelating agent moiety and covalently attaching the chelating agent moiety (and the conjugated radionuclide) to the antibody or antibody fragment through a reaction of the reactive functional group with the corresponding reactive functional group of the antibody (or a fragment thereof). Examples known to those skilled in the art of attaching the chelating agent moiety to an antibody or a fragment thereof to suitable reactive functional groups, such as primary amine groups, hydroxyl groups, and / or cysteine ​​amino acids (see, for example, Denardo...) et al. , 1998, Clin Cancer Res. 4(10):2483-90; Peterson et al. ,1999, Bioconjug. Chem. 10(4):553-7; and Zimmerman et al. , 1999, Nucl. Med. Biol. 26(8):943-50).

[0223] Transgenic mice expressing mSOD1 are referred to interchangeably as Tg or ALS in this article and in the figures, while wild-type mice are referred to interchangeably as Wt or CTL in this article.

[0224] The terms “subject,” “individual,” or “patient” refer to any subject, particularly a mammalian subject, such as a person for whom treatment is expected to be diagnosed, prognostic, preventive, or monitored (e.g., a patient with ALS). The terms “subject,” “individual,” and “patient” are used interchangeably herein.

[0225] As used in this article, the term “about” in the context of quantitative measurement means ±10% of the indicated amount. For example, within the range of ±10%, “about 5 mg” can mean 4.5 to 5.5 mg.

[0226] As used herein, the term "treat" or "treatment" refers to therapeutic treatment and prophylactic or preventative measures aimed at preventing or mitigating (alleviating) undesirable physiological changes or disturbances, such as the development of heart failure. Beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms, reduction of disease severity, stabilization (i.e., non-deterioration) of the disease state, delay or slowing of disease progression, improvement or mitigation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. Those requiring treatment include individuals who already have a condition or disorder, those predisposed to developing a condition or disorder, or those seeking to prevent the manifestation of a condition or disorder.

[0227] Pharmaceutically acceptable carriers and routes of administration can be obtained from relevant literature known to those skilled in the art. The pharmaceutical compositions of this invention can be formulated according to methods well known in the art; see, for example, Remington: The Science and Practice of Pharmacy (2000) by the University of Sciences in Philadelphia, ISBN 0-683-306472, Vaccine Protocols. 2nd Edition by Robinson. et al.Humana Press, Totowa, New Jersey, USA, 2003; Banga, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems. 2nd Edition by Taylor and Francis. (2006), ISBN: 0-8493-1630-8. Examples of suitable drug carriers are well known in the art and include phosphate-buffered saline solutions, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions, etc. Compositions containing such carriers can be formulated using known conventional methods. These drug compositions can be administered to subjects at appropriate doses. The administration of suitable compositions can be affected by different modes of transport. Examples include compositions containing pharmaceutically acceptable carriers administered via oral, intranasal, rectal, topical, intraperitoneal, intravenous, intramuscular, subcutaneous, subdermal, transdermal, intrathecal, and intracranial methods. Aerosol formulations such as nasal spray formulations comprise purified aqueous solutions or other solutions of active agents containing preservatives and isotonic agents. Such formulations are preferably adjusted to a pH and isotonic state compatible with the nasal mucosa. The invention also envisions pharmaceutical compositions for oral administration, such as single-domain antibody molecules (e.g., “nanobody™”). Such oral formulations may be in tablet, capsule, powder, liquid, or semi-solid form. Tablets may include a solid carrier, such as gelatin or an adjuvant. Formulations for rectal or vaginal administration may be presented as suppositories with a suitable carrier; see also O'Hagan. et al. See Nature Reviews, Drug Discovery 2(9) (2003), 727-735. Further guidance on formulations suitable for various dosing types can be found in Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, PA, 17th ed. (1985) and its corresponding updates. For a brief review of drug delivery methods, see Langer, Science 249 (1990), 1527-1533.

[0228] Furthermore, unless otherwise stated, the terms and expressions used herein to characterize the invention are given as defined in WO2012 / 080518 A1, particularly in the “I. Definitions” section on pages 10 to 30, the disclosure of which is expressly incorporated herein by reference. This also applies to general embodiments of antibodies, etc., disclosed in WO 2012 / 080518 A1.

[0229] This specification contains numerous references. All references cited herein (including those cited in this application, authorized patents, published patent applications, including background sections and manufacturer's specifications, etc.) are hereby expressly invoked; however, no cited reference is acknowledged as prior art relating to this invention.

[0230] A more complete understanding can be obtained by referring to the following specific embodiments, which are provided for illustrative purposes only and are not intended to limit the scope of the invention.

[0231] Example The overall goal of the experiments described below is to develop and validate the derivatives of AP-101 and use them in conjunction with... 89 A Zr-labeled radiotracer was used to detect misfolded SOD-1 deposits via PET imaging. This radiotracer will be used for the early diagnosis, follow-up of treatment response, and / or disease monitoring of ALS, thus becoming the first diagnostic tool available for ALS.

[0232] The specific objectives of the experiment described in this article are as follows: Objective-1: To prepare AP-101-based... 89 Zr PET tracers, and preferably developed for generating selected 89 Automated process for Zr-DFO-AP-101 PET tracer: using a cartridge-based module 89 Automation of the Zr-DFO-AP-101 will reduce operator dosing, improve reproducibility, and meet GMP-compliant quality standards. Two [specifications / developments] have been developed. 89 Zr-AP-101 derived PET tracer: 89 Zr-DFO-AP-101 and deglycosylated 89 Zr-DFO-AP-101(Degly- 89 Zr-DFO-AP-101 was used to reduce the uptake of radioimmunoconjugates by immune cells expressing Fc-γ receptors (FcγR) in non-target tissues such as the liver and spleen.

[0233] Objective-2 Assessment 89 Zr-DFO-AP-101 demonstrated its efficacy in early detection of aggregated SOD1 via PET imaging in appropriate animal models. To identify transgenic (Tg, [B6.Cg-Tg(SOD1)) expressing m-SOD1 aggregates during the symptomatic and pre-symptomatic phases... The optimal mouse age for detecting misfolded SOD1 aggregation in G93A)1Gur / J)]) mice and 89The optimal time following Zr-DFO-AP-101 administration was determined using longitudinal imaging. 89 Zr-DFO-AP-101 and Degly- 89 Zr-DFO-AP-101 was used in a control imaging experiment in wild-type (Wt) mice.

[0234] Objective-3 Determined 89 Zr-DFO-AP-101 and Degly- 89 Can Zr-DFO-AP-101 be used for early diagnosis and follow-up of treatment response in ALS? (B) Estimate the selected... 89 The expected human dose (OLINDA) of the Zr-DFO-AP-101 PET tracer. A blockade study was conducted using unlabeled AP-101 (100 mg / kg) to evaluate the bioavailability and efficacy of unlabeled AP-101. 89 The effect of Zr-DFO-AP-101 binding to mSOD1 aggregates. Using Degly- 89 A dose determination study of Zr-DFO-AP-101 was conducted in Wt mice to estimate the expected human injection dose for use in PET diagnostics. C57BL6 mice were used as the model mice. In adult males and females, 89 The effective measured doses of Zr-DFO-AP-101 in humans are estimated to be 0.171 mSv / MBq and 0.237 mSv / MBq, respectively (see [reference needed]). Figure 23 (and the description of the mouse dosing experiment in Example 8). Based on the preliminary results of the Phase I clinical trial described in Example 10, it is estimated that... 89 The effective dose of Zr-DFO-AP-101 in humans ranges from about 0.4 mSv / MBq to about 0.65 mSv / MBq depending on sex, with a particularly effective dose of 0.48 mSv / MBq in a male human model and 0.54 mSv / MBq in a female human model.

[0235] The following describes 89 Zr-DFO-AP-101 and its analogues Degly- 89 Preparation and characterization (stability, shelf life) of Zr-DFO-AP-101 and evaluation of their potential for detecting mSOD1 aggregates by PET imaging.

[0236] Materials and Methods: Preparation of DFO-AP-101 Sample preparationAP-101 (20 mg / mL, 1 mL) was centrifuged using four 50 kDa molecular weight cutoff centrifuges (Amicon® Ultra-05) to remove all additives and washed four times with PBS (trace metal base, 300 µL each) pH 7.2 at 5000 rpm for 10 min. A final wash was performed with NaHCO3 0.1 M, pH 8–9. The sample (~100–150 µL) was then combined into another 50 kDa molecular weight cutoff centrifuge (Amicon® Ultra-05) and centrifuged to a final volume of ~150–200 µL. mAb AP-101 was diluted with NaHCO3 0.1 M solution to a volume of ~500–600 µL and then transferred to 5 mL screw-top vials (Fisherbrand) using a 200 µL micropipette. Concentrations below 2 mg / mL reduce the efficiency and reproducibility of the conjugation reaction; therefore, use AP-101 concentrations of 2 mg / mL or higher.

[0237] DFO combination The AP-101 solution (500–600 µL in 0.1 M NaHCO3) was incubated with three equivalents of SCN-Bz-DFO (15 µL of 1.1 mg SCN-Bz-DFO dissolved in 100 µL of DMSO). The resulting mixture was kept at 37°C for 1 hour. The DMSO concentration was maintained below 2% to avoid alteration of AP-101 properties. The conjugate was then purified ten times using the centrifugation and filtration technique described above, with two centricons and PBS (trace metal base, 200 µL each) at pH 7.2. The sample (~100–150 µL) was then combined into 5 mL screw-top vials (Fisherbrand) and stored at 4°C. HPLC was performed on a BioSep-SEC S3000 column using a standard mobile phase (0.1 M PBS + 0.025% NaN3) at a flow rate of 1 mL / min at 280 nm using size exclusion chromatography (SEC). The mean amount of DFO per molecule of AP-101 was determined using a modified isotope dilution assay, similar to Holland's method. et al.As described in J Nucl Med. (2010);51(8):1293-1300, and especially in the supplementary material on page 33 / 46, this document (including its supplementary material) is incorporated herein by reference in its entirety. The concentration of the conjugate was determined spectrophotometrically using a biquinoline carboxylic acid (BCA) protein assay (He. F. (2011) Bio-101: e44. DOI:10.21769 / BioProtoc.44), which is also incorporated herein by reference in its entirety. The half-life of AP-101 in serum is approximately 9.5 days, and the shelf life of DFO-AP-101 at 2–8ºC is approximately 2 years, as shown in the HPLC curves performed immediately after purification and after 2 years (see [link to HPLC curves]). Figure 24 ).

[0238] Clinical study preparation described in Example 8 Can obtain 89 Zr-labeled antibodies or antibody-conjugates, for example, by initially using... 89 Zr-oxalate form 89 Zr, as described by Alnahwi et al. (2018), particularly by the method described in Section 2. Materials and Methods, pages 2-6, the disclosure of which is incorporated herein, is conjugated with an antibody or antibody conjugate to be labeled, wherein, preferably, the antibody conjugate is DFO-AP-101. For example, Zr obtained by said method 89 Zr-oxalate is preferably first captured on a quaternary methyl ammonium (QMA) anion exchange column. To automate this process, subsequent steps can be performed, for example, on a miniAllinOne (MiniAIO) cartridge module (Ans, Belgium), as described in Alnahwi et al. (2018), specifically in section 2.4. 89 The disclosures in this section of the Automated Kit Module and Separation Chemistry for Zr-Oxalate are incorporated herein by reference.

[0239] This automated generation method preferably includes the following steps. 89 Zr-oxalate is transferred (captured) onto an anion exchange column (e.g., a quaternary methylammonium (QMA) column). A solution containing HCl is then added to the column to... 89 Zr-oxalate is converted into less toxic form 89Zr chloride is eluted from the column. Preferably, for this purpose, so-called HCl metal trace analysis, i.e., a 99.999% pure HCl solution based on trace metal analysis, is used. Preferably, a NaCl-HCl solution is used for this purpose, wherein the most preferred solution is a 500 mM NaCl-137 mM HCl solution. The volume of the NaCl-HCl solution is preferably from about 0.25 mL to about 2.5 mL, more preferably from about 0.5 mL to about 1.5 mL or 1 mL, and most preferably about 0.5 mL. 89 The Zr-chloride eluent is collected from the column in a container (e.g., a vial). If it is available from other sources... 89 Zr chloride or toxicity ratio 89 Another type of low Zr-oxalate 89 Zr-salts can also be used to begin the methods described herein. The vial / container is then transferred to the reactor, i.e., where the temperature of subsequent reactions can be specifically controlled. The pH of the eluent is adjusted to obtain a pH in the range of approximately 6.5-8.0. For pH adjustment, sodium bicarbonate solution and HEPES buffer solution are preferably used. Preferred antibodies or antibody conjugates are added, and DFO-AP-101 is also preferably added to the pH-adjusted eluent. The specific additions can be based on the reaction used. 89 The amount of Zr-oxalate is used to select the amount of antibody or antibody conjugate. For example, about 1 mg to about 6 mg, about 2 mg to about 5 mg, about 2.5 mg to about 4 mg, about 3 mg to about 3.5 mg of antibody or antibody conjugate, preferably DFO-AP-101, is added. The resulting solution is then heated in a reactor for a period of time. The resulting solution is heated in the reactor from about 34ºC to about 42ºC, preferably from about 35ºC to about 40ºC, more preferably from about 36ºC to about 38ºC for about 15 minutes to about 90 minutes, preferably from about 20 minutes to about 60 minutes, more preferably from about 30 minutes to about 50 minutes, and even more preferably from about 40 minutes to about 50 minutes. Most preferably, the resulting solution is heated to about 37ºC for about 45 minutes. During the heating of the solution in the reactor, another desalting size exclusion column is adjusted. A column packed with gel filtration resin is preferably used in this step, wherein the resin is preferably Sephadex G-25. In this step, a PD-10 column (GE Healthcare) is most preferably used. Conditioning includes rinsing the column with brine (a mixture of NaCl and water), wherein rinsing is preferably repeated, preferably three times. A 0.9% brine solution is preferably used, and rinsing is preferably performed at a rate of 1.5 mL / min. The amount of brine used in each rinse is preferably about 10 mL. After the radiolabeling reaction is carried out in the reactor, the generated... 89The Zr-DFO-AP-101 solution was transferred to a conditioning column and eluted again with saline. The volume of saline used for elution should be less than 10 mL, preferably 4 mL (0.9%). The eluent was collected in a new vial, and the obtained eluent volume was adjusted to the desired final volume. Preferably, the collected volume was adjusted by adding saline. 89 The Zr-DFO-AP-101 solution was brought to a final volume of approximately 10 mL. Afterward, it was filtered. 89 Zr-DFO-AP-101 solution, and can be used for intended administration to patients. Therefore, 89 The Zr-DFO-AP-101 solution is preferably prepared in a solvent containing 0.9% saline and 10 mg gentianic acid to prevent radioactive decomposition. In experiments conducted according to the present invention, the obtained... 89 Zr-DFO-AP-101 exhibits high radiochemical purity of 90% or higher, with a radiochemical yield greater than approximately 60%. Radiochemical impurities (such as...) 88 Zr and 88 Y) is at an extremely low level and below 25 within 29 hours of the end-of-synthesis (EOS) shelf life.

[0240] Preparation of Degly-DFO-AP-101 To generate deglycosylated DFO-AP-101, an immobilized GlycINATOR rotating column was used for removal. N- Glycan, the rotating column contains GlycINATOR enzyme covalently coupled to agarose beads GlycINATOR® (EndoS2). Each column was equilibrated with 3 x 300 µL of 1M PBS (trace metals) and centrifuged at 200 g for 1 min. DFO-AP-101 (3 mg, 300 µL) was added to each column (0.5 mg per column). The volume of each column was adjusted to 300 µL of PBS, and the solution was incubated at room temperature for 2 h by end-to-end mechanical mixing. After centrifugation at 1000 g for 1 min, Degly-DFO-AP-101 was collected into different reaction vials / tubes. For maximum yield, 300 µL of 1M PBS was added to the GlycINATOR rotating column, followed by centrifugation to recover residual Degly-DFO-AP-101; this step was repeated 3 times. After GlycINATOR digestion, the crude deglycosylated antibody was characterized by HPLC on SEC as described above. The conjugate was then purified three times using the centrifugation filtration technique described above, with two centricons and PBS (trace metal base, 300 µL each) at pH 7.2. The sample (~100–150 µL) was then combined into another 30 kDa molecular weight cutoff centrifuge filter (Amicon® Ultra-05) and centrifuged to a final volume of ~150–200 µL. The sample (~100–150 µL) was then combined into 5 mL screw-top vials (Fisherbrand) and stored at 4ºC. The concentration of the conjugate was measured spectrophotometrically using the BCA protein assay (He. F. 2011). The average amount of DFO per molecule of AP-101 was determined using a modified isotope dilution assay, such as Holland (Holland... et al. As described in (2010), this document is incorporated herein by reference in its entirety. Specific activity was determined according to the procedures we previously reported (Alnahwi et al. According to the determination in 2018, the document is incorporated herein in its entirety.

[0241] Radiolabeling and determination of the average number of chelating agents per antibody Normally, according to Alnahwi et al. , Appl. Sci. 8 (2018):1579(Alnahwi et al. The procedure described in the article (2018, as included in its full text here) on pages 2-9, and using 89 Y-suppressed targets produce 1-2 GBq in a cyclotron. 89 Zr (oxalate) 2. [The text abruptly ends here.] 89 ZrCl4 was loaded onto an ion exchange column and distilled in 0.5–1 mL of 1 M HCl.89 Eluting in ZrCl4 form, with a recovery of 95%. 400 µL of [unclear - possibly a specific solution] was neutralized with 110–120 µL of 2 M Na2CO3. 89 ZrCl4 (400-500 MBq) to adjust 89 The pH of a ZrCl4 solution is 7-7.5. Towards neutralization... 89 DFO-AP-101 (0.5–1 mg) in 0.5 M NaOAc at pH 6.5 was added to ZrCl4 solution, and the volume was adjusted to obtain a DFO-AP-101 concentration of at least 0.2 µg / µL, with a final pH of 7–7.2. The radiolabeling reaction was carried out at 37ºC for 45 min. The radiolabeling yield of the reaction was assessed by radiometric TLC using 50 mM DTPA (pH 5–6) as the mobile phase, and counted on a TLC plate detector. The final radiochemical purity was determined using radiometric TLC and SDS-PAGE.

[0242] Stability Study Shelf life of PET tracers For the preparation 89 Zr-DFO-AP-101 and Degly- 89 Stability studies were conducted on Zr-DFO-AP-101 (used alone in 1M PBS, pH 7.2, or stored at 4ºC for 9–10 days in the presence of gentianic acid (GA, 2.5 mg / mL). Free ions were monitored by radioactive TLC. 89 The presence of Zr was detected by analyzing the solution with SDS-PAGE and exposure to autoradiography film. 89 Zr metabolites and / or aggregates.

[0243] In vivo stability study In PBS 89 Zr-DFO-AP-101 (175–200 MBq) was incubated for different time periods in 500 µL of fresh mouse plasma and PBS (1 / 1) at 37ºC. Stability was directly monitored by radioactive ITLC using 50 mM DTPA as the elution buffer to track free plasma. 89 The existence of Zr.

[0244] SDS-PAGE analysis DFO-AP-101 and Degly-DFO-AP-101 were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Briefly, 4–20 ng of antibody (6–30 µL of 0.6 mg / mL stock solution) was combined with 0–16 µL of Tris / glycine / SDS buffer and 4 µL of 4X Laemmli protein sample buffer. For denaturation conditions, 1 µL of 1,4-dithiothreitol (DTT) was added, and the mixture was then denatured by heating in boiling water for 5 min using a heating block. Subsequently, each sample was loaded with an appropriate molecular weight marker (PageRuler™ pre-stained protein ladder, ThermoFisher Scientific) onto a 4–15% pre-prepared polyacrylamide gel (Bio-Rad) and run at 120 V for 1 h in Tris / glycine / SDS buffer. The completed gel was stained with Coomassie Brilliant Blue solution for 1 h and then destained overnight in a destaining solution (10% acetic acid, 10% methanol). 89 Zr-DFO-AP-101 and Degly- 89 For Zr-DFO-AP-101, unstained radioactive gels were placed on a layer of Saran wrap and exposed to a Molecular Dynamics screen for 30–60 min. The information on the screen was then read using a Storm fluorescent screen imager (Amersham Biosciences). The images were analyzed using Fiji software, and a protein ladder pattern on the gel was overlaid on the images for further analysis.

[0245] Animal care and experimental conditions This animal study has been approved by the University of Sherbrooke's IACUC and complies with the guidelines of the Canadian Council for Animal Care (CCAC). Females expressing the ALS genotype Tg[B6.Cg-Tg(SOD1)] G93A) 1Gur / J)] mice and their control group Wt [C56BL / 6J] were purchased from Jackson (610 Main Street Bar Harbor, ME 04609). Upon arrival, these mice were between 51 and 58 days old and had been housed at CIMS Animal Care until 126 days of age. Upon arrival, the mice were examined to ensure they were healthy and that each group was of uniform appearance and size. Throughout the study, the mice were housed in groups of up to four mice per cage on ventilated racks (Techniplast SealSafe Plus.Techniplast Ventilation Unit) in a controlled environment room. The cages were lined with corncob bedding and included enrichment materials (plastic domes, EnviroDri® nesting material, and wooden blocks). Cages were changed weekly. Throughout the study, the mice had free access to water and food (LabDiet 5008) (standard label). The mice were observed daily throughout the study to assess ALS symptom performance (weight loss, hanging and walking tests, such as Hatzipetros). et al. (As described in J Vis Exp. 104 (2015):53257).

[0246] Radioactive tracer administration Under isoflurane anesthesia (induction with 2-3% isoflurane, maintenance with 1-2.5%, 1-1.5 L oxygen / min), a catheter (30G needle / PE10 tube) is inserted into the tail vein of the test mice to administer the radioactive tracer (20-30 MBq / 0.2 mL saline 0.9%). 89 Zr-DFO-AP-101 89 Zr-DFO-AP-101 with 100 mg / kg AP-101 or Degly- 89 Zr-DFO-AP-101 were co-injected. The number of imaging days following radiotracer administration was optimized when collecting the first results, with the final group imaging only on days 7 and 10. Once the radiotracer was administered, the mice were placed in cages to recover from anesthesia and then returned to the rearing facility.

[0247] PET Imaging and Image Analysis PET imaging was performed on days 7 and 10 following administration of the radiotracer. Imaging was performed on a LabPET-2 PET scanner (https: / / imagingrt.com / ). Aperture: 72 mm. Axial FOV: 50 mm. Transverse FOV: 60 mm. Spatial resolution: 0.74 mm (isotropic at 5 mm from the center). Detection efficiency (250–650 keV): 3.3% for small animal imaging. Preparation and PET imaging were performed under anesthesia via nasal cone delivery of isoflurane (induction with 2–3% isoflurane, followed by 1–2.5% 1–1.5 L oxygen / min), with the level of anesthesia adjusted as needed during PET imaging. Ophthalmic gel was applied to the eyes to prevent dryness. Mice were placed within the field of view of the PET imager for 30 min of static acquisition, followed by moving the imager bed into the CT module of the LabPET-8 scanner for 2 min of CT acquisition. During PET imaging, mice were kept warm using pulsed heated air maintained at 32ºC, and ambient temperature and respiratory rate were monitored throughout the imaging process (PCSAM. 1025T monitoring and gating system, SA Instruments Inc.). Once the imaging process was complete, the mice were placed in cages to recover from anesthesia and then returned to the rearing facility.

[0248] Aggregate identification and data processing PET data were acquired in list mode and reconstructed on a 200×200×168 grid using 16 iterations and 8 subsets of the 3D Ordered Subset Expectation Maximization (OSEM) algorithm, with a voxel size of 0.3×0.3×0.3 mm. 3 All PET images are for 89 Zr physical radionuclide decay and random coincidence were corrected for. Using PMOD software (version 3.8, PMOD Technologies Ltd., Zurich, Switzerland), PET images of each animal were registered to their corresponding CT images via rigid matching transformation.

[0249] Figure 1The steps for identifying mSOD1 aggregates and quantifying their radiotracer content are presented. Once reconstructed, PET images are visually examined to identify potential mSOD1 aggregates that appear as discrete spots of radiotracer within the spinal cord. If these spots are deemed valid, a 3D region of interest (ROI) is plotted to include the entire mSOD1 spot. Validity criteria are that the spot has a well-defined boundary within the spinal cord and is not contaminated by radiotracer spillage from the anterior vertebrae. Once validated, the radiotracer content of the mSOD1 spot is expressed as %ID / g. Assuming a density of 1 g / mL, the weight (in grams) of the mSOD1 aggregate is estimated based on the ROI volume. Next, the spot radiotracer content is compared to that found in a similar ROI (internal reference) plotted within the same mouse and to an ROI (external reference) traced at the same location in the same group of control mice. Each experimental group ( 89 Zr-DFO-AP-101, blocked by 100 mg / kg AP-101 89 Zr-DFO-AP-101 and deglycosylated 89 Zr-DFO-AP-101 reports the following data: number of mice in the group, total number of spots identified, number of mice with spots, percentage of mice with spots, number of spots per mouse, spot intensity (%ID / g), internal reference intensity (%ID / g), external reference intensity (%ID / g), and the ratio of spots to internal and external references. The mSOD1 spot-to-internal ratio is considered the most relevant value because it represents the ability to distinguish mSOD1 spots from their true background.

[0250] The mean radiotracer content in the spinal cord and anterior vertebrae was measured by tracing a thin region of interest (ROI) that runs through the length of these tissues. Figure 2 From these contoured ROIs, the mean %ID / g values ​​measured within the spinal cord and ventral vertebrae were compared with the "spinal cord to vertebral ratio". Other ROIs were drawn in the femoral head and spleen to evaluate the injection. 89 Zr-DFO-AP-101 and Degly- 89 The average %ID / g value in these tissues after Zr-DFO-AP-101 ( Figure 3 ).

[0251] Correction of spillover effect Once the region of interest (ROI) is located, tracking is then performed around these spots, and the radioactive tracer content is measured using the recovery factor corrected for partial volume effect (PVE), and corrected for the presence of free tracers in the anterior vertebrae. 89 Zr caused by overflow ( Figure 4 Recovery coefficient was obtained using a micro-hotspot phantom. Figure 5 The radioactivity content of aggregated m-SOD1 is expressed as %ID / g.

[0252] Biological distribution Following the last PET scan (day 10), mice were perfused to remove blood, and the spinal cord and tissues of interest were extracted to measure their radioactivity levels. Under isoflurane anesthesia, mice were injected with 45... o The tissue was placed at an angle, head up, on a perfusion plate in the blood collection tray. The chest cavity was then opened to access the heart, and a catheter (25 G) was inserted into the left ventricle. Perfusion solution (PBS containing 10 U / mL heparin) was delivered via a perfusion pump (20 mL @ 5 mL / min), and the right atrial appendage was cut open with small scissors. The sampled tissue was rinsed with saline, aspirated, and weighed. The radioactivity was then measured using a Hidex (HIDEX AMG, Gamble Technologies Limited, Mississauga, Canada) gamma counter. The measurements were then corrected for radioactive decay. Results are expressed as a percentage of the injected dose per gram of tissue (%ID / g). The spinal cord and brain were preserved (in 4% PFA for 24 h, stored in PBS) for histopathological analysis.

[0253] Example 1: - 89 Zr-DFO-AP-101 and Degly 89 Synthesis and Characterization of Zr-DFO-AP-101 By optimizing the lysine residue at AP-101 89 Zr-chelating agent (DFO-SCN) conjugation was used to prepare large quantities of DFO-AP-101 to obtain a DFO:AP-101 ratio of 1.1:1.0 (Table 1, n=7). nat The Zr-DFO-AP-101 conjugate maintains high selectivity and affinity for misfolded SOD1 (EC). 50 =0.5 nM), and similar to unmodified AP-101 ( Figure 6 The purity of DFO-AP-101 was confirmed by HPLC analysis, during which narrow peaks of the conjugate were observed. Figure 7 A). SDS-PAGE analysis confirmed the absence of aggregates. Figure 7 B). The radiolabeled yield is greater than 97%, and the apparent specific activity is 0.66 GBq / mg.

[0254] Table 1. Characterization of DFO-AP-101 and Degly-DFO-AP-101 (related to EC) 50 Value, see also Figure 6 )

[0255] To reduce the uptake of radioimmunoconjugates by FcγR-expressing immune cells in non-target tissues (such as the liver and spleen), a deglycosylated analog of DFO-AP-101 was considered. Deglycosylation of DFO-AP-101 was performed using the EndoS2 kit (deGlycIT spinning column from Genovis AB, Sweden, comprising an IgG-specific endoglycosidase of a complex N-glycan acting on the Fc-glycosylation site of IgG, wherein the endoglycosidase is covalently coupled to agarose beads). Loss of DFO chelates was observed during deglycosylation: an average of 0.6 DFOs per antibody was obtained by isotope dilution techniques (Table 1). No trace aggregates were observed on the purified Degly-DFO-AP-101. Figure 7 B). Gel electrophoresis was used to monitor the removal of glycans under reducing conditions, and this was confirmed by the downward shift of immunoglobulin heavy chain migration at ~55 kD. Figure 7 B). To achieve high radiolabeled yield and apparent specific activity. 89 The specific activity of Zr Degly-DFO-AP-101 (0.32 ± 0.11 GBq / mg) was slightly lower than 89 Zr-DFO-AP-101. This can be explained by the lower DFO:AP-101 ratio. Compared to unmodified AP-101, Degly- nat Zr-DFO-AP-101 has a low affinity for misfolded SOD1 (EC). 50 =2.4 nM), but its EC50 value is similar to... nat Similar to Zr-DFO-AP-101.

[0256] Example 2: Degly 89 Stability of Zr-DFO-AP-101 89 Zr-DFO-AP-101 was stable for over 10 days at 4ºC when formulated in PBS and for 7 days in mouse plasma. Only trace amounts of free Zr were detected after 10 days of incubation in plasma. 89 Zr (<5%). Degly- 89 Zr-DFO-AP-101 remained stable in PBS at 4ºC in both the presence and absence of GA, which is used due to its antioxidant and radioprotective properties (Joshi). et al. 2012)( Figure 9 It is worth noting that, 89 Zr-DFO chelating agents are mainly found in Degly- 89On the immunoglobulin heavy chain at ~55kD of Zr-DFO-AP-101.

[0257] Example 3: Evaluation of Use 89 Zr-DFO-AP-101 and Degly- 89 Feasibility of Zr-DFO-AP-101 for PET imaging of spinal cord mSOD1 aggregates A total of 47 mice were used in this study (30 Tg mice and 17 Wt mice). Figure 10 The distribution of mice in different experimental groups and the study timeline are presented. This study lasted several months and used different radioactive tracer products. This longitudinal study aimed to evaluate... 89 The optimal age and time for PET imaging after Zr-DFO-AP-101 administration. 89 Imaging was performed on 91, 110, and 126-day-old Tg and Wt mice on days 1, 4, 7, and 10 after Zr-DFO-AP-101 administration. Figure 10 ).

[0258] Based on the results obtained, it was decided to set the PET imaging age to 126 days and limit PET imaging to [specific timeframe]. 89 Days 7 and 10 after Zr-DFO-AP-101 administration. It is also noteworthy that most of the spots observed on day 7 remained on day 10. The method for analyzing PET images was modified based on the presence of [missing information - likely a specific trait or feature] in the ventral portion of the vertebrae in Tg and Wt mice. 89 Zr-DFO-AP-101, considering radioactive spillover into the spinal cord.

[0259] During PET imaging, Tg mice were smaller in size compared to the reference Wt group, which can be explained by their ALS phenotype. In fact, the body weight of 126-day-old Tg mice (25±2 g) was significantly lower than that of Wt mice (30±2 g). Figure 11 Compared to Wt mice (0.59 ± 0.06 MBq / g), the dose of radiotracer administered to Tg mice was significantly higher, normalized to body weight (MBq / g) (0.69 ± 0.07 MBq / g). Figure 12 As shown. However, 89 Zr-DFO-AP-101 has high specific activity and high reproducibility, and all doses of the radiotracer administered are sufficient to obtain high-quality PET images.

[0260] Although PET imaging was performed on days 7 and 10 after administration of the radiotracer, the data analysis focused on the results from day 10 because this was the time when the highest mSOD1 spot uptake values ​​were observed. Figure 12 ).

[0261] for 89 Zr-DFO-AP-101 (6 spots on day 7 compared to 18 spots on day 10) and for Degly- 89 Zr-DFO-AP-101 (6 spots on day 7 vs. 12 spots on day 10), the number of spots detected on day 10 (including false positive spots) was more significant than on day 7. Some of these were “false positives,” spots that appeared in control or blockade mice. All but one mouse observed false (+) spots on day 7 did not reappear on day 10. Figure 13 The distribution of m-SOD1 spots in the spinal cord of mice on day 10 is shown. All spots were located between the T10 and L2 vertebrae, with the majority located between the T11 and T13 thoracic vertebrae. 89 Zr-DFO-AP-101: 12 / 14, 85% relative to Degly- 89 Zr-DFO-AP-101: 9 / 11, 82%).

[0262] In order to evaluate 89 The specificity of Zr-DFO-AP-101 was demonstrated by co-injecting a group of mice (13 Tg, 4 Wt) with 100 mg / kg of natural AP-101 to block... 89 The binding of Zr-DFO-AP-101 with mSOD1. The results are as follows... Figure 13 and 19 As shown. Co-injection (blocking) with natural AP-101 significantly reduced the number of detected mSOD1 spots; under blocking conditions, this number decreased from [previous value]. 89 The percentage of Zr-DFO-AP-101 decreased from 67% (6 / 9) to 15% (5 / 13). When used alone... 89 The intensity of the spots also decreased from 8.05±1.30 %ID / g to 5.73±0.28 when Zr-DFO-AP-101 or in the presence of excess AP-101. 89 Zr-DFO-AP-101 (4.62±1.06) and blocking 89 The ratio of Zr-DFO-AP-101 (4.70±0.15) remained similar to that of the internal reference.

[0263] Discover 89 Zr-DFO-AP-101 accumulates in the ventral bone of the vertebrae, which is problematic because radiation from the vertebrae will spill into the spinal cord, masking potential mSOD1 spots. 89 The development of Zr-DFO-AP-101 aims to reduce the non-specific uptake of radiotracers in the vertebrae. Figure 13 , 14 Compared with 1989 The ability of Zr-DFO-AP-101 and its deglycosylated analogues to detect mSOD1 spots.

[0264] Assuming all Tg mice possessed mSOD1 aggregates, deglycosylation increased the efficacy of the radiotracer in detecting mSOD1 aggregates from 67% (6 out of 9 mice) to 100% (7 out of 7 mice). Figure 14 Deglycosylation also significantly increased the average intensity of the detected spots, from 89 The concentration of Zr-DFO-AP-101 increased from 8.05 ± 1.30 %ID / g to Degly- 89 The concentration of Zr-DFO-AP-101 was 13.14 ± 2.80 %Id / g (two-tailed Student's t-test, p = 0.01). However, Degly- 89 The spot ratio of Zr-DFO-AP-101 (1.92±0.34) to the internal reference was only slightly higher than that of the internal reference. 89 Zr-DFO-AP-101 (1.80±0.40), and the difference was not statistically significant (two-tailed Student's t-test, p=0.08 ns). Figure 15 For Degly- 89 The higher spinal cord background observed with Zr-DFO-AP-101 may be due to its increased uptake in the vertebrae. Figure 16 ).

[0265] In short, 89 Zr-DFO-AP-101 can specifically bind to mSOD1 aggregates, as demonstrated by co-injection of blocking doses of natural AP-101. The main limitation is that its accumulation in the abdominal vertebrae leads to radioactive spillover into the spinal cord, potentially masking some mSOD1 spots. The presence of radioactivity in the vertebrae is attributed to binding to FcγR, which promotes deglycosylation. 89 Development of Zr-DFO-AP-101. Deglycosylation did not reduce the uptake of radiotracers in the vertebrae, but led to higher uptake of radiotracers in mSOD1 aggregates.

[0266] Example 4: 89 Zr-DFO-AP-101 and Degly- 89 Spinal cord and vertebral uptake of Zr-DFO-AP-101 The effects of spinal cord and vertebrae on the spinal cord and vertebrae of Wt and Tg mice under all experimental conditions were measured and compared. 89 Intake of Zr-DFO-AP-101. Figure 16 The spinal cord pairs of different groups of mice were given. 89 Overview of Zr-DFO-AP-101 intake.

[0267] It is important to note that even with a narrower ROI, values ​​measured in the spinal cord in both Tg and Wt mice can be contaminated by radioactive tracers within the vertebrae. Furthermore, in Tg mice, the ROI may pass through mSOD1 aggregates containing the radioactive tracer, thus increasing the mean %ID / g value. To avoid these issues in some mice, we also tracked discrete ROIs in areas free of mSOD1 and spilled radioactivity.

[0268] Figure 3 and 17 The effects of radiotracer administration on Tg and Wt mice on day 10 were compared. 89 Zr-DFO-AP-101 and Degly- 89 Intake of Zr-DFO-AP-101. In the spinal cord and vertebrae of Tg mice. 89 The concentration of Zr-DFO-AP-101 was significantly higher in mice than in Wt mice (p=0.01). ), Figure 17 AB and Figure 3 E, F. However, the ratio of spinal cord to ventral vertebral bone was similar in Tg and Wt mice (p=0.21) Figure 17 C).

[0269] Blocking with 100 mg / kg AP-101 significantly reduced spinal cord injury (p=0.0002). ) and vertebrae (p<0.0001) )right 89 Intake of Zr-DFO-AP-101 ( Figure 17 (AB). Blocking also significantly improved the ratio of the spinal cord to the ventral vertebrae (p=0.03). In Wt mice, blocking reduced spinal cord... 89 The uptake of Zr-DFO-AP-101 was significantly reduced (p=0.053). However, blocking the uptake in Wt mice was also effective. 89 Zr-DFO-AP-101 had no significant effect on vertebral uptake (p=0.16) or the spinal cord to vertebral ratio (p=0.31). Liver uptake was increased in both Tg and Wt mice, but under the blockade condition, the increase in liver uptake was more significant in Wt mice. Figure 16 ).

[0270] In the experiments described in this article, it was observed that... 89 Accumulation of Zr-DFO-AP-101 in the anterior vertebral region. The accumulation of the radioactive tracer in the vertebral region was initially attributed to free... 89 The existence of Zr was later refuted, and the reason for its existence was attributed to... 89The binding of Zr-DFO-AP-101 heavy chain glycans to FcγR on immune cells (Vivier) et al. (2019). This promotes deglycosylation. 89 The development and in vivo validation of Zr-DFO-AP-101 aimed to reduce off-target uptake in vivo (i.e., reduce uptake by bone, liver, and spleen), improve the contrast between the target and healthy organs, and obtain higher quality PET images.

[0271] In Tg mice, Degly- 89 Zr-DFO-AP-101 showed the spinal cord (p<0.0001). ) and vertebrae (p<0.0001) Intake increased significantly, but did not improve the ratio of spinal cord to vertebrae (p=47). Figure 17 and Figure 3 (Continued from 2)). In accepting Degly- 89 In Wt mice with Zr-DFO-AP-101, a lower spinal cord and vertebral background was observed, resulting in better contrast between Wt and Tg mice. We also observed better contrast when using Degly- 89 The results found in Zr-DFO-AP-101 Tg mice hardly support our initial hypothesis. In fact, deglycosylation led to a sharp increase in the uptake of the radiotracer in the vertebrae, indicating the presence of mSOD1 in bone or bone marrow and specific uptake in these tissues.

[0272] Example 5: Femoral head and spleen 89 Zr-DFO-AP-101 and Degly- 89 Intake of Zr-DFO-AP-101 The spleen and femoral head are clearly visible on PET images. Figure 3 Furthermore, it was found that the content of their radioactive tracers varied with experimental conditions. Figure 3 A summary of measurements taken in the spleen and femoral head. 89 Zr-DFO-AP101 and Degly- 89 Average %ID / g value of Zr-DFO-AP101.

[0273] The observed results for femoral head uptake and vertebral resorption were similar. Blocking this in Tg mice led to... 89 Zr-DFO-AP-101 showed a significant decrease in femoral head uptake (p<0.0001). ), while when using Degly- 89 A significant increase in uptake was observed during PET imaging with Zr-DFO-AP-101 (p=0.0004). ()( Figure 3 G).

[0274] and 89 Compared to Zr-DFO-AP-101 intake, the presence of excessive AP-101... 89 Zr-DFO-AP-101 and Degly- 89 Zr-DFO-AP-101 both significantly reduced spleen uptake (blockade p=0.0003). Deglycosylation p<0.0001 ), Figure 3 H. It is worth noting that non-target tissue uptake of the tracer will increase its radiation burden, and its effects must be assessed through computational dosimetry.

[0275] Example 6: 89 Zr-DFO-AP-101 and Degly- 89 Biodistribution of Zr-DFO-AP-101 On day 10, after the final PET scan, the mice were euthanized, and tissues of interest were harvested after perfusion. Residual activity (%ID / g) was measured using a gamma counter. The results for the selected tissues are as follows: Figure 18 , 19 As shown.

[0276] For tissues visible on PET images, %ID / g measured by PET is correlated with %ID / g measured by tissue sampling and counting. This is an assessment for the spinal cord, bone (femoral head in PET relative to the tibia in biodistribution), and spleen. Figure 18 Apart from the low tissue concentration of radiotracers in the spinal cord, there was a significant correlation between %ID / g values ​​measured by PET imaging or biodistribution (bone: R). 2 =0.34, p<0.0001 And spleen: R 2 =0.71 p<0.0001 ).

[0277] On day 10, Tg mice were administered IV. 89 Following Zr-DFO-AP101, the radioactive tracer was primarily concentrated in the spleen, blood, bones, and liver. Figure 8 A similar biodistribution pattern was observed in Wt mice, but with lower tissue concentrations of the radiotracer. For blood (p=0.05) ), lung (p=0.03) ) and muscle (p=0.0007) The difference between Tg mice and Wt mice was statistically significant.

[0278] AP-101 blockade significantly reduced most tissues and blood (p<0.0001). ), bone (p=0.002) ), spleen (p=0.002) Lung (p=0.01) ) and muscle (p=0.05) )right 89 Intake of Zr-DFO-AP-101, Figure 8 However, blocking increases the amount of [something] in the liver. 89 Zr-DFO-AP-101 uptake (p<0.0001) This was even more pronounced in Wt mice (p=0.0008). These results are consistent with PET image analysis. Figure 16 ).

[0279] and 89 Compared to Zr-DFO-AP-101, Degly- 89 Zr-DFO-AP-101 showed increased bone uptake (p=0.09 ns), while significantly reduced in most other tissues, such as blood (p=0.0003 ns). ), spleen (p=0.0002) ), heart (p=0.0008) Lung (p=0.0001) Kidney (p=0.01) ) and muscle (p=0.002) ). 89 Deglycosylation of Zr-DFO-AP-101 had no significant effect on the concentration of radiotracers in the liver.

[0280] Example 7: Detection of misSOD1 in bone and articular cartilage tissue of SOD1 G93A transgenic mice Hematoxylin was used, as was AP-101, and the commercially available misSOD1 antibody (B8H10 from MÉDIMAPS, Gros-Louis) was used. et al. (J. Neurochem., 113 (2010), 1188-1199) stained sections of dorsal and ventral vertebrae and the femoral head joint region from SOD1 G93A transgenic and wild-type mice (see [reference needed]). Figure 22 In addition, control staining was performed using a 1 nM isotype control antibody. No immune response was detected using either the isotype control antibody or in wild-type mice. In SOD1 G93 transgenic mice, misfolded SOD1 was detected in bone and joint cross-sections using AP-101 and B8H10 antibodies.

[0281] Summary of mouse experiment results A long-term goal of the experiments described in Examples 1-6 is to verify the use of... 89 The feasibility of using Zr's AP-101 radiotracer for PET imaging of mSOD1 aggregates in ALS patients serves as an example of a tracer for immunoimaging of mSOD1 in subjects. Preclinical evaluations in Tg and Wt mice demonstrated the feasibility of using... 89 Zr-DFO-AP-101 is capable of recognizing discrete mSOD1 aggregates in the spinal cord of Tg mice. However, a considerable amount was found in the anterior vertebral portion. 89 Zr-DFO-AP-101. Uptake by the spleen and some vertebrae is attributed to the binding of AP-101 heavy chain glycans to FcγR on bone marrow T and B lymphocytes, which promotes development... 89 Deglycosylated analogues of Zr-DFO-AP-101.

[0282] Deglycosylation increased the efficiency of radioactive tracer detection of mSOD1 aggregates from 67% to 100%. Both radioactive tracers were found to bind to spinal cord mSOD1 aggregates. Using Degly- 89 Zr-DFO-AP-101 yielded the highest uptake and detected number of mSOD1 aggregates. However, deglycosylation also significantly increased the uptake of the radiotracer in the vertebrae and femoral heads, indicating the presence of mSOD1 in bone and cartilage tissues and its specific uptake in these tissues. Furthermore, in the case of Degly- 89 Lower spinal cord and vertebral background was observed in Zr-DFO-AP-101-treated Wt mice, leading to the use of this Zr-DFO-AP-101-based... 89 The contrast between Wt and Tg mice was better with the Zr AP-101 radiotracer. For these reasons, both radiotracers were suitable, but Degly-89Zr-DFO-AP-101 appeared less stable and prone to aggregation, while Degly- 89 Zr-DFO-AP-101 appears to be the most suitable radiotracer for clinical applications and was therefore selected for human clinical studies. The presence of radioactivity in the vertebrae has less impact on humans compared to mice because of the difference in spinal size, and clinical PET / CT offers higher resolution and better ability to differentiate lesions.

[0283] Furthermore, misfolded SOD1 was detected in bone and articular cartilage tissues of two ALS animal models, SOD1 G93A and SOD1 G37R transgenic mice, suggesting a potential role in the decline in bone health observed in ALS patients and ALS model mice (Caplliure-Llopis). et al., (2020) and Zhu et al. (2015), and combined with previous findings on the potential of antibodies targeting mutated or misfolded SOD1 in treating ALS-related symptoms, such as prolonging lifespan of treated SOD1 transgenic animals, significantly delaying their weight loss and improving motor neuron loss (see WO2012 / 080518 A1, particularly in Example 9), it is suggested that the potential of using antibodies targeting pathologically misfolded / aggregated SOD1 species for treatment is also for treating bone and / or joint-related diseases, particularly those showing impaired bone and / or joint function in affected structures associated with the presence of misfolded SOD1.

[0284] Example 8: Dosage estimation based on mouse biodistribution data The safety, tolerability, and pharmacokinetics of AP-101 have been evaluated in two clinical trials. A multicenter, open-label, single-escalation (SAD) phase 1 study was completed in November 2020 (https: / / classic.clinicaltrials.gov / ct2 / show / NCT03981536). In this study, 100 mg, 500 mg, and 2500 mg doses of AP-101 were tested in humans (initially enrolled 9 ALS patients). Even at the highest dose (single 2500 mg), no events causing clinically significant safety or tolerability problems occurred; specifically, no serious adverse events (SAEs) or infusion reactions related to the test antibody were observed. The observed AEs were related to the lumbar puncture procedure used for antibody administration. The second study is a multicenter, randomized, double-blind, placebo-controlled phase 2a trial (https: / / classic.clinicaltrials.gov / ct2 / show / NCT05039099), initiated in September 2021, with 63 patients (63 participants) with fALS and sALS, and an estimated completion date of July 30, 2023. To date, no SAEs or toxicities related to AP-101 have been reported in this study.

[0285] As described herein, the inventors have optimized the conjugation of lysine residues on AP-101. 89 Zr-chelating agent (DFO-SCN) was used to obtain a 1:1 DFO:AP-101 ratio. The radiolabeled yield was greater than 97%, and the apparent specific activity was 0.656 GBq / mg. 89 Zr-DFO-AP-101 showed stability for over 10 days when formulated in PBS, saline, or mouse plasma. Tracking via PET / CT imaging was evaluated in a limited number of animals as described in the foregoing examples. 89The feasibility of Zr-DFO-AP-101 spinal uptake was demonstrated. Three crucial steps essential to the entire project were achieved: - 89 Zr-DFO-AP-101 is stable in vitro and in plasma; - Nat Zr-DFO conjugates retain their affinity and specificity for misfolded SOD1; and - 89 Zr-DFO-AP-101 exhibits specific uptake of the spinal cord in a mouse ALS model (see, for example, Example 4 and...). Figure 3 , 17 18).

[0286] Dosing studies in C57BL5 mice have enabled the estimation of expected human injection doses for diagnostic use with PET / CT (see Purpose-3 section at the beginning of Examples).

[0287] Specifically, animal biodistribution data obtained through standard organ dissection methods were used to calculate... 89 Estimated human radiation dose from Zr-DFO-AP-101.

[0288] In order to estimate 89 The dosage determination of Zr-DFO-AP-101 was performed in biodistribution studies in 126-day-old female and male C57BL6 mice. Each mouse was injected via tail vein. 89 Zr-DFO-AP-101: Population-1 (female n=20, weight 21.6 sdm 1.8 g, dose 0.86 sdm 0.05 MBq, euthanized at 2h and on days 1, 2, 7, and 10); Population-2 (male n=20, weight 33.3 sdm 1.6 g, dose 0.84 sdm 0.04 MBq, euthanized at 2h and on days 1, 2, 7, and 10), euthanized on day 10. Data are not corrected for radioactive decay and correspond to actual tissue radioactivity burden at each time point. Biodistribution data are as follows. Figure 23 As shown.

[0289] Dosimetry was calculated based on the residence time of the radiotracer using OLIDA software (OLINDA - Organ-Level Internal Dosimetry Code, Version 2.2.3 - Copyright Vanderbilt University - 2012). Human equivalent organ %ID was estimated based on the %ID / g values ​​of mouse organs listed in OLIDA. PRISM software (GraphPadPrism version 9.25 for MacOS, GraphPad Software, San Diego, California USA) was used. www.graphpad.com The time-activity curve is fitted to a single-exponential or double-exponential model. The residence time (in hours) is obtained by integrating the fitted time-activity curve and scaled according to the organ mass of the selected model (mouse or human) used for dosimetry calculation. Radiation dose includes from 89 The contributions from Zr emission of beta and gamma rays also include contributions from the activity within an organ itself, as well as contributions from neighboring organs and the rest of the body.

[0290] The observed maximum radiation doses in females relative to males were 0.0535 mSv / MBq in the lungs and 0.0515 mSv / MBq in the liver. Biodistribution results showed that radiation was observed in the lungs 2 hours after injection (pi). 89 High uptake of Zr-DFO-AP-101, but it decays rapidly over time. However, 89 Zr-DFO-AP-101 uptake in the liver is slightly higher than in the lungs and remains stable over time, which may lead to dose-assay-calculated spillover activity in the lungs. Female mice... 89 The estimated Zr-DFO-AP-101 dose (0.237 mSv / MBq) was higher in male mice than in female mice (0.171 mSv / MBq) (Table 2). It was found that the effective dose in females was approximately 20-40% higher than in males, and this is true for many radiopharmaceuticals (Stabin MG, 1997). This is consistent with other human-based... 89 These values ​​are acceptable compared to Zr tracers. In fact, these values ​​are calculated based on animal data. 89 The effective dose of Zr-trastuzumab (in females) was 0.44 mSv / MBq, and in males it was 0.39 mSv / MBq, which was found to be safe for phase 0-1 studies (Laforest et al. 2016). Based on several human studies, 89 The estimated radiation dose to patients receiving Zr-trastuzumab PET is approximately 0.54 mSv / MBq. https: / / www.ninds.nih.gov / news- events / directors-messages / all-directors-messages / fda-approval-amyotrophic- lateral-sclerosis-als-drug-represents-progress-were-not-done?search-term= amyotrophic%20lateral%20sclerosis%20als%20fact%20sheet.%20Accessed:%20June%202023 ; and Laforest et al. 2016.

[0291] Preliminary results from the Phase I clinical trial described in Example 10 (the study design is described in Example 8) show that these estimates have been confirmed by the study data, with an effective dose of 0.54 mSv / MBq for the female human model and 0.48 mSv / MBq ICRP for the male human model.

[0292] Although the value is higher 18 F-FDG PET, but based on 89 Zr PET tracers are safe for clinical use (Heskamp et al. 2017, Stabin 1997, Laforest et al. 2016, Dijkers et al. 2010, Farr et al. 2021, Yoon et al. 2020, Veldhuijzen et al. 2018, Oosting et al. 2016, Even et al. 2017).

[0293] The toxicity of AP-101 was measured in monkeys. This biocompound demonstrated safety and met the requirements for clinical trial applications. Due to the matching chemical / biological properties, it is expected that... Nat Zr-DFO-AP-101 and 89 The same applies to Zr-DFO-AP-101.

[0294] Table 2. Extrapolated activity per unit dose 89 Zr-DFO-AP-101 Human Organ Dose Estimation [mSv / MBq]

[0295] Example 9: PET imaging in the Phase 1 clinical trial of Example 10 89 Generation of Zr-DFO-AP-101 By 89 Zr was combined with DFO-AP-101 to obtain [the desired product]. 89 Zr-labeled antibodies. 89 Zr can be, for example, as 89 Zr-oxalate is obtained as described in Section 2. Materials and Methods, pages 2-6 of Alnahwi et al. (2018), and in the abbreviations used in Example 1 above. The Zr-oxalate obtained thus... 89Zr-oxalate is captured on a quaternary methylammonium (QMA) anion exchange column. To automate this process, subsequent steps can be performed, for example, on a miniAllinOne (MiniAIO) cartridge module (Ans,Belgium), as described in Alnahwi et al. (2018), specifically in section 2.4. 89 Automated box-type modules and separation chemistry for Zr-oxalate.

[0296] The generation method includes the following steps. 89 Zr-oxalate was transferred (captured) onto a quaternary methylammonium (QMA) anion exchange column. Then, 0.5 mL of a 500 mM NaCl-137 mM HCl solution was added to the column. The eluent was collected from the column into a vial. The vial was then transferred to a reactor. The pH of the eluent was adjusted using sodium bicarbonate solution and HEPES buffer to achieve a pH in the range of 6.5–8.0. Approximately 3 mg of DFO-AP-101 was also added to the pH-adjusted eluent. The resulting solution was then warmed to 37ºC for 45 min. During the warming of the solution in the reactor, the PD-10 column (GE Healthcare) was conditioned by rinsing it three times with 10 mL of 0.9% saline at 1.5 mL / min. After the radiolabeling reaction was completed in the reactor, the generated... 89 The Zr-DFO-AP-101 solution was transferred to a conditioned PD-10 column and eluted with 4 mL of 0.9% saline. The eluent was collected in a new vial, and the volume was adjusted to approximately 10 mL by adding saline. The solution was then filtered. 89 Zr-DFO-AP-101 solution.

[0297] The obtained 89 Zr-DFO-AP-101 exhibits high radiochemical purity of 90% or higher, with a radiochemical yield greater than approximately 60%. Radiochemical impurities ( 88 Zr and 88 Y) is at an extremely low level and is below 2% within 29 hours of the end of synthesis (EOS).

[0298] To prepare the solution for patient injection, the solution obtained as described above is prepared in a solvent comprising 0.9% saline and 10 mg gentianic acid. This addition helps prevent radiation decomposition. As described above, the injection thus prepared is stable for 29 hours after EOS. The calculated dose of DFO-AP-101 is approximately 1.5 mg per injection. The final injection volume is selected to conform to the prescribed amounts of HEPES or gentianic acid, which should not exceed 200 µg / injection and 5 mg / injection, respectively.

[0299] Generated by the above method 89 The Zr-DFO-AP-101 conjugate solution exhibits the following specifications: Appearance - clear and colorless; pH 4.5-8.0; maximum final product of 10 mg; Radiochemical properties - RT ± 10% vs STD; Radiochemical purity of 90% or higher as determined by HPLC; Radiochemical purity of 90% or higher as determined by instantaneous thin-layer chromatography (iTLC); Radionuclide purity and identification of 98% or higher; Radionuclide impurities not exceeding 2%. The filter used for product purification has passed a filter integrity test exceeding 50 psi. As mentioned above, the amount of HEPES buffer does not exceed 200 µg / injection, and gentianic acid does not exceed 5 mg / injection. 89 The Zr-DFO-AP-101 solution is sterile, and its pyrogen content does not exceed 175 EU / vol (17.5 EU / mL). The radionuclide identification half-life is approximately 4235-5175 minutes (i.e., approximately 70.58-86.25 hours). Pre-injection checks [ 89 Clarity of Zr-DFO-AP-101 conjugate solution before injection.

[0300] Example 10: Used for evaluation 89 A single-center, phase I, open-label study of the biodistribution, pharmacokinetics, and safety of Zr-DFO-AP-101 in healthy volunteers and ALS patients. The new clinical trial described in this article will be for the purpose of evaluating... 89 A single-center, open-label study of the biodistribution, pharmacokinetics, and safety of Zr-DFO-AP-101 PET radiotracer in healthy volunteers and ALS patients.

[0301] Table 3 below summarizes the research objectives and endpoints:

[0302] Table 3 – Research Objectives and Endpoints Specifically, the following outcomes are to be measured: - Primary outcome measurement: 1. Incidence of adverse events (AEs) and serious adverse events (SAEs). The time range is from day 0 (post-injection) to day 14 (end of study). The incidence will be determined. 89 The number of new or worsening adverse events (AEs) and serious adverse events following administration of Zr-DFO-AP-101 (compared to baseline / before administration).

[0303] 2. 89Biodistribution of Zr-DFO-AP-101. Time range from before injection dose to 2 hours, 1, 3, 7, and 10 days after injection dose. Biodistribution will be evaluated using whole-body PET imaging. 3. 89 Dosage determination of Zr-DFO-AP-101 in humans. The time range is as described in point 2 above. Organ activity concentrations (liver, kidney, blood, spleen, and other organs) were measured by plotting regions of interest on PET images.

[0304] -Secondary outcome measurement: 1. Cmax. The time range is again before administration and 2 hours, 1, 3, 7, and 10 days after administration. Cmax in plasma after injection will be measured. 89 Maximum concentration of Zr-DFO-AP-101 over time 2. Area under the curve (AUC). Time range: As shown in 1. Measurement of plasma concentration after injection. 89 AUC of Zr-DFO-AP-101 over time.

[0305] 3. Duration of stay. Time range: as shown in 1 and 2 above. Measurement 89 The residence time of Zr-DFO-AP-101 in patient plasma (1 / 2).

[0306] 4. Excretion. Time range: as shown in section -3 above. Measurements will be taken. 89 The concentration of Zr-DFO-AP-101 in participants' urine over time.

[0307] -Other pre-specified outcome measurements: 1. Differential markers and uptake. Time ranges were again defined as before administration and 2 hours, 1, 3, 7, and 10 days after administration. Target organ / tissue ratios in ALS patients relative to healthy volunteers were assessed.

[0308] Research Design Overall Design This study is a phase I clinical trial, a two-part, single-center, open-label study conducted in healthy volunteers (Part A) and patients diagnosed with ALS (Part B). The primary objective is to evaluate... 89 Safety and biodistribution of Zr-DFO-AP-101 in healthy volunteers and ALS patients.

[0309] Research Visit After a screening period of up to 28 days, eligible participants will proceed to CRCHUS for all study evaluations according to the event schedule (Table 4). On day 0, a single intravenous dose will be administered. 89Zr-DFO-AP-101 40 MBq, with 45-minute whole-body PET / CT scans performed before injection and two hours after injection of the radiotracer. Physical examination, ECG, vital signs checks, and blood / urine sample collection will be performed before and after injection of the radiotracer (Day 0). Further PET scans will be performed on days 1, 3, 7, and 10 after injection of the radiotracer. The collection time can be adjusted as needed, but should not exceed 60 minutes. Participants will be contacted for their final follow-up approximately 14 days after administration of the study product. Blood and urine samples (if available) will be collected and their radioactivity measured at each PET / CT scan. If the ALS patient's condition does not allow for multiple visits, one or two visits may be skipped, but at least three scans should be performed, with a final telephone visit 14 days later.

[0310] Research Products 89 Zr-DFO-AP-101 is an imaging agent produced by a combination of radiopharmaceuticals and biological compounds. AP-101 is a biological compound that acts as a monoclonal antibody that specifically recognizes and binds to human misfolded SOD1. DFO is conjugated to AP-101 as zirconium-89 (… 89 Zr chelating agent, 89 Zr is a positron emitter (with a half-life of 3.4 days) and can be detected in PET / CT scans.

[0311] Number of participants A total of twelve patients, eight healthy volunteers (four women and four men), and four ALS-confirmed patients (+) will be enrolled. The aim is to enroll at least one patient with a SOD1 gene mutation and one patient with a sporadic mutation in the ALS-confirmed patient group. Those who do not complete the study will be replaced.

[0312] End of study definition The study ends upon completion of the final study visit and any applicable follow-up by the last patient. “Trial end” refers to the date of the last patient’s last visit or last scheduled procedure.

[0313] Dosage basis The dosage range (30-40 MBq) in this study was consistent with other clinical studies. The amount of protein administered (less than 2 mg) was significantly lower than the therapeutic dose of AP-101.

[0314] Study population Selection criteria 1. Age: - For healthy participants: male or female subjects aged 50 or older - For ALS patients: Male or female subjects aged 18 years and older 2. Can maintain a supine position for up to 45 minutes without respiratory support.

[0315] 3.A) For ALS patients, a definitive diagnosis of ALS is made according to the El-Escorial criteria (Brooks et al., 2000).

[0316] B) For healthy participants: No neurological conditions (confirmed by physical examination) 4. Have sufficient intravenous access to allow for blood sampling. 5. Reliable and willing to be on call at any time during the research period, and willing to follow CRCHUS-specific research procedures.

[0317] Exclusion criteria 1. Currently enrolled or enrolled within the past 12 weeks: any other clinical trial involving the off-label use of the investigational drug or drug or device, or any other type of medical research deemed scientifically or medically incompatible with this study.

[0318] 2. Female participants who are pregnant or breastfeeding; or women of reproductive age (<50 years) and sexually active men who do not wish to use recognized effective methods of contraception. All female participants under 50 years of age will be confirmed as not pregnant by a pregnancy test (serum hCG) at screening and on day 0 of CRCHUS.

[0319] For participants in purely homosexual relationships or those who are infertile (surgical sterilization [e.g., tubal obstruction, hysterectomy, bilateral salpingectomy] or physiologically unable to conceive, or who have been postmenopausal and have not menstruated for at least 12 months), the contraceptive requirement does not apply. Conversely, sexually active participants (women under 50 years of age or any man with a female partner of reproductive age) must use at least one method of contraception from screening until 30 days after injection: hormonal contraceptives (oral, transdermal patch, vaginal, or injectable); intrauterine devices (IUDs) with or without hormones; condoms or vasectomy (“barrier” method) performed >30 days prior to screening; diaphragms or cervical caps; abstinence.

[0320] 3. Plans to perform surgery or other non-invasive procedures during the study period (up to 14 days after injection). 4. Having a progressive medical condition, including but not limited to any cardiovascular, liver, respiratory, blood, endocrine, mental or nervous system disease, seizures, or any clinically significant laboratory abnormalities at screening and first visit (D0) that, in the physician's opinion, indicate a medical problem that would prevent participation in the study.

[0321] 5. Having one of the following conditions (for both patient groups): a) Having liver disease at the time of screening, such as hepatic encephalopathy, abnormal liver laboratory test results (ALT or AST ≥3×ULN or total bilirubin ≥2×ULN) and hematological abnormalities.

[0322] B) Having severe chronic kidney disease at the time of screening (e.g., estimated glomerular filtration rate [eGFR] <30 mL / min / 1.73 m or requiring chronic dialysis).

[0323] c) Suffering from severe mental illness.

[0324] d) Diagnosed with other neurodegenerative diseases (such as Parkinson's disease, Alzheimer's disease, etc.).

[0325] e) Having a serious infection or a known inflammatory process at screening or D0.

[0326] f) Based on patient self-reported alcohol or drug abuse g) History of allergy to related atopic drugs or antibodies; H) Abnormal blood pressure (supine position), defined as diastolic blood pressure >90 or <45 mmHg and / or systolic blood pressure >160 or <90 mmHg. At the researchers' discretion, a retest may be performed during the screening visit if the initial blood pressure measurement is abnormal within 2 hours.

[0327] 6. For ALS patients: a) Had a tracheotomy due to ALS symptoms.

[0328] b) Nasal intermittent positive airway pressure (NIPPV) for >4 hours during daytime wakefulness to treat ALS-related symptoms.

[0329] c) There are other causes of neuromuscular weakness.

[0330] 7. The patient has received treatment with a biologic agent (such as a monoclonal antibody, including marketed drugs and AP-101) within 3 months or 5 half-lives (whichever is longer) prior to the administration of the study drug.

[0331] 8. Have received any blood or blood products within 3 months prior to screening.

[0332] 9. Unable to communicate reliably with researchers.

[0333] 10. Unwilling or unable to provide written informed consent.

[0334] 11. The doctor or his / her representative believes that the candidate is unsuitable for inclusion in the study.

[0335] Screening failed For individuals who do not meet the participation criteria for this study (failed screening), researchers may decide to re-screen them once, subject to sponsor approval. If participants are re-screened, they will be assigned a new participant number and will be required to sign a new informed consent form (ICF).

[0336] Intervention Product administration All participants will receive a single intravenous dose of 40 MBq ± 10% 89 Zr-DFO-AP-101. Immediately prior to injection, the patient was measured using an appropriate radiocalibration system (see “Study Visits,” “Safety Assessment,” and Table 4 for details).

[0337] Allocation method, blinded This is a non-randomized study; eligible participants will be enrolled and receive the study product in sequence. This is an open-label study and is not blinded.

[0338] Dosage adjustment Dosage levels, sampling schedules, and procedural times (e.g., PK sample collection time) may be adjusted based on safety or PK data observed during the study. Visits to ALS patients will be reduced after reviewing the first 3–5 healthy participants. However, healthy participants will attend all scheduled visits.

[0339] Concomitant therapy Generally, concomitant medications should be avoided; however, over-the-counter (OTC) medications (e.g., acetaminophen for headaches) may be used at the researcher's discretion. If concomitant medications (excluding OTC medications) are required, the researcher will decide, in consultation with the sponsor, whether to enroll or continue the patient study. Any medications used during the study will be documented.

[0340] Research evaluation and procedures Efficacy evaluation This is a Phase I study, therefore no efficacy evaluation is being conducted.

[0341] Security assessment According to CIMS-CRCHUS, the primary safety endpoint is the number of serious and non-serious adverse events. Safety assessments were performed at each study visit. Statistical comparisons between the two groups (healthy group and ALS group) were not performed for the safety endpoint.

[0342] All treatments, medications, and investigational procedures related to adverse events (AEs) will be listed. Summary statistics for AEs and subacute acute exacerbations (SAEs) will be provided. Safety assessments may include laboratory tests, vital signs, ECG, physical examinations, and any automatically reported events. Parameters will be listed and summarized using standard descriptive statistics. Additional analyses will be performed as needed.

[0343] Although the medical director may have previously assessed each AE in a timely manner, a safety committee will still be established to assess the causality and management of all AEs. The committee will determine whether the event is likely related to the study, is unintended and serious, and whether the SAE should be reported to regulatory agencies. If so, and if life-threatening or related to death, the sponsor will submit a full SAE report to the Central REB within 7 days, or, in other cases, within 15 calendar days. Therefore, the sponsor will report this potentially relevant and unintended SAE to Health Canada and the supplier. A Development Safety Update Report (DSUR) will also be submitted to the supplier annually.

[0344] CRCHUS has facilities that help ensure participant safety. Its proximity to the hospital and CHUS emergency room makes CRCHUS an ideal location for this Phase I study. Two neurologists are involved in the study and will join the safety committee. They, or at least one of the physicians, will conduct a physical examination of each patient for screening and will be on-site for each patient during Day 0 (the day of injection) should any issues arise. Medical staff will be at the patient's bedside throughout Day 0 (from injection until 2 hours after injection). A safety cart is available near the examination room in case of any problems. If a patient develops health problems after participating in the study (up to the last visit), the physician will refer them to a specialist for treatment.

[0345] Dosage measurement Dosimetry will be performed according to the method previously described by Laforest et al. (2016). To obtain organ activity concentrations, regions of interest (ROIs) will be drawn on PET images for the following organs / tissues: liver, kidney, blood (left ventricular chamber), spleen, muscle, subcutaneous fat, and red bone marrow. To assess red bone marrow activity concentrations, the mean activity concentration measured in the lumbar vertebrae will be used. Total activity for each organ will be calculated as the product of activity concentration and standard adult organ weight. Decay-corrected data from all patients will be processed to obtain organ-time activity curves. Organ dwell time will be derived by fitting analytical integrals. Lemmens will be used. et al.The method described in (2006) measures the patient's total blood volume. To assess the residence time in the rest of the body, the sum of the residence times observed in organs is subtracted from the maximum theoretical residence time. OLINDA / EXM version 2.2.3 software is used to obtain human dose estimates based on residence time.

[0346] Pharmacokinetics If applicable, blood and urine samples will be collected with each PET scan. Radioactivity in the blood and urine will be measured; any remaining sample material after analysis will be destroyed. Pharmacokinetic parameters will be summarized by dose level using descriptive statistics. Mean and individual data will be presented. 89 Zr-DFO-AP-101 serum concentration-time activity curve.

[0347] Accidental discovery Whether healthy individuals or participants with ALS, any incidental findings were referred to their primary physician or specialist for appropriate medical follow-up, depending on their condition. Incidental findings may be observed during PET / CT imaging, through physical examination, or blood / urine laboratory evaluation.

[0348] Sample quantity determination The sample size was not determined based on the primary endpoint.

[0349] Statistical analysis Statistical analysis for this study will be conducted by the sponsor or its designated personnel. Safety analysis will be performed regardless of whether all enrolled patients have completed all protocol requirements. For continuous variables, summary statistics will include the number of patients, mean, median, standard deviation, minimum, and maximum. Categorical endpoints will be summarized using patient number, frequency, and percentage.

[0350] Preliminary results of the Phase I clinical trial Following the method previously described by Laforest et al. (Laforest R et al.) Mol Imaging Biol. 2016;18(6):952-959), dose measurements were performed on five healthy controls (3 women and 2 men). To obtain organ activity concentrations, regions of interest (ROIs) for the following organs / tissues were plotted on PET images (see 2016;18(6):952-959). Figure 27 and its section above. Description ): Liver, kidneys, heart (left ventricle), spleen, bones, bladder.

[0351] Total activity for each organ was calculated as the product of the active concentration and the standard adult organ weight. Decay-corrected data from all patients were processed to obtain organ-time-activity curves, as shown in Figures 26A and 26B. Organ residence time was derived through fitted analytical integration. To assess residence time in the rest of the body, the sum of residence times observed in organs was subtracted from the maximum theoretical residence time. Human dose estimates were obtained using OLIDA / EXM version 2.2.3 software based on patient residence time and effective dose, as shown in Table 6 below.

[0352] Dosing was performed as described. In short, regions of interest (ROIs) were plotted to obtain organ activity concentrations in different organs / tissues on PET images: for example, liver, kidneys, blood (heart, especially the left ventricular chamber), spleen, bone, and bladder. Time-percentage-of-injection dose profiles were evaluated to conform to... 89 Beavering of Zr-DFO-AP-101 injection. Total activity in each organ was calculated as the product of active concentration and organ weight. Organ residence time was obtained by fitting analytical integrals. To assess residence time in the rest of the body, the sum of residence times observed in organs was subtracted from the maximum theoretical residence time.

[0353] Table 6: 89 Organ radiation dose of Zr-DFO-AP-101 [mSv / MBq]

[0354] The total urinary excretion rate was 4%, and the hepatobiliary excretion rate was 2%. Two hours after injection, 44 ± 5% of the radiotracer remained in the blood pool, with an effective half-life of ± 53 hours. Based on sex-specific dosimetry, the liver was the dose-limiting organ, with doses of 2.65 mSv / MBq (female) and 2.24 mSv / MBq (male). The effective doses were 0.54 mSv / MBq (female) and 0.48 mSv / MBq (male).

[0355] In summary, as demonstrated in the above embodiments, the labeled antibody conjugates of the present invention are safe to use, and their clearance is primarily through the body, with early excretion via urine. Therefore, this tracer can be safely used in patients. Data to be obtained in ALS patients will validate its ability to measure mSOD1 levels in vivo for diagnostic and treatment monitoring.

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Claims

1. A labeled antibody conjugate for use in a method for diagnosing amyotrophic lateral sclerosis (ALS) or monitoring ALS drug treatment and / or progression in a subject, wherein, The method includes in vivo immunoimaging of misfolded SOD1 in a subject, and the labeled antibody conjugate comprises: (a) An antibody that specifically binds to misfolded SOD1; and (b) Detection marker conjugated to the antibody.

2. The labeled antibody conjugate for the use according to claim 1, wherein, The detection marker is a radionuclide.

3. The labeled antibody conjugate for the use according to claim 2, wherein, The radioactive nuclide is 89 Zr.

4. A labeled antibody conjugate for use according to any one of the preceding claims, wherein, The detection marker is conjugated to the antibody via a bifunctional chelating agent or a cofactor, preferably wherein the bifunctional chelating agent or cofactor is randomly conjugated to a lysine residue of the antibody.

5. The labeled antibody conjugate for the use according to claim 4, wherein, The bifunctional chelating agent includes p -SCN-Bn deferoxamine (SCN-Bn-DFO).

6. A labeled antibody conjugate for use according to any one of the preceding claims, wherein, The antibody is characterized in that its variable region, i.e., the binding domain, includes six CDRs of a variable heavy chain (VH) and a variable light chain (VL), wherein: (a) VH-CDR1 comprises the amino acid sequence of SEQ ID NO: 3 or a variant thereof, wherein the variant comprises one or two amino acid substitutions. (b) VH-CDR2 comprises the amino acid sequence of SEQ ID NO: 4 or a variant thereof, wherein the variant comprises one or two amino acid substitutions. (c) VH-CDR3 comprises the amino acid sequence of SEQ ID NO: 5 or a variant thereof, wherein the variant comprises one or two amino acid substitutions. (d) VL-CDR1 comprises the amino acid sequence of SEQ ID NO: 8 or a variant thereof, wherein the variant comprises one or two amino acid substitutions. (e) VL-CDR2 comprises the amino acid sequence of SEQ ID NO: 9 or a variant thereof, wherein the variant comprises one or two amino acid substitutions, and (f) VL-CDR3 includes the amino acid sequence of SEQ ID NO: 10 or a variant thereof, wherein the variant includes one or two amino acid substitutions.

7. A labeled antibody conjugate for use according to any one of the preceding claims, wherein, The antibody is characterized in that its variable region, i.e., the binding domain, includes a VH chain and a VL chain, wherein... (a) The VH chain comprises the amino acid sequence shown in SEQ ID NO: 1 or 2 or a variant thereof, wherein the variant comprises one or more amino acid substitutions; and (b) The VL chain comprises the amino acid sequence shown in SEQ ID NO: 6 or 7 or a variant thereof, wherein the variant comprises one or more amino acid substitutions; Preferably, the amino acid sequences of the VH and VL chains have at least 90% identity with SEQ ID NO: 1 or 2 and 6 or 7, respectively.

8. A labeled antibody conjugate for use according to any one of the preceding claims, wherein, The antibody includes - Two heavy chains, each of which includes the amino acid sequence shown in SEQ ID NO: 14; and - Two light chains, each light chain comprising the amino acid sequence shown in SEQ ID NO:

15.

9. A labeled antibody conjugate for use according to any one of the preceding claims, wherein, The antibody is AP-101.

10. A labeled antibody conjugate for use according to any one of the preceding claims, wherein, The antibody is glycosylated or deglycosylated.

11. A labeled antibody conjugate for use according to any one of the preceding claims, wherein, The imaging includes positron emission tomography (PET), single-photon emission computed tomography (SPECT), or a combination thereof with computed tomography (CT).

12. A medicament for treating a subject with ALS, said subject (i) having been diagnosed with or having developed ALS and / or (ii) being monitored for efficacy and / or disease progression of the medicament for ALS by a method defined in any one of the preceding claims, preferably wherein said medicament is selected from riluzole (Rilutek®), edaravone (Radicava®), terfuscin (BIIB067), AP-101, dextromethorphan hydrobromide and quinidine sulfate (Nuedexta®), remisente (CY5031), arelomemo, levosimendan, fasudil, pyrimethamine (Daraprim). TM ), rapamycin, baclofen (Gablofen®, Kemstro®, Lioresal®), diazepam (Diastat®, Valium®), amitriptyline (Elavil®), trihexyphenidyl, Scopoderm® (scopolamine patch), glycopyrronium bromide (Robinul®), piracetam (BNJ441, ALXN1210 or Ultomiris®), and eculizumab (Soliris®).

13. A labeled antibody conjugate as defined in any one of the preceding claims.

14. The labeled antibody conjugate according to claim 13, wherein, The labeled antibody conjugate is suitable for in vivo immunoimaging of misfolded SOD1 in subjects.

15. The labeled antibody conjugate according to claim 13 or 14, wherein, The antibody conjugate is 89 Zr-DFO-AP-101, and includes (a) AP-101, as an antibody that specifically binds to misfolded SOD1; (b) p -SCN-Bn deferoxamine (SCN-Bn-DFO), as the bifunctional chelating agent, and (c) 89 Zr, as the radioactive nuclide.

16. An antibody that specifically binds to misfolded SOD1, comprising a bifunctional chelating agent, preferably wherein the bifunctional chelating agent is p -SCN-Bn deferoxamine (SCN-Bn-DFO).

17. An imaging composition comprising a labeled antibody conjugate according to any one of claims 13 to 15, preferably further comprising NaCl and gentianic acid, at a pH of 4.5-8.

0.

18. The imaging composition according to claim 17, comprising a 10 mL solution of NaCl (0.9%) and gentianic acid (10 mg) in a sealed vial, containing about 10 mg of a labeled antibody conjugate and about 300 to 500 MBq, preferably wherein the amount of the labeled antibody conjugate is greater than 97% relative to the amount of unlabeled antibody and / or wherein, The labeled antibody conjugate has an apparent specific activity of about 0.656 GBq / mg, and optionally, the injection solution can be diluted with NaCl before injection.

19. A diagnostic composition or kit, comprising: - The labeled antibody conjugate according to any one of claims 13 to 15; - The imaging composition according to claim 17 or 18; or - The antibody according to claim 16, optionally further comprising a detection marker, preferably comprising a radionuclide, preferably wherein, The radioactive nuclide is 89 Zr; And optionally, a description for use with the method as defined in any one of claims 1 to 11.

20. A method for in vivo immunoimaging of superoxide dismutase (SOD1), wherein the superoxide dismutase (SOD1) is used as a marker of amyotrophic lateral sclerosis (ALS) or as a marker of the efficacy of ALS drug treatment in a subject, the method comprising: (a) administering to a subject the labeled antibody conjugate according to any one of claims 13 to 15; and (b) Detect the presence of labeled antibody conjugates in the subject by imaging.

21. The method of claim 20, further comprising a radiographic imaging method of the subject after administration of the labeled antibody conjugate; and the preparation of a radiographic image of the subject for detecting the labeled antibody conjugate; wherein, The radiographic images are used to diagnose the presence of misfolded and / or aggregated SOD1 in the subject.

22. A method for generating an image of a human body, preferably a method for generating an image of a human body of an ALS patient, comprising administering a labeled antibody conjugate according to any one of claims 13 to 15 to the human body, and generating an image of at least a portion of the human body to which the labeled antibody conjugate has been distributed.

23. The method according to any one of claims 20 to 22, wherein, The labeled antibody conjugate is present in or provided by the imaging composition according to claim 17 or 18.

24. The method according to any one of claims 20 to 23, comprising contacting cells, tissues, or organs with the labeled antibody conjugate, wherein, The labeled antibody conjugate was administered at a target dose in the range of 10-100 MBq; And X-ray images were prepared for detecting the labeled antibody conjugates.

25. The method according to claim 24, wherein, The labeled antibody conjugate was administered at a target dose in the range of approximately 40 MBq ± 10%.

26. The method according to any one of claims 20 to 25, wherein, The labeled antibody conjugate is administered at a target dose significantly lower than the therapeutic dose of the antibody.

27. The method according to claim 26, wherein, The labeled antibody conjugate is administered at a target dose, wherein the amount of antibody protein is less than 2 mg.

28. The method according to any one of claims 20 to 27, wherein, The labeled antibody conjugate was administered via intravenous injection.

29. The method according to claim 28, wherein, The final volume for injection will be based on the HEPES or gentianic acid content, with specifications of ≤200 µg / injection and ≤5 mg / injection, respectively.

30. The method according to any one of claims 20 to 29, wherein, The labeled antibody conjugate is administered in a single dose.

31. The method according to any one of claims 20 to 30, wherein, The imaging method is performed at a time after administration of the labeled antibody conjugate, wherein the time is selected from a group consisting of 1 hour, 2 hours, 3 hours, and 24 hours.

32. The method according to claim 31, wherein, The imaging method was performed 2 hours after injection.

33. The method according to claim 31 or 32, wherein, The imaging method was also performed 1, 3, 7 and / or 10 days after injection.

34. The method according to any one of claims 20 to 33, wherein, The imaging method is selected from the group consisting of planar imaging, positron emission tomography (PET), single-photon computed tomography (SPECT), and combinations thereof with computed tomography (CT).

35. The method according to any one of claims 20 to 34, wherein, Image the regions of interest (ROIs) of selected organs / tissues, including the liver, kidneys, blood (left ventricular chamber), spleen, muscles, subcutaneous fat, red bone marrow, bone, joints, and cartilage.

36. The method according to any one of claims 20 to 35, wherein, The labeled antibody conjugate is administered to predict the effectiveness of the therapeutic agent in treating diseases caused by and / or associated with the presence of misfolded and / or aggregated SOD1.

37. The method of claim 36, wherein, The therapeutic drug is selected from the drugs defined in claim 9.

38. The method according to any one of claims 20 to 37, wherein, The method of administering the labeled antibody conjugate to diagnose ALS or monitor the effectiveness of therapeutic treatment for ALS includes performing radiographic imaging on the subject after administration of the labeled antibody conjugate. And to produce X-ray images of the subjects for detecting the labeled antibody conjugates; wherein the X-ray images are used for diagnosing ALS and for assessing the effectiveness of therapeutic treatment for ALS.

39. Use of an anti-SOD1 antibody or an equivalent SOD1 binding molecule for targeting misfolded and / or aggregated SOD1 in the bones and / or joints of a subject, preferably wherein the antibody is an AP-101 antibody.

40. The anti-SOD1 antibody or equivalent SOD1 binding molecule for the use according to claim 39, wherein, The anti-SOD1 antibody or equivalent SOD1 binding molecule is a labeled antibody conjugate as defined in any of the preceding claims.

41. 89 Zr or 89 Zr-DFO is used for immunoimaging of misfolded and / or aggregated SOD1 in subjects, preferably for use in labeled antibody conjugates or methods according to any of the preceding claims and / or for early diagnosis of ALS and / or for monitoring therapeutic interventions for ALS.

42. Use of the antibody or labeled antibody conjugate according to any one of claims 13 to 16 in the production of an imaging composition for use in any one of claims 20 to 38.

43. A tool for preparing the product comprising the one described in claim 15 89 A method for preparing a solution of the Zr-DFO-AP-101 antibody conjugate includes the following steps: (a) will 89 Zr chloride solution was combined with DFO-AP-101 to obtain a reaction solution; (b) The reaction solution is heated to a temperature of approximately 37ºC for approximately 45 minutes; (c) will 89 Zr-DFO-AP-101 is recovered from the reaction solution to the recovery solution; and preferably... (d) By adding salt water 89 The Zr-DFO-AP-101 recovery solution is adjusted to the final volume, preferably wherein... The final volume is approximately 10 mL; and preferably... (e) Filter the 89 Zr-DFO-AP-101 solution.

44. The method according to claim 43, wherein, In step (a) 89 Zr chloride solution is provided through the following steps: (a1) provides 89 Zr-oxalate solution; (a2) Add a solution containing HCl to the solution in (a1); and (a3) Collect from the solution of (a2) including 89 Solution of Zr chloride.

45. The method according to claim 43 or 44, further comprising the following steps: (f) The filtered product obtained in step (e) is reacted by adding 0.9% saline and 10 mg gentianic acid. 89 The Zr-DFO-AP-101 solution was formulated into a composition, preferably wherein the final volume of the composition is about 10 mL.

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