Radiolabeled compounds for detecting steap1

Abstract

The present invention relates to anti-STEAP1 constructs for detecting STEAP1 in a subject. The present invention further relates to anti-STEAP1 (Fab')2 fragments that are radiolabeled. The constructs can be used to image STEAP1 expression, for example, in positron emission tomography.

Description

Technical Field

[0001] This invention relates to anti-STEAP1 constructs for detecting STEAP1 in humans. The invention further relates to a radiolabeled anti-STEAP1 (Fab')2 fragment. These constructs can be used, for example, to image STEAP1 expression in positron emission tomography (PET). Instructions for electronically submitted text files

[0002] This application contains a sequence list that has been electronically submitted in XML format, and the entire sequence list is hereby incorporated by reference. A computer-readable copy of the sequence list was created on December 10, 2024, named 10289-WO01-SEC_ST26.xml, and is 41,366 bytes in size. Background Technology

[0003] Prostate cancer (PCa) is the most frequently diagnosed malignancy and a leading cause of cancer-related deaths in men worldwide, with over 1.4 million new cases and approximately 345,000 deaths in 2020 (Siegel et al., CA Cancer J Clin 2023;73(1):17-48; and Sung et al., CA Cancer J Clin 2021;71(3):209-249). Androgen deprivation therapy (ADT) has been the cornerstone of prostate cancer treatment. Although most PCa patients initially respond to ADT, the duration of response varies, and approximately 10%–50% of patients eventually progress to metastatic castration-resistant prostate cancer (mCRPC), which has a high mortality rate (Akaza et al., J Glob Oncol. 2018 Sep;4:1-12). Current treatment prospects for mCRPC encompass a range of approaches, including androgen receptor-targeted therapy, chemotherapy, and more recently, PSMA-targeted radioligand therapy (177Lu-PSMA-617). While these treatments offer benefits, the ongoing pursuit of improved overall survival and duration of response necessitates the development of treatment options enhanced with better biomarkers and patient selection strategies. Summary of the Invention

[0004] This invention provides an anti-STEAP1 construct, wherein the anti-STEAP1 construct is used... 64 Cu is radiolabeled. In one embodiment, the anti-STEAP1 construct is a (Fab')2 fragment. In one embodiment, the anti-STEAP1 construct further comprises at least one nota. In one embodiment, the construct comprises one nota. In one embodiment, the construct comprises two notas. In one embodiment, at least one nota comprises...64 Cu. In one embodiment, each NOTA contains 64 Cu. In one embodiment, the anti-STEAP1 construct comprises HCDR1 containing SEQ ID NO: 1, HCDR2 containing SEQ ID NO: 2, HCDR3 containing SEQ ID NO: 3, LCDR1 containing SEQ ID NO: 4, LCDR2 containing SEQ ID NO: 5, and LCDR3 containing SEQ ID NO: 6. In one embodiment, the anti-STEAP1 construct comprises HCVR containing SEQ ID NO: 7 and LCVR containing SEQ ID NO: 8. In one embodiment, the anti-STEAP1 construct comprises a heavy chain (HC) fragment containing SEQ ID NO: 9 and a light chain (LC) fragment containing SEQ ID NO: 10.

[0005] This invention provides an anti-STEAP1 construct, wherein the anti-STEAP1 construct is used... 64 Cu is radiolabeled, and the anti-STEAP1 construct is an antibody. In one embodiment, the anti-STEAP1 antibody comprises HCDR1 containing SEQ ID NO: 1, HCDR2 containing SEQ ID NO: 2, HCDR3 containing SEQ ID NO: 3, LCDR1 containing SEQ ID NO: 4, LCDR2 containing SEQ ID NO: 5, and LCDR3 containing SEQ ID NO: 6. In one embodiment, the anti-STEAP1 antibody comprises HCVR containing SEQ ID NO: 7 and LCVR containing SEQ ID NO: 8. In one embodiment, the anti-STEAP1 antibody comprises HC containing SEQ ID NO: 37 and LC containing SEQ ID NO: 10. In one embodiment, the anti-STEAP1 antibody comprises HC containing SEQ ID NO: 27 and LC containing SEQ ID NO: 28.

[0006] This invention provides an anti-STEAP1 construct, wherein the anti-STEAP1 construct is used... 89 Zr is radiolabeled. In one embodiment, the anti-STEAP1 construct is a (Fab')2 fragment. In one embodiment, the anti-STEAP1 construct further comprises at least one DFO or DFO In one embodiment, the construct contains a DFO or DFO In one embodiment, the construct contains two DFOs or DFOs. In one embodiment, at least one DFO or at least one DFO Include 89Zr. In one embodiment, each DFO or DFO Include 89 Zr. In one embodiment, the anti-STEAP1 construct comprises HCDR1 containing SEQ ID NO: 1, HCDR2 containing SEQ ID NO: 2, HCDR3 containing SEQ ID NO: 3, LCDR1 containing SEQ ID NO: 4, LCDR2 containing SEQ ID NO: 5, and LCDR3 containing SEQ ID NO: 6. In one embodiment, the anti-STEAP1 construct comprises HCVR containing SEQ ID NO: 7 and LCVR containing SEQ ID NO: 8. In one embodiment, the anti-STEAP1 construct comprises HC containing SEQ ID NO: 9 and LC containing SEQ ID NO: 10.

[0007] This invention provides an anti-STEAP1 construct, wherein the anti-STEAP1 construct is used... 89 Zr is radiolabeled, and the anti-STEAP1 construct is an antibody. In one embodiment, the anti-STEAP1 antibody comprises HCDR1 containing SEQ ID NO: 1, HCDR2 containing SEQ ID NO: 2, HCDR3 containing SEQ ID NO: 3, LCDR1 containing SEQ ID NO: 4, LCDR2 containing SEQ ID NO: 5, and LCDR3 containing SEQ ID NO: 6. In one embodiment, the anti-STEAP1 antibody comprises HCVR containing SEQ ID NO: 7 and LCVR containing SEQ ID NO: 8. In one embodiment, the anti-STEAP1 antibody comprises HC containing SEQ ID NO: 37 and LC containing SEQ ID NO: 10. In one embodiment, the anti-STEAP1 antibody comprises HC containing SEQ ID NO: 27 and LC containing SEQ ID NO: 28.

[0008] This invention provides a polynucleotide encoding the amino acid sequence of a construct of this invention. This invention provides a vector comprising the polynucleotide of this invention. This invention provides a mammalian cell comprising the vector of this invention. In one embodiment, the mammalian cell comprises two vectors of this invention.

[0009] This invention provides a method for detecting STEAP1 in a subject, wherein the method includes administering the anti-STEAP1 construct of the invention to the subject. In one embodiment, the subject has cancer. In one embodiment, the subject has a solid tumor. In one embodiment, the subject has a solid tumor expressing STEAP1. In one embodiment, the subject has prostate cancer. In one embodiment, the subject has metastatic castration-resistant prostate cancer. In one embodiment, the subject has metastatic castration-sensitive prostate cancer.

[0010] This invention provides an anti-STEAP1 construct for use in detecting STEAP1 in a subject. In one embodiment, the subject has cancer. In one embodiment, the subject has a solid tumor. In one embodiment, the subject has a solid tumor expressing STEAP1. In one embodiment, the subject has prostate cancer. In one embodiment, the subject has metastatic castration-resistant prostate cancer. In one embodiment, the subject has metastatic castration-sensitive prostate cancer.

[0011] This invention provides the use of the anti-STEAP1 construct of the present invention in the manufacture of an imaging agent for detecting cancer. This invention also provides the use of the anti-STEAP1 construct of the present invention in the manufacture of an imaging agent for treating cancer. In one embodiment, the cancer is a solid tumor. In one embodiment, the cancer is a solid tumor expressing STEAP1. In one embodiment, the cancer is prostate cancer. In one embodiment, the cancer is metastatic castration-resistant prostate cancer. In one embodiment, the subject has metastatic castration-sensitive prostate cancer.

[0012] In one embodiment, STEAP1 is detected by positron emission tomography.

[0013] In one embodiment, STEAP1 was detected in the prostate of the subject. In another embodiment, STEAP1 was detected at the metastatic site.

[0014] In one embodiment, a cancer treatment agent is further administered to the subject. In one embodiment, a prostate cancer treatment agent is further administered to the subject. In one embodiment, the prostate cancer treatment agent binds to STEAP1. In one embodiment, the prostate cancer treatment agent is an inhibitor of STEAP1. In one embodiment, the prostate cancer treatment agent causes the killing of cancer cells expressing STEAP1. In one embodiment, the prostate cancer treatment agent is salinomycin. In one embodiment, the subject has been diagnosed with STEAP1 expression.

[0015] This invention provides an anti-STEAP1 construct for use as a diagnostic biomarker.

[0016] This invention provides a diagnostic biomarker comprising the anti-STEAP1 construct of this invention.

[0017] This invention provides a kit comprising the anti-STEAP1 construct of this invention.

[0018] This invention provides a method for treating a subject diagnosed with cancer, wherein the method includes administering the anti-STEAP1 construct of the invention to the subject, imaging STEAP1 expression in the subject, diagnosing the subject with cancer, and administering a cancer therapeutic agent to the subject. In one embodiment, the subject has elevated STEAP1 expression in the subject's tumor. In one embodiment, the cancer therapeutic agent is salinomycin.

[0019] This invention provides a method for treating a subject diagnosed with cancer, wherein the method includes administering the anti-STEAP1 construct of the invention to the subject, imaging STEAP1 expression in the subject, and administering a cancer therapeutic agent to the subject. In one embodiment, the subject has elevated STEAP1 expression in the subject's tumor. In one embodiment, the cancer therapeutic agent is salinomycin. Attached Figure Description

[0020] Figure 1A and 1B The binding of NOTA-(CAR1)-conjugated anti-STEAP1 (Fab')2 cells was depicted. C4-2B Luc cells were conjugated with NOTA-(Fab')2 (CAR1) at concentrations increasing up to 1 μM. Figure 1A ) or cold-labeled 63Cu-NOTA-(Fab')2(CAR1) Figure 1B The cells were incubated with unconjugated (Fab')2 at 4°C for 1 hour. After incubation with the AF647-conjugated anti-human IgG (Fab')2 specific secondary antibody, cell-bound antibodies were detected by flow cytometry, and the median fluorescence intensity (MFI) of the AF647 channel at increasing concentrations of the corresponding molecule was shown.

[0021] Figure 2A and 2B Depicting DFO (CAR1) or DFO (CAR)-conjugated anti-STEAP1 (Fab')2 cells were bound. C4-2B Luc cells were conjugated with DFO-(Fab')2 (CAR1) at increasing concentrations up to 3 μM. Figure 2A ) or DFO -(Fab')2(CAR1)( Figure 2BThe cells were incubated with unconjugated (Fab')2 at 4°C for 1 hour. After incubation with the AF647-conjugated anti-human IgG (Fab')2 specific secondary antibody, cell-bound antibodies were detected by flow cytometry, and the median fluorescence intensity (MFI) of the AF647 channel at increasing concentrations of the corresponding molecule was shown.

[0022] Figure 3A and 3B The binding of Nota (CAR2) conjugated anti-STEAP1 (Fab')2 cells was depicted. C4-2B Luc ( Figure 3A ) or 22Rv1 Luc ( Figure 3B Cells were incubated at 4°C for 1 hour with increasing concentrations of NOTA-(Fab')2 (CAR2) up to 1 μM (compared to unconjugated (Fab')2). Cell-bound antibodies were detected by flow cytometry after incubation with an AF647-conjugated anti-human IgG (Fab')2-specific secondary antibody, and the median fluorescence intensity (MFI) of the AF647 channel at increasing concentrations of the corresponding molecule was displayed.

[0023] Figure 4A and 4B The binding of DFO (CAR2) conjugated anti-STEAP1 (Fab')2 cells was depicted. C4-2B Luc ( Figure 4A ) or 22Rv1 Luc ( Figure 4B Cells were incubated at 4°C for 1 hour with DFO-(Fab')2 (CAR2) at concentrations increased up to 1 μM (compared to unconjugated (Fab')2). Cell-bound antibodies were detected by flow cytometry after incubation with an AF647-conjugated anti-human IgG (Fab')2-specific secondary antibody, and the median fluorescence intensity (MFI) of the AF647 channel at increased concentrations of the corresponding molecule was displayed.

[0024] Figures 5A-5H The binding dynamics of the anti-STEAP1 (Fab')2:human STEAP1 interaction and the sensor plots of the 1:1 model fit were depicted. Figure 5A Unjoined; Figure 5B NOTA-CAR1; Figure 5C DFO-CAR1; Figure 5D DFO -CAR1; Figure 5E NOTA-CAR2; Figure 5F DFO-CAR2; Figure 5G 63Cu-NOTA CAR1; Figure 5H ) Buffer blank negative control.

[0025] Figure 6 Mean (and standard deviation) pharmacokinetic curves were plotted in female BALB / c nude mice after administration of 10 mg / kg NOTA-conjugated anti-STEAP1 (Fab')2 or DFO-conjugated anti-STEAP1 (Fab')2.

[0026] Figure 7 The mean (and SD) pharmacokinetic curves of cynomolgus monkeys after administration of 1.125 mg / kg were plotted.

[0027] Figure 8A and 8B Depicting STEAP1-positive xenografts 64 Cu-NOTA-Fab'2 CAR1 and CAR2 PET / CT imaging. 22Rv1 STEAP1 high-density cells were implanted into the right shoulder of male balb / c nude mice. Approximately 200 uCi of radiotracer was injected via the tail vein. 64 Cu-NOTA-Fab'2 CAR1 ( Figure 8A ) or CAR2 ( Figure 8B Images were acquired 22 h (for CAR1) or 24 h (for CAR2) post-injection. The uptake of the radiotracer by each tumor was quantified by calculating the percentage of injected dose per gram of tissue (%ID / g). 64 Both Cu-NOTA-Fab'2 CAR1 and CAR2 were successfully localized to STEAP1 high xenograft tumors and exhibited considerable tumor uptake.

[0028] Figure 9A and 9B Depicting STEAP1 negative xenografts 64 Cu-NOTA-Fab'2 CAR1 and CAR2 PET / CT imaging. 22Rv1 STEAP1 negative cells were implanted into the right shoulder of male balb / c nude mice. Approximately 200 uCi of radiotracer was injected via the tail vein. 64 Cu-NOTA-Fab'2 CAR1 ( Figure 9A ) or CAR2 ( Figure 9B Images were acquired 22 h (for CAR1) or 24 h (for CAR2) post-injection. The uptake of the radiotracer by each tumor was quantified by calculating the percentage of injected dose per gram of tissue (%ID / g). As expected, very low tumor uptake was observed for both molecules.

[0029] Figure 10A and 10B Depicting STEAP1 high xenografts 89Zr-DFO-Fab'2 CAR1 and CAR2 PET / CT imaging. 22Rv1 STEAP1 high-density cells were implanted into the right shoulder of male balb / c nude mice. Approximately 200 uCi of radiotracer was injected via the tail vein. 89 Zr-DFO-Fab'2 –CAR1 ( Figure 10A ) or CAR2 ( Figure 10B Images were acquired 22 h (for CAR1) or 24 h (for CAR2) post-injection. The uptake of the radiotracer by each tumor was quantified by calculating the percentage of injected dose per gram of tissue (%ID / g). 89 Both Zr-DFO-Fab'2 CAR1 and CAR2 are located in STEAP1 high xenograft tumors and exhibit low tumor uptake.

[0030] Figure 11A and 11B PET / CT imaging of 89Zr-DFO-Fab'2 CAR1 and CAR2 in STEAP1-negative xenografts was performed. 22Rv1 STEAP1-negative cells were implanted into the right shoulder of male balb / c nude mice. Approximately 200 uCi of radiotracer was injected via the tail vein. 89 Zr-DFO-Fab'2 –CAR1 ( Figure 11A ) or CAR2 ( Figure 11B Images were acquired 22 h (for CAR1) or 24 h (for CAR2) post-injection. The uptake of the radiotracer by each tumor was quantified by calculating the percentage of injected dose per gram of tissue (%ID / g). No significant tumor uptake was observed for either 89Zr-DFO-Fab'2-CAR1 or CAR2.

[0031] Figure 12A and 12B Depicting STEAP1-positive and STEAP1-negative xenografts 89 Zr-DFO -Fab'2CAR1 PET imaging. 22Rv1 STEAP1 high-density cells (A) and 22Rv1 STEAP1-KO cells (B) were implanted into the right shoulder region of male balb / c nude mice. Approximately 200 uCi of radiotracer was injected via the tail vein, and images were acquired 24 h post-injection. Radiotracer uptake by each tumor was indicated by the percentage of injected dose per gram of tissue (%ID / g). 89 Zr-DFO -Fab'2 CAR2 is localized in vivo to STEAP1-positive xenograft tumors, but tumor uptake is very low in STEAP1-negative xenografts.

[0032] Figure 13 Tumor uptake of various labeled constructs in vivo was depicted. Approximately 200 uCi of radiotracer was administered to STEAP1-high and STEAP1-negative tumor-bearing mice, and imaging was performed 22–24 h later. Tumor uptake was quantified by measuring the percentage of injected dose per gram of tissue (%ID / g).

[0033] Figure 14A B and C describe the IRF measurement ( Figure 14A ), beads used in the measurement ( Figure 14B ) and measured readings ( Figure 14C An overview of ( ). Detailed Implementation

[0034] Prostatic six-transmembrane epithelial antigen-1 (STEAP1) has emerged as an emerging therapeutic target for prostate cancer due to its membrane localization and overexpression in mCRPC (Gomes et al., Urol Oncol [Urologic Oncology] 2014;32(1):53.e23-9). The strategic integration of molecular imaging techniques has the potential to facilitate decision-making and improve treatment outcomes in the dynamic context of mCRPC management by enabling patient selection and response monitoring of STEAP1-targeted therapies.

[0035] While immunohistochemistry (IHC) is valuable and widely used, it has several drawbacks compared to radioactive tracers used for patient selection, such as PET tracers. IHC sample collection is invasive and often causes pain and discomfort to patients. Furthermore, IHC assays typically generate information from a limited number of tissue samples collected at a single time point, limiting the dynamic assessment of target antigen expression and treatment response. Particularly in bone samples (nearly 90% of men with mCRPC have bone metastases), decalcification methods can impair the accuracy of IHC results due to potential alterations in the epitopes recognized by IHC antibodies. In contrast, imaging agents offer a non-invasive approach to generating real-time systemic information to guide clinical decision-making.

[0036] A zirconium-89 labeled anti-STEAP-1 antibody has been disclosed (PCT Publication No. WO 2011153346), and an anti-STEAP1 PET imaging agent using a zirconium-89 labeled monoclonal antibody has been investigated in a phase I / II clinical trial (see, for example, O'Donoghue et al., Mol Pharm. [Molecular Pharmacology] 2019 July 1;16(7):3083-3090). However, it appears that no further clinical studies have been conducted using this imaging agent. Therefore, there is a clinical need for an alternative imaging method for the non-invasive, accurate, and efficient detection of STEAP1-positive lesions in mCRPC.

[0037] Copper-64 ( 64 The half-life of Cu is 12.7 hours, and it decays via electron capture (44%), positron (β+) emission (17%, 0.655 MeV), and β (β-) emission (39%, 0.573 MeV). Positron (β+) emission can be used to perform positron emission tomography (PET) imaging studies, and the half-life is ideal for imaging peptides and small proteins, allowing for imaging up to 24 hours post-injection. Zirconium-89 ( 89 Zr has a half-life of 3.27 days and decays via electron capture (77%) and β+ emission (17%, 0.396 MeV). Its long half-life makes it well-suited for full-length antibody imaging, as it allows imaging up to 7–10 days post-injection.

[0038] The (Fab')2 molecule exhibits faster clearance from the body while retaining the basic specificity and affinity of the intact antibody, which is desirable for imaging agents (see, for example, Freise et al., Molec. Immunology, Vol. 67(2), Part A (October 2015)). Interestingly, as part of this invention, anti-STEAP1 Fab molecules were tested compared to the (Fab')2 molecule, and the Fab molecule showed poorer binding to STEAP1, while the (Fab')2 molecule retained its binding to STEAP1.

[0039] Since the predicted human biological half-life of (Fab')2 is modeled as 12 hours, an attempt was made to use... 89 Zr-labeled anti-STEAP1 (Fab')2 fragments allow imaging approximately 10 days. This interval allows time for the radiolabeled compound (e.g., a PET tracer) to clear from the blood, achieving a good tumor-to-background ratio and good quantification. 64 Cu may allow imaging for up to 24 hours. Interestingly, both... 89 Zr-labeled (Fab')2 derivatives DFO-(Fab')2 and DFO (Fab')2 successfully enabled STEAP1+ tumor imaging; however, tumor uptake was significantly lower than expected. 64 The intake achieved by Cu-NOTA- (Fab')2.

[0040] This document discloses a radiolabeled anti-STEAP1 antibody construct that can be used as an imaging agent (e.g., a PET tracer). In one embodiment, the construct is a (Fab')2 fragment. In another embodiment, the construct contains... 89 Zr. In one embodiment, the build contains 64 Cu.

[0041] As used herein, "anti-STEAP1 construct" refers to a protein that specifically binds to STEAP1. In one embodiment, the anti-STEAP1 construct is an antibody. In one embodiment, the anti-STEAP1 construct is a (Fab')2 fragment. In one embodiment, the antibody or (Fab')2 fragment contains the heavy and light chain CDRs of the Fab of salinomycin that binds to STEAP1. In one embodiment, the (Fab')2 fragment uses... 64 Cu labeling.

[0042] As measured by surface plasmon resonance techniques (e.g., BIACore, GE Healthcare, Uppsala, Sweden) or kinetic exclusion assays (KinExA, Sapidyne, Boise, Idaho), when antigen-binding proteins are ≤10 -7 When an antigen-binding protein binds to its antigen, the protein is said to "specifically bind" to the antigen.

[0043] As used herein, an "antibody" is an immunoglobulin molecule comprising two heavy chains (HC) and two light chains (LC) linked by disulfide bonds. The amino-terminal portion of each LC and HC includes a variable region of approximately 100–120 amino acids, primarily responsible for antigen recognition via the CDRs contained therein. The CDRs are separated from more conserved regions called frame regions ("FRs"). Each LCVR and HCVR consists of three CDRs and four FRs arranged in the following order from amino-terminus to carboxyl-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the LC are referred to as "LCDR1, LCDR2, and LCDR3," and the three CDRs of the HC are referred to as "HCDR1, HCDR2, and HCDR3." The CDRs contain the majority of residues that specifically interact with the antigen. Therefore, the functional ability of an antibody to bind to a specific antigen is largely influenced by the amino acid residues within the six CDRs. The amino acid allocation to the CDR domains within the LCVR and HCVR regions of the antibody of this invention is based on the well-known Kabat numbering convention (Kabat et al., Ann. NY Acad. Sci. 190:382-93 (1971); Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242 (1991)).It is understandable that other numbering rules can also be used, such as those of Chothia et al. ("Canonical structures for the hypervariable regions of immunoglobulin", Journal of Molecular Biology, 196, 901-917 (1987); Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), and / or North et al. ("A New Clustering of Antibody CDR Loop Conformations", Journal of Molecular Biology, 406, 228-256 (2011)). The IgG Fc region contains two CH2 and two CH3 domains.

[0044] In one embodiment, the antibody of the present invention comprises a sequence containing a C-terminal lysine. Alternatively, the antibody comprises HC without a C-terminal lysine. Furthermore, the N-terminal glutamine and / or N-terminal glutamate of HC or HCVR can be converted to pyroglutamic acid. Additionally, the N-terminal glutamine and / or N-terminal glutamate of HC can be converted to pyroglutamic acid, and the sequence may lack a C-terminal lysine. All forms are contemplated for the antibody of the present invention.

[0045] The (Fab')2 fragment contains the antibody LC, antibody HCVR, and CH1 domains, as well as at least a portion of the hinge region. Therefore, the (Fab')2 fragment is essentially an antibody without the Fc region. The (Fab')2 fragment can be generated by digesting the antibody with a pepsinase, thereby removing the antibody's Fc region. Methods for preparing the (Fab')2 fragment are known in the art (see, for example, Khawli et al., Hybrid Hybridomics. [Hybrioma and Hybridomics] Feb 2002;21(1):11-8).

[0046] As used in this article, "chelating agent" refers to an agent that can bind with radioactive metal ions, such as... 64 Cu or 89Zr forms coordination complexes, enabling it to stably bind to molecules targeting moieties such as antibodies or antibody fragments (e.g., (Fab')2).

[0047] Chelating agents can be conjugated to constructs using methods known in the art. For example, chelating agents can be conjugated via random lysine conjugation. Exemplary random lysine conjugations are described in the examples herein. Chelating agents can also be conjugated via site-specific conjugation (e.g., conjugation with cysteine). Exemplary site-specific conjugations are also described in the examples herein.

[0048] The phrase "chelator to antibody ratio" or "CAR" generally refers to the amount of chelating agent attached to a construct, which can be, for example, a (Fab')2 fragment or an antibody. The use of the phrase CAR does not necessarily mean that the chelating agent is attached to an antibody as defined herein, but rather that the chelating agent can be attached to a protein, such as a (Fab')2 fragment. DFO (deferoxamine) and DFO It can be used 89 Examples of Zr chelating agents, and those known in the art (see, for example, Raavé et al., Eur J Nucl Med Mol Imaging, 46, 1966–1977 (2019); and Chomet et al., Eur J Nucl Med Mol Imaging, 48, 694–707 (2021)). NOTA (2,2′,2”-(1,4,7-triazacyclononane-1,4,7-triyl)triacetic acid) and DOTA (2,2′,2”,2”'-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid) are available for use. 64 Examples of Cu chelators are known in the art (see, for example, Lee et al., Diagnostics 2023, 13, 2649). For example, “CAR1” refers to the average CAR1, which means that some constructs may have 0 chelators attached, some constructs may have two chelators attached, but on average, CAR1 is 1.

[0049] Positron emission tomography (PET) is a non-invasive imaging test that detects radioactivity in a subject's body. It involves administering a PET tracer, a radiolabeled compound containing a radioactive isotope that emits positrons and binds to a target. The radioactive isotope decays, emitting positrons. When these emitted positrons encounter electrons within the body, they annihilate, producing two gamma-ray photons traveling in opposite directions. Special detectors around the body capture the path and timing of these photons. A computer can then use this information to generate a 3D image of the source of the annihilation event. PET imaging can be used for the diagnosis, staging, and evaluation of the effectiveness of therapies and treatment planning in cancer (see, for example, Krarup et al., Seminars in Nuclear Medicine, Vol. 52; 6 (November 2022)).

[0050] Radiolabeling refers to the process of incorporating a radioactive isotope into a compound (e.g., Fab'2) to make them detectable during imaging and / or therapy. Radioactive isotopes that can be conjugated with the compounds of this invention include copper-64 (…). 64 Cu), Zirconium 89 ( 89 Zr), Fluorine-18 ( 18 F), Gallium-68 ( 68 Ga), iodine 123 / 131 ( 123 I, 131 I), carbon 11 ( 11 C), Scandium-44 ( 44 Sc), Iodine-124 ( 124 I) and yttrium-86 ( 86 Y) and lutetium 177 ( 177 Lu). The radiolabeling is particularly useful for PET imaging. Other radiolabeling methods that can also be used for imaging and therapy include... 3 H, 11 C 14 C 18 F, 32 P, 35 S, 64 Cu、 68 Ga、 86 Y、 89 Zr、 99 Tc, 111 In、 123 I, 124 I, 125 I, 131 I, 133 Xe, 177 Lu、 211 At、 225 Ac、 212 Pb and213 Bi (see, for example, U.S. Patent No. 10,730,944; U.S. Patent No. 8,771,966; PCT Publication No. WO / 2015055318; and PCT Publication No. WO / 2016086021).

[0051] The constructs of the present invention or pharmaceutical compositions comprising them may be administered via parenteral routes, non-limiting examples of which are subcutaneous and intravenous administration. The constructs of the present invention may be administered to subjects in single or multiple doses with pharmaceutically acceptable carriers, diluents, or excipients. Pharmaceutical compositions of the present invention may be prepared by methods well known in the art (e.g., Remington: The Science and Practice of Pharmacy, 22nd ed. (2012), A. Loyd et al., Pharmaceutical Press), and may comprise the constructs disclosed herein, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0052] The anti-STEAP1 construct of the present invention can be administered as part of a treatment plan for a subject with prostate cancer. In one embodiment, the anti-STEAP1 construct is administered together with a therapeutic agent that can be used to treat cancer. The anti-STEAP1 construct may be administered prior to the administration of the therapeutic agent. The anti-STEAP1 construct may be administered after treatment with the therapeutic agent. As used interchangeably herein, “treatment and / or treating and / or treating” is intended to refer to all processes in which there may be a slowing, interruption, prevention, control, cessation, or reversal of the progression of the disorder described herein, but does not necessarily mean the complete elimination of all disorder symptoms. Treatment includes administering a therapeutic agent to treat a disease or condition in a person who will benefit from the activity of the therapeutic agent, and includes: (a) inhibiting further progression of the disease; and (b) alleviating the disease, i.e., causing the disease or disorder to subside or reducing its symptoms or complications.

[0053] “Subject” refers to a person. In one embodiment, the subject has cancer. In one embodiment, the subject has prostate cancer. In one embodiment, the subject has metastatic resistant prostate cancer. In one embodiment, the subject has metastatic castration-sensitive prostate cancer.

[0054] The constructs of the present invention are envisioned to be used to detect STEAP1 in cancer. The constructs of the present invention are envisioned to be used to detect STEAP1 in solid tumors. The constructs of the present invention are envisioned to be used to detect STEAP1 in metastatic sites. In one embodiment, the constructs of the present invention are envisioned to be used to detect STEAP1 in solid tumors with elevated STEAP1 expression. In one embodiment, STEAP1 expression in the tumor is higher than in non-cancer cells. In one embodiment, the cancer is lung cancer. In one embodiment, the cancer is non-small cell lung cancer (adenocarcinoma and squamous cell carcinoma). In one embodiment, the cancer is gastric cancer. In one embodiment, the cancer is esophageal cancer. In one embodiment, the cancer is colorectal cancer. In one embodiment, the cancer is hepatocellular carcinoma. In one embodiment, the cancer is pancreatic ductal adenocarcinoma. In one embodiment, the cancer is prostate cancer. In one embodiment, the cancer is metastatic castration-resistant prostate cancer. In one embodiment, the cancer is metastatic castration-sensitive prostate cancer. In one embodiment, the cancer is Ewing sarcoma.

[0055] Metastatic sites are parts of the body where cancer cells have spread beyond the original tumor site. For example, in prostate cancer, common metastatic sites include bones, lymph nodes, lungs, and liver. Other metastatic sites include the adrenal glands, brain, breast, eyes, kidneys, muscles, pancreas, salivary glands, and spleen.

[0056] It is also envisioned that the constructs of the present invention can detect STEAP1 in other mammalian species, such as cynomolgus monkeys.

[0057] Salullitamine (AMG 509) is an XmAb® 2+1 T-cell conjugate (TCE) molecule designed to direct T effector cells to cells expressing STEAP1. The first human study of salullitamine in patients with metastatic castration-resistant prostate cancer (mCRPC) was designed to evaluate its safety, tolerability, pharmacokinetics (PK), and antitumor activity as a monotherapy or in combination with other therapies administered intravenously or subcutaneously. Preliminary efficacy results observed with salullitamine were numerically superior to those reported for other TCEs in prostate cancer. Salullitamine kills tumor cells expressing high levels of STEAP1.

[0058] The compounds of the present invention can be used as part of a treatment regimen for a cancer therapeutic agent. In one embodiment, the cancer therapeutic agent is a prostate cancer therapeutic agent. In another embodiment, the prostate cancer therapeutic agent is salinomycin. The compounds of the present invention can be used to determine the STEAP1 expression level in the prostate of a subject, thereby providing information on STEAP1 expression before and after treatment with a prostate cancer therapeutic agent (e.g., salinomycin) for prostate cancer.

[0059] The constructs of this invention can be used to detect STEAP1 in conjunction with the administration of cancer therapeutic agents.

[0060] A cancer therapeutic agent is a drug or molecule that can be used to treat cancer. Depending on the type of cancer and other factors such as the stage of the disease, the subject's physician may determine the cancer therapeutic agent to be administered to the subject. In one embodiment, the cancer therapeutic agent is a drug or molecule that binds to STEAP1. Non-limiting examples include antibody-drug conjugates, T-cell conjugate molecules, antibodies, and bispecific antibodies.

[0061] For example, a prostate cancer therapeutic agent refers to a drug or molecule that can be used to treat prostate cancer. In one embodiment, a prostate cancer therapeutic agent is a drug or molecule that binds to STEAP1. In one embodiment, a prostate cancer therapeutic agent is salimethrin. Salulmetamine is described in PCT Publication No. WO / 2020010079. In one embodiment, a prostate cancer therapeutic agent comprises one or more sequences given in any one of SEQ ID NO 11-36. In one embodiment, a prostate cancer therapeutic agent is described in PCT Publication No. WO / 2020010079, which is incorporated herein by reference in its entirety.

[0062] The compounds of this invention can be used as imaging agents for cancers such as prostate cancer. Such imaging agents can also be used as diagnostic biomarkers for cancers such as prostate cancer. These diagnostic biomarkers can detect and / or confirm diseases such as prostate cancer. The diagnostic biomarkers can also identify subjects with increased STEAP1 expression. Subjects with increased STEAP1 expression (compared to STEAP1 expression in non-cancer cells) are considered to have a more favorable response to molecules capable of killing STEAP1-expressing cancer cells (such as salinomycin). The diagnostic biomarkers can also be used to monitor STEAP1 expression during and after treatment with anticancer therapies (such as salinomycin).

[0063] The imaging agent can be a PET tracer, in which a subject is injected with the PET tracer, and the subject is subsequently scanned (PET scan) to detect STEAP1 in the subject. Clinicians can then recommend a treatment plan based at least in part on the subject's STEAP1 expression. Such a treatment plan may include the administration of a prostate cancer treatment agent, such as salinomycin. The compounds of the present invention can also be used during the treatment of a subject. In one embodiment, the use can be used to determine the subject's STEAP1 expression during or after treatment. Therefore, clinicians are able to determine the effectiveness of treatment (e.g., the use of salinomycin) in a subject (e.g., a prostate cancer patient). Thus, based on STEAP1 expression, clinicians can modify or maintain the treatment plan. Example

[0064] Example 1: Anti-STEAP1(FAB')2 (CAR1) cell binding

[0065] To determine the appropriate conjugation of anti-STEAP1 (Fab')2 to DFO, DFO Or NOA chelating agent or use 63 Does the Cu cold-labeling of the anti-STEAP1 (Fab')2 NOTA molecule affect binding affinity in STEAP1-expressing cell lines? The binding affinity of the conjugated or cold-labeled molecule to ECMO (electrode binding affinity) was investigated. 50 Comparison with unconjugated anti-STEAP1 (Fab')2.

[0066] To determine whether it is NOTA (CAR1), DFO (CAR1), or DFO (CAR1) conjugation or cold labeling counteracted the effect of STEAP1(Fab')2 binding to cells expressing STEAP1. C4-2B Luc cells were conjugated with increased concentrations (up to 1 µM) of NOTA-(Fab')2 (CAR1), DFO-(Fab')2 (CAR1), or DFO. -(Fab')2 (CAR1), or cold label 63 Cu-NOTA-(Fab')2 (CAR1), or unconjugated (Fab')2, was incubated at 4°C for 1 hour. After incubation with an anti-human IgG (Fab')2 specific secondary antibody conjugated with AF647, cell-bound antibody was detected by flow cytometry, and the median fluorescence intensity (MFI) of the AF647 channel was displayed at increasing concentrations of the corresponding molecule. Results for the NOTA-(Fab')2 molecule are as follows... Figure 1A and 1B As shown. DFO or DFO The results for the (Fab')2 molecule are as follows: Figure 2A and 2B As shown. Molecular binding EC 50 The values ​​are shown in Table 1 and Table 2.

[0067] Similar experiments were performed in cell lines with various STEAP1 surface expression levels (determined by DAKO Qifikit).

[0068] Table 1. Cell binding to anti-STEAP1 NOTA(CAR1) (Fab')2 molecules as determined by flow cytometry.

[0069]

[0070] Table 2. Cells identified by flow cytometry and their correlation with anti-STEAP1 DFO or DFO (CAR1) (Fab')2 molecules Combine.

[0071]

[0072] These data indicate that in cell lines with variable STEAP1 surface expression, whether it is NOTA (CAR1), DFO (CAR1), or DFO... (CAR1) conjugation or cold labeling maintains binding to anti-STEAP1 (Fab')2 cells.

[0073] Example 2: Anti-STEAP1(FAB')2 (CAR2) cell binding

[0074] To determine the effect of NOA (CAR2) or DFO-(Fab')2 (CAR2) conjugation on the binding of STEAP1 (Fab')2 to cells expressing STEAP1, C4-2B Luc or 22Rv1 Luc cells were incubated at increased concentrations (up to 1 μM) of NOA-(Fab')2 (CAR2) or unconjugated (Fab')2 at 4°C for 1 hour. As described above, cell-bound antibodies were detected by flow cytometry, and the results are shown in Figure 3 (NOTA) and Figure 4 (DFO). Molecule binding EC 50 The values ​​are shown in Tables 3 (NOTA) and 4 (DFO). These data indicate that anti-STEAP1(Fab')2 cell binding is maintained in cell lines after conjugation with either NOA (CAR2) or DFO (CAR2).

[0075] Table 3. Cell binding to anti-STEAP1 NOTA (CAR2) (Fab')2 molecules as determined by flow cytometry.

[0076]

[0077] Table 4. Cell binding to anti-STEAP1 DFO (CAR2) (Fab')2 molecules as determined by flow cytometry.

[0078]

[0079] Example 3: CAR determination

[0080] To determine the optimal chelator:antibody ratio (“CAR”), the radiolabeling efficiency, specific activity, radiochemical purity, yield, serum stability, and retention of immunoreactivity of the radiolabeled molecules anti-STEAP1(Fab')2 CAR2 and anti-STEAP1(Fab')2 CAR1 were evaluated. Retention of immunoreactivity was determined by immunoreactivity fraction (IRF) assay.

[0081] Immunoreactivity score (IRF) measurement

[0082] Immunoreactivity fraction (IRF) assay is a quality control method for measuring the binding (to target) fraction of a compound, which is crucial for meeting release standards for compounds used in preclinical and clinical settings. An overview of the assay is shown in Figure 14. Bead preparation, IRF assay protocol, and data analysis are described below.

[0083] Preparation of STEAP1 antigen-coated beads and control beads To prepare antigen huSTEAP1-tagged coated beads (ACB), huSTEAP1-tags and anti-tag magnetic beads (Thermo Fisher Scientific) were mixed at a ratio of 120 µg huSTEAP1-tag / 200 µL beads in a final volume of 1 mL of ice-cold buffer containing 0.005% TBS-GDN. After constant shaking at 1000 rpm for 1 hour at 4°C, the beads (200 µL) were stored and washed once with 1 mL of 0.005% TBS-GDN-BSA 1%. The beads were then resuspended in 600 µL of 0.005% TBS-GDN-BSA 1% and aliquoted into 80 µL vials. The settled beads (20 µL / vial) were stored at -80°C for long-term use.

[0084] To prepare negative control (uncoated) beads, the anti-tag magnetic beads were washed once with buffer containing 0.005% TBS-GND and 1% BSA. The precipitated beads (200 µL) were then resuspended in 600 µL of 0.005% TBS-GND and 1% BSA and aliquoted into 80 µL vials. The precipitated beads (20 µL / vial) were stored at -80°C for long-term use.

[0085] IRF Measurement Protocol Immediately after the PET tracer synthesis was completed, the immunoreactivity of each radiolabeled test specimen was assessed using STEAP1 antigen-coated beads (ACB). An immunoreactivity score >90% for the radiolabeler indicates that the targeting function of the test specimen remains intact after radiolabeling.

[0086] The test sample used for IRF determination is: 89 Zr-labeled DFO-(Fab')2-CAR1, 89 Zr-labeled DFO-(Fab')2-CAR2, 89 DFO marked with Zr - (Fab')2-CAR1、 64 Cu-labeled NOTA-(Fab')2-CAR1 or 64Cu-labeled NOTA-(Fab')2-CAR2. A negative control (uncoated beads) group is also included. Assay buffer TBS (20 mM Tris-Cl, pH 7.5 + 150 mM NaCl) + 0.005% GDN + 1% BSA was prepared and stored at room temperature. Beads were provided in 20 µL aliquots. The vials were rapidly thawed prior to assay.

[0087] Incubate vials containing beads and radioligands at 4°C for 1 hour on a shaker / vibrator. After incubation, centrifuge the vials at 100 G for 15 seconds at 4°C, then place them on a magnetic rack for 3 minutes. Collect the supernatant into the "Supernatant" vial, wash the beads with 100 µL of assay buffer, and collect the supernatant into the "Wash Buffer" vial. Wash the beads a second time with 100 µL of assay buffer, and collect the supernatant into the same "Wash Buffer" vial. Resuspend the beads in 100 µL of assay buffer and transfer them to a new vial labeled "Binding". Perform gamma counts on the vials: binding; supernatant; wash buffer; reference (3 reference standards); background (3 background vials).

[0088] IRF (%) is calculated as follows: (bound / (bound + supernatant + washing solution)) 100%

[0089] use 89 Zr marks DFO-Fab´2-CAR1 and DFO -(Fab')2-CAR1, labeling efficiency and radiochemical purity >99%. Stability tests showed that both labeled materials were stable in PBS at 4°C for up to 48 hours (maximum duration tested) (radiochemical purity (“RCP”) >98%). Both labeled materials showed a slight decrease in RCP after 48 hours in human and mouse serum at 37°C (approximately 91% RCP for DFO-Fab'2-CAR1, and approximately 91% for DFO-Fab'2-CAR1). -(Fab')2-CAR1 has an IRF of approximately 97%. Both radiotracers meet the IRF release criteria (DFO-Fab'2-CAR1 has an IRF of 93.7%, DFO...). -Fab'2-CAR1 was 96.4% (Table 5).

[0090] Similarly, using 64 Cu-labeled NOA-Fab2-CAR1 conjugates exhibited labeling efficiency and radiochemical purity >99%. The purified labeled material was stable in PBS at 4°C (RCP >99%) and in human and mouse serum at 37°C (RCP >98%) for up to 24 hours (maximum duration tested). This radiotracer meets the IRF release criteria (IRF 95.9%) (Table 5).

[0091] Table 5. Anti-STEAP1 CAR1 NOTA, DFO and DFO (Fab')2 characteristics.

[0092]

[0093] Although higher CAR values ​​may lead to increased specific activity, target specific activity was achieved with radiolabeled molecules of anti-STEAP1 (Fab')2 CAR1, making it unnecessary to increase the number of CARs (Tables 6 and 7).

[0094] Table 6. Anti-STEAP1 89 Comparison of the properties of Zr-DFO-(Fab')2 CAR1 and CAR2.

[0095]

[0096] These data indicate that Zr-89-labeled radiotracers under CAR1 or CAR2 exhibit fairly good radiolabeling efficiency, specific activity, radiochemical purity, yield, and serum stability. Both radiotracers used in the in vivo biodistribution studies met IRF publication criteria.

[0097] Table 7. Anti-STEAP1 64 Comparison of the properties of Cu-NOTA-(Fab')2 CAR1 and CAR2.

[0098]

[0099] These data indicate that under CAR1 or CAR2 64 Cu-labeled radiotracers exhibited quite good radiolabeling efficiency, specific activity, radiochemical purity, and yield. Both radiotracers used in in vivo biodistribution studies met IRF publication criteria.

[0100] Overall, the use of STEAP1-(Fab')2-CAR1 radiolabeled molecules ( 64 Cu or 89 Zr-labeled CARs achieved specific activity against the target, allowing for in vitro assessments without the need to increase the number of CARs.

[0101] Example 4: Adhesive (FAB')2 binding affinity

[0102] To evaluate the conjugation of anti-STEAP1 (Fab')2 to 64 Cu or 89 Does Zr affect the binding affinity with STEAP1? The KD binding affinity of unconjugated anti-STEAP1 (Fab')2 was determined and compared with that of DFO under CAR1 or CAR2 conditions. The affinity of the NOA-conjugated strain for STEAP1 (Fab')2 was compared. Additionally, the affinity of NOA-CAR1 was measured.63 Cu (cold-labeled). All KD binding affinities were measured in a Sartorius Octet HTX instrument.

[0103] Binding affinity was analyzed using a Sartorius Octet HTX instrument (Sartorius, Inc., Bohemia, NY #18-5119) equipped with a streptavidin (SAX) biosensor. All Octet binding assays, baselines, and dilutions were performed using Octet assay buffer (10 mM TRIS pH 7.5, 150 mM NaCl, 1 mM CaCl2, 0.08% GDN, 0.10 mg / mL BSA). Recombinant human STEAP1-tag (GDN) protein was generated as described in Nolan-Stevaux et al., CancerDiscov (October 2023).

[0104] (Fab')2 molecules were immobilized using an anti-human IgG (Fab')2 goat polyclonal antibody (Jackson ImmunoResearch, West Grove, PA, #109-005-006), which was biotinylated to 2.4 biotin / molecule using Calbiochem Innolink Biotin 354S (#203119) according to kit instructions. Black flat-bottomed 384-well plates or black tilted-bottom plates were used for the method steps. The acquisition rate was set to "High Sensitivity Kinetics" in 96-point tip mode. Each interaction tested was generated by a column of eight biosensors; each (Fab')2 molecule was immobilized onto the eight biosensors in the column to a fixed level, and the first six biosensors were incubated with a soluble human STEAP1 dilution series, while the remaining two biosensors were incubated with assay buffer and used for reference subtraction. Testing 7 (Fab')2 molecules and buffer blanks corresponds to immersing 8 biosensors in 8 columns or 64 SAX biosensors in various wells in a single experiment.

[0105] Octet binding assay was performed using a seven-step method: (1) assay buffer, 60 seconds; (2) 5 nM biotinylated anti-human IgG (Fab')2 goat polyclonal antibody (2.4 biotin / molecule) for 2000 seconds to reach a loading level of 3.8 nm; (3) assay buffer, 60 seconds; (4) 10 nM anti-STEAP1 (Fab')2 molecule or negative control antibody (one column of 8 SAX biosensors for each (FAB')2 molecule or buffer blank control); the final (Fab')2 loading level was 1.1 – 1.2 nm; (5) assay buffer, 60 seconds; (6) association step: 8 biosensors per column: human STEAP1 (1-339)-tag (GDN) 100 nM, 33.3 nM, 11.1 nM, 3.70 nM, 1.23 nM, 0.41 (7) Dissociation step: Measure buffer, 5400 seconds.

[0106] Raw data were processed using Genedata Screener v19 (using the SPR affinity package) and standard Octet data processing: for each biosensor column, the signals from two reference biosensors were averaged and subtracted from the remaining six biosensors. The Y-axis was aligned with the previous baseline, and inter-step corrections were performed using a dissociation step; Savitzky-Golay filtering was used to reduce high-frequency noise, and the association and dissociation steps of each interaction pair were separated into sensor maps. The processed data for each group were then globally fitted using a 1:1 combination model to obtain measurements of the association rate constant (ka) and dissociation rate constant (kd). The KD affinity was then calculated as the ratio of the rate constant kd / ka.

[0107] In this assay, unconjugated anti-STEAP1 (Fab')2 bound to human STEAP1 with a KD affinity of 2.6 nM. All conjugated anti-STEAP1 (Fab')2 molecules bound to human STEAP1 with a KD affinity less than twice that of the unconjugated form, indicating that conjugation does not significantly affect the binding affinity of anti-STEAP1 (Fab')2 to the human STEAP1 receptor (Table 8). Furthermore, the sensor plots depicting the binding kinetics and the 1:1 model fits to these interactions were indistinguishable between unconjugated and conjugated (Fab')2 molecules, further demonstrating the presence of NOA, DFO, and DFO in CAR1 and CAR2 conjugations. It has no significant effect on the binding kinetics (Figure 5).

[0108] Table 8. Dynamics of the measured association rate constant (ka), dissociation rate constant (kd), and calculated equilibrium dissociation constant (KD) Mechanical and affinity values.

[0109]

[0110] Example 5: Pharmacokinetics and In Vivo Stability

[0111] The aim of this study was to characterize the pharmacokinetics of the STEAP1 compound and assess its in vivo stability following a single intravenous bolus administration (5 mL / kg of unlabeled CAR2 (Fab')2 molecules at a dose of 2 mg / mL, or 10 mg / kg) in female BALB / c nude mice (n=9 mice / group). Three unlabeled CAR2 molecules were included in this study: unconjugated CAR2 (Fab')2 (“unconjugated”), (Fab')2 CAR2 with DFO conjugation (“DFO”), and (Fab')2 CAR2 with NOA conjugation (“NOTA”). The unconjugated compound was administered in 10 mM NaOAc; 9% sucrose; pH 5.2 (A52Su). The DFO compound was administered in 0.1 M NaOAc, pH 5.9. The NOA compound was administered in 0.1 M NH4OAc, pH 6.0.

[0112] Serum samples were collected at 5 minutes, 1, 2, 4, 8, 24, 48, 72, 96, and 120 hours post-drug administration. Animals were arranged in groups of three, staggered. All plasma samples were stored at approximately -70°C (±10°C) until transferred for subsequent analysis.

[0113] Quantification of (Fab')2 CAR2 molecules in mouse serum was performed using an electrochemiluminescence immunoassay assay, employing a biotinylated anti-idiotype mAb interacting with the anti-STEAP1 binding domain as a capture reagent and a mouse anti-human IgG Fab assay reagent. Analyte concentrations were interpolated from the standard curve using the target protein, and data analysis was performed in a non-GLP Watson L assay.

[0114] Analysis was performed using Phoenix Winonlin. Data on serum drug concentrations versus relative nominal time after administration were used for all pharmacokinetic analyses. Mean (and standard deviation) curves are shown below. Figure 6 As shown. Non-compartmental analysis was performed. AUC values ​​were calculated using linear logarithmic-linear trapezoidal interpolation and used to estimate clearance and distribution volume, as shown in Table 9. Terminal half-life is also reported in Table 9. Pharmacokinetic curves and pharmacokinetic parameters show the different exposures of each of the three molecules evaluated in this study.

[0115] Table 9. Total pharmacokinetic parameters after IV administration of (Fab')2 CAR2 10 mg / kg to female BALB / c mice Knot.

[0116]

[0117] Example 6: Pharmacokinetic Analysis of Crab-Eating Mammals

[0118] The aim of this study was to determine the pharmacokinetics of the Fab'2 NOTA CAR1 conjugate when administered intravenously to cynomolgus monkeys. This study included three experimentally unexperimented male cynomolgus monkeys from Asia (2.7 to 2.9 years old), weighing between 1.8 and 2.0 kg at administration. All monkeys received a single intravenous bolus of 0.75 mg / mL unlabeled Fab'2 NOTA CAR1 conjugate at a dose of 1.125 mg / kg in 0.1 M NH4OAc buffer (pH 5.9). Serum samples were collected at 5 minutes, 1, 2, 4, 8, 12, 16, 24, 48, 72, 96, 120, and 168 hours post-administration.

[0119] Quantification of Fab'2 NOTA CAR1 conjugates in cynomolgus monkey serum was performed using an electrochemiluminescence immunoassay assay, employing a biotinylated anti-idiotype mAb interacting with the anti-STEAP1 binding domain as a capture reagent and a mouse anti-human IgG Fab assay reagent. Analyte concentrations were interpolated from the standard curve using the target protein, and data analysis was performed in a non-GLP Watson LIMS.

[0120] Further analysis was performed. Data on serum drug concentrations versus relative nominal time after administration were used for all pharmacokinetic analyses. Non-compartmental analysis was performed for each individual animal. AUC values ​​were calculated using linear logarithmic-linear trapezoidal interpolation and used to estimate clearance and distribution volume, as shown in Table 10. Terminal half-life is also reported in Table 9. Mean (and standard deviation) curves are shown below. Figure 7 As shown.

[0121] Table 10. Mean pharmacokinetics of 1.125 mg / kg Fab'2 NOTA CAR1 conjugate administered to cynomolgus monkeys parameter.

[0122]

[0123] Example 7. In vivo imaging

[0124] To determine the efficacy in tumor-bearing mice 64 Cu-labeled anti-STEAP1 Fab'2 and 89 The uptake of Zr-labeled anti-STEAP1 Fab'2 molecules was assessed, and the biodistribution of each molecule was evaluated in mice with STEAP1-high (STEAP1-overexpressing 22Rv1 Luc cells) and STEAP1-negative (STEAP1-knockout 22Rv1 Luc cells) tumors.

[0125] Male Balb / c nude mice were subcutaneously inoculated with 5 x 10 spores in the right shoulder.6 A high number of STEAP1-positive or STEAP1-negative cells. When the tumor size reaches 150–400 mm. 3 At that time, the mice were divided into STEAP1 high or STEAP1 negative groups, with 4 mice in each group.

[0126] Includes five types of constructs: 64 Cu-NOTA-Fab'2-CAR1 64 Cu-NOTA-Fab'2-CAR2 89 Zr-DFO-Fab'2-CAR1 89 Zr-DFO-Fab'2-CAR2 and 89 Zr-DFO -Fab'2-CAR1.

[0127] Each construct was labeled on the day of injection, and all products met the release criteria for radiochemical purity (≥90%).

[0128] On the day of injection, one of the constructs was intravenously injected into each mouse at approximately 200 uCi. All groups were imaged by PET / CT in a 4-bed mouse chamber. 89 Zr-labeled imaging agents were used for 30-minute static acquisitions at 2, 22–24, 46, and 74 h, and for… 64 Cu-labeled imaging agents were acquired at 2, 22–24 h, and 46 h. Following each PET acquisition, CT was performed for anatomical co-registration and attenuation and scattering correction. Blood samples were collected for gamma counting after the 22–24 h imaging timepoint. Immediately after the last imaging timepoint, all animals in the study were humanely euthanized, and tissues were collected for ex vivo radiometric analysis via gamma counting.

[0129] Tumor uptake of each imaging agent was determined by calculating the percentage (%ID / g) of the injected dose per gram in the xenograft tumor at the indicated time points post-injection. Specific tumor uptake was determined by comparing the %ID / g values ​​in STEAP1-high and STEAP1-negative xenografts.

[0130] Compare 64 Cu-NOTA-Fab'2-CAR1 and 64 Tumor uptake of Cu-NOTA-Fab'2-CAR2 in STEAP1-high (Fig. 8) and STEAP1-negative (Fig. 9) xenografts. 64 Cu-NOTA-Fab'2-CAR1 and 64Cu-NOTA-Fab'2-CAR2 was successfully localized to STEAP1-positive xenograft tumors with considerable tumor uptake (mean %ID / g = 11.9 for CAR1 and 11.8 for CAR2). Very low tumor uptake was observed in STEAP1-negative xenografts (mean %ID / g = 3.5 for CAR1 and 3.3 for CAR2), consistent with the specific tumor uptake observed in STEAP1-positive tumors.

[0131] Compare 89 Zr-DFO-Fab'2-CAR1 and 89 Tumor uptake of Zr-DFO-Fab'2-CAR2 in STEAP1-high (Fig. 10) and STEAP1-negative (Fig. 11) xenografts. Although both molecules are localized in STEAP1-high xenografts, tumor uptake is low.

[0132] Comparison of STEAP1 levels with STEAP1-negative xenografts 89 Zr-DFO -Fab'2 CAR1 tumor uptake. 89 Zr-DFO -Fab'2 CAR1 was successfully localized to tumors with high STEAP1 (%ID / g = 7.9 for high STEAP1 and 2.7 for STEAP1 negative).

[0133] In short, 64 Cu-NOTA-Fab'2-CAR1 and CAR2 successfully enabled STEAP1 to be highly effective in tumor imaging, with almost identical tumor uptake of the two molecules. 89 Zr-labeled Fab'2 derivatives DFO-Fab'2 and DFO -Fab'2 successfully enabled STEAP1+ tumor imaging; however, tumor uptake was significantly lower than expected. 64 Cu-NOTA-Fab'2. DFO -Fab'2 is superior to DFO-Fab'2. Quantitative results are as follows: Figure 13 As shown.

[0134] In addition to tumor uptake, all five imaging agents showed rapid clearance from the blood, observed from low concentrations in the blood at 22 h in ex vivo data. For example, in the STEAP1 high group, blood... 64 Cu-NOTA-Fab'2 has 1.32 ± 0.04% ID / g, while 89Zr-DFO-Fab'2 has 0.48 ± 0.13% ID / g. 89 Zr-DFO -Fab'2 is 1.1 ± 0.04% ID / g.

[0135] Among the organs cleared, the kidneys showed the highest uptake of the imaging agent across all groups. The highest renal uptake occurred during drug administration. 89 The Zr-labeled imaging agent group (18.5%–65.4% ID / g at 74 h) showed the lowest uptake at the time of administration. 64 Cu-labeled imaging agent group (17.1%–21.4% ID / g at 46 h). Additionally, during administration... 64 In the Cu-labeled imaging agent group, hepatic uptake was high (12.8%–15.1% ID / g at 46 h), while after administration... 89 The levels were relatively low in the Zr-labeled imaging agents (1.8%–3.9% ID / g at 74 h).

[0136] Example 8. Molecular Engineering

[0137] The STEAP1 (Fab')2 molecule was engineered using various conjugations and radiolabeling techniques, and its activity was evaluated. Engineered forms included STEAP1 (Fab')2 conjugated to NOA. 64 Cu, and STEAP1 (Fab')2 via DFO or DFO Join to 89 Zr. Conjugation was performed via random lysine conjugation. To recombinantly generate STEAP1 (Fab')2, a four-amino acid peptide PCPP was inserted into the HC of huSTEAP1_21019 (VH:N67Q) after HC P230 (EU number). Therefore, the hinge region of STEAP1 (Fab')2 is EPKSCDKTHTCPPCPPCPP.

[0138] Synthesis of STEAP1 (Fab')2-NOTA CAR1 conjugate :

[0139] The solution buffer of STEAP1 (Fab')2 (12 mg, 14.759 mg / mL, in 10 mM NaOAc, 9% sucrose, pH 5.2) was replaced with HEPES buffer (0.1 M HEPES, pH 8.5) using an Amicon Ultra-15 centrifuge filter unit (30 K MWCO). A stock solution of NOTA-NHS in DMSO (15 mM, 5 eq, 0.041 mL, from CheMatech, c 100) was added to the resulting solution (6 mL), and the reaction mixture was heated to 37 °C with stirring for 90 min. More NOTA-NHS solution (2 eq, 0.016 mL) was added, and the mixture was heated to 37 °C with stirring for another 90 min. The mixture was cooled to room temperature and buffer-exchanged to 0.1 M NH4OAc (pH 5.9) using an Amicon Ultra-15 centrifuge filter unit (30K MWCO) to obtain a Nota conjugate solution (4 mL, 2.9 mg / mL). The product was analyzed on an Agilent 6230 LC / TOF, and the Nota:(Fab')2 ratio was calculated to be 1.13 based on a triplicate MS dataset. Data were analyzed using PMI software.

[0140] Synthesis of STEAP1 (Fab')2-NOTA CAR2 conjugate :

[0141] The solution buffer of STEAP1 (Fab')2 (6 mg, 14.759 mg / mL, in 10 mM NaOAc, 9% sucrose, pH 5.2) was exchanged for HEPES buffer (0.1 M HEPES, pH 8.5) using an Amicon Ultra-15 centrifuge filter unit (30 K MWCO). A stock solution of NOTA-NHS in DMSO (10 mM, 3 eq, 0.018 mL, from CheMatech, c 100) was added to the resulting solution (6 mL), and the reaction mixture was heated to 37 °C with stirring for 90 min. More NOTA-NHS solution (3 eq, 0.018 mL) was added, and the mixture was heated to 37 °C with stirring for 1 h. More NOTA-NHS solution (3 eq, 0.018 mL) was added, and the mixture was heated to 37 °C with stirring for 1 h, then cooled to 4 °C overnight. The mixture buffer was exchanged to 0.1 M NH4OAc (pH 6) using an Amicon Ultra-15 centrifugal filter unit (30 K MWCO). The reaction was repeated with 6 mg STEAP1 (Fab')2, and the products were combined to obtain a Nota conjugate solution (4.2 mL, 2.9 mg / mL). The product was analyzed on an Agilent 6230 LC / TOF, and the Nota:(Fab')2 ratio was calculated to be 2.36 based on a triplicate MS dataset. Data were analyzed using PMI software.

[0142] Synthesis of STEAP1 (Fab')2-DFO CAR1 conjugate :

[0143] The solution buffer of STEAP1 (Fab')2 (8 mg, 14.759 mg / mL, in 10 mM NaOAc, 9% sucrose, pH 5.2) was replaced with HEPES buffer (0.1 M HEPES, pH 8.5) using an Amicon Ultra-15 centrifuge filter unit (30K MWCO). A stock solution of p-SCN-Bn-DFO in DMSO (6.67 mM, 3 eq, 0.038 mL, from Macrocyclics, B-705) was added to the resulting solution (5.5 mL), and the reaction mixture was heated to 37°C with stirring for 1 hour. More p-SCN-Bn-DFO in DMSO (3 eq, 0.038 mL) was added, and the mixture was heated to 37°C with stirring for another 90 minutes. The mixture was cooled to room temperature for 2 hours and then buffer-exchanged to 0.1 M NaOAc (pH 5.9) using an Amicon Ultra-15 centrifuge filter unit (30K MWCO) to obtain a DFO conjugate solution (2.5 mL, 2.7 mg / mL). The product was analyzed on an Agilent 6230 LC / TOF, and the DFO:(Fab')2 ratio was calculated to be 1.2 based on a triplicate MS dataset. Data were analyzed using PMI software.

[0144] Synthesis of STEAP1 (Fab')2-DFO CAR2 conjugate :

[0145] The solution buffer of STEAP1 (Fab')2 (6 mg, 14.759 mg / mL, in 10 mM NaOAc, 9% sucrose, pH 5.2) was replaced with HEPES buffer (0.1 M HEPES, pH 8.5) using an Amicon Ultra-15 centrifuge filter unit (30K MWCO). A stock solution of p-SCN-Bn-DFO in DMSO (8 mM, 3 eq, 0.023 mL, from Macrocyclics, B-705) was added to the resulting solution (12 mL), and the reaction mixture was heated to 37°C with stirring for 90 min. More p-SCN-Bn-DFO in DMSO (3 eq, 0.046 mL) was added hourly, and the mixture was heated to 37°C with stirring for another 4 h. The mixture was cooled to 4°C overnight and buffer-exchanged to 0.1 M NaOAc (pH 5.9) using an Amicon Ultra-15 centrifugal filter unit (30K MWCO). The reaction was repeated with 12 mg STEAP1 (Fab')2, and the products were combined to obtain a DFO conjugate solution (4.2 mL, 5.5 mg / mL). The product was analyzed on an Agilent 6230 LC / TOF, and the DFO:(Fab')2 ratio was calculated to be 2.63 based on a triplicate MS dataset. Data were analyzed using PMI software.

[0146] STEAP1 (Fab')2-DFO Synthesis of conjugates

[0147] The solution buffer of STEAP1 (Fab')2 (8 mg, 14.759 mg / mL, in 10 mM NaOAc, 9% sucrose, pH 5.2) was exchanged for HEPES buffer (0.1 M HEPES, pH 8.5) using an Amicon Ultra-15 centrifuge filter unit (30K MWCO). DFO was added to the resulting solution (5.5 mL). - A stock solution of NCS in DMSO (8 mM, 3 eq, 0.032 mL, from Abcr, AB573544) was added, and the reaction mixture was heated to 37°C with stirring for 1 hour. More DFO was added to the DMSO. -NCS (3 eq, 0.032 mL), and heat the mixture to 37°C with stirring for 90 minutes. Add more DFO to the DMSO every hour. -NCS (1 eq, 0.010 mL), over 4 hours. The mixture buffer was exchanged for 0.1 M NaOAc (pH 5.9) using an Amicon Ultra-15 centrifuge filter unit (30K MWCO) to obtain DFO. The conjugate solution (2.5 mL, 2.6 mg / mL) was analyzed on an Agilent 6230 LC / TOF instrument, and the DFO was calculated based on a triplicate MS dataset. The (Fab')2 ratio is 1.03. Data was analyzed using PMI software.

[0148] Example 10: Cell Binding

[0149] C4-2B Luc cells (0.25 x 10⁶ / 100 μL) were incubated at 4°C for 1 h with increasing concentrations (up to 1 µM) of anti-STEAP1 mAb, anti-STEAP1 (Fab')2, or anti-STEAP1 Fab. Cell-bound antibody was detected by flow cytometry after incubation at 4°C for 30 min with a secondary antibody conjugated to 5 μg / mL AF647, and the median fluorescence intensity (MFI) of the AF647 channel at increasing concentrations of the corresponding molecule is shown (top). The MFI (AF647) from 300 nM primary antibody was plotted in a histogram and compared only with the secondary antibody.

[0150] The results showed that the EC50 value of mAb was 15.68 pM, that of (Fab')2 was 4.322 pM, and that of Fab was 207,907 pM. These data indicate that Fab cannot bind STEAP1, while mAb and (Fab')2 can bind STEAP1.

[0151] Example 11: Site-specific conjugation

[0152] To prepare the construct via site-specific conjugation, a cysteine ​​mutation was performed on LC to enable site-specific conjugation to engineer anti-STEAP1 (Fab')2.

[0153] Synthesis of STEAP1 (Fab')2 D88C-NOTA CAR2 conjugate:

[0154] Add 1 mM cysteamine and 2.5 mM cysteamine in 40 mM HEPES buffer (pH 8.5) to a STEAP1 (Fab')2 D88C solution (20 mg in 10 mM NaOAc, 9% sucrose, pH 5.2). Incubate the mixture at room temperature for 1 hour and dilute the solution with 100 mM NaOAc buffer (pH 5.0). Purify the mixture by cation exchange chromatography and collect the fraction containing the product. Use an Amicon Ultra-15 centrifuge filter unit (30K MWCO) to exchange the concentrate buffer for 10 mM NaOAc, 9% sucrose, pH 5.2 buffer to obtain 1.5 mL of cysteamine-capped Fab'2 (10.6 mg / mL).

[0155] Add TPPTS aqueous solution (3.85 mM, 4 eq, 0.049 mL) to a solution of bis(cysteamine)-terminated Fab'2 (10 mM NaOAc, 9% sucrose, pH 5.2, 4.5 mg) and incubate the mixture at room temperature for 1 hour. Then, exchange the reaction mixture buffer for PBS by rotational concentration using an Amicon Ultra-15 centrifuge filter unit (30 K MWCO). Add DHAA aqueous solution (4 mM, 10 eq., 0.112 mL) to the resulting solution and incubate the mixture at room temperature for 2 hours. Then, evenly distribute the mixture into two vials. After DHAA oxidation, add a stock solution of maleimide-monoamide-NOTA in DMSO (10 mM, 4 eq, 0.008 mL, from Macrocyclics, B-622) to one of the vials containing the solution. Then, stir the resulting mixture at room temperature for 1 hour. The reaction mixture buffer was replaced with 0.1 M NaOAc (pH 5.9) buffer to obtain the Fab'2 D88C-NOTA CAR2 conjugate (0.8 mL, 2.3 mg / mL).

[0156] Synthesis of STEAP1 (Fab')2 D88C-DFO CAR2 conjugate:

[0157] Following DHAA oxidation, a stock solution of DFO-maleimide in DMSO (10 mM, 4 equivalents, 0.008 mL, from Macrocyclics, B-772) was added to one of the vials containing the Fab'2 solution, and the mixture was kept at room temperature with stirring for 1 hour. The reaction mixture was then exchanged for 0.1 M NaOAc (pH 5.9) buffer to give the Fab'2D88C-DFO CAR2 conjugate (0.8 mL, 2.36 mg / mL).

[0158] Nota and DFO conjugates were prepared and tested in a binding assay to determine whether the conjugation affected cell binding. The binding assay evaluated the binding of the constructs to C4-2B luc cells (STEAP1 high-cells) and 22Rv1 luc cells (STEAP1 low-cells).

[0159] These data indicate that the conjugation of Fab'2 with NOA or DFO does not affect its binding to cells.

[0160] Table 11. Cell binding assays of constructs engineered via site-specific conjugation.

[0161] sequence

[0162]

[0163]

[0164] .

Claims

1. A STEAP1-resistant construct, wherein the STEAP1-resistant construct is used 64 Cu is radioactively labeled.

2. The anti-STEAP1 construct as claimed in claim 1, wherein the anti-STEAP1 construct is a (Fab')2 fragment.

3. The anti-STEAP1 construct as claimed in claim 1 or claim 2, wherein the anti-STEAP1 construct further comprises at least one NOA.

4. The anti-STEAP1 construct as described in claim 3, wherein the construct contains a NOTA.

5. The anti-STEAP1 construct as described in claim 3, wherein the construct comprises two Nota.

6. The anti-STEAP1 construct as described in any one of claims 3-5, wherein at least one Nota contains 64 Cu.

7. The anti-STEAP1 construct according to any one of claims 1-6, wherein the anti-STEAP1 construct comprises HCDR1 containing SEQ ID NO: 1, HCDR2 containing SEQ ID NO: 2, HCDR3 containing SEQ ID NO: 3, LCDR1 containing SEQ ID NO: 4, LCDR2 containing SEQ ID NO: 5, and LCDR3 containing SEQ ID NO:

6.

8. The anti-STEAP1 construct according to any one of claims 1-7, wherein the anti-STEAP1 construct comprises HCVR containing SEQ ID NO: 7 and LCVR containing SEQ ID NO:

8.

9. The anti-STEAP1 construct according to any one of claims 1-8, wherein the anti-STEAP1 construct comprises HC containing SEQ ID NO: 9 and LC containing SEQ ID NO:

10.

10. An anti-STEAP1 construct, wherein the anti-STEAP1 construct is used 64 Cu is radiolabeled, and the anti-STEAP1 construct is an antibody containing HC with SEQ ID NO: 37 and LC with SEQ ID NO:

10.

11. A method for detecting STEAP1 in a subject, wherein the method comprises administering to the subject an anti-STEAP1 construct as described in any one of claims 1-10.

12. The method of claim 11, further comprising detecting STEAP1 by positron emission tomography.

13. The method of claim 11 or 12, wherein STEAP1 is detected in the prostate of the subject.

14. The method of claim 11 or 12, wherein STEAP1 is detected at the transfer site.

15. The method of any one of claims 11-14, wherein the subject is further administered a cancer treatment agent.

16. The method of claim 15, wherein the cancer therapeutic agent is salinomycin.

17. The anti-STEAP1 construct according to any one of claims 1-10, for use in detecting STEAP1 in a subject.

18. Use of the anti-STEAP1 construct as described in any one of claims 1-10 for the manufacture of an imaging agent for the treatment of cancer.

19. The use as described in claim 17 or 18, wherein the subject has cancer.

20. The use as described in claim 17 or 18, wherein the subject has prostate cancer.

21. The anti-STEAP1 construct as described in any one of claims 1-10, for use as a diagnostic biomarker.

22. A diagnostic biomarker comprising an anti-STEAP1 construct as described in any one of claims 1-10.

23. A kit comprising the anti-STEAP1 construct as described in any one of claims 1-10.

24. A method of treating a subject diagnosed with cancer, wherein the method comprises administering to the subject an anti-STEAP1 construct as described in any one of claims 1-10, imaging STEAP1 expression in the subject, and administering a cancer therapeutic agent to the subject.

25. The method of claim 24, wherein the subject has elevated STEAP1 expression in the subject's cancer.

26. The method of claim 24 or 25, wherein the cancer therapeutic agent is salinomycin.

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