Conjugates and use of same for theranostics

Abstract

The present invention is directed to a conjugate including: (i) an antigen-binding polypeptide having a specific binding affinity to prostate specific membrane antigen (PSMA); and (ii) a benzylguanidine prosthetic group including a radionuclide, wherein (i) and (ii) are covalently bound. Further provided are a composition including the conjugate, and use of same, such as in a theranostic method.

Description

CONJUGATES AND USE OF SAME FOR THERANOSTICSREFERENCE TO AN ELECTRONIC SEQUENCE EISTING

[0001] The contents ofthe electronic sequence listing (BGU-DUKE-P-0141-PCT.xml; size: 6,027 bytes; and date of creation: September 25, 2024) is herein incorporated by reference in its entirety.FIELD OF INVENTION

[0002] The present invention is in the field of inter alia radiolabeled molecules.BACKGROUND

[0003] Prostate-specific membrane antigen (PSMA) is an attractive target for the imaging and therapy of prostate cancers with its levels linked to disease progression and transition to metastatic castration-resistant prostate cancer (mCRPC). For decades, researchers have explored various strategies to target PSMA ranging from small molecule inhibitors to antibodies. Regarding small molecule PSMA inhibitors, [,77Lu]PSMA-617 (Pluvicto) was approved by FDA in 202.2 while the proteins targeting PSMA developed to date have been intact monoclonal antibodies (mAbs). These mAbs have prolonged serum half-lives, leading to increased risk of undesired side effects. The large size of mAbs also hinders tumor penetration reducing drug delivery efficiency. On the contrary, rapid blood clearance of small molecule inhibitors usually necessitates multi-dose regimens that can potentially lead to increased toxicity in patients.

[0004] With the objective of reducing side effects while still delivering effective treatment, there is a growing interest in single domain antibody fragments (sdAbs), also known as nanobodies or VHH. Camelie-derived sdAbs are fragments of heavy chain-only antibodies with notable characteristics including high affinity, moderate size (12-15 kDa), and better tumor penetration making them an excellent platform for cancer imaging and therapy. There are a few reports describing radiolabeled PSMA -targeted sdAbs providing proof of concept but not w ith properties likely to merit clinical translation. Believing that a PSMA-targeted sdAb with more favorable characteristics is needed, the inventors recently developed four high affinity PSMA-targeting sdAbs that bind to an epitope on PSMA different from that recognized by small molecule inhibitors . Of these, NB7 exhibited the best combination of high PSMA affinity and tumor cell internalization.

[0005] Regarding the choice of radionuclide for PSMA targeted radiotherapy, the current approach has been to utilize211At, which emits a single alpha particle per decay. This allows simplification of dosimetry calculations and minimizing the risks of off-target side effects from daughterradionuclides that confound alternate alpha-emitters, notably225Ac. Moreover, astatine belongs to the same chemical group as iodine, making it possible to adapt radioi odi nation chemistry and use radioiodinated analogues to facilitate the development of211At-iabeled therapeutics. Previously, the inventors reported promising results for several211At-labeled small molecule PSMA inhibitors including tumor growth suppression. Likewise, three211At-labeled analogues - [211At]PSMAl, [211At]PSMA5 and [211At]PSMA6 based on the structure of PSMA-1007 have recently been developed. Although results have been encouraging in some cases, none of these compounds have advanced to clinical trial.

[0006] Thus, there is still a great need for a theranostically applicable radiolabeled conjugate of sdAb-radionuciide capable of specifically targeting PSMA with properties appropriate for clinical translation.SUMMARY

[0007] The current invention, in some embodiments, relies on the examination of two versions of NB7 sdAb - without or with His6-tag (termed “NB7H6”) - conjugated to four guanidino- substituted prosthetic agents: A-succinimidyl 4-guanidinometliyl 3-[*I]iodobenzoate ([125'131I]SGMIB; 1,3,4 isomer), A-succinimidyl 3-guanidinomethyl 5-[*I]iodobenzoate (iso- [125,131T]SGMIB; 1 ,3,5 isomer), A-succinimidyl 3-[211At]astato-4-guanidinomethyI benzoate ([211At]SAGMB) and A-succinimidyl 3-[211At]astato-5-guanidinomethyl benzoate (iso- [211At]SAGMB), In vitro and in vivo behavior of these NB7 sdAb radio-conjugates was assessed in PSMA+PC3 PIP and PSMA- PC3 flu cells and PSMA+PC3 PIP xenograft models.

[0008] The present invention, in some embodiments, is based, at least in part, on the findings showing that NB7 radio-conjugates exhibited good accumulation in PSMA-positive xenografts with rapid clearance from kidney and other normal tissues. Accordingly, the inventors conclude that NB7 is a potentially useful scaftbkl for developing PSMA-targeted theranostics with different characteristics than current small molecule and antibody-based approaches.

[0009] According to the first aspect, there is provided a conjugate comprising: (i) an antigenbinding polypeptide having a specific binding affinity to prostate specific membrane antigen (PSMA); and (ii) a benzylguanidine prosthetic group comprising a radionuclide, wherein the (i) and (ii) are covalently bound.

[0010] According to another aspect, there is provided a composition comprising a theranostically effective amount of the conjugate of the invention, and a theranostically acceptable carrier.

[0011] According to another aspect, there is provided a combination comprising the conjugate of the invention and at least one agent having specific binding affinity to a catalytic site of the PSMA, for use in diagnosis, treatment, or both, of a PSMA-associated disease or disorder, in a subject in need thereof.

[0012] According to another aspect, there is provided a method for imaging PSMA in a biological sample, the method comprising contacting a biological sample comprising PSMA with an effective amount of the conjugate of the invention or the composition of the invention, and detecting the presence of the PSMA in the biological, thereby imaging PSMA in the biological sample.

[0013] According to another aspect, there is provided a method for detecting PSMA in a subject in need thereof, the method comprising administering to the subject an effective amount of the conjugate of the invention or the composition of the invention, and detecting the presence of the PSMA in the subject.

[0014] According to another aspect, there is provided a method for preparing the conjugate of the invention, the method comprising contacting the antigen-binding polypeptide having a specific binding affinity to PSMA with an effective amount of a benzylguanidine prosthetic group precursor comprising a radionuclide under conditions suitable for formation of a covalent bond between the antigen-binding polypeptide having a specific binding affinity to PSMA and the guanidino- substituted prosthetic group precursor comprising radionuclide, thereby preparing the conjugate.

[0015] In some embodiments, the radionuclide comprises any one of: an alpha emitter, a beta emitter, a gamma emitter, and any combination thereof.

[0016] In some embodiments, the radionuclide comprises any one of: Iodine (I), Astatine (At), and both.

[0017] In some embodiments, I comprises any one of:125I,131I,123I,124I and any combination thereof.

[0018] In some embodiments, At is21'At.

[0019] In some embodiments, the benzylguanidine prosthetic group comprising the radionuclide is selected from the group consisting of: 4-guanidinomethyl 3-[*I]iodobenzoate, 3-guanidinomethyl 5-[*I]iodobenzoate, 3-[211At]astato-4-guanidinomethyl benzoate, 3-[211At]astato-5- guanidinomethyl benzoate, and any combination thereof, wherein *1 is selected from the group consisting of:125I,131I,1231,124I, and any combination thereof.

[0020] In some embodiments, the covalently bound is via an amide bond or via succinimidethioether.

[0021] In some embodiments, the antigen-binding polypeptide is characterized by binding constant (Ka) of at least 104Molar"1sec"1to the PSMA.

[0022] In some embodiments, the antigen-binding polypeptide is characterized by dissociation constant (KD) of 10 to 100 pM to the PSMA.

[0023] In some embodiments, the PSMA comprises the amino acid sequence set forth in SEQ ID NO: 5.

[0024] In some embodiments, the specific binding affinity is to a non-catalytic site of the PSMA.

[0025] In some embodiments, the antigen-binding polypeptide is characterized by molecular weight of less than 25 kDa.

[0026] In some embodiments, the antigen-binding polypeptide is a single-domain antibody (sdAb).

[0027] In some embodiments, the antigen-binding polypeptide comprises the three heavy chain complementary-determining regions (CDRs-H) comprising: CDR-H1 comprising the amino acid sequence GYTDSNYYMS (SEQ ID NO: 1), CDR-H2 comprising the amino acid sequence GVNTGRGSTSYADSVKG (SEQ ID NO: 2), and CDR-H3 comprising the amino acid sequence AACHFCDSLPKTQDEYIL (SEQ ID NO: 3).

[0028] In some embodiments, the antigen-binding polypeptide comprises the amino acid sequence: QVQLQESGGGSVQAGGSLRLSCTAPGYTDSNYYMSWFRQAPGKEREWVAGVNTGRGS TSYADSVKGRFTISQDNAKNTMFLQMNSLKPEDTAIYYCAV AACHFCDSLPKTQDEYIL WGQGTQVTVSSAAAYPYDVPDYGS (SEQ ID NO: 4).

[0029] In some embodiments, the composition further comprises at least one agent having specific binding affinity to a catalytic site of the PSMA.

[0030] In some embodiments, the at least one agent is a small molecule.

[0031] In some embodiments, the at least one agent is YF2.

[0032] In some embodiments, the composition is for use in treatment of a disease, diagnosis thereof, or both, in a subject in need thereof.

[0033] In some embodiments, the conjugate is formulated within a first pharmaceutical composition and the at least one agent is formulated within a second pharmaceutical composition.

[0034] In some embodiments, the biological sample is derived or obtained from a subject.

[0035] In some embodiments, the subject is afflicted with a PSMA-associated disease or disorder.

[0036] In some embodiments, the PSMA-associated disease or disorder is prostate cancer.

[0037] In some embodiments, the id benzylguanidine prosthetic group precursor comprising the radionuclide is selected from the group consisting of: A’-succinimidyl 4-guanidinomethyl 3-[*I] iodobenzoate ([*I]SGMIB: 1,3,4 isomer), A'-succinimidyl 3 -guanidinomethyl 5-[*I] iodobenzoate (iso-[*I]SGMIB; 1,3,5 isomer), A-succinimidyl 3-[211At]astato-4- guanidinomethyl benzoate ([211At]SAGMB) and A’-succinimidyl 3-[211At]astato-5- guanidinomethyl benzoate (iso-[211At]SAGMB), wherein *1 is selected from the group consisting of:125I,131I,1231,124I, and any combination thereof.

[0038] In some embodiments, the conditions comprise subjection to: pH of about 8.5, room temperature for a period of 5-60 minutes, or both.

[0039] In some embodiments, the method further comprises a step comprising isolating, purifying, or both, the prepared conjugate.

[0040] In some embodiments, the method further comprises a step before the contacting comprising conjugating the guanidino-substituted prosthetic group precursor to the radionuclide.

[0041] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0042] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE FIGURES

[0043] Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description together with thedrawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

[0044] Figs. 1A-1B include vertical bar graphs showing in vitro binding and specificity in paired- label format of iso-[125I]SGMIB-NB7 and iso-[131I]SGMIB-NB7H6 in PC3 PIP cells (n = 3). (1A) Total cellular uptake (Left) and internalization (Right) levels at 1, 2, 4 and 24 h after incubation at 37 °C. Values are expressed as percentages of applied radioactivity. (IB) Retention of125’131I activity after incubation at 37 °C following 1 h incubation at 4 °C at various time points. Membrane-bound and internalized activity are expressed as percentages of initially cell-bound radioactivity after an initial incubation at 4 °C.

[0045] Figs. 2A-2B include vertical bar graphs showing in vitro binding and specificity in paired- label format of [211At]SAGMB-NB7H6 and [131I]SGMIB-NB7H6 in PC3 PIP cells (n = 3). (2A) Total cellular uptake (Left) and internalization (Right) levels at 1, 2, 4 and 21 h after incubation at 37 °C. Values are expressed as percentages of applied radioactivity (2B) Retention of125’131I activity after incubation at 37 °C following 1 h incubation at 4 °C at various time points. Membrane-bound and internalized activity are expressed as percentages of initially cell-bound radioactivity after an initial incubation at 4 °C.

[0046] Fis. 3 includes vertical bar graphs showing uptake in selected organs from the single-label biodistribution ofiso - [125I ]SGMIB-NB7H6 in athymic mice with subcutaneous PC3 PIP xenografts (n = 5).

[0047] Fig. 4 includes vertical bar graphs showing uptake in selected organs from a paired-label biodistribution of iso-[125I]SGMIB-NB7 and [131I]SGMIB-NB7 in athymic mice with subcutaneous PC3 PIP xenografts (n = 5).

[0048] Fig. 5 includes vertical bar graphs showing uptake in selected organs from a paired-label biodistribution of [211At]SAGMB-NB7H6 and [131I]SGMIB-NB7H6 in athymic mice bearing subcutaneous PC3 PIP xenografts (n = 5).

[0049] Fig. 6 includes vertical bar graphs showing paired-label uptake of [125I]SGMIB-NB7H6 and [131I]YF2 in selected organs in athymic mice with subcutaneous PC3 PIP xenografts (n = 5).

[0050] Fig. 7 includes vertical bar graphs showing tumor-to-normal-tissue ratios calculated from the biodistribution of [125I]SGMIB-NB7H6 and [131I]YF2 in athymic mice with subcutaneous PC3 PIP xenografts (n = 5).DETAILED DESCRIPTION

[0051] According to a first aspect, there is provided a conjugate comprising: (i) a polypeptide having a specific binding affinity to prostate specific membrane antigen (PSMA); and (ii) a guanidino-substituted prosthetic group.

[0052] In some embodiments, the guanidino-substituted prosthetic group comprises a radionuclide. In some embodiments, the guanidino-substituted prosthetic group comprises or is a benzylguanidine prosthetic group.

[0053] As used herein, the term “benzylguanidine prosthetic group” refers to a chemical moiety that includes a benzyl group attached to a guanidine functional group. It is often used in bioconjugation to attach radionuclides to biomolecules, enhancing their ability to target specific biological structures for diagnostic or therapeutic purposes.

[0054] In some embodiments, the polypeptide having a specific binding affinity to PSMA and the guanidino-substituted prosthetic group are bound. In some embodiments, bound comprises covalently bound.

[0055] As used herein, the expression or term “guanidino-substituted prosthetic group” refers to a chemical moiety that includes a guanidino functional group, which is characterized by the presence of a carbon atom double-bonded to a nitrogen atom and single-bonded to two other nitrogen atoms. This group is often used in bioconjugation to attach radionuclides to biomolecules, enhancing their ability to target specific biological structures, such as proteins or antigens, for diagnostic or therapeutic purposes, or both.

[0056] As used herein, the term “radionuclide” refers to an atom with an unstable nucleus that undergoes radioactive decay, emitting radiation in the form of alpha or beta particles, or gamma rays, or any combination thereof. Radionuclides are used in various applications, including medical imaging and therapy, where they serve as tracers or treatment agents due to their radioactive properties.

[0057] Radionuclides are common and would be apparent to one of ordinary skill in the art.

[0058] Types of radionuclides commonly used in medical and scientific applications, include, but are not limited to: Alpha Emitters, such as:211At (Astatine-211),223Ra (Radium-223),212Pb (Lead- 212), and225Ac (Actinium-225); Beta Emitters:131I (Iodine-131),90Y (Yttrium-90), and177Lu (Lutetium- 177); and Gamma Emitters:99mTc (Technetium-99m),123I (Iodine-123), and111In (Indium- 111).

[0059] In some embodiments, the radionuclide comprises: an alpha emitter, a beta emitter, a gamma emitter, or any combination thereof.

[0060] As used herein, the term “alpha emitter” refers to a type of radionuclide that undergoes radioactive decay by emitting alpha particles. Alpha particles consist of two protons and two neutrons, making them relatively large and positively charged. Due to their size and charge, alpha particles have a limited range and are typically used in targeted therapies where localized radiation is needed, such as in cancer treatment.

[0061] As used herein, the term “beta emitter” refers to a type of radionuclide that undergoes radioactive decay by emitting beta particles, which are high-energy, high-speed electrons or positrons. Beta emitters are used in various medical and scientific applications, including cancer treatment, due to their ability to penetrate tissues and deliver targeted radiation and when also emitting a gamma ray, for diagnostic imaging.

[0062] As used herein, the term “gamma emitter” refers to a type of radionuclide that undergoes radioactive decay by emitting gamma rays, which are high-energy electromagnetic waves. Gamma emitters are commonly used in medical imaging because gamma rays can penetrate deep into tissues, allowing for effective diagnostic imaging of various conditions.

[0063] In some embodiments, the radionuclide comprises: Iodine (I), Astatine (At), or both.

[0064] In some embodiments, Iodine (I) comprises:125I,13'I,123I,124I, or any combination thereof.

[0065] In some embodiments, At comprises or is211At.

[0066] In some embodiments, the guanidino-substituted prosthetic group the radionuclide comprises: 4-guanidinomethyl 3-[*I]iodobenzoate, 3-guanidinomethyl 5-[*I]iodobenzoate, 3- [211At]astato-4-guanidinomethyl benzoate, 3-[211At]astato-5-guanidinomethyl benzoate, or any combination thereof, wherein *1 is selected from the group consisting of:125I,131I,123I, and any combination thereof.

[0067] In some embodiments, the guanidino-substituted prosthetic group comprising the radionuclide comprises 4-guanidinomethyl 3-[125I]iodobenzoate. In some embodiments, the guanidino-substituted prosthetic group comprising the radionuclide comprises 4-guanidinomethyl 3-[131I]iodobenzoate. In some embodiments, the guanidino-substituted prosthetic group comprising the radionuclide comprises 4-guanidinomethyl 3-[123I]iodobenzoate.

[0068] In some embodiments, the guanidino-substituted prosthetic group comprising the radionuclide comprises 3-guanidinomethyl 5-[125I]iodobenzoate. In some embodiments, the guanidino-substituted prosthetic group comprising the radionuclide comprises 3-guanidinomethyl5-[131I]iodobenzoate. In some embodiments, the guanidino-substituted prosthetic group comprising the radionuclide comprises 3-guanidinomethyl 5-[123I]iodobenzoate.

[0069] In some embodiments, the guanidino-substituted prosthetic group comprising the radionuclide comprises 3-[211At]astato-4-guanidinomethyl benzoate. In some embodiments, the guanidino-substituted prosthetic group comprising the radionuclide comprises 3-[211At]astato-5- guanidinomethyl benzoate.

[0070] The term "prostate specific membrane antigen" or PSMA, as used herein, refers to glutamate carboxypeptidase II, also known as N-acetyl-L-aspartyl-L-glutamate peptidase I (NAALADase I or NAAG peptidase). As a non-limiting example, human PSMA has the UniProt accession no. Q04609.

[0071] In some embodiments, PSMA comprises or consists of the amino acid sequence: MWNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEATNITPKHNM KAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLLS YPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNYA RTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGV KSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIG YYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVTRIY NVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAWHEIVRSFGTLKKEGWRPRRTI LFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVH NLTKELKSPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRAR YTKNWETNKFSGYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLP FDCRDYAVVLRKYADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLQDFD KSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDI ESKVDPSKAWGEVKRQIYVAAFTVQAAAETLSEVA (SEQ ID NO: 5).

[0072] The terms "antibody" and "antigen-binding polypeptide" (also referred to as an “immunoglobulin” or "Ig") refer to a polypeptide or group of polypeptides that include at least one binding domain that is specific for one antigen. In certain embodiments, the use of a chimeric antibody or a humanized antibody is also encompassed by the invention.

[0073] In some embodiments, the term "antibody fragments" refers to a portion of an intact antibody, preferably comprising the antigen binding region thereof.

[0074] The term "single-domain antibody" as used herein refers to an antibody fragment consisting of a single variable domain (VHH). Single-domain antibody is a smaller functional fragment of the antibody that also can bind a specific antigen. In some embodiments, the single-domain antibodyhas beter tissue penetration than conventional antibodies and therefore they are beneficial for clinical / diagnostic use.

[0075] In some embodiments, the single-domain antibody of the present invention comprises three complementary-determining regions (CDRs).

[0076] In some embodiments, the term "complementary-determining region" refers to variable heavy chain. In some embodiments, the variable heavy chain comprises an amino acid sequence capable of binding a specific PSMA.

[0077] Kabat et al. defined a numbering system for variable domain sequences that is applicable to any antibody. The skilled artisan can unambiguously assign this system of "Kabat numbering" to any variable domain sequence, without reliance on any experimental data beyond the sequence itself. As used herein, "Kabat numbering" refers to the numbering system set forth by Kabat et al, U.S. Dept, of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0078] In some embodiments, the antigen-binding polypeptide comprises three heavy chain CDRs (CDR-H) comprising CDR-H1 comprising the amino acid sequence GYTDSNYYMS (SEQ ID NO: 1), CDR-H2 comprising the amino acid sequence GVNTGRGSTSYADSVKG (SEQ ID NO:2), and CDR-H3 comprising the amino acid sequence AACHFCDSLPKTQDEYIL (SEQ ID NO:3).

[0079] In some embodiments, the antigen-binding polypeptide comprises the amino acid sequence: QVQLQESGGGSVQAGGSLRLSCTAPGYTDSNYYMSWFRQAPGKEREWVAGVNTGRGS TSYADSVKGRFTISQDNAKNTMFLQMNSLKPEDTAIYYCAV AACHFCDSLPKTQDEYIL WGQGTQVTVSSAAAYPYDVPDYGS (SEQ ID NO: 11).

[0080] In some embodiments, covalently bound is via an amide bond. In some embodiments, covalently bound is via succinimide-thioether. In some embodiments, covalently bound is via an amide bond or a succinimide-thioether.

[0081] In some embodiments, the molar ratio between (ii) and (i) (of the conjugate of the invention) is between 1: 1 and 1 :2, 1 : 1 and 1:2, 1: 1 and 1:3, 1: 1 and 1:4, 1: 1 and 1:5, 1: 1 and 1:6, or 1: 1 and 1:7. Each possibility represents a separate embodiment of the invention.

[0082] In some embodiments, the antigen-binding polypeptide has a specific binding affinity to PSMA.

[0083] As used herein, the term “specific binding affinity” refers to the non-covalent physical association between two entities, such as a ligand and a receptor, where the association issignificantly stronger than with other moieties in the environment. This is often quantified by the equilibrium dissociation constant (KD), with lower values indicating higher affinity. Specific binding is crucial for targeting specific biological structures in diagnostic and therapeutic applications.

[0084] Methods and means for determining binding affinity are common and would be apparent to one of ordinary skill in the art. Non-limiting examples for such methods include, but are not limited to, Surface Plasmon Resonance (SPR), Isothermal Titration Calorimetry (ITC), Fluorescence Polarization (FP), Enzyme-Linked Immunosorbent Assay (ELISA), Radioimmunoassay (RIA), Biolayer Interferometry (BLI), Microscale Thermophoresis (MST), Equilibrium Dialysis, and Affinity Chromatography, to name a few.

[0085] In some embodiments, the binding of two or more entities may be considered specific if the equilibrium "dissociation constant", KD, is less than 10 ’ M, less than 104M, less than 105M, less than 106M, less than 107M. less than 108M, less than 109M, less than 1019M, less than 101 1M, or less than 1012M, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the binding of two or more entities may be considered specific if the equilibrium "dissociation constant", KD, is 1010M - 103M, 1012M - 104M. Each possibility represents a separate embodiment of the invention. In some embodiments, specific binding can be accomplished by a plurality of weaker interactions. Calculation of a peptide's dissociation constant (KD) is known to a skilled artisan and is also show in the Examples section herein below.

[0086] As used herein, the term "binding constant", or "association constant", refers to a special case of the equilibrium constant (Ka), which is the inverse of the dissociation constant (KD). It quantifies the strength of the binding interaction between two molecules, with higher values indicating stronger binding affinity. As used herein, the term “dissociation constant (KD) refers to a measure of the affinity between two molecules, such as a ligand and a receptor. It represents the concentration at which half of the binding sites are occupied. A lower KDvalue indicates a higher affinity, meaning the molecules bind more tightly to each other.

[0087] Methods for determining "binding constant", or "association constant" are common, such as described herein.

[0088] In some embodiments, the antigen-binding polypeptide is characterized by binding constant (Ka) of at least 103Molar1sec"1(M"1s"1), at least 2x l03M"1s’1, at least 104M’1s’1, at least 2x l04M’1s’1, at least 105M’1s’1, or at least 5 x 105M’1s’1to PSMA, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.

[0089] In some embodiments, the antigen-binding polypeptide is characterized by dissociation constant (KD) of less than 10 pM, less than 50 pM, less than 500 pM, less than 15 nM, less than 50 nM, or less than 500 nM to PSMA, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the antigen-binding polypeptide is characterized by dissociation constant (KD) of 10 pM to 100 pM, 1 pM to 200 pM, 15 pM to 250 pM, 0.1 pM to 1,000 pM, or 50 pM to 500 pM. Each possibility represents a separate embodiment of the invention.

[0090] In some embodiments, the polypeptide binds to a non-catalytic site of PSMA. In one embodiment, the polypeptide binds to an extracellular domain of PSMA.

[0091] As used herein, the terms “peptide”, "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues. In another embodiment, the terms "peptide", "polypeptide" and "protein" as used herein encompass native peptides, peptidomimetics (typically including non-peptide bonds or other synthetic modifications) and the peptide analogues peptoids and semipeptoids or any combination thereof.

[0092] In some embodiments, a peptide consists of 2 to 50 amino acids and are smaller than proteins. They often serve as signaling molecules or hormones. In some embodiments, a polypeptide comprises more than 50 amino acids. In some embodiments, a polypeptide folds into a specific structure and / or may function independently or as part of a larger protein complex. In some embodiments, a protein is a complex molecule composed of one or more polypeptide chains. In some embodiments, the one or more polypeptide chains is / are folded into a specific three- dimensional structure. Proteins perform a wide range of functions in the body, including catalyzing biochemical reactions, providing structural support, and regulating cellular processes.

[0093] In some embodiments, the antigen-binding polypeptide binding to PSMA is characterized by allowing further interaction to PSMA. In some embodiments, the antigen-binding polypeptide binding to PSMA is characterized by retaining PSMA enzyme activity. Methods of determining PSMA activity are known in the art. Non-limiting examples of such methods include but are not limited to, Enzyme Assays, Radiolabeled Substrate Assays, Fluorescence-Based Assays, Chromogenic Substrate Assays, Western Blotting, Immunohistochemistry, and Flow Cytometry, to name a few.

[0094] In some embodiments, the antigen-binding polypeptide further comprises a tag motif. In some embodiments, the tag motif is positioned in the N’- or C’-terminus of the antigen-binding polypeptide. In some embodiments, the tag motif comprises at least six amino acids. In some embodiments, the tag motif comprises at least six repeats of the same amino acid. In someembodiments, the amino acid is or comprises Histidine. In some embodiments, the antigen-binding polypeptide comprises a poly histidine (His)-tag.

[0095] In some embodiments, the conjugate is characterized by fast or faster clearance from a nontarget tissue, such as compared to a target tissue, e.g., atumor. In some embodiments, the conjugate is characterized by fast or faster clearance from a non-target tissue compared to the clearance from a target tissue. In some embodiments, a target tissue is or comprises a cancerous or malignant tissue. In some embodiments, a target tissue comprises a tumor. In some embodiments, a target tissue comprises a tumor of the prostate gland. In some embodiments, a target tissue is or comprises a cancerous or malignant prostate tissue. In some embodiments, a non-target tissue comprises or is a benign tissue. In some embodiments, a non-target tissue is or comprises kidney, stomach, salivary gland, lacrimal gland, thyroid, or any combination thereof.

[0096] In some embodiments, the conjugate is characterized by increased tumor to kidney ratio compared to a control. In some embodiments, the increased tumor to kidney ratio is determined 2- 6 hours after administration. In some embodiments, the composition is characterized by increased tumor to lacrimal gland ratio compared to a control. In some embodiments, the increased tumor to lacrimal gland ratio is determined 0.5 to 1.5 hours after administration, 2 to 6 hours after administration, or both.

[0097] In some embodiments, the conjugate or a composition comprising thereof is characterized by a tumor to kidney ratio of 3 to 10. In some embodiments, the conjugate or a composition comprising thereof is characterized by a tumor to kidney ratio of 3 to 10 determined 2-6 hours after administration.

[0098] In some embodiments, the conjugate or a composition comprising thereof is characterized by a tumor to kidney ratio of 10 to 30, 20 to 60, 15 to 60, or 40 to 60. Each possibility represents a separate embodiment of the invention. In some embodiments, the conjugate or a composition comprising thereof is characterized by a tumor to kidney ratio of 15 to 25 determined 0.5-2 hours after administration. In some embodiments, the conjugate or a composition comprising thereof is characterized by a tumor to kidney ratio of 25 to 55 determined 2-6 hours after administration.

[0099] In some embodiments, a control comprises a sample obtained or derived from a subject administered with a PSMA inhibitor. In some embodiments, control comprises a sample obtained or derived from a subject administered with a PSMA small molecule inhibitor. In some embodiments, control comprises a sample obtained or derived from a subject administered with the PSMA inhibitor YF2 (Feng et al., (2024); “A third generation PSMA-targeted agent [211At]YF2: Synthesis and in vivo evaluation.” Nucl Med Biol 134-135(5)108916).

[0100] Tumor to tissue ratio, e.g., tumor to kidney, may be determined such as exemplified herein. Briefly, tissues are weighed and counted for radioactivity along with injection standards to permit calculation percent injected dose (ID) per organ, ID / g of tissue and tumor-to-tissue ratios. All radioactivity measurements were decay corrected. In some embodiments, the method comprises collecting a biopsy of a target tissue, e.g., tumor and of a non-target tissue, e.g., kidney. In some embodiments, the method comprises determining the injected dose (ID) per organ (ID / g) in the target tissue and the non-target tissue. In some embodiments, the method comprises determining the ratio between the ID / g of the target tissue and the ID / g of the non-target tissue, thereby determining the tumor to tissue ratio or the target to non-target ratio.Compositions

[0101] According to another aspect, there is provided a composition comprising the conjugate of the invention. In some embodiments, the composition comprises an effective amount of the conjugate of the invention. In some embodiments, an effective amount comprises a therapeutically effective amount. In some embodiments, an effective amount comprises a diagnostically effective amount. In some embodiments, an effective amount comprises a theranostically effective amount.

[0102] As used herein, the term “theranostic” refers to a treatment approach that combines therapeutic and diagnostic capabilities in a single agent or system. It is designed to diagnose, deliver targeted therapy, monitor the response to treatment, often using the same molecular platform, to provide personalized and efficient patient care, or any combination thereof.

[0103] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the composition further comprises a diagnostically acceptable carrier. In some embodiments, the composition further comprises a theranostically acceptable carrier.

[0104] In some embodiments, the composition further comprises at least one agent having specific binding affinity to a catalytic site of PSMA. In some embodiments, the at least one agent is a small molecule. In some embodiments, at least one agent is YF2, such as described herein or published by Feng et al., (2024) Nuclear Medicine and Biology “A THIRD GENERATION PSMA- TARGETED AGENT [211AT]YF2: SYNTHESIS AND IN VIVO EVALUATION”.

[0105] In some embodiments, there is provided a combination comprising the conjugate of the invention and at least one agent having specific binding affinity to a catalytic site of PSMA. In some embodiments, the combination is for use in diagnosis, treatment, or both, of a PSMA- associated disease or disorder, in a subject in need thereof.

[0106] In some embodiments, the conjugate is formulated within a first pharmaceutical composition and the at least one agent is formulated within a second pharmaceutical composition.

[0107] In some embodiments, the composition is for use in treatment of a disease, diagnosis thereof, or both, in a subject in need thereof.

[0108] In some embodiments, the composition is for use in the preparation of a theranostic agent for treatment of a disease, diagnosis thereof, or both, in a subject in need thereof.

[0109] In some embodiments, the conjugate of the invention or a composition comprising thereof is for use in the manufacture of a pharmaceutical composition for the treatment, diagnosis, theranostic or prophylaxis of cancer or neurological disorder.

[0110] In some embodiments, the composition further comprises at least additional therapeutic ingredient(s), imaging agent(s), or any combination thereof.

[0111] In some embodiments, the composition is characterized by fast or faster clearance from a non-target tissue. In some embodiments, the composition is characterized by increased tumor to kidney ratio compared to a control.

[0112] The compositions of the invention can be formulated in the form of a pharmaceutically acceptable salt of the polypeptide of the invention or their analogs thereof. Pharmaceutically acceptable salts include those salts formed with free amino groups such as salts derived from nontoxic inorganic or organic acids such as hydrochloric, phosphoric, acetic, oxalic, tartaric acids, and the like, and those salts formed with free carboxyl groups such as salts derived from non-toxic inorganic or organic bases such as sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, and the like. In one embodiment, pharmaceutical compositions of the present invention are manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0113] The term "pharmaceutically acceptable" means suitable for administration to a subject, e.g., a human. For example, the term "pharmaceutically acceptable" can mean approved by a regulatory agency of the Federal or a state government or listed in the U. S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents. Water is a preferred carrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates or phosphates. Antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; and agents for the adjustment of tonicity such as sodium chloride or dextrose are also envisioned. The carrier may constitute, in total, from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.

[0114] The compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, gels, creams, ointments, foams, pastes, sustained-release formulations and the like. The compositions can be formulated as a suppository, with traditional binders and carriers such as triglycerides, microcrystalline cellulose, gum tragacanth or gelatin. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in: Remington's Pharmaceutical Sciences" by E.W. Martin, the contents of which are hereby incorporated by reference herein. Such compositions will contain a therapeutically effective amount of the active agent and the antigen-binding polypeptide of the invention, preferably in a substantially purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.

[0115] An embodiment of the invention relates to the conjugate or a composition comprising thereof being presented in unit dosage form and is prepared by any of the methods well known in the art of pharmacy. In an embodiment of the invention, the unit dosage form is in the form of a tablet, capsule, lozenge, wafer, patch, ampoule, vial or pre-filled syringe. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the nature of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses can be extrapolated from dose-response curves derived from in-vitro or in-vivo animal model test bioassays or systems.

[0116] Depending on the location of the tissue of interest, the conjugate of the invention (or a composition comprising thereof) can be supplied in any manner suitable for the provision of the conjugate of the invention to cells within the tissue of interest. Thus, for example, a composition comprising the conjugate of the invention can be introduced, for example, into the systemiccirculation, which will distribute the conjugate to the tissue of interest. Alternatively, a composition can be applied topically to the tissue of interest (e.g., injected, or pumped as a continuous infusion, or as a bolus within a tissue, applied to all or a portion of the surface of the skin, etc.).

[0117] In some embodiments, the conjugate of the invention (or a composition comprising thereof) is administered via oral, rectal, vaginal, topical, nasal, ophthalmic, transdermal, subcutaneous, intramuscular, intraperitoneal or intravenous routes of administration. The route of administration of the pharmaceutical composition will depend on the disease or condition to be treated. Suitable routes of administration include, but are not limited to, parenteral injections, e.g., intradermal, intravenous, intramuscular, intralesional, subcutaneous, intrathecal, and any other mode of injection as known in the art. Although the bioavailability of the conjugate administered by other routes can be lower than when administered via parenteral injection, by using appropriate formulations it is envisaged that it will be possible to administer the compositions of the invention via transdermal, oral, rectal, vaginal, topical, nasal, inhalation and ocular modes of treatment. In addition, it may be desirable to introduce the pharmaceutical compositions of the invention by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir.

[0118] For topical application, the conjugate of the invention (or a composition comprising thereof) can be combined with a pharmaceutically acceptable carrier, an imaging agent, and one or more therapeutic agents, so that an effective dosage is delivered, based on the desired activity. The carrier can be in the form of, for example, and not by way of limitation, an ointment, cream, gel, paste, foam, aerosol, suppository, pad or gelled stick.

[0119] For oral applications, the pharmaceutical composition may be in the form of tablets or capsules, which can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose; a disintegrating agent such as alginic acid, Primogel, or com starch; a lubricant such as magnesium stearate; or a glidant such as colloidal silicon dioxide. When the dosage unit form is a capsule, it can contain, in addition to materials of the above type, a liquid carrier such as fatty oil. In addition, dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar, shellac, or other enteric agents. The tablets of the invention can further be film coated.

[0120] For purposes of parenteral administration, solutions in sesame or peanut oil or in aqueous propylene glycol can be employed, as well as sterile aqueous solutions of the corresponding water- soluble salts. Such aqueous solutions may be suitably buffered, if necessary, and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. These aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal injection purposes.

[0121] The compositions of the present invention are generally administered in the form of a pharmaceutical composition comprising the antigen-binding polypeptide of this invention together with a pharmaceutically acceptable carrier or diluent. Thus, the compositions of this invention can be administered either individually or together in any conventional oral, parenteral or transdermal dosage form.

[0122] Pharmaceutical compositions according to embodiments of the invention may contain 0. l%-95% of the conjugate of the invention. In any event, the composition or formulation to be administered may contain a quantity of the conjugate of the invention and further active and / or imaging agents according to embodiments of the invention in an amount effective to treat or diagnose the condition or disease of the subject being administered.

[0123] The compositions also comprise preservatives, such as benzalkonium chloride and thimerosal and the like; chelating agents, such as EDTA sodium and others; buffers such as phosphate, citrate and acetate; tonicity agents such as sodium chloride, potassium chloride, glycerin, mannitol and others; antioxidants such as ascorbic acid, acetylcystine, sodium metabisulfote and others; aromatic agents; viscosity adjustors, such as polymers, including cellulose and derivatives thereof; and polyvinyl alcohol and acid and bases to adjust the pH of these aqueous compositions as needed. The compositions may also comprise local anesthetics or other actives.

[0124] In addition, the compositions may further comprise binders (e.g., acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone), disintegrating agents (e.g., cornstarch, potato starch, alginic acid, silicon dioxide, croscarmellose sodium, crospovidone, guar gum, sodium starch glycolate), buffers (e.g., Tris-HCI., acetate, phosphate) of various pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts), protease inhibitors, surfactants (e.g., sodium lauryl sulfate), permeation enhancers, solubilizing agents (e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite, butylated hydroxyanisole), stabilizers (e.g., hydroxypropyl cellulose, hydroxypropylmethyl cellulose), viscosity increasing agents (e.g., carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum), sweeteners (e.g., aspartame, citric acid), preservatives (e.g., Thimerosal, benzyl alcohol, parabens), lubricants (e.g., stearic acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate), flow-aids (e.g., colloidal silicon dioxide), plasticizers (e.g., diethyl phthalate, triethyl citrate), emulsifiers (e.g., carbomer, hydroxypropyl cellulose,sodium lauryl sulfate), polymer coatings (e.g., poloxamers or poloxamines), coating and fdm forming agents (e.g., ethyl cellulose, acrylates, polymethacrylates) and / or adjuvants.

[0125] The conjugate of the invention (or a composition comprising thereof) can be delivered in a controlled release system. Thus, an infusion pump can be used to administer the antigen-binding polypeptide such as the one that is used, for example, for delivering insulin or chemotherapy to specific organs or tumors. In one embodiment, the conjugate of the invention (or a composition comprising thereof) is administered in combination with a biodegradable, biocompatible polymeric implant, which releases the conjugate of the invention (or a composition comprising thereof) over a controlled period of time at a selected site. Examples of preferred polymeric materials include, but are not limited to, polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, copolymers and blends thereof (See, Medical applications of controlled release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Fla., the contents of which are hereby incorporated by reference in their entirety). In yet another embodiment, a controlled release system can be placed in proximity to a therapeutic target, thus requiring only a fraction of the systemic dose.

[0126] In one embodiment, compositions of the present invention are presented in a pack or dispenser device, such as an FDA approved kit, which contain one or more unit dosage forms containing the active ingredient. In one embodiment, the pack or dispenser device is accompanied by instructions for administration.

[0127] In one embodiment, it will be appreciated that the conjugate of the invention (or a composition comprising thereof) can be provided to the individual with active agents to achieve an improved therapeutic effect as compared to treatment without a targeting agent. In another embodiment, measures (e.g., dosing and selection of the complementary agent) are taken to adverse side effects which are associated with combination therapies.

[0128] A "therapeutically effective amount" of the active agent and / or the conjugate of the invention is the amount sufficient to provide a beneficial effect to the subject to which the composition is administered. More specifically, a therapeutically effective amount means an amount of the active agent and / or the conjugate of the invention effective to prevent, alleviate or ameliorate tissue damage or symptoms of a disease of the subject being treated.

[0129] In some embodiments, preparation of effective amount or dose can be estimated initially from in vitro assays. In one embodiment, a dose can be formulated in animal models and such information can be used to more accurately determine useful doses in humans.

[0130] In one embodiment, toxicity and therapeutic efficacy of the active / targeting agents described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. In one embodiment, the data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. In one embodiment, the dosages vary depending upon the dosage form employed and the route of administration utilized. In one embodiment, the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. [See e.g., Fingl, et al., (1975) "The Pharmacological Basis of Therapeutics", Ch. 1 p. l],

[0131] In one embodiment, depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved. In one embodiment, the amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc. In one embodiment, compositions including the preparation of the present invention formulated in a compatible pharmaceutical carrier are also prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.Methods for treatment and diagnosis (theranostics)

[0132] According to another aspect, there is provided a method for imaging PSMA in a biological sample. In some embodiments, the method comprises contacting a biological sample comprising PSMA with an effective amount of the conjugate of the invention or a composition comprising thereof, and detecting the presence of PSMA in the biological, thereby imaging PSMA in the biological sample.

[0133] In some embodiments, the biological sample is derived or obtained from a subject. In some embodiments, the biological sample comprises: Blood, Urine, Tissue biopsy, Saliva, Cerebrospinal fluid, Bone marrow, Sputum, Swabs (e.g., nasal, throat), Hair, Skin scrapings, or any combination thereof. In some embodiments, the biological sample comprises any bodily fluid, such as: Blood, Urine, Saliva, Cerebrospinal fluid, Synovial fluid, Amniotic fluid, Breast milk, Sweat, Tears, Semen, and any combination thereof.

[0134] In some embodiments, a tissue biopsy comprises a biopsy of a prostate gland.

[0135] According to another aspect, there is provided a method for detecting PSMA in a subject in need thereof.

[0136] In some embodiments, the method comprises administering to the subject an effective amount of the conjugate of the invention or a composition comprising thereof, and detecting the presence of PSMA in the subject.

[0137] In some embodiments, the subject is afflicted with a PSMA-associated disease or disorder. In some embodiments, the PSMA-associated disease or disorder is prostate cancer.

[0138] In another embodiment, the present invention provides a method for targeting PSMA by contacting a sample comprising PSMA with an antigen-binding polypeptide of the invention, thereby targeting PSMA.

[0139] In one embodiment, the present invention provides a method for treating, diagnosing, prognosticating or determining the suitability for treatment of a subject suffering from a PSMA- associated disorder, the method comprising administering to the subject a pharmaceutical composition comprising an effective amount of the antigen-binding polypeptide of the invention, a cytotoxic agent or a theranostic agent, and a pharmaceutical acceptable carrier, thereby treating diagnosing, prognosticating or determining the suitability for treatment of a subject suffering from a PSMA-associated disorder in the subject.

[0140] In one embodiment, there is provided a method for imaging PSMA in a subject, such as a subject suffering from or suspected to suffer from a PSMA-associated disorder, the method comprising administering to the subject a composition comprising an effective amount of the antigen-binding polypeptide of the invention, and an imaging agent; and detecting the PSMA in the subject, thereby imaging PSMA in a subject.

[0141] In some embodiments, the imaging techniques are selected from, without being limited thereto, computed X-ray tomography (CT), ultrasound (US), and magnetic resonance imaging (MRI), planar imaging, positron emission tomography (PET), single-photon emission computed tomography (SPECT), fluorescence and radio assays, cytofluorimetry, and fluorescence activated cell sorting. The principles of such techniques can be found in immunochemistry handbooks, for example: A Johnstone and R. Thorpe, hnmunochemistry in practice, 2nd Edition (1987), Blackwell Scientific publications, Oxford London Edinburgh Boston Palo Alto Melbourne.

[0142] Non-limiting exemplary embodiments demonstrate the diagnosis of prostate tumors in vivo by near infra-red (NIR) imaging after 24 hours from the administration of the antigen-binding polypeptide conjugated to a fluorescent label.

[0143] In one embodiment, the method further comprises determining the relative percentage of the PSMA subpopulations by the administration of antigen-binding polypeptide.

[0144] In some embodiments, the conjugate of the invention can be used in conjunction with at least one additional therapeutic treatment modality, including surgery, cryosurgery, radiation, thermotherapy, hormone treatment, chemotherapy, immunotherapy, vaccines, and any combination thereof.

[0145] In some embodiments, the at least one additional therapeutic agent can include any agent (e.g., molecule, drug, pharmaceutical composition, etc.) capable of preventing, inhibiting, or arresting the symptoms and / or progression of a disease.

[0146] In some embodiments, the at least one additional therapeutic agent is selected from, but not limited to: a chemotherapeutic agent (e.g., methotrexate, cisplatin and paclitaxel), an anti- oncogenic agent, an anti-angiogenic agent, a tumor suppressor agent, an anti-microbial agent, or an expression construct comprising a nucleic acid encoding a therapeutic protein.

[0147] In some embodiments, the PSMA-associated disorder is prostate cancer.

[0148] In some embodiments, the PSMA-associated disorder is a neurological disorder. In some embodiments, the neurological disorder is selected from, but not limited to: Parkinson disease, Alzheimer disease, Huntington disease, amyotrophic lateral sclerosis (ALS), and schizophrenia.Methods of preparation

[0149] According to another aspect, there is provided a method for preparing the conjugate of the invention.

[0150] In some embodiments, the method comprises contacting the antigen-binding polypeptide having a specific binding affinity to PSMA with an effective amount of a guanidino-substituted prosthetic group precursor. In some embodiments, the guanidino-substituted prosthetic group precursor comprises a radionuclide. In some embodiments, contacting is under conditions suitable for formation of a covalent bond between the antigen-binding polypeptide having a specific binding affinity to PSMA and the guanidino-substituted prosthetic group precursor.

[0151] In some embodiments, a guanidino-substituted prosthetic group precursor, such as comprising a radionuclide is selected from: A-succinimidyl 4-guanidinomethyl 3-[’I]iodobenzoate ([*I]SGMIB; 1,3,4 isomer), N-succinimidyl 3-guanidinomethyi 5-[*I]iodobenzoate (iso- [*I]SGMIB; 1 ,3,5 isomer), N-succinimidyl 3-[211At]astato-4-guanidinomethyl benzoate ([211At]SAGMB) and A-succinimidyl 3-[211At]astato-5-guanidinomethyl benzoate (iso- [211At]SAGMB), wherein *1 is selected from the group consisting of:125I,131I,123I,124I, or any combination thereof.

[0152] In some embodiments, suitable conditions, such as for formation of a covalent bond, e.g., between an antigen-binding polypeptide having a specific binding affinity to PSMA and a guanidino-substituted prosthetic group precursor, comprise subjection to pH of: about 7.0, about 7.5 about, 8.0, about 8.5, about 9.0, at a temperature of 22 °C to about 28 °C, about 23 °C to about 28 °C, about 24 °C to about 28 °C, or about 25 °C to about 28 °C, for a period of 5-60 minutes, 10- 60 minutes, 15-60 minutes, 20-60 minutes, 25-60 minutes, 15-40 minutes, 5-50 minutes, or any combination thereof. Each possibility represents a separate embodiment of the invention.

[0153] In some embodiments, suitable conditions comprise subjecting to room temperature. In some embodiments, suitable conditions comprise subjecting to ambient temperature.

[0154] In some embodiments, the method further comprises a step comprising isolating, purifying, or both, the prepared conjugate.

[0155] In some embodiments, the method further comprises a step before or preceding the contacting comprising conjugating a guanidino-substituted prosthetic group precursor to a radionuclide. In some embodiments, conjugating a guanidino-substituted prosthetic group precursor to a radionuclide is performed under conditions suitable for formation of a bond between the guanidino-substituted prosthetic group precursor and the radionuclide. In some embodiments, a bond comprises or is a covalent bond.

[0156] Methods and means such for conjugating a guanidino-substituted prosthetic group precursor and a radionuclide can be found in Choi et al., (2014) ‘W-SUCCINIMIDYL GUANIDINOMETHYL IODOBENZOATE PROTEIN RADIOHALOGENATION AGENTS: INFLUENCE OF ISOMERIC SUBSTITUTION ON RADIOLABELING AND TARGET CELL RESIDUALIZATION” Nuclear Medicine and Biology 41:802-812, which is incorporated herein by reference in its entirety.General

[0157] As used herein the term “about” refers to ± 10 %.

[0158] The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to" . The term “consisting of’ means “including and limited to”. The term "consisting essentially of' means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0159] The word “exemplary” is used herein to mean “serving as an example, instance or illustration”. Any embodiment described as “exemplary” is not necessarily to be construed aspreferred or advantageous over other embodiments and / or to exclude the incorporation of features from other embodiments.

[0160] The word “optionally” is used herein to mean “is provided in some embodiments and not provided in other embodiments”. Any particular embodiment of the invention may include a plurality of “optional” features unless such features conflict.

[0161] As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0162] Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0163] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

[0164] As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0165] As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.

[0166] In those instances where a convention analogous to "at least one of A, B, and C, etc." is used, in general such a construction is intended in the sense one having skill in the art wouldunderstand the convention (e.g., "a system having at least one of A, B, and C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

[0167] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0168] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.EXAMPLES

[0169] Generally, the nomenclature used herein, and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New Y ork (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996).

[0170] Reference is now made to the following examples which, together with the above descriptions, illustrate the invention in a non-limiting fashion.Materials and MethodsGeneral

[0171] Chemicals and reagents were purchased from Sigma Aldrich (St. Louis, MO) or ThermoFisher (Waltham, MA) unless stated otherwise. Sodium [125I]iodide in 0.1 N NaOH (629 GBq / mg, 12.95 GBq / mL) was purchased from Perkin-Elmer Life and Analytical Sciences (Boston, MA). Sodium [131I]iodide (936 GBq / mg, 236.097 GBq / mL) in 0.04 M sodium thiosulfate, 0.2 M NaOH and 0.2 M sodium carbonate was obtained from International Isotopes Inc. (Idaho Falls, ID). Astatine-211 was produced on the Duke University CS-30 Cyclotron via the209Bi(a, 2n)211At reaction by bombarding natural bismuth metal targets with 28 MeV a-particles as reported. The Boc -protected tin precursors 1,3,4-SGMTB and its 1,3,5-SGMTB were synthesized as previously reported. The NB7 sdAb without His6-tag was produced by BioIntron Biological Inc. (Shanghai, China). SDS-PAGE performed upon arrival indicated two bands - one corresponding to an sdAb monomer (-75%) and the other -25% with a molecular weight consistent with an sdAb dimer - and was used without further purification. The NB7 sdAb with His6-tag at the C-terminus (NB7H6) was produced and characterized in Ben-Gurion University of the Negev, Beer-Sheva, Israel. Details of the synthesis and radiolabeling of the small molecule PSMA inhibitor YF2, a DOTAGA analogue of compound 3 reported in Mease et al., have been published by Feng et al., (2024); “A third generation PSMA-targeted agent [211At]YF2: Synthesis and in vivo evaluation.” Nucl Med Biol 134-135(5)108916. High performance liquid chromatography (HPLC) was performed using an Agilent Technologies 1260 Affinity HPLC system equipped with two-solvent modules. Normal phase HPLC was employed for the purification of radiohalogenated products. For this, a 4.6 x 250 mm Zorbax Rx-SIL 5 pm column (Agilent Technologies, Santa Clara, CA) was eluted with a gradient consisting of hexanes and ethyl acetate, each containing 0.2 % acetic acid. Radiolabeled sdAbs were purified using PD-10 desalting columns obtained from GE Healthcare (Picastaway, NJ).Cells and cell culture conditions

[0172] The cell lines used include PC3 PIP and PC3 flu cell lines (originally developed by Dr. W. Heston (Cleveland, OH)). These cell lines, namely PSMA-expressing (PC3 PIP) and nonexpressing (PC3 flu) prostate cancer cell lines, were screened for mycoplasma contamination using the MycoAlert Mycoplasma detection kit (Lonza). The passage numbers typically ranged from P20 to P40. PC3 PIP cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum,Pen-Strep (10 mL / L), plasmocin (5 mg / L) and puromycin (20 mg / L) (InvivoGen, San Diego CA). Adding puromycin to PC3 PIP cells serves the purpose of maintaining PSMA expression. PC3 flu cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum and Pen-Strep (5 mL / L). Cells were cultured at 37 °C in a humidified incubator under 5% CO2.Radiolabeling ofNB7 sdAbs

[0173] The prosthetic agents were synthesized by the radiohalogenation of their respective tin precursors as reported previously. Briefly, a solution of A-chlorosuccinimide (NCS) in MeOH (0.2 mg / mL) containing 0.6-1% (v / v) acetic acid was added to a vial containing 50 pg tin precursor and 37-74 MBq (1-2 mCi) of125I or131I. For211At labeling, a solution of 74-148 (2-4 mCi)211At in NCS / MeOH was added to a half-dram vial containing 50 pg tin precursor, followed by the addition of acetic acid (1% v / v). The mixture was stirred at room temperature for 30 min and injected onto a normal phase HPLC column that was eluted with a linear gradient of 30 to 70% ethyl acetate / hexanes containing 0.2% AcOH at a flow rate of 1.5 mL / min. HPLC fractions containing the radiohalogenated product were collected and solvents evaporated to dryness under a stream of argon gas. The Boc groups from the protected intermediates were removed by treatment with trifluoroacetic acid (TFA; 100 pL) at room temperature for 20 min. TFA was evaporated using an argon stream followed by the addition and evaporation of 3 x 100 pL of ethyl acetate to ensure complete removal of TFA. The NB7 or NB7H6 sdAbs were conjugated with the radiolabeled prosthetic agents as previously reported. Briefly, -100 pg of sdAb in 100 pL 0.1 M borate buffer pH 8.5 was added to about 1-2 mCi of the dry prosthetic agents in a half-dram vial, and the mixture was kept at room temperature for 20 min. The radiolabeled sdAb was isolated by gel filtration over a PD10 column. The sdAb concentration was determined using a NanoDrop microvolume spectrophotometer (Thermo Fisher). The radiochemical purity of radiohalogenated sdAb was determined by SDS-PAGE / phosphor imaging.Saturation binding assays

[0174] The binding affinity of NB7 radioconjugates and specificity for PSMA was determined by saturation binding assays using PSMA+PC3 PIP and PSMA PC3 flu cell lines. Briefly, cells were plated in 24-well plates at a density of 1 x 104cells / well / mL and incubated overnight at 37 °C. On the next day, cells were acclimatized at 4 °C for 30 min and then 0.1-100 nM of the NB7 radioconjugates were added to PC3 PIP cells, followed by incubation at 4 °C for 2 h. The medium containing unbound radioactivity was then removed, the cells were washed twice with ice-cold RPMI 1640 media and solubilized by 1% SDS in deionized H2O. The supernatants were collected and counted on an automated gamma counter. Nonspecific binding was determined in parallelassays as above but using PSMA-negative PC3 flu cells. The data were fitted using GraphPad Prism software to determine Kd values. Experiments were repeated three times.In vitro cell uptake and internalization

[0175] Uptake and internalization of radiolabeled NB7 sdAbs were measured in a paired-label format. Briefly, both PC3 PIP and PC3 flu cells (5 x 105cells / well / 2 mL medium) were plated in 6-well plates the day prior to the study. For cellular binding studies, cells were incubated with 11.1— 22.2 kBq (0.3-0.6 pCi) of NB7 radioconjugates at 37 °C for 1, 2, 4 and either 24 h (radioiodine) or 21 h (211At). The medium containing unbound radioactivity was removed, and the cells were washed twice with cold RPMI 1640 media. The cells were then treated with 2 x 0.05 M glycine buffer, pH 2.8, and the supernatants were collected as surface-bound radioactivity. The cells were solubilized with 1 mL 0.1% SDS and counted to determine internalized fraction. To determine retention of radioactivity after binding, cells were acclimatized at 4 °C for 30 min, after which 0.3- 0.5 pg NB7 radioconjugate was added and then incubated at 4 °C for 1 h. The unbound fraction was removed, cells were supplemented with fresh medium, and then incubated at 37 °C for different times. At each time point, the supernatant (‘effluxed’ fraction) was removed and counted for radioactivity. Then the cells were treated with 2 x 0.05 M glycine buffer, pH 2.8, and the supernatants were collected as the membrane-bound fraction. Subsequently, cells were solubilized with 1 mb of 0. 1 % SDS and counted to determine the internalized fraction. Finally, the inventors evaluated whether NB7 sdAb and the small molecule PSMA inhibitor YF2 competed for binding to PSMA. The uptake and internalization of [125I]SGMIB-NB7 and [131I]YF2 both in paired-label and single-label formats was determined using protocols as above but doing the assay just for one time point - 2 h. In all experiments, nonspecific uptake was assessed using PC3 flu cells and all studies were performed in triplicate.Biodistribution studies

[0176] All experiments involving animals were carried out in accordance with the guidelines and regulations of the Institutional Animal Care and Use Committee (IACUC) of Duke University and described in approved protocol number A204-21-10. For biodistribution studies, 4-6-week old male athymic nude mice (25 g; Jackson Labs) were inoculated subcutaneously in the flank with 3 x 106PC3 PIP cells. When the tumors reached 100-500 mm3, groups of five mice were injected with the radioconjugate(s) via the tail vein. At 1, 4, and at 24 h (radioiodine) or 21 h (211At) post injection, blood and urine samples were collected and the animals were necropsied. Tissues were weighed and counted for radioactivity along with injection standards to permit calculation of percent injected dose (ID) per organ, ID / g and tumor-to-tissue ratios of the radioactivity. Allradioactivity measurements were decay corrected. In a preliminary study, the biodistribution of iso- [125I]SGMIB-NB7H6 (1 pCi; 1 pg) was evaluated at 1, 4 and 24 h in single-label format primarily to determine whether the presence of a His6-tag resulted in excessive retention of activity in the kidney as has been reported for sdAbs labeled with radiometals. In the first paired-label study, the tissue distribution of [131I]SGMIB-NB7 (5 pCi; 4 pg) and iso-[125I]SGMIB-NB7 (2.5 pCi; 4 pg) were compared. In the second paired-label experiment, mice received both [131I]SGMIB-NB7H6 (122 kBq / 3.3 pCi; 4 pg) and [211At]SAGMB-NB7H6 (122 kBq / 3.3 pCi; 4 pg). The third paired- label experiment was performed to directly compare the biodistribution of the small molecule PSMA inhibitor YF2 and the PSMA-targeted sdAb. Mice were injected intravenously with 148 kBq (5 pCi; 4 pg) of [125I]SGMIB-NB7H6 and 148 kBq (5 pCi; 0.02 pg) of [131I]YF2. Biodistribution was performed as above at 1 and 4 h post injection.Statistical analyses

[0177] Results are presented as mean ± SD. For experiments done in paired-label format, a two- tail, paired Student's / -test was used to evaluate the significance of differences. A P value of <0.05 was considered statistically significant.EXAMPLE 1Radiolabeling

[0178] The yield for prosthetic agent conjugation to NB7 was 20-31% for iso-[*I]SGMIB, 23- 33% for [*I]SGMIB, and 15-50% for [211At]SAGMB. The specific activity of the labeled sdAbs were similar for125I and131I and were: [*I]SGMIB-NB7 (70-90 MBq / mg), iso-[*I]SGMIB-NB7 (70-110 MBq / mg), [*I]SGMIB-NB7H6 (70-110 MBq / mg) and iso-[*I] SGMIB-NB7H6 (60-100 MBq / mg). The specific activity of [211At] SAGMB-NB7H6 was about 60-100 MBq / mg. SDS- PAGE / phosphor imaging was performed to evaluate the radiochemical purity of radiohalogenated sdAbs immediately after PD10 purification. A single radioactive band at approximately 16 kDa was observed for [*I]SGMIB-NB7H6, iso-[*I]SGMIB-NB7H6 and [211At]SAGMB NB7H6, consistent with the molecular weight of NB7 sdAb. Two radioactive bands were observed for [*I]SGMIB-NB7 and isoo-|* I|SGMIB-NB7. corresponding to both the expected sdAb monomer (75%) as well as a second species (25%) of unknown origin that was likely a dimer.EXAMPLE 2Saturation binding assays

[0179] Saturation binding assays on PC3 PIP and PC3 flu cells (for nonspecific uptake) revealed Kd values for PSMA-specific binding of 6.4 ± 2.0 nM and 1.4 ± 0.4 nM for [131I]SGMIB-NB7 and [131I]SGMIB-NB7H6, respectively. The Kd value was 4.0 ± 0.5 nM for [211At]SAGMB NB7H6.EXAMPLE 3In vitro cell uptake and internalization

[0180] Paired -label assays were performed to evaluate the cellular uptake and internalization of NB7 and NB7H6. No significant differences in either parameter were observed between [*I]SGMIB and iso-[*I]SGMIB. For example, total cellular binding was about 24% of input activity with ~7% internalized into PC3 PIP cells for both [131I]SGMIB-NB7 and iso-|125I ]SG] MIB- NB7 after 2 h incubation at 37 °C. Similarly, no significant differences in cell uptake and internalization were observed between [131I]SGMIB-NB7H6 and iso-[125I]SGMIB-NB7H6. It should be noted that cell uptake (about 32% of input activity at 2 h) was higher than observed with the NB7 radioconjugates lacking a His6-tag. In all cases, uptake by PSMA PC3 flu cells at 2 h was <0.3 %, confirming the PSMA specificity of radiolabeled NB7 uptake. In the retention assays, about 12-14 % of the input activity was taken up by the cells after 1 h incubation at 4 °C for both [131I]SGMIB-NB7 and AO-[125I]SGMIB-NB7. The percentage of initially bound activity that was internalized was about 26-31% with no significant differences detected between the two prosthetic agent isomers. Similar to the NB7 constructs without His6-tags, [131I]SGMIB-NB7H6 and iso- [125I]SGMIB-NB7H6 showed no isomer-related differences in cell uptake or internalization; however initial uptakes were 20-24% of input counts, about twice those observed for the corresponding NB7 radioconjugates. Given the differences in cell uptake noted above, paired-label assays were performed to directly compare the in vitro behavior of NB7 with and without a His6- tag at the C-terminus (Fig. 1). After 2 h incubation at 37 °C, uptake by PC3 PIP cells for iso- [131I]SGMIB-NB7H6 was 51.6 ± 1.1% compared with 42.8 ± 2.2% for iso-[125I]SGMIB-NB7 in PC3 PIP cells (P = 0.003). However, at 24 h, the cell uptake of the two isomer conjugates was not significantly different (iso-[131I]SGMIB-NB7H6, 54.0 ± 0.9%; iso-[125I]SGMIB-NB7, 53.2 ± 0.9%; P > 0.05). In the retention assay, iso-|131I]SGMIB-NB7H6 also showed a slightly higher uptake (23.7 ± 1.5%) compared with that for iso-[125I]SGMIB-NB7 (20.4 ± 1.6%) after 1 h incubation at 4 °C, which was significant by a paired t-test (P < 0.001). In contrast, the percentage of initially bound activity that remained internalized at 4 h and 24 h was slightly higher for iso-[125I]SGMIB-NB7 compared to iso- [131I]SGMIB-NB7H6 (P < 0.05). In order to evaluate potential effects of substituting radioiodine with211At, paired-label studies were performed to compare [131I]SGMIB-NB7H6 and [211At]SAGMB-NB7H6. When the radioconjugates were incubated with PC3 PIP cells at 37 °C, no significant differences in cellular uptake or internalization were observed between131I and211At (Fig. 2A). For example, after 2 h incubation, cell uptake of [131I]SGMIB- NB7H6 was 31.5 ± 0.7% of input activity compared with 31.2 ± 1.0% for [211At]SAGMB-NB7H6. Likewise, no significant differences were observed between [131I]SGMIB-NB7H6 and [211At]SAGMB-NB7H6 in the internalization and retention assay (Fig. 2B). To investigate whether NB7 and the PSMA small molecule inhibitor YF2 compete for binding to PSMA, [125I]SGMIB- NB7H6 and [131I]YF2 were incubated with PC3 PIP cells in single- and paired-label format at 37 °C for 2 h. No significant differences in cell uptake or internalization levels between [125I]SGMIB- NB7H6 and [131I]YF2 when the radiotracers were incubated individually or together (P > 0.05). In addition, in the retention assay (1 h initial incubation at 4 °C) 24.8 ± 0.3% of initially cell-bound [125I]SGMIB-NB7H6 remained internalized at 2 h in the presence of [131I]YF2 compared with 25.8 ± 0.5% in the single-label assay. Overall, co-incubation with [131I]YF2 had no significant effect on the cell uptake and internalization of [125I]SGMIB-NB7H6 (P > 0.05).EXAMPLE 4Biodistribution studies

[0181] Results from the single-label biodistribution study of iso-[125I]SGMIB-NB7H6 in athymic mice with subcutaneous PC3 PIP xenografts are summarized in Fig. 3. Tumor uptake was highest at 1 h post injection (7.2 ± 1.5% ID / g), and only declined slightly to 5.2 ± 1.2% ID / g at 24 h. Radioactivity levels in kidneys were high at 1 h (72.5 ± 21.2% ID / g), but rapidly cleared to <0.2 ± 0.0% ID / g at 24 h. Dehalogenation was minimal, as evidenced by thyroid and stomach accumulation of <0.25 % ID / organ at all-time points. The uptake in salivary and lacrimal glands was <1.5 % ID / g at 1 h, which declined to <0.5 % ID / g by 4 h. The results from a paired-label study comparing the biodistribution of [131I]SGMIB-NB7 and iso-|125I]SGMIB-NB7 are summarized in Fig. 4. At all-time points, tumor uptake of the two NB7 radioconjugates was not significantly different. Kidney activity levels of both tracers cleared rapidly and were similar at 1 h. However, at later time points, [131I]SGMIB-NB7 exhibited better kidney clearance with 0.3 ± 0.0 % ID / g seen at 24 h compared with 1.3 ± 0.3 % ID / g for iso-[125I]SGMIB-NB7 (P < 0.05). Stomach, thyroid, salivary gland, and lacrimal gland activity levels were low for both isomer conjugates; however, they were consistently lower for [131I]SGMIB-NB7 with the differences significant (P < 0.05) in salivary gland at 1 and 4 h. Another paired-label study was performed to evaluate the tissuedistribution of [211At]SAGMB-NB7H6 in direct comparison to that for [131I]SGMIB-NB7H6. No significant differences in tumor and kidney activity levels were observed up to 21 h, the time when injected211Athad decayed to -10% ofinjected activity (Fig. 5). However, notable differences were observed in stomach, thyroid, and lacrimal / salivary glands, where21'At activity levels were more than twice those for co-administered [131I]SGMIB-NB7H6. Higher211At uptake levels were also seen in lungs, liver, and spleen. In order to compare the PSMA-targeting ability ofNB7 sdAb with a small molecule PSMA inhibitor, a paired-label biodistribution of [125I] SGMIB-NB7H6 in tandem with [131I]YF2 was performed at 1 and 4 h. Tumor uptake of [125I]SGMIB-NB7H6 was 2-3-fold lower (P < 0.05) than that for co-administered [131I]YF2 at both time points (Fig. 6). However, renal activity levels of [125I]SGMIB-NB7H6 were 4- and 84-fold lower than those observed for [131I]YF2 at 1 and 4 h, respectively; (P < 0.05). Additionally, uptake of [125I]SGMIB-NB7H6 was lower than [131I]YF2 in thyroid, stomach, salivary glands and lacrimal glands (P < 0.05 except for thyroid at 1 h and stomach at 4 h). Tumor-to-kidney ratios at 1 and 4 h for [125I]SGMIB-NB7H6 rapidly increased from 0.2 ± 0.0 at 1 h to 5.8 ± 4.0 at 4 h, values that were higher than those observed for [131I]YF2 (1 h: 0.1 ± 0.0; P < 0.02; 4 h: 0.2 ± 0.1; P < 0.02) (Fig. 7).Discussion

[0182] Herein, the inventors have evaluated NB7 as a PSMA-targeted vector in part because sdAbs offer an intermediately sized molecule between PSMA inhibitors like PSMA-I&T and intact antibodies like J591. Combinations of these two targeting vectors are now being investigated clinically with the goal of minimizing toxicity to normal tissue. Perhaps a PSMA-targeted sdAb alone, with its intermediate properties, might provide a simpler way of achieving this goal. Alternatively, an sdAb would provide a more rapidly diffusing alternative to an intact mAb in therapeutic cocktail with a small molecule PSMA inhibitor. Because NB7 binds to a different site on PSMA than PSMA inhibitors, this should be feasible. Moreover, NB7 exhibits an avidity effect due to its simultaneous interaction with two PSMA monomers so that if one site is detached from PSMA, the other site may remain bound. Regarding selection of a radiolabeling strategy for NB7, previous studies have documented that residualizing radiohalogenated prosthetic agents offer significant advantages compared with radiometals for labeling sdAbs. For example, head-to-head studies have demonstrated comparable tumor uptake with both labeling approaches but considerably higher kidney retention of radioactivity with radiometals. Specifically, the inventors have shown that SGMIB and iso-SGMIB, as well as their211At-labeled analogues, were excellent residualizing prosthetic agents for labeling HER2 -targeted sdAbs. These radiohalogenated sdAbs demonstrated excellent tumor uptake, minimal in vivo dehalogenation and high tumor-to-normal- tissue ratios. In addition to or central objective of evaluating the potential utility of NB7 as ascaffold for developing PSMA-targeted theranostics, the inventors also wished to evaluate variables of potentially broader impact on radiopharmaceutical design: the effects of a His6-tag on kidney activity levels of an sdAb after SGMIB labeling and whether the choice of SGMIB isomer used for radiohalogenation had a significant effect on its in vivo behavior. With radiometals, the presence of His6-tag can lead to high and prolonged kidney levels when used to label sdAbs, which for therapy, could result in dose limiting renal toxicity. For example, in a study conducted by D'Huyvetter et al., evaluating 2Rs 15d, an anti-HER2 sdAb, labeled with177Lu, kidney radioactivity levels decreased 88 % for untagged [177Lu]2Rsl5d compared with that for Myc-His6-tagged [177LU] 2Rsl5d. Similarly, Chatalic et al. reported that another anti-PSMA sdAb, JVZ-007 radiolabeled with11'in displayed substantially higher renal radioactivity levels when Myc-His6- Tag was present at its C-terminus. This has been attributed to the high polarity of the C-terminal of the sdAb with a His6-tag. In contrast, radioactivity levels in the kidneys were similar from 1 to 24 h for AO-[125I]SGMIB-NB7 and iso-[125I]SGMIB-NB7H6 despite the presence of a His6-tag in NB7H6. While the reason for this observation needs to be investigated, the inventors speculate that differences in the renal retention of labeled catabolites generated from proteins labeled with SGMIB-like prosthetic agents and radiometals likely plays a role. When small proteins are labeled with radiometals via either DOTA or DTPA bifunctional chelates, radiometal-chelate-E-lysine catabolites are generated, and these species are retained at high levels in the kidney. On the other hand, the main catabolites generated from proteins labeled using SGMIB are 4-guanidinomethyl- 3-iodobenzoic acid (GMIBA) and its glycine conjugate, both of which rapidly pass though the kidney into the urine. The inventors speculate that the increased polarity induced by the His6-tag leads to increased uptake of sdAbs in the kidney within a few minutes, leading to generation of more radiolabeled catabolites which are retained in the kidney for radiometals and rapidly excreted for SGMIB. This was one of the factors guiding the current selection of21'At as the preferred a- emitter for labeling sdAbs instead of a radiometal like225Ac that when used for sdAb labeling results in high and prolonged kidney activity levels. The inventors note that there is a second factor that could account for the increased kidney retention of radiometals when a His6-tag is present. Although divalent cations such as Ni2+are used most frequently in immobilized metal affinity chromatography, trivalent cations including Ga(III) and Fe(III) as well as Zr(IV) also have been used successfully. Thus, it seems plausible that when sdAb-chelate conjugates are reacted with radiometals like177Lu and225Ac, unanticipated labeling at the His6-tag could occur. This could lead to release of the metal in vivo as well as the generation of different catabolites, which might contribute to enhanced kidney retention of the radiometal. Experiments are planned to investigate these possibilities. The 1,3,5-iso-SGMIB isomer originally was developed to improve precursor and radiolabeling synthesis efficiencies by providing less steric hindrance than the 1,3,4- SGMIBisomer, and this was achieved. When the biodistribution of an intact mAb, trastuzumab, labeled using [131I]SGMIB and iso-|l 25I | SGMIB were compared in paired-label format, no significant differences in tissue distribution were observed. In a subsequent study using HER2 -targeted 5F7 sdAb done in single-label format, the 1,3,5-isomer conjugates for both131I and211At exhibited higher tumor uptake and lower activity levels in thyroid and stomach than their 1,3,4-isomer counterparts. However, the sdAb comparison was not done in paired-label format so the well- known differences that can occur among groups of experimental animals could have played a role. For this reason, the inventors performed paired-label experiments to assess whether changing the position of the guanidinomethyl group in the SGMIB template would affect the residualizing ability of their NB7 radioconjugates. Paired-label studies in PC3 PIP cells demonstrated that total cell uptake and internalization of the two isomer conjugates were nearly identical; consistent with this, no significant isomer dependent differences in PC3 PIP xenograft uptake were observed in vivo. Radioactivity cleared rapidly from normal tissues with both SGMIB conjugates; however, when small differences were significant by paired t-test, activity levels were higher for the iso- [125I]SGMIB-NB7 conjugate. Thus, while both isomers performed well, iso-SGMIB appeared to offer an advantage with anti-HER2 sdAb 5F7. It is plausible that the disparity may be due to differential catabolism of two isomeric conjugates and the differing susceptibility of labeled catabolites towards dehalogenation. Several factors could play a role including microenvironment of the labeling site(s) on NB7 and 5F7, different intracellular routing / processing of HER2- and PSMA-targeted sdAbs, and strain of the mice (athymic for NB7 and SCID for 5F7). Finally, the 5F7 experiments were done in single-label format so differences in variables such as tumor size and HER2 expression level could have played a role. NB7 radioconjugates exhibited excellent binding affinity to the PSMA+ PC3 PIP cell lines with Kd values in the low nanomolar range compared with <0.1 nM affinity to the PSMA molecule as measured previously by surface plasmon resonance. Such differences between affinity to a molecule when in pure form and presented on a cell are not uncommon. The binding affinity for the [131I]SGMIB conjugate ofNB7H6 was better than that for NB7 (1.4 ± 0.4 nM versus 6.4 ± 2.0 nM), which might reflect differences in their purification method. Nonetheless, cell binding and internalization in vitro, and tumor accumulation in vivo were similar for NB7 constructs with and without a His6-tag. There are two previous reports on the development of radiolabeled sdAbs for targeting PSMA. Evazalipour et al. reported a Kd of 4.6 nM for PSMA30, their best sdAb, in unlabeled form but the affinity for "mTc-labeled PSMA30 was not given. Chatalic et al. obtained a Kd value of 27.4 nM for the111In-labeled PSMA targeted sdAb JVZ-007-c-myc-his. With the caveat that different cell lines and xenografts were used in these studies, the affinity values the inventors obtained for the current radiolabeled NB7 sdAbs were comparable or considerably better. Moreover, the NB7 radioconjugates described herein alsoexhibited substantially higher cell uptake and internalization than those reported for the99mTc- and 'in-labeled anti-PSMA sdAb. Importantly, tumor targeting for the NB7 radioconjugates was considerably higher than those reported for99mTc-labeled PSMA30 (1.57 ± 0.38 % ID / g at 90 min) and "'in-labeled JVZ-007-cys (3.70 ± 0.29 % ID / g at 4 h). In addition, renal activity levels for the radiohalogenated NB7 conjugates were considerably lower except in the case of11'in-labeled JVZ- 007-cys when co-administered with lysine and gelofusine. Although131I-labeled sdAbs are currently being evaluated as theranostics in patients, the long-term translational objective of this work is to develop a211At-labeled NB7 conjugate for targeted alpha particle therapy. The current in vitro studies demonstrated that the levels of cellular uptake and intracellular trapping for [211A t]SAGMB-NB7H6 were virtually identical to those for [131I]SGMIB-NB7H6. Moreover, in vivo studies revealed no notable differences in tumor uptake and kidney clearance between [211At]SAGMB-NB7H6 and its131I-labeled analogue at any time point. However,211At activity levels were significantly higher than those for13'l in thyroid, stomach, and lacrimal and salivary gland, tissues known to sequester iodide and also [211At]astatide. On the other hand, the uptake of211At in the thyroid after injection of [211At]SAGMB-NB7H6 was <0.3 % ID at all-time points suggesting that the absolute level of deastatination in vivo was low. It is worth noting that the thyroid and stomach accumulation of211At after injection of [211At]SAGMB-NB7H6 are comparable to those reported previously for [211At]SAGMB-5F7 conjugates, which have been shown to be very effective therapeutically at a single dose in preclinical models. Finally, it should be noted that blocking agents such as saturated solution of potassium iodide are available and are effective at preventing tissue uptake of [211At]astatide and have been used clinically for this purpose . Molecular docking simulation analysis indicated that NB7 binds to human PSMA far from its catalytic active site and at a different site than PSMA small molecule inhibitors suggesting that the two PSMA targeting strategies should not compete. Head-to-head comparisons were performed to confirm this and to directly compare [125I]SGMIB-NB7 and the recently developed small molecule PSMA inhibitor, YF2. The inventors note that the cross reactivity of NB7 and YF2 with murine PSMA has not been determined. No discernible differences in PSMA+ PC3 PIP cell uptake and internalization were observed when the compounds were incubated with the cells together or individually. On the other hand, notable differences were observed in mice with PSMA+PC3 PIP xenografts where [131I]YF2 exhibited 2.5-fold higher uptake than co-administered [125I]SGMIB- NB7. However, the radioactivity levels in kidney for [125I]SGMIB-NB7 were substantially lower than [131I]YF2 with about 2-fold, 4-fold and 84-fold differences observed at 1, 4 and 24 h postinjection, respectively. Renal radiation toxicity after treatment with radiolabeled small molecule PSMA inhibitors can be problematic. Thus, NB7 sdAbs labeled with these guanidino-substituted residualizing prosthetic agents could provide an attractive treatment option, either alone or intandem with a small molecule PSMA, for achieving tumor control while delivering less radiation dose to the kidneys. Preclinical therapeutic efficacy experiments are being planned to evaluate these possibilities.Conclusion

[0183] In summary, the inventors have shown that PSMA-targeted sdAb NB7 could be labeled with radioiodine and211At using the guanidino-bearing residualizing prosthetic agents SGMIB and SAGMB with excellent retention of affinity. Biodistribution studies in athymic mice with PSMA+ PC3 PIP xenografts demonstrated rapid tumor accumulation and clearance from kidneys, accompanied by minimal uptake in other non-targeted organs. A paired-label biodistribution study demonstrated that with SGMIB labeling, that presence of a C-terminal His6-tag on NB7 did not result in significant increase of radioiodine levels in kidneys. In a second head-to-head comparison of NB7 labeled using SGMIB and iso -SGMIB prosthetic agents, no isomer-dependent differences in tumor uptake or meaningful normal tissue levels were observed. In a final paired-label experiment, [125I]SGMIB-NB7 exhibited a 2.5-fold lower tumor uptake than [131I]YF2 but considerably more favorable tumor-to-kidney uptake ratios. Given that its biological properties are compatible with the physical characteristics of21'At. evaluation of the therapeutic potential of211At-labeled NB7 appears warranted.

[0184] All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such a reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

Claims

CLAIMSWhat is claimed is:

1. A conjugate comprising: (i) an antigen-binding polypeptide having a specific binding affinity to prostate specific membrane antigen (PSMA); and (ii) a benzylguanidine prosthetic group comprising a radionuclide, wherein said (i) and (ii) are covalently bound.

2. The conjugate of claim 1, wherein said radionuclide comprises any one of: an alpha emitter, a beta emitter, a gamma emitter, and any combination thereof.

3. The conjugate of claim 1 or 2, wherein said radionuclide comprises any one of: Iodine (I), Astatine (At), and both.

4. The conjugate of claim 3, wherein said I comprises any one of:125I,131I,123I,124I and any combination thereof.

5. The conjugate of claim 3 or 4, wherein said At is21'At.

6. The conjugate of any one of claims 1 to 5, wherein said benzylguanidine prosthetic group comprising said radionuclide is selected from the group consisting of: 4-guanidinomethyl 3- [*I]iodobenzoate, 3-guanidinomethyl 5-[*I]iodobenzoate, 3-[211At]astato-4-guanidinomethyl benzoate, 3-[211At]astato-5-guanidinomethyl benzoate, and any combination thereof, wherein *1 is selected from the group consisting of:125I,13'I,123I,124I, and any combination thereof.

7. The conjugate of any one of claims 1 to 6, wherein said covalently bound is via an amide bond or via succinimide-thioether.

8. The conjugate of any one of claims 1 to 7, wherein said antigen-binding polypeptide is characterized by binding constant (Ka) of at least 104Molar"1sec"1to said PSMA.

9. The conjugate of any one of claims 1 to 8, wherein said antigen-binding polypeptide is characterized by dissociation constant (KD) of 10 to 100 pM to said PSMA.

10. The conjugate of any one of claims 1 to 9, wherein said PSMA comprises the amino acid sequence set forth in SEQ ID NO: 5.

11. The conjugate of any one of claims 1 to 10, wherein said specific binding affinity is to a non-catalytic site of said PSMA.

12. The conjugate of any one of claims 1 to 11, wherein said antigen-binding polypeptide is characterized by molecular weight of less than 25 kDa.

13. The conjugate of any one of claims 1 to 12, wherein said antigen-binding polypeptide is a single-domain antibody (sdAb).

14. The conjugate of any one of claims 1 to 13, wherein said antigen-binding polypeptide comprises the three heavy chain complementary-determining regions (CDRs-H) comprising: CDR-H1 comprising the amino acid sequence GYTDSNYYMS (SEQ ID NO: 1), CDR-H2 comprising the amino acid sequence GVNTGRGSTSYADSVKG (SEQ ID NO: 2), and CDR-H3 comprising the amino acid sequence AACHFCDSLPKTQDEYIL (SEQ ID NO: 3).

15. The conjugate of any one of claims 1 to 14, wherein said antigen-binding polypeptide comprises the amino acid sequence:QVQLQESGGGSVQAGGSLRLSCTAPGYTDSNYYMSWFRQAPGKEREWVAGVNTGRGS TSYADSVKGRFTISQDNAKNTMFLQMNSLKPEDTAIYYCAV AACHFCDSLPKTQDEYIL WGQGTQVTVSSAAAYPYDVPDYGS (SEQ ID NO: 4).

16. A composition comprising a theranostically effective amount of the conjugate of any one of claims 1 to 15, and a theranostically acceptable carrier.

17. The composition of claim 16, further comprising at least one agent having specific binding affinity to a catalytic site of said PSMA.

18. The composition of claim 17, wherein said at least one agent is a small molecule.

19. The composition of claim 17 or 18, wherein said at least one agent is YF2.

20. The composition of any one of claims 16 to 19, for use in treatment of a disease, diagnosis thereof, or both, in a subject in need thereof.

21. A combination comprising the conjugate of any one of claims 1 to 15 and at least one agent having specific binding affinity to a catalytic site of said PSMA, for use in diagnosis, treatment, or both, of a PSMA-associated disease or disorder, in a subject in need thereof.

22. The combination for use according to claim 21, wherein said conjugate is formulated within a first pharmaceutical composition and said at least one agent is formulated within a second pharmaceutical composition.

23. The combination for use according to claim 21 or 22, wherein said at least one agent is a small molecule.

24. The combination for use according to any one of claims 21 to 23, wherein said at least one agent is YF2.

25. A method for imaging PSMA in a biological sample, the method comprising contacting a biological sample comprising PSMA with an effective amount of the conjugate of any one of claims 1 to 15 or the composition of claim 16, and detecting the presence of said PSMA in said biological, thereby imaging PSMA in the biological sample.

26. The method of claim 25, wherein said biological sample is derived or obtained from a subject.

27. A method for detecting PSMA in a subject in need thereof, the method comprising administering to said subject an effective amount of the conjugate of any one of claims 1 to 15 or the composition of claim 16, and detecting the presence of said PSMA in the subject.

28. The method of claim 26 or 27, wherein said subject is afflicted with a PSMA-associated disease or disorder.

29. The method of claim 28, wherein said PSMA-associated disease or disorder is prostate cancer.

30. A method for preparing the conjugate of any one of claims 1 to 15, the method comprising contacting said antigen-binding polypeptide having a specific binding affinity to PSMA with an effective amount of a benzylguanidine prosthetic group precursor comprising a radionuclide under conditions suitable for formation of a covalent bond between said antigen-binding polypeptide having a specific binding affinity to PSMA and said guanidino-substituted prosthetic group precursor comprising radionuclide, thereby preparing the conjugate.

31. The method of claim 30, wherein said benzylguanidine prosthetic group precursor comprising said radionuclide is selected from the group consisting of: A’-succinimidyl 4- guanidinomethyl 3-[*I]iodobenzoate isomer), A’-succinimidyl 3- guanidinomethyl 5-[*I]iodobenzoate ( isomer), A’-succinimidyl 3- [211At]astato-4-guanidinomethyl benzoate-succinimidyl 3-[2 !1At]astato- 5-guanidinomethyl benzoate (iso-[211At]SAGMB), wherein *1 is selected from the group consisting of:125I,131I,1231,124I, and any combination thereof.

32. The method of claim 30 or 31, wherein said conditions comprise subjection to: pH of about 8.5, room temperature for a period of 5-60 minutes, or both.

33. The method of any one of claims 30 to 32, further comprising a step comprising isolating, purifying, or both, the prepared conjugate.

34. The method of any one of claims 30 to 33, further comprising a step before said contacting comprising conjugating said guanidino-substituted prosthetic group precursor to said radionuclide.

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