PSMA8 nanobody compositions and methods of use thereof

Abstract

Provided herein are nanobodies that specifically bind to PSMA8 and various compositions of such nanobodies. Also provided are method of using the nanobodies in therapeutics and diagnostics for cancer.

Description

Attorney Docket No. 090723-1544373-MDA25-056BPCTPSMA8 NANOBODY COMPOSITIONS AND METHODS OF USE THEREOFCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of United States Provisional Patent Application Serial No. 63 / 759,829, filed February 18, 2025, and United States Provisional Patent Application Serial No. 63 / 788,564, filed April 14, 2025. The entire contents of these applications are incorporated herein by this reference as if fully set forth herein.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing, which has been submitted herewith and is hereby incorporated by reference in its entirety. The .xml copy, created on February 18, 2026, is named “MDA25-056BPCT-Sequence_listing” and is 8,210 bytes in size.BACKGROUND

[0003] Cancer has a great impact on society and is one of the leading causes of death worldwide. For example, over 2 million new cases of cancer were diagnosed and over 600,000 cancer-related deaths occurred in 2024 in the United States alone. Numerous treatment options are available for treating patients with cancer, but many of the current treatments available have the undesirable effect of damaging healthy cells or tissue in addition to treating the cancer cells. Targeted treatments, which recognize and destroy cancer cells, while sparing healthy cells and tissues can achieve this purpose. Therapeutic monoclonal antibodies have been developed to recognize specific biomarkers on certain cancer cells. These antibodies can be used to target cancer cells for destruction, for example, through the delivery of radionuclides or cytotoxic drugs. Due to the size of traditional monoclonal antibodies, it can be difficult for antibodies to penetrate non-vascularized tissue and solid tumors, and to bind to surface antigens that contain grooves or cavities. Thus additional targeted therapeutic treatments would be advantageous.SUMMARY

[0004] The Summary is provided to introduce a selection of concepts that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used as an aid in limiting the scope of the claimed subject matter.1USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCT

[0005] In one aspect, a nanobody is provided. In some embodiments, the nanobody comprises a CDR1 comprising SEQ ID NO: 2, a CDR2 comprising SEQ ID NO: 3, and a CDR3 comprising SEQ ID NO: 4. In some embodiments, the nanobody has at least 92% identity to SEQ ID NO: 1. In certain embodiments, the nanobody comprises SEQ ID NO: 1. In some embodiments, the nanobody is conjugated or fused to an imaging agent, a cytotoxic agent, a metal, or a reactive moiety. In some embodiments, the nanobody is conjugated to an imaging agent. The imaging agent is a fluorophore or a radioactive moiety. In certain embodiments, the nanobody is conjugated or fused to a radioactive moiety. The radioactive moiety may be selected from Zr-89, Cu-64, F-18, Y-90, Lu-177, At-211, Ac-225, or Pb-212. In some embodiments, the nanobody is an immune conjugate. In certain embodiments, the nanobody is a nanobody-drug conjugate.

[0006] In another aspect, a pharmaceutical composition is provided. The pharmaceutical composition comprises the isolated nanobody of any one of the embodiments described above and a pharmaceutically acceptable carrier.

[0007] In yet another aspect, a method of treating cancer in a patient is provided. In some embodiments, the method of treating a cancer in a patient comprises administering, to the patient, an anti-tumor effective amount of the pharmaceutical composition comprising the isolated nanobody of any one of the embodiments described above and a pharmaceutically acceptable carrier. In some embodiments, the composition comprises the isolated nanobody conjugated to a therapeutic agent. In some embodiments, the therapeutic agent is at least one of a cytotoxic agent, a chemotherapeutic agent, an immunosuppressive agent, or a radioactive moiety. In some embodiments, the cancer has been determined to express an elevate level of PSMA8 relative to a healthy tissue. In certain embodiments, the cancer is selected from a group consisting of a lung cancer, a stomach cancer, a blood cancer, an ovarian cancer, a bladder cancer, a melanoma, a prostate cancer, a breast cancer, a glioma, and a lymphoma. In some embodiments, the patient has previously failed to respond to an immune checkpoint inhibitor. In some embodiments, the patient has relapsed. In certain embodiments, the method further comprises administering at least a second anti-cancer therapy. In certain embodiments, the second anti-cancer therapy is a chemotherapy, molecular targeted therapy, immunotherapy, radiotherapy, radioimmunotherapy, phototherapy, gene therapy, surgery, hormonal therapy, epigenetic modulation, anti -angiogenic therapy or cytokine therapy.

[0008] In another aspect, a method of detecting a presence of PSMA8 in a biological sample is provided. In some embodiments, the method comprises contacting the sample with the nanobody described herein and detecting an amount of binding of the isolated nanobody as a 2USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCTdetermination of the presence of PSMA8 in the sample. In some embodiments, the biological sample comprises cancer cells. In some embodiments, the biological sample comprises a tumor sample of a tumor from a subject.

[0009] In another aspect, a method of imaging a tumor in a subj ect with a PSMA8-expressing cancer is provided. The method comprises administering to the subject the isolated nanobody as described herein conjugated to an imaging label, and detecting the imaging label in the subject to obtain an image of the tumor. In some embodiments, the imaging label is a fluorophore or a radioactive moiety. In some embodiments, the radioactive moiety is Zr-89, Cu-64, F-18, Y-90, Lu-177, At-211, Ac-225, or Pb-212.

[0010] In yet another aspect, a method of monitoring response of a subject with a PSMA8-expressing cancer to cancer therapy. In some embodiments, the method comprises detecting a presence of a tumor in the subject at a first time point by administering the nanobody as described herein conjugated to a first radioactive moiety and obtaining a first image of the tumor; and treating the subject by administering a therapeutically effective amount of the nanobody as described herein conjugated to a second radioactive moiety; and detecting the presence or absence of the tumor in the subject at a second time point after the subject has been treated with anti-cancer therapy by administering the nanobody conjugated to the first radioactive moiety and obtaining a second image of the tumor; and comparing the first image to the second image to determine whether a change in tumor size has occurred. In some embodiments, the first radioactive moiety and the second radioactive moiety are independently selected from a group consisting of89Zr,1311,1251,1231,1UI, "mTc,90Y,186Re,188Re,32P,153Sm,67Ga,2O1T1,77Br,18F,161Tb,225Ac,161Tb / 225Ac,177Lu,134Ce,140Nd,169Er,134Ce / 134La, and140Nd / 140Pr. In some embodiments, the method includes detecting the presence or absence of the tumor in the subject at a third time point after the subject has been treated with anti-cancer therapy by administering the nanobody conjugated to the first radioactive moiety and obtaining a third image of the tumor; and comparing the first image to the second image to determine whether a change in tumor size has occurred. In some embodiments, the anti-cancer therapy is a chemotherapy, molecular targeted therapy, immunotherapy, radiotherapy, radioimmunotherapy, phototherapy, gene therapy, surgery, hormonal therapy, epigenetic modulation, anti -angiogenic therapy or cytokine therapy.3USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCTBRIEF DESCRIPTION OF THE DRAWINGS

[0011] The present application includes the following figures. The figures are intended to illustrate certain embodiments and / or features of the compositions and methods, and to supplement any description(s) of the compositions and methods. The figures do not limit the scope of the compositions and methods, unless the written description expressly indicates that such is the case.

[0012] FIG. 1 provides BLI OCTET assay results for an exemplary PSMA8 nanobody (Dl-7-93, NB31), demonstrating the ability of Dl-7-93 to bind to PSMA8.

[0013] FIGS. 2A-2C are histograms of fluorescence-activated cell sorting (FACS) assay results demonstrating the validation of an exemplary PSMA8 nanobody (Dl-7-93, NB31).FIG. 2A is a histogram showing FACS assay results for a high PSMA8 expressing cell line, H2195 (a small cell lung cancer cell line), after incubation with Dl-7-93 or after no treatment.FIG. 2B is a histogram showing FACS assay results for a high PSMA8 expressing cell line, SNU-1 (a gastric cancer cell line), after incubation with Dl-7-93 or after no treatment. FIG.2C is a histogram showing FACS assay results for a low PSMA8 expressing cell line, THP-1 (a leukemia cancer cell line), after incubation with Dl-7-93 or after no treatment.

[0014] FIGS. 3A-3B are histograms showing FACS assay results demonstrating the selective localization of an exemplary PSMA8 nanobody (Dl-7-93, NB31) in a human gastric cancer (SNU1) cell line-derived xenograft model. SNU1 is a high PSMA8 expressing cell line.FIG. 3A shows FACS assay results for a single cell suspension from an ex vivo tumor sample from the mice, 72 hours post-injection with Dl-7-93 or control (dye). FIG. 3B shows FACS assay results for a single cell suspension from an ex vivo bone marrow sample from the mice, 72 hours post-injection with Dl-7-93 or control (dye).

[0015] FIG. 4 is a histogram showing FACS assay results demonstrating the validation of an exemplary PSMA8 nanobody (Dl-7-93, NB31) in a human pancreatic (PANC-1) cell line-derived xenograft model. PANC-1 is a cell line that does not express PSMA8. The histogram shows results for a single cell suspension from an ex vivo tumor sample from mice, 72 hours after injection with Dl-7-93 or control (dye), demonstrating the nanobody does not localize to tumors that do not express PSMA8.

[0016] FIGS. 5A-5D are confocal microscopy images demonstrating the internalization of an exemplary PSMA8 nanobody, Dl-7-93, following a 2-hour (FIGS. 5A-5B) or 24-hour incubation (FIGS.5C-5D) with SNU-1 cells. FIGS.5A and 5C show SNU-1 cells stained with DAPI and lysosome stain. FIGS. 5B and 5D show SNU-1 cells after incubation with an4USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCTexemplary nanobody (Dl-7-93, conjugated to the CF647 fluorophore) after 2-hours (FIG. 5B) and after 24-hours (FIG. 5D).DETAILED DESCRIPTION

[0017] The following description recites various aspects and embodiments of the present compositions and methods. No particular embodiment is intended to define the scope of the compositions and methods. Rather, the embodiments merely provide non-limiting examples of various compositions and methods that are at least included within the scope of the disclosed compositions and methods. The description is to be read from the perspective of one of ordinary skill in the art; therefore, information well known to the skilled artisan is not necessarily included.I. Introduction

[0018] Peptides from testis-specific proteasome subunit alpha-type 8 (PSMA8) were discovered on the surface of certain cancer cell lines using mass spectrometry. Due to its high expression in different cancer types and minimal expression in normal tissues, the effective and specific targeting of PSMA8 is of interest in anti-cancer drug development.

[0019] Provided herein are nanobodies that bind specifically to PSMA8. Also provided herein are various compositions of such nanobodies and methods of their use. Because the nanobodies have high binding affinity to PSMA8, provided herein are methods of using the nanobodies or nanobody compositions of this disclosure for diagnostic and / or therapeutic purposes for cancer, as well as for detecting PSMA8 protein.II. Definitions

[0020] Unless otherwise defined, all terms of art, notations, and other scientific or medical terms or terminology used herein are intended to have the meanings commonly understood by those of ordinary skill in the art. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not be construed as representing a substantial difference over the definition of the term as generally understood in the art.

[0021] Articles “a” and “an” are used herein to refer to one or to more than one (i.e., at least one) of the grammatical object of the article. By way of example, “an element” means at least one element and can include more than one element.

[0022] The use herein of the terms “including,” “comprising,” or “having,” and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof as well as additional elements. Embodiments recited as “including,” “comprising,” or “having” certain5USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCTelements are also contemplated as “consisting essentially of and “consisting of those certain elements. As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations where interpreted in the alternative (“or”).

[0023] As used herein, the transitional phrase “consisting essentially of’ (and grammatical variants) is to be interpreted as encompassing the recited materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention. See, e.g., In re Herz, 537 F.2d 549, 551-52 (CCPA 1976) (emphasis in the original); see also MPEP § 2111.03. Thus, the term “consisting essentially of’ as used herein should not be interpreted as equivalent to “comprising.”

[0024] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.

[0025] The terms “about” and “approximately” as used herein shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20% (%); preferably, within 10%; and more preferably, within 5% of a given value or range of values. Any reference to “about X” or “approximately X” specifically indicates at least the values X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, and 1.05X. Thus, expressions “about X” or “approximately X” are intended to teach and provide written support for a claim limitation of, for example, “0.98X.” Numerical quantities given herein are approximate unless stated otherwise, meaning that the term “about” or “approximately” can be inferred when not expressly stated. When “about” is applied to the beginning of a numerical range, it applies to both ends of the range.

[0026] The term “theranostic” or “theranostics” as used herein shall generally mean a medical technique that combines diagnosis and treatment to treat cancer or another disease or condition. In nuclear medicine, the method includes the use of radioactive compounds to image biologic phenomena (by detecting expression of specific disease targets such as cell surface receptors), and then the use of specifically designed agents to deliver ionizing radiation to the 6USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCTtissues that express these targets. For example, theranostics may refer to a two-pronged approach to diagnosing and treating cancer through the use of radiotracers. A “theranostic pair” refers to two agents that bind to a target cell, one agent that is used to image the target cell and one agent that is used to deliver ionizing radiation to the target cell. For example, a theranostic pair may include a first radionuclide conjugated nanobody for imaging a target cell to which the nanobody binds and a second radionuclide conjugated nanobody for delivering ionizing radiation to the target cell.III.PSMA8 Nanobody CompositionsPSMA8 Nanobodies

[0027] In one aspect, the present disclosure provides nanobodies that bind specifically to proteasome subunit alpha-type 8 (PSMA8) (also known as PSMA7L, proteasome 20 S subunit alpha 8, and proteasome subunit alpha type-7-like). A nanobody can be described by the antigen to which it specifically binds. For example, as used herein, the terms “PSMA8 nanobody” and “anti-PSMA8 nanobody” both refer to a nanobody that specifically binds PSMA8. As used herein, the term “nanobody” or “single-domain antibody” encompasses, but is not limited to, single-domain antibodies derived from any type of antibody that specifically bind to a target antigen. For example, nanobodies or single-domain antibodies (sdAbs) can be engineered from heavy chain antibodies found in camelids or cartilaginous fishes. In other embodiments, the sdAb can be split from dimeric variable domains of common antibodies, such as from humans or mice, into monomers. In some embodiments, nanobodies may be derived from light chains of the dimeric variable domains and may bind specifically to target epitopes.

[0028] Nanobodies may be derived from any species including, but not limited to mouse, rat, guinea, pig, human, camel, llama, fish, shark, goat, rabbit, and bovine. A sequence derived from a heavy chain antibody naturally devoid of light chain is known herein as a VHH or nanobody to distinguish it from the conventional VH of four chain immunoglobulins as described above. Such a VHH molecule can be derived from antibodies raised in Camelidae species (e.g., camel, llama, dromedary, alpaca, and guanaco) or other species besides Camelidae.

[0029] A nanobody includes only a single domain variable region and typically has a molecular mass of about 15 kDa. Variable regions of nanobodies differ in sequence and are used in the binding and specificity of each particular nanobody for its particular antigen. However, the variability is not usually evenly distributed through the variable domains of nanobodies. It is typically concentrated in three segments called complementarity determining7USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCTregions (CDRs) or hypervariable regions in the variable domain. The more highly conserved portions of the variable domains are called the framework regions (FRs). The nanobodies each comprise four FRs, largely adopting a P-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the P-sheet structure. The CDRs in each chain are held together in close proximity by the FRs and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of nanobodies. Nanobodies are characterized by the absence of the light chain constant and variable domains found in common antibody structures, such as human antibodies. While the constant region of a common antibody is not involved directly in binding of the antibody to an antigen, the constant region exhibits various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity. Thus, a nanobody cannot produce cytotoxic effects such as antibody dependent cell cytotoxicity (ADCC) or cell-dependent cytotoxicity (CDC). A nanobody, which includes the VH (VHH or variable domain of new antigen receptor (VNAR)), comprises three CDRs and four FRs, arranged in the following order (from N-terminus to C-terminus): FR1 -CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4. The CDRs are involved in antigen-binding and confer antigen specificity and binding affinity to the nanobody. (See Kabat et al. (1991) Sequences of Proteins of Immunological Interest 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD.) CDR sequences of the nanobody may be designated as CDR1, CDR2, and CDR3. Nanobodies include four hallmark amino acids in the framework-2 region, and a longer third antigen-binding loop. For therapeutic applications, the camelid specific amino acid sequences in the framework have to be mutated to their human heavy chain variable equivalent (e.g., humanized). In some embodiments, the nanobody described herein is a humanized nanobody.

[0030] By contrast, native common antibodies are usually heterotetrametric glycoproteins, composed of two identical light (L) chains and two identical heavy (H) chains, adding up to a total molecular mass of 150 kDa. Typically, each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH or VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL or VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains. The light 8USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCTchains of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda (1), based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. In some embodiments, the nanobody, as described herein, may be derived from antibodies, or antigen binding fragments.

[0031] The term “epitope,” as used herein, means a component of an antigen capable of specific binding to a nanobody. Such components optionally comprise one or more contiguous amino acid residues and / or one or more non-contiguous amino acid residues. Epitopes frequently consist of surface-accessible amino acid residues and / or sugar side chains and can have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. An epitope can comprise amino acid residues that are directly involved in the binding, and other amino acid residues, which are not directly involved in the binding. The epitope to which an antigenbinding protein binds can be determined using known techniques for epitope determination such as, for example, testing for binding to antigen variants with different point mutations.

[0032] As used herein, the terms “binds specifically to,” “specifically binds to,” “specific for,” “targets,” “binds selectively to,” and “selective for” PSMA8 or an isoform or an epitope of a PSMA8 protein, and the like, mean binding that is measurably different from a non-specific or non-selective interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule. Specific binding can also be determined by competition with a control molecule that is similar to the target, such as an excess of non-labeled target. In that case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by the excess non-labeled target.

[0033] In some embodiments, the nanobody comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence of the variable region of SEQ ID NO:1. In some embodiments, the variable region sequence of the nanobody is set forth in Table 1. In some embodiments, the CDR sequences in the variable domain listed in Table 1 are indicated by bold and underlined text. In some embodiments, the nanobody comprises a variable region having at least 90%9USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCTsequence identity (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 1.Table 1. Amino acid sequence of a PSMA8 nanobody.

[0034] The amino acid sequences of the CDRs and framework regions can be determined using various well-known definitions in the art, e.g., Kabat, Chothia, international ImMunoGeneTics database (IMGT), AbM, and observed antigen contacts (“Contact”). In some embodiments, CDRs are determined according to the IMGT definition. See Brochet et al., 2008, Nucl. Acids Res. 36:W503-508. In some embodiments, CDRs are determined by a combination of Kabat, Chothia, and / or Contact CDR definitions.

[0035] In some embodiments, the nanobodies as provided in this disclosure comprise the CDR1, CDR2, and CDR3 sequences listed in Table 2.Table 2. Complementarity determining regions (CDRs) of select PSMA8 nanobody.

[0036] The skilled artisan would understand that any of the CDRs described herein can be combined in any combination to create an isolated nanobody. In some embodiments, provided herein is an isolated nanobody, wherein the nanobody comprises a CDR1 amino acid sequence comprising SEQ ID NO: 2, a CDR2 amino acid sequence comprising SEQ ID NO: 3, and a CDR3 amino acid sequence comprising SEQ ID NO: 4.

[0037] In some embodiments, provided herein is an isolated nanobody, wherein the nanobody comprises a sequence having at least 90% sequence identity (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 1. In some embodiments, provided herein is an isolated nanobody, wherein the nanobody comprises SEQ ID NO: 1.

[0038] In some embodiments, provided herein is an isolated nanobody, wherein the nanobody comprises a sequence having at least 90% sequence identity (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 1 and comprising a CDR1 amino acid sequence comprising SEQ ID NO: 2, a CDR2 amino acid10USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCTsequence comprising SEQ ID NO: 3, and a CDR3 amino acid sequence comprising SEQ ID NO: 4.

[0039] In each case, where a specific amino acid sequence is recited, embodiments comprising a sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the recited sequence are also provided.

[0040] The amino acid residue sequences provided herein are set forth in single-letter amino acid code which can be used interchangeably with three-letter amino acid code. An amino acid refers to any monomer unit that can be incorporated into a peptide, polypeptide, or protein. The twenty natural or genetically encoded alpha-amino acids are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamine (Gin or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (He or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Vai or V). The structures of these twenty natural amino acids are shown in, e.g., Stryer et al., 2002, Biochemistry, 5thed., Freeman and Company. The term amino acid also includes unnatural amino acids, modified amino acids (e.g., having modified side chains and / or backbones), and amino acid analogs.

[0041] As with all peptides, polypeptides, and proteins, including fragments thereof, it is understood that additional modifications in the amino acid sequence of the embodiments of the PSMA8-specific nanobody described herein, can occur that do not alter the nature or function of the nanobody. Such modifications include conservative amino acids substitutions, such that each recited sequence optionally contains one or more conservative amino acid substitutions. The list provided below identifies groups that contain amino acids that are conservative substitutions for one another; these groups are exemplary as other conservative substitutions are known to those of skill in the art:1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E);3) Asparagine (N), Glutamine (Q);4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);7) Serine (S), Threonine (T); and8) Cysteine (C), Methionine (M).11USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCT

[0042] By way of example, when an aspartic acid at a specific residue is mentioned, also contemplated is a conservative substitution at the residue, for example, glutamic acid. Nonconservative substitutions, for example, substituting a proline with glycine or substituting a lysine with an asparagine, are also contemplated.

[0043] In some embodiments, the isolated nanobody has an identical sequence to the sequence produced by the methods described herein and, in the Examples, below. In some embodiments, the sequence of the isolated nanobody comprises one or more modifications, e.g., amino acid substitutions, deletions, or insertions.

[0044] In some embodiments, a nanobody can also comprise, e.g., a scaffold protein. These proteins are generally obtained through combinatorial chemistry-based adaptation of preexisting antigen-binding proteins. For example, the binding site of human transferrin for human transferrin receptor can be diversified using the system described herein to create a diverse library of transferrin variants, some of which have acquired affinity for different antigens. See, e.g., Ali et al., 1999, J. Biol. Chem. 274:24066-73. The portion of human transferrin not involved with binding the receptor remains unchanged and serves as a scaffold, like framework regions of antibodies, to present the variant binding sites. The libraries are then screened, as an antibody library is screened, and in accordance with the methods described herein, against a target antigen of interest to identify those variants having optimal selectivity and affinity for the target antigen. See, e, g., Hey et al., 2005, TRENDS Biotechnol. 23(I0):5I4-522. The same principles can be applied to PSMA8 epitopes according to the present disclosure.

[0045] One of ordinary skill in the art would appreciate that the scaffold protein can include, e.g., all or part of the Z domain of S. aureus protein A, human transferrin, human tenth fibronectin type III domain, kunitz domain of a human trypsin inhibitor, human CTLA-4, an ankyrin repeat protein, a human lipocalin (e.g., anticalins, such as those described in, e.g., International Application Publication No. WO2015 / 104406), human crystallin, human ubiquitin, or a trypsin inhibitor from E. elaterium.

[0046] In some embodiments, the nanobody can be conjugated to a heterologous moiety. The heterologous moiety can be, e.g., a heterologous polypeptide, a therapeutic agent (e.g., a toxin or a drug), a photosensitizer, or a detectable label such as, but not limited to, a radioactive label, an enzymatic label, a fluorescent label, a heavy metal label, a luminescent label, or an affinity tag such as biotin or streptavidin. In some embodiments, the heterologous moiety is a nanobody that specifically binds to a different target, and such a conjugated nanobody is a type of nanobody composition that can be referred to as a bispecific nanobody. Additional suitable 12USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCTheterologous polypeptides include, e.g., an antigenic tag (e.g., FLAG (DYKDDDDK) (SEQ ID NO: 5), polyhistidine (6-His; HHHHHH (SEQ ID NO: 6)), hemagglutinin (HA; YPYDVPDYA (SEQ ID NO: 7)), glutathione-S-transferase (GST), or maltose-binding protein (MBP)) for use in purifying the nanobody. Heterologous polypeptides also include polypeptides (e.g., enzymes) that are useful as diagnostic or detectable markers, for example, luciferase, a fluorescent protein (e.g., green fluorescent protein (GFP)), or chloramphenicol acetyl transferase (CAT). A suitable photosensitizer (also referred to as a photoabsorber is, for example, IRDye700DX (IR700). Suitable radioactive labels include, e.g.,32P,33P,14C,125I,1311,35S, and3H. Suitable fluorescent labels include, without limitation, fluorescein, fluorescein isothiocyanate (FITC), green fluorescent protein (GFP), DyLight™ 488, phycoerythrin (PE), propidium iodide (PI), PerCP, PE-Alexa Fluor® 700, Cy5, allophycocyanin, and Cy7. Luminescent labels include, e.g., any of a variety of luminescent lanthanide (e.g., europium or terbium) chelates. For example, suitable europium chelates include the europium chelate of di ethylene triamine pentaacetic acid (DTP A) or tetraazacyclododecane-l,4,7,10-tetraacetic acid (DOTA). Enzymatic labels include, e.g., alkaline phosphatase, CAT, luciferase, and horseradish peroxidase. Another labeling technique which may result in greater sensitivity consists of coupling the nanobody to low molecular weight haptens. These haptens can then be specifically altered by means of a second reaction. For example, it is common to use haptens such as biotin, which reacts with avidin, or dinitrophenol, pyridoxal, or fluorescein, which can react with specific antihapten antibodies. Additional acceptable heterologous moieties are described below in Section VI.

[0047] Two proteins (e.g., a nanobody and a heterologous moiety) can be cross-linked using any of a number of known chemical cross linkers. Examples of such cross linkers are those that link two amino acid residues via a linkage that includes a “hindered” disulfide bond. In these linkages, a disulfide bond within the cross-linking unit is protected (by hindering groups on either side of the disulfide bond) from reduction by the action, for example, of reduced glutathione or the enzyme disulfide reductase. One suitable reagent, 4-succinimidyloxycarbonyl-a-methyl-a(2-pyridyldithio) toluene (SMPT), forms such a linkage between two proteins utilizing a terminal lysine on one of the proteins and a terminal cysteine on the other. Heterobifunctional reagents that cross-link by a different coupling moiety on each protein can also be used. Other useful cross-linkers include, without limitation, reagents which link two amino groups (e.g., N-5-azido-2-nitrobenzoyloxysuccinimide), two sulfhydryl groups (e.g., 1,4-bis-maleimidobutane), an amino group and a sulfhydryl group (e.g., m-13USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCTmaleimidobenzoyl-N-hydroxysuccinimide ester), an amino group and a carboxyl group (e.g., 4-[p-azidosalicylamido]butylamine), and an amino group and a guanidinium group that is present in the side chain of arginine (e.g., p-azidophenyl glyoxal monohydrate).

[0048] Methods for conjugating a fluorescent label (sometimes referred to as a fluorophore) or other heterologous moiety to a protein (e.g., a nanobody) are known in the art of protein chemistry. For example, fluorophores can be conjugated to free amino groups (e.g., of lysines) or sulfhydryl groups (e.g., cysteines) of proteins using succinimidyl (NHS) ester or tetrafluorophenyl (TFP) ester moieties attached to the fluorophores. In some embodiments, the fluorophores can be conjugated to a heterobifunctional cross-linker moiety such as sulfo-SMCC. Suitable conjugation methods involve incubating a nanobody protein with the fluorophore under conditions that facilitate binding of the fluorophore to the protein. See, e.g., Welch and Redvanly, (2003), Handbook of Radiopharmaceuticals: Radiochemistry and Applications, John Wiley and Sons.

[0049] In some embodiments, the nanobody can be modified, e.g., with a moiety that improves the stabilization and / or retention of the nanobody in circulation, e.g., in blood, serum, or other tissues. For example, the nanobody described herein can be PEGylated in a similar manner to that of antibodies as described in, e.g., Lee et aP 1999, Bioconjug. Chem. 10(6): 973-78; Kinstler et al., 2002, Advanced Drug Deliveries Reviews 54:477-485; and Roberts et al., 2002, Advanced Drug Delivery Reviews 54:459-476, or HESylated (Fresenius Kabi, Germany) (see, e.g., Pavisic et al., 2010, Int. J. Pharm. 387(1-2): 110-119). The stabilization moiety can improve the stability, or retention of, the nanobody by at least 1.5 fold (e.g., at least 2, 5, 10, 15, 20, 25, 30, 40, or 50 fold, or more).

[0050] In some embodiments, the nanobody described herein can be glycosylated. In some embodiments, a nanobody described herein can be subjected to enzymatic or chemical treatment, or produced from a cell, such that the nanobody has reduced or absent glycosylation. Similarly, nanobodies may be produced or isolated from a variety of cells. The cells may produce nanobodies having different patterns or levels of glycosylation. The nanobody may also be partially glycosylated, i.e. having less glycosylation than fully glycosylated nanobodies, with certain glycosylation sites being glycosylated and other (potential) glycosylation sites being non-glycosylated.Radiolabels

[0051] In some embodiments, the disclosed PSMA8 nanobodies are conjugated to a radiolabel. The radiolabel may be selected, for example, from a group consisting of277Ac,211At,128Ba,131Ba,7Be,204Bi,205Bi,206Bi,76Br,77Br,82Br,109Cd,47Ca,nC,14C,36C1,48Cr,51Cr,14USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCT62Cu,64Cu,67Cu,165Dy,155EU,18F,153Gd,66Ga,67Ga,68Ga,72Ga,198Au,3H166Ho,mIn,113mIn,115mIn,123I,125I,131I,189Ir,191mIr,192Ir,194Ir,52Fe,55Fe,59Fe,177Lu,150,191m’1910s,109Pd,32P,&"65Zn,89Zr. In some embodiments, the radioactive moiety is selected from a group consisting of161Tb,225Ac,161Tb / 225Ac,89Zr,177Lu,134Ce,140Nd,169Er,134Ce / 134La, and140Nd / 140Pr. In some embodiments, the radioisotope is89Zr,131I,125I,123I,inI, "mc,90Y,186Re,188Re,32P,153Sm,67Ga,2O1T1,77Br, or18F. In some embodiments, more than one radiolabeled nanobody may be produced according to methods that are generally known in the art. For example, in some embodiments, the radiolabeled nanobody as described herein may include a PSMA8-specific nanobody conjugated to [89Zr], [161Tb], or [225Ac], In some embodiments, a nanobody described herein is used in the methods described below, which may include administering a first radiolabeled nanobody for imaging a tumor in a subject and subsequently administering a second radiolabeled nanobody for treatment of a subject. In some embodiments, the [89Zr], [161Tb], and [225Ac] radiolabeled nanobodies may independently be used for imaging and therapeutic treatment of a subject with cancer. In some embodiments, the first radiolabeled nanobody and second radiolabeled nanobody may include the same radiolabel (e.g., both [89Zr]). In some embodiments, the radiolabel for each nanobody is different. Such procedures for labeling biological agents with the radioactive isotopes are generally known in the art.

[0052] The PSMA8 radiolabeled nanobody as described herein may rapidly internalize in a PSMA8+ tissue. Given the internalization characteristics as described further below, more than one radiolabeled nanobody may be generated as described above. The different isotopes used in the radiolabeled nanobody for treatment may individually have unique mechanisms of damage. For example, a [161Tb]-labeled nanobody may have a dual emission of medium energy beta particles and Auger electrons. Conversely, a [225Ac]-labeled nanobody may emit high linear alpha particles. In some embodiments, a combined treatment including a [161Tb]-labeled nanobody and a [225Ac]-labeled nanobody may produce a competitive environment of the beta and alpha particles with the Auger electrons, potentially reducing toxicity of the radiolabeled nanobody treatment options. In some embodiments, the radiolabeled nanobody described herein may be generated at least in part based on the type of particle and electron emission from the isotopes. In some embodiments, treatment plans may be generated based on the emitted particles from the isotopes. Thus, described herein may include patient specific and tumor specific radiolabeled nanobody and methods of treating such cancer.15USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCT

[0053] Techniques for conjugating a therapeutic moiety (e.g., a radioactive moiety) to a PSMA8-specific nanobody may be similar to the techniques for conjugating a therapeutic moiety to an antibody, such methods are well known. See, for example, Arnon et al., 1985, Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56; Hellstrom et al., 1987, Controlled Drug Delivery (2nd Ed.), Robinson etal. (eds.), pp. 623-53; Thorpe, 1985, Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506; “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy” In: Monoclonal Antibodies For Cancer Detection And Therapy, (Baldwin etal. eds.), pp. 303-316 (1985), and Thorpe et al., 1982, Immunol. Rev. 62:119-158. Similarly, one skilled in the art may modify known techniques for conjugating a therapeutic moiety to an antibody for the purpose of producing a nanobody conjugate.

[0054] In some embodiments, a radioactive label can be directly conjugated to the amino acid backbone of the nanobody. Alternatively, the radioactive label can be included as part of a larger molecule (e.g.,125I in meta-[125I]iodophenyl-N-hydroxysuccinimide ([125I]mIPNHS), which binds to free amino groups to form meta-iodophenyl (mIP) derivatives of relevant proteins (see, e.g., Rogers et al., 1997, J. NucL Med. 38:1221-29) or chelate (e.g., to DOTA or DTP A), which is in turn bound to the protein backbone. Methods of conjugating the radioactive labels, or larger molecules / chelates containing them, to the nanobody are known in the art. Such methods involve incubating the proteins with the radioactive label under conditions (e.g., pH, salt concentration, and / or temperature) that facilitate binding of the radioactive label or chelating agent to the protein (see, e.g., U.S. Patent No. 6,001,329). In some embodiments, chelating agents may include diethylene triamine pentaacetic acid (DTPA) or tetraazacyclododecane- 1,4,7, 10-tetraacetic acid (DOTA), deferoxamine (DFO), or 1,4,7,10-tetraazacyclododecane (DO3A).

[0055] In some embodiments, the PSMA8-specific radiolabeled nanobody described herein may include a linking group that may bind specifically to the amino acids of the nanobody and further bind to the chelating agent. Methods of conjugating the chelating group for radioactive labeling are known in the art.

[0056] Also provided herein are host cells that have been engineered to express and secrete a PSMA8 nanobody as described in this disclosure. In some embodiments, the cells are animal or human cells, and can be autologous, heterologous, or xenogeneic. In certain embodiments, the cells can be immortalized.

[0057] Appropriate host cells for the expression of nanobodies include yeast, bacteria, insect, plant, and mammalian cells. Of particular interest are bacteria such as E. coli, fungi such as 16USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCTSaccharomyces cerevisiae an Pichia pastoris, insect cells such as SF9, mammalian cell lines (e.g., human cell lines), as well as primary cell lines. Also provided herein are populations of any such cells.

[0058] In some embodiments, a nanobody can be expressed in, and purified from, transgenic animals (e.g., transgenic mammals). For example, a nanobody be produced in transgenic nonhuman mammals and isolated.

[0059] One method of producing proteins comprising the provided nanobody is to link two or more peptides or polypeptides together by protein chemistry techniques. For example, peptides or polypeptides can be chemically synthesized using currently available laboratory equipment using either Fmoc (9-fluorenylmethyl-oxycarbonyl) or Boc (tertbutyloxycarbonoyl) chemistry (Applied Biosystems, Inc.; Foster City, CA). Those of skill in the art readily appreciate that a peptide or polypeptide corresponding to the nanobody provided herein, for example, can be synthesized by standard chemical reactions. For example, a peptide or polypeptide can be synthesized and not cleaved from its synthesis resin whereas the other fragment of a nanobody can be synthesized and subsequently cleaved from the resin, thereby exposing a terminal group that is functionally blocked on the other fragment. By peptide condensation reactions, these two fragments can be covalently joined via a peptide bond at their carboxyl and amino termini, respectively, to form a nanobody or fragment thereof. (Grant GA, 1992, Synthetic Peptides: A User Guide. W.H. Freeman and Co., N.Y.; Bodansky M and Trost B., Ed., 1993, Principles of Peptide Synthesis. Springer Verlag Inc., NY). Alternatively, the peptide or polypeptide can by independently synthesized in vivo. Once isolated, these independent peptides or polypeptides may be linked to form a nanobody via similar peptide condensation reactions.

[0060] For example, enzymatic ligation of cloned or synthetic peptide segments can allow relatively short peptide fragments to be joined to produce larger peptide fragments, polypeptides, or whole protein domains (Abrahmsen et al., 1991, Biochemistry, 30:4151). Alternatively, native chemical ligation of synthetic peptides can be utilized to synthetically construct large peptides or polypeptides from shorter peptide fragments. This method consists of a two-step chemical reaction (Dawson et al., 1994, Science, 266:776 779). The first step is the chemoselective reaction of an unprotected synthetic peptide a thioester with another unprotected peptide segment containing an amino terminal Cys residue to give a thioester linked intermediate as the initial covalent product. Without a change in the reaction conditions, this intermediate undergoes spontaneous, rapid intramolecular reaction to form a native peptide bond at the ligation site. Application of this native chemical ligation method to the total 17USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCTsynthesis of a protein molecule is illustrated by the preparation of human interleukin 8 (IL-8) (Baggiolini etal., 1992, FEB S Let. 307:97-101; Clark etal, 1994, J. Biol. Chem. 269:16075; Clark et al., 1991, Biochemistry 30:3128; Rajarathnam et al., 1994, Biochemistry 33 :6623-30).

[0061] Alternatively, unprotected peptide segments can be chemically linked where the bond formed between the peptide segments as a result of the chemical ligation is an unnatural (nonpeptide) bond (Schnolzer et al., 1992, Science 256:221). This technique has been used to synthesize analogs of protein domains as well as large amounts of relatively pure proteins with full biological activity (deLisle et al, 1992, Techniques in Protein Chemistry IV. Academic Press, New York, pp. 257-267).

[0062] Following expression, the nanobody can be isolated. A nanobody can be isolated or purified in a variety of ways known in the art depending on what other components are present in the sample. Standard purification methods include electrophoretic, molecular, immunological, and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography. Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. See, e.g., Scopes, 1994, Protein Purification, 3rdedition, Springer-Verlag, New York City, New York. The degree of purification necessary varies depending on the desired use. In some instances, no purification of the expressed nanobody is necessary.

[0063] Methods for determining the yield or purity of a purified nanobody are known in the art and include, e.g., Bradford assay, UV spectroscopy, Biuret protein assay, Lowry protein assay, amido black protein assay, high pressure liquid chromatography (HPLC), mass spectrometry (MS), and gel electrophoretic methods (e.g., using a protein stain such as Coomassie Blue or colloidal silver stain).IV. Pharmaceutical Compositions and Formulations

[0064] The PSMA8-specific nanobodies described herein, as well as the various molecules comprising said nanobodies are suitable for administration in vitro or in vivo as part of a composition comprising a carrier or delivery vehicle (such as a liquid, for example, such as phosphate-buffered saline prepared in a research lab). In some embodiments, such compositions can also be pharmaceutically acceptable compositions. In some embodiments, the compositions comprise a PSMA8-specific nanobody of the present disclosure and a pharmaceutically acceptable carrier (excipient). A pharmaceutically acceptable carrier (excipient) is a material that is not biologically or otherwise undesirable, i.e., the material is administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with the other components of the pharmaceutical composition in which it 18USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCTis contained. The carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject. The compositions may further comprise a diluent, solubilizer, emulsifier, preservative, and / or adjuvant to be used with the methods disclosed herein. Such compositions can be used, for example, in a subject with cancer or non-cancerous malignancy that would benefit from any of the PSMA8-specific nanobody described herein.

[0065] Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy, 2P1Edition, Philip P. Gerbino, ed., Lippincott Williams & Wilkins (2006). In certain embodiments, acceptable formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed. In certain embodiments, the formulation material(s) are for subcutaneous and / or intravenous administration. In certain embodiments, the formulation comprises an appropriate amount of a pharmaceutically-acceptable salt to render the formulation isotonic. In certain embodiments, the pharmaceutical composition can contain formulation materials for modifying, maintaining, or preserving, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. In certain embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen- sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta- cyclodextrin); fillers; monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants. In 19USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCTcertain embodiments, the optimal pharmaceutical composition is determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format and desired dosage. See, for example, Remington: The Science and Practice of Pharmacy, 22ndEdition, Lloyd V. Allen, Jr., ed., The Pharmaceutical Press (2014). In certain embodiments, such compositions may influence the physical state, stability, rate of in vivo release and / or rate of in vivo clearance of the PSMA8-specific nanobodies described herein.

[0066] In certain embodiments, the primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature. For example, in certain embodiments, a suitable vehicle or carrier can be sterile water for injection, physiological saline solution, buffered solutions like Ringer’s solution, dextrose solution, or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. In certain embodiments, the saline comprises isotonic phosphate-buffered saline. In certain embodiments, neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. In certain embodiments, pharmaceutical compositions comprise a pH controlling buffer such phosphate-buffered saline or acetate-buffered saline. In certain embodiments, a composition comprising a PSMA8-specific nanobody disclosed herein can be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (see Remington: The Science and Practice of Pharmacy, 22ndEdition, Lloyd V. Allen, Jr., ed., The Pharmaceutical Press (2014)) in the form of a lyophilized cake or an aqueous solution. Further, in certain embodiments, a composition comprising a PSMA8 specific nanobody disclosed herein can be formulated as a lyophilizate using appropriate excipients. In some instances, appropriate excipients may include a cryopreservative, a bulking agent, a surfactant, or a combination of any thereof. Exemplary excipients include one or more of a polyol, a disaccharide, or a polysaccharide, such as, for example, mannitol, sorbitol, sucrose, trehalose, and dextran 40. In some embodiments, the cryopreservative may be sucrose or trehalose. In some embodiments, the bulking agent may be glycine or mannitol. In one example, the surfactant may be a polysorbate such as, for example, polysorbate-20 or polysorbate-80.

[0067] In certain embodiments, the pharmaceutical composition can be selected for parenteral delivery (e.g., through injection by intravenous, intraperitoneal, intracerebral (intra-parenchymal), intracerebral, intraventricular, intramuscular, subcutaneous, intra-ocular, intraarterial, intraportal, or intralesional routes). Preparations for parenteral administration can be in the form of a pyrogen-free, parenterally acceptable aqueous solution (i.e., water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media)20USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCTcomprising a PSMA8-specific nanobody in a pharmaceutically acceptable vehicle. Preparations for parenteral administration can also include non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Parenteral vehicles include sodium chloride solution, Ringer’s dextrose, dextrose and sodium chloride, lactated Ringer’s, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer’s dextrose), and the like. Preservatives and other additives are optionally present such as, for example, antimicrobials, anti-oxidants, chelating agents, inert gases, and the like. In certain embodiments, the preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes, that can provide for the controlled or sustained release of the product which can then be delivered via a depot injection. In certain embodiments, hyaluronic acid can also be used, and can have the effect of promoting sustained duration in the circulation. In certain embodiments, implantable drug delivery devices can be used to introduce the desired molecule.

[0068] In certain embodiments, the compositions can be selected for inhalation or for delivery through the digestive tract, such as orally. Compositions for oral administration include powders or granules, suspension or solutions in water or non-aqueous media, capsules, sachets, or tables. In certain embodiments, the compositions can be formulated as a dry powder for inhalation. In certain embodiments, an inhalation solution can be formulated with a propellant for aerosol delivery. In certain embodiments, solutions can be nebulized. Pulmonary administration is further described in International Application Publication No. WO / 1994 / 020069, which describes pulmonary delivery of chemically modified proteins. Thickeners, flavorings, diluents, emulsifiers, dispersing aids, or binders are optionally desirable.

[0069] In certain embodiments, the compositions can be selected for topical delivery. Formulations for topical administration include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners, and the like are optionally necessary or desirable.

[0070] In certain embodiments, the formulation components are present in concentrations that are acceptable to the site of administration. In certain embodiments, buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8. For example, the pH may be 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6,21USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCT7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5. In some instances, the pH of the pharmaceutical composition may be in the range of 6.6-8.5 such as, for example, 7.0-8.5, 6.6-7.2, 6.8-7.2, 6.8-7.4, 7.2-7.8, 7.0-7.5, 7.5-8.0, 7.2-8.2, 7.6-8.5, or 7.8-8.3. In some instances, the pH of the pharmaceutical composition may be in the range of 5.5-7.5 such as, for example, 5.5-5.8, 5.5-6.0, 5.7-6.2, 5.8-6.5, 6.0-6.5, 6.2-6.8, 6.5-7.0, 6.8-7.2, or 6.8-7.5. In some instances, the pH of the pharmaceutical composition may be in the range of 4.0-5.5 such as, for example, 4.0-4.3, 4.0-4.5, 4.2-4.8, 4.5-4.8, 4.5-5.0, 4.8-5.2, or 5.0-5.5.

[0071] In certain embodiments, a pharmaceutical composition can comprise an effective amount of a PSMA8-specific nanobody in a mixture with non-toxic excipients suitable for the manufacture of tablets. In certain embodiments, by dissolving the tablets in sterile water or other appropriate vehicle, solutions can be prepared in unit-dose form. In certain embodiments, suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.

[0072] Additional pharmaceutical compositions can be selected by one skilled in the art, including formulations involving a PSMA8-specific nanobody in sustained- or controlled-delivery formulations. In certain embodiments, techniques for formulating a variety of other sustained- or controlled-delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. See, for example, International Application Publication No. WO 1993 / 015722, which describes the controlled release of porous polymeric microparticles for the delivery of pharmaceutical compositions. In certain embodiments, sustained-release preparations can include semipermeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Sustained release matrices can include polyesters, hydrogels, polylactides (see, e.g., U.S. Patent No. 3,773,919; U.S. Patent No. 5,594,091; U.S. Patent No. 8,383,153; U.S. Patent No.4,767,628; International Application Publication No. WO1998 / 043615, Calo etal., 2015, Eur. Polymer J. 65:252-67 and European Patent No. EP 058,481), including, for example, chemically synthesized polymers, starch based polymers, and polyhydroxyalkanoates (PHAs), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al, 1993, Biopolymers 22:547-56), poly (2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed. Mater. Res. 15:167-277; and Langer, 1982, Chem. Tech. 12:98-105), ethylene vinyl acetate (Hsu & Langer, 1985, J. Biomed. Materials Res. 19(4):445-60), or poly-D(-)-3 -hydroxybutyric acid (European Patent No. EP0133988). In certain embodiments, sustained release compositions can also include liposomes, which can be prepared by any of several methods 22USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCTknown in the art. (See, e.g., Eppstein et al., 1985, Proc. Natl. Acad. Sci. USA 82:3688-92; European Patent No. EP 036,676; and U.S. Patent Nos. 4,619,794 and 4,615,885).

[0073] The pharmaceutical composition to be used for in vivo administration typically is sterile. In certain embodiments, sterilization is accomplished by filtration through sterile filtration membranes. In certain embodiments, where the composition is lyophilized, sterilization using this method can be conducted either prior to or following lyophilization and reconstitution. In certain embodiments, the composition for parenteral administration can be stored in lyophilized form or in a solution. In certain embodiments, parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[0074] In certain embodiments, once the pharmaceutical composition has been formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. In certain embodiments, such formulations can be stored either in a ready-to-use form or in a form (e.g., lyophilized) that is reconstituted prior to administration.

[0075] In certain embodiments, kits are provided for producing a single-dose administration unit. In certain embodiments, the kit can contain both a first container having a dried nanobody composition and a second container having an aqueous formulation. In certain embodiments, kits containing single and multi -chambered pre-filled syringes are included.

[0076] In certain embodiments, the effective amount of a pharmaceutical composition comprising any of the nanobody compositions described herein to be employed therapeutically depends, for example, upon the therapeutic context and objectives. One skilled in the art will appreciate that the appropriate dosage levels for treatment, according to certain embodiments, vary depending, in part, upon the molecule delivered, the indication for which the PSMA8-specific nanobody is being used, the route of administration, and the size (body weight, body surface or organ size) and / or condition (the age and general health) of the patient. The clinician can titer the dosage and modify the route of administration to obtain the optimal therapeutic effect.

[0077] The clinician can also select the frequency of dosing, taking into account the pharmacokinetic parameters of the PSMA8-specific nanobody in the formulation used. In certain embodiments, a clinician administers the composition until a dosage is reached that achieves the desired effect. In certain embodiments, the composition can therefore be administered as a single dose or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via, for example, an implantation device or catheter. Further refinement of the appropriate dosage is routinely made 23USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCTby those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. In certain embodiments, appropriate dosages can be ascertained through use of appropriate dose-response data.

[0078] The term “unit dose” or “dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the therapeutic composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and treatment regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the effect desired. The actual dosage amount of a composition of the present disclosure administered to a patient or subject can be determined by physical and physiological factors, such as body weight, the age, health, and sex of the subject, the type of disease being treated, the extent of disease penetration, previous or concurrent therapeutic interventions, idiopathy of the patient, the route of administration, and the potency, stability, and toxicity of the particular therapeutic substance. For example, a dose may comprise from about 1 pg / kg / body weight to about 1000 mg / kg / body weight (this such range includes intervening doses) or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 pg / kg / body weight to about 100 mg / kg / body weight, about 5 pg / kg / body weight to about 500 mg / kg / body weight, etc., can be administered.

[0079] In certain embodiments, the PSMA8-specific nanobody, or molecules comprising the PSMA8-specific nanobody, can be administered at a dose of 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, or 10 mg / kg once every other day at least four times. An exemplary treatment regime may include administration once per day, once per week, twice a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months, or once every three to 6 months. In some cases, the treatment comprises administering a PSMA8-specific nanobody, or molecules comprising the PSMA8-specific nanobody, according to one of the aforementioned dosing regimens for a first period and another of the aforementioned dosing regimens for a second period. In some cases, the treatment discontinues for a period of time before the same or a different dosing regimen is resumed. For example, a patient may be on a PSMA8-specific nanobody dosing regimen for two weeks, off for a week, on for another two weeks, and so on. Dosage regimens for the PSMA8-specific nanobody of this disclosure include 0.1 mg / kg body weight, 0.3 mg / kg body weight, 2 mg / kg body weight, 3 mg / kg body weight, or 10 mg / kg via intravenous administration, with the PSMA8-specific nanobody being given using one of the following24USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCTdosing schedules: (i) every four weeks for six dosages, then every three months; (ii) every three weeks; or (iii) 3 mg / kg body weight once followed by 1 mg / kg body weight every three weeks.

[0080] In some embodiments, a radiolabeled PSMA8-specific nanobody or molecule comprising the PSMA8-specific nanobody can be administered at a dose of 1 pCi to 300 mCi (e.g., 1 pCi-300 mCi, 10 pCi-250 mCi, 50 pCi-200 mCi, 100 pCi-300 mCi, 150 pCi-300 mCi, 200 pCi-300 mCi, 100 pCi-150 mCi, 100 pCi-200 mCi, 100 pCi-250 mCi, 1 to 200 pCi, 200 to 400 pCi, 400 to 600 pCi, 600 to 800 pCi, 800 pCi to 1 mCi, 1 to 100 mCi, 100 to 300 mCi, 150 to 250 mCi, 150 to 200 mCi, 200 pCi to 250 mCi, etc. and every range within). In certain embodiments, the radiolabeled PSMA8-specific nanobody or molecule comprising the PSMA8-specific nanobody can be administered at a dose of 1 pCi to 200 mCi. In some embodiments, the radiolabeled PSMA8-specific nanobody can be administered at a dose of about 1 pCi to 300 mCi in a single dose, in dose intervals, or over a number of days or weeks (e.g., 1 day, 2 days, 3 days, 4 days, or 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks). In some embodiments, the dose concentration may be dependent upon the conjugated nanobody as described above. In some embodiments, an exemplary treatment regime may include administration once per day, once per week, twice a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months, or once every three to 6 months. In some cases, the treatment comprises administering the radiolabeled PSMA8-specific nanobody or molecule comprising the radiolabeled PSMA8-specific nanobody according to one of the aforementioned dosing regimens for a first period and another of the aforementioned dosing regimens for a second period. In some cases, the treatment discontinues for a period of time before the same or a different dosing regimen resumes. For example, a patient may be on a radiolabeled PSMA8-specific nanobody dosing regimen for two weeks, off for a week, on for another two weeks, and so on. Dosage regimens for the radiolabeled PSMA8-specific nanobody of this disclosure include 0.1 mg / kg body weight, 0.3 mg / kg body weight, 2 mg / kg body weight, 3 mg / kg body weight, or 10 mg / kg via intravenous administration, with the radiolabeled PSMA8-specific nanobody being given using one of the following dosing schedules: (i) every four weeks for six dosages, then every three months; (ii) every three weeks; (iii) 3 mg / kg body weight once followed by 1 mg / kg body weight every three weeks. Dosage regimens for the radiolabeled PSMA8-specific nanobody of this disclosure include, for example, 0.1 mg / kg body weight 1 pCi, 10 pCi, 20 pCi, 30 pCi, 40 pCi, 50 pCi, 100 pCi, 150 pCi, 200 pCi, 250 pCi, or 300 pCi via intravenous administration (or any value within 1 pCi to 300 mCi), with the radiolabeled PSMA8-specific nanobody being25USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCTgiven using one of the following dosing schedules: (i) every four weeks for six dosages, then every three months; (ii) every three weeks; (iii) higher dosage once followed by lower dosage every three weeks.

[0081] In another embodiment, unit dose forms comprising a PSMA8-specific nanobody as described in this disclosure are provided. A unit dose form can be formulated for administration according to any of the routes described in this disclosure. In some embodiments, the unit dose form is formulated for intravenous or intraperitoneal administration. In another embodiment, pharmaceutical packages comprising unit dose forms of a PSMA8-specific nanobody, or of molecules comprising the PSMA8-specific nanobody, are provided.

[0082] In some instances, the PSMA8 nanobody may be an isolated PSMA8-specific nanobody as described in this disclosure. The term “isolated,” as used with reference to a protein (or nucleic acid), denotes that the protein (or nucleic acid) is essentially free of other cellular components with which it is associated in the natural state. In certain aspects, isolated polypeptides according to the present disclosure can consist essentially of the isolated polypeptide. It is preferably in a homogeneous state. Purity and homogeneity are typically determined using analytical chemistry techniques such as electrophoresis (e.g., polyacrylamide gel electrophoresis) or chromatography (e.g., high performance liquid chromatography). In some embodiments, an isolated protein (or nucleic acid) is at least 85% pure, at least 90% pure, at least 95% pure, or at least 99% pure.

[0083] In some instances, the PSMA8-specific nanobody may be a formulated into virus-like particles (VLPs). VLPs comprise viral protein(s) derived from the structural proteins of a virus. Methods for making and using virus like particles are described in, for example, Garcea and Gissmann, 2004, Current Opinion in Biotechnology 15:513-7.

[0084] In some instances, the PSMA8 nanobody may be a formulated into subviral dense bodies (DBs). DBs transport proteins into target cells by membrane fusion. Methods for making and using DBs are described in, for example, Pepperl-Klindworth et al., 2003, Gene Therapy 10:278-84.V. Kits and Packaging

[0085] The PSMA8-specific nanobodies, or molecules or cells comprising the PSMA8-specific nanobodies, disclosed herein may be used for the preparation of a kit (e.g., a diagnostic test kit or kit for the treatment of a patient). In some embodiments, kits are provided for carrying out any of the methods described herein. The kits of this disclosure may comprise a carrier container being compartmentalized to receive in close confinement one or more containers26USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCTsuch as vials, tubes, and the like, each of the containers comprising one of the separate elements to be used in the method.

[0086] In some embodiments, one of the containers may comprise a PSMA8-specific nanobody as described in this disclosure that is, or can be, detectably labeled. The kit may also have containers containing buffer(s) and / or a container comprising a reporter-means, for example, a biotin-binding protein, such as avidin or streptavidin, bound to a reporter molecule, such as an enzymatic or fluorescent label. For example, a kit for imaging a tumor in a subject with a PSMA8 expressing cancer is provided herein. In some embodiments, the kit comprises a container containing a labeled PSMA8-specific nanobody. In some embodiments, the kit comprises separate containers containing a PSMA8-specific nanobody and a detectable label.

[0087] A PSMA8-specific nanobody, or molecule or cell comprising the PSMA8-specific nanobody, as described in this disclosure for use in treating cancer patients, may be delivered in a pharmaceutical package or kit to doctors, healthcare providers, treatment facilities, or cancer patients. Such packaging is intended to improve patient convenience and compliance with the treatment plan. Typically, the packaging comprises paper (cardboard) or plastic. In some embodiments, the kit or pharmaceutical package further comprises instructions for use (e.g., for administering according to a method as described herein).

[0088] In some embodiments, a pharmaceutical package or kit comprises unit dose forms of a PSMA8-specific nanobody or molecule or cell comprising the PSMA8-specific nanobody. In some embodiments, the pharmaceutical package or kit further comprises unit dose forms of one or more of a chemotherapeutic agent, a cytotoxic agent, a radiotherapeutic agent, or an immunotherapeutic agent.

[0089] In one embodiment, the kit or pharmaceutical package comprises a PSMA8-specific nanobody, or a molecule or cell comprising the PSMA8-specific nanobody, in a defined, therapeutically effective dose in a single unit dosage form or as separate unit doses. The dose and form of the unit dose (e.g., pre-filled syringe, tablet, capsule, immediate release, delayed release, etc.) can be any doses or forms as described herein.

[0090] In one embodiment, the kit or pharmaceutical package includes doses suitable for multiple days of administration, such as one week, one month, or three months.

[0091] In certain embodiments, kits are provided for producing a single-dose administration unit. In certain embodiments, kits containing single or multi-chambered pre-filled syringes are included. In certain embodiments, kits containing one or more containers of a formulation described in this disclosure are included.27USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCTVI. Methods of UseA. Methods of Detecting PSMA8

[0092] Methods for detecting the presence of PSMA8 expressing cells in a biological sample are provided. In some embodiments, the methods include: (a) contacting said sample with a composition comprising an isolated PSMA8-specific nanobody as described in this disclosure; and (b) detecting an amount of binding of the isolated nanobody or antigen-binding portion thereof as a determination of the presence of PSMA8 expressing cells. In some embodiments, the biological sample comprises a tissue or tumor sample. In some embodiments, the biological sample comprises blood, plasma, serum, cerebrospinal fluid, or a tissue sample. The terms “contacted” and “exposed,” when applied to a biological sample (such as a cell), are used herein to describe the process by which a PSMA8-specific nanobody is delivered to a biological sample (such as a target cell) or placed in direct juxtaposition with the biological sample (such as the target cell).

[0093] In some embodiments, PSMA8 expression in cells (e.g., cells from a tumor or tissue sample) can be examined by using one or more routine biochemical analyses. In some embodiments, PSMA8 expression is determined by detecting protein expression using methods such as Western blot analysis, flow cytometry, and immunohistochemistry staining using a PSMA8-specific nanobody as described in this disclosure. Examples of types of immunoassays that can utilize the nanobody according to the present disclosure are competitive and noncompetitive immunoassays in either a direct or indirect format. For example, the immunoassay that can utilize the nanobody according to the present disclosure may be the same or similar to immunoassays employed for antibodies. Examples of such immunoassays are the radioimmunoassay (RIA) and the sandwich (immunometric) assay. Detection of antigens using the nanobody according to the present disclosure can be done utilizing immunoassays which are run in either the forward, reverse, or simultaneous modes, including immunohistochemical assays on physiological samples. Those of skill in the art will know, or can readily discern, other immunoassay formats without undue experimentation. In some embodiments, PSMA8 expression is determined by detecting mRNA levels using methods such as RT-PCR, RNA sequencing, microarray analysis, and Northern blot analysis. In some instances, a combination of these methods may be used.

[0094] The nanobody according to the present disclosure can be bound to many different carriers and used to detect the presence of PSMA8 expressing cells. Examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, agarose and magnetite. The nature of the 28USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCTcarrier can be either soluble or insoluble for purposes according to the present disclosure. Those skilled in the art will know of other suitable carriers for binding nanobodies according to the present disclosure, or will be able to ascertain such, using routine experimentation.

[0095] For purposes of this disclosure, PSMA8 protein may be detected by the provided nanobody when the PSMA8 protein is present in biological fluids and tissues from a subject. Any sample containing a detectable amount of PSMA8 protein can be used. A sample can be a liquid such as , blood, serum, urine, saliva, cerebrospinal fluid, or the like; a solid or semisolid such as tissues, feces, or the like; or, alternatively, a solid tissue such as those commonly used in histological diagnosis.

[0096] In some instances, a threshold amount of PSMA8 protein expression is used to characterize PSMA8 expression as either high or low. A high level of PSMA8 protein expression refers to a measure of PSMA8 protein expression above a particular threshold. For example, the threshold may be a normal, an average, or a median amount of PSMA8 protein expression as measured in a particular set of samples, referred to as a reference population. In some instances, the reference population may be a population of normal / healthy subjects (i.e., subjects not diagnosed with a cancer or other non-cancerous malignancy) and the samples may be one or more biological samples from the normal / healthy subjects. In other instances, the reference population may be a population of subjects having a particular condition or type of cancer (the same type of condition or cancer that the subject being assessed has). A low level of PSMA8 expression refers to the converse of the above. For example, the threshold may be determined by identifying two distinct subgroups in the reference population by dividing samples around a mathematically determined point, such as, without limitation, a median, thus creating a subgroup whose measure is high (i.e., higher than the median) and another subgroup whose measure is low. In certain embodiments, the cancer may originate in the pancreas, colon, rectum, or lung. In other embodiments, the cancer may originate in the bladder, blood, bone, bone marrow, brain, breast, esophagus, duodenum, small intestine, large intestine, gum, head, kidney, liver, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testis, tongue, or uterus.

[0097] Also provided are methods of imaging a tumor or tissue in a subject with a PSMA8-expressing cancer, the method comprising administering to the subject an isolated nanobody that binds specifically to PSMA8 that is conjugated to an imaging label and detecting the imaging label in the subject (such as those described in preceding sections of this disclosure). Imaging methods may be used to assess, for example, tumor size in a subject with cancer and changes in tumor size over or after the course of a treatment administered to the subject. The 29USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCTmethods may be useful to assess response of the subject to an administered treatment. In some instances, the methods may be useful to grade the subject’s condition or cancer.

[0098] Also provided are methods of monitoring response of a subject with a PSMA8 expressing cancer to therapy. The methods include administering to the subject a PSMA8-specific nanobody conjugated to an imaging label at a first time point prior to the subject before the subject receives therapy, detecting the imaging label in the subject to obtain a first image of the tissue, administering to the subject a PSMA8-specific nanobody conjugated to an imaging label at a second time point after the subject receives therapy, detecting the imaging label in the subject to obtain a second image of the tissue; and comparing the first image to the second image to determine whether a change has occurred. In some instances, the steps of administering to the subject a PSMA8-specific nanobody conjugated to an imaging label at a first time point after the subject receives therapy, detecting the imaging label in the subject to obtain a second image of the subject; and comparing the first image to the second image to determine whether a change has occurred may be repeated at a third time point (or additional time points) after the subject receives therapy. The subject may be evaluated in one or more of the following periods: prior to beginning of treatment; during the treatment; or after one or more elements of the treatment have been administered. Evaluation may include evaluating the need for further treatment, e.g., evaluating whether a dosage, frequency of administration, or duration of treatment should be altered. It can also include evaluating the need to add or drop a selected therapeutic modality, e.g., adding or dropping any of the treatments for the cancer described herein. In some embodiments, the evaluation is performed at least one (1) hour, e.g., at least 2, 4, 6, 8, 12, 24, or 48 hours, or at least 1 day, 2 days, 4 days, 10 days, 13 days, 20 days or more, or at least 1 week, 2 weeks, 4 weeks, 10 weeks, 13 weeks, 20 weeks or more, after an administration.B. Methods of Treatment

[0099] Also provided herein are methods to treat, inhibit, or delay progression of a disease or disorder associated with elevated levels of PSMA8, such as cancer. In some embodiments, the subject has cancer. In some embodiments, methods of treatment according to the present disclosure can include administering to a subject compositions comprising, consisting of, or consisting essentially of the PSMA8-specific nanobody (such as those described in Section III. above) as a monotherapy or part of a combinatorial therapy regime (a treatment schedule comprising more than one treatment modality).

[0100] Functioning of PSMA8 (or PSMA8 signaling) may be reduced by a PSMA8-specific nanobody (or a molecule or composition comprising or encoding the PSMA8 nanobody) as 30USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCTdescribed in this disclosure. Methods of treating a subject with a cancer, condition, or disorder are provided, the methods comprising administering to the subject a therapeutically effective amount of a composition comprising (or consisting of or consisting essentially of) a PSMA8-specific nanobody (or a molecule comprising or encoding the PSMA8 nanobody) as described in this disclosure. In some embodiments, the methods comprise administering to a subject a therapeutically effective amount of a composition comprising an isolated PSMA8-specific nanobody (or a molecule comprising or encoding the PSMA8 nanobody) described herein. The composition may further comprise a pharmaceutically acceptable carrier as described above in Section IV.

[0101] As used throughout, the term “subject” does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered. As used herein, “patient” or “subject” may be used interchangeably and includes human and veterinary subjects. The PSMA8-specific nanobodies described herein are useful for treating cancer in humans, including, without limitation, pediatric and geriatric populations, and in animals, e.g., veterinary applications. In some embodiments, the subject is a human. In some embodiments, the subject has or is suspected to have cancer or other non-cancerous malignancy associated with upregulated PSMA8 expression. In some embodiments, the subject is diagnosed with a cancer.

[0102] Also provided are methods of treatment to reduce one or more symptoms of a cancer (e.g., a cancerous or non-cancerous malignancy), by modulating PSMA8 signaling as a result of administration of compositions as described herein comprising the nanobody as discussed in Section III above. As used herein the terms “cancer” and “tumor” are used to indicate malignant tissue. The term “cancer” is also used to refer to the disease associated with the presence of malignant tumor cells in an individual, and the term “tumor” is used herein to refer to a plurality of cancer cells that are physically associated with each other. Cancer cells are malignant cells that give rise to cancer, and tumor cells are malignant cells that can form a tumor and thereby give rise to cancer. The term “cancer,” as used herein, may be used to describe a solid tumor, metastatic cancer, or non-metastatic cancer. The term also encompasses a circulating tumor cell. In certain embodiments, the cancer may originate in the lung, stomach, brain, or blood. In other embodiments, the cancer may originate in the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, rectum, skin, stomach, testis, tongue, or uterus. In other embodiments, the cancer is a metastasis into the brain from a31USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCTperipheral tumor. In certain embodiments, the cancer may be small cell lung cancer, glioma, gastric cancer, or ovarian cancer.

[0103] “Treating,” “treatment,” and the like may refer to any indicia of success in the treatment or amelioration of cancer. Treating or treatment of cancer refers to ameliorating cancer in a subject or any one or more symptoms thereof. The term ameliorating refers to any therapeutically beneficial result in the treatment of cancer, lessening in the severity or progression, promoting remission or durations of remission, or curing thereof. Thus, treating or treatment includes ameliorating at least one physical parameter or symptom. Treating or treatment includes modulating the cancer, either physically (e.g., stabilization of a discernible symptom) or physiologically (e.g., stabilization of a physical parameter) or both. For example, “treating” or “treatment” with respect to cancer includes the administration of an agent to impede growth of a cancer, to do one or more of the following: cause a cancer to shrink by weight or volume (i.e., shrink from a first weight or volume to a second weight or volume, wherein the second weight or volume is less than the first), delay or prevent metastasis, extend the expected survival time of the subject, or extend the expected time to progression of the tumor, or the like. Thus, in the disclosed methods, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of the cancer. For example, a method for treating a cancer in a subject by administering a pharmaceutical composition as described in this disclosure is considered to be a treatment if there is a 10% reduction in one or more symptoms of the cancer in a subject as compared to a control. Thus the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to native or control levels. The effect of treatment can be compared to an individual or pool of individuals not receiving the treatment, or to the same patient prior to treatment or at a different time during treatment. It is understood that treatment does not necessarily refer to a cure or complete ablation of the cancer or symptoms of the cancer.

[0104] The term “administer,” as used herein, refers to a method of delivering agents, compounds, or compositions to the desired site of biological action. The pharmaceutical compositions (e.g., as described above) are prepared for administration in a number of ways, including but not limited to injection, ingestion, transfusion, implantation, or transplantation, depending on whether local or systemic treatment is desired, and on the area to be treated. The preparation of such pharmaceutically acceptable compositions is within the ability of one skilled in the art. The compositions are administered via any of several routes of administration, including topical, oral, parenteral, intravenous, intra-articular, intraperitoneal, intracerebral 32USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCT(intra-parenchymal), intracerebral, intraventricular, intramuscular, subcutaneous, intraarterial, intraportal, intracavity, intralesional, transdermal, intradermal, intrahepatical, intrathecal, intracranial, rectal, transmucosal, intestinal, intra-ocular or ocular, otic, nasal, inhalation, or intrabronchial delivery, or any other method known in the art. In some embodiments, the PSMA8-specific nanobody is administered intravenously, or through local injection. In certain embodiments, administered is by bolus injection, continuously by infusion, by sustained release system, or by implantation device. In certain embodiments, individual elements of a combination therapy (as discussed below) may be administered by different routes. In certain embodiments, the composition can be administered locally, e.g., during surgery or topically. Optionally local administration is via implantation of a membrane, sponge, or another appropriate material onto which the desired molecule has been absorbed or encapsulated. In certain embodiments, where an implantation device is used, the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, timed-release bolus, or continuous administration. Further discussion on routes of administration and formulations is provided in Section IV.

[0105] In some instances, the PSMA8-specific nanobody can be administered as an isolated protein. In some instances, the PSMA8-specific nanobody can be administered via virus-like particles or by subviral dense bodies. Virus-like particles and subviral dense bodies may be formulated as described herein and as known in the art. In some instances, the PSMA8-specific nanobody can be administered by tegument aggregates. Methods for making and using tegument aggregates are described, for example, in International Publication No. WO 2006 / 110728.

[0106] As used herein, the term “therapeutically effective amount” or “effective amount” refers to an amount of a therapeutic composition that, when administered to a subject, is effective to treat a disease or disorder such that the symptoms of the disease or disorder are ameliorated, or the likelihood of the disease or disorder developing or progressing is decreased. A therapeutically effective amount is not, however, a dosage so large as to cause adverse side effects, such as hyperviscosity syndromes, pulmonary edema, congestive heart failure, and the like. A suitable dose of a therapeutic composition as described herein, which dose is capable of treating a cancer in a subject, can depend on a variety of factors including the particular therapeutic composition used and whether it is used concomitantly with other therapeutic agents. Other factors affecting the dose administered to the subject include, e.g., the type or extent of cancer. For example, a subject that has had a previous cancer (e.g., a subject with relapsed or recurrent cancer) may require administration of a different dosage of a PSMA8- 33USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCTspecific nanobody than a subject who has not previously had cancer. Generally, a therapeutically effective amount may vary with the subject’s age, condition, and sex, as well as the extent of the disease in the subject and can be determined by one of skill in the art. Other factors can include, e.g., other medical disorders concurrently or previously affecting the subject, the age and general health of the subject, the genetic disposition of the subject, diet, time of administration, the route of administration, and the size (body weight, body surface, or organ size), the rate of excretion, drug combination, and any other additional therapeutics that are administered to the subject. It should also be understood that a specific dosage and treatment regimen for any particular subject also depends upon the judgment of the treating medical practitioner (e.g., doctor or nurse) as described above in Section IV. A therapeutically effective amount is also one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects. The dosage of the therapeutically effective amount may be adjusted by the individual physician or veterinarian in the event of any complication. In some instances, a therapeutically effective amount may vary from about 0.01 mg / kg to about 50 mg / kg, preferably from about 0.1 mg / kg to about 20 mg / kg, most preferably from about 0.2 mg / kg to about 2 mg / kg, in one or more dose administrations daily, for one or several days. In some embodiments, the doses can be about 1, about 0.5, about 0.1, about 0.05, or about 0.01 mg / kg, or any intervening dose between about 0.01 mg / kg and 1 mg / kg. In some instances, the PSMA8-specific nanobody is administered for 2 to 5 or more consecutive days. In some instances, the PSMA8-specific nanobody is administered to a subject at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.

[0107] The clinician also selects the frequency of dosing, taking into account the pharmacokinetic parameters of the PSMA8-specific nanobody in the formulation used. Such pharmacokinetic parameters are well known in the art, / .<?., the rate of absorption, bioavailability, metabolism, clearance, and the like (see, e.g., Hidalgo- Aragones, 1996, J. Steroid Biochem. Mol. Biol. 58:611-17; Groning, 1996, Pharmazie 51:337-41; Fotherby, 1996, Contraception 54:59-69; Johnson, 1995, J. Pharm. Sci. 84:1144-46; Rohatagi, 1995, Pharmazie 50:610-13; Brophy, 1983, Eur. J. Clin. Pharmacol. 24:103-08; the latest Remington's, supra). In certain embodiments, a clinician administers the composition until a dosage is reached that achieves the desired effect. In certain embodiments, the composition can therefore be administered as a single dose or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via, for example, an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely 34USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCTperformed by them. In certain embodiments, appropriate dosages can be ascertained through use of appropriate dose-response data.

[0108] Toxicity and therapeutic efficacy of the PSMA8-specific nanobody can be determined by known pharmaceutical procedures in cell cultures or experimental animals (e.g., animal models of any of the cancers described herein). These procedures can be used, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio LD50 / ED50. A PSMA8-specific nanobody that exhibits a high therapeutic index is preferred. While nanobody compositions that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such constructs to the site of affected tissue and to minimize potential damage to normal cells and, thereby, reduce side effects.

[0109] The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of a PSMA8-specific nanobody can lie generally within a range of circulating concentrations of the PSMA8-specific nanobody that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For the PSMA8-specific nanobody as described herein, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the EC50 (i.e., the concentration of the construct - e.g., nanobody - which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography. In some embodiments, e.g., where local administration is desired, cell culture or animal models can be used to determine a dose required to achieve a therapeutically effective concentration within the local site.

[0110] Suitable human doses of the PSMA8-specific nanobody described herein can further be evaluated in, e.g., Phase I dose escalation studies. See, e.g., van Gurp et al. (2008) Am J Transplantation 8(8): 1711-1718; Hanouska et al. (2007) Clin Cancer Res 13(2, part 1):523-531; and Hetherington et al. (2006) Antimicrobial Agents and Chemotherapy 50(10): 3499-3500.

[0111] In some embodiments, a PSMA8-specific nanobody described herein can be administered to a subject as a monotherapy (i.e., a standalone therapy not used in conjunction with other therapies). Alternatively, the PSMA8-specific nanobody can be administered in 35USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCTconjunction with other therapies for the cancer (combination therapy, also referred to as combinatorial therapy). For example, the composition can be administered to a subject at the same time, prior to, or after, a second non-PSMA8 -nanobody therapy. In some embodiments, the PSMA8-specific nanobody and the one or more additional active agents (of the second or additional therapies of the combinatorial therapy regimen) are administered at the same time. Optionally, the PSMA8-specific nanobody is administered first in time and the one or more additional active agents are administered second in time. In some embodiments, the one or more additional active agents are administered first in time and the PSMA8-specific nanobody is administered second in time. Optionally, the PSMA8-specific nanobody and the one or more additional agents are administered simultaneously in the same or different routes.

[0112] A PSMA8-specific nanobody as described herein can replace or augment a previously or currently administered therapy. For example, upon treating with a PSMA8-specific nanobody, administration of the one or more additional active agents can cease or diminish, e.g., be administered at lower levels or dosages. In some embodiments, administration of the previous therapy can be maintained. In some embodiments, a previous therapy is maintained until the level of the PSMA8-specific nanobody reaches a level sufficient to provide a therapeutic effect.

[0113] In some embodiments, the PSMA8-specific nanobody is conjugated to a photosensitizer (also referred to as a photoabsorber). In some instances, the method of treatment comprises using the anti-PSMA8 nanobody conjugate in near infrared photoimmunotherapy (NIR-PIT for the subject. NIR-PIT is a two-part therapy that typically utilizes a monoclonal antibody (mAb) conjugated to a photoabsorbing dye, such as (but not limited to) IRDye700DX (IR700), which is then activated by NIR light (e.g., see Kobayashi et al., Acc. Chem. Res. 52:2332-2339, 2019). After intravenous injection to the subject, the antibody-photoabsorber conjugate (APC) binds to its target overexpressed on the surface of cancer cells. Then, NIR light at 690 nm is delivered by laser to excite IR700 on the APC, leading to rapid, highly selective, lethal damage to the cell membrane. In some embodiments, the NIR-PIT may be applied to the nanobodies described herein, and may be referred to as a nanobody-photoabsorber conjugate (NPC). In some embodiments, the method of treatment comprises administering an anti-PSMA8 NPC to the subject followed by administering NIR to the subject. The NIR may for instance be applied externally onto the subject, or internally via a catheter or similar device.

[0114] In some instances, the provided methods may include administering a PSMA8-specific nanobody and a second form of cancer therapy to the subject (i.e., a combination or 36USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCTcombinatorial therapy). The second form of cancer therapy may include a cytotoxic agent, a chemotherapeutic agent, an immunosuppressive agent (including immune checkpoint inhibitors), or radiation therapy. In some embodiments, the second form of cancer therapy is an antibody (e.g., a monoclonal antibody).

[0115] The methods and compositions, including combination therapies, enhance the therapeutic or protective effect, and / or increase the therapeutic effect of another anti-cancer. Therapeutic and prophylactic methods and compositions can be provided in a combined amount effective to achieve the desired effect, such as the killing of a cancer cell and / or the inhibition of cellular hyperproliferation. This process may involve contacting the cells with both a nanobody and a second therapy. A tissue, tumor, or cell can be contacted with one or more compositions or pharmacological formulation(s) comprising one or more of the agents (e.g., nanobody or an anti-cancer agent), or by contacting the tissue, tumor, and / or cell with two or more distinct compositions or formulations, wherein one composition provides 1) a nanobody, 2) an anti-cancer agent, or 3) both a nanobody and an anti-cancer agent. Also, it is contemplated that such a combination therapy can be used in conjunction with chemotherapy, radiotherapy, surgical therapy, immunotherapy, or radioimmunotherapy. Exemplary anticancer agents (also referred to below as therapeutic agents) include chemotherapeutic agents, radiotherapeutic agents, and immunotherapeutic agents, as well as combinations thereof. The terms “contacted” and “exposed,” when applied to a cell in this context, are used herein to describe the process by which a therapeutic construct and a chemotherapeutic or radiotherapeutic agent are delivered to a target cell or are placed in direct juxtaposition with the target cell. In some embodiments, to achieve cell killing, the PSMA8-specific nanobody and the chemotherapeutic, radiotherapeutic agent, or immunotherapeutic agent are delivered to a cell in a combined amount effective to kill the cell or prevent it from dividing.

[0116] A nanobody may be administered before, during, after, or in various combinations relative to another anti-cancer treatment. The administrations may be in intervals ranging from concurrently to minutes to days to weeks. In embodiments where the nanobody is provided to a patient separately from another anti-cancer agent, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the two compounds would still be able to exert an advantageously combined effect on the patient. In such embodiments, it is contemplated that one may provide a patient with the nanobody therapy and the anti-cancer therapy within about 6 to 72 hours, about 6 to 48 hours, or about 6 to 24 hours of each other and, more particularly, within about 6-12 hours of each other. In some situations, it may be desirable to extend the time period for treatment significantly where 37USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCTseveral days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) lapse between respective administrations.

[0117] In certain embodiments, a course of treatment will last 1-90 days or more (including intervening days). It is contemplated that one agent may be given on any day of day 1 to day 90 (including intervening days) or any combination thereof, and another agent is given on any day of day 1 to day 90 (including intervening days) or any combination thereof. Within a single day (24-hour period), the patient may be given one or multiple administrations of the agent(s). Moreover, after a course of treatment, it is contemplated that there is a period of time at which no anti-cancer treatment is administered. This time period may last 1-7 days, 1-5 weeks, and / or 1-12 months, or more, or any time period within these ranges (including intervening days), depending on the condition of the patient, such as their prognosis, strength, health, etc. It is expected that the treatment cycles would be repeated as necessary.

[0118] In some embodiments, the PSMA8-specific nanobody can be labeled, conjugated, or fused with a therapeutic agent or diagnostic agent (such as an imaging agent). The linkage can be covalent or noncovalent (e.g., ionic). In some embodiments, the PSMA8-specific nanobody (or radiolabeled PSMA8-specific nanobody) is conjugated to a therapeutic agent. The therapeutic agent may be at least one of a cytotoxic agent, a chemotherapeutic agent, or an immunosuppressive agent, as well as combinations thereof. In this way, the PSMA8-specific nanobody delivered to the subject can be multifunctional, in that it exerts one therapeutic effect by binding to the PSMA8 protein and a second therapeutic effect by delivering a supplemental therapeutic agent. In some embodiments, such a nanobody is referred to as a nanobody-drug conjugate (NDC) or immunoconjugate. Nanobody drug conjugates are useful for the local delivery of therapeutic agents, particularly cytotoxic or cytostatic agents, i.e., drugs to kill or inhibit tumor cells in the treatment of cancer allows targeted delivery of the drug moiety to tumors, and intracellular accumulation therein, where systemic administration of these unconjugated drug agents may result in unacceptable levels of toxicity to normal cells as well as the tumor cells sought to be eliminated. In some embodiments, cytotoxic agents include but are not limited to toxins, for example, plant and bacterial toxins, small molecules, peptides, polypeptides, and proteins. Genetically engineered fusion proteins, in which genes encoding a nanobody including the Fv region, or peptides can be fused to the genes encoding a toxin to deliver a toxin to the target cell, are also provided. As used herein, a target cell or target cells are PSMA8 positive cells.

[0119] Examples of toxins or toxin moi eties include diphtheria, ricin, streptavidin, and modifications thereof. Additional examples include paclitaxel, cisplatin, carboplatin,38USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCTcytochalasin B, gramicidin D, ethidium bromide, emetine, etoposide, tenoposide, colchicin, dihydroxy anthracin di one, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, decarbazine), alkylating agents (e.g., mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine). Cytotoxic peptides such as auristatin (antineoplastic) peptides auristatin E (AE) and monomethylauristatin (MMAE), which are synthetic analogs of dolastatin, may also be conjugated to the PSMA8-specific nanobody.

[0120] The therapeutic agent can act extracellularly, for example by initiating or affecting an immune response (for example, through activation of cell-surface receptors that initiated intracellular signaling cascades within a cell that can then impact other cells of the immune system associated with immune responses), or it can act intracellularly, either directly by translocating through the cell membrane or indirectly by, for example, affecting transmembrane cell signaling. The therapeutic agent is optionally cleavable from the PSMA8-specific nanobody. Cleavage can be autolytic, accomplished by proteolysis, or affected by contacting the cell with a cleavage agent.

[0121] As referred to herein, a chemotherapeutic agent is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include erlotinib (such as TARCEVA®, Genentech / OSI Pharm.), bortezomib (such as VELCADE®, Millenium Pharm.), fulvestrant (such as FASLODEX®, AstraZeneca), sutent (such as SU11248, Pfizer), letrozole (such as FEMARA®, Novartis), imatinib mesylate (such as GLEEVEC®, Novartis), PTK787 / ZK222584 (Novartis), oxaliplatin (such as Eloxatin®, Sanofi), 5 -fluorouracil (5-FU), leucovorin, rapamycin (also known as sirolimus) (such as RAPAMUNE®, Wyeth), lapatinib (such as TYKERB®, GSK572016, GlaxoSmithKline), lonafamib (such as SCH 66336), sorafenib (such as BAY43-9006, Bayer Labs.), capecitabine (such as XELODA®, Roche), docetaxel (such as TAXOTERE®), and gefitinib (such as IRESSA®, Astrazeneca), AG1478, AG1571 (such as SU 5271; Sugen Inc.), alkylating agents such as thiotepa and cyclosphosphamide (such as CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan 39USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCTand piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancrati statin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, particularly calicheamicin yi1and calicheamicin Oi1); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, anthramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (such as ADRIAMYCIN®, including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; antiadrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; Trametes Versicolor polysaccharide-K (Krestin, PSK) (JHS 40USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCTNatural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2', 2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; cytarabine (cytosine arabinoside, “Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel (such as TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™ (a Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, IL)), and doxetaxel (such as TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chloranbucil; gemcitabine (such as GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; vinorelbine (such as NAVELBINE®); novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluorometlhylomithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0122] Chemotherapeutic agents, as used herein, also refers to (i) anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, and toremifene (such as FARESTON®); (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrol acetate (such as MEGASE®), exemestane (such as AROMASIN®), formestanie, fadrozole, vorozole (such as RIVISOR®), letrozole (such as FEMARA®), and anastrozole (such as ARIMIDEX®); (iii) anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) aromatase inhibitors; (v) protein kinase inhibitors; (vi) lipid kinase inhibitors; (vii) antisense oligonucleotides, particularly those which inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras; (viii) VEGF receptor and angiogenesis inhibitors (including ribozymes such as ANGIOZYME®) and a HER2 expression inhibitor; (ix) vaccines such as gene therapy vaccines, for example, ALLOVECTIN-7® vaccine (plasmid / lipid complex containing the DNA sequences encoding HLA-B7 and 132 microglobulin), LEUVECTIN® vaccine (plasmid DNA expression vector encoding interleukin-2 (IL-2) complexed with a lipid delivery vehicle (DMRIE / DOPE)), and VAXID® vaccine (patient-specific naked DNA vaccine); IL-2 or aldesleukin (such as 41USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCTPROLEUKIN®); topoisomerase 1 inhibitors (such as TOPOTECAN®); gonadotropinreleasing hormone antagonists (such as ABARELIX®); (x) anti-angiogenic agents such as bevacizumab (such as AVASTIN®, Genentech); and (xi) pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0123] In some embodiments, the treatment methods provided herein may further comprise administering an immunosuppressive agent such as an immune checkpoint inhibitor as part of the method. These treatments work by “taking the brakes off’ the immune system (are immunosuppressive), allowing it to mount a stronger and more effective attack against cancer. Several different types of checkpoint inhibitors, targeting different checkpoints or “brakes” on immune cells, are currently in use. Immune checkpoint proteins that may be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), CCL5, CD27, CD38, CD8A, CMKLR1, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, also known as CD152), CXCL9, CXCR5, glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR), HLA-DRB 1, ICOS (also known as CD278), HLA-DQA1, HLA-E, indoleamine 2,3 -dioxygenase 1 (IDO1), killer-cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAG-3, also known as CD223), Mer tyrosine kinase (MerTK), NKG7, 0X40 (also known as CD134), programmed death 1 (PD-1), programmed death-ligand 1 (PD-L1, also known as CD274), PDCD1LG2, PSMB 10, ST A Tl, T cell immunoreceptor with 1g and ITIM domains (TI GIT), T-cell immunoglobulin domain and mucin domain 3 (TIM-3), and V-domain 1g suppressor of T cell activation (VISTA, also known as C10orf54). In particular, the immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4. Exemplary immunosuppressive agents are PD-1 inhibitors (such as nivolumab and pembrolizumab), PD-L1 inhibitors (such as atezolizumab, durvalumab, and avelumab), and CTLA-4 inhibitors (such as ipilimumab). In one example, the second form of cancer therapy comprises a PD-L1 inhibitor, a PD-1 inhibitor, or a CTLA4 inhibitor. In some instances, combinations of such inhibitors can be administered. In some instances, the PD-L1 inhibitor, the PD-1 inhibitor, and / or the CTLA4 inhibitor may be an inhibitory antibody that binds specifically to PD-L1, PD-1, or CTLA4, respectively.

[0124] In some instances, the treatment methods provided herein may further comprise administering radiation therapy to the subject. Radiation therapy uses high-energy radiation to shrink tumors and kill cancer cells. X-rays, gamma rays, and charged particles are types of radiation used for cancer treatment. The radiation may be delivered by a machine outside the body (external-beam radiation therapy), or it may come from radioactive material placed in the body near cancer cells (internal radiation therapy, also called brachytherapy). Systemic 42USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCTradiation therapy uses radioactive substances, such as radioactive iodine, that travel in the blood to kill cancer cells.

[0125] In some embodiments of the treatment methods, PSMA8 expression (e.g., in cancer cells) can be examined by using one or more routine biochemical analyses before, during, or after treatment. In some embodiments, PSMA8 expression is determined by detecting protein expression using methods such as mass spectrometry, Western blot analysis, flow cytometry, or immunohistochemistry staining. In some embodiments, such methods comprise use of a PSMA8-specific nanobody (e.g., as described in this disclosure). In some embodiments, PSMA8 expression is determined by detecting mRNA levels using methods such as RT-PCR, RNA sequencing, microarray analysis, and Northern blot analysis. In some embodiments, a combination of these methods may be used, or additional methods known in the art may also be used. Suitable methods are described in more detail in Section VI.

[0126] In some embodiments, the therapeutic PSMA8-specific radiolabeled nanobody may be administered with a second therapeutic radiolabeled PSMA8 nanobody. In some embodiments, the second therapeutic radiolabeled PSMA8 nanobody comprises a radiolabel selected from a group consisting of161Tb,225Ac277Ac,211At,128Ba,131Ba,7Be,204Bi,205Bi,206Bi,76Br,77Br,82Br,109Cd,47Ca,nC,14C,36C1,48Cr,51Cr,62Cu,64Cu,67Cu,165Dy,155Eu,18F,153Gd,66Ga,67Ga,68Ga,72Ga,198Au,3H166Ho,mIn,113mIn,115mIn,123I,125I,131I,189Ir,191mIr,192Ir,194Ir,52Fe,55Fe,59Fe,177Lu,150,191m’1910s,109Pd,32P,33P,42K,226Ra,186Re,188Re,82mRb,153Sm,46Sc,47Sc,72Se,75Se,105Ag,22Na,24Na,89Sr,35S,38S,177Ta,96Tc, "mTc,2O1T1,2O2T1,113Sn,117mSn,121Sn,166Yb,169Yb,175Yb,88Y,90Y,62Zn, and65Zn. In some embodiments, the radiolabeled nanobody as described herein may generate a dual emission of medium energy beta particles and Auger electrons that may interact favorably with the second theranostic that may emit alpha particles. In some embodiments, the methods described herein may include administration of a PSMA8-specific [161Tb]-radiolabeled nanobody in combination with a PSMA8-specific [225Ac]-radiolabeled nanobody or PSMA8-specific [177Lu]-radiolabeled nanobody. In some embodiments, the selected combination as described herein may result in a reduced toxicity as compared to a single PSMA8-specific radiolabeled nanobody.C. Diagnostic and Prognostic Methods

[0127] In another aspect, provided are prognostic and diagnostic methods for cancer, based on detection and / or quantitation of PSMA8 using a PSMA8-specific nanobody as described in this disclosure.

[0128] In some embodiments, provided are methods of assessing eligibility of a subject for inclusion in or exclusion from a clinical trial of or treatment with a PSMA8 targeted therapy 43USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCTusing a PSMA8 nanobody. The method comprises (a) measuring in a biological sample from a subject the amount of PSMA8; (b) determining if the subject has a cancer characterized as having a high level of PSMA8 expression; and (c) indicating that the subject is eligible for a clinical trial of or treatment with a PSMA8 targeted therapy if the subject’s sample is characterized as having a high level of PSMA8 expression, i.e., above a predetermined threshold or that the subject is ineligible for a clinical trial of treatment with the PSMA8 targeted therapy if the subject’s sample is characterized as having a low level of PSMA8 expression, i.e., below a predetermined threshold. In some embodiments, the amount of PSMA8 in the sample from the subject is measured using a PSMA8-specific nanobody as described herein. In some embodiments, the amount of PSMA8 in the sample is measured using, a method of detecting PSMA8 as described in Section VI. In some instances, the threshold level is a median amount of PSMA8 determined in a reference population of patients having the same kind of condition or cancer as the subject. In another instance, the threshold level is an optimal amount of PSMA8 determined in a reference population of patients having the same kind of condition or cancer as the subject. “Optimal cutoff’ as used herein, refers to the value of a predetermined measure on subjects exhibiting certain attributes that allow the best discrimination between two categories of an attribute. For example, finding a value for an optimal cutoff that allows one to best discriminate between two categories (subgroups) of patients for determining at least one of overall survival, time to disease progression, progression-free survival, and likelihood to respond to treatment (e.g., for cancer, based on clinical assessment using the RECIST criteria, e.g., Eisenhauer, E.A., et al., 2009, Eur. J. Cancer 45:228-247, or the like as recognized in the medical field). Optimal cutoffs are used to separate the subjects with values lower than or higher than the optimal cutoff to optimize the prediction model, for example, without limitation, to maximize the specificity of the model, maximize the sensitivity of the model, maximize the difference in outcome, or minimize the p-value from hazard ratio or a difference in response.

[0129] In another aspect, provided are methods to diagnose cancer in a subject. Specifically, the diagnosis may be of a PSMA8-expressing cancer. The method may comprise measuring in a sample from a subject the amount of PSMA8 and diagnosing the subject with cancer if the amount of PSMA8 expression in the sample is high. In some instances, the method may comprise (a) measuring in a sample from a subject the amount of PSMA8 using a PSMA8 nanobody; and (b) determining if the subject has cancer characterized as having a high level of PSMA8 expression. Conversely, if the amount of PSMA8 expression in the sample is low level, the subject may not be diagnosed with cancer or may not be diagnosed with a PSMA844USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCTexpressing cancer. In some instances, the amount of PSMA8 in the sample is measured using a PSMA8-specific nanobody as described herein. In some embodiments, the amount of PSMA8 in the sample is measured using a method of detecting PSMA8 as described in Section VI.

[0130] In some instances, to diagnose cancer in a subject, or to characterize a subject’s cancer, a biopsy is typically taken from a subject having an abnormal tissue growth, such as a tumor. Samples may be formalin-fixed, paraffin-embedded tissue samples obtained from the subject’s cancer (tumor). In other instances, such as where circulating tumor cells are to be assessed, the sample from the subject is a blood, plasma, or lymph sample. Typically, the tissue or cells of the patient sample are examined under a microscope to confirm the diagnosis and / or assess information about the tumor. In some cases, additional tests may need to be performed on the proteins, DNA, and / or mRNA of the cells in the ample to verify the diagnosis or characterization.

[0131] The monitoring methods may include administering to the subject a PSMA8-specific nanobody conjugated to an imaging label at a first time point prior to the subject before the subject receives therapy, detecting the imaging label in the subject to obtain a first image of the sample or tumor, administering to the subject a PSMA8-specific nanobody conjugated to an imaging label at a second time point after the subject receives therapy, detecting the imaging label in the subject to obtain a second image of the sample or tumor; and comparing the first image to the second image to determine whether a change (e.g., tumor size) has occurred. In some instances, the steps of administering to the subject a PSMA8-specific nanobody conjugated to an imaging label at a first time point after the subject receives therapy, detecting the imaging label in the subject to obtain a second image of the sample tumor; and comparing the first image to the second image to determine whether a change has occurred may be repeated at a third time point (or additional time points) after the subject receives therapy.

[0132] In one embodiment, a subject is administered a labeled PSMA8 nanobody or antigenbinding fragment thereof as described in this disclosure that is conjugated to an imaging agent. The labeled PSMA8 nanobody or antigen-binding fragment thereof is allowed to incubate in vivo and bind to PSMA8 in the subject’s tissues (or an ex vivo tissue sample). The imaging label is thereby localized to relevant cells or tissues, and the localized imaging label is detected using an appropriate imaging device as known to those skilled in the art.

[0133] The imaging agent may carry a bioluminescent or chemiluminescent label. Such labels include polypeptides known to be fluorescent, bioluminescent or chemiluminescent, or that act as enzymes on a specific substrate (reagent), or can generate a fluorescent, bioluminescent or chemiluminescent molecule. Examples of bioluminescent or 45USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCTchemiluminescent labels include luciferases, aequorin, obelin, mnemiopsin, berovin, a phenanthridinium ester, and variations thereof and combinations thereof. A substrate for the bioluminescent or chemiluminescent polypeptide may also be used in imaging. For example, the chemiluminescent polypeptide can be luciferase and the reagent luciferin. A substrate for a bioluminescent or chemiluminescent label can be administered before, at the same time (e.g., in the same formulation), or after administration of the agent. The imaging agent can include a radionuclide, such as the ones described above.

[0134] The imaging agent may include a paramagnetic compound, such as a polypeptide chelated to a metal (e.g., a metalloporphyrin). The paramagnetic compound may also include a monocrystalline nanoparticle, e.g., a nanoparticle including a lanthanide (e.g., Gd) or iron oxide; or a metal ion such as a lanthanide. Examples of elements that are useful in magnetic resonance imaging include gadolinium, terbium, tin, iron, or isotopes thereof.

[0135] Whole body imaging techniques using the radioisotope labeled nanobody as described herein can be used for locating diseased cells and tissues (e.g., primary tumors and tumors which have metastasized). In some cases, the labeled agents for locating the tumor tissue or cells are administered intravenously. The bio-distribution of the label can be monitored by scintigraphy, and accumulations of the label are related to the presence of PSMA8 or other tumor markers. Whole body imaging techniques are described in, e.g., U.S. Patent Nos. 4,036,945 and 4,311,688. In embodiments wherein imaging is performed on the patient, the physician may administer a preselected dose that may be based on concentrations known by those skilled in the art. In some embodiments, the PSMA8-specific radiolabeled nanobody may be administered at a concentration to deliver about 100 pCi to 400 pCi.

[0136] An image according to this disclosure can be generated by computer assisted tomography (CAT or CT), magnetic resonance spectroscopy (MRS) image, magnetic resonance imaging (MRI), positron emission tomography (PET), single-photon emission computed tomography (SPECT), or bioluminescence imaging (BLI) or equivalent.

[0137] Computer assisted tomography (CAT) and computerized axial tomography (CAT) systems and devices well known in the art can be used to generate an image. (See, for example, U.S. Pat. Nos. 6,151,377; 5,946,371; 5,446,799; 5,406,479; 5,208,581; and 5,109,397.) The imaging methods may also utilize animal imaging modalities, such as MicroCAT™ (ImTek, Inc.).

[0138] Magnetic resonance imaging (MRI) systems and devices well known in the art can be used for imaging. For a description of MRI methods and devices, see, for example, U.S. Pat.46USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCTNo. 6,151,377. MRI and supporting devices are commercially available, for example, from Bruker Medical GMBH; Caprius; Esaote Biomedica; Fonar; GE Medical Systems (GEMS); Hitachi Medical Systems America; Intermagnetics General Corporation; Lunar Corp.; MagneVu; Marconi Medicals; Philips Medical Systems; Shimadzu; Siemens; Toshiba America Medical Systems; including imaging systems, by, e.g., Silicon Graphics.

[0139] Positron emission tomography imaging (PET) systems and devices well known in the art can be used for imaging. For example, an imaging method of this disclosure may use the system designated Pet VI located at Brookhaven National Laboratory. For descriptions of PET systems and devices, see, for example, U.S. Pat. Nos. 6,151,377. Animal imaging modalities such as micro-PETs (Concorde Microsystems, Inc.) can also be used.

[0140] Single-photon emission computed tomography (SPECT) systems and devices well known in the art can be used for imaging. (See, for example, U.S. Pat. Nos. 6,115,446; 6,072,177; 5,608,221; 5,600,145; 5,210,421; 5,103,098) Imaging methods may also use animal imaging modalities, such as micro-SPECTs.

[0141] Sensitive photon detection systems can be used to detect bioluminescent and fluorescent proteins externally; see for example, Contag, 2000, Neoplasia 2:41-52; and Zhang, 1994, Clin. Exp. Metastasis, 12:87-92. The imaging methods of the disclosure can be practiced using any such photon detection device, for example, an intensified charge-coupled device (ICCD) camera coupled to an image processor. Photo detection devices are also commercially available from Xenogen, Hamamatsue.

[0142] Disclosed herein are materials, compositions, and methods that can be used for, can be used in conjunction with or can be used in preparation for the disclosed embodiments. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutations of these compositions may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a method is disclosed and discussed, and a number of modifications that can be made to a number of molecules included in the method are discussed, each and every combination and permutation of the method, and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of this disclosure including, but not limited to, steps in methods using the disclosed compositions. Thus, if there are various additional steps that can be performed, it is understood that each of these additional steps can be performed with any specific method steps or 47USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCTcombination of method steps of the disclosed methods, and that each such combination or subset of combinations is specifically contemplated and should be considered disclosed.

[0143] Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference in their entireties. The following description provides further non-limiting examples of the disclosed compositions and methods.EXAMPLESThe following examples are offered to illustrate, but not to limit, the claimed invention.Example 1. Materials and MethodsPSMA8 Peptide

[0144] PSMA8 was identified as being expressed on the surface of several cancer cell lines (e.g., H2195 (small cell lung cancer cells) and SNU1 (gastric cancer cells)) but not on normal cells. Camels were immunized to produce nanobodies that bind the PSMA8 peptide, using a fusion protein comprising a hexameric version of the PSMA8 peptide (SEQ ID NO:8) coexpressed with a human Fc domain. In addition, the same construct was used to facilitate purification and screening of candidate nanobodies.Bio-Layer Interferometry (BLI) for Nanobody Screening

[0145] For the BLI assays, the Fc-conjugated PSMA8 peptide immunogens were captured on anti-human-Fc sensors. After loading, the baseline signal was then recorded for 120 seconds in IX kinetics Buffer. The sensors were then immersed into wells containing a PSMA8 nanobody at various concentrations (400 nM, 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM and 6.25 nM) for 300 seconds (association phase), followed by immersion in IX Kinetics Buffer for an additional 600 seconds (dissociation phase). The background signal was measured using a PSMA8 immunogen loaded reference sensos immersed into a IX Kinetics buffer during both association and dissociation phases, which was subtracted from the signal obtained from sensors dipped into the nanobody solutions. Kinetic analyses were performed at least twice with an independently prepared analyte dilution series. The data was processed and fitted using the Octet® Analysis Studio data analysis software. Mean k-on, k-off values were determined by averaging all binding curves that matched the theoretical fit with an R2value of >=0.95. Flow Cytometry Assays

[0146] Nanobodies were conjugated to the CF647 fluorophore using a lysine-based conjugation technique. The conjugated nanobodies were incubated with cell lines of interest48USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCT(H2195, SNU-1 : PSMA8hlghand THP-1 : PSM8low) for a period of 1 hour on ice before being run on a CytoFLEX flow cytometer.Cell line derived xenograft (CDX) model

[0147] All animal experiments were approved by and conformed to the relevant regulatory standards of the Institutional Animal Care and Use Committee at the MDACC. CDX mouse models were generated to assess the binding affinity of the PSMA8 nanobody (DI -7-93). Twelve-week-old female nude mice were inoculated subcutaneously with either 3.5xl06SNU-1 cells or 3.5xl06PANC-1 cells. After tumors were established, mice received either the nanobody conjugated to a near-infrared dye or a dye-only control. 72 hours after injection, mice were euthanized, and cells from the tumor and bone marrow were dissociated into single-cell suspensions and analyzed by flow cytometry.Example 2. Validation of Nanobody Candidates.

[0148] Thirty-seven nanobodies were identified in an initial screening for binding to the PSMA8 peptide used for immunization of the camels. Of the 37 identified nanobodies, Dl-7-93 exhibited consistent binding to PSMA8 in additional binding studies (FIG. 1). In these experiments, anti-Human-Fc sensors were loaded with the PSMA8 immunogen (SEQ ID NO:8) used for generating nanobodies against PSMA8 protein. The loaded sensors were dipped in solutions of varying concentrations (400 nM, 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM and 6.25 nM) of the nanobody. Their interaction was assessed, and subject to BLI analysis. The data was processed and fitted using the Octet® Analysis Studio software. Only Association and Dissociation steps are shown in Figure 1, divided by a vertical dotted line. Solid horizontal lines depict lines of best fit from the analysis software. The nonlinear regression fits from 1:1 global analysis are shown as horizontal lines. Detailed kinetic analysis provided KD, Ka, and Kdis values, corroborating binding behaviors, and specificities (Table 5). These findings collectively underscore the successful recombinant production and functionality of the PSMA8 nanobody.Table 5. Affinities of the lead nanobody

[0149] Following affinity studies, cell binding was then conducted using flow cytometry analyses (FIGS. 2A-2C). DI -7 -93 nanobody, conjugated to the CF647 fluorophore, was incubated with several cell lines of interest (H2195 (small cell lung cancer cells, PSMA8hlgh),49USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCTSNU-1 (gastric cancer cells, PSMA8hlgh), and THP-1 (leukemia cells, PSM8low)) for a period of 1 hour on ice. The cells were then run on a CytoFLEX flow cytometer. Rightward shift of the histograms when compared to the untreated control cells demonstrated positive nanobody binding on the cell surface for all three cancer cell lines tested.

[0150] Additional cell binding was conducted in a cell-line derived xenograft model (FIGS.3A-3B). DI -7 -93 nanobody, conjugated to a near infrared dye, or a control (dye alone) was injected into mice inoculated with SNU-1 (gastric cancer cells, PSMA8hlgh) cells. After 72 hours, cells from the tumor and bone marrow were dissociated into a single cell suspension and analyzed on a CytoFLEX flow cytometer. A rightward shift of the histograms when compared to the control cells showed the nanobody binding on the cell surface for the ex vivo tumor sample (FIG.3A). This binding demonstrates the selective localization and target engagement of the nanobody in vivo. No observable right shift was detected for the ex vivo bone marrow sample (FIG. 3B), demonstrating that the nanobody has minimal off-target accumulation and a low likelihood of bone marrow-associated toxicity. Cell binding was also studied in a PANC-1 CDX model (FIG. 4). The PANC-1 tumor cells (which do not express PSMA8) were isolated from the mice post 72-hour incubation with DI -7-93 or control (dye alone). The cells demonstrated no observable shift in the signal, demonstrating that DI -7-93 only localizes in tumor cells due to target expression.

[0151] Following flow cytometry analysis above, experiments were conducted to assess the internalization and localization of the nanobodies in SNU-1 cells (FIGS. 5A-5D). DI -7-93 nanobodies were conjugated to the CF647 red fluorophore using a lysine-based conjugation technique as in the flow cytometry analyses. To assess the ability of the nanobodies to internalize into tumor cells, the conjugated nanobodies were incubated with PSMA8hlghhuman gastric cancer SNU-1 cells for a fixed time-period at 37 °C. The cells were also counter-stained with the nuclear stain Hoechst (blue) and LysoTracker® probe (green) for staining lysosomes (FIGS. 5A, 5C). The cells were imaged on a Nikon confocal microscope at various timepoints.FIGS. 5B and 5D show the cellular internalization and localization of DI -7-93 following a 2 hour incubation (FIG. 5B) and a 24 hour incubation (FIG. 5D). Notably, DI -7-93 is internalized within 24 hours, and localization occurs in areas of the cells including but not limited to the lysosomes.50USl 155116975 1Atorney Docket No. 090723-1544373-MDA25-056BPCTSEQUENCES51USl 155116975 1

Claims

Attorney Docket No. 090723-1544373-MDA25-056BPCTWHAT IS CLAIMED:

1. An isolated nanobody, comprising:(i) a CDR1 comprising SEQ ID NO: 2;(ii) a CDR2 comprising SEQ ID NO: 3; and(iii) a CDR3 comprising SEQ ID NO: 4.

2. The isolated nanobody of claim 1, wherein the nanobody has at least 92% identity to SEQ ID NO: 1.

3. The isolated nanobody of claim 1, wherein the nanobody comprises SEQ ID NO: 1.

4. The nanobody of any one of claims 1-3, wherein the nanobody is conjugated or fused to an imaging agent, a cytotoxic agent, a metal, or a radioactive moiety.

5. The nanobody of any one of claims 1-4, wherein the nanobody is conjugated to an imaging agent, and wherein the imaging agent is a fluorophore or a radioactive moiety.

6. The nanobody of claim 4 or 5, wherein the nanobody is conjugated or fused to a radioactive moiety, and wherein the radioactive moiety is Zr-89, Cu-64, F-18, Y-90, Lu-177, At-211, Ac-225, orPb-212.

7. The nanobody of any one of claims 1-4, wherein the nanobody is an immune conjugate.

8. The nanobody of any one of claims 1-4, wherein the nanobody is a nanobody-drug conjugate.

9. A pharmaceutical composition comprising the isolated nanobody of any one of claims 1-8 and a pharmaceutically acceptable carrier.

10. A method of treating cancer in a patient, comprising administering to the patient an anti-tumor effective amount of the pharmaceutical composition of claim 9.

11. The method of claim 10, wherein the composition comprises the isolated nanobody conjugated to a therapeutic agent.

12. The method of claim 11, wherein the therapeutic agent is at least one of a cytotoxic agent, a chemotherapeutic agent, an immunosuppressive agent, or a radioactive moiety.52USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCT13. The method of any one of claims 10-12, wherein the cancer has been determined to express an elevated level of PSMA8 relative to a healthy tissue.

14. The method of any one of claims 10-13, wherein the cancer is selected from a group consisting of a lung cancer, a stomach cancer, a blood cancer, an ovarian cancer, a bladder cancer, a melanoma, a prostate cancer, a breast cancer, a glioma, and a lymphoma.

15. The method of any one of claims 10-14, wherein the patient has previously failed to respond to an immune checkpoint inhibitor.

16. The method of claim 15, wherein the patient has relapsed.

17. The method of any one of claims 10-16, further comprising administering at least a second anti-cancer therapy.

18. The method of claim 17, wherein the second anti-cancer therapy is a chemotherapy, molecular targeted therapy, immunotherapy, radiotherapy, radioimmunotherapy, phototherapy, gene therapy, surgery, hormonal therapy, epigenetic modulation, anti-angiogenic therapy, or cytokine therapy.

19. A method of detecting a presence of PSMA8 in a biological sample comprising:(a) contacting the sample with the nanobody of any one of claims 1 to 3; and(b) detecting an amount of binding of the isolated nanobody as a determination of the presence of PSMA8 in the sample.

20. The method of claim 19, wherein the biological sample comprises cancer cells.

21. The method of claim 19, wherein the biological sample comprises a tumor sample of a tumor from a subject.

22. A method of imaging a tumor in a subj ect with a PSMA8-expressing cancer, the method comprising:(a) administering to the subject the isolated nanobody of any one of claims 1 to 3 conjugated to an imaging label; and(b) detecting the imaging label in the subject to obtain an image of the tumor.

23. The method of claim 22, wherein the imaging label is a fluorophore or a radioactive moiety.53USl 155116975 1Attorney Docket No. 090723-1544373-MDA25-056BPCT24. The method of claim 23, wherein the radioactive moiety is Zr-89, Cu-64, F-18, Y-90, Lu-177, At-211, Ac-225, or Pb-212.

25. A method of monitoring response of a subject with a PSMA8 expressing cancer to cancer therapy, the method comprising:(a) detecting a presence of a tumor in the subject at a first time point by administering the nanobody of any one of claims 1 to 3 conjugated to a first radioactive moiety and obtaining a first image of the tumor; and(b) treating the subject with an anti-cancer therapy comprising administering a therapeutically effective amount of the nanobody of any of claims 1 to 3 conjugated to a second radioactive moiety;(c) detecting the presence or absence of the tumor in the subject at a second time point after the subject has been treated with anti-cancer therapy by administering the nanobody conjugated to the first radioactive moiety and obtaining a second image of the tumor; and (d) comparing the first image to the second image to determine whether a change in tumor size has occurred.

26. The method of claim 25, wherein the first radioactive moiety and the second radioactive moiety are independently selected from a group consisting of89Zr,131I,125I,123I,1UI, "mTc,90Y,186Re,188Re,32P,153Sm,67Ga,2O1T1,77Br,18F,161Tb,225Ac,161Tb / 225Ac,177Lu,134Ce,140Nd,169Er,134Ce / 134La, and140Nd / 140Pr.

27. The method of claim 25 or 26, wherein steps (c) to (d) are repeated at a third time point after the subject receives anti-cancer therapy.

28. The method of any one of claims 25-27, wherein the anti-cancer therapy comprises a chemotherapy, molecular targeted therapy, immunotherapy, radiotherapy, radioimmunotherapy, phototherapy, gene therapy, surgery, hormonal therapy, epigenetic modulation, anti-angiogenic therapy, or cytokine therapy.54USl 155116975 1

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