Antibody-based human lamp-1 composition and its uses

Abstract

Polypeptides, such as antibodies, nanobodies, and antigen-binding fragments thereof, and the use of these polypeptides to label cell surface LAMP-1. Polypeptides of the invention can be used to label and track tumor cells in imaging studies, as well as to treat a wide variety of cancers and inflammatory diseases.

Description

[0001] ANTIBODY-BASED HUMAN LAMP-1 COMPOSITION AND ITS USES

[0002] Field of the Invention

[0003] The invention relates to polypeptides, such as antibodies, nanobodies, and antigen-binding fragments thereof, capable of binding to lysosomal associated membrane proteins (LAMPs), such as LAMP-1 . The polypeptides of the invention can be used in the field of cancer therapy and inflammatory diseases as a diagnostic, theranostic, or therapeutic tool.

[0004] Cross-Reference to Related Applications

[0005] This application claims benefit of U.S. Provisional Application No. 63 / 602,198 filed November 22, 2023, and U.S. Provisional Application No. 63 / 549,177 filed February 2, 2024, the contents of which are incorporated by reference.

[0006] Statement As To Federally Funded Research

[0007] This invention was made with government support under 5R01 DK123143, 5R01 CA21 1223, and 5K08CA249047 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0008] Background of the Invention

[0009] Cancer is a major cause of morbidity and mortality worldwide, with a predicted increase in its global burden over the following two decades (1 , 2). Epithelial carcinomas of the breast, colon, prostate, and pancreas are among the leading causes of cancer incidence and mortality, with an estimated combined incidence of 40% of all new cancer cases and more than four million deaths in 2020 (3). Imaging is a substantial part of cancer diagnosis and management. There is an increasing need to develop novel imaging agents for the accurate localization and characterization of tumors and guiding patient selection for the newer generation of cancer therapies (4). Positron emission tomography (PET) is an essential contributor to the field by providing information on tumor characteristics and treatment response at the cellular and molecular levels (5-7). ImmunoPET is a growing modality, combining the sensitivity of PET with the precise targeting ability of monoclonal antibodies (mAbs) and mAb-based derivatives (8). An immunoPET probe targeting a common highly expressed antigen among a wide range of adenocarcinomas is currently lacking and can be significantly helpful for precision imaging and targeted therapy of these burdensome cancers.

[0010] Lysosomes are crucial organelles for maintaining cellular homeostasis. Lysosomal count, activity, and localization in the cell are highly regulated in response to altered cell and microenvironment conditions (9). In addition to their long-known digestive role in phagocytosis and autophagy, lysosomes are involved in metabolic activity, signaling, and remodeling of the extracellular matrix (ECM) (9, 10). A body of literature is available on the implications of the lysosomal system in cancer cell survival and adaptation (1 1 -13). Lysosomal associated membrane proteins (LAMPs) are a family of transmembrane proteins with a highly glycosylated and sialylated N-terminal facing the lumen of lysosomes and a short cytoplasmic C-terminal. LAMP-1 and LAMP-2 are two members of the family studied in more detail. LAMP-1 was initially considered a structural protein, protecting the lysosomal membrane integrity from hydrolytic enzymes and the acidic pH of the lumen (14, 15).

[0011] However, further studies have shown additional roles of LAMP-1 in the lysosome activity, especially in transit, docking, and fusion of lysosomes to the plasma membrane (PM) for exocytosis (16, 17), which leads to the redistribution of LAMP-1 to the PM with the N-terminal domain facing ECM (18-20).

[0012] The involvement of LAMP-1 in cancer progression, invasion, and metastasis has been suggested in carcinomas of the colon, breast, pancreas, prostate, and other malignancies such as glioblastoma, melanoma, and esophageal squamous cell carcinoma (21 -30). Furthermore, an increase in the proportion of cell surface displayed LAMP-1 compared to total cellular expression is suggested to increase the risk of metastasis (21 , 28, 31 ).

[0013] However, given that there is a paucity of literature focusing on LAMP-1 for non-invasive imaging and targeted cancer therapy, there is a need in the art to develop such tools.

[0014] Summary of the Invention

[0015] In one aspect, the invention, in general, features an anti-LAMP-1 antibody or nanobody composition. In some embodiments, the nanobody includes CDR1 (GLIFSINSMG) (SEQ ID NO: 1 ), CDR2 (VHNGDST) (SEQ ID NO: 2), and CDR3 (NARDPRGGHLWNY) (SEQ ID NO: 3) of NB132.

[0016] In some embodiments, the nanobody includes QVQLQESGGGLVQAGGSLRLSCTASGLIFSINSMGWYRQVPGKQRELVASVHNGDSTYSADSVKG RFTISVDDAKNMVYLQMNDLEPEDTAVYYCNARDPRGGHLWNYWGQGTQVTVSS (NB132) (SEQ ID NO: 4).

[0017] In some embodiments, the amino acid sequences of the nanobody includes one or more CDRs selected from the group consisting of:

[0018] (a) CDR1 : GLIFSINSMG (SEQ ID NO: 1 ); CDR2: VHNGDST (SEQ ID NO: 2); and CDR3: NARDPRGGHLWNY (SEQ ID NO: 3) of NB132;

[0019] (b) CDR1 : GLIFRINVMG (SEQ ID NO: 5); CDR2: ITHGGST (SEQ ID NO: 6); and CDR3: NAADSAGSNMNAKWSY (SEQ ID NO: 7) of NB121 ;

[0020] (c) CDR1 : GDIFSIDRMG (SEQ ID NO: 9); CDR2: IFLSDGET (SEQ ID NO: 10); and CDR3: WSYTTTNS (SEQ ID NO: 11 ) of NB103;

[0021] (d) CDR1 : GIIFRINDMG (SEQ ID NO: 13); CDR2: ATMDDST (SEQ ID NO: 14); and CDR3: YAGRSEKWAY (SEQ ID NO: 15) of NB63;

[0022] (e) CDR1 : ETIFNANTMY (SEQ ID NO: 17); CDR2: ITTRGATD (SEQ ID NO: 18); and CDR3: NVFIFGVDY (SEQ ID NO: 19) of NB185;

[0023] (f) CDR1 : GFTFRNYAMR (SEQ ID NO: 21 ); CDR2: ISSGGDATS (SEQ ID NO: 22); and CDR3: ATSDFNI (SEQ ID NO: 23) of NB191 ;

[0024] (g) CDR1 : GLIFRINDMG (SEQ ID NO: 25); CDR2: EATDDSTS (SEQ ID NO: 26); and CDR3: YAGRSGKWDY (SEQ ID NO: 27) of NB189;

[0025] (h) CDR1 : GSISSIDRMG (SEQ ID NO: 29); CDR2: LSTSGDILT (SEQ ID NO: 30); and CDR3: WSYDFHNY (SEQ ID NO: 31 ) of NB95;

[0026] (i) CDR1 : GIIFRINDMG (SEQ ID NO: 13); CDR2: ATMDDSTN (SEQ ID NO: 33); and CDR3: YAGRSEKWAY (SEQ ID NO: 15) of NB56;

[0027] (j) CDR1 : GIIFRINDMG (SEQ ID NO: 13); CDR2: ATTDDSTN (SEQ ID NO: 35); and CDR3: YAGRSEKWAY (SEQ ID NO: 15) of NB173;

[0028] (k) CDR1 : GIIFRINAMA (SEQ ID NO: 37); CDR2: IGQDDSTT (SEQ ID NO: 38); and CDR3: NAADRVPYERWSY (SEQ ID NO: 39) of NB109; and

[0029] (l) CDR1 : GIIFRINDMG (SEQ ID NO: 13); CDR2: ETIDGSRN (SEQ ID NO: 41 ); and CDR3: NAARESGGRILWSY (SEQ ID NO: 42) of NB128.

[0030] In some embodiments, the amino acid sequence of the nanobody composition is selected from the group consisting of:

[0031] (a)QVQLQESGGGLVQAGGSLRLSCTASGLIFSINSMGWYRQVPGKQRELVASVHNGDSTYS ADSVKGRFTISVDDAKNMVYLQMNDLEPEDTAVYYCNARDPRGGHLWNYWGQGTQVTVSS (NB132) (SEQ ID NO: 4);

[0032] (b)QVQLQESGGGLVQAGGSLRLSCAASGLIFRINVMGWYRQAPGKERELVAQITHGGSTNY ADSVKGRFTVSRDDAENTVDLQMNSLKPEDTAVYYCNAADSAGSNMNAKWSYWGQGTQVTVSS (NB121 ) (SEQ ID NO: 8);

[0033] (c)QVQLQESGGGLVQAGGSLRLSCAASGDIFSIDRMGWFRQTPGKERELVASIFLSDGETKY GDFVKGRFTISRDNAKNAVYLQMNSLKPEDTAVYYCWSYTTTNSWGQGTQVTVSS (NB103) (SEQ ID NO: 12);

[0034] (d)QVQLQESGGGLAQAGGSLRLSCTASGIIFRINDMGWYRQAPGKQRELVAVATMDDSTNY ADSVKGRFTVSRDSAKNSVYLQMNSLRPEDTAVYYCYAGRSEKWAYWGQGTQVTVSS (NB63) (SEQ ID NO: 16);

[0035] (e)QVQLQESGGGLVQAGGSLRLSCAVPETIFNANTMYWYRRAPGKEREWVAVITTRGATDY ADSVKGRFTISRDNAKNTLNLEMNSLKPEDTAVYYCNVFIFGVDYWGQGTQVTVSS (NB185) (SEQ ID NO: 20);

[0036] (f)QVQLQESGGGLVQPGGSLRLSCAASGFTFRNYAMRWVRQGPGNGLEGVSTISSGGDAT SYADSVTGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCATSDFNIRGQGTQVTVSS (NB191 ) (SEQ ID NO: 24);

[0037] (g)QVQLQESGGGLAQAGGSLRLSCTASGLIFRINDMGWYRQAPGKQRELVAVEATDDSTSY ADSVKGRFTVSRDSAKNTVYLQMNSLKPEDTAVYYCYAGRSGKWDYWGQGTQVTVSS (NB189) (SEQ ID NO: 28);

[0038] (h)QVQLQESGGGLVQAGGSLTLSCSVSGSISSIDRMGWFRQAPGKERELVAVLSTSGDILTY ADDVKGRFTISRDDAMNTVSLQMNSLKPEDTAVYYCWSYDFHNYWGQGTQVTVSS (NB95) (SEQ ID NO: 32);

[0039] (i)QVQLQESGGGSVQVGESLTLSCTASGIIFRINDMGWYRQAPGKQRELVAVATMDDSTNY ADSVKGRFTVSRDSAKNSVYLQMNSLRPEDTAVYYCYAGRSEKWAYWGQGTQVTVSS (NB56) (SEQ ID NO: 34);

[0040] (j)QVQLQESGGGLAQAGGSLRLSCTASGIIFRINDMGWYRQAPGKQRELVAVATTDDSTNYA DSVKGRFTVSRDSAKNSVYLQMNSLRPEDTAVYYCYAGRSEKWAYWGQGTQVTVSS (NB173) (SEQ ID NO: 36);

[0041] (k)QVQLQESGGGLAQAGESLRLSCVASGIIFRINAMAWYRQDPGKQRELVAAIGQDDSTTYA DSVKGRFAISRDNANDTVYLQMNSLKPEDTSVYYCNAADRVPYERWSYWGQGTQVTVSS (NB109) (SEQ ID NO: 40); and

[0042] (l)QVQLQESGGGSAQPGGSLRLSCAASGIIFRINDMGWYRQAPGKHREMVAVETIDGSRNY GDSVKGRFTISRDSANDTVYLEMNNLKPEDTAVYYCNAARESGGRILWSYWGQGTQVTVSS (NB128) (SEQ ID NO: 43).

[0043] In some embodiments, the nanobody is conjugated to a diagnostic agent including a radionuclide, a contrast agent, a fluorescent agent, a chemiluminescent agent, a bioluminescent agent, a paramagnetic ion, an enzyme, or a photoactive diagnostic agent.

[0044] In some embodiments, the radionuclide includes89Zr,3H,11C,14C,18F,32P,35S,36CI,51Cr,52Fe,57Co,58Co,59Fe,64Cu,67Cu,67Ga,68Ga,75Se,76Br,77Br,89Zr,90Y, "mTc,111In,123l,124l,125l,131l,152Eu,153Sm,166Ho,177Lu,186Re,188Re,201TI,203Pb,210At,211At, 212Bi,213Bi,149Tb,160Tb,161Tb, or225Ac.

[0045] In some embodiments, the nanobody is conjugated to desferrioxamine (DFO).

[0046] In another aspect, the invention features a nucleic acid molecule (e.g., a DNA, RNA, or mRNA molecule) encoding any of the aforementioned sequences.

[0047] In another aspect, the invention features a composition, including a nanobody which is specific for and binds directly to a LAMP epitope, wherein the diseased state LAMP epitope is present in greater amounts in a diseased tissue than in a normal tissue, and wherein the nanobody is conjugated to an active agent.

[0048] In some embodiments, the active agent is linked to N-terminus of the nanobody.

[0049] In some embodiments, the active agent is linked to C-terminus of the nanobody.

[0050] In some embodiments, the active agent is an imaging probe.

[0051] In some embodiments, the imaging probe is selected from fluorophores, immunohistochemical tracers, PET tracers, near-infrared (NIR) probes, single-photon emission computed tomography (SPECT) probes, magnetic particle imaging (MRI) probes, and radioisotopes.

[0052] In some embodiments, the nanobody includes a sequence set forth in SEQ ID NOs: 1 -43 and their derivatives with a sequence identity to the original sequences.

[0053] In another aspect, the invention features a composition, including a peptide including a sequence set forth in SEQ ID NOs: 1 -43 or fragment thereof and a pharmaceutically acceptable carrier.

[0054] In some embodiments, the peptide is conjugated to an active agent.

[0055] In some embodiments, the peptide is a monoclonal antibody, a humanized antibody, a chimeric antibody, a human antibody, or antibody fragment.

[0056] In some embodiments, the fragment thereof is a CDR.

[0057] In another aspect, the invention features a composition, including a nanobody including a sequence set forth in SEQ ID NOs: 1 -43 or fragment thereof and a pharmaceutically acceptable carrier.

[0058] In some embodiments, the nanobody is conjugated to an active agent.

[0059] In some embodiments, the nanobody is conjugated to a monoclonal antibody, a humanized antibody, a chimeric antibody, a human antibody, or other antibody fragment. In another aspect, the invention features a method, including administering to a subject having a tumor or other disease state a composition of any one of aforementioned compositions, in an effective amount to deliver the active agent to the tumor or to a region affected by the disease state.

[0060] In some embodiments, the method involves determining the presence or absence of one or more LAMP proteins characteristic of a diseased state in a subject and determining whether the subject has a disease.

[0061] In some embodiments, the presence or absence of one or more LAMP proteins is determined by imaging.

[0062] In some embodiments, the diseased state is a cancer.

[0063] In some embodiments, the cancer is breast or colon cancer.

[0064] In some embodiments, the LAMP proteins are detected using one or more nanobodies that specifically bind to the LAMP proteins.

[0065] In another aspect, the invention features a method, including administering to a subject having a tumor a composition of any of the aforementioned compositions, wherein the nanobody includes a detectable label, in an effective amount to bind to and identify tumor margins of the tumor.

[0066] In some embodiments, the nanobody specifically binds the diseased state LAMP epitope with a binding affinity in the nM range, as measured by surface plasmon resonance.

[0067] In some embodiments, the nanobody includes a sequence set forth in SEQ ID NOs: 1 -43 and their derivatives with a sequence identity to the original sequences.

[0068] In another aspect, the invention features a method, including administering to a subject having a tumor or other disease state a composition of aforementioned compositions, wherein the nanobody is linked to an active agent, in an effective amount to deliver the active agent to the tumor or other disease site.

[0069] In some embodiments, the nanobody specifically binds the diseased state LAMP epitope with a binding affinity in the nM range, as measured by surface plasmon resonance.

[0070] In some embodiments, the nanobody includes a sequence set forth in SEQ ID NOs: 1 -43 and their derivatives with a sequence identity to the original sequences.

[0071] In another aspect, the invention features use of a nanobody described herein in PET imaging.

[0072] In another aspect, the invention features use of a nanobody described herein in theranostics.

[0073] In another aspect, the invention features use of a nanobody described herein to treat LAMP-

[0074] 1 + cancers or to treat an inflammatory condition characterized by overexpression of LAMP-1 .

[0075] In some embodiments, the cancer is selected from the group consisting of breast cancer, colon cancer, esophageal cancer, stomach cancer, liver cancer, bladder cancer, pancreatic cancer, and prostate cancer.

[0076] In some embodiments, the nanobody binds to the same epitope as or competes for binding to LAMP-1 with an antibody that includes a CDR sequence according to any one of the aforementioned nanobodies.

[0077] In some embodiments, the nanobody is conjugated to at least one diagnostic agent selected from the group consisting of a radionuclide, a contrast agent, a fluorescent agent, a chemiluminescent agent, a bioluminescent agent, a paramagnetic ion, an enzyme, and a photoactive diagnostic agent. In some embodiments, the diagnostic agent is a radionuclide including89Zr,3H,11C,14C,18F,

[0078] In some embodiments, the radionuclide is89Zr and the use further includes PET imaging.

[0079] In some embodiments, the nanobody is conjugated to an paramagnetic ion selected from the group consisting of chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III), and erbium (III).

[0080] In another aspect, the invention features use anti-LAMP-1 antibody, antibody fragment, or nanobody for diagnostic, theranostic, or therapeutic purposes in the treatment of LAMP+ cancers and inflammatory conditions characterized by overexpression of LAMP-1 .

[0081] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0082] Definitions

[0083] To facilitate the understanding of the invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by those of ordinary skill in the areas relevant to the invention.

[0084] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a molecule” optionally includes a combination of two or more such molecules, and the like.

[0085] As used herein, the terms “antibody” and “Ab” refer to a molecule that specifically binds to, or is immunologically reactive with, a particular antigen and includes at least the variable domain of a heavy chain, and normally includes at least the variable domains of a heavy chain and of a light chain of an immunoglobulin. Antibodies may be monoclonal, polyclonal, genetically engineered, and otherwise modified forms of antibodies, including but not limited to chimeric antibodies, humanized antibodies, heteroconjugate antibodies, and antigen-binding fragments of antibodies, including e.g., Fab', F(ab')2, Fab, Fv, rlgG, and scFv fragments. Moreover, unless otherwise indicated, the term “monoclonal antibody” (mAb) is meant to include both intact molecules, as well as antibody fragments (e.g., Fab and F(ab')2 fragments) that are capable of specifically binding to a target protein. Fab and F(ab')2 fragments lack the Fc fragment of an intact antibody, clear more rapidly from the circulation of the animal, and may have less non-specific tissue binding than an intact antibody (32).

[0086] As used herein, the term “antigen-binding fragment” refers to one or more fragments of an immunoglobulin that retain the ability to specifically bind to a target antigen. The antigen-binding function of an immunoglobulin can be performed by fragments of a full-length antibody. The antibody fragments can be a Fab, F(ab’)2, scFv, SMIP, diabody, a triabody, an affibody, a nanobody, an aptamer, or a domain antibody. Examples of binding fragments encompassed of the term “antigenbinding fragment” of an antibody are (i) a Fab fragment, a monovalent fragment consisting of the Vi_, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb (33) including VH and VL domains; (vi) a dAb fragment that consists of a VH domain; (vii) a dAb that consists of a VH or a VL domain; (viii) an isolated complementarity determining region (CDR); and (ix) a combination of two or more isolated CDRs which may optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single-chain Fv (scFv); 34, 35). These antibody fragments can be obtained using conventional techniques known to those of skill in the art, and the fragments can be screened for utility in the same manner as intact antibodies. Antigen-binding fragments can be produced by recombinant DNA techniques, enzymatic, or chemical cleavage of intact immunoglobulins, or, in certain cases, by chemical peptide synthesis procedures known in the art.

[0087] As used herein, the terms “complementarity determining region” and “CDR” refer to a hypervariable region found both in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FRs). As is appreciated in the art, the amino acid positions that delineate a hypervariable region of an antibody can vary, depending on the context and the various definitions known in the art. Some positions within a variable domain may be viewed as hybrid hypervariable positions in that these positions can be deemed to be within a hypervariable region under one set of criteria while being deemed to be outside a hypervariable region under a different set of criteria. One or more of these positions can also be found in extended hypervariable regions. The invention includes antibodies comprising modifications in these hybrid hypervariable positions. The variable domains of native heavy and light chains each comprise four framework regions that primarily adopt a p-sheet configuration, connected by three CDRs, which form loops that connect, and in some cases form part of, the p-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions in the order FR1 -CDR1 - FR2-CDR2-FR3-CDR3-FR4 and, with the CDRs from the other antibody chains, contribute to the formation of the target binding site of antibodies (see Kabat et al, Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, Md. 1987; incorporated herein by reference). As used herein, numbering of immunoglobulin amino acid residues is done according to the immunoglobulin amino acid residue numbering system of Kabat et al, unless otherwise indicated.

[0088] As used herein, the terms “conservative mutation,” “conservative substitution,” and “conservative amino acid substitution” refer to a substitution of one or more amino acids for one or more different amino acids that exhibit similar physicochemical properties, such as polarity, electrostatic charge, and steric volume. These properties are summarized for each of the 20 naturally- occurring amino acids in Table 1 below.

[0089] Table 1. Representative physicochemical properties of naturally-occurring amino acids

[0090] From this table it is appreciated that the conservative amino acid families include, e.g., (i) G, A, V, L, I, P, and M; (ii) D and E; (iii) C, S, and T; (iv) H, K, and R; (v) N and Q; and (vi) F, Y, and W. A conservative mutation or substitution is therefore one that substitutes one amino acid for a member of the same amino acid family (e.g., a substitution of Ser for Thr or Lys for Arg).

[0091] As used herein, the term “conjugate” refers to a compound formed by the chemical bonding of a reactive functional group of one molecule with an appropriately reactive functional group of another molecule. For example, the polypeptides of the disclosure may be conjugated to a diagnostic agent such as a radionuclide or paramagnetic ion. As used herein, the term “epitope” refers to a portion of an antigen molecule to which an antibody specifically binds. Epitopes usually consist of chemically active (such as polar, non-polar, or hydrophobic) surface groupings of moieties such as amino acids or polysaccharide side chains, and can have specific three-dimensional structural characteristics, as well as specific charge characteristics. An epitope may be a linear epitope or a conformational epitope.

[0092] As used herein, the terms “framework region” and “FR” refer to amino acid residues that are adjacent to the CDRs. FR residues may be present in, for example, human antibodies, rodent-derived antibodies (e.g., murine antibodies), humanized antibodies, primatized antibodies, chimeric antibodies, antibody fragments (e.g., Fab fragments), single-chain antibody fragments (e.g., scFv fragments), antibody domains, and bispecific antibodies, among others.

[0093] As used herein, the term “nanobody” refers to a polypeptide having a single monomeric variable domain and has a molecular weight of between about 12 kDa and about 15 kDa. A nanobody is able to selectively bind to a specific antigen.

[0094] As used herein, the term “paramagnetic ion” refers to an ion that contains at least one unpaired electron. Paramagnetic ions are attracted by a magnetic field. Examples of paramagnetic ions that can be used in conjunction with the polypeptides of the disclosure are chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III), and erbium (III).

[0095] As used herein, the term “percent (%) sequence identity” with respect to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to the nucleic acids or amino acids in the reference polynucleotide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the capabilities of those of skill in the art, for instance, using publicly available computer software, such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, percent sequence identity values may be generated using the sequence comparison computer program BLAST. As an illustration, the percent sequence identity of a given nucleic acid or amino acid sequence, A, to, with, or against a given nucleic acid or amino acid sequence, B, (which can alternatively be phrased as a given nucleic acid or amino acid sequence, A that has a certain percent sequence identity to, with, or against a given nucleic acid or amino acid sequence, B) is calculated as follows:

[0096] 100 multiplied by (the fraction X / Y) where X is the number of nucleic acids or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in that program’s alignment of A and B, and where Y is the total number of nucleic acids in B. It will be appreciated that where the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, the percent sequence identity of A to B will not equal the percent sequence identity of B to A.

[0097] As used herein, the terms “positron emission tomography imaging” and “PET imaging” refer to an imaging test that uses radioactive substances to visualize and measure changes in metabolic activity of the cells of body tissues.

[0098] As used herein, the term “radionuclide” refers to an unstable chemical element that releases radiation as it becomes more stable. Radionuclides are used in imaging tests, such as PET imaging. Examples of radionuclides that can be used in conjunction with the polypeptides of the disclosure are3H,11C,14C,18F,32P,35S,36CI,51Cr,52Fe,57Co,58Co,59Fe,64Cu,67Cu,67Ga,68Ga,75Se,76Br,77Br,89Zr,90Y, "mTc,111In,123l,124l,125l,131l,152Eu,153Sm,166Ho,177Lu,186Re,188Re,201TI,203Pb,210At,211At,212Bi,213Bi,149Tb,160Tb,161Tb, and225Ac.

[0099] As used herein, the terms “subject” and “patient” refer to an organism that receives treatment for a particular disease or condition as described herein (such as cancer or an inflammatory condition). Examples of subjects and patients include mammals, such as humans, receiving treatment for a disease or condition described herein.

[0100] As used herein, the term “theranostics” refers to a medical technology that combines therapeutics and diagnostics. For example, PET imaging is used to identify and visualize tumor cells in a subject, as well as to determine the therapeutic strategy for the subject.

[0101] As used herein, the terms “treat,” “treatment,” and “treating” refer to therapeutic treatment, in which the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the progression of a cancer (e.g., breast cancer, colon cancer, esophageal cancer, stomach cancer, liver cancer, bladder cancer, pancreatic cancer, and prostate cancer) or an inflammatory condition. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e. , not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Those in need of treatment include those already with the condition or disorder, as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

[0102] Brief Description of the Figures

[0103] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0104] FIG. 1 is a schematic representation of lysosomal exocytosis and autophagy. Lysosomes move along actin filaments via interaction with a motor myosin and dock at the plasma membrane (PM), a process mediated by LAMP-1 . During exocytosis, cancer cells secrete lysosomal contents extracellularly to remodel and degrade the extracellular matrix (ECM). Chemotherapy agents that are weak bases accumulate in the acidic lysosomes, and their efflux via exocytosis can lead to chemoresistance. Exosomes packed with drug metabolites and invasive signaling molecules are excreted by cancer cells and induce the transformation of resident fibroblasts and macrophages of the tumor microenvironment into cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs).

[0105] FIG. 2A shows representative immunofluorescence staining of LAMP-1 (red channel) in major carcinomas and their respective normal tissues. Nuclei are stained with DAPI (blue channel). FIG. 2B shows LAMP-1 fluorescent signal intensity in malignant cores (blue) and normal cores (red). Green: kidney; Purple: lymph node, **“: P < 0.0001 . FIG. 2C shows LAMP-1 fluorescent signal intensity in prostate, pancreas, colon, breast, uterus, skin, lymph node, and kidney. A significantly higher LAMP-1 fluorescence was observed in prostate, pancreas, colon, breast, and uterine endometrium carcinomas (red) compared to normal tissues (blue). The opposite pattern was observed in renal cell carcinoma.

[0106] * P < 0.05, “: P< 0.001 . FIGS. 2D-2F are bar graphs showing dedicated TMAs for breast, colon, and prostate.

[0107] FIG. 3A shows pan-cancer LAMP-1 RNA expression in tumors (red) and normal tissues (blue). FIG. 3B shows a positive significant correlation between LAMP-1 and ERK-2 expression. FIG. 3C shows a positive significant correlation between LAMP-1 and EGFR expression. FIG. 3D shows higher LAMP-1 expression in pan-cancer doxorubicin non-responders compared to responders.

[0108] FIG. 4A shows H&E staining of breast ductal carcinoma. FIGS. 4B-4C show pathologist annotation based on histological features and UMAP analysis. FIGS. 4D-4E show LAMP-1 expression pattern and UMAP analysis. FIGS. 4F-4G show UMAP analysis demonstrating the distribution of barcodes in ductal and non-ductal regions. FIG. 4H shows comparison of Iog2 expression of LAMP-1 in different groups. FIG. 41 shows barcodes with high / low LAMP-1 expression that are negative for immune markers after re-clustering of ductal regions. FIG. 4J shows difference in LAMP-1 expression in barcodes with high / low expression that are negative for immune markers. FIG. 4K shows ranked expression of significantly altered genes in high LAMP-1 expression barcodes compared to low expression, and UMAP analysis of LAMP-1 expression. FIG. 4L shows results of KEGG pathway analysis. FIGS. 4M-4P show GSEA enrichment analysis using different databases, including GO, HALLMARK, and oncogenic signature.

[0109] FIG. 5A shows LAMP-1 + cell population across major normal organs and tumors (cell surface and total) in MC38 tumor-bearing mice. FIG. 5B shows LAMP-1 + cell population across CD45- cells (parenchymal cells) in major normal organs and tumors. FIG. 5C shows LAMP-1 + cell population across CD45+ cells (immune cells) in major normal organs and tumors. FIG. 5D shows CD45+ cells in major normal organs and tumors.

[0110] FIGS. 6A-6B show representative89Zr-DFO-LAMP-1 MIP PET / CT images of MDA-MB-231 and Caco2 on day 1 , day 3, and day 7. Red arrows point at the tumor. FIG. 6C shows tumor to background ratio (TBR) over the course of the experiment in MDA-MB-231 (purple) and Caco2 (orange) demonstrating a sustained increase in tumor tracer localization. FIG. 6D is a set of bar graphs demonstrating that tumor SUVmax of89Zr-DFO-LAMP-1 (blue) was significantly higher than89Zr-DFO-lgG (green) at day 7 in both models. **: P < 0.001 , *** P < 0.0001 . FIGS. 6E-6F show representative89Zr-DFO-lgG MIP PET / CT images of MDA-MB-231 and Caco2 on day 1 , day 3, and day 7. Red arrows point at the tumor. FIGS. 6G-6H are bar graphs demonstrating that temporal tumor and blood pool uptake in MDA-MB-231 and Caco2 showed a sustained increase in tumor SUVmax, while blood pool uptake minimally changed. “ : P < 0.001 , ***: P < 0.0001 .

[0111] FIGS. 7A-7B show biodistribution of89Zr-DFO-LAMP-1 (blue) and89Zr-DFO-lgG (red) in tumor and major organs in MDA-MB-231 and Caco2. High tumor uptake and low normal organ retention of89Zr-DFO-LAMP-1 are demonstrated. FIG. 8A shows the top significant down-regulated signatures in Hallmark gene sets for RNAseq analysis. The blue highlights down-regulated signatures. FIGS. 8B-8D show GSEA analysis on signatures of epithelial mesenchymal transition, apoptosis, and autophagy.

[0112] FIG. 9A is a set of bar graphs showing alteration in the population of LAMP-1 + cells by increasing temperature from 4 °C to 37 °C in human breast, colon, and pancreas adenocarcinoma cell lines. FIG. 9B shows LAMP-1 + population in murine breast, colon, and pancreas adenocarcinoma cell lines. Orange: positive, Blue: negative.

[0113] FIG. 10A is a schematic representation of DFO conjugation and89Zr labeling. FIG. 10B shows binding affinity by SPR. Representative SPR sensorgrams show the response over time (resonance units, RU) during the association and disassociation binding phases of immobilized anti- LAMP-1 IgG Ab to recombinant human LAMP-1 (1 :1 fit is shown as black curves). The anti-LAMP-1 IgG binds LAMP-1 at an affinity of KD=1 .78 ± 0.84 nM. FIGS. 10C-10D show representative radio- TLC chromatograms on89Zr-DFO-LAMP-1 of crude radioimmunoconjugate and after purification, which resulted in filtering the unbound89Zr.

[0114] FIG. 11 shows representative immunofluorescence staining of LAMP-1 (red) in extracted xenografts of MDA-MB-231 and Caco2. Cell nuclei are stained with DAPI (blue).

[0115] FIG. 12 is a graphic representation of the screening of specific single positive Nb clones by enzyme-linked immunosorbent assay (ELISA). Individual Nb colonies from a bacterial library were screened for LAMP-1 recognition by ELISA. Nb clones with high absorbance ratio relative to the negative control of more than 3 were considered positive and sequenced.

[0116] FIG. 13A shows the four candidates of Nbs targeting LAMP-1 that were selected based on amino acid sequence. Diversified framework regions (FR) and complementarity determining regions (CDRs) are indicated. FIG. 13B is an SDS-PAGE gel stained with Coomassie Blue showing the purified Nbs — termed NB121 , NB103, NB132, and NB63. High purity and expected size of ~15 kDa are shown. All purified Nbs are expressed with the C-terminal sequence AAAYPYDVPDYGSHHHHHH corresponding to HA tag, GS linker, and a His tag.

[0117] FIG. 14 shows mass spectrometry results of the purified Nbs. The molecular weight and purity of the four Nbs were analyzed by MALDI-TOF. Peaks corresponding to singly and doubly charged protonated molecular ions of the Nbs are indicated. This spectrum presents a single peak at m / z ~15 kDa, corresponding to the singly charged molecular ion, [M+H]+.

[0118] FIGS. 15A-15D show that the Nbs bind to LAMP-1 with nanomolar affinity. The binding affinity of each Nb to recombinant human LAMP-1 protein was determined by using SPR spectroscopy on a Biacore T200 instrument. A 1 :1 kinetic model fitted on the sensorgrams was used to calculate the affinity (KD). In vitro binding affinity of the purified Nbs to immobilized LAMP-1 was in the nanomolar range: NB103 KD=1 .7 nM, NB132 KD=8.5 nM, NB63 KD=23.79 nM, NB121 KD=N / A.

[0119] FIGS. 16A-16B are confocal images of the Nbs and LAMP-1 co-localization in live cells. FIG. 16A shows that NB132 (red) has internalized into high LAMP-1 -expressing stable Dubca cells and colocalize with LAMP-1 (green) in the cytoplasm in small circular structures, possibly representing endocytic vesicles. FIG. 16B shows that NB121 (red) was also observed inside MDA-MB-231 cells and co-localizes with LAMP-1 (green) in the cytoplasm, suggesting internalization following interaction with cell surface LAMP-1 . The nucleus was stained with DAPI. Scale bar = 10 pm.

[0120] FIG. 17 shows in vivo optical imaging of fluorescent-labeled Nbs targeting LAMP-1 . An in vivo mice Matrigel model was used to assess Nbs targeting of LAMP-1 protein. Athymic nude mice were injected subcutaneously with 0.1 mL of recombinant human LAMP-1 protein (1 pg or 5 pg) mixed with Matrigel (1 :1 ) to the right upper flank or with 0.1 mL of Matrigel only on the left flank as negative control. Four mice were then injected intravenously with 25 pg of either NB132 or NB103 labeled with NHS-ester AlexaFluor 647. One non-injected mouse served as negative control. The labeled Nbs were specifically detected in LAMP-1 + site 1 hour post-injection (LAMP-1 site indicated by white arrows). The fluorescent signal shows specific uptake on the right flank (LAMP-1 +) and not on the left flank.

[0121] FIG. 18 shows the results of PET / CT imaging of89Zr-labeled anti-LAMP-1 Nb using in vivo mice Matrigel model. Athymic nude mice were injected subcutaneously with recombinant human LAMP-1 protein (5 pg) mixed with Matrigel to the left upper flank, and Matrigel only injected to the right flank as negative control.89Zr-labeled NB103 (300 pCi) was injected intravenously, showing specific uptake on the left flank (LAMP-1 site indicated by white arrows) and not on the right flank, followed by a washout through the kidneys. Images were obtained 2.5 hours post-injection.

[0122] FIG. 19 shows radio thin-layer chromatograph (TLC) analysis of non-specific89Zr-labeled anti- LAMP-1 NB103. Labeling efficiency was measured by iTLC with 50 mM DTPA, pH 5.0 as an eluant, and was shown to be more than 99%.

[0123] FIG. 20 lists sequences of anti-LAMP-1 NB185, NB191 , NB189, NB95, NB56, NB173, NB109, and NB128. FRs and CDRs of each sequence are indicated.

[0124] Detailed Description

[0125] The present invention provides anti-LAMP-1 antibodies, nanobodies, or antigen-binding fragments thereof and their use in the diagnosis, theranostics, and treatment of a cancer or an inflammatory condition in a subject.

[0126] LAMP-1 polypeptides

[0127] LAMP-1 is mainly expressed on the lysosomal membrane and its cell surface localization is suggested to be involved in cancer progression and metastasis. Thus, it would be advantageous to have a PET tracer targeting human LAMP-1 . One is provided herein.

[0128] The nanobody compositions of the invention have a CDR sequence selected from:

[0129] (a) NB132 - CDR1 : GLIFSINSMG (SEQ ID NO: 1 ); CDR2: VHNGDST (SEQ ID NO: 2); and CDR3: NARDPRGGHLWNY (SEQ ID NO: 3);

[0130] (b) NB121 - CDR1 : GLIFRINVMG (SEQ ID NO: 5); CDR2: ITHGGST (SEQ ID NO: 6); and CDR3: NAADSAGSNMNAKWSY (SEQ ID NO: 7);

[0131] (c) NB103 - CDR1 : GDIFSIDRMG (SEQ ID NO: 9); CDR2: IFLSDGET (SEQ ID NO: 10); and CDR3: WSYTTTNS (SEQ ID NO: 1 1 );

[0132] (d) NB63 - CDR1 : GIIFRINDMG (SEQ ID NO: 13); CDR2: ATMDDST (SEQ ID NO: 14); and CDR3: YAGRSEKWAY (SEQ ID NO: 15);

[0133] (e) NB185 - CDR1 : ETIFNANTMY (SEQ ID NO: 17); CDR2: ITTRGATD (SEQ ID NO: 18); and CDR3: NVFIFGVDY (SEQ ID NO: 19);

[0134] (f) NB191 - CDR1 : GFTFRNYAMR (SEQ ID NO: 21 ); CDR2: ISSGGDATS (SEQ ID NO: 22); and CDR3: ATSDFNI (SEQ ID NO: 23);

[0135] (g) NB189 - CDR1 : GLIFRINDMG (SEQ ID NO: 25); CDR2: EATDDSTS (SEQ ID NO: 26); and CDR3: YAGRSGKWDY (SEQ ID NO: 27);

[0136] (h) NB95 - CDR1 : GSISSIDRMG (SEQ ID NO: 29); CDR2: LSTSGDILT (SEQ ID NO: 30); and CDR3: WSYDFHNY (SEQ ID NO: 31 );

[0137] (i) NB56 - CDR1 : GIIFRINDMG (SEQ ID NO: 13); CDR2: ATMDDSTN (SEQ ID NO: 33); and CDR3: YAGRSEKWAY (SEQ ID NO: 15);

[0138] (j) NB173 - CDR1 : GIIFRINDMG (SEQ ID NO: 13); CDR2: ATTDDSTN (SEQ ID NO: 35); and CDR3: YAGRSEKWAY (SEQ ID NO: 15);

[0139] (k) NB109 - CDR1 : GIIFRINAMA (SEQ ID NO: 37); CDR2: IGQDDSTT (SEQ ID NO: 38); and CDR3: NAADRVPYERWSY (SEQ ID NO: 39); and

[0140] (l) NB128 - CDR1 : GIIFRINDMG (SEQ ID NO: 13); CDR2: ETIDGSRN (SEQ ID NO: 41 ); and CDR3: NAARESGGRILWSY (SEQ ID NO: 42).

[0141] The nanobody compositions of the invention have a full amino acid sequence selected from: a) NB132:

[0142] QVQLQESGGGLVQAGGSLRLSCTASGLIFSINSMGWYRQVPGKQRELVASVHNGDSTYSAD SVKGRFTISVDDAKNMVYLQMNDLEPEDTAVYYCNARDPRGGHLWNYWGQGTQVTVSS (SEQ ID NO: 4); b) NB121 :

[0143] QVQLQESGGGLVQAGGSLRLSCAASGLIFRINVMGWYRQAPGKERELVAQITHGGSTNYAD

[0144] SVKGRFTVSRDDAENTVDLQMNSLKPEDTAVYYCNAADSAGSNMNAKWSYWGQGTQVTVSS (SEQ ID NO: 8); c) NB103:

[0145] QVQLQESGGGLVQAGGSLRLSCAASGDIFSIDRMGWFRQTPGKERELVASIFLSDGETKYGD

[0146] FVKGRFTISRDNAKNAVYLQMNSLKPEDTAVYYCWSYTTTNSWGQGTQVTVSS (SEQ ID NO: 12); d) NB63:

[0147] QVQLQESGGGLAQAGGSLRLSCTASGIIFRINDMGWYRQAPGKQRELVAVATMDDSTNYAD

[0148] SVKGRFTVSRDSAKNSVYLQMNSLRPEDTAVYYCYAGRSEKWAYWGQGTQVTVSS (SEQ ID NO: 16); e) NB185:

[0149] QVQLQESGGGLVQAGGSLRLSCAVPETIFNANTMYWYRRAPGKEREWVAVITTRGATDYAD

[0150] SVKGRFTISRDNAKNTLNLEMNSLKPEDTAVYYCNVFIFGVDYWGQGTQVTVSS (SEQ ID NO: 20); f) NB191 :

[0151] QVQLQESGGGLVQPGGSLRLSCAASGFTFRNYAMRWVRQGPGNGLEGVSTISSGGDATSYA

[0152] DSVTGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCATSDFNIRGQGTQVTVSS (SEQ ID NO: 24); g) NB189:

[0153] QVQLQESGGGLAQAGGSLRLSCTASGLIFRINDMGWYRQAPGKQRELVAVEATDDSTSYAD

[0154] SVKGRFTVSRDSAKNTVYLQMNSLKPEDTAVYYCYAGRSGKWDYWGQGTQVTVSS (SEQ ID NO: 28); h) NB95:

[0155] QVQLQESGGGLVQAGGSLTLSCSVSGSISSIDRMGWFRQAPGKERELVAVLSTSGDILTYAD

[0156] DVKGRFTISRDDAMNTVSLQMNSLKPEDTAVYYCWSYDFHNYWGQGTQVTVSS (SEQ ID NO: 32); i) NB56:

[0157] QVQLQESGGGSVQVGESLTLSCTASGIIFRINDMGWYRQAPGKQRELVAVATMDDSTNYADS VKGRFTVSRDSAKNSVYLQMNSLRPEDTAVYYCYAGRSEKWAYWGQGTQVTVSS (SEQ ID NO: 34); j) NB173:

[0158] QVQLQESGGGLAQAGGSLRLSCTASGIIFRINDMGWYRQAPGKQRELVAVATTDDSTNYADS VKGRFTVSRDSAKNSVYLQMNSLRPEDTAVYYCYAGRSEKWAYWGQGTQVTVSS (SEQ ID NO: 36); k) NB109:

[0159] QVQLQESGGGLAQAGESLRLSCVASGIIFRINAMAWYRQDPGKQRELVAAIGQDDSTTYADS

[0160] VKGRFAISRDNANDTVYLQMNSLKPEDTSVYYCNAADRVPYERWSYWGQGTQVTVSS (SEQ ID NO: 40); and l) NB128:

[0161] QVQLQESGGGSAQPGGSLRLSCAASGIIFRINDMGWYRQAPGKHREMVAVETIDGSRNYGDS

[0162] VKGRFTISRDSANDTVYLEMNNLKPEDTAVYYCNAARESGGRILWSYWGQGTQVTVSS (SEQ ID NO: 43).

[0163] Exemplary sequences of CDRs and nanobodies of the invention are shown in Table 2 below.

[0164] Table 2. Exemplary Sequences of an anti-LAMP-1 nanobody

[0165] LAMP-1 conjugates

[0166] In some embodiments, it may be desirable to conjugate the polypeptide (e.g., polypeptide, antibody, nanobody, or antigen-binding fragment thereof) to a second molecule. Anti-LAMP-1 polypeptides, antibodies, nanobodies, and antigen-binding fragments thereof can be conjugated to other molecules at either the N-terminus or C-terminus of a light or heavy chain of the polypeptide using any one of a variety of established conjugation strategies that are well-known in the art. Examples of pairs of reactive functional groups that can be used to covalently tether an anti-LAMP-1 polypeptide, antibody, nanobody, or antigen-binding fragment thereof to another molecule are thiol pairs, carboxylic acids and amino groups, ketones and amino groups, aldehydes and amino groups, thiols and alpha, betaunsaturated moieties (such as maleimides or dehydroalanine), thiols and alpha-halo amides, carboxylic acids and hydrazides, aldehydes and hydrazides, and ketones and hydrazides.

[0167] Anti-LAMP-1 polypeptides, antibodies, nanobodies, and antigen-binding fragments thereof can be covalently appended directly to another molecule by chemical conjugation as described. Alternatively, fusion proteins containing anti-LAMP-1 polypeptides, antibodies, nanobodies, and antigen-binding fragments thereof can be expressed recombinantly from a cell (e.g., a eukaryotic cell or prokaryotic cell). This can be accomplished, for example, by incorporating a polynucleotide encoding the fusion protein into the nuclear genome of a cell (e.g., using techniques described herein or known in the art). Optionally, anti- LAMP-1 polypeptides, antibodies, nanobodies, and antigen-binding fragments thereof of the invention can be joined to a second molecule by forming a covalent bond between the polypeptide and a linker. This linker can then be subsequently conjugated to another molecule, or the linker can be conjugated to another molecule prior to ligation to the anti-LAMP-1 polypeptide, antibody, nanobody, and antigenbinding fragment thereof. Examples of linkers that can be used for the formation of a conjugate are polypeptide linkers, such as those that contain naturally-occurring or non-naturally-occurring amino acids. In some embodiments, it may be desirable to include D-amino acids in the linker, as these residues are not present in naturally-occurring proteins and are thus more resistant to degradation by endogenous proteases. Fusion proteins containing polypeptide linkers can be made using chemical synthesis techniques, such as those described herein, or through recombinant expression of a polynucleotide encoding the fusion protein in a cell (e.g., a prokaryotic or eukaryotic cell). Linkers can be prepared using a variety of strategies that are well known in the art, and depending on the reactive components of the linker, can be cleaved by enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under basic conditions, oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage (36).

[0168] Labeled anti-LAMP- 1 polypeptides

[0169] In some embodiments, anti-LAMP-1 polypeptides, antibodies, nanobodies, and antigen-binding fragments thereof may be conjugated to another molecule (e.g., an epitope tag) for the purpose of purification or detection. Examples of such molecules that are useful in protein purification include those that present structural epitopes capable of being recognized by a second molecule. This is a common strategy that is employed in protein purification by affinity chromatography, in which a molecule is immobilized on a solid support and exposed to a heterogeneous mixture containing a target protein conjugated to a molecule capable of binding the immobilized compound. Examples of epitope tag molecules that can be conjugated to anti-LAMP-1 polypeptides, antibodies, nanobodies, and antigenbinding fragments thereof for the purposes of molecular recognition are maltose-binding protein, glutathione-S-transferase, a poly-histidine tag, a FLAG-tag, a myc-tag, human influenza hemagglutinin (HA) tag, biotin, and streptavidin. Conjugates containing the epitopes presented by these molecules are capable of being recognized by such complementary molecules as maltose, glutathione, a nickel- containing complex, an anti-FLAG antibody, an anti-myc antibody, an anti-HA antibody, streptavidin, or biotin, respectively. For example, one can purify an anti-LAMP-1 polypeptide, antibody, nanobody, and antigen-binding fragment thereof of the invention that has been conjugated to an epitope tag from a complex mixture of other proteins and biomolecules (e.g., DNA, RNA, carbohydrates, phospholipids, etc.) by treating the mixture with a solid-phase resin containing a complementary molecule that can selectively recognize and bind the epitope tag of the anti-LAMP-1 polypeptide, antibody, nanobody, and antigenbinding fragment thereof. Examples of solid-phase resins include agarose beads, which are compatible with purifications in aqueous solution.

[0170] An anti-LAMP-1 polypeptide of the invention can also be covalently appended to a fluorescent molecule, e.g., to detect the antibody, nanobody, or antigen-binding fragment thereof by fluorimetry and / or by direct visualization using fluorescence microscopy. Exemplary fluorescent molecules that can be conjugated to polypeptides of the invention include green fluorescent protein, cyan fluorescent protein, yellow fluorescent protein, red fluorescent protein, phycoerythrin, allophycocyanin, hoescht, 4',6-diamidino-2-phenylindole (DAPI), propidium iodide, fluorescein, coumarin, rhodamine, tetramethylrhoadmine, and cyanine. Additional examples of fluorescent molecules suitable for conjugation to polypeptides of the invention are well-known in the art and have been described in detail in, e.g., U.S. Patent Nos. 7,417,131 and 7,413,874, each of which is incorporated by reference herein.

[0171] Anti-LAMP-1 polypeptides containing a fluorescent molecule are particularly useful for monitoring the cell-surface localization properties of polypeptides, such as polypeptides, antibodies, nanobodies, and antigen-binding fragments thereof of the invention. For instance, one can expose cultured mammalian cells (e.g., T-reg cells) to anti-LAMP-1 polypeptides, antibodies, nanobodies, and antigen-binding fragments thereof of the invention that have been covalently conjugated to a fluorescent molecule and subsequently analyze these cells using conventional fluorescent microscopy techniques known in the art. Confocal fluorescent microscopy is a particularly powerful method for determining cell-surface localization of anti-LAMP-1 polypeptides, antibodies, nanobodies, and antigen-binding fragments, as individual planes of a cell can be analyzed in order to distinguish antibodies or fragments thereof that have been internalized into a cell’s interior, e.g., by receptor- mediated endocytosis, from those that are bound to the external face of the cell membrane. Additionally, cells can be treated with anti-LAMP-1 antibodies conjugated to a fluorescent molecule that emits visible light of a particular wavelength (e.g., fluorescein, which fluoresces at about 535 nm) and an additional fluorescent molecule that is known to localize to a particular site on the T-reg cell surface and that fluoresces at a different wavelength (e.g., a molecule that localizes to CD25 and that fluoresces at about 599 nm). The resulting emission patterns can be visualized by confocal fluorescence microscopy and the images from these two wavelengths can be merged in order to reveal information regarding the location of the anti-LAMP-1 polypeptide, antibody, nanobody, or antigen-binding fragment thereof on the T-reg cell surface with respect to other receptors.

[0172] Bioluminescent proteins can also be incorporated into a fusion protein for the purposes of detection and visualization of an anti-LAMP-1 polypeptide. Bioluminescent proteins, such as Luciferase and aequorin, emit light as part of a chemical reaction with a substrate (e.g., luciferin and coelenterazine). Exemplary bioluminescent proteins suitable for use as a diagnostic sequence and methods for their use are described in, e.g., U.S. Patent Nos. 5,292,658, 5,670,356, 6,171 ,809, and 7,183,092, each of which is herein incorporated by reference. Anti-LAMP-1 polypeptides, antibodies, nanobodies, or antigen-binding fragments thereof labeled with bioluminescent proteins are a useful tool for the detection of antibodies of the invention following an in vitro assay. For instance, the presence of an anti-LAMP-1 antibody that has been conjugated to a bioluminescent protein can be detected among a complex mixture of additional proteins by separating the components of the mixture using gel electrophoresis methods known in the art (e.g., native gel analysis) and subsequently transferring the separated proteins to a membrane in order to perform a Western blot. Detection of the anti-LAMP-1 antibody among the mixture of other proteins can be achieved by treating the membrane with an appropriate Luciferase substrate and subsequently visualizing the mixture of proteins on film using established protocols.

[0173] The polypeptides (e.g., polypeptides, antibodies, nanobodies, and antigen-binding fragments thereof) of the invention can also be conjugated to a molecule comprising a radioactive nucleus, such that an antibody or fragment thereof of the invention can be detected by analyzing the radioactive emission pattern of the nucleus. Alternatively, an anti-LAMP-1 antibody or fragment thereof can be modified directly by incorporating a radioactive nucleus within the antibody during the preparation of the protein. Radioactive isotopes of methionine (35S), nitrogen (15N), or carbon (13C) can be incorporated into antibodies or fragments thereof of the invention by, e.g., culturing bacteria in media that has been supplemented with nutrients containing these isotopes. Optionally, tyrosine derivatives containing a radioactive halogen can be incorporated into an anti-LAMP-1 antibody or fragment thereof by, e.g., culturing bacterial cells in media supplemented with radiolabeled tyrosine. It has been shown that tyrosine functionalized with a radioactive halogen at the C2 position of the phenol system are rapidly incorporated into elongating polypeptide chains using the endogenous translation enzymes in vivo (U.S. Patent No. 4,925,651 ; incorporated herein by reference). The halogens include fluorine, chlorine, bromine, iodine, and astatine. Additionally, anti-LAMP-1 antibodies, nanobodies, or antigen-binding fragments thereof can be modified following isolation and purification from cell culture by functionalizing antibodies or fragments thereof of the invention with a radioactive isotope. The halogens represent a class of isotopes that can be readily incorporated into a purified protein by aromatic substitution at tyrosine or tryptophan, e.g., via reaction of one or more of these residues with an electrophilic halogen species. Examples of radioactive halogen isotopes are18F,75Br,77Br,122l,123l,124l,125l,129l,131l, or211At.

[0174] Another alternative strategy for the incorporation of a radioactive isotope is the covalent attachment of a chelating group to the anti-LAMP-1 polypeptide (e.g., polypeptide, antibody, nanobody, or fragment thereof). Chelating groups can be covalently appended to an anti-LAMP-1 polypeptide by attachment to a reactive functional group, such as a thiol, amino group, alcohol, or carboxylic acid. Examples of chelating agents that can be used in conjunction with the polypeptide of the disclosure are DFO (Desferrioxamine B), NOTA (1 ,4,7-triazacyclononanetriacetic acid), NOTAGA (1 ,4,7-triazacyclononane, 1 -glutaric acid,4,7-acetic acid), DOTA (1 ,4,7,10-tetraazacyclododecane- 1 ,4,7,10-tetraacetic acid), DOT AGA (dodeca-1 -glutaric acid-1 ,4,7,10-tetraamine-triacetic acid), NODAGA (1 , 4, 7-triazacyclononane-1 -glutaric acid-4, 7-diacetic acid), EDTA (ethylene diamine tetraacetic acid), DTPA (diethylene triaminepentaacetic acid), CDTA (cyclohexyl-1 ,2-diaminetetraacetic acid), EGTA (ethyleneglycol-O,O'-bis(2-aminoethyl)-N,N,N',N'-tetraacetic acid), HBED (N,N- bis(hydroxybenzyl)-ethylenediamine-N,N'-diacetic acid), TTHA (triethylene tetramine hexaacetic acid), HEDTA (hydroxyethyidiamine triacetic acid), TETA (1 ,4,8,1 1 -tetraazacyclotetradecane-N,N',N",N"'- tetraacetic acid), and TCMC (1 ,4,7,10-tetraaza-1 ,4,7,10-tetra- (2-carbamoyl methyl)-cyclododecane). In a preferred embodiment, the chelating agent is DFO.

[0175] The chelating groups can then be modified to contain any of a variety of metallic radioisotopes. Examples of radiolabels are3H,11C,14C,18F,32P,35S,36CI,51Cr,52Fe,57Co,58Co,59Fe, embodiment, the radiolabel is89Zr and the chelating agent is DFO.

[0176] In some embodiments, it may be desirable to covalently conjugate the polypeptides (e.g., polypeptides, antibodies, nanobodies, or antigen-binding fragments thereof) of the invention with a chelating group capable of binding a metal ion from heavy elements or rare earth ions, such as Gd3+, Fe3+, Mn3+, or Cr2+. Conjugates containing chelating groups that are coordinated to such paramagnetic metals are useful as in MRI imaging applications. Paramagnetic metals include, but are not limited to, chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and erbium (III).

[0177] In this way, anti-LAMP-1 antibodies can be detected by MRI spectroscopy. For instance, one can administer anti-LAMP-1 antibodies or fragments thereof conjugated to chelating groups bound to paramagnetic ions to a mammalian subject (e.g., a human patient) in order to monitor the distribution of the antibody following administration. This can be achieved by administration of the antibody to a patient by any of the administration routes described herein, such as intravenously, and subsequently analyzing the location of the administered antibody by recording an MRI of the patient according to established protocols.

[0178] Anti-LAMP-1 polypeptides (e.g., polypeptides, antibodies, nanobodies, or antigen-binding fragments thereof) can additionally be conjugated to other molecules for the purpose of improving the solubility and stability of the protein in aqueous solution. Examples of such molecules are PEG, PSA, bovine serum albumin (BSA), and human serum albumin (HSA), among others. For instance, one can conjugate an anti-LAMP-1 antibody or fragment thereof to carbohydrate moieties in order to evade detection of the antibody or fragment thereof by the immune system of the patient receiving treatment. This process of hyperglycosylation reduces the immunogenicity of therapeutic proteins by sterically inhibiting the interaction of the protein with B-cell receptors in circulation. Alternatively, anti-LAMP-1 antibodies or fragments thereof can be conjugated to molecules that prevent clearance from human serum and improve the pharmacokinetic profile of antibodies of the invention. Exemplary molecules that can be conjugated to or inserted within anti-LAMP-1 antibodies or fragments thereof of the invention so as to attenuate clearance and improve the pharmacokinetic profile of these antibodies and fragments are salvage receptor binding epitopes. These epitopes are found within the Fc region of an IgG immunoglobulin and have been shown to bind Fc receptors and prolong antibody half-life in human serum. The insertion of salvage receptor binding epitopes into anti-LAMP-1 antibodies or fragments thereof can be achieved, e.g., as described in US Patent No. 5,739,277; incorporated herein by reference.

[0179] Antibody-drug conjugates (ADCs)

[0180] In some embodiments, anti-LAMP-1 polypeptides, antibodies, nanobodies, and antigen-binding fragments thereof may be conjugated to another molecule (e.g., a drug) for the purpose of treating a cancer and / or inflammatory condition.

[0181] Antibody-drug conjugates (ADCs) are targeted chemotherapeutic molecules which combine properties of both antibodies and cytotoxic drugs by targeting potent cytotoxic drugs to antigen-expressing tumor cells (37), thereby enhancing the therapeutic index by maximizing efficacy and minimizing off-target toxicity (38, 39).

[0182] The anti-LAMP-1 polypeptide (e.g., polypeptide, antibody, nanobody, or antigen-binding fragment thereof) of the invention may be covalently attached to a drug moiety through a linker to form an ADC. The ADC may selectively deliver an effective dose of a drug to tumor tissue whereby greater selectivity, i.e. a lower efficacious dose, may be achieved while increasing the therapeutic index.

[0183] The drug moiety of the ADC may include any compound, moiety, or group that has a cytotoxic or cytostatic effect. Drug moieties may impart their cytotoxic and cytostatic effects by mechanisms including but not limited to tubulin binding, DNA binding or intercalation, and inhibition of RNA polymerase, protein synthesis, and / or topoisomerase. Exemplary drug moieties include, but are not limited to, a maytansinoid, dolastatin, auristatin, calicheamicin, pyrrolobenzodiazepine (PBD), nemorubicin and its derivatives, PNU- 159682, anthracycline, duocarmycin, vinca alkaloid, taxane, trichothecene, CC1065, camptothecin, elinafide, and stereoisomers, isosteres, analogs, and derivatives thereof that have cytotoxic activity.

[0184] The ADCs may be prepared by several routes employing organic chemistry reactions, conditions, and reagents known to those skilled in the art.

[0185] Methods of detection, diagnosis, and treatment

[0186] Anti-LAMP-1 polypeptides (e.g., polypeptides, antibodies, nanobodies, or antigen-binding fragments thereof) of the invention are useful in the detection or diagnosis of a wide array of cancers and inflammatory conditions. Anti-LAMP-1 polypeptides (e.g., polypeptides, antibodies, nanobodies, or antigen-binding fragments thereof) can be administered to a mammalian subject, such as a human, suffering from a cancer (e.g., breast cancer, colon cancer, esophageal cancer, stomach cancer, liver cancer, bladder cancer, pancreatic cancer, and prostate cancer) or an inflammatory condition to detect tumor cells.

[0187] Anti-LAMP-1 polypeptides of the invention can be administered to a mammalian subject (e.g., a human) suffering from a cancer or inflammatory condition in order to improve the condition of the patient by promoting the immune response against cancer cells and tumorogenic material. Antibodies of the invention can be administered to a subject, e.g., via any of the routes of administration described herein. Polypeptides of the invention can also be formulated with excipients, biologically acceptable carriers, and may be optionally conjugated to, admixed with, or co-administered separately (e.g., sequentially) with additional therapeutic agents, such as anti-cancer agents.

[0188] Cancers that can be treated by administration of polypeptides (e.g., polypeptides, antibodies, nanobodies, or antigen-binding fragments thereof) of the invention include such cancers as leukemia, lymphoma, liver cancer, bone cancer, lung cancer, brain cancer, bladder cancer, gastrointestinal cancer, breast cancer, cardiac cancer, cervical cancer, uterine cancer, head and neck cancer, gallbladder cancer, laryngeal cancer, lip and oral cavity cancer, ocular cancer, melanoma, pancreatic cancer, prostate cancer, colorectal cancer, testicular cancer, and throat cancer. Particular cancers that can be treated by administration of antibodies or antigen-binding fragments thereof of the invention include, without limitation, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), adrenocortical carcinoma, AIDS-related lymphoma, primary CNS lymphoma, anal cancer, appendix cancer, astrocytoma, atypical teratoid / rhabdoid tumor, basal cell carcinoma, bile duct cancer, extrahepatic cancer, ewing sarcoma family, osteosarcoma and malignant fibrous histiocytoma, central nervous system embryonal tumors, central nervous system germ cell tumors, craniopharyngioma, ependymoma, bronchial tumors, burkitt lymphoma, carcinoid tumor, primary lymphoma, chordoma, chronic myeloproliferative neoplasms, colon cancer, extrahepatic bile duct cancer, ductal carcinoma in situ (DCIS), endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, extracranial germ cell tumor, extragonadal germ cell tumor, fallopian tube cancer, fibrous histiocytoma of bone, gastrointestinal carcinoid tumor, gastrointestinal stromal tumors (GIST), testicular germ cell tumor, gestational trophoblastic disease, glioma, childhood brain stem glioma, hairy cell leukemia, hepatocellular cancer, langerhans cell histiocytosis, hodgkin lymphoma, hypopharyngeal cancer, islet cell tumors, pancreatic neuroendocrine tumors, wilms tumor and other childhood kidney tumors, langerhans cell histiocytosis, small cell lung cancer, cutaneous T-cell lymphoma, intraocular melanoma, merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer, midline tract carcinoma, multiple endocrine neoplasia syndromes, multiple myeloma / plasma cell neoplasm, myelodysplastic syndromes, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-hodgkin lymphoma (NHL), non-small cell lung cancer (NSCLC), epithelial ovarian cancer, germ cell ovarian cancer, low malignant potential ovarian cancer, pancreatic neuroendocrine tumors, papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, rectal cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, kaposi sarcoma, rhabdomyosarcoma, sezary syndrome, small intestine cancer, soft tissue sarcoma, throat cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, urethral cancer, endometrial uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Waldenstrom macroglobulinemia.

[0189] Inflammatory conditions that can be treated by administration of polypeptides (e.g., polypeptides, antibodies, nanobodies, or antigen-binding fragments thereof) of the invention include autoimmune diseases such as inflammatory bowel disease, inflammatory arthritis, interstitial lung disease, sarcoidosis, and multiple sclerosis, among others. In some embodiments, the inflammatory condition is characterized by overexpression of LAMP-1 .

[0190] A physician having ordinary skill in the art can readily determine an effective amount of an anti-LAMP-1 polypeptide for administration to a mammalian subject (e.g., a human) in need thereof. For example, a physician could start prescribing doses of a polypeptide of the invention at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. Alternatively, a physician may begin a treatment regimen by administering an anti-LAMP-1 antibody or antibody fragment at a high dose and subsequently administer progressively lower doses until a therapeutic effect is achieved (e.g., a reduction in the volume of one or more tumors, a decrease in the population of T-reg cells, or remission of a cell proliferation disorder). In general, a suitable daily dose of an antibody or antigen-binding fragment thereof of the invention will be an amount of the antibody which is the lowest dose effective to produce a therapeutic effect. A polypeptide, antibody, nanobody, or antigen-binding fragment thereof of the invention may be administered by injection, e.g., by intravenous, intramuscular, intraperitoneal, or subcutaneous injection, optionally proximal to the site of the target tissue (e.g., a tumor). A daily dose of a therapeutic composition of an antibody or antigen-binding fragment thereof of the invention may be administered as a single dose or as two, three, four, five, six or more doses administered separately at appropriate intervals throughout the day, week, month, or year, optionally, in unit dosage forms. While it is possible for an antibody or fragment thereof of the invention to be administered alone, it may also be administered as a pharmaceutical formulation in combination with excipients, carriers, and optionally, additional therapeutic agents.

[0191] Polypeptides (e.g., polypeptides, antibodies, nanobodies, or antigen-binding fragments thereof) of the invention can be monitored by any of a variety of methods known in the art. For instance, a physician may monitor the response of a mammalian subject (e.g., a human) to treatment with an antibody, nanobody, antibody fragment, or polypeptide of the invention by analyzing the volume of one or more tumors in the patient. For example, polypeptides (e.g., polypeptides, antibodies, nanobodies, or antigen-binding fragments thereof) of the invention may be capable of reducing tumor volume by between 1% and 100% (e.g., 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%). Alternatively, a physician may monitor the responsiveness of a subject (e.g., a human) to treatment with anti-LAMP-1 polypeptides, antibodies, nanobodies, or antigen-binding fragments thereof of the invention by analyzing the T-reg cell population in the lymph of a particular subject. For instance, a physician may withdraw a sample of blood from a mammalian subject (e.g., a human) and determine the quantity or density of a population of T-reg cells (e.g., CD4+ CD25+ FOXP3+ T-reg cells or CD17+ T-reg cells) using established procedures, such as fluorescence activated cell sorting.

[0192] Imaging techniques

[0193] Labeled polypeptides (e.g., polypeptides, antibodies, nanobodies, or antigen-binding fragments thereof) of the invention can be used as diagnostic agents and in various imaging techniques, including Positron Emission Tomography (PET), Single Photon Emission Computed Tomography (SPECT), Computed Tomography (CT), Magnetic Resonance Imaging (MRI), Infra-Red and Near Infra-Red Imaging (IR / NIR), Ultrasound (US), and fluorescence-activated cell sorting (FACS). These polypeptides may also be conjugated to a chemotherapeutic for targeted therapeutic delivery to tumors exhibiting LAMP-1 overexpression.

[0194] Pharmaceutical compositions

[0195] Therapeutic compositions containing an anti-LAMP-1 polypeptide, such as a polypeptide, antibody, nanobody, or antigen-binding fragment thereof of the invention can be prepared using methods known in the art. For example, such compositions can be prepared using, e.g., physiologically acceptable carriers, excipients or stabilizers {Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980); incorporated herein by reference), and in a desired form, e.g., in the form of lyophilized formulations or aqueous solutions. The compositions can also be prepared so as to contain the active agent (e.g., an anti-LAMP-1 antibody or fragment thereof) at a desired concentration. For example, a pharmaceutical composition of the invention may contain at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%) active agent by weight (w / w).

[0196] Additionally, an active agent (e.g., an anti-LAMP-1 antibody or fragment thereof of the invention) that can be incorporated into a pharmaceutical formulation can itself have a desired level of purity. For example, an antibody or antigen-binding fragment thereof of the invention may be characterized by a certain degree of purity after isolating the antibody from cell culture media or after chemical synthesis, e.g., of an antibody fragment by established solid-phase peptide synthesis methods or native chemical ligation as described herein. An anti-LAMP-1 antibody of the invention may be at least 10% pure prior to incorporating the antibody into a pharmaceutical composition (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or 100% pure).

[0197] Pharmaceutical compositions of anti-LAMP-1 polypeptides (e.g., polypeptides, antibodies, nanobodies, or antigen-binding fragments thereof) of the invention can be prepared for storage as lyophilized formulations or aqueous solutions by mixing the antibody having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers typically employed in the art, e.g., buffering agents, stabilizing agents, preservatives, isotonifiers, non-ionic detergents, antioxidants, and other miscellaneous additives. See, e.g., Remington's Pharmaceutical Sciences, 16th edition (Osol, ed. 1980; incorporated herein by reference). Such additives must be nontoxic to the recipients at the dosages and concentrations employed.

[0198] Buffering agents

[0199] Buffering agents help to maintain the pH in the range which approximates physiological conditions. They can be present at concentration ranging from about 2 mM to about 50 mM. Suitable buffering agents for use with anti-LAMP-1 polypeptides (e.g., polypeptides, antibodies, nanobodies, or antigen-binding fragments thereof) of the invention include both organic and inorganic acids and salts thereof such as citrate buffers {e.g., monosodium citrate-disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-monosodium citrate mixture, etc.), succinate buffers (e.g., succinic acid- monosodium succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid-disodium succinate mixture, etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaric acid- potassium tartrate mixture, tartaric acid-sodium hydroxide mixture, etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture, fumaric acid-disodium fumarate mixture, monosodium fumarate-disodium fumarate mixture, etc.), gluconate buffers (e.g., gluconic acid-sodium gluconate mixture, gluconic acid-sodium hydroxide mixture, gluconic acid-potassium gluconate mixture, etc.), oxalate buffer (e.g., oxalic acid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassium oxalate mixture, etc.), lactate buffers (e.g., lactic acid-sodium lactate mixture, lactic acid-sodium hydroxide mixture, lactic acid-potassium lactate mixture, etc.) and acetate buffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodium hydroxide mixture, etc.). Additionally, phosphate buffers, histidine buffers, and trimethylamine salts such as Tris can be used.

[0200] Preservatives

[0201] Preservatives can be added to a composition of the invention to retard microbial growth, and can be added in amounts ranging from 0.2%-l% (w / v). Suitable preservatives for use with anti-LAMP- 1 polypeptides (e.g., polypeptides, antibodies, nanobodies, or antigen-binding fragments thereof) of the invention include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalconium halides (e.g., chloride, bromide, and iodide), hexamethonium chloride, and alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, and 3-pentanol. Isotonicifiers sometimes known as “stabilizers” can be added to ensure isotonicity of liquid compositions of the invention and include polhydric sugar alcohols, for example trihydric or higher sugar alcohols, such as glycerin, arabitol, xylitol, sorbitol and mannitol. Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which solubilizes the therapeutic agent or helps to prevent denaturation or adherence to the container wall. Typical stabilizers can be polyhydric sugar alcohols (enumerated above); amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol and the like, including cyclitols such as inositol; polyethylene glycol; amino acid polymers; sulfur containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, a-monothioglycerol and sodium thiosulfate; low molecular weight polypeptides (e.g., peptides of 10 residues or fewer); proteins such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone monosaccharides, such as xylose, mannose, fructose, glucose; disaccharides such as lactose, maltose, sucrose and trisaccharides such as raffinose; and polysaccharides such as dextran. Stabilizers can be present in the range from 0.1 to 10,000 weights per part of weight active protein.

[0202] Detergents

[0203] Non-ionic surfactants or detergents (also known as “wetting agents”) can be added to help solubilize the therapeutic agent as well as to protect the therapeutic protein against agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stressed without causing denaturation of the protein. Suitable non-ionic surfactants include polysorbates (20, 80, etc.), polyoxamers (184, 188, etc.), pluronic polyols, and polyoxyethylene sorbitan monoethers (TWEEN®- 20, TWEEN®-80, etc.). Non-ionic surfactants can be present in a range of about 0.05 mg / mL to about 1 .0 mg / mL, for example about 0.07 mg / mL to about 0.2 mg / mL. Additional miscellaneous excipients include bulking agents (e.g., starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbic acid, methionine, vitamin E), and cosolvents.

[0204] Other pharmaceutical carriers

[0205] Alternative pharmaceutically acceptable carriers that can be incorporated into a composition of the invention may include dextrose, sucrose, sorbitol, mannitol, starch, rubber arable, potassium phosphate, arginate, gelatin, potassium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oils. A composition containing an anti-LAMP-1 polypeptide of the invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, and a preservative. Details of suitable pharmaceutically acceptable carriers and formulations can be found in Flemington's Pharmaceutical Sciences (19th ed., 1995), which is incorporated herein by reference.

[0206] Routes of administration and dosing

[0207] Anti-LAMP-1 polypeptides (e.g., polypeptides, antibodies, nanobodies, or antigen-binding fragments thereof) of the invention can be administered to a mammalian subject (e.g., a human) by a variety of routes such as orally, transdermally, subcutaneously, intranasally, intravenously, intramuscularly, intraocularly, intratumorally, parenterally, topically, intrathecal ly and intracerebroventricularly. The most suitable route for administration in any given case will depend on the particular antibody or antigen-binding fragment administered, the patient, pharmaceutical formulation methods, administration methods (e.g., administration time and administration route), the patient's age, body weight, sex, severity of the diseases being treated, the patient’s diet, and the patient’s excretion rate.

[0208] The effective dose of an anti-LAMP-1 polypeptide, antibody, nanobody, or antigen-binding fragment thereof of the invention can range from about 0.0001 to about 100 mg / kg of body weight per single (e.g., bolus) administration, multiple administrations or continuous administration, or to achieve a serum concentration of 0.0001 -5000 pg / mL serum concentration per single (e.g., bolus) administration, multiple administrations or continuous administration, or any effective range or value therein depending on the condition being treated, the route of administration and the age, weight, and condition of the subject. In certain embodiments, e.g., for the treatment of cancer, each dose can range from about 0.0001 mg to about 500 mg / kg of body weight. For instance, a pharmaceutical composition of the invention may be administered in a daily dose in the range of 0.001 -100 mg / kg (body weight). The dose may be administered one or more times (e.g., 2-10 times) per day, week, month, or year to a mammalian subject (e.g., a human) in need thereof.

[0209] Therapeutic compositions can be administered with medical devices known in the art. For example, in an embodiment, a therapeutic composition of the invention can be administered with a needleless hypodermic injection device, such as the devices disclosed in U.S. Pat. Nos. 5,399,163; 5,383,851 ; 5,312,335; 5,064,413; 4,941 ,880; 4,790,824; or 4,596,556. Examples of well-known implants and modules useful in the invention are U.S. Pat. No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Pat. No. 4,486,194, which discloses a therapeutic device for administering medicaments through the skin; U.S. Pat. No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and U.S. Pat. No. 4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art.

[0210] Kits containing anti-LAMP-1 polypeptides

[0211] This invention also includes kits that contain anti-LAMP-1 polypeptides (e.g., polypeptides, antibodies, nanobodies, or antigen-binding fragments thereof). The kits provided herein may contain any of the anti-LAMP-1 polypeptides described above, as well as any of the polynucleotides encoding these polypeptides, vectors containing these polypeptides, or cells engineered to express and secrete polypeptides of the invention (e.g., prokaryotic or eukaryotic cells). A kit of this invention may include reagents that can be used to produce the compositions of the invention (e.g., anti-LAMP-1 polypeptides, such as polypeptides, antibodies, nanobodies, or fragments thereof, conjugates containing anti-LAMP-1 polypeptides, polynucleotides encoding anti-LAMP-1 polypeptides, vectors containing these polypeptides). Optionally, kits of the invention may include reagents that can induce the expression of anti-LAMP-1 antibodies within cells (e.g., mammalian cells), such as doxycycline or tetracycline. In other cases, a kit of the invention may contain a compound capable of binding and detecting a fusion protein that contains an anti-LAMP-1 polypeptide and an epitope tag. For instance, in such cases a kit of the invention may contain maltose, glutathione, a nickel-containing complex, an anti-FLAG antibody, an anti-myc antibody, an anti-HA antibody, biotin, or streptavidin.

[0212] Kits of the invention may also include reagents that are capable of detecting an anti-LAMP-1 polypeptide, antibody, nanobody, or antigen-binding fragment thereof directly. Examples of such reagents are secondary antibodies that selectively recognize and bind particular structural features within the Fc region of an anti-LAMP-1 antibody of the invention. Kits of the invention may contain secondary antibodies that recognize the Fc region of an anti-LAMP-1 antibody and that are conjugated to a fluorescent molecule. These antibody-fluorophore conjugates provide a tool for analyzing the localization of anti-LAMP-1 antibodies, e.g., in a particular tissue or cultured mammalian cell using established immunofluorescence techniques. In some embodiments, kits of the invention may include additional fluorescent compounds that exhibit known sub-cellular localization patterns. These reagents can be used in combination with another antibody-fluorophore conjugate, e.g., one that specifically recognizes a different receptor on the cell surface in order to analyze the localization of an anti-LAMP-1 antibody relative to other cell-surface proteins.

[0213] Kits of the invention may also contain a reagent that can be used for the analysis of a patient’s response to treatment by administration of anti-LAMP-1 polypeptides (e.g., polypeptides, antibodies, nanobodies, or antigen-binding fragments thereof) of the invention. For instance, kits of the invention may include an anti-LAMP-1 polypeptide, such as a polypeptide, antibody, nanobody, or antibody fragment, and one or more reagents that can be used to determine the quantity of T-reg cells in a blood sample withdrawn from a subject (e.g., a human) that is undergoing treatment with an antibody of the invention. Such a kit may contain, e.g., antibodies that selectively bind cell-surface antigens presented by T-reg cells, such as CD4 and CD25. Optionally, these antibodies may be labeled with a fluorescent dye, such as fluorescein or tetramethylrhodamine, in order to facilitate analysis of a population of T-reg cells by fluorescence-activated cell sorting (FACS) methods known in the art. Kits of the invention may optionally contain one or more reagents that can be used to quantify a population of tumor-reactive T-lymphocytes in order to determine the effectiveness of an anti-LAMP- 1 polypeptide of the invention in restoring tumor-infiltrating lymphocyte proliferation. For instance, kits of the invention may contain an antibody that selectively binds cell-surface markers on the surface of a cytotoxic T-cell, such as CD8 or CD3. Optionally, these antibodies may be labeled with fluorescent molecules so as to enable quantitation by FACS analysis.

[0214] A kit of the invention may also contain one or more reagents useful for determining the affinity and selectivity of an anti-LAMP-1 polypeptide, antibody, nanobody, or antigen-binding fragment thereof of the invention for one or more peptides derived from LAMP-1 (e.g., a peptide containing the sequence of any one of SEQ ID NOs: 4 and 33-43). For instance, a kit may contain an anti-LAMP-1 antibody and one or more reagents that can be used in an ELISA assay to determine the Kn ot an antibody of the invention for one or more peptides that present a LAMP-1 epitope in a conformation similar to that of the epitope in the native protein. A kit may contain, for example, a microtiter plate containing wells that have been previously conjugated to avidin, and a library of LAMP-1 -derived peptides, each of which is conjugated to a biotin moiety. Such a kit may optionally contain a secondary antibody that specifically binds to the Fc region of an anti-LAMP-1 antibody of the invention, and the secondary antibody may be conjugated to an enzyme (e.g., horseradish peroxidase) that catalyzes a chemical reaction that results in the emission of luminescent light.

[0215] Kits of the invention may also contain anti-LAMP-1 polypeptides (e.g., polypeptides, antibodies, nanobodies, or antigen-binding fragments thereof) of the invention and reagents that can be conjugated to such a polypeptide, including those previously described (e.g., a cytotoxic agent, a fluorescent molecule, a bioluminescent molecule, a molecule containing a radioactive isotope, a molecule containing a chelating group bound to a paramagnetic ion, etc.). These kits may additionally contain instructions for how the conjugation of an anti-LAMP-1 polypeptide of the invention to a second molecule, such as those described above, can be achieved.

[0216] A kit of the invention may also contain a vector containing a polynucleotide that encodes an anti-LAMP-1 polypeptide, antibody, nanobody, or antigen-binding fragment thereof, such as any of the vectors described herein. Alternatively, a kit may include mammalian cells (e.g., CHO cells) that have been genetically altered to express LAMP-1 polypeptides (e.g., polypeptides, antibodies, nanobodies, or fragments thereof) from the nuclear genome of the cell. Such a kit may also contain instructions describing how expression of the LAMP-1 polypeptide, antibody, nanobody, or fragment thereof from a polynucleotide can be induced, and may additionally include reagents (such as, e.g., doxycycline or tetracycline) that can be used to promote the transcription of these polynucleotides. Such kits may be useful for the manufacture of anti-LAMP-1 antibodies or antigen-binding fragments thereof of the invention.

[0217] Other kits of the invention may include tools for engineering a prokaryotic or eukaryotic cell (e.g., a CHO cell or a BL21 (DE3) E. coli cell) so as to express an anti-LAMP-1 polypeptide, antibody, nanobody, or fragment thereof of the invention from the nuclear genome of the cell. For example, a kit may contain CHO cells stored in an appropriate media and optionally frozen according to methods known in the art. The kit may also provide a vector containing a polynucleotide that encodes a nuclease (e.g., such as the CRISPER / Cas, zinc finger nuclease, TALEN, ARCUS™ nucleases described herein) as well as reagents for expressing the nuclease in the cell. The kit can additionally provide tools for modifying the polynucleotide that encodes the nuclease so as to enable one to alter the DNA sequence of the nuclease in order to direct the cleavage of a specific target DNA sequence of interest. Examples of such tools are primers for the amplification and site-directed mutagenesis of the polynucleotide encoding the nuclease of interest. The kit may also include restriction enzymes that can be used to selectively excise the nuclease-encoding polynucleotide from the vector and subsequently re-introduce the modified polynucleotide back into the vector once the user has modified the gene. Such a kit may also include a DNA ligase that can be used to catalyze the formation of covalent phosphodiester linkages between the modified nuclease-encoding polynucleotide and the target vector. A kit of the invention may also provide a polynucleotide encoding an anti-LAMP-1 polypeptide, antibody, nanobody, or fragment thereof, as well as a package insert describing the methods one can use to selectively cleave a particular DNA sequence in the genome of the cell in order to incorporate the polynucleotide encoding an anti-LAMP-1 polypeptide into the genome at this site. Optionally, the kit may provide a polynucleotide encoding a fusion protein that contains an anti-LAMP-1 polypeptide, antibody, nanobody, or antigen-binding fragment thereof and an additional polypeptide, such as, e.g., those described herein.

[0218] Examples

[0219] Below we describe the usefulness of LAMP-1 as an imaging target in cancer. We assessed LAMP-1 expression levels in various adenocarcinomas and used an anti-N terminal LAMP-1 monoclonal antibody labeled with Zirconium-89 (89Zr) to image mouse models of human breast and colon adenocarcinoma.

[0220] The following examples are put forth so as to provide those of ordinary skill in the art with a description of how the compositions and methods described herein can be used and evaluated and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention.

[0221] Example 1. Development of PET Tracer Targeting Human LAMP-1

[0222] Objective

[0223] The objective of this study was to develop an antibody-based PET tracer targeting human LAMP-1 and evaluate the probe in preclinical breast and colon adenocarcinoma models.

[0224] Materials and Methods

[0225] Cancer Cell Lines and Cell Culture

[0226] All human and murine cancer cell lines were purchased from American Type Culture Collection (ATCC), except for MC38 cells (Kerafast, Boston, MA, USA) and HY15549 (a kind gift from Dr. Kenneth Tanabe, Massachusetts General Hospital, Boston, MA, USA). Human cancer cell lines used include MDA-MB-231 (breast), Caco2 (colon), H441 (lung), BXPC3, SU86.86, and CAPAN2 (pancreas). Murine cell lines used include HY15549 (pancreas), 4T1 (breast), MC38 (colon), and KL205 (lung).

[0227] Cells were grown using DMEM (Thermo Fisher) or RPMI (ATCC) medium according to the vendor’s recommendation, supplemented with 10% fetal bovine serum (Thermo Fisher) and 1% penicillin / streptomycin (Thermo Fisher) at 37°C and 5% CO2 incubator. Mycoplasma screening by PCR was performed regularly, and cells were discarded after 15 passages. Xenograft and Allograft Models

[0228] The Institutional Animal Care and Use Committee (IACUC) approved all experimental procedures and animal studies. Mouse models of human breast, colon, and lung adenocarcinoma were generated with the subcutaneous injection of 2 x106cells in a 1 :1 (v:v) ratio in Matrigel (Corning) into the left shoulder of 6-8 week old athymic nude mice (Charles River Laboratories). Female mice were used for the MDA-MB-231 breast cancer model, and male mice were used for the Caco-2 colon cancer model. The animals were followed until tumor size reached 150 mm3and were then randomly assigned to the experimental LAMP-1 probe or control IgG probe group (n=7 per group)

[0229] To measure the expression of LAMP-1 in tumors compared to normal organs, we used an immunocompetent C57BL / 6 mouse model to ensure the expression of LAMP-1 in organs with rich immune cell presence is taken into account. Syngeneic allograft MC38 tumors were implanted subcutaneously in the left shoulder of male 6-8 week old male C57BL / 6 mice (Jackson laboratories), using 1 x 106cells in a 1 :1 ratio in Matrigel. When the tumor size reached 150-250 mm3, mice were sacrificed. Tumors and major organs were harvested and processed on ice for flow cytometry (n=3).

[0230] Flow cytometry

[0231] For in vitro expression assessment, cells were dissociated using a non-enzymatic cell dissociation solution (30-2103; ATCC) for minimal disruption of the cell surface proteins.

[0232] Human cell lines were stained with AlexaFluor 647 (AF 647)-conjugated clone D2D11 LAMP- 1 antibody (9091 S; Cell Signaling) and murine cell lines with APC-conjugated anti-mouse LAMP-1 antibody (121614; BioLegend), for 30 minutes, followed by three washes in PBS containing 10% FBS. Next, cells were stained with Zombie Aqua fixable viability dye (BioLegend) for 10 minutes. After washing with PBS, cells were fixed in paraformaldehyde 2% and run on the LSRFortessa X20 flow cytometer (BD Biosciences). Data were acquired using BD FACSDIVA software (BD Biosciences).

[0233] To examine the in vivo expression of LAMP-1 in normal organs in tumor-bearing mice, the entire tumor, liver, lung, spleen, and kidney were harvested. Portions of the small bowel at the duodenum section and thigh muscle were also separated. Organs were cut into small pieces, further dissociated with physical pressure, and passed through a 70-pm cell strainer to remove cell debris. Cold phosphate-buffered saline (PBS) containing 10% FBS (Gibco-lnvitrogen) was used to wash the filter and collect filtered cells. Bone marrow cells were harvested from both femurs. The cells were centrifuged and resuspended in RBC lysis buffer (BioLegend) for 5 minutes. The pellet was centrifuged and incubated with PBS containing 10% FBS and FC block (anti-mouse CD16 / 32) (1 :1000, BioLegend) for 10 minutes. Cells were stained with APC-conjugated anti-mouse LAMP-1 antibody (121614; BioLegend), processed, and according to the same protocol described above. All flow cytometry data were analyzed using FlowJo software (version 10.1 ).

[0234] Immunofluorescence Staining of Human Tissue Samples

[0235] All human tissue microarrays (TMAs) were purchased from TissueArray.Com LLC (formerly US Biomax Inc.). Initially, we stained three mixed TMAs of multiple-organ cancers and normal tissue (CTRL142, CTRL143, and BCN601 a) to screen and evaluate the overall expression of LAMP-1 across a wide range of cancers and their related normal tissue. Samples were deparaffinized and rehydrated. Antigen retrieval was performed by the standard heat-based antigen retrieval method (40). Blocking nonspecific binding was performed using Dako serum-free Protein Block (Agilent Technologies) for one hour at room temperature. Samples were incubated with clone D2D11 rabbit anti-human LAMP-1 (9091 S; Cell Signaling) overnight at 4°C. The following day, stained slides were washed three times with PBST and incubated with AF 647-conjugated alpaca anti-rabbit IgG secondary antibody (611 -605-215; Jackson Immunoresearch) for one hour at room temperature. Slides were washed three times and incubated for five minutes at room temperature with DAPI for nuclei staining. After applying ProLong Gold antifade mounting media (P10144; Thermo Fisher), a coverslip was fixed to each slide using clear nail polish. Microscopic imaging was performed in the far- red channel for LAMP-1 and the blue channel for DAPI, using the Biotek Cytation 5 Cell Imaging Multi-Mode Reader. Manual semiquantitative immunofluorescence analysis was carried out by measuring mean and median fluorescence intensity in the far-red channel after drawing regions of interest based on fluorescence in the blue channel (DAPI) to exclude non-specific signal from acellular structures, using Fiji ImageJ software.

[0236] Next, to further investigate expression among a larger sample size for each type of adenocarcinoma and related normal tissue, we chose dedicated TMAs for colon (CO1507), breast (BR1191 ), prostate (PR781 ), pancreas (PA1003) and lung (LCN721 , LC706b) adenocarcinomas. These slides were processed and immunostained according to the same protocol. Fixed stained slides were then handed to the Tissue Microarrays & Imaging (TMI) Core of Dana-Farber / Harvard Cancer Center at Brigham and Women's Hospital Specialized Pathology Services Facility. The core scanned tissue slides using the Aperio ScanScope System. The TMA images were analyzed with the Bioimage Analysis Software Qupath (41 ). This tool allows for performing tissue segmentation, calculating the metrics, and labeling the cores with the metadata for further processing of the results. Tissue segmentation was performed based on the brightfield images carefully registered to the DAPI and far-red fluorescence channels to label the regions of the images containing tissue regardless of the presence of fluorescence. Then, the areas with very low cellularity based on the signal in the DAPI channel or areas showing nonspecific fluorescence were omitted from regions of interest for analysis. The intensity of the fluorescent emission in the far-red channel was then calculated for each region of interest. The difference between the signal intensity values in normal and malignant tissues in each organ was evaluated using the non-parametric Mann-Whitney U test. A Shapiro test of normality was performed prior to the analysis. The statistical analysis was performed using the statistical package of SciPy (42).

[0237] Bioconjugation and Radiolabeling

[0238] All the buffers were treated with chelex 100 resin (Sigma Aldrich) to ensure metal-free conditions. Rabbit anti-human LAMP-1 mAb (NBP2-89844; Novus Biological) was conjugated via amine-isothiocyanate reaction to p-SCN-Bn-deferoxamine (DFO) chelator (Macrocyclics) before radiolabeling with89Zr. The concentration of LAMP-1 mAb was adjusted to 2.5 mg / ml in 500 pL of HEPES buffer (0.5 M; pH = 7.0). The pH was adjusted to 8.8-9.0 by adding aliquots of Sodium carbonate buffer (0.2 M), and a solution of DFO in dry DMSO was added to the antibody mixture. The pH was readjusted to 8.8-9.0, and the reaction was incubated for one hour at 37°C on an agitating heat block at 350 rpm. The conjugated mAb was purified by size exclusion chromatography using PD Miditrap G-25 columns (Cytivia) with HEPES (0.5 M) as the mobile phase. A 4-fold molar excess of DFO to mAb ratio was employed to ensure minimal LAMP-1 mAb binding affinity disruption.89Zr in oxalic acid (1 M) was purchased from 3D Imaging LCC with a specific activity of ~11 1 GBq / pmol and chemical purity of > 98%. During radiolabeling, ~150 MBq (~4 mCi) of89Zr were buffered with HEPES (1 M) until the pH was adjusted to 6.8-7.5. DFO-LAMP-1 was added to the radioactive solution, and the reaction was left to proceed for one hour at 37 °C on an agitating heat block at 350 rpm.89Zr- DFO-LAMP-1 was purified by PD Miditrap G-25 columns using 0.9% sterile saline with 5 mg / ml gentisic acid as the mobile phase. To determine radiochemical purity, 2 pL samples of the radiolabeling reaction and the purified fractions were spotted into instant thin-layer chromatography (iTLC) strips (SGI0001 ; Agilent Technologies), run with 50 mM DTPA (pH = 5.5) and read on a radio- TLC scanner. The labeled antibody remained at the origin (F?r<0.1 ), whereas free89Zr was chelated and moved with the solvent front (Ffr >0.9). The radiolabeling yield of the reaction was calculated by dividing the amount of radioactivity initially added to the antibody solution by the amount of radioactivity isolated with the purified89Zr-DFO-LAMP-1 radioimmunoconjugate. The specific activity was also calculated by dividing the radioactivity isolated with the purified radioimmunoconjugate by the initial mass of DFO-LAMP-1 in the reaction. The number of chelators per mAb molecule was measured by labeling a 10 pL aliquot of the unpurified DFO-conjugated mAb with89Zr and then determining the proportion of89Zr -DFO-LAMP-1 mAb versus free89Zr -DFO by iTLC and multiplying this fraction by the molar ratio used in the reaction according to the available literature (43).

[0239] The rabbit IgG control antibody (I5006; Millipore Sigma) was conjugated and labeled according to the same protocol.

[0240] Binding Studies

[0241] The binding kinetics of anti-LAMP-1 mAb with human LAMP-1 protein (R&D Systems) were analyzed by surface plasmon resonance (SPR) using a BIACORE T200 instrument (Cytiva, formally known as GE Healthcare) at 25 °C using a PBS buffer supplemented with 0.05% Tween 20 as the running solution. The antibody was covalently immobilized onto a CM5 sensor chip (Cytiva) by the standard amine coupling method, as recommended by the manufacturer. LAMP-1 protein at concentration series of 0-50 nM (at two-fold dilutions) was injected over the channels at a flow rate of 30 pl / min for 300 seconds and allowed to dissociate for another 600 seconds before regeneration with 10 mM glycine-HCI pH 2.5. Sensorgrams were corrected with appropriate blank references and fitted with Biacore Evaluation software using a 1 :1 binding model to retrieve association rate constants (ka), dissociation rate constants (kd), and equilibrium dissociation constants (KD, a measurement of affinity).

[0242] PET / CT Imaging PET imaging was performed 24-, 72-, and 168-hours post-injection of approximately 7.5 MBq of either89Zr-DFO-LAMP-1 or89Zr-DFO-lgG via tail-vein injection. For imaging, mice were anesthetized (isoflurane inhalation), and PET / CT images were acquired on a rodent Argus PET scanner (Sedecal). PET acquisition was performed in whole body mode with two bed positions for 20 minutes per bed position, followed by CT acquisition. Images were reconstructed using a 3D-MLEM algorithm (4 iterations and 20 subsets) and corrected for scatter and randoms. Images were fused and processed using VivoQuant software version 4.0 patch 3 (InviCRO). A 3D region of interest (ROI) was manually drawn around the tumor and cardiac blood pool using CT images for anatomic reference. The software calculated mean and max standardized uptake values (SUVs) for each ROI. Representative PET / CT images were extracted using the maximum intensity projection visualization tool for each imaging time point.

[0243] In Vivo Biodistribution

[0244] Following the last PET / CT scan time point 168 hours post probe injection, mice from experimental and control groups were euthanized according to institutionally approved protocols. Blood samples and the major organs were collected, weighed, and their decay-corrected radioactive contents were measured using a Wizard-2 y counter (PerkinElmer). Results of the biodistribution studies are presented as percent injected dose per gram of tissue (%l D / g).

[0245] Histopathologic Analysis

[0246] After the last imaging timepoint and biodistribution studies, harvested tumors were fixed in 10% neutral-buffered formalin overnight and submitted to the Specialized Histopathology Services of the MGH Pathology Core for processing and sectioning. Slides were stained and imaged according to the protocol previously described in the immunofluorescent (IF) staining of human tissue samples and processed by Fiji Imaged software.

[0247] Genome and transcriptome analysis

[0248] We used the online databases TNMplot.com and Gepia2 to compare the gene expression of LAMP-1 in normal and malignant samples. TNMplot.com is an integrated database using available transcriptome-level datasets across all genes in real-time generated by either gene arrays from the Gene Expression Omnibus of the National Center for Biotechnology Information (NCBI-GEO) or RNA- seq from The Cancer Genome Atlas (TCGA), Therapeutically Applicable Research to Generate Effective Treatments (TARGET), and The Genotype-Tissue Expression (GTEx) repositories (44). The database automatically uses the Mann-Whitney U test to compare each cancer with the respective normal tissue. We also used the online transcriptome-level validation tool ROC plotter, which integrates transcriptome data with therapy response and progression-free survival in breast, colon, and ovarian cancer patients (45). We investigated the correlation between LAMP-1 expression and response to anthracycline regimens using relapse-free survival at five years.

[0249] Enrichment plot data was obtained from PC3 prostate cancer cell line data (GSM2059432) available at NCBI-GEO (46). Spatial transcriptomic analysis

[0250] We obtained pre-processed spatial transcriptomic data from a Human Breast Cancer: Ductal Carcinoma in Situ, Invasive Carcinoma (FFPE) sample using the Visium platform technology from 10x Genomics, CA, USA (see 10xgenomics.com / resources / datasets / human-breast-cancer-ductal- carcinoma-in-situ-invasive-carcinoma-ffpe-1 -standard-1 -3-0). The Space Ranger pipeline was utilized to process and normalize the raw sequencing data, and the obtained expression data were normalized using the Log2 function. The normalized data were then analyzed by mapping the expression data to the H&E image.

[0251] Results

[0252] Human adenocarcinoma tissue samples highly express LAMP- 1

[0253] Three mixed TMAs composed of multiple human adenocarcinoma and normal tissues were screened by the IF method (FIG. 2A). Malignant cores combined had significantly higher LAMP-1 fluorescent intensity compared to normal cores (p-value<0.0001 ) (FIG. 2B). Mean fluorescent signal intensity of prostate (2871 .62 ± 1104.78 vs. 948.12 ± 497.98, p-value<0.05), pancreas (1785.69 vs. 948.12 ± 497.98, p-value<0.05), colon (2597.12 ±1 828.96 vs. 304 ± 128.49, p-value<0.01 ), uterus (1662.53 ± 830 vs. 494.25 ± 95.81 , p-value<0.01 ) and breast (3076.87 ± 621 .011 vs. 380.15 ± 25.05, p-value<0.05) were significantly higher compared to their corresponding normal tissue. The same pattern was observed for skin (1666.25 ± 830 vs. 224.37 ± 95.81 ). However, the difference was not statistically significant due to the lower number of normal cores. In contrast, normal kidney samples showed a reversed pattern and slightly higher expression than renal carcinoma (FIG. 2C).

[0254] Next, we assessed additional TMAs specific to colon, breast, and prostate cancer and their corresponding normal tissue. A similar trend to the mixed TMAs was observed here, with significantly higher signal intensity in cancerous cores than normal cores for the colon, breast, and prostate (p- value<0.0001 ). (FIGS. 2D-2F). No significant difference was observed between mean signal intensity based on grade and clinical stage.

[0255] Bioinformatic analysis

[0256] Pan-cancer RNAseq analysis showed higher LAMP-1 expression in malignant samples, with the exception of kidney samples (FIG. 3A). Positive correlations between LAMP-1 and ERK2 (R=0.45, p-value<0.001 , FIG. 3B) and EGFR (R=0.5, p-value<0.001 , FIG. 3C) were observed.

[0257] Silencing LAMP-1 led to downregulation in the signatures of epithelial-mesenchymal transition (EMT), autophagy, and apoptosis pathways (data not shown).

[0258] Spatial transcriptome sequencing data and subsequent analyses (FIGS. 4A-4P) revealed significantly higher LAMP-1 expression in ductal carcinoma regions compared to other regions and immune cells (P=0.008 and P<0.05, respectively). Genes differentially expressed between high and low LAMP-1 expression groups showed alterations in the extracellular matrix (ECM) matrix, growth factor binding, enzyme-linked receptor protein signaling pathways, and oncogenic signatures. We explored the functional differences between ductal carcinoma cells with high expression of LAMP-1 and those with low expression of LAMP-1 to determine the phenotype / tumorigenesis behavior. We divided the cancer cells into two subgroups based on the level of LAMP-1 expression: high expression (Iog2 expression more than 4.5) and low expression (Iog2 expression less than 2) (FIGS. 4I-4J). We then conducted a gene differential expression analysis to detect the most significant genes that were up-or down-regulated. We identified 239 genes that were significantly altered in cells with high expression of LAMP-1 , with three genes up-regulated and 236 genes down- regulated. We applied these genes and their Iog2-fold change to evaluate the KEGG and GSEA pathway analyses. Our KEGG pathway analysis revealed that the most significant pathways between these two subgroups were ECM-receptor interaction, antigen processing and presentation, protein digestion and absorption, phagosome, and focal adhesion (FIG. 4L). Additionally, the GSEA enrichment analysis using GO, HALLMARK, and oncogenic signature databases revealed that cells with high expression of LAMP-1 exhibited significant alterations in the ECM matrix, and growth factor binding regarding molecular function, response to transforming growth factor and enzyme-linked receptor protein signaling pathways, regarding biological processes, coagulation, and epithelial- mesenchymal transition, regarding hallmark gene set of GSEA, and BMI, KRAS, as well as RB, regarding oncogenic signature (FIGS. 4M-4P).

[0259] LAMP- 1 expression in normal organs is lower than in tumors in vivo

[0260] We measured LAMP-1 cell surface expression in subcutaneous tumors and healthy organs in MC-38 tumor-bearing mice. Flow cytometry data revealed that LAMP-1 expression in normal organs, except for small bowel, was significantly lower than in tumors (liver: 17.07% ± 2.71 , lung :31 .47%±3.72, bone marrow: 21 .6% ± 8.25, kidney: 44.27%± 3.98, muscle: 24.47%± 9.05, spleen:16.77%± 7.801 , small bowel: 82.57%± 7.45 vs. tumor: 80.93%± 2.83, p-value<0.05 for all) (FIG. 5A).

[0261] To investigate the overall cellular expression of LAMP-1 and compare the cell surface and cytoplasmic localization, the above experiment was repeated after cellular PM permeabilization, which allows antibody access to LAMP-1 in both the cytoplasm and cell surface. The tumoral expression increased to 88.97%± 5.79, which reflects that a significant portion of LAMP-1 is expressed on the PM in the tumor cells.

[0262] Breast, colon, and pancreatic adenocarcinoma cells highly express LAMP- 1 in vitro

[0263] To choose the optimal cell lines for in vivo imaging, we measured the level of LAMP-1 cell surface expression in human (FIG. 9A) and murine (FIG. 9B) breast, colon, and pancreas cancer cell lines by flow cytometry. For human cell lines, experiments were performed at two different temperatures (4 °C and 37 °C) with the aim of better understanding dynamic changes in LAMP-1 cell surface expression. In all tested human cells, the cell surface LAMP-1 expression at 37 °C increased by 30-40%, compared to 4 °C (FIG. 9A). More than 90% of cells were LAMP-1 + in all tested murine cell lines (FIG. 9B). Radiotracer preparation and characterization

[0264] Human monoclonal anti-LAMP-1 antibody (LAMP-1 mAb) was conjugated to DFO, then radiolabeled with89Zr4+to yield89Zr-DFO-LAMP-1 mAb (FIG. 10A). The reaction of the antibody with a 4-fold molar excess of p-SCN-Bn-DFO resulted in the substitution of approximately 1 .70 DFO chelators per antibody molecule. SPR showed a high binding affinity of LAMP-1 mAb to human LAMP-1 protein (KD=1 .78 ± 0.84 nM, FIG. 10B). Radiochemical purity was 95.85% (FIGS. 10C-10D), with a radiolabeling efficiency of 85.35%.

[0265] PET / CT imaging

[0266] 89Zr-DFO-LAMP-1 PET / CT imaging showed antibody localization in the subcutaneous xenografts as early as 24 hours after injection in both models, with minimal antibody accumulation in normal organs (FIGS. 6A-6B). Tumor radiotracer accumulation increased in subsequent scan time points, peaking at 168 hours post-injection. The blood pool signal gradually decreased, consistent with blood pool clearance and temporal tumor radiotracer accumulation, as reflected by a constant gradual increase in the tumor to background ratio (FIG. 6C). The SUVmean of tumors was significantly higher than the blood pool at 72 hours and 168 hours post-injection in both MDA-MB-231 and Caco2 models (Table 3, p-value<0.01 ; FIG. 6D). The same pattern was observed for SUVmax Of MDA-MB-231 (72h: 9.25 ± 3.37 vs. 3.62 ± 1 .18, 168h: 12.96 ± 5.68 vs. 2.53 ± 0.82, p-value<0.01 ) and Caco2 (72h: 5.91 ± 1.11 vs. 2.35 ± 0.99, 168h: 8.53 ± 3.03 vs. 2.69 ± 1 .54, p-value<0.001 ) respectively (FIGS. 6G- 6H).

[0267] In contrast, the89Zr-DFO-lgG tumor uptake had only minimal or no temporal increase (FIGS. 6E-6F, Table 3, p-value for temporal comparison not significant). In both models, the89Zr-DFO-LAMP- 1 tumor uptake was significantly higher than the respective control group at every time point (Table 3).

[0268] Table 3. The PET Quantitative Values in MDA-MB-231 and Caco2 Xenografts

[0269] Biodistribution studies

[0270] Biodistribution studies seven days after the administration of the probes showed significantly higher uptake of the89Zr-DFO-LAMP-1 mAb compared to the IgG control probe by the breast (21 ,79±5.77 vs. 7.69±3.90, p-value<0.001 , FIG. 7A) and colon xenografts (11 ,75±4.69 vs. 1 .65±0.52, p-value<0.001 , FIG. 7B). In the colon adenocarcinoma model, radiotracer retention in the normal colon was significantly lower than in the tumor (1 .07±0.37 vs. 11 .76±4.69, p-value<0.0001 ). As expected, a high level of uptake was detected in the liver and spleen as the principal organs involved in antibody clearance and biodistribution.

[0271] Ex vivo analysis of tumor xenografts

[0272] After the last imaging timepoint, tumors were extracted for ex vivo analysis by IF staining. The tissue sections of both models showed a high level of specific staining for LAMP-1 (FIG. 11).

[0273] Conclusion

[0274] In this study, we developed an anti-LAMP-189Zr-DFO-mAb PET tracer. We have demonstrated that LAMP-1 expression can be used as an imaging biomarker for the non-invasive detection of breast and colon adenocarcinomas. Tumors were visible on PET / CT as early as 24 hours post-injection, and significant tracer accumulation and retention continued up to 7 days after injection. In contrast, the IgG uptake remained stable during the same period, reflecting specific uptake and retention regardless of probe size and perfusion. Ex-vivo analysis of tumors on biodistribution studies and IF staining confirmed the imaging findings. By performing LAMP-1 IF staining on multiple samples of human colon, breast, pancreas, and prostate, carcinomas and genomic analysis, we showed the expandability of utilizing LAMP-1 -based immunoPET as a general imaging probe for adenocarcinomas.

[0275] Suggested mechanisms of LAMP-1 facilitating tumor invasion, metastasis, and treatment resistance include increased exocytosis to alter ECM and efflux of lysosomotropic chemotherapy agents, regulating cell-to-cell adhesion and signaling, and lastly, promoting autophagy. However, despite the suggested explanations, the exact role of LAMP-1 in the mentioned processes is still unclear (11 , 12, 47-49). A demographic summary of the suggested roles of LAMP-1 in cancer progression and how anti-LAMP-1 radioconjugates can be incorporated in modulating these pathways for progression control is demonstrated in FIGS. 8A-8D.

[0276] LAMP-1 is one of the mediators of lysosome binding to actin filaments and their eventual docking to PM during exocytosis. Even though this physical binding is through the cytoplasmic C- terminal tail of LAMP-1 , it is suggested that alteration in glycan composition of the N-terminal domain leads to structural changes of the C-terminal and binding regulation (11 ). Neuraminidase 1 (NEU1 ) is a lysosomal sialidase that suppresses exocytosis by cleaving LAMP-1 sialic acids and shortening its half-life (50). NEU1 suppression was correlated with extensive exocytosis, leading to an invasive, migratory, and chemoresistant phenotype in human sarcoma models (12). Cisplatin-resistant ovarian cancer cells were found to release more exosomes with increased LAMP-1 and drug metabolite content and almost lacking intracellular LAMP-1 expression compared to the non-resistance cell line (47). A similar lysosomal secretory profile was observed in ovarian and glioblastoma cancer cells under hypoxia, which also correlated with a real-time downregulation of NEU1 for ovarian cancer cells (51 , 52). Lower uptake and increased doxorubicin efflux were observed in the MDA-MB-231 TNBC cell line as well. The same study reported higher LAMP-1 expression in TNBC breast cancer patients who were nonresponders or had shorter relapse-free survival (53). Interestingly, a study on predicting chemoresistance to microtubule-modulating drugs on more than 50 human cancer cell lines showed that the LAMP-1 expression correlated with chemoresistance to all three agents studied and silencing LAMP-1 restored chemosensitivity by suppressing drug efflux (48).

[0277] Modifying the cell surface glycosylation is a critical step in mediating cancer cell adhesion to endothelium and cells of the metastasis recipient organs (54). The expression of p1 ,6 branched N- oligosaccharides correlates with disease progression and the probability of metastasis in many cancers by providing a platform for further modifications, including the substitution of poly N- acetyllactosamine (polylacNAc), which together has a high affinity to bind lectins in ECM, basal membrane and cells (55). LAMP-1 is a major carrier p1 ,6 branched N-oligosaccharides substituted with polylacNAc and translocation of LAMP-1 to the cell surface leads to significantly higher expression of these galectin ligands on metastatic cells’ PM (27, 55, 56).

[0278] The role of autophagy in cancer initiation, progression, and treatment response is complex and dependent on many factors (57). Evidence suggests that after the initial establishment of the malignancy, autophagy can facilitate the survival and proliferation of cancer cells in unfavorable environments and energy-deprivation settings, both in the primary and metastatic sites (58, 59). LAMP-1 and LAMP-2 are involved in lysosomal interaction and fusion with endosomes, autophagosomes, and appropriate lysosomal function (60). Ubiquitin-like protein 4A (UBL4A) suppressed autophagy and epithelial-to-mesenchymal (EMT) transition by binding to LAMP-1 and disrupting lysosomal function in the late stage of autophagy in pancreatic ductal adenocarcinoma (PDAC). This observation makes LAMP-1 an exciting target for combination therapy with lysosomotropic agents for dual suppression of autophagy (61 , 62).

[0279] As mentioned earlier, the current body of literature shows alterations of LAMP-1 expression in a diverse group of malignancies, including cancers of the breast, colon, pancreas, prostate, ovary, and brain, which altogether have a significant burden on the health system. In addition, in some of these cancers, such as TNBC, PDAC, and glioblastoma, the number and effectiveness of the available therapeutic options are already limited (63-65). Therefore, there is an increasing unmet demand for developing novel targeted therapy agents and related biomarkers for patient selection and treatment response monitoring in combination with available standard-of-care options. LAMP-1 -based theranostics have the unique potential to serve this purpose as a shared target in many cancers. However, despite all the promising evidence, there is still a lot of unclarity in the knowledge on the regulation and downstream effects of LAMP-1 and how it fits in complex cancer biology. One finding that reflects this is a controversy in the survival outcomes of LAMP-1 overexpression among different cancers. Higher TNM staging and worse survival outcomes are reported for breast, gynecological, colorectal, prostate, esophageal, and head and neck cancers (25, 29, 30, 66-68).

[0280] In contrast, the opposite trend is reported in PDAC and gliomas despite the observed overexpression of LAMP-1 in all these cancers (24, 27, 61 , 69). Our results from the IF signal intensity of the TMAs for the breast, colon, prostate, and lung are in agreement with the available genomic data and the published literature. Whereas for PDAC, our results were controversial. Despite the significantly higher expression of the LAMP-1 gene in PDAC, our TMA samples failed to reflect this. This can be the result of using TMAs containing small sections of the tissue blocks to analyze target protein expression may not be the best representative of the whole tumor. This is especially important in the case of the pancreas, where there is a significant difference between LAMP-1 expression in ductal and stromal cells, and the proportion of each cell type in the core can affect the final signal intensity. In the normal pancreas, moderate to strong Lamp-1 immunoreactivity was reported in the islet cells and macrophages, and faint staining in the cytoplasm of acinar cells. Ductal cells of the normal pancreas were negative for Lamp-1 . In PDAC tissues, Lamp-1 immunoreactivity was intensively present in the cytoplasm of cancer cells, macrophages, and some lymphocytes. Lamp-1 was present primarily at the luminal side of the ductal carcinoma cells (27). This dilemma highlights the need to investigate the expression of LAMP-1 in each cancer grade and stage in a broad group of patients with a complementary robust genomic and proteomic analysis as well, which goes beyond the scope of our current study.

[0281] The ability of mAbs to specifically target tumor antigens makes them excellent candidates for the theranostic approach to diagnosis, therapy efficacy evaluation, and targeted delivery of ionizing radiation to the tumor. We observed a significant increase in the LAMP-1 signal on flow cytometry with increasing temperature to 37 °C, reflecting the potential internalization of the antibody after binding to the PM-expressed LAMP-1 , which is promising for the effective targeted delivery of therapeutic compounds for radionuclide therapy and antibody drug conjugate (ADC) specific cargo delivery. Furthermore, this ability can potentially facilitate dual therapy, sensitizing tumors to lysosomotropic chemotherapy agents, and improving the delivery of lysosome-targeted drugs to induce cell death by activating downstream cell death pathways. The retention and delayed washout of our probe make it an appropriate option for the continued delivery of radiation to the malignant lesions if used as a theranostic agent.

[0282] One of the main limitations of our study was lacking a negative control xenograft model, which is due to generally moderate to high expression levels of LAMP-1 in commercially available adenocarcinoma cell lines and the lack of commercially available and stable LAMP-1 knock-out cell lines. To overcome this challenge, we used IgG from rabbit serum as our control probe to confirm the specific binding of our LAMP-1 probe compared to non-specific IgG. Using in-vivo blocking with cold antibody before PET probe injection was a less feasible alternative due to the lack of access to high quantities of the commercially available antibody required for adequate blocking.

[0283] Despite the promising preliminary evidence of mAb-based immunoPET of LAMP-1 , the big size of the full structure mAbs extends the circulation time and delays the appropriate accumulation in the tumor for producing a high-quality image compared to smaller size probes. This phenomenon necessitates using long half-life radionuclides such as89Zr and limits the feasibility of routine use in the clinic. Using smaller antibody-based vectors such as nanobodies has the advantage of faster kinetics due to their significantly smaller size while maintaining the high-affinity properties of mAbs. This characteristic facilitates radiolabeling with short half-life radionuclides and, therefore, more straightforward clinical application. Furthermore, the renal excretion of nanobodies can overcome the limited use of full-structure mAb-based LAMP-1 probes for detecting hepatic and splenic lesions due to their high background signal as the main organs involved in mAb excretion.

[0284] This study showcases the merit of LAMP-1 in cancer imaging. Our findings suggest that LAMP-1 can serve as a potentially valuable biomarker to image a wide range of adenocarcinomas, from a lower-grade colon adenocarcinoma model to a high-grade TNBC breast adenocarcinoma model, with high specificity. Given the interaction of LAMP-1 with tumor growth, invasion, and drug resistance pathways, monitoring response to anticancer therapies targeting these pathways using LAMP-1 immunoPET can further elaborate the role of LAMP-1 in cancer biology and investigate the effectiveness of a combined treatment regimen with anti-LAMP1 pharmaceuticals for improved outcomes of the available treatments. Further investigations are warranted to assess LAMP-1 -based radiopharmaceuticals’ performance in the theranostic approach to these malignancies, especially for cancers with high mortality and limited therapeutic options such as PDAC and TNBC.

[0285] Example 2. Generation of Anti-LAMP-1 Nanobodies

[0286] Objective

[0287] The objective of this study was to generate and purify nanobodies targeting human LAMP-1 .

[0288] Materials and Methods

[0289] Animal Procedures

[0290] Animal experiments complied with ethical regulations and were approved by Institutional Animal Care and Use Committee. Extensive efforts were made to minimize the number and suffering of animals used in this study.

[0291] Construction of VHH library

[0292] The protocol for Nb generation was adapted from Vincke (70). Briefly, a Llama (Lama glama, an American Camelid) was immunized seven times with approximately 1x108Human LAMP-1 - expressing cells to generate antigen-specific antibodies. The RNA from Llama lymphocytes was then isolated and converted to cDNA. The cDNA sequence encoding the antigen binding fragment (VHH) of heavy chain only antibodies was then amplified and cloned into a phage display vector to construct a VHH library also known as Nanobodies (Nb’s). The pool of amplified Nb’s DNA in the phage-display vector was transformed into TG1 E. coll electrocompetent cells to generate a library of 109transformants.

[0293] Cell-based panning with Phage-display

[0294] The Nb’s library in TG1 E. coli cells was infected with 1.0x1011M13K07 helper-phages to create a Nb-displaying bacteriophage library for screening large number of clones in a simple and systematic fashion. The in vitro panning phage-display was performed against viable, adherent LAMP-1 -expressing cells in a total of 5 subsequent rounds starting with a negative screen followed by a positive screen. The final clones of the library population contained single Nb clones that were used for a second screening by a Periplasmic enzyme-linked immunosorbent assay (ELISA).

[0295] Selection of high-affinity Nb binders

[0296] After five rounds of panning, Nbs-containing colonies were transformed into a naive TG1 E. coll culture and spread on LB-ampicillin glucose plates to further evaluation of binding to LAMP-1 by using Periplasmic-ELISA. Small scale expressed Nb’s were prepared as follows: 198 colonies were randomly picked and grown in 24-deep well plates with 2 ml TB medium. The production of each Nb was induced and exported to the E. Coll periplasm space. The Periplasmic Nb’s from each colony were tested for antigen recognition by incubation of each clone with LAMP-1 -expressing cells in comparison to LAMP-1 negative cells in a 96-well plate. Detection of Nb binding was performed by using a mouse anti-HA antibody and secondary HRP-anti-mouse IgG, followed by adding 100 pl TMB-ELISA Substrate Solution to each well. The reaction was stopped by adding 1 M sulphuric acid, and the absorbance of optical density (OD450 nm) was read by a Microplate Reader. Nb clones with high absorbance ratio relative to the negative control of more than 3 were considered positive and were selected for sequencing.

[0297] Recombinant expression and purification of Nb’s

[0298] Based on sequencing data of 41 Nb clones, four clones were selected (NB132, NB121 , NB103, and NB63) and were further expressed in WK6 E. Coll culture (2L) and purified by using affinity chromatography on Ni-NTA gravitational beads. The eluted Nb’s size and purity was evaluated by SDS-PAGE gel mass spectrometry (MALDI-TOF), confirming the expected size of ~15 kDa and >95% purity.

[0299] The amino acids sequences of the four clones are as follows and their CDR sequences for each of the four nanobodies are described in FIG. 13A and below.

[0300] 1 ) NB132: QVQLQESGGGLVQAGGSLRLSCTASGLIFSINSMGWYRQVPGKQRELVASVHNGDSTYSADSVKG RFTISVDDAKNMVYLQMNDLEPEDTAVYYCNARDPRGGHLWNYWGQGTQVTVSS

[0301] CDR1 is GLIFSINSMG; CDR2 is VHNGDST; CDR3 is NARDPRGGHLWNY.

[0302] 2) NB121 : QVQLQESGGGLVQAGGSLRLSCAASGLIFRINVMGWYRQAPGKERELVAQITHGGSTNYADSVKGR FTVSRDDAENTVDLQMNSLKPEDTAVYYCNAADSAGSNMNAKWSYWGQGTQVTVSS

[0303] CDR1 is GLIFRINVMG; CDR2 is ITHGGST; CDR3 is NAADSAGSNMNAKWSY. 3) NB103: QVQLQESGGGLVQAGGSLRLSCAASGDIFSIDRMGWFRQTPGKERELVASIFLSDGETKYGDFVKG RFTISRDNAKNAVYLQMNSLKPEDTAVYYCWSYTTTNSWGQGTQVTVSS

[0304] CDR1 is GDIFSIDRMG; CDR2 is IFLSDGET; CDR3 is WSYTTTNS.

[0305] 4) NB63: QVQLQESGGGLAQAGGSLRLSCTASGIIFRINDMGWYRQAPGKQRELVAVATMDDSTNYADSVKGR FTVSRDSAKNSVYLQMNSLRPEDTAVYYCYAGRSEKWAYWGQGTQVTVSS

[0306] CDR1 is GIIFRINDMG; CDR2 is ATMDDST; CDR3 is YAGRSEKWAY.

[0307] All purified nanobodies were expressed with the C-terminal sequence AAAYPYDVPDYGSHHHHHH corresponding to HA tag, GS linker, and a Hisx6 tag, for detection or purification purposes. Alternatively, the sequence AAALPETGGHHHHHH corresponding to Sortase-tag and Hisx6 tag was used for site-specific labeling and purification purposes.

[0308] Binding Studies

[0309] The binding kinetics of NB121 , NB103, NB132, and NB63 were analyzed by SPR as described above in Example 1 .

[0310] Confocal Imaging

[0311] Confocal images of the Nbs and LAMP-1 were performed in Dubca cells and MDA-MB-231 cells using techniques known in the field to assess co-localization. The nucleus was stained with DAPI.

[0312] In vivo Optical Imaging

[0313] In vivo optical imaging of fluorescent-labeled Nbs targeting LAMP-1 was conducted. An in vivo mice Matrigel model was used to assess Nbs targeting of LAMP-1 protein. Athymic nude mice were injected subcutaneously with 0.1 mL of recombinant human LAMP-1 protein (1 pg or 5 pg) mixed with Matrigel (1 :1 ) to the right upper flank, or with 0.1 mL of Matrigel only on the left flank as negative control. Four mice were then injected intravenously with 25 pg of either NB132 or NB103 labeled with NHS-ester AlexaFluor 647. One non-injected mouse served as negative control.

[0314] Nanobody Conjugation

[0315] For specific and accurate diagnostic imaging or therapy of LAMP-1 + tumors or inflammation sites, the nanobodies can be conjugated to a chelating agent. The chelator DFO was employed and the Nbs were functionalized with it as follows.

[0316] Site-specific conjugation of Nb’s was carried out via Sortase (SrtA) enzyme reaction. The SrtA-mediated conjugation was optimized with Nb’s with a C-terminal sequence of LPETGG and chelators (GGG)-DFO, (GGG)-azide-DFO or (GGG)-azide-DOTA as the substrates. To functionalize the nanobodies with the chelator of choice, a mixture of Nb (2-4 mg / mL), chelator (1-2 mM) and sortase 7M (of 5-7 mM) was incubated overnight at 4 °C followed by purification by a PD-10 column and further removal of Sortase by Ni-NTA gravitational beads.

[0317] To radiolabel the DFO functionalized nanobody with89Zr for imaging purposes, the conjugated Nb can be coupled with Dibenzocyclooctyne (DBCO) polyethylene glycol (PEG) to increase its overall mass by a click reaction. The DBCO-PEG (20kDa) was incubated with the nanobody functionalized with axide-DFO substrate (approximately 1 -2 mg / mL) to reach 1 :1 mole equivalents compared to the nanobody. An89Zr-oxalate solution was diluted with 300 pL of Chelexed Hepes or PBS and -200 pL of DFO-functionalized nanobody (0.5-2.0 mg) was added in Hepes / NaCI buffer pH 7.5. The reaction pH was increased to 6.8-7.5 with sodium carbonate (2 M) and the mixture was incubated for 60 min at 25 °C using a thermomixer shaking at 350 rpm.

[0318] For radiotherapy purposes the DOTA-nanobody was labeled with177Lu as follows. A177Lu chloride solution was diluted with 200 pL of 1 M Ammonium Acetate pH 5, and -300 pL of DFO- functionalized nanobody (0.5-2.0 mg) was added in Hepes / NaCI buffer pH 7.5. The pH was verified to be 5-5.5 and the mixture was incubated for 60 min at 37 °C using a thermomixer shaking at 600 rpm.

[0319] PET / CT Imaging

[0320] PET / CT imaging of89Zr-labeled anti-LAMP-1 Nb was performed using in vivo mice Matrigel model. Athymic nude mice were injected subcutaneously with recombinant human LAMP-1 protein (5 pg) mixed with Matrigel to the left upper flank, and Matrigel only injected to the right flank as negative control.89Zr-labeled NB103 (300 pCi) was injected intravenously, followed by a washout through the kidneys. Images were obtained 2.5 hours post-injection.

[0321] TLC analysis

[0322] TLC analysis of non-specific89Zr-labeled anti-LAMP-1 NB103 was performed as described above in Example 1 . Labeling efficiency was measured by iTLC with 50 mM DTPA, pH 5.0 as an eluant, and was shown to be more than 99%.

[0323] Additional Results

[0324] Selection of high-affinity Nb binders

[0325] A graphic representation of the screening of specific single positive Nb clones by ELISA is shown in FIG. 12.

[0326] Recombinant expression and purification of Nb’s

[0327] Purified NB121 , NB103, NB132, and NB63 have the expected size of -15 kDa (FIG. 13B).

[0328] The mass spectrometry results of the purified NB121 , NB103, NB132, and NB63 are shown in FIG. 14. The single peak at m / z -15 kDa corresponds to the singly charged molecular ion, [M+H]+.

[0329] Binding Studies The Nbs bind to LAMP-1 with nanomolar affinity (FIGS. 15A-15D). In vitro binding affinity of the purified Nbs to immobilized LAMP-1 was in the nanomolar range: NB103 KD=1 .7 nM, NB132 KD=8.5 nM, NB63 KD=23.79 nM, NB121 KD=N / A.

[0330] Confocal Imaging

[0331] NB132 was internalized into high LAMP-1 -expressing Dubca cells and co-localized with LAMP- 1 in the cytoplasm in small circular structures, possibly representing endocytic vesicles (FIG. 16A).

[0332] NB121 was also observed inside MDA-MB-231 cells and co-localized with LAMP-1 in the cytoplasm, suggesting internalization following interaction with cell surface LAMP-1 (FIG. 16B).

[0333] In vivo Optical Imaging

[0334] In vivo optical imaging showed that the labeled Nbs are specifically detected in LAMP-1 + site 1 - hour post-injection (LAMP-1 site indicated by arrows; FIG. 17). The fluorescent signal showed specific uptake on the right flank (LAMP-1 +) and not on the left flank.

[0335] PET / CT Imaging

[0336] 89Zr-labeled NB103 was detected on the left flank (LAMP-1 site indicated by arrows) and not on the right flank (FIG. 18).

[0337] TLC analysis

[0338] Results of TLC analysis are shown in FIG. 19.

[0339] Conclusion

[0340] These anti-LAMP-1 nanobodies can be used as theranostic tools for diagnosis and specific radiotherapy by targeting LAMP-1 + cancers and for diagnosis of inflammation sites where LAMP-1 is overexpressed.

[0341] These nanobodies can be conjugated to a chelating agent, functionalized, and radiolabeled. The labeled nanobodies of the invention can be used as diagnostic agents and in various imaging techniques, including, for example, Positron Emission Tomography (PET), Single Photon Emission Computed Tomography (SPECT), Computed Tomography (CT), Magnetic Resonance Imaging (MRI), Infra-Red and Near Infra-Red Imaging (IR / NIR), Ultrasound (US), and fluorescence-activated cell sorting (FACS). These nanobodies may also be conjugated to a chemotherapeutic for targeted therapeutic delivery (ADC) to tumors exhibiting LAMP-1 overexpression. The radioactive label (e.g., isotope) for imaging or therapy can be selected from this group:3H,1 1C,14C,18F,32P,35S,36CI,51Cr,52Fe,57Co,58Co,

[0342] One of skill in the art can employ already know techniques, methods and protocols, including the method described in Example 1 above for labeling the anti-LAMP-1 monoclonal antibody, to label the nanobodies. References

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[0413] Other Embodiments

[0414] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.

[0415] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the invention that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims. Other embodiments are within the claims.

[0416] What is claimed is:

Claims

CLAIMS1 . An anti-LAMP-1 antibody or nanobody composition.

2. The composition of claim 1 , wherein the nanobody comprises CDR1 (GLIFSINSMG) (SEQ ID NO: 1 ), CDR2 (VHNGDST) (SEQ ID NO: 2), and CDR3 (NARDPRGGHLWNY) (SEQ ID NO: 3) of NB132.

3. The composition of claim 1 , wherein the nanobody comprises QVQLQESGGGLVQAGGSLRLSCTASGLIFSINSMGWYRQVPGKQRELVASVHNGDSTYSADSVKG RFTISVDDAKNMVYLQMNDLEPEDTAVYYCNARDPRGGHLWNYWGQGTQVTVSS (NB132) (SEQ ID NO: 4).

4. The composition of claim 1 , wherein the amino acid sequences of the nanobody comprises one or more CDRs selected from the group consisting of:(a) CDR1 : GLIFSINSMG (SEQ ID NO: 1 ); CDR2: VHNGDST (SEQ ID NO: 2); and CDR3: NARDPRGGHLWNY (SEQ ID NO: 3) of NB132;(b) CDR1 : GLIFRINVMG (SEQ ID NO: 5); CDR2: ITHGGST (SEQ ID NO: 6); and CDR3: NAADSAGSNMNAKWSY (SEQ ID NO: 7) of NB121 ;(c) CDR1 : GDIFSIDRMG (SEQ ID NO: 9); CDR2: IFLSDGET (SEQ ID NO: 10); and CDR3: WSYTTTNS (SEQ ID NO: 11 ) of NB103;(d) CDR1 : GIIFRINDMG (SEQ ID NO: 13); CDR2: ATMDDST (SEQ ID NO: 14); and CDR3: YAGRSEKWAY (SEQ ID NO: 15) of NB63;(e) CDR1 : ETIFNANTMY (SEQ ID NO: 17); CDR2: ITTRGATD (SEQ ID NO: 18); and CDR3: NVFIFGVDY (SEQ ID NO: 19) of NB185;(f) CDR1 : GFTFRNYAMR (SEQ ID NO: 21 ); CDR2: ISSGGDATS (SEQ ID NO: 22); and CDR3: ATSDFNI (SEQ ID NO: 23) of NB191 ;(g) CDR1 : GLIFRINDMG (SEQ ID NO: 25); CDR2: EATDDSTS (SEQ ID NO: 26); and CDR3: YAGRSGKWDY (SEQ ID NO: 27) of NB189;(h) CDR1 : GSISSIDRMG (SEQ ID NO: 29); CDR2: LSTSGDILT (SEQ ID NO: 30); and CDR3: WSYDFHNY (SEQ ID NO: 31 ) of NB95;(i) CDR1 : GIIFRINDMG (SEQ ID NO: 13); CDR2: ATMDDSTN (SEQ ID NO: 33); and CDR3: YAGRSEKWAY (SEQ ID NO: 15) of NB56;(j) CDR1 : GIIFRINDMG (SEQ ID NO: 13); CDR2: ATTDDSTN (SEQ ID NO: 35); and CDR3: YAGRSEKWAY (SEQ ID NO: 15) of NB173;(k) CDR1 : GIIFRINAMA (SEQ ID NO: 37); CDR2: IGQDDSTT (SEQ ID NO: 38); and CDR3: NAADRVPYERWSY (SEQ ID NO: 39) of NB109; and(l) CDR1 : GIIFRINDMG (SEQ ID NO: 13); CDR2: ETIDGSRN (SEQ ID NO: 41 ); and CDR3: NAARESGGRILWSY (SEQ ID NO: 42) of NB128.

5. The composition of claim 1 , wherein the amino acid sequence of the nanobody composition is selected from the group consisting of:(a)QVQLQESGGGLVQAGGSLRLSCTASGLIFSINSMGWYRQVPGKQRELVASVHNGDSTYS ADSVKGRFTISVDDAKNMVYLQMNDLEPEDTAVYYCNARDPRGGHLWNYWGQGTQVTVSS (NB132) (SEQ ID NO: 4);(b)QVQLQESGGGLVQAGGSLRLSCAASGLIFRINVMGWYRQAPGKERELVAQITHGGSTNY ADSVKGRFTVSRDDAENTVDLQMNSLKPEDTAVYYCNAADSAGSNMNAKWSYWGQGTQVTVSS (NB121 ) (SEQ ID NO: 8);(c)QVQLQESGGGLVQAGGSLRLSCAASGDIFSIDRMGWFRQTPGKERELVASIFLSDGETKY GDFVKGRFTISRDNAKNAVYLQMNSLKPEDTAVYYCWSYTTTNSWGQGTQVTVSS (NB103) (SEQ ID NO: 12);(d)QVQLQESGGGLAQAGGSLRLSCTASGIIFRINDMGWYRQAPGKQRELVAVATMDDSTNY ADSVKGRFTVSRDSAKNSVYLQMNSLRPEDTAVYYCYAGRSEKWAYWGQGTQVTVSS (NB63) (SEQ ID NO: 16);(e)QVQLQESGGGLVQAGGSLRLSCAVPETIFNANTMYWYRRAPGKEREWVAVITTRGATDY ADSVKGRFTISRDNAKNTLNLEMNSLKPEDTAVYYCNVFIFGVDYWGQGTQVTVSS (NB185) (SEQ ID NO: 20);(f)QVQLQESGGGLVQPGGSLRLSCAASGFTFRNYAMRWVRQGPGNGLEGVSTISSGGDAT SYADSVTGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCATSDFNIRGQGTQVTVSS (NB191 ) (SEQ ID NO: 24);(g)QVQLQESGGGLAQAGGSLRLSCTASGLIFRINDMGWYRQAPGKQRELVAVEATDDSTSY ADSVKGRFTVSRDSAKNTVYLQMNSLKPEDTAVYYCYAGRSGKWDYWGQGTQVTVSS (NB189) (SEQ ID NO: 28);(h)QVQLQESGGGLVQAGGSLTLSCSVSGSISSIDRMGWFRQAPGKERELVAVLSTSGDILTY ADDVKGRFTISRDDAMNTVSLQMNSLKPEDTAVYYCWSYDFHNYWGQGTQVTVSS (NB95) (SEQ ID NO: 32);(i)QVQLQESGGGSVQVGESLTLSCTASGIIFRINDMGWYRQAPGKQRELVAVATMDDSTNY ADSVKGRFTVSRDSAKNSVYLQMNSLRPEDTAVYYCYAGRSEKWAYWGQGTQVTVSS (NB56) (SEQ ID NO: 34);(j)QVQLQESGGGLAQAGGSLRLSCTASGIIFRINDMGWYRQAPGKQRELVAVATTDDSTNYADSVKGRFTVSRDSAKNSVYLQMNSLRPEDTAVYYCYAGRSEKWAYWGQGTQVTVSS (NB173) (SEQ ID NO: 36);(k)QVQLQESGGGLAQAGESLRLSCVASGIIFRINAMAWYRQDPGKQRELVAAIGQDDSTTYA DSVKGRFAISRDNANDTVYLQMNSLKPEDTSVYYCNAADRVPYERWSYWGQGTQVTVSS (NB109) (SEQ ID NO: 40); and(l)QVQLQESGGGSAQPGGSLRLSCAASGIIFRINDMGWYRQAPGKHREMVAVETIDGSRNY GDSVKGRFTISRDSANDTVYLEMNNLKPEDTAVYYCNAARESGGRILWSYWGQGTQVTVSS (NB128) (SEQ ID NO: 43).

6. The composition of claim 1 , wherein the nanobody is conjugated to a diagnostic agent comprising a radionuclide, a contrast agent, a fluorescent agent, a chemiluminescent agent, a bioluminescent agent, a paramagnetic ion, an enzyme, or a photoactive diagnostic agent.

7. The composition of claim 6, wherein the radionuclide comprises89Zr,3H,11C,14C,18F,32P,35S,36CI,51Cr,52Fe,57Co,58Co,59Fe,64Cu,67Cu,67Ga,68Ga,75Se,76Br,77Br,89Zr,90Y, "mTc,1111n, 123| 124| , 1251, 131 1 , 152E u, 153S m, 166|_|O, 177[_u, 186Re, 188Re, 201T| , 203pb, 210At, 21 1At,212Bi,213Bi,149Tb,160Tb,161Tb, or225Ac.

8. The composition of claim 1 , wherein the nanobody is conjugated to desferrioxamine (DFO).

9. A nucleic acid molecule encoding any of the sequences of claims 2-5.

10. A composition, comprising a nanobody which is specific for and binds directly to a LAMP epitope, wherein the diseased state LAMP epitope is present in greater amounts in a diseased tissue than in a normal tissue, and wherein the nanobody is conjugated to an active agent.1 1 . The composition of claim 10, wherein the active agent is linked to N-terminus of the nanobody.

12. The composition of claim 10, wherein the active agent is linked to C-terminus of the nanobody.

13. The composition of any one of claims 10-12, wherein the active agent is an imaging probe.

14. The composition of claim 13, wherein the imaging probe is selected from fluorophores,immunohistochemical tracers, PET tracers, near-infrared (NIR) probes, single-photon emission computed tomography (SPECT) probes, magnetic particle imaging (MRI) probes, and radioisotopes.

15. The composition of any one of claims 10-14, wherein the nanobody comprises a sequence set forth in SEQ ID NOs: 1 -43 and their derivatives with a sequence identity to the original sequences.

16. A composition, comprising a peptide comprising a sequence set forth in SEQ ID NOs: 1 -43 or fragment thereof and a pharmaceutically acceptable carrier.

17. The composition of claim 16, wherein the peptide is conjugated to an active agent.

18. The composition of claim 16, wherein the peptide is a monoclonal antibody, a humanized antibody, a chimeric antibody, a human antibody, or antibody fragment.

19. The composition of claim 16, wherein the fragment thereof is a CDR.

20. A composition, comprising a nanobody comprising a sequence set forth in SEQ ID NOs: 1 -43 or fragment thereof and a pharmaceutically acceptable carrier.21 . The composition of claim 20, wherein the nanobody is conjugated to an active agent.

22. The composition of claim 20, wherein the nanobody is conjugated to a monoclonal antibody, a humanized antibody, a chimeric antibody, a human antibody, or other antibody fragment.

23. A method, comprising administering to a subject having a tumor or other disease state a composition of any one of claims 1 -8 and 10-22, in an effective amount to deliver the active agent to the tumor or to a region affected by the disease state.

24. The method of claim 23, wherein the method involves determining the presence or absence of one or more LAMP proteins characteristic of a diseased state in a subject and determining whether the subject has a disease.

25. The method of claim 24, wherein the presence or absence of one or more LAMP proteins is determined by imaging.

26. The method of claim 23, wherein the diseased state is a cancer.

27. The method of claim 26, wherein the cancer is breast or colon cancer.

29. The method of claim 24, wherein the LAMP proteins are detected using one or more nanobodies that specifically bind to the LAMP proteins.

30. A method, comprising administering to a subject having a tumor a composition of any one of claims 1 -8 and 10-22, wherein the nanobody comprises a detectable label, in an effective amount to bind to and identify tumor margins of the tumor.31 . The method of claim 30, wherein the nanobody specifically binds the diseased state LAMP epitope with a binding affinity in the nM range, as measured by surface plasmon resonance.

32. The method of claim 30, wherein the nanobody comprises a sequence set forth in SEQ ID NOs: 1 -43 and their derivatives with a sequence identity to the original sequences.

33. A method, comprising administering to a subject having a tumor or other disease state a composition of any one of claims 1 -8 and 10-22, wherein the nanobody is linked to an active agent, in an effective amount to deliver the active agent to the tumor or other disease site.

34. The method of claim 33, wherein the nanobody specifically binds the diseased state LAMP epitope with a binding affinity in the nM range, as measured by surface plasmon resonance.

35. The method of claim 33, wherein the nanobody comprises a sequence set forth in SEQ ID NOs: 1 -43 and their derivatives with a sequence identity to the original sequences.

36. Use of a nanobody of claim 4 or claim 5 in PET imaging.

37. Use of a nanobody of claim 4 or claim 5 in theranostics.

38. Use of a nanobody of any one of claims 1 -5 to treat LAMP-1 + cancers or to treat an inflammatory condition characterized by overexpression of LAMP-1 .

39. The use of claim 38, wherein the cancer is selected from the group consisting of breastcancer, colon cancer, esophageal cancer, stomach cancer, liver cancer, bladder cancer, pancreatic cancer, and prostate cancer.

40. The use of claim 38, wherein the nanobody binds to the same epitope as or competes for binding to LAMP-1 with an antibody that comprises a CDR sequence according to any one of claims 1 -5.41 . The use of claim 38, wherein the nanobody is conjugated to at least one diagnostic agent selected from the group consisting of a radionuclide, a contrast agent, a fluorescent agent, a chemiluminescent agent, a bioluminescent agent, a paramagnetic ion, an enzyme, and a photoactive diagnostic agent.

42. The use of claim 41 , wherein the diagnostic agent is a radionuclide comprising89Zr,3H,11C,14C,18F,32P,35S,36CI,51Cr,52Fe,57Co,58Co,59Fe,64Cu,67Cu,67Ga,68Ga,75Se,76Br,77Br,90Y, "mTc,111In,123l,124l,125l,131l,152Eu,153Sm,166Ho,177Lu,186Re,188Re,201TI,203Pb,210At,211At, 212Bi,213Bi,149Tb,160Tb,161Tb, or225Ac.

43. The use of claim 42, wherein the radionuclide is89Zr and the use further comprises PET imaging.

44. The use of claim 41 , wherein the nanobody is conjugated to an paramagnetic ion selected from the group consisting of chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III), and erbium (III).

45. Use of an anti-LAMP-1 antibody, antibody fragment, or nanobody for diagnostic, theranostic, or therapeutic purposes in the treatment of LAMP+ cancers and inflammatory conditions characterized by overexpression of LAMP-1 .