Fluorescently labeled antibodies

Abstract

Described herein are tumor-specific antibody and fluorophore conjugates for detecting, localizing and imaging of various tumors in fluorescence image-guided surgery. Also disclosed are methods for detecting, localizing and imaging a solid tumor before or during a tumor resection surgery using the antibody-fluorophore conjugates.

Description

[0001] Atorney Docket No.: 116437-1547609 (005WO1) FLUORESCENTLY LABELED ANTIBODIES

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 770,157, filed on March 11, 2025, which is incorporated herein by reference in its entirety.

[0003] REFERENCE TO A SEQUENCE LISTING SUBMITTED AS XML VIA EFS-WEB The instant application contains a Sequence Listing that has been filed electronically in .xml format and is hereby incorporated by reference in its entirety. Said .xml copy, created on March 10. 2026, is named CYH-005W01 2026 03 11 Sequence Listing.xml and is 29,163 bytes in size.

[0004] BACKGROUND

[0005] There is an unmet clinical need in the care of patients with certain ty pes of cancer for beter preoperative and intraoperative tumor imaging techniques. Furthermore, accurate preoperative staging of tumor remains a challenge for clinicians, as there remains a high rate of both local and distant recurrence after surgical resection. Improved techniques of delineating accurate tumor margins at the time of surgery7and, also identifying small volume metastatic disease, would help improve outcomes for patients with cancer.

[0006] SUMMARY

[0007] Disclosed herein are antibody-fluorophore conjugates with high fluorescence intensity7, tumor-labeling specificity7, and beneficial signal differentiation from background tissues as compared to conventional fluorophores. The disclosed technology7can be applied to cancer diagnosis, cancer prognosis, cancer treatment, tumor imaging, and imaging assisted surgery.

[0008] Accordingly, the present disclosure includes an antibody-fluorophore conjugate comprising a tumor-specific antibody conjugated to a near-infrared (NIR) fluorophore, wherein the NIR fluorophore is a modified heptamethine cyanine compound of the following structural formula:

[0009]

[0010] The present disclosure also includes a method of detecting a solid tumor in a subject, comprising: a) administering to the subject an antibody-fluorophore conjugate comprising a tumor-specific antibody conjugated to a near-infrared (NIR) fluoroph ore, wherein the NIR fluorophore is a modified heptamethine cyanine compound of the following structural formula:

[0011]

[0012] b) detecting the antibody after administration thereby detecting or localizing the solid tumor.

[0013] BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 shows the absorbance and emission spectra for M5A-IRDye800CW, M5A-Compound I, and M5A-Compound II in phosphate buffered saline (PBS).

[0015] Figure 2 shows images of vials containing serial dilutions of antibody fluorophore conjugates M5A-IRDye800CW, M5A-Compound I. and M5A-Compound II. Panel A shows images of the samples exposed to white light. Panel B shows images of the samples exposed to NIR fluorescence at 800 nm. Panel C shows images of the color-intensity overlay.

[0016] Figure 3 shows a graph of fluorescence intensity7versus protein concentration of antibody fluorophore conjugates M5A-IRDye800CW, M5A-Compound I, and MSA-Compound II at different concentrations of the antibody fluorophore conjugates.Figure 4 shows in-vivo images of nude mice bearing subcutaneous LS174-Luc (human colorectal cancer) treated with antibody fluorophore conjugates. Panel A shows the images using the M5A-IRDye800CW antibody fluorophore conjugate. Panel B shows images using the M5A-Compound I antibody fluorophore conjugate. Panel C shows images using the M5A-Compound II antibody fluorophore conjugate.

[0017] Figure 5 shows a graph of tumor background ratio versus time calculated from in-vivo images of nude mice bearing subcutaneous LS174-Luc (human colorectal cancer) treated with M5A-IRDye800CW, M5A-Compound I, and M5A-Compound II conjugates.

[0018] Figure 6A-B show graphs plotting the mean fluorescence intensity over time from in-vivo images of nude mice bearing subcutaneous LS174-Luc (human colorectal cancer) treated with M5A-IRDye800CW, M5A-Compound I, and M5A-Compound II conjugates. FIG. 6A, shows the mean fluorescence intensity of the tumor site over time. FIG. 8B, shows the mean fluorescence intensity of the background over time.

[0019] Figure 7 shows a graph of mean fluorescence intensity at all major organs demonstrating the biodistribution of M5A-IRDye800CW, M5A-Compound I, and MSA-Compound II conjugates in nude mice bearing subcutaneous LS174-Luc (human colorectal cancer).

[0020] Figure 8 shows near infrared (NIR) fluorescence images of nude mice bearing subcutaneous or orthotopic human colorectal cancer (LS174T-luc) treated with M5A-IRDye800CW, M5A-Compound I. and M5A-Compound II conjugates. Panel A shows fluorescence images acquired when optimizing for detection of IRDye800. Panel B shows fluorescence images acquired when optimizing for detection of indocyanine green (ICG). Panel C shows fluorescence images when optimizing for detection of lower wavelength dyes. All fluorescence images were quantified using ImageJ software.

[0021] Figure 9 shows a graph of mean fluorescence intensity over time collected from nude mice bearing subcutaneous or orthotopic human colorectal cancer (LS174T-luc) treated with M5A-IRDye800CW, M5A-Compound I, and M5A-Compound II conjugates by a Pearl Trilogy Animal Imager.

[0022] Figure 10 shows a graph of mean fluorescence intensity over time collected from nude mice bearing subcutaneous or orthotopic human colorectal cancer (LS174T-luc) treated with M5A-IRDye800CW, M5A-Compound I, and M5A-Compound II conjugates by a Indocyanine Green (ICG) laparoscope.

[0023] Figure 11 shows a graph of mean fluorescence intensity over time collected from nude mice bearing subcutaneous or orthotopic human colorectal cancer (LS174T-luc) treatedwith M5A-IRDye800CW, M5A-Compound I, and M5A-Compound II conjugates by a targetagent laparoscope.

[0024] Figure 12 shows a graph of tumor to background ratio versus time collected from in-vivo images of nude mice bearing subcutaneous or orthotopic human colorectal cancer (LS174T-luc) treated with M5A-IRDye800CW, M5A-Compound I, and M5A-Compound II conjugates by a Pearl Trilogy Animal Imager.

[0025] Figure 13 shows a graph of tumor to background ratio versus time calculated from in-vivo images of nude mice bearing subcutaneous or orthotopic human colorectal cancer (LS174T-luc) treated with M5A-IRDye800CW, M5A-Compound I, and M5A-Compound II conjugates by a Indocyanine Green (ICG) laparoscope.

[0026] Figure 14 shows a graph of tumor to background ratio versus time collected from in-vivo images of nude mice bearing subcutaneous or orthotopic human colorectal cancer (LS174T-luc) treated with M5A-IRDye800CW, M5A-Compound I, and M5A-Compound II conjugates by a target-agent laparoscope.

[0027] Figure 15 shows in-vivo near infrared (NIR) fluorescence images of Athymic mice bearing subcutaneous flank tumors at different timepoints after intravenous injection of anti-TAG72 (huCC49) antibody conjugated to Compound I. Panel A shows images for a Athymic mouse bearing an OV-90 subcutaneous flank tumor. Panel B shows images for a Athymic mouse bearing an ID8-ST+ subcutaneous flank tumor. Panel C shows images for a Athymic mouse bearing an 0VCAR3 subcutaneous flank tumor.

[0028] Figure 16 shows a graph of the mean fluorescence intensity over time for a Athymic mouse bearing the OV90-GFP SQ ovarian cancer cell line treated with anti-TAG72 (huCC49) antibody conjugated to Compound I relative to background.

[0029] Figure 17 shows a graph of the mean fluorescence intensity over time for a Athymic mouse bearing the ID8-mST-luc SQ ovarian cancer cell line treated with anti-TAG72 (huCC49) antibody conjugated to Compound I relative to background.

[0030] Figure 18 shows a graph of the mean fluorescence intensity over time for a Athymic mouse bearing the 0VCAR3-luc SQ ovarian cancer cell line treated with anti-TAG72 (huCC49) antibody conjugated to Compound I relative to background.

[0031] Figure 19 shows a graph of tumor to background ratios calculated from in-vivo near infrared (NIR) fluorescence images of Athymic mice bearing OV-90, ID8-ST+, and 0VCAR3 subcutaneous flank tumors at different timepoints after being treated with anti-TAG72 (huCC49) antibody conjugated to Compound I.Figure 20 shows images of tissue sections from nude mice bearing LS174T tumors after treatment with M5A-IRDye800CW, M5A-Compound I, and M5A-Compound II conjugates and analyzed by hematoxylin and eosin (H&E) and immunohistochemistry (IHC).

[0032] Figure 21 shows fluorescence images of nude mice bearing LS174T tumors and treated with M5A-IRDye800CW, M5A-Compound I, and M5A-Compound II conjugates. Column A, shows images of mice after treatment exposed to white light. Column B, shows images of mice after treatment exposed to Bio luminescence. Column C, shows images of mice after treatment exposed to merged NIR fluorescence at 800 nm. Column D, shows images of mice after treatment exposed to NIR fluorescence at 800 nm only.

[0033] DETAILED DESCRIPTION

[0034] Definitions

[0035] While various some aspects and aspects of the present invention are shown and described herein, such some aspects and aspects are provided by way of example only.

[0036] Numerous variations, changes, and substitutions will now7occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to some aspects described herein may be employed.

[0037] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular examples described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular examples only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0038] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subj ect matter described. All documents, or portions of documents, cited in the application including, without limitation, patents, patent applications, articles, books, manuals, and treatises are hereby expressly incorporated by reference in their entirety for any purpose.

[0039] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. See, e.g., Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 3nd ed., J. Wiley & Sons (New York, NY 2006); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 4thed., Cold Springs Harbor Press (Cold Springs Harbor, NY 2014). Any methods, devices and materials similar or equivalent to those described herein can be used. The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.The term “antibody” is used according to its commonly know n meaning in the art. An antibody molecule contains two identical pairs of polypeptide chains, each pair having one light chain and one heavy chain. Each light chain and heavy chain in turn consists of two regions: a variable (“V”) region, involved in binding the target antigen, and a constant (“C”) region that interacts with other components of the immune system. The light and heavy chain variable regions (also referred to herein as light chain v ariable (VL) domain and heavy chain variable (VH) domain, respectively) come together in 3-dimensional space to form a variable region that binds the antigen (for example, a receptor on the surface of a cell). Within each light or heavy chain variable region, there are three short segments called the complementarity determining regions (“CDRs”). The six CDRs in an antibody variable domain (three from the light chain and three from the heavy chain) fold up together in 3-dimensional space to form the actual antibody binding site which binds the target antigen. The position and length of the CDRs have been precisely defined by Kabat, E. et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Sen ices, 1983, 1987. The part of a variable region not contained in the CDRs is called the framework ("FR"), which forms the environment for the CDRs.

[0040] The terms variable light chain (VL), variable light chain (VL) domain or light chain variable region and variable heavy chain (VH), variable heavy chain (VH) domain or heavy chain variable region refer to these light and heavy chain regions, respectively. The terms variable light chain (VL), variable light chain (VL) domain and light chain variable region as referred to herein may be used interchangeably. The terms variable heavy chain (VH), variable heavy chain (VH) domain and heavy chain variable region as referred to herein may be used interchangeably.

[0041] An “antibody variant” as provided herein refers to a polypeptide capable of binding to an antigen and including one or more structural domains (e.g., light chain variable domain, heavy chain variable domain) of an antibody or fragment thereof. Non-limiting examples of antibody variants include single-domain antibodies or nanobodies, monospecific F(ab)2, bispecific F(ab)2. trispecific F(ab)2, monovalent IgGs, single chain variable fragments (scFv), membnane tethered agonist (mTa). bispecific antibodies, bispecific diabodies, trispecific triabodies, scFv- Fc, minibodies, IgNAR, V-NAR, hdgG, VhH, or peptibodies. Further non-limiting examples of antibody variants known in the art include antibodies produced by cartilaginous fish or camelids. A general description of antibodies from camelids and the variable regions thereof and methods for their production, isolation, and use may be found in references WO97 / 49805 and WO 97 / 49805 which are incorporated by reference herein in their entirety and for all purposes. Likewise, antibodies from cartilaginous fish andthe variable regions thereof and methods for their production, isolation, and use may be found in W02005 / 118629, which is incorporated by reference herein in its entirety and for all purposes.

[0042] The terms “CDR LI”, “CDR L2” and “CDR L3” as provided herein refer to the complementarity determining regions (CDR) 1, 2, and 3 of the variable light (L) chain of an antibody. The variable light chains provided herein can include in N-terminal to C- terminal direction a CDR LI, a CDR L2 and a CDR L3. Likewise, the terms "‘CDR Hl”, “CDR H2” and “CDR H3” as provided herein refer to the complementarity determining regions (CDR) 1, 2, and 3 of the variable heavy (H) chain of an antibody. The variable heavy chains provided herein can include in N-terminal to C-terminal direction a CDR Hl, a CDR H2 and a CDR H3.

[0043] The terms “FR LI”, “FR L2”, “FR L3” and “FR L4” as provided herein are used according to their common meaning in the art and refer to the framework regions (FR) 1, 2, 3 and 4 of the variable light (L) chain of an antibody. In some aspects, the variable light chain provided herein includes in N-terminal to C-terminal direction a FR LI, a FR L2, a FR L3 and a FR L4. Likewise, the terms “FR Hl”. “FR H2”, “FR H3” and “FR H4” as provided herein are used according to their common meaning in the art and refer to the framework regions (FR) 1, 2, 3 and 4 of the variable heavy (H) chain of an antibody. In some aspects, the variable heavy chain provided herein includes in N-terminal to C-terminal direction a FR Hl, a FR H2, a FR H3 and a FR H4.

[0044] A single-chain variable fragment (scFv) is typically a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker peptide of 10 to about 25 amino acids. The linker may usually be rich in glycine for flexibility, as well as serine or threonine for solubility. The linker can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. An scFv may be provided as a membrane-bound or membrane-tethered version comprising a hinge and / or transmembrane domain. For example, a membrane-tethered agonist (mTa) is a membrane bound scFv agonist (i.e., an scFv which binds to and induces activity of a target).

[0045] A “chimeric antibody” is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and / or species, or an entirely different molecule which confers new' properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, and the like; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.The term ‘‘humanized” and “humanized antibody” refers to an antibody containing structural elements of a human antibody (the acceptor) and the antigen binding site of a nonhuman antibody (the donor). “Humanized antibodies” contain a minimal number of residues from the non-human antibody. For instance, they may contain only the CDR regions of the non-human antibody, or only those residues that make up the hypen ariable regions of the non-human antibody. They may also contain certain residues from outside the variable regions of the non-human polypeptide, such as residues that are necessary to mimic the structure of the non-human antibody or to minimize steric interference. In addition, humanized antibodies may contain residues that do not correspond to either the human or the non-human antibodies.

[0046] For preparation of suitable antibodies of the invention and for use according to the invention, e.g., recombinant, monoclonal, or polyclonal antibodies, many techniques known in the art can be used (see, e.g., Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985); Coligan, Current Protocols in Immunology (1991); Harlow & Lane, Antibodies. A Laboratory Manual (1988); and Goding, Monoclonal Antibodies: Principles and Practice (2d ed. 1986)). The genes encoding the heavy and light chains of an antibody of interest can be cloned from a cell, e.g., the genes encoding a monoclonal antibody can be cloned from a hybridoma and used to produce a recombinant monoclonal antibody. Gene libraries encoding heavy and light chains of monoclonal antibodies can also be made from hybridoma or plasma cells. Random combinations of the heavy and light chain gene products generate a large pool of antibodies with different antigenic specificity7(see, e.g., Kuby, Immunology7(3rd ed. 1997)). Techniques for the production of single chain antibodies or recombinant antibodies (U.S. Patent 4,946.778, U.S. Patent No. 4,816.567) can be adapted to produce antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms such as other mammals, may be used to express humanized or human antibodies (see, e.g., U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633.425; 5,661,016, Marks et al., Bio / Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et al., Nature Biotechnology 14:845-51 (1996); Neuberger, Nature Biotechnology 14:826 (1996); and Lonberg & Huszar, Intern. Rev. Immunol. 13:65-93 (1995)). Alternatively, phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens (see, e.g.. McCafferty7et al., Nature 348:552-554 (1990); Marks et al., Biotechnology 10:779-783 (1992)). Antibodies can also be made bispecific, i.e., able to recognize two different antigens (see, e.g., WO 93 / 08829, Traunecker et al., EMBO J.10:3655-3659 (1991); and Suresh et al., Methods in Enzymology 121 :210 (1986)).

[0047] Antibodies can also be heteroconjugates, e.g., two covalently joined antibodies, or immunotoxins (see, e.g., U.S. Patent No. 4,676,980 , WO 91 / 00360; WO 92 / 200373; and EP 03089).

[0048] Methods for humanizing or primatizing non-human antibodies are well known in the art (e.g., U.S. PatentNos. 4,816,567; 5,530,101; 5,859,205; 5,585,089; 5,693,761;

[0049] 5.693,762; 5,777.085; 6,180.370; 6,210,671; and 6,329,511; WO 87 / 02671; EP Patent Application 0173494; Jones et al. (1986) Nature 321 :522; and Verhoyen et al. (1988) Science 239: 1534). Humanized antibodies are further described in, e.g., Winter and Milstein (1991) Nature 349:293. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers (see, e.g., Morrison et al., PNAS USA, 81 :6851- 6855 (1984), Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Morrison and Oi, Adv. Immunol., 44:65-92 (1988), Verhoeyen et al., Science 239: 1534- 1536 (1988) and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992), Padlan, Molec. Immun., 28:489- 498 (1991); Padlan, Molec. Immun., 31(3): 169-217 (1994)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Patent No. 4,816.567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non- human species. In practice, humanized antibodies are ty pically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. For example, polynucleotides comprising a first sequence coding for humanized immunoglobulin framew ork regions and a second sequence set coding for the desired immunoglobulin complementarity determining regions can be produced synthetically or by combining appropriate cDNA and genomic DNA segments. Human constant region DNA sequences can be isolated in accordance with well known procedures from a variety of human cells.

[0050] The term “fluorescence” refers to the phenomenon where a compound absorbs light at one w avelength and then emits light at a longer wavelength, i.e., it absorbs light of one color and emits light of a different color. The emission is due to the elections in the compound being excited to a higher energy state and then releasing that energy as light when returning to their ground state, a process that may happen quickly and only while the excitation light is present.The term “fluorophore” refers to a fluorescent compound that can absorb photons in its ground state, leading to a fluorescence emission and vibration relaxation in the excited state.

[0051] The term ‘"fluorescence image-guided surgery’7and “FIGS” refers to the combination of advanced imaging platforms with targeted fluorescent agents to enhance neoplasms and improve their intraoperative detection by the surgeon. One technique is to covalently bind an organic dye to a monoclonal antibody of a known tumor-specific antigen. As described in U.S. Patent No. 10.758,632 B2, which is incorporated herein by reference, one such tumor biomarker that is being developed preclinically is anti-carcinoembryonic antigen (CEA) antibodies to target and label human colorectal cancer in nude mouse models. Mouse and chimeric (mouse / human) antibodies against CEA have been strongly labeled with fluorescent dyes and may be capable of enhancing visualization of submillimeter tumor deposits and successful FIGS. IRDye800 can be broadly used in clinical setting with IR800 optical detection units. This technique has been applied to human clinical trials to localize squamous cell carcinoma of the head and neck using a cetuximab-IRDye800 conjugate.

[0052] The terms “surgical resection,” “resection” and “surgery” refers to a procedure that removes all or part of an organ or tissue.

[0053] The term “solid tumor” and “tumor” refers to an abnormal mass of tissue that forms when cells grow and divide more than they should or do not die when they should. Tumors may be benign (not cancer) or malignant (cancer). Benign tumors may grow large but do not spread into, or invade, nearby tissues or other parts of the body. Malignant tumors can spread into, or invade, nearby tissues. They can also spread to other parts of the body through the blood and lymph systems.

[0054] The term “radionuclide” refers to an unstable form of an atom with an excess of neutrons or protons in its nucleus which may cause it to spontaneously release radiation, e.g.. alpha, beta, or gamma rays, to become stable through a process called radioactive decay. The term “tumor-specific” refers to the targeting of a characteristic that is only present on cancer cells and not found on normal cells in the body or expressed at higher levels in malignant cells compared to normal cells of that tissue ty pe (i.e., epidermal growth factor receptor (EGFR). human epidermal growth factor receptor 2 (Her2). For example, the characteristic may be a particular molecule, protein, or antigen.

[0055] The terms “conjugate,” “conjugation,” or “conjugated” refer to being joined together. As may be appreciated by one of ordinary skill in the art, two or more compounds may be joined together by forming chemical bonds by sharing electrons (i.e., covalent bonding), or by transferring electrons between them (i.e., ionic bonding).The terms “chelating” or “chelating agent” refer to a complexer which builds a stable link between a radio-metal (radionuclide) and a carrier molecule.

[0056] The term “patient” or “subject” refers to a living organism suffering from or prone to a disease or condition that can be treated by administration of a composition or pharmaceutical composition as provided herein. Non-limiting examples include humans, other mammals, bovines, rats, mice, dogs, monkeys, goat, sheep, cows, deer, and other nonmammalian animals. In some aspects, a patient is human.

[0057] The terms “dose” and “dosage” are used interchangeably herein. A dose refers to the amount of active ingredient given to an individual at each administration. The dose will vary depending on a number of factors, including the range of normal doses for a given therapy, frequency of administration; size and tolerance of the individual; severity of the condition; risk of side effects; and the route of administration. One of skill will recognize that the dose can be modified depending on the above factors or based on therapeutic progress. The term “dosage form” refers to the particular format of the pharmaceutical or pharmaceutical composition, and depends on the route of administration. For example, a dosage form can be in a liquid form for nebulization, e.g., for inhalants, in a tablet or liquid, e.g., for oral delivery, or a saline solution, e.g., for injection.

[0058] The term “administering” as used herein means oral administration, administration as a suppository, topical contact, intravenous, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini- osmotic pump, to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intraarteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc. By “co-administer” it is meant that a composition described herein is administered at the same time, just prior to, or just after the administration of one or more additional therapies, for example cancer therapies such as chemotherapy, hormonal therapy, radiotherapy, or immunotherapy. The compounds can be administered alone or can be co-administered to a subject. Co-administration is meant to include simultaneous or sequential administration of the compounds individually or in combination (more than one compound). Thus, the preparations 205 10 152025 30 can also be combined, when desired, with other active substances (e.g. to reduce metabolic degradation). The compositions of the present invention can be delivered by transdermally.by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.

[0059] The compositions of the present invention may additionally include components to provide sustained release and / or comfort. Such components include high molecular weight, anionic mucomimetic polymers, gelling polysaccharides and finely-divided drug carrier substrates. These components are discussed in greater detail in U.S. Pat. Nos. 4,911,920; 5.403,841; 5,212.162; and 4,861,760. The compositions of the present invention can also be delivered as microspheres for slow release in the body. For example, microspheres can be administered via intradermal injection of drug-containing microspheres, which slowly release subcutaneously (see Rao, J. Biomater Sci. Polym. Ed. 7:623-645, 1995; as biodegradable and injectable gel formulations (see, e.g.. Gao Pharm. Res. 12:857-863, 1995); or, as microspheres for oral administration (see, e.g., Eyles, J. Pharm. Pharmacol. 49:669-674, 1997). In some aspects, the formulations of the compositions of the present invention can be delivered using liposomes which fuse with the cellular membrane or are endocytosed, i.e., by employing receptor ligands attached to the liposome, that bind to surface membrane protein receptors of the cell resulting in endocytosis. By using liposomes, particularly where the liposome surface carries receptor ligands specific for target cells, or are otherwise preferentially directed to a specific organ, one can focus the delivery of the compositions of the present invention into the target cells in vivo. (See, e.g., Al -Muhammed, J.

[0060] Microencapsul. 13:293-306, 1996; Chonn. Cnrr. Opin. Biotechnol. 6:698-708, 1995; Ostro, Am. J. EIosp. Pharm. 46: 1576-1587, 1989). The compositions of the present invention can also be delivered as nanoparticles.

[0061] As used herein, the term “pharmaceutically acceptable” is used synonymously with “physiologically acceptable” and “pharmacologically acceptable”. A pharmaceutical composition will generally comprise agents for buffering and preservation in storage, and can include buffers and carriers for appropriate delivery, depending on the route of administration.

[0062] “Pharmaceutically acceptable excipient” and “pharmaceutically acceptable carrier” refer to a substance that aids the administration of an active agent to and absorption by a subject and can be included in the compositions of the present invention without causing a significant adverse toxicological effect on the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, lactated Ringer’s, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer's solution), alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethycellulose,polyvinyl pyrrolidine, and colors, and the like. Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and / or aromatic substances and the like that do not deleteriously react with the compounds of the invention. One of skill in the art will recognize that other pharmaceutical excipients are useful in the present invention.

[0063] I. Antibodv-Fluorophore Conjugates and Compositions

[0064] Fluorescence image-guided surgery (FIGS) using tumor-specific probes can enhance the effectiveness of oncologic surgeries. One ty pe of tumor-specific probes is an antibody conjugated to a fluorescent dye, e.g.. a fluorophore. The choice of fluorophore can impact the overall pharmacokinetic properties of the final molecule, affecting the clinical applicability and success of the probes.

[0065] The present disclosure provides fluorophore-conjugated tumor-specific antibodies such as anti-carcinoembryonic antigen (anti-CEA) antibodies and / or anti-tumor-associated glycoprotein 72 (anti-TAG-72) antibodies and / or anti-prostate-specific membrane antigens (anti-PSMA) for detection and surgical resection of cancers overexpressing these antigens. The disclosed fluorophore-antibody conjugates demonstrate improved contrast for fluorescence guided oncologic surgeries.

[0066] The antibody -fluorophore conjugates can include a tumor-specific antibody conjugated to a near-infrared (NIR) fluorophore, wherein the NIR fluorophore may be a modified heptamethine cyanine compound of the following structural formula:

[0067]

[0068]

[0069] The choice of fluorophore can significantly impact the overall pharmacokinetic properties of the final molecule, which in turn can affect the clinical applicability and success of the agent. LI-COR's IRDye800CW (LI-COR Environmental, Lincoln, Nebraska) is the most widely utilized NIR dye in combination with monocolonal antibodies (mAbs) in preclinical and clinical studies (e.g., Cetuximab-IRDye800, and Panitumumab-IRDye800, Bevacizumab-IRDye800, Trastuzumab-IRDye800). The IRDye800 molecule is extremely hydrophobic. In-vivo, this leads to aggregation that results in significant liver accumulation and rapid serum elimination as the degree of labeling (DOL) increases. This restricts the potential to attach more fluorophores, limiting the overall brightness of the molecule.

[0070] Beneficially, the modified heptamethine cyanine dyes described herein were designed with the placement of a quaternary amine at the central carbon and triethylene glycol substituents on the indolenine positions. The inventors surprisingly and unexpectedly discovered these structural changes result in a linear increase in fluorescence emission as a function of labeling density (up to DOL 5-6) with no evidence of aggregation when conjugated to other targeting probes. In-vivo studies with the anti-carcinoembryonic antigen (anti-CEA) M5A mAb conjugated to these dyes has demonstrated improved tumor targeting with enhanced fluorescence signal intensity, decreased background noise, and liver uptake.

[0071] In some aspects, the present disclosure provides antibody-fluorophore conjugates, wherein the modified heptamethine cyanine compound may comprise one of the follow structural formulas:

[0072]

[0073] Compound I Compound II

[0074] In some aspects, the antibody-fluorophore conjugates disclosed herein may comprise a tumor-specific antibody that may be targeted markers that specifically bind to antigens present on tumor cells. For example, the tumor-specific antibodies may allow for precise visualization and identification of tumors within a tissue sample via imagine techniques as disclosed herein.

[0075] Anti-carcinoembryonic antigen (anti -CEA) antibodies and humanized anti-CEA antibodies are disclosed in, e.g., U.S. Pat. Nos. 7,776,330 and 7,273,608, the entire contents of which are incorporated herein by reference. These radionuclide labeled antibodies exhibit high levels of binding affinity and specificity for CEA. Moreover, the humanized antibody has exhibited the ability to specifically target tumors that express CEA in-vivo, making it a potentially powerful tool for the detection and treatment of such tumors. In some embodiments, the humanized anti-CEA T84.66-M5A antibody disclosed in the above-mentioned US Patents can be used. It is within the purview of one skilled in the art to make necessary modification of this antibody, for example, to facilitate the antibody conjugating to a suitable fluorophore without significantly lowering the binding affinity and specificity for CEA and without significantly decreasing the ability to specifically target CEA-expressing tumors in vivo. In some embodiments, modified humanized anti-CEA antibodies or fragments thereof can be used as long as the binding affinity and specificity for CEA in vivo are not significantly compromised.

[0076] The methods and technology7disclosed herein can be carried out by using any tumorspecific antibody which may be conjugated to a fluorophore, labeled with a radioactive isotope, or both. Depending on the specific tumor type or the specific biomarker for the tumor, one can choose a suitable antibody for detection and localization of the tumor. Such antibodies may include but are not limited to anti-Carcinoembryonic antigen (anti-CEA)antibodies, anti-tumor-associated glycoprotein 72 (anti-TAG-72) antibodies, and anti-prostate-specific membrane antigen (anti-PSMA) antibodies. It is within the purview of one of ordinary skill in the art to use humanized antibodies or monoclonal antibodies to maximize the specific binding and tumor detection thereby optimizing the tumor imaging effects. In some aspects, the tumor-specific antibody may be a humanized anti-CEA T84.66-M5A antibody. The tumor-specific antibody may be a humanized anti-TAG-72 huCC49 antibody. The tumor-specific antibody may be a humanized anti-PSMA J591 antibody.

[0077] Specificity may be influenced by the heavy chain and light chain variable domains as they form the antigen-binding site of the antibody, i.e., the specific regions that recognize and bind to the tumor tissues. In some aspects, the antibody-fluorophore conjugates disclosed herein may comprise a tumor-specific antibody comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO: 1, a CDR H2 as set forth in SEQ ID NO:2, and a CDRH3 as set forth in SEQ ID NO:3; and wherein the light chain variable domain comprises: a CDR LI as set forth in SEQ ID NO:4, a CDR L2 as set forth in SEQ ID NO: 5, and a CDR L3 as set forth in SEQ ID NO:6, see Table 1. Optionally, the heavy chain variable domain comprise SEQ ID NO:25 and the light chain variable domain comprises SEQ ID NO:26. Optionally, the antibody-fluorophore conjugates may comprise a tumor-specific antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises: SEQ ID NO: 19; and wherein the light chain comprises: SEQ ID NO:20 which are as described in the informal sequence listing.

[0078] Table 1. CDRs of Anti-CEA M5A Antibody

[0079]

[0080] In some aspects, the antibody-fluorophore conj ugates may comprise a tumor-specific antibody comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO:7, a CDR H2 as set forth in SEQ ID NO:8, and a CDRH3 as set forth in SEQ ID NO:9; and wherein the light chain variable domain comprises: a CDR LI as set forth in SEQ ID NO: 10,a CDR L2 as set forth in SEQ ID NO: 11, and a CDR L3 as set forth in SEQ ID NO: 12, see Table 2. Optionally, the heavy chain variable domain comprises SEQ ID NO:27 and the light chain variable domain comprises SEQ ID NO:28. Optionally, the antibody-fluorophore conjugates may comprise a tumor-specific antibody comprises a heavy chain and alight chain, wherein the heavy chain comprises: SEQ ID NO:21; and wherein the light chain comprises: SEQ ID NO:22, which are as described in the informal sequence listing.

[0081] Table 2. CDRs of anti-TAG-72 huCC49 Antibody

[0082]

[0083] In some aspects, the antibody-fluorophore conjugates may comprise a tumor-specific antibody comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO: 13, a CDR H2 as set forth in SEQ ID NO: 14, and a CDRH3 as set forth in SEQ ID NO: 15; and wherein the light chain variable domain comprises: a CDR LI as set forth in SEQ ID NO: 16, a CDR L2 as set forth in SEQ ID NO: 17. and a CDR L3 as set forth in SEQ ID NO: 18, see Table 3. Optionally, the antibody-fluorophore conjugates may comprise tumorspecific antibody comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises: SEQ ID NO:23; and wherein the light chain variable domain comprises: SEQ ID NO:24, which are as described in the informal sequence listing. Optionally, the antibody-fluorophore conjugates may comprise a tumor-specific antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises: SEQ ID NO:30; and wherein the light chain comprises: SEQ ID NO:29, which are as described in the informal sequence listing.

[0084] Table 3. CDRs of anti-PSMA J591 Antibody

[0085]

[0086]

[0087] In some aspects, the antibody-fluorophore conjugates described herein may comprise a tumor-specific antibody which may be further labeled with a radionuclide. For example, the radionuclide may comprise fluorine-18 (F-18), phosphorus-32 (P-32), phosphorus-33 (P-33), titanium-45 (Ti-45), scandium-47 (Sc-47), iron-52 (Fe-52), iron-59 (Fe-59), copper-62 (Cu-62). copper-64 (Cu-64), copper-67 (Cu-67), gallium-67 (Ga-67), gallium-68 (Ga-68). arsenic-77 (As-77), yttrium-86 (Y -86), yttrium-90 (Y-90), zirconium-89 (Zr-89), strontium-89 (Sr-89), technetium-94 (Tc-94), technetium-95 (Tc-95), technetium-99m (Tc-99m), molybdenum-99 (Mo-99), palladium-105 (Pd-105), rhodium-105 (Rh-105), silver-111 (Ag-111), indium-ill (In-111), iodine- 123 (1-123), iodine- 124 (1-124), iodine- 125 (1-125), iodine-131 (1-131). praseodymium-142 (Pr-142), praseodymium- 143 (Pr-143), promethium-149 (Pm-149), samarium-153 (Sm-153), terbium-161 (Tb-161), dysprosium- 166 (Dy-166), holmium- 166 (Ho- 166), erbium- 169 (Er- 169), lutetium- 175 (Lu- 175), lutetium- 177 (Lu- 177), rhenium-186 (Re-186), rhenium-188 (Re-188), rhenium-189 (Re-189), iridium-194 (Ir-194), gold-198 (Au-198), gold-199 (Au-199), astatine-211 (At-211), lead-203 (Pb-203), lead-211 (Pb-211), lead-212 (Pb-212), bismuth-212 (Bi-212), bismuth-213 (Bi-213), radium-223 (Ra-223), actinium-225 (Ac-225), or combinations thereof. In some aspects, the radionuclides may be zirconium-89 (Zr-89), copper-64 (Cu-64), indium-111 (In-111), technetium-99m (Tc-99m), or combinations thereof.

[0088] In some aspects, the radionuclide may be attached to the tumor-specific antibody via a chelating agent. For example, the chelating agent may comprise 1,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid (DOTA), desferrioxamine B (DFO),diethylenetriaminepentaacetic acid (DTP A), ethylenediamine-di (O-hydroxyphenylacetic acid) (EDHPA), 2,2' dipyridyl (DIPY). l,4,7-lriazacyclononane-l,4,7-triacetic acid (NOTA), and tri ethylenetetramine (TETA). In some aspects, the chelating agent is 1,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid (DOTA).

[0089] A pharmaceutical composition including an antibody -fluorophore conjugate provided herein and a pharmaceutically acceptable excipient is also provided. Pharmaceutical compositions of the antibody-fluorophore conjugates can be prepared in accordance with methods well known and routinely practiced in the art. See, e.g., Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20th ed., 2020; and Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York. 1978. Pharmaceutical compositions are preferably manufactured under GMP conditions.

[0090] Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, or acetate at a pH typically of 5.0 to 8.0, most often 6.0 to 7.0; salts such as sodium chloride, potassium chloride, etc. to make isotonic; antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers such as polysorbate 80, amino acids such as glycine, carbohydrates, chelating agents, sugars, and other standard ingredients known to those skilled in the art (Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20th ed.. 2020).

[0091] A pharmaceutical composition including an antibody or antibody variant (e.g., scFv) as described herein can be administered by a variety of methods known in the art. The route and / or mode of administration vary depending upon the desired results. In some aspects, administration is intravenous, intramuscular, intraperitoneal, or subcutaneous, or administered proximal to the site of the target. Pharmaceutically acceptable excipients can be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).

[0092] II. Fluorescence Image-Guided Surgery (FIGS)

[0093] Fluorescence image-guided surgery’ (FIGS) combines advanced imaging platforms with targeted fluorescent agents to enhance neoplasms and improve their intraoperative detection by the surgeon. One technique may be to covalently bind organic dye to a monoclonal antibody of a know n tumor-specific antigen.

[0094] In some aspects, the present disclosure relates to a method of detecting a solid tumor in a subject, comprising: a) administering to the subject an antibody-fluorophore conjugate comprising a tumor-specific antibody conjugated to a near-infrared (NIR) fluorophore,wherein the NIR fluorophore is a modified heptamethine cyanine compound of the following structural formula:

[0095]

[0096] b) detecting the antibody after administration thereby detecting or localizing the solid tumor.

[0097] In some aspects, the method described herein may comprise a modified heptamethine cyanine compound of the follow structural formulas:

[0098]

[0099] The success of a curative surgery for cancer may be dependent on the complete removal of all cancer cells. Increasing the surgeon's ability to detect cancer before and during the operation has the dual benefit of identification of disease that can be fully resected and recognizing locally advanced disease that cannot. FIGS has the potential to advance the ability to recognize where an individual's disease is rather than relying on an understanding of where it most likely should be. With applications in staging laparoscopy, tumor localization, real-time assessment of margins during FIGS, and surveillance for local recurrence, fluorescence tumor labeling is an essential tool for improving outcomes in surgical oncology. Tumor visualization by the surgeon can be enhanced through fluorescent antibody targeting.

[0100] In some aspects, the method described herein comprises a tumor-specific antibody. The tumor-specific antibody may be a humanized anti-carcinoembiyonic antigen (anti-CEA) T84.66-M5A antibody. The tumor-specific antibody may be a humanized anti-tumor-associated glycoprotein 72 (anti-TAG-72) huCC49 antibody. The tumor-specific antibody may be a humanized anti-prostate-specific membrane antigen (anti-PSMA) J591 antibody.

[0101] Patient outcome following surgical resection of a tumor may be enhanced by increased specificity of tumor-specific antibodies i.e., enabling a surgeon to distinguish tumor tissue from healthy tissue. In some aspects, the antibody-fluorophore conjugates disclosed herein may comprise a tumor-specific antibody comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO: 1, a CDR H2 as set forth in SEQ ID NO:2, and a CDRH3 as set forth in SEQ ID NO:3; and wherein the light chain variable domain comprises: a CDR LI as set forth in SEQ ID NO:4, a CDR L2 as set forth in SEQ ID NO:5, and a CDR L3 as set forth in SEQ ID NO:6, see Table 1. Optionally, the heavy chain variable domain compriseSEQ ID NO:25 and the light chain variable domain comprises SEQ ID NO:26. Optionally, the antibody -11 uorophore conjugates may comprise a tumor-specific antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises: SEQ ID NO: 19; and wherein the light chain comprises: SEQ ID NO:20 which are as described in the informal sequence listing.

[0102] In some aspects, the antibody-fl uorophore conjugates may comprise a tumor-specific antibody comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO:7, a CDR H2 as set forth in SEQ ID NO: 8, and a CDRH3 as set forth in SEQ ID NO: 9; and wherein the light chain variable domain comprises: a CDR LI as set forth in SEQ ID NO: 10, a CDR L2 as set forth in SEQ ID NO: 11, and a CDR L3 as set forth in SEQ ID NO: 12. see Table 2. Optionally, the heavy chain variable domain comprises SEQ ID NO:27 and the light chain variable domain comprises SEQ ID NO:28. Optionally, the antibody-fluorophore conjugates may comprise a tumor-specific antibody comprises a heavy chain and alight chain, wherein the heavy chain comprises: SEQ ID NO:21; and wherein the light chain comprises: SEQ ID NO:22, which are as described in the informal sequence listing.

[0103] In some aspects, the antibody-fluorophore conjugates may comprise a tumor-specific antibody comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO: 13, a CDR H2 as set forth in SEQ ID NO: 14, and a CDRH3 as set forth in SEQ ID NO: 15; and wherein the light chain variable domain comprises: a CDR LI as set forth in SEQ ID NO: 16, a CDR L2 as set forth in SEQ ID NO: 17, and a CDR L3 as set forth in SEQ ID NO: 18, see Table 3. Optionally, the antibody-fluorophore conjugates may comprise tumorspecific antibody comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises: SEQ ID NO:23; and wherein the light chain variable domain comprises: SEQ ID NO:24, which are as described in the informal sequence listing. Optionally, the antibody-fluorophore conjugates may comprise a tumor-specific antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises: SEQ ID NO:30; and wherein the light chain comprises: SEQ ID NO:29, which are as described in the informal sequence listing.

[0104] Tumor identification may be enhanced by labeling tumor-specific antibodies with radionuclides. Radionuclides may allow surgeons to visualize that exact location of a tumor, i.e., allowing for highly targeted detection and thereby providing crucial information for the diagnosis and treatment of patients. In some aspects, the antibody-fluorophore conjugates may comprise a tumor-specific antibody which may be further labeled with a radionuclide.For example, the radionuclide may comprise fluorine-18 (F-18), phosphorus-32 (P-32), phosphorus-33 (P-33), titanium-45 (Ti-45), scandium-47 (Sc-47), iron-52 (Fe-52), iron-59 (Fe-59), copper-62 (Cu-62), copper-64 (Cu-64), copper-67 (Cu-67). gallium-67 (Ga-67), gallium-68 (Ga-68), arsenic-77 (As-77), yttrium-86 (Y-86), ytrium-90 (Y-90), zirconium-89 (Zr-89), strontium-89 (Sr-89), technetium-94 (Tc-94), technetium-95 (Tc-95), technetium-99m (Tc-99m), molybdenum-99 (Mo-99), palladium-105 (Pd-105), rhodium-105 (Rh-105), silver-111 (Ag-111), indium-i ll (In-111), iodine-123 (1-123), iodine-124 (1-124), iodine-125 (1-125), iodine-131 (1-131), praseodymium- 142 (Pr-142), praseodymium- 143 (Pr-143), promethium- 149 (Pm-149), samarium-153 (Sm-153), terbium-161 (Tb-161), dysprosium- 166 (Dy-166), holmium-166 (Ho-166), erbium-169 (Er-169), lutetium-175 (Lu-175), lutetium-177 (Lu-177), rhenium-186 (Re-186), rhenium-188 (Re-188), rhenium-189 (Re-189), indium-194 (Ir-194), gold-198 (Au-198), gold-199 (Au-199), astatine-211 (At-211), lead-203 (Pb-203), lead-211 (Pb-211), lead-212 (Pb-212), bismuth-212 (Bi-212), bismuth-213 (Bi-213), radium-223 (Ra-223), actinium-225 (Ac-225), or combinations thereof. In some aspects, the radionuclides may be zirconium-89 (Zr-89), copper-64 (Cu-64), indium- 111 (In-111), technetium-99m (Tc-99m). or combinations thereof.

[0105] In some aspects, the radionuclide may be attached to the tumor-specific antibody via a chelating agent. For example, the chelating agent may be selected from, including, but not limited to, l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid (DOTA), desferrioxamine B (DFO), diethylenetriaminepentaacetic acid (DTP A), ethylenediamine-di (O-hydroxyphenylacetic acid) (EDHPA), 2,2' dipyridyl (DIPY), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), and tri ethylenetetramine (TETA). In some aspects, the chelating agent is l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid (DOTA).

[0106] In some aspects, the solid tumor may be cancer. In some aspects, the cancer may be colorectal cancer, pancreatic cancer, gastric cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, and / or thyroid cancer. The cancer may be colorectal cancer. The cancer may be ovarian cancer.

[0107] The humanized tumor-specific antibody such as an anti-CEA, anti-T AG-72, or anti-PSMA antibody can be conjugated to a variety of labels, particularly fluorescent labels and / or radioactive labels, and administered to a subject for detection or localization of a tumor expressing CEA, TAG-72, or PSMA by subcutaneous, peritoneal, intravenous, intravascular, intramuscular, intradermal or transdermal injection, among other methods. These labels maybe linked to the humanized antibodies by covalent binding, affinity binding, intercalation, coordinate binding, or complexation, among other methods.For detection and localization of tumors expressing CEA, TAG-72, or PSMA, the humanized antibody conjugate disclosed herein may be administered at a dose sufficient for detection by a scanning device. In some aspects, the dose may be about 1 mg to about 60 mg per human dose. This dosage will be dependent on the type of label being used. The type of scanning device to be used will vary depending on the label being used. For detection of a tumor expressing CEA, TAG-72, or PSMA, the humanized antibody conjugate disclosed herein may be prepared as a formulation within pharmaceutically acceptable media. This formulation may include physiologically tolerable liquids, gels, solid carriers, diluents, adjuvants, or excipients, or some combination thereof.

[0108] In some aspects, the method relates to detecting the antibody-fluorophore conjugates using a scanning device. Detecting may comprise detecting the antibody-fluorophore conjugate using a scanning device to identify tumors. Detecting may comprise imaging a tumor to direct surgical resection of a tumor. Detecting may be done before surgery.

[0109] Detecting may be done during surgery7. Detecting may be done before and during surgery7. In some aspects, the scanning may be done before surgery to detect the radionuclide and during surgery to detect the fluorophore. In some aspects, the scanning device may be a PEARL® Near-Infrared Small Animal Imager (LI-COR Environmental, Lincoln, Nebraska), a STRYKER® 1688 Advanced Imagining Modalities (AIM) 4K Platform (Stry ker, Kalamazoo, Michigan), a STRYKER® 1788 Platform for Minimally Invasive Surgery (Stryker, Kalamazoo. Michigan), or combinations thereof.

[0110] The technology disclosed herein may increase a surgeon's ability to detect and localize cancer during the operation, and therefore it has the dual benefit of identification of disease that can be fully resected and recognizing locally advanced disease that cannot. FIGS allows precise localization of an individual's disease rather than rely ing on an understanding of where it most likely should be. With applications in staging laparoscopy, tumor localization, real-time assessment of margins during FIGS, and surveillance for local recurrence, fluorescence tumor labeling is an essential tool for improving outcomes in surgical oncology.

[0111] The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.

[0112] Thus, as will be apparent to those of skill in the art upon reading this disclosure, each of the individual examples described and illustrated herein has discrete components andfeatures which may be readily separated from or combined with the features of any of the other several examples without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0113] Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference in their entireties.

[0114] EXAMPLES

[0115] Example 1 : Enhanced Fluorescence of Anti-CE A Antibodies for Visualizing Colorectal Cancers using M5A Antibody Fluorophore Conjugates

[0116] Fluorescence image-guided surgery (FIGS) using tumor-specific probes may enhance the effectiveness of oncologic surgeries. The choice of fluorophore may impact the overall pharmacokinetic properties of the final molecule, affecting the clinical applicability and success of the probes. As disclosed herein, two modified heptamethine cyanine dyes were identified which, when linked to a humanized antibody that targets carcinoembryonic antigen (“anti-CEA”), improve tumor uptake, fluorescence intensity, and contrast for colorectal cancer visualization.

[0117] The humanized anti-CEA hT84.66-M5A (M5A) monoclonal antibody (mAb) was conjugated to the following dyes: blank, i.e., the line with a peak indicated at 280 nm (FIG.

[0118] 1); IRDye® 800CW (“IRDye800CW”) from LI-CORbio™ (LI-COR Environmental, Lincoln, Nebraska), i.e., the line with a peak indicated at 780 nm (excitation 774 nm, emission 789 nm; FIG. 1 ); Compound I, i.e, the line with a peak indicated at 766 (excitation 766 nm; emission 788 nm; FIG. 1 ); and Compound II, i.e., the line with a peak indicated at 804 (excitation 804 nm, emission 830 nm; FIG. 1 ). Conjugation was performed at a 10 to 40-fold molar excess of fluorophore. The product was purified using size exclusion high performance liquid chromatography (SEC-HPLC) using a next-generation chromatography (NGC) system controlled and recorded using ChromLab software (Bio-Rad ) connected to a Superdex 200 10 / 300 column (Cytiva, Wilmington, DE).

[0119] The antibody -fluorophore conjugates were purified by diafiltration using an Amicon ultrafiltration stirred cell (Millipore) equipped with a 30 kDa molecular weight cut-off membrane. Following buffer exchange with 15-20 diavolumes of phosphate buffered saline (PBS, pH 7.2), the antibody was recovered and sterile-filtered. The protein concentration and degree of labeling (DOL) w ere determined via absorbance spectrophotometry using established equations (Equations 1 and 2). Antibody aggregation and the presence of free dye (i.e., fluorophore) were assessed by high-performance liquid chromatography-size exclusion chromatography (HPLC-SEC) using a Superdex 200 column (Cytiva). The NGCchromatography system (Bio-Rad) monitored absorbance at 280 nm and 774 nm and was equipped with an external fluorescence detector (FP4020, Jasco Inc., Easton, MD, USA) set to the peak excitation and emission wavelengths for each conjugate. The DOL was calculated using the equation:

[0120] DOL = (A(Dye max) X 8(Ab)) / [S(Dye)x(A(280) - Correction Factor X A(Dyemax)]) Equation 1,

[0121] The antibody concentration (mg / mL) was determined using the equation:

[0122] Concentration (mg / ml) = Dilution Factor x MW(Ab) x [(A<280) - Correction Factor x A(Dye max )) / s(Dye)] Equation 2,

[0123] where MW(Ab) = 150,000, A(Dyemax) corresponds to the absorption maxima for each dye (766 nm for Compound I, 780 nm for IRDye800CW, and 804 nm for Compound II), £<Ab) = 210,000

[0124]

[0125] 8(CompoundI) = 188,000, 8(FNIR800CW) = 270,000, 8(Compound II) = 125,000. The correction factor used was 0.03 for Compound I and IRDye800CW, and 0.1 for Compound II.

[0126] The fluorescence properties of the dyes, i.e., Labs - wavelength absorption, Lem -wavelength emission, 8 - extinction coefficient, and <T>i - quantum yield are shown in Table 1.

[0127] Table 1.

[0128]

[0129] To determine baseline fluorescence in-vitro imaging of the antibody fluorophore conjugates were obtained at different concentrations. Aliquots of M5A-IRDye800CW, M5A Compound I, and M5A Compound II conjugates were serially diluted with phosphate buffered saline (PBS) from 1.5 mg / mL to 1.0 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.12 mg / mL, 0.06 mg / mL, 0.03 mg / mL, and 0.0015 mg / mL. Using the Pearl® Near-Infrared Small Animal Imager from LI-CORbio™ (“Pearl Trilogy Animal Imager’') (LI-COR Environmental, Lincoln, Nebraska), images were recorded in white light mode FIG. 2, Panel A; NIR fluorescence at 800 nm FIG. 2, Panel B; and a color intensity overlay FIG. 2, Panel C. Visually, the M5A-Compound I conjugate showed the brightest fluorescence signal at allconcentrations, see FIG. 2, Panel A-C. Additionally, this finding was further demonstrated by quantification of fluorescence comparing fluorescence intensity to protein concentration, FIG. 3. As demonstrated in FIG. 3, the M5A-Compound I conjugate provided nearly a tenfold increase in fluorescence over the M5A-IRDye800CW conjugate. In contrast, the MSA-Compound II conjugate demonstrated comparable fluorescence intensity to the M5A-IRDye800CW conjugate. The reduced fluorescence of Compound II compared to Compound I may be due to the scanning device used, i.e., the Pearl Trilogy Animal Imager. For example, the Pearl Trilogy Animal Imager with an 800 nm filter (excitation 785 nm / emission 820 nm) is designed specifically for the IRDye800CW (excitation 775 nm, emission 796 nm), while compound II (excitation 804 nm, emission 830 nm) has fluorescence properties similar to ICG. The small wavelength differences may have resulted in significant fluorescence intensity differences.

[0130] To analyze the in-vivo activity, 50 pg of the M5A-fluorophore conjugates were intravenously administered to nude mice bearing subcutaneous LS174-Luc (human colorectal cancer) tumors. As shown in FIG. 4, serial NIR fluorescent images of the mice were obtained using the Pearl Trilogy Animal Imager, at the following time points: 4 hours, 24 hours. 48 hours, and 72 hours. Based on the measured absorption, emission, extinction coefficient, and quantum yield, M5A-IRDye800CW would be expected to have the highest brightness. In vivo data demonstrates that compound I had a significantly brighter signal and compound II had a moderately brighter signal. For example, data shows the M5A-Compound I conjugate (FIG.

[0131] 4, Panel B) demonstrated significantly increased fluorescence at the tumor site, while MSA-Compound II conjugate (FIG. 4, Panel C) demonstrated moderately improved fluorescence as compared to the M5A-IRDye800CW conjugate (FIG. 4, Panel A). This demonstrates the beneficial and unexpected tumor labeling of the described conjugates.

[0132] Generally, attempts to increase fluorescence intensity over the tumor result in an increase in fluorescence intensity at the background, i.e., no improvement in contrast.

[0133] Surprisingly, the M5A-Compound I and Compound II conjugates achieve both increased fluorescence intensity and contrast. For example, tumor background analyses demonstrated over a two-fold increase in tumor to background ratio over time, FIG. 5, and highest mean fluorescence intensity, FIG. 6, for the M5A-Compound I conjugate as compared to the M5A-IRDye800CW conjugate. In contrast to the in-vitro results, the M5A-Compound II conjugate demonstrated comparable to improved maximal fluorescence intensity at the tumor (FIG. 4, Panel C) as compared to the M5A-IRDye800CW conjugate (FIG. 4, Panel A) while also providing a significant one-to-two-fold increase in contrast in tumor to background ratioFIG. 5. This demonstrates the described conjugates provide beneficial tumor labeling as compared to conventional dyes.

[0134] The cause of the increased in-vivo fluorescence activity was investigated by analyzing the pharmacokinetics of the disclosed conjugates. Optical biodistribution studies were performed on necropsy specimen at 96 hours using the Pearl Trilogy7Small Animal Imager. Quantification of fluorescence demonstrated both the M5 A-Compound I and MSA-Compound II conjugates had comparable to decreased fluorescence signal at all major organs except the spleen and pancreas, see FIG. 7, demonstrating beneficial biodistribution.

[0135] Beneficially, the studies demonstrated that the described modified heptamethine cyanine fluorophores linked to humanized anti-CEA antibody M5A are useful for improved fluorescence intensity7and contrast under in-vitro and in-vivo tumor imaging when compared to the standard NIR fluorophore IRDye800CW.

[0136] Example 2: Optimization of Fluorophore and Imaging Devices for Fluorescence Image-Guided Surgery

[0137] Fluorescence image-guided surgery7(FIGS) using tumor-specific probes can enhance the effectiveness of cancer surgery. A critical limitation may be that the pre-clinical devices used to validate the probes in developmental scientific research have different optical channels from fluorescence imaging devices used in medical practice, i.e., the operating room. Beneficially, the fluorescent optical properties of the conjugates described herein yvere investigated and validated using three different NIR fluorescence imaging devices.

[0138] To investigate this interplay, a humanized anti-CEA hT84.66-M5A (M5A) monoclonal antibody (mAb) was conjugated to IRDye800CW, Compound I, or Compound II. Nude mice bearing subcutaneous or orthotopic human colorectal cancer (LS174T-luc) received 50 pg of the M5A-conjugates intravenously and were imaged at serial timepoints after injection, i.e., 4 hours, 24 hours, 48 hours, 72 hours, and 96 hours. NIR fluorescent images FIG. 8 were acquired yvith the Pearl® Near-Infrared Small Animal Imager from LI-CORbio™ (“Pearl Trilogy Animal Imager”) (LI-COR Environmental, Lincoln, Nebraska) (Panel A), the Stryker® 1688 Advanced Imagining Modalities (AIM) 4K Platform (“Stry ker 1688 laparoscope”) (Stryker. Kalamazoo, Michigan) (Panel B). and the Stryker® 1788 Platform for Minimally Invasive Surgery7(“Stryker 1788 laparoscope”) (Stryker, Kalamazoo, Michigan) (Panel C). Regions of interest yvere marked over tumor and normal adjacent tissue. Fluorescence signal was quantified using ImageJ software. Beneficially, this demonstrates M5 A-Compound I and M5 A-Compound II conjugates can be detected using available clinical imagining devices which support translation. All three conjugates demonstrated strong localization over all orthotropic sigmoid colon tumors. Hoyvever, theM5A-IRDye800CW conjugate had higher background signal and also a significant off target liver signal leading to a lower tumor-to-background ratio of 7.49, as shown in FIG. 21. Column C and D. Compound I had a higher fluorescence intensity and a lower background leading to both a higher mean fluorescence intensify (MFI) value of 2.38 and a higher tumor to background ratio (TBR) value of 11.58 as compared to the IRDye800CW conjugate and the highest TBR overall. Compound II had a lower fluorescence intensify, but a lower background value and had a higher TBR value of 9.89 as compared to the IRDye800CW conjugate.

[0139] Fluorescence intensify overtime, which indicates conjugate concentration or activity at the tumor, was investigated. As shown in FIG.9, using the Pearl Trilogy Animal Imager, optimized for detection of IRDye800CW, the M5A-Compound I conjugate had the highest fluorescence intensify at all time points, followed by the M5A-IRDye800CW and the M5A-Compound II conjugates, respectively. However, when using the Stryker 1688 laparoscope, FIG. 10, optimized for detection of indocyanine green (ICG), the MSA-Compound II conjugate had the highest fluorescence intensify. Finally, using the Stryker 1788 laparoscope, FIG. 11, which was developed specifically to detect lower wavelength NIR dyes used in tumor-specific fluorescence imaging, the M5A-Compound I and M5A-IRDye800CW conjugates demonstrated the greatest fluorescence intensify. Surprisingly, images with all three probes illustrate that one of the disclosed conjugates, M5A-Compound I or M5A-Compound II, always outperformed the standard M5A-IRDye800CW currently used in practice.

[0140] Second, the tumor to background ratio, which shows the degree of increased signal emitted from a tumor compared to the surrounding normal tissue, i.e., how well it can be distinguished, was investigated. Surprisingly, as compared to the M5A-IRDye800CW conjugate, both the M5A-Compound I and M5A-Compound II conjugates demonstrated increased tumor to background ratio up to the 48-hour time point, see FIGS. 12-14. MSA-Compound II demonstrated the highest contrast when paired with the imagining device that is specific for its wavelength.

[0141] As described herein, there is a need for fluorescent imaging devices to be precisely paired to fluorophore emission w avelength to optimize the fluorescence signal for tumortargeting probes. Surprisingly, the M5A-Compound I conjugate described herein outperformed the M5A-IRDye800CW, even when using the Pearl Trilogy Animal Imager optimized for detection of IRDye800CW, in both mean fluorescence intensify, FIG. 9, and tumor to background ratio, FIG. 12.Example 3: Enhanced Surgical Precision in Ovarian Cancer Using Anti-TAG72 Antibodies Conjugates

[0142] The unmet need for enhanced fluorescence image-guided surgery (FIGS) may be especially apparent in ovarian cancer where the tumor can spread over the surface of multiple organs, complicating resection. As described herein, the tumor-associated glycoprotein 72 (TAG-72) antibody linked to Compound I was investigated for visualization of ovarian cancer in-vivo. The humanized anti-TAG72 antibody (hCC49) was conjugated to Compound I (excitation 776 nm, emission 788 nm) at a 15-fold molar conjugate excess, purified by ultrafiltration, and degree of labeling (DOL) was measured by spectroscopy at 280 nm and 680 nm, and using Equation 1 and 2 as described herein. Athymic mice with subcutaneous human ovarian cancers (OV-90, OVCAR3, and ID8) received 75 ug of the huCC49-Compound I conjugate intravenously.

[0143] Mice were serially imaged at the following time points: 24 hours, 48 hours, 72 hours, and 96 hours. NIR fluorescence imaging was performed using the Pearl® Near-Infrared Small Animal Imager from LI-CORbio™ (“Pearl Trilogy Animal Imager”) (LI-COR Environmental. Lincoln, Nebraska). Imaging was performed using the Pearl Trilogy Small Animal Imager at 800 nm and white light mode. Representative images demonstrate the same animal over time with the near-infrared fluorescence image overlaid on the white light images co-registered. FIG. 15, Panel A shows a mouse with OV-90 human ovarian cancer.

[0144] FIG. 15, Panel B shows a mouse with OVCAR3 human ovarian cancer. FIG. 15, Panel C shows a mouse with ID8 human ovarian cancer. As is shown, strong visual fluorescence was demonstrated in all three ovarian cancers over the tested time range, see FIG. 15, Panels A-C. Therefore, the disclosed conjugates specifically and brightly label the tumor across the three human ovarian cancer cell types tested. These images demonstrate the described conjugate provides surprising and unexpected labeling fluorescence intensity over time, providing beneficial identification for FIGS applications as described below.

[0145] The duration of fluorescence may be important, as a greater duration allows a surgeon to more accurately identify and resect cancerous tissue during surgery, thereby decreasing removal of healthy tissue and increasing patient outcome. Fluorescence imagining was performed using the Pearl Trilogy Animal Imager at 24 hours, 48 hours, 72 hours, and 96 hours. Fluorescence intensify' was measured over the tumor and adjacent subcutaneous tissue and signal was quantified at the regions of interest using the LI-COR Image Studio software. Beneficially, peak fluorescence intensity was found at 24 to 48 hours in all cancer lines, while retaining comparable fluorescence intensity throughout 96 hours with the background signal steadily decreasing over time yielding an increase in tumor-to-background ratios(TBR), see FIGS. 16-18. Specifically, the TBR for OV-90, ID8, and 0VCAR3 cell lines increased progressively over 24 to 96 hours. For example, as shown in FIG. 16, OV-90 demonstrated a steady rise (TBR: 3.7 to 6.2). As shown in FIG. 17. ID8 showed the highest values across all timepoints (TBR: 5.1 to 8.8). As shown in FIG. 18, 0VCAR3 exhibited the lowest (TBR: 2.9 to 4.1). Summarily, FIG. 19 shows the tumor to background ratio steadily increased after administration of the huCC49-Compound I conjugate in all cancer cell lines over time, demonstrating a broad window of imaging with comparable fluorescence intensity over time and a strong contrast.

[0146] Example 4: Tissue Analysis

[0147] Tissue specificity of the disclosed antibody fluorophore conjugates was further examined via histological investigation. Mice bearing LS174T tumors orthotopically implanted over the sigmoid colon were treated with M5A-IRDye800CW, M5A-Compound I, and M5A-Compound II conjugates. After whole body fluorescence imaging, the tumor and adjacent sigmoid colon were harvested, fixed, and sectioned for H&E and IHC staining, FIG.

[0148] 20. IHC staining demonstrated strong peroxidase staining over the tumor for all M5A conjugates without staining over the adjacent tissue providing microscopic confirmation of tumor-specific over expression of the human carcinoembryonic antigen.

[0149] Nude mice bearing LS174T tumors orthotopically implanted over the sigmoid colon were treated with M5A-IRDye800CW, M5A-Compound I, and M5A-Compound II conjugates. Forty-eight (48) hours after intravenous injection of the compounds and 15 minutes after intraperitoneal luciferase injection, mice were sacrificed and imaged under white light, bioluminescence, and near infrared (800nm) wavelength using the Pearl trilogy small animal imager. There w as strong localization of all 3 probes over all orthotropic sigmoid colon tumors. However the LICOR-IR800 probe had higher background signal and also a significant off target liver signal leading to a lower tumor-to-background ratio.

[0150] Compound 1 had a higher fluorescence intensity and a lower background leading to both a higher MFI value and a higher TBR value compared to the LICOR probe and the highest TBR overall. Compound II had a low er fluorescence intensity, but a low er background value and had a higher TBR value than the LICOR probe.

[0151] Informal Sequence Listing

[0152] CDRs of Anti-CEA M5A Antibody

[0153] Heavy Chain CDRs

[0154] SEQ ID NO: 1 KDTYMH

[0155] SEQ ID NO: 2 RIDPANGNSKYSEQ ID NO: 3 APFGYYVSDYAMAY

[0156] Light Chain CDRs

[0157] SEQ ID NO: 4 RAGES VDIFGVGFLH

[0158] SEQ ID NO: 5 RASNLES

[0159] SEQ ID NO: 6 QQTNEDPYT

[0160] CDRs of Anti- TAG- 72 huCC49 IDEC antibody

[0161] Heavy Chain

[0162] SEQ ID NO: 7 DHAIH

[0163] SEQ ID NO: 8 YFSPGNDDFKYNERFKG

[0164] SEQ ID NO: 9 SLNMAY

[0165] Light Chain

[0166] SEQ ID NO: 10 KSSQSLLYSGNQKNYLA

[0167] SEQ ID NO: 11 WASARES

[0168] SEQ ID NO: 12 QQYYSYPLT

[0169] CDRs of ANTI-PSMA J591 monoclonal antibody

[0170] Heavy Chain

[0171] SEQ ID NO: 13 GYTFTEYTIH

[0172] SEQ ID NO: 14 NINPNNGGTTYNQKFED

[0173] SEQ ID NO: 15 GWNFDY

[0174] Light Chain

[0175] SEQ ID NO: 16 KASQDVGTAVD

[0176] SEQ ID NO: 17 WASTRHT

[0177] SEQ ID NO: 18 QQYNSYPLT

[0178] Anti CEA M5A Heavy Chain: SEQ ID NO: 19 Full Length EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYMHWVRQAPGKGLEWVARIDPAN GNSKYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAPFGYYVSDYAMAY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0179] Anti-CEA M5A Light Chain:

[0180]

[0181] DIQLTQSPSSLSASVGDRVTITCRAGESVDIFGVGFLHWYQQKPGKAPKLLIYRASN LESGVPSRFSGSGSRTDFTLTISSLQPEDFATYYCQQTNEDPYTFGQGTKVEIKRTVA APSVFIFPPSDEQLKSGAASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0182] Anti-TAG-72 huCC49 IDEC Heavy Chain; SEQ ID NO: 21 Full Length QVQLVQSGAEVVKPGASVKISCKASGYTFTDHAIHWVKQNPGQRLEWIGYFSPGN DDFKYNERFKGKATLTADTSASTAYVELSSLRSEDTAVYFCTRSLNMAYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0183] Anti-TAG-72 huCC49 IDEC Light Chain: SEQ ID NO: 22 Full Length DIVMSQSPDSLAVSLGERVTLNCKSSQSLLYSGNQKNYLAWYQQKPGQSPKLLIYW ASARESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYSYPLTFGAGTKLELK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0184] ANTI-PSMA J591 monoclonal antibody Variable heavy chain SEQ ID NO:23 EVQLQQSGPELVKPGTSVRISCKTSGYTFTEYTIHWVKQSHGKSLEWIGNINPNNG GTTYNQKFEDKATLTVDKSSSTAYMELRSLTSEDSAVYYCAAGWNFDYWGQGTT LTVSS

[0185] ANTI-PSMA J591 monoclonal antibody Variable light chain SEQ ID NO:24 DIVMTQSHKFMSTSVGDRVSIICKASQDVGTAVDWYQQKPGQSPKLLIYWASTRHT GVPDRFTGSGSGTDFTLAITNVQSEDLADYFCQQYNSYPLTFGAGTKLEIK

[0186] Anti CEA M5A Heavy Chain Variable Domain SEQ ID NO:25 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYMHWVRQAPGKGLEWVARIDPAN GNSKYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAPFGYYVSDYAMAY WGQGTLVTVSS

[0187] Anti CEA M5A Light Chain Variable Domain SEQ ID NO:26DIQLTQSPSSLSASVGDRVTITCRAGESVDIFGVGFLH WYQQKPGKAPKLLIYRASNLES GVPSRFSGSGSRTDFTLTISSLQPEDFATYYCQQTNEDPYT FGQGTKVEIK

[0188] Anti- TAG-72 huCC49 IDEC Heavev Chain Variable Domain SEO ID NO: 27 QVQLVQSGAEVVKPGASVKISCKASGYTFTDHAIHWVKQNPGQRLEWIGYFSPGND DFKYNERFKGKATLTADTSASTAYVELSSLRSEDTAVYFCTRSLNMAYWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSS

[0189] Anti- TAG- 72 huCC49 IDEC Light Chain Variable Domain SEO ID NO: 28 DIVMSQSPDSLAVSLGERVTLNCKSSQSLLYSGNQKNYLAWYQQKPGQSPKLLIYW ASARESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYSYPLT FGAGTKLELK

[0190] ANTI-PSMA J591 monoclonal antibody Light chain Full Length Sequence SEQ ID NO:29 DIVMTQSHKFMSTSVGDRVSIICKASQDVGTAVDWYQQKPGQSPKLLIYWASTRHT GVPDRFTGSGSGTDFTLAITNVQSEDLADYFCQQYNSYPLTFGAGTKLEIKRTVAAPS VFIFPPSDEQLKSGAASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0191] ANTI-PSMA J591 monoclonal antibody Heavy chain Full Length Sequence SEQ ID NQ:30 EVQLQQSGPELVKPGTSVRISCKTSGYTFTEYTIHWVKQSHGKSLEWIGNINPNNGGT TYNQKFEDKATLTVDKSSSTAYMELRSLTSEDSAVYYCAAGWNFDYWGQGTTLTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Claims

Attorney Docket No.: 116437-1547609 (005WO1) WHAT IS CLAIMED IS:

1. An antibody-fluorophore conjugate comprising a tumor-specific antibody conjugated to a near-infrared (NIR) fluorophore, wherein the NIR fluorophore is a modified heptamethine cyanine compound of the following structural formula:

2. The antibody-fluorophore conjugate of claim 1, wherein the modified heptamethine cyanine compound is:

3. The antibody-fluorophore conjugate of claim 1, wherein the modified heptamethine cyanine compound is:

4. The antibody-fluorophore conjugate of any one of claims 1 to 3, wherein the tumor-specific antibody is a humanized anti-CEA T84.66-M5A antibody.

5. The antibody-fluorophore conjugate of any one of claims 1 to 3, wherein the tumor-specific antibody is a humanized anti-T AG-72 huCC49 antibody.

6. The antibody-fluorophore conjugate of any one of claims 1 to 3, wherein the tumor-specific antibody is a humanized anti-PSMA J591 antibody.

7. The antibody-fluorophore conjugate of any one of claims 1 to 3, wherein the tumor-specific antibody comprises a heavy chain variable domain and a lightchain variable domain, wherein the heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO: 1, a CDR H2 as set forth in SEQ ID NO:2, and a CDRH3 as set forth in SEQ ID NO:3; and wherein the light chain variable domain comprises: a CDR LI as set forth in SEQ ID NO:4, a CDR L2 as set forth in SEQ ID NO: 5, and a CDR L3 as set forth in SEQ ID NO:6.

8. The antibody-fluorophore conjugate of any one of claims 1 to 3, wherein the tumor-specific antibody comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO: 7, a CDR H2 as set forth in SEQ ID NO: 8, and a CDRH3 as set forth in SEQ ID NON; and wherein the light chain variable domain comprises: a CDR LI as set forth in SEQ ID NOTO, a CDR L2 as set forth in SEQ ID NO: 11, and a CDR L3 as set forth in SEQ ID NO: 12.

9. The antibody-fluorophore conjugate of any one of claims 1 to 3, wherein the tumor-specific antibody comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO: 13, a CDR H2 as set forth in SEQ ID NO: 14, and a CDRH3 as set forth in SEQ ID NO: 15; and wherein the light chain variable domain comprises: a CDR LI as set forth in SEQ ID NO: 16, a CDR L2 as set forth in SEQ ID NO:

17. and a CDR L3 as set forth in SEQ ID NO: 18.

10. The antibody-fluorophore conjugate of claim 7, wherein the heavy chain variable domain comprises: SEQ ID NO: 19; and wherein the light chain variable domain comprises: SEQ ID NO:20.

11. The antibody-fluorophore conjugate of claim 8, wherein the heavy chain variable domain comprises: SEQ ID NO:25; and wherein the light chain variable domain comprises: SEQ ID NO:26.

12. The antibody-fluorophore conjugate of claim 9, wherein heavy chain variable domain comprises: SEQ ID NO:27; and wherein the light chain variable domain comprises: SEQ ID NO:28.

13. The antibody-fluorophore conjugate of any one of claims 1 to 12, wherein the tumor-specific antibody is further labeled with a radionuclide.

14. The antibody-fluorophore conjugate of claim 13, wherein the radionuclide is lluorine-18 (F-18), phosphorus-32 (P-32), phosphorus-33 (P-33), titanium-45 (Ti-45). scandium-47 (Sc-47), iron-52 (Fe-52), iron-59 (Fe-59). copper-62 (Cu-62), copper-64 (Cu-64), copper-67 (Cu-67), gallium-67 (Ga-67), gallium-68 (Ga-68), arsenic-77 (As-77), yttrium-86 (Y-86), yttrium-90 (Y-90), zirconium-89 (Zr-89), strontium-89 (Sr-89), technetium-94 (Tc-94), technetium-95 (Tc-95), technetium-99m (Tc-99m), molybdenum-99 (Mo-99), palladium-105 (Pd-105), rhodium-105 (Rh-105), silver-111 (Ag-111), indium-ill (In-111), iodine-123 (1-123), iodine-124 (1-124), iodine-125 (1-125), iodine-131 (1-131), praseodymium- 142 (Pr-142), praseodymium- 143 (Pr-143), promethium- 149 (Pm-149), samarium-153 (Sm-153), terbium-161 (Tb-161), dysprosium- 166 (Dy-166), holmium-166 (Ho-166), erbium-169 (Er-169), lutetium-175 (Lu-175), lutetium-177 (Lu-177), rhenium-186 (Re-186), rhenium-188 (Re-188), rhenium-189 (Re-189), iridium-194 (Ir- 194), gold-198 (Au-198), gold-199 (Au-199), astatine-211 (At-211), lead-203 (Pb-203), lead-211 (Pb-211), lead-212 (Pb-212), bismuth-212 (Bi-212), bismuth-213 (Bi-213), radium-223 (Ra-223), actinium-225 (Ac-225), or combinations thereof.

15. The antibody-fluorophore conjugate of claim 13 or claim 14, wherein the radionuclide is attached to the tumor-specific antibody via a chelating agent.

16. The antibody-fluorophore conjugate of claim 15. wherein the chelating agent is l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid (DOT A), desferrioxamine B (DFO), diethylenetriaminepentaacetic acid (DTP A), ethylenediamine-di (O-hydroxyphenylacetic acid) (EDHPA), 2,2' dipyridyl (DIPY), l,4,7-triazacyclononane-l,4,7-triacetic acid (NOTA), and triethylenetetramine (TETA).

17. A method of detecting a solid tumor in a subject, comprising:a) administering to the subject an antibody-fluorophore conjugate comprising a tumor-specific antibody conjugated to a near-infrared (NIR) fluorophore, wherein the NIR fluorophore is a modified heptamethine cyanine compound of the following structural formula:b) detecting the antibody after administration thereby detecting or localizing the solid tumor.

18. The method of claim 17, wherein the modified heptamethine cyanine compound is:

19. The method of claim 17, wherein the modified heptamethine cyanine compound is:

20. The method of any one of claims 17 to 19, wherein the tumor-specific antibody is a humanized anti-CEA T84.66-M5A antibody.

21. The method of any one of claims 17 to 19, wherein the tumor-specific antibody is a humanized anti-TAG-72 huCC49 antibody.

22. The method of any one of claims 17 to 19, wherein the tumor-specific antibody is a humanized anti-PSMA J591 antibody.

23. The antibody-fluorophore conjugate of any one of claims 17 to 19, wherein the tumor-specific antibody comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO: 1, a CDR H2 as set forth in SEQ ID NO:2, and a CDRH3 as set forth in SEQ ID NOT; and wherein the light chain variable domain comprises: a CDR LI as set forth in SEQ ID NO:4, a CDR L2 as set forth in SEQ ID NO: 5, and a CDR L3 as set forth in SEQ ID NO:6.

24. The antibody-fluorophore conjugate of any one of claims 17 to 19, wherein the tumor-specific antibody comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO:7, a CDR H2 as set forth in SEQ ID NO:8, and a CDRH3 as set forth in SEQ ID NO:9; and wherein the light chain variable domain comprises: a CDR LI as set forth in SEQ ID NOTO, a CDR L2 as set forth in SEQ ID NOT 1, and a CDR L3 as set forth in SEQ ID NO: 12.

25. The antibody-fluorophore conjugate of any one of claims 17 to 19, wherein the tumor-specific antibody comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO: 13, a CDR H2 as set forth in SEQ ID NO: 14, and a CDRH3 as set forth in SEQ ID NO: 15; and wherein the light chain variable domain comprises: a CDR LI as set forth in SEQ ID NO: 16, a CDR L2 as set forth in SEQ ID NO: 17, and a CDR L3 as set forth in SEQ ID NOT 8.

26. The antibody-fluorophore conjugate of claim 23, wherein the tumorspecific antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises: SEQ ID NO: 19; and wherein the light chain comprises: SEQ ID NO:20.

27. The antibody-fluorophore conjugate of claim 24, wherein the tumorspecific antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises: SEQ ID NO:25; and wherein the light chain comprises: SEQ ID NO:26.

28. The antibody-fluorophore conjugate of claim 25, wherein the tumorspecific antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises: SEQ ID NO:27; and wherein the light chain comprises: SEQ ID NO:28.

29. The method of any one of claims 17 to 28, wherein the tumor-specific antibody is further labeled with a radionuclide.

30. The method of claim 29, wherein the radionuclide is fluorine- 18 (F-18), phosphorus-32 (P-32), phosphorus-33 (P-33), titanium-45 (Ti-45), scandium-47 (Sc-47), scandium-75 (Sc-75), iron-52 (Fe-52), iron-59 (Fe-59), copper-62 (Cu-62), copper-64 (Cu-64). copper-67 (Cu-67), gallium-67 (Ga-67). gallium-68 (Ga-68), arsenic-77 (As-77), yttrium-86 (Y-86), yttrium-90 (Y-90), zirconium-89 (Zr-89), strontium-89 (Sr-89), technetium-94 (Tc-94), technetium-95 (Tc-95), technetium-99m (Tc-99m), molybdenum-99 (Mo-99), palladium-105 (Pd-105), rhodium-105 (Rh-105), silver-111 (Ag-111), indium-111 (In-111), iodine-123 (1-123), iodine-124 (1-124). iodine-125 (1-125), iodine-131 (1-131), praseodymium- 142 (Pr-142), praseodymium- 143 (Pr-143), promethium- 149 (Pm-149), samarium-153 (Sm-153), terbium-161 (Tb-161), dysprosium- 166 (Dy-166), holmium-166 (Ho-166), erbium-169 (Er-169), lutetium-175 (Lu-175), lutetium-177 (Lu-177), rhenium-186 (Re-186), rhenium-188 (Re-188), rhenium-189 (Re-189), iridium-194 (Ir-194), gold-198 (Au-198), gold-199 (Au-199), astatine-211 (At-211). lead-203 (Pb-203). lead-211 (Pb-211). lead-212 (Pb-212), bismuth-212 (Bi-212), bismuth-213 (Bi-213), radium-223 (Ra-223), actinium-225 (Ac-225), or combinations thereof.

31. The method of claim 29 or claim 30, wherein the radionuclide is attached to the tumor-specific antibody via a chelating agent.

32. The method of claim 31, wherein the chelating agent is 1,4,7,10-tetraazacyclododecane- 1.4.7.10-tetraacetic acid (DOTA), or desferrioxamine (DFO), diethylenetriaminepentaacetic acid (DTP A), ethylenediamine-di (O-hydroxyphenylacetic acid) (EDHPA), 2,2' dipyridyl (DIPY), l,4,7-triazacyclononane-l,4,7-triacetic acid (NOTA), and triethylenetetramine (TETA).

33. The method of any one of claims 17 to 32, wherein the solid tumor is cancer.

34. The method of claim 33, wherein the cancer is colorectal cancer, pancreatic cancer, gastric cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, and / or thyroid cancer.

35. The method of any one of claims 17 to 34, wherein the detecting comprises detecting the antibody-fluorophore conjugate using a scanning device, wherein thescanning device is a PEARL® Near-Infrared Small Animal Imager, a STRYKER® 1688 Advanced Imagining Modalities (AIM) 4K Platform, a STRYKER® 1788 Platform for Minimally Invasive Surgery, or combinations thereof.

36. The method of any one of claims 17 to 35, wherein the detecting comprises imaging a tumor to direct surgical resection of the tumor.

Images