HUMANIZED ANTI-TF ANTIGEN ANTIBODIES

Abstract

Humanized monoclonal antibodies (mAbs) or fragments thereof that bind specifically to the human Thomsen-Friedenreich tumor antigen (TF) are provided. Three different variable heavy chains and three different variable light chains are provided and can be combined to make a total of 25 different combinations of heavy and light chains. Methods for using the mAbs and their fragments for cancer therapy and diagnostic imaging are also provided, including methods for producing the mAbs and their fragments. In vitro cell cultures expressing the mAbs and their fragments, as well as the necessary kits, are also provided.

Description

HUMANIZED ANTI-TF ANTIGEN ANTIBODIES Cross-reference with related applications This application claims priority over U.S. Provisional Application No. 61 / 981,240 filed on April 18, 2014, the description of which is incorporated herein by reference. Field of invention This description generally refers to humanized antibodies that recognize the Thomsen-Friedenreich (TF) human tumor antigen and methods of using monoclonal antibodies. Background of the invention During carcinogenesis, alterations occur in the biosynthesis of cell surface carbohydrate structures, and several different carbohydrates linked to either proteins or lipids have been recognized as tumor-associated antigens. The Thomsen-Friedenreich (TF) disaccharide (Gal313GalNAca) is typically O-linked to serine or threonine residues. TF-Ag (also known as T antigen) has been associated with several human carcinomas, including those found in the pancreas, colon, and breast, and on this basis has been termed a marker of pan-carcinoma. TF-Ag is hidden from the immune system in normal adult tissues by extension with larger glycan chains. In cancer, the cellular glycation machinery is APfrnnn / Lznz / E / YiAi can be altered, leading to the truncation of these chains and the exposure of the TF antigen. New monoclonal antibodies and antibody fragments thereof that can target TF-Ag are desirable and are provided by the present description. Brief description of the invention This description comprises, in various forms, compositions and methods for the therapy of TF+ cancers. In some forms, the description comprises partially humanized monoclonal antibodies (mAbs) or fragments thereof that bind specifically to TF-Ag. The mAb or fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises a sequence selected from the group consisting of: (SEQ ID NO:7) (H1); EVQLLESGAELKKPGASVKVSCKASGYTFTTYWMHW VRQAPGQG LEWMGFISPNTDYTEYNQKFRDRVTLTADKSSSTAYMELSSLTSEDTAVY YCARSFIGYNFDFWGQGTTVTVSS (SEC ID NO:8) (H2); y EVQLVESGAEVKKPGASVKVSCKASGYTFTTYWMHWVKQAPGQG LEW IGFIS PNTDYTE YNQKFRDKATMTADTS ISTAYM ELSRLRSDDTAVY YCARSFIGYNFDFWGQGTTLTVSS (SEC ID NO:9) (H3) y combinaciones de las mismas. The class follows the group that consists of: RChnnn / 1 ζπζ / ε / υιλι DVVMTQSPLSLPLVTLGQPASISCRSSQTIVYSNGNTYLEWFQQRPG QSPRLLI YKVSNRFSGVPDRFSGSGSGTDFTLKISR VE AED VGVYYCFQG SHVPFTFGSG TKLEIK (SEC ID NO:10) (L1); L2DIVMTQTPLSLPVTLGQPASISRSSQTIVYSNGNTYLEW FQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVG VYY CFQGSHVPFTFGSG TKLEIK (S EC ID NO: 1 1) (L2); y DVVMTQSPLSLPLVTLGQPASISCRSSQTIVYSNGNTYLEWYLQRPG QSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISR VE AED VGVYYCFQG SHVPFTFGSG TKLEIK (SEC ID NO:12) (L3); and combinations of mismas. The additional versions H2, H3, L2, and L3 were also developed and analyzed. These include the following: Heavy variable region H2a: QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYWMHWVRQAPGQG LEWMGFISPNTDYTEYNQKFRDRVTITADKSTSTAYMELSSLRSEDTAVY YCARSFIGYNFDFWGQGTTVTVS (SEQ ID NO:13) Heavy variable region H3a: EGQLLESGAELAKPGASVKMSCKASGYTFTTYWMHWVKKRPGQGL EWIGFISPNTDYTEYNQKFRDKATLTADKSSTTAYMQLSSLTSDDSAVYY CARSFIGYNFDFWGQGTTLTVSS (SEC ID NO:14) Light variable region L2a: DIVMTQS P LSL P VTPG EP AS ISCRSSQTIVYSNGNTYLEWYLQKPGQ SPQLLI YKVSNRFSGVPDRFSGSGSGTDFTLKISR VE A EDVGVYYCFQGS HVPFTFGSGTKVDIK (SEC ID NO:15) Light variable region L3a: RPfrnnn / Lznz / E / YiAi ELVMTQTPLSLPVNLGDQASISCRSSQTIVYSNGNTYLEWYLQKPG QSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEADDLGVYYCFQG SHVPFTFGSGTKLEIK (SEO ID NO:16). The heavy and variable chain combinations included in the present description are: H1 -L1; H1-L2; H1-L2a; H1-L3; H1-L3a; H2L1; H2-L2; H2-L2a; H2-L3; H2-L3a; H3-L1; H3-L2; H3-L2a; H3-L3; H3L3a;H2a-L1; H2a-L2; H2a-L2a; H2a-L3; H2a-L3a; H3a-L1; H3a-L2; H3a-L2a; H3a-L3 and H3a-L3a. In some formulations, the mAb comprises a constant region of human IgG. In other formulations, the mAb or its TF-Ag binding fragment is conjugated to an agent selected from a group consisting of chemotherapeutic drugs, toxins, and radioactive isotopes. In another aspect, the description comprises a method for cancer prophylaxis and / or therapy in an individual, wherein the cancer comprises cancer cells expressing TF-Ag. The method comprises administering to the individual one or more mAbs or fragments as described above, wherein the growth or survival, or metastasis, or a combination thereof, of the cancer cells in the individual is inhibited following administration. In another aspect, pharmaceutical compositions comprising the partially humanized mAb fragment thereof are provided. In another aspect, the description provides a cell culture APfrnnn / Lznz / E / YiAi in vitro, where the cells in the cell culture express the mAb or partially humanized fragments of the same. In another aspect, the description provides polynucleotide sequences encoding the mAbs and their TF-Ag binding fragments, expression vectors comprising such polynucleotides, and in vitro cell cultures comprising such expression vectors. In some embodiments, the mAb or its TF-Ag binding fragment is encoded by more than one expression vector. In some embodiments, methods for manufacturing the mAbs and TF-Ag binding fragments thereof are provided and generally comprise the expression of the mAbs or TF-Ag fragments or binding combinations thereof in an in vitro cell culture and the separation of the mAbs or TF-Ag binding fragments, or combinations thereof, from the cell culture. Kits comprising the mAbs and TF-Ag binding fragments thereof are also provided. Brief description of the figures Figure 1 shows the amino acid sequence alignments of a heavy variable JAA-F11 and the three VH variant regions (H1, H2, and H3) included in this description. Bold indicates identical amino acids between JAA-F11 and the engineered, humanized H chains; bold and shaded amino acids indicate differences between the engineered humanized chains and mouse JAA-F11. Alanine at position 72 is shown in bold and RChnnn / 1 ζπζ / ε / υιλι (italicized) is a mouse remnant that was retained to avoid steric clashes. Complementarity-determining regions (CDRs) are in italics. Numbering follows the Kabat system. The sequence mJAAF11 is SEO ID NO: 17. The sequence H1 is SEO ID NO: 7; the sequence H2 is SEC ID NO: 8; the sequence H3 is SEC ID NO: 9. Figure 2 shows the amino acid sequence alignments of light variable MJAA-F11 (JAA-F11VH) and three VL variant regions (L1, L2, and L3). Bold indicates mouse amino acids or amino acids identical to the mouse sequence; bold and shaded amino acids are differences between the engineered humanized JAA-F11 variants and mouse JAA-F11. The leucine at position 51, shown in bold and italics, is a mouse remnant that was retained to avoid steric clashes. The mJAAF11 sequence is SEC ID NO: 17. The L1 sequence is SEC ID NO: 10; the L2 sequence is SEC ID NO: 11; and the L3 sequence is SEC ID NO: 12. Figure 3. Determination of relative affinity using MJAA-F11 inhibition by ELISA to evaluate the binding of each h JAAF11 and the chimeric antibody for TF-Ag-BSA. Error bars represent ± 1 standard error. Figure 4. Binding of hJAA-F11, chimeric JAA-F11, and mouse JAA-F11 (50 mg / ml) to 10 breast cancer cell lines. The cell lines include triple-negative (HCC 70, BT 549, MDA-MB-231, MDAMB-468, DU 4475), ER / P R-positive (HCC 1419, AU 565, MDA-kb2), and RPfrnnn / Lznz / E / YiAi H E R2-positive (CAMA-1, HCC 1428). For all cell lines analyzed above, binding to I1JAA-F11, chimeric JAA-F11, or mouse JAA-F11 (at 50 pg / ml) was significantly higher (p<0.05) (Student's t-test) than that of TF-Ag-negative control myeloma. Each error bar represents ±1 standard deviation. Figure 5. The chimeric antibody and the humanized JAA-F11 antibodies, except for H3L3, caused a small (~6–11%) but statistically significant inhibition of tumor cell growth. No increase in cell proliferation was observed. No significant result was observed for either antibody at 2 or 1 µg / ml. Figure 6A. Mouse, chimeric, and four hJAA-F1 construct ADCC activity against a TF-Ag-positive human breast cancer cell line (BT 549). Results are presented as percent cell lysis in antibody-treated cells compared to 100% cell lysis. H2L2 and H3L3 showed statistically higher ADCC activity than the chimeric or mouse antibodies (p < 0.05) in all cases in the human cell line. This is a representative average of all experiments with PBMCs from donor #1 and donor #2 (more than three independent experiments). Error bars represent ±1 standard error. Figure 6B. Mouse, chimeric, and 4 hJAA-F11 construct ADCC activity against a TF-Ag (BT 549) positive human breast cancer cell line in donor #1 PBMCs. The results RChnnn / 1 zoz / ε / uli presents cell lysis as a percentage in antibody-treated cells compared to 100% cell lysis. H2L2 and H3L3 showed statistically higher ADCC activity than chimeric or mouse antibodies (p < 0.05) in all cases in the human cell line. This is the average of at least 3 independent experiments. Error bars represent ± 1 standard error. Figure 6C. Mouse, chimeric, and four hJAA-F1 construct ADCC activity against a TF-Ag (BT 549)-positive human breast cancer cell line in Donor#2 PBMCs. Results are presented as percentage cell lysis in antibody-treated cells compared to 100% cell lysis. H2L2 and H3L3 showed statistically higher ADCC activity from chimeric or mouse antibodies (p < 0.05) in all cases against the human cell line. This is the average of at least three independent experiments. Error bars represent ±1 standard error. Figure 6D. Mouse, chimeric, and three hJAA-F11 construct ADCC activity against the TF-Ag-positive 4T1 mouse breast cancer cell line. Results are presented as cell lysis percentage in antibody-treated cells compared to 100% cell lysis. H3L3 and the chimeric constructs showed statistically higher mouse antibody ADCC activity (p < 0.05). Error bars represent ±1 standard error. ADCC was performed only once with donor PBMCs #3. Figure 7. Humanized, chimeric, and mouse JAA-F11 do not induce complement-dependent cytotoxicity (CDC). CDC activity Mouse, chimeric, and two hJAA-F11 constructs RPfrnnn / Lznz / E / YiAi were assayed against the human breast cancer cell line HCC 1428. Results are presented as the percentage of cell lysis in antibody-treated cells compared to 100% cell lysis. Positive control: LDH-positive cells included in the reagent kit showed lysis. Error bars represent ±1 standard error. Figure 8. Internalization of mouse, chimeric, and 4 hJAA-F11 antibodies using an enzyme immunoassay to measure surface binding. Internalization was analyzed by incubating 4T1 mouse breast cancer cells with antibodies at 37°C or 4°C. As expected, the pattern was the opposite of the ADCC pattern shown by H3L3 in Figure 20, but unexpectedly, H2L2 induced ADCC and was internalized. Mouse antibodies, H2L2, chimeric antibodies, H2L3, and H1L1 were all significantly internalized (p < 0.05). Error bars represent ±1 standard error. Figure 9. MicroPET images showing the immunolocalization of hJAA-F11 antibodies in a mouse carrying the 4T1 tumor. Figure 10. MicroPET images showing the localization of 124Free Iodine to the thyroid in a mouse bearing the 4T1 tumor. Detailed description This description includes distinct humanized monoclonal antibodies (mAbs) and fragments thereof that specifically recognize TF-Ag, methods for manufacturing the mAbs, and methods for using the mAbs for prophylactic and / or therapeutic purposes and for diagnostic imaging. RPfrnnn / Lznz / E / YiAi The amino acid sequences of the mAbs provided in this description were developed using a novel approach to modify the amino acid sequences in the mAb produced by a hybridoma deposited at the ATCC with catalog number CRL2381, such that the mAb framework regions incorporate both murine and human immunoglobulin (Ig) sequences in a guided manner that maintains specificity and reduces immunogenicity. The hybridoma produces an mAb known as JAAF11. The modifications introduced to the JAA-F11 sequence within the murine framework regions result in a set of three distinct heavy chains and three distinct variable chains that can be combined to produce 25 distinct mAbs suitable for combating various cancer types involving TF-Ag-expressing cancer cells.Furthermore, mAbs possess desirable characteristics that make them particularly well-suited to preferentially induce distinct anticancer mechanisms in an individual. For example, depending on the choice of heavy and light chains, they can provide enhanced antibody-dependent cytotoxicity stimulation, complement-dependent cytotoxicity, or mAb internalization. The description modalities include the CDR sequences of the JAA-F11 mAb. These are: VH string: CDR1: SGYTFTTYWMH; (SEC ID NO:1); CDR2: FISPNTDYTEYNQKFRD; (SEC ID NO:2); CDR3: RSFIGYNFDFWGQGT; (SEC ID NO:3); and VL string: CDR1: RPfrnnn / Lznz / E / YiAi 1 CRSSQTIVYSNGNTYLEW; (SEQ ID NO:4); CDR2: KVSNRFSGVPD; (SEQ ID NO:5); and CDR3: CFQGSHVPFTGSG; (SEQ ID NO:6). The CDR sequences are placed within the context of the modified in-frame sequences. Consequently, the mAbs and their TF-Ag binding fragments comprise a selected heavy chain of: H1 - EVQLVESGAEVKKPGASVKVSCKASGYTFTTYWMHWVRQA PGQGLEWMGFISPNTDYTEYNQKFRDRVTMTADTSISTAYMELSRLRSD DTAVYYCARSFIGYNFDFWGQGTLVTVSS (SEQ ID NO:7); H2 - EVQLLESGAELKKPGASVKVSCKASGYTFTTYWMHWVRQ APGQGLEWMGFISPNTDYTEYNQKFRDRVTLTADKSSSTAYMELSSLTS EDTAVYYCARSFIGYNFDFWGQGTTVTVSS (SEQ ID NO:8); H3 - EVQLVESGAEVKKPGASVKVSCKASGYTFTTYWMHWVKQ APGQGLEWIGFISPNTDYTEYNQKFRDKATMTADTSISTAYMELSRLRSD DTAVYYCARSFIGYNFDFWGQGTTLTVSS (SEC ID NO:9) and combinations thereof; and a selected light chain from the group consisting of: L1 - DVVMTQSPLSLPVTLGQPASIS CRSSQTIVYSNGNTYLEW FQQRPGQSPRLLIY KVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYY CFQGSHVPFTFGSG TKLEIK (SEQ ID NO:10); L2 - DIVMTQTPLSLPVTLGQPASIS CRSSQTIVYSNGNTYLEW FQQRPGQSPRLLIY KVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYY CFQGSHVPFTFGSG TKLEIK (SEQ ID NO:1 1); RPfrnnn / Lznz / E / YiAi 2 L3 - DVVMTQSPLSLPVTLGQPASIS CRSSQTIVYSNGNTYLEW YLQRPGQSPRLLIY KVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYY CFQGSHVPFTFGSG TKLEIK (SEC ID NO:12) and combinations thereof. In addition to the preceding sequences, the following heavy and light chains were made and analyzed in certain combinations: Heavy variable region H2a: QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYWMHW VRQAPGQG LEWMGFISPNTDYTEYNQKFRDRVTITADKSTSTAYMELSSLRSEDTAVY YCARSFIGYNFDFWGQGTTVTVS (SEQ ID NO:13) Heavy variable region H3a: EGQLLESGAELAKPGASVKMSCKASGYTFTTYWMHW VKKRPGQGL EWIGFISPNTDYTEYNQKFRDKATLTADKSSTTAYMQLSSLTSDDSAVYY CARSFIGYNFDFWGQGTTLTVSS (SEC ID NO:14) Light variable region L2a: DIVMTQS P LSL PVTPGEP AS ISCRSSQTI VYSNGNTYLEW YLQKPGQ SPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCFQGS HVPFTFGSGTKVDIK (SEC ID NO:15) Light variable region L3a: ELVMTQTPLSLPVNLGDQASISCRSSQTIVYSNGNTYLEWYLQKPG QSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEADDLGVYYCFQG SHVPFTFGSGTKLEIK (SEQ ID NO:16). RFfrnnn / Lznz / E / YiAi The description covers the mAbs and TF-Ag binding fragments thereof comprising amino acid sequences comprising or consisting of the preceding sequences. In some embodiments, the description comprises mAbs and TF-Ag binding fragments thereof, wherein the frame sequences and CDR sequences consist of SEC ID NO:7, SEC ID NO:8, SEC ID NO:9, SEC ID NO:10, SEC ID NO:11, SEC ID NO:12, SEC NO:13, SEC ID NO:14, SEC ID NO:15, and SEC ID NO:16. Por consiguiente, las siguientes 25 combinaciones de cadena ligera y pesada están abarcadas por esta descripción: H1 - L1; H1-L2; H1-L2a; H1-L3; H1-L3a; H2-L1; H2-L2; H2-L2a; H2-L3; H2-L3a; H3-L1; H3-L2; H3-L2a; H3-L3; H3-L3a; H2a-L1; H2a-L2; H2a-L2a; H2a-L3; H2a-L3a; H3a-L1; H3a-L2; H3a-L2a; H3a-L3 y H3a-L3a. In some formulations, the description includes mAbs and TF-Ag binding fragments comprising a combination of H1 L1, H2 L2, H3 L3, H2 L3, H2ay L2a, and H3ay L3a. In this description, in addition to the results for the H and L chain combinations as described below, H2a-L2a and H3a-L3a were made and tested, and the results for these two combinations were similar to those reported for H2-L2 and H3-L3, respectively. The H2a-L2a and H3a-L3A combinations were made as a result of subcloning procedures, allowing the selection of additional subclones with even more enhanced TF-Ag binding characteristics, as determined by the highest reactivity in enzyme immunoassays (EIAs). Of these, the best for the Rffrnnn / Lznz / E / YiAi 4 internal I i zac i ó n are H2L2 and H2aL2a, while the best for ADCC are H2L2, H2aL2a, H3L3 and H3aL3a. Representative VH and VL sequences are further described by means of Figures 1 and 2. In particular, Figure 1 provides the sequence alignments of the heavy variable JAA-F11 amino acids and the three VH variant regions (H1, H2, and H3) included in this description. Bold indicates identical amino acids between JAA-F11 and engineered, humanized H chains; bold and shaded amino acids indicate differences between engineered humanized chains and mouse JAA-F11. The alanine at position 72 shown in bold and italics is a mouse residue that was withheld to avoid steric clashes. CDRs are in italics. Numbering is according to the Kabat system. It is therefore acknowledged that, while the mAbs described herein refer to humanized versions that do not comprise certain murine residues, they may thus be considered partially humanized. Figure 2 shows the amino acid sequence alignments of light variable MJAA-F11 (JAA-F11VH) and three VL variant regions (L1, L2, and L3). Bold indicates mouse amino acids or amino acids identical to the mouse sequence; amino acids in bold and shaded represent differences between the engineered humanized JAAF11 variants and mouse JAA-F11. The leucine at position 51, shown in bold and italics, is a mouse remnant that was retained to avoid steric clashes. RChnnn / 1 ζπζ / ε / υιλι 5. From Figures 1 and 2, it can be seen that the humanized H and L chains currently presented comprise differences from the JAA-F11 mouse antibody and differences between them, but certain murine amino acids have been conserved to maintain the specificity of the humanized mAbs. The humanized mAb fragments described herein are also included in the invention. Examples of suitable antibody fragments include Fab, Fab', F(ab')2, scFv, and Fv fragments. Several techniques have been developed for the production of antibody fragments and are included within the scope of this description. In some embodiments, the mAbs or fragments thereof are produced in host cells by means of recombinant expression vectors. This description includes all polynucleotide sequences encoding the amino acid sequences described herein, expression vectors comprising such polynucleotide sequences, and in vitro cell cultures comprising such expression vectors. In some embodiments, the cell cultures are eukaryotic cells. In some embodiments, the cell cultures are mammalian cells. In some embodiments, the cells are CHO cells.The kits comprising the mAbs and / or TF-Ag binding fragments thereof and / or cell cultures expressing the mAbs and / or TF-Ag binding fragments thereof are provided by this description. In general, the kits comprise one or more sealed containers holding the mAbs and / or TF-Ag binding fragments thereof, or cells expressing them. Instructions for use are provided. APfrnnn / Lznz / E / YiAi 6 mAbs and / or TF-Ag binding fragments for therapeutic and / or imaging purposes may be included in the kits. In some embodiments, the description includes a method for manufacturing the mAbs or TF-Ag binding fragments thereof, comprising culturing cells containing an expression vector or other polynucleotide sequence encoding the mAb or TF-Ag binding fragments thereof, enabling the expression of the mAbs or TF-Ag binding fragments thereof, and the separation of the mAbs or TF-Ag binding fragments thereof from the cell culture. The nucleotide sequences encoding the mAbs or TF-Ag binding fragments thereof can be expressed using any suitable expression vector, many of which are known in the art and / or commercially available. In one embodiment, the heavy and light chains are expressed in a single expression vector, such as a plasmid.In another embodiment, the heavy and light chains are expressed on separate plasmids within the same cell, after which the expressed heavy and light chains form the conventional mAb structure. The mAbs or TF-Ag binding fragments thereof can be isolated and / or purified using conventional techniques, given the benefit of the present description. In another aspect, the description provides a method for inhibiting the growth of cancer cells in an individual, and / or inhibiting the metastasis of cancer cells in an individual, whose cancer cells express TF-Ag molecules. The method comprises the RPfrnnn / Lznz / E / YiAi 7. Administration to an individual of a therapeutic amount of humanized mAbs and / or fragments thereof, wherein the administration inhibits the growth and / or inhibits the metastasis of TFAg-expressing cancer cells. In the modalities, the practice of the method of the invention reduces the volume of a tumor and / or reduces the formation of metastatic foci or secondary tumors. In the modalities, the method is provided to an individual in need thereof. In the modalities, the individual in need has been diagnosed with, is suspected of having, or is at risk of developing, or has a recurrence of cancer. In the modalities, a therapeutically effective amount of a mAb or TF-Ag-binding fragment thereof is used.The term therapeutically effective as used herein means that the amount of mAb or TF-Ag binding fragment thereof administered is sufficient to inhibit the growth, survival and / or metastasis of TF+ cancer cells. In several formulations, mAbs and / or humanized fragments thereof can be conjugated to a chemotherapeutic agent to enable the agent to target cancer cells by binding to cells expressing TF-Ag. Chemotherapeutic agents useful in generating such antibody conjugates include enzymatically active toxins and fragments thereof. Suitable enzymatically active toxins include diphtheria A chain, non-binding active fragments of diphtheria toxin, and exotoxin A chain (of RPfrnnn / Lznz / E / YiAi 8 Pseudomonas aeruginosa), ricin A chain, abrin A chain, modecin A chain, alpha sarcin, Aleurites fordii proteins, diantin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Saponaria officinalis inhibitor, gelonin, mitogilin, restrictocin, fenomycin, enomycin, and trichothecenes. Chemotherapeutic agents may be covalently bound to the mAb or TF-Ag binding fragment thereof by any suitable chemical conjugation approach. In the modalities, the chemotherapeutic agent may comprise a fusion protein segment with the mAb or TF-Ag binding fragment. In another aspect, the description provides a method for identifying metastatic foci, tumors, or combinations thereof in an individual, where the foci or metastatic tumors comprise cells expressing TF-Ag. The method comprises the steps of administering individual humanized mAbs and / or fragments thereof, where the mAbs and / or humanized fragments thereof have been conjugated to a detectable marker, and detecting the detectable marker to identify the metastatic foci, tumors, or combinations thereof. Therefore, the mAbs and / or humanized fragments thereof can be conjugated to a detectable marker, such as a radioactive agent. A variety of radioactive isotopes are available for the conjugation of JAA-F11 mAbs such that the cells to which the JAA monoclonal antibodies bind RPfrnnn / Lznz / E / YiAi 9 F11 cells can be selectively photographed or destroyed. For the selective destruction of TF-Ag-expressing cells, JAA-F11 mAbs can be conjugated to a highly radioactive atom, such as In11 1, At21 1, 1131, 1125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212, and radioactive isotopes of Lu. When mAbs and / or humanized fragments thereof are used for the identification of TF-Ag-expressing cells in metastatic foci or tumors, they may comprise a radioactive atom for gamma studies, e.g. Tc99m (metastable technetium-99), 1123, or a spin marker for nuclear magnetic resonance (NMR) (also known as magnetic resonance imaging, or MRI), such as 1123, 1131, 1124, F19, C13, N15, O17 or gadolinium (III) or manganese (II). Labeled mAbs and / or humanized fragments thereof can be injected into patients diagnosed with or suspected of having metastatic disease to identify metastatic foci and / or tumors. The information from such imaging can be used to diagnose or stage the patient's disease status. The marker can be selected according to the imaging system to be used. For example, Indiol-11, Technetium-99, or Iodine-131 can be used for planar scans or single-photon emission computed tomography (SPECT). Positron-emitting markers such as Fluorine-19, Iodine-123, and Iodine-124 can be used in positron emission tomography. Paramagnetic ions such as Gadolinium(III) or RPfrnnn / Lznz / E / YiAi manganese(II) markers are used in magnetic resonance imaging (MRI). Localization of the marker within a particular tissue allows for the identification of metastatic foci or tumors comprising cells that express TF-Ag. A marker concentration at a specific site that is higher than the background concentration allows for the identification of the presence of metastatic cells. In a preferred modality, after administration of labeled mAbs and / or humanized fragments thereof, a sufficient period of time is allowed to elapse to allow for the elimination of unbound mAbs and / or humanized fragments thereof from the individual, such that the background marker concentration is significantly reduced. Therapeutic formulations comprising conjugated or unconjugated humanized mAbs and / or fragments thereof may be prepared by mixing with pharmaceutically acceptable vehicles, excipients, or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. The vehicles, excipients, or stabilizers are non-toxic to the recipients at the doses and concentrations used and include phosphates such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low-sodium polypeptides RChnnn / 1 ζπζ / ε / υιλι molecular weight (less than approximately 10 residues); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN®, PLURONICS®, or PEG. mAbs and / or humanized fragments can be combined with other chemotherapeutic agents in pharmaceutical compositions. mAbs and / or humanized TF-binding fragments thereof can be administered by any suitable route, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal. Parenteral infusions include intramuscular, intravenous, intra-arterial, intraperitoneal, intralymphatic, or subcutaneous administration. In addition, mAbs and / or humanized fragments thereof can be appropriately administered by pulse infusion, for example, with decreasing doses of the antibody. Preferably, dosage is administered by injection, most preferably intravenous or subcutaneous injection, depending in part on whether administration is short-term or chronic. In certain modalities, mAbs and / or humanized fragments thereof are administered to an individual diagnosed with or suspected of having breast cancer. RPfrnnn / Lznz / E / YiAi of colon, prostate, ovarian, bladder or other TF Ag positive cancers to inhibit metastasis or to inhibit the growth of cancer cells. Other compounds, such as chemotherapeutic agents, immunosuppressants, and / or cytokines, may also be administered. Combined administration may include co-administration using separate formulations or a single pharmaceutical formulation, and may also include sequential administration in any order, preferably with a time interval during which both (or all) of the active agents exert their biological activities simultaneously. mAbs and / or humanized fragments thereof may be administered to a human or other animal according to the treatment methods described above in a quantity sufficient to inhibit metastasis and / or the growth of TFAg-expressing cells. It will be recognized by someone skilled in the art that the form and character of the pharmaceutically acceptable vehicle or diluent will be dictated by the amount of active ingredient with which it is to be combined, the route of administration, and other well-known variables, such as the size of the person and the stage of the disease. Example 1 The following example describes the manufacture and use of humanized mAbs from this description and the physical and functional properties of the mAbs. Design and construction of a humanized JAA-F11 βΓ^ηηη / Lznz / E / YiAi Subsequently, the sequences of the variable regions of mouse JAA-F11 were verified, and the CDRs were defined to maintain specificity and affinity for TF-Ag. The included CDRs were defined according to both Kabat et al.

[139] and Chothia et al.

[141] based on sequence and structural variability, respectively. Two amino acids are considered to be in contact with each other in a protein structure if they both contain a pair of atoms separated by 6 Å or less

[138] . X-ray crystallography and computational carbohydrate threading studies of amino acid residues in mouse JAA-F11

[142] identified residues within 5–6 Å from the binding site. These amino acids, as well as two cysteine ​​residues at positions L23 and L88 of the light chain that are important for the conformational structure, were included in the final overall definition of the CDRs used in the humanization of JAA-F11. Selection of human frames Three different approaches were applied to select the human acceptor antibody framework for the heavy and light chains of the JAAF11 variable region. Three heavy chain variants (H1, H2, H3) and three light chain variants (L1, L2, L3) were designed. Any of the three heavy chains (VH) can be paired with any of the three light chains (VL) to create a total of nine possible hJAA-F11 VH / VL combination variants. To create these variants, a BLAST search was performed comparing the VH and VL sequences of mJAA-F11 separately. RPfrnnn / Lznz / E / YiAi (mouse antibody) against human immunoglobulin sequences. The 10 best homologous human VH and VL sequences were aligned with the corresponding variable regions of mJAA-F11 using the Seaview sequence alignment program. For the first variants, H1 and L1, the amino acid sequence of the frame regions was selected based on the most frequently observed amino acid at each position among the top 10 most homologous human IgG variable sequences. For variants H2 and L2, the amino acid sequence of the frame regions was selected as follows: if the amino acid in any of the 10 human sequences matched the corresponding amino acid in mouse immunoglobulin, then that amino acid was selected; in the remaining positions, the amino acid most frequently observed in the human sequences was selected. For the third variant, H3 and L3, the amino acid sequence of the frame region contained three mouse amino acids on each side of each CDR, while the remainder of the sequence was composed of the most frequently observed amino acids in the top 10 human sequences. A chimeric JAA-F11 was also constructed, consisting of the complete mouse JAA-F11 variable region joined to human constant regions. The chimeric version was created to verify that the correct mouse variable regions had been cloned and sequenced, and also to serve as a positive control in the evaluation of humanized JAA-F11 antibodies. It is expected that the chimeric RChnnn / 1 ζοζ / ε / υιλι maintains the same junction characteristics as the mouse JAA-F11, while having the human constant region. Evaluation of hJAA-F11 models The originally proposed hJAA-F11 constructs were evaluated for conformational effects at the binding site. The three heavy-chain variants (H1, H2, H3) had an alanine-arginine replacement at position 72, which could potentially cause severe steric interactions with surrounding amino acids. A leucine at position 51 in the light-chain variants (L1, L2, L3) was replaced by arginine, which could result in steric collisions with surrounding amino acid side chains. The arginine residue was removed and replaced with the original mouse JAA-F11 framework residue, alanine 72 (VH) and leucine 51 (VL), as shown in Figures 1 and 2. Alignment comparisons between the mouse JAA-F11 heavy and light variable amino acid sequences and the hJAA-F11 constructs are shown in Figures 1 and 2.Experts in the technique will also recognize the differences in the amino acid sequences presented in H2a, H3a, L2a and L3a, compared to the other H and L sequences. Immunogenicity prediction of hJAA-F11 variants The immunogenicity predicted by the T20 score of our humanized variants (grafted with CDRs) is expected to be very low (Table 1). The T20 score is used to measure the humanization of variable region sequences of monoclonal antibodies. This scoring system was developed by Gao et al. RPfrnnn / Lznz / E / YiAi [Monoclonal antibody humanness score and its applications. 2013. BMC Biotechnology, 13:55], using a database of over 38,000 human antibody sequences. In this method, a BLAST protein is generated from this database, and the humanized antibody from the test is compared to these human sequences. The humanized antibody is compared to the 20 best human antibody matches by BLAST, and the similarity to these sequences is calculated. The highest possible score is 100 (most similar to human). The validation of this method is shown when Gao et al. tested this immunogenicity scoring method on more than 90 antibodies that have been used clinically in humans and found that antibodies with T20 scores for the FR and CDR sequences above 80 were not immunogenic, while T20 scores for the FR sequences only, above 85, were not immunogenic. Using the T20 values ​​of current hJAA-F11 variants, very low immunogenicity is expected in patients. This demonstrates that CDR-grafted antibodies are more human and are expected to be less immunogenic than chimeric antibodies, that the H1L1 variant is the most human of the CDR-grafted variants, and that all hJAA-F11 variants are expected to have low immunogenicity. Table 1. Evaluation of the immunogenicity of the hJAA-F11 constructs. *Score >80 for FR and CDR sequences is not immunogenic in humans. **Score >85 for FR sequences only is not immunogenic in humans. APfrnnn / Lznz / E / YiAi RPfrnnn / Lznz / E / YiA JAA-F11 Variants T20 Score (FR & CDR) T20 Score (FR) Heavy Chain Light Chain Heavy Chain Light Chain Chimerics 67 78 77 86 H1 L1 83 88 97 96 H2L2 76 87 90 95 H3L3 78 86 90 93 Expression and production of humanized and chimeric AA-F11 variants The hJAA-F11 and chimeric VH and VL genes were cloned into two different mammalian expression vectors containing the human lgG1 heavy chain constant region (6307 pAH) and the human kappa light chain constant region (6714 pAN), respectively, to produce plasmids containing the complete lgG1 and kappa genes. The correct sequence and orientation of each of the cloned VH and VL regions into the 6307 pAH and 6714 pAN expression vectors were verified by sequencing. After stable co-transfection of the two vectors into CHO-K1 cells, stable clones expressing either humanized or chimeric hJAA-F11 were selected based on resistance to neomycin using the antibiotic G418 and expression of either the chimeric or hJAA-F11. hJAA-F11 candidates were screened using an ELISA for anti-TF-Ag antibody, following established techniques. Clones derived from the co-transfection of each combination variant of humanized H / L JAA-F11 and chimeric JAA-F11 that showed the highest TF-Ag reactivity were selected for further characterization as described herein. The humanized and chimeric JAA-F11 constructs were purified from culture supernatants by protein A column chromatography. A chimeric antibody has also been produced, as well as H1 L1, H2L2, H3L3 and H2L3, H2aL3a and H3aL3a.Taking into account the benefit of the present description, the person skilled in the art can easily produce the remaining H and L chain antibody combinations that are included in the present invention. Analysis of chemical specificity by glycan matrix After initial screening, the chemical specificity of hJAA-F11 and chimeric JAA-F11 variants was determined using a glycan array by the Functional Glycomics Consortium. These data were compared with those obtained for mouse JAA-F11. Glycan array analysis is an indirect immunofluorescence method for determining the glycan-binding reactivity of lectins and antibodies. A glycan array was previously used to demonstrate the chemical specificity of mouse JAA-F11. This method was used to analyze the reactivity of antibody candidates with 610 different glycans. The hJAA-F11 H1L1, H2L2, H3L3, and chimeric antibodies showed the same fine binding specificity as the mouse antibody, including limited binding to Gaipi-3GalNAc-a linkage structures. RChnnn / Lznz / E / YiAi (TF-Ag) and the lack of binding to sialylated α2-3 structures. The glycan matrix shows that out of 610 glycans, the humanized, chimeric antibodies and mouse JAA-F11 only bind to TF-Ag and four other TF-Ag saccharide-containing structures. The four additional saccharides bound to mouse JAA-F11, chimeric constructs, and hJAA-F11, up to 440 or 610 analyzed saccharides, should not be biologically problematic for JAA-F11 tumor targeting. Neu5Aca2-6(Gaipi-3GalNAcp, G IcN Aοβ 1-6(Gal β 1-3) Unsubstituted OtINAβ and Galβ 1-4OIONAΑοβ 1-6(θ3ΐβ 1-3)GalNAβ have only been found in excreted fluids, cancer, or other diseases. Nβυ5Αοβ2-6 OtINAβ 1-3GalNAc is not a naturally occurring structure and is not found in humans. The specificity of the humanized JAA-F11 constructs appears to be the same as, or even improved upon compared to, the chimeric and mouse antibodies. For example, H2L3 shows statistically (p < 0.05 by ANOVA) less binding with any TF-Ag addition, and H1L1 and H2L2 do not allow a disaccharide to add to the C-6 hydroxyl of GalNAc, which is statistically different (p < 0.05) from the other antibodies. The lack of binding to over 600 negative saccharides in the matrix indicates the likely orientation of all these antibodies. Important examples of closely related saccharides that do not bind are the structures linked to OθIβI3GalNAc-beta, indicating that the antibody would bind to tumor tissues and not to the GM1 ganglioside of the central nervous system. RPfrnnn / Lznz / E / YiAi asialo-GM 1 of NK cells, the GD1 of glycolipids or asialo GM1 of peripheral nervous tissue (these are beta-linked). The well-known elongation of TF-Ag in normal tissues is carried out by pi-3N-acetylglucosaminyltransferase adding GlcNAcpi-3 to Gal, forming G IcN Aoοβ1-3Gaip 1-3Gal N Aca-S P8 or by a 2-3 sialyltransferase forming Neu5Aca2-3Gaip 1 -3GalNAc (sialyl-TF) or with a second sialyltransferase forming Neu5Ac2-3Gaipi-3 (Neu5Aca2-6) GalNAc (disia I i I - TF), which do not bind to mouse, chimeric or humanized JAA-F11. Therefore, normal tissue is not expected to bind to JAA-F11 or hJAA-F11 constructs. Relative affinity analysis The relative binding affinities of the four hJAAF11 and chimeric antibodies to TF-Ag were determined by comparing the ability of the mouse antibody to compete with the humanized antibodies in an enzyme immunoassay. In this assay, 3 g of each humanized Ab were mixed with serial dilutions of the mouse anti-TF-Ag antibody (mJAA-F11). The binding of the anti-TF-Ag antibody to the TF-Ag-coated plate was measured using a species-specific anti-human IgG. The amount of mouse mJAA-F11 required to inhibit 1 g of hJAA-F11 at 50% was extrapolated and taken as a measure of relative affinity. The greater the amount of mouse antibody required for inhibition, the greater the relative affinity of the antibody. The results are summarized in Figure 3 and Tables 2 and 3. Table 2 shows the mean maximum inhibitory concentration of JAARChnnn / 1 ζπζ / ε / υιλι The required IC50 for mouse F11 antibodies to compete with 1 pg of this humanized antibody was determined. H2L2 showed the highest affinity for TF-Ag among the antibodies, with an IC50 of 2.31 pg of mouse antibody. Table 7 shows the p-values ​​calculated for each antibody compared to each other using the Tukey post-hoc ANOVA test. This difference in affinity for H2L2 antibody was statistically significant (p < 0.05) when compared to each of the hJAA-F11 and chimeric antibodies, except for H2L3, where it approached significance (p = 0.057) (Table 3). H2L3 was not significantly different from H1L1, H2L2, H3L3, or the chimeric antibody. The relative affinities of H1L1, H3L3, and the chimeric antibody were not significantly different from each other. Table 2. Relative affinity of hJAA-F11 and chimeric JAA-F11 antibodies for TF-Ag. IC50, Relative affinity for TF-Ag: pg mJAA-Fl 1 required for 50% inhibition of 1 pg of each hJAA-Fl 1 0 chimeric H1 L1 H2L2 H2L3 H3L3 Chimeric 1.11 ± 0.18a 2.31 ± 0.20 1.64 ± 0.39a 1.28 ± 0.41a 1.14 ± 0.30a The average of three independent experiments is shown as +.1 SD. Table 3. ANOVA analysis was performed on replicates of the CI50 assay to compare the differences between CI50 values ​​of different antibodies. RChnnn / 1 ζπζ / ε / υιλι Significant PcO.O (shaded) p-value (Tukey) H2L2 H2L3 H3L3 H1 L1 H2L2 0.057 0.002 0 00 H2L3 0.057 0.330 0.071 H3L3 0.002 0.33 0.908 H1 L1 0.00 0.071 0.908 Chimeric 0.00 0.093 0.948 1.00 Analysis of the biological reactivity, specificity and activity of hJAA-F1 1 3hJAA-F1 1 and chimeric JAA-F11 antibodies bind to human tumor cell lines. The binding of hJAA-F11 and chimeric JAA-F11 antibodies to several human breast tumor cell lines was assessed by whole-cell ELISA. The TF-Ag-positive mouse mammary tumor 4T1 cell line served as a positive control, while the P3-X63-Ag8 myeloma cell line served as a TF-Ag-negative control. Assays were performed on three different days, and 3 to 4 cell lines were tested at one time point in four replicates with the control cell lines. A cell line was considered positive if the relative binding due to hJAA-F11 or chimeric JAA-F11 (at 50 pg / ml) was significantly higher (p = 0.05) than that of the TF-Ag-negative control myeloma. The results are shown in Figure 4. The cancer cell lines analyzed consisted of different subgroups of breast cancer: hormone receptor-positive or negative, HER2-positive or negative, and triple-negative. The estrogen receptor (ER) and progesterone receptor (PR)-positive cell lines tested were CAMA-1 and HCC-1428. Two HER2 / neu receptor-positive cell lines, HCC-1419 and UA-565, were also tested. The triple-negative breast cancer (TNBC) cell lines tested were HCC-70, MDA-MB-231, MDA-MB-468, DU4475, and BT-549. One cell line, MDA-KB2, expresses the androgen receptor but is estrogen receptor-negative. These breast cancer cell lines had been previously screened using the mouse JAA-F11 assay, and all were positive for TF-Ag expression.In this description, the results show that hJAA-F11 antibodies and chimeric JAA-F11 antibodies bind to all 10 breast cancer cell lines examined, confirming TF-Ag expression in these cell lines. Currently, there is no specific treatment for TNBC among the different breast cancer subgroups. The data obtained indicate a therapeutic role for TF-Ag in targeting aggressive TNBC and the use of hJAA-F11 antibodies to treat and improve survival in breast cancer patients regardless of recipient status. Table 4 summarizes the breast cancer types and TF-Ag expression in each cell line tested. Table 4. Table of breast cancer cell lines tested in whole cell ELISA. RPfrnnn / Lznz / E / YiAi The table shows the hormone receptor, Her2 / neu receptor, and TF-Ag expression. Triple negative (bold), ER / PR positive (italicized), and Her2-positive (bold and italicized). MDA-kb2* is negative for estrogen receptors and expresses the androgen receptor. RPfrnnn / Lznz / E / YiAi Cell line Estrogen receptor Progesterone receptor Her2 / neu receptor TF-Ag expression HCC-70 - - - + BT-549 - - - + MDA-MB-231 - - - + MDA-MB-468 - - - + DU-4475 - - - + CAMA-1 + + - + HCC-1428 + + - + HCC-1419 - - + + AU 565 - - + + MDA-kb2* - - - + Humanized JAA-F11 inhibits the proliferation of cancer cells in vitro Because some anti-TFAg antibodies have been shown to cause tumor cell proliferation, it was important to determine the effect of different constructs on cell proliferation. The MTT assay, which measures metabolic activity, was selected as a surrogate for the direct tritiated thymidine proliferative assay. The effect of humanized, chimeric, and mouse JAA-F11 antibodies on cancer cell growth compared to a control without antibodies was determined at 4 µg / ml of the antibodies using known methods. The data are shown in Figure 5. The chimeric antibody and the humanized JAA-F11 antibodies, with the exception of H3L3, caused a small (~6–11%) but statistically significant inhibition of tumor cell growth. No increase in cell proliferation was observed. Humanized JAA-F11 induces ADCC Antibody-dependent cell-mediated cytotoxicity (ADCC) was performed in human breast tumor cell lines using the non-radioactive CytoTox 96 cytotoxicity assay (Promega, Madison, Wl), based on the lactate dehydrogenase release (LDH) assay, according to standard techniques with the following adaptations. Fresh human peripheral blood mononuclear cells were isolated from anticoagulated EDTA whole blood for use with the humanized antibody and mouse 4T1 breast cancer cells in an effector at a target ratio of 100:1. LDH release was used to quantify cytotoxicity. Figure 6A shows the amount of ADCC facilitated by mouse, chimeric, H1L1, and H3L3 antibodies compared in a human breast cancer cell line, BT 549. The experiments were repeated three more times. In all experiments, H2L2 and H3L3 showed significantly more (p<0.5) ADCC than either the chimeric or mouse antibody in the cell lines. RChnnn / 1 zoz / e / uli human. More than 20% of the TF-Ag-containing BT549 and HCC70 target cells were targeted by the H2L2 and H3L3 antibodies at 200 pg / ml in a 100:1 effector-to-target (E:T) ratio. Similarly, in all experiments in the 4T1 mouse cell line, although all tested antibodies induced less than 10% ADCC, the H3L3 antibody showed significantly more (p<0.05) ADCC than either the chimeric or mouse antibody. This indicates that the H2L2 and H3L3 antibodies are currently the best option for immunotherapy. In contrast, the mouse JAA-F11 did not show statistically significant ADCC capabilities in any of the assays. The ADCC results from three donors of the individual PBMCs are as shown in Figures 6B, 6C, and 6D. The humanized JAA-F11 does not induce CDC The ability of hJAA-F11 and chimeric antibodies to mediate complement-dependent cytotoxicity (CDC) was determined by the lactate dehydrogenase release (LDH) assay using HCC-1428 human breast cancer cells as target cells. The positive LDH control supplied with the reagent kit was used as a positive control and showed lysis. Mouse, chimeric, H1L1, H3L3, and H2L3 JAAF11 antibodies did not induce complement-dependent cytotoxicity because no lysis occurred, as shown in Figure 7. Humanized JAA-F11 is internalized into cancer cells The internalization of humanized and chimeric JAA-F11 in 4T1 breast tumor cells was determined by two methods, one RPfrnnn / Lznz / E / YiAi enzyme immunoassay with measured and compared binding surface after cell incubation at either 4°C or 37°C and an immunofluorescence microscopy method using LAMP-1 (lysosomal membrane-associated protein, a lysosomal marker) and DAPI staining for nuclear staining. In the enzyme immunoassay, as shown in Figure 8, mouse, H2L2, chimeric, H2L3, and H1L1 antibodies were significantly internalized with p-values ​​of 0.001, 0.002, 0.001, 0.002, and 0.014, respectively. However, the H3L3 antibody did not show significant internalization (p = 0.16) and that is expected since H3L3 showed significantly more (p<0.5) ADCC than the chimeric or mouse JAA-F11. In immunofluorescence experiments, consistent with the enzyme immunoassay, the chimeric mouse antibodies H2L2 and H2L3 showed internalization and co-localization with LAMP-1, while H3L3 showed membrane staining indicating no internalization. These data confirmed the results obtained using the surface enzyme immunoassay. It might be expected that antibodies exhibiting higher ADCC activity would show lower internalization, as the presence of antibodies on the cell surface is necessary for ADCC function. Internalization assays were performed on the chimeric hJAAF11 and MJAA-F11 antibodies. H3L3 showed agreement with this expectation, exhibiting low levels of internalization but relatively high ADCC activity. H1L1, H2L3, the chimeric antibodies, and mJAA-F11 all showed statistically significant internalization and poor ADCC function. An unexpected result was observed for the antibody RChnnn / 1 ζοζ / ε / υιλι H2L2 induced high ADCC similar to H3L3 but showed the highest percentage of internalization activity compared to chimeric antibodies and other humanized antibodies. The immunofluorescent internalization experiment was initially performed using 5 pg / ml of antibody and was repeated using antibody concentrations of 0.1 pg / ml. The antibodies were internalized even at this low concentration. The data indicate that, due to the high percentage and rapid rate of internalization, the chimeric H2L2 and H2L3 constructs have the potential to be used as antibody-drug conjugates. Glycan specificity, relative affinity internalization, and ADCC and CDC data are summarized in Table 5 below. Table 5. Summary of internalization, ADCC and glycan specificity of JAA-F11 from mouse, humanized and chimeric. RPfrnnn / Lznz / E / YiAi JAAF11 ADCC CDC Variants Internalization Specificity Affinity Range Mouse No No Yes No Chimeric No No Yes No 3 H1L1 Yes No No Yes 3 H2L2 Yes No Yes Yes 1 H2L3 No No Yes Yes 2 H3L3 Yes No No No 3 MicroPET imaging MicroPET imaging was performed serially in each mouse injected with the 1,24Iodo-hJAA-F11 H2L2 antibody and with free 1,24Iodo. Imaging was performed at 24, 48, 72, 96, 168, and 192 hours. Figure 9 shows a coronal view of a mouse injected with the 1,24Iodo-hJAA-F11 H2L2 antibody at different time points, while Figure 10 shows a control mouse injected with free 1,24Iodo. Tumor uptake of the radiolabeled antibody was observed at 24, 48, 72, and 96 hours post-injection. Antibody uptake was also observed in the spleen at 48 hours (Figure 9). The negative control mouse that has the 4T1 TF-Ag positive tumor, which received 124 free Iodine only, showed no localization to any organ or tumor except the thyroid during the entire study (Figure 10). The fully murine JAA-F11 antibody has great potential for passive humoral immunotherapy and drug-conjugate therapy in breast cancer patients. However, since the use of mouse antibodies in humans has been shown to be limited by the development of human anti-murine antibody (HAMA) responses and a short half-life in patients, humanization is desirable to decrease the immunogenicity of the mouse JAA-F11 antibody and also allow it to remain in circulation for a longer period. It will be recognized from the above description of the results that this description provides, among other aspects, the description APfrnnn / Lznz / E / YiAi describes a novel approach to humanizing mouse JAA-F11 mAb and compares and contrasts between mouse, chimeric, and humanized JAA-F11 antibodies. The mJAA-F11 was humanized in part by CDR grafting. As will be recognized by those skilled in the technique, traditionally, CDR grafting humanization uses a single human antibody acceptor framework [Jones, et al. Replacing the complementarity-determining regions in a human antibody with those from a mouse. 1986. Nature 321: 522-525]. Therefore, the human structural sequences are selected from existing human germline genes. In contrast, in the present description, three novel and different approaches have been used to select the human acceptor antibody framework for the JAAF11 variable region heavy and light chains, generating three heavy chain variants (H1, H2, H3) and three light chain variants (L1, L2, L3).As discussed above, any of the 3 heavy chains (VH) can be associated with any of the 3 light chains (VL) to create a total of 9 possible combined hJAA-F11 VH / VL variants. A chimeric JAAF11 having the full mouse variable region and a human lgG1 and kappa constant regions was also generated and used as a control. One concern was whether the approach used in defining CDRs and FR regions would reduce the immunogenicity of humanized antibodies. The T20 scoring method developed by Gao et al. [Monoclonal antibody humanness score and its [RPfrnnn / Lznz / E / YiAi applications. 2013. BMC Biotechnology, 13:55] and validated with the analysis of 90 antibodies that have been used in patients, was used to predict the immunogenicity of humanized JAA-F11 variants. The T20 scores of the humanized JAA-F11 variants are all superior (improved) (> 85) to the chimeric JAA-F11 (<85) indicating that the humanization of heavy and light variable chains by current methods created hJAAF11 variants that will be less immunogenic than the chimeric variant. The JAA-F11 mouse was humanized and five antibodies were produced using CHO-K1 cells, a chimeric construct, and four constructs: hJAA-F11, H1L1, H2L2, H3L3, and H2L3. Therefore, given the benefit of the present description, the person skilled in the art could make use of any of the other four possible combinations of VH and VL. Humanization of antibodies by CDR grafting can cause a decrease in affinity or loss of antigen binding, which may be due to the CDR conformation and the antigen-binding site being negatively affected by changes in some amino acid residues of the β-sheet in the humanized antibody. CDRs are composed of residues that interact with β-sheet regions and other CDRs. In addition to the interaction of CDR amino acids with sequentially close amino acids, some β-sheet residues that can affect antigen binding directly or indirectly include Vernier zone residues and residues at the VL / VH interface. Vernier zone residues are residues in the β-sheet β-sheet that are based on the CDRs and provide a foundation. RPfrnnn / Lznz / E / YiAi for the formation of loop structures. The Vernier residues in the light chain are at positions 2, 4, 35, 36, 46, 48, 49, 64, 67, 69 and 71, while those in the heavy chain are identified at positions 2, 27, 28, 29, 30, 47, 48, 49, 67, 69, 71, 73, 78, 93, 94 and 103. The residues at the VL / VH interface have been identified by Chothia et al. [Chothia C, et al. The packing of variable domains. 1989. J. Mol. Biol. 186:651-63] and are found in positions 34, 36, 38, 44, 46, 87, 89, 91, 96 and 98 of the light chain and in positions 35, 37, 39, 45, 47, 91, 93, 95, 100-100K and 103 for the heavy chain. We have demonstrated that the methods used in this description for selecting the human FR and CDRs did not negatively alter the affinity of humanized JAA-F11 for TF-Ag, relative to chimeric and mouse JAA-F11. When defining the CDRs, residues within 5 Å to 6 Å of the binding site were included to ensure that affinity and specificity for TF-Ag were maintained. These residues are located at positions H31, H32, H33, H35, H50, H52, H53, H54, H95, H96, H97, H98, and H100 on the heavy chain and at positions L27d, L28, L30, L32, L34, L50, L89, L91, L92, and L96 on the light chain. However, relative affinity studies showed that while all humanized antibodies maintained affinity for TF-Ag, differences in affinity were observed among the antibodies. These studies revealed that antibody H2L2 had the highest affinity, followed by antibody H2L3. H2L2 had the highest relative affinity for TF-Ag. RChnnn / Lznz / E / YiAi among the antibodies, requiring more than 2 mg of mouse antibody for the inhibition of 1 mg of H2L2 antibody. The H2L3 antibody also had higher affinity than H1L1 and the chimeric antibody, although not significantly, while the relative affinities of H1L1, H3L3, and the chimeric antibody for TF-Ag were not significantly different from each other. These improvements in affinity can be attributed to changes in some residues between the different humanized variants. The differences between the variants are listed below in Tables 6 and 6B. Table 6A. Differences between the H1, H2, H3 heavy variables and mouse sequences. APfrnnn / Lznz / E / YiAi RPfrnnn / Lznz / E / YiAi POSITION H1 H2 H3 Ratón 5 Valine Leucine Valine Leucine 11 Valine Leucine Valine Leucine 38* Arginine Arginine Lysine Lysine 4g*** Methionine Methionine Isoleucine Isoleucine 66* Arginine Arginine Lysine Lysine 67*** Valine Valine Alanine Alanine 69*** Methionine Leucine Methionine Leucine 73** Threonine Lysine Threonine Lysine 75 Isoleucine Serine Isoleucine Serine 82b Arginine Serine Arginine Serine 83 Arginine Threonine Arginine Threonine 85 Aspartate Glutamate Aspartate Glutamate 108* Leucine Threonine Threonine Threonine 109* Valine Valine Leucine Leucine 12 Lysine Lysine Lysine Alanine 20 Valine Valine Valine Methionine 40 Alanine Alanine Alanine Arginine 76 Serine Serine Serine Threonine 81 Glutamate Glutamate Glutamate Glutamine ** indicates the position of the Vernier zone * indicates that this position is within 4 amino acids of our modified CDR Table 6B. Differences between L1, L2, L3 slight variables Rffrnnn / ίζηζ / Ε / γΐΛ mouse sequences POSITION L1 L2 L3 Mouse 1 Aspartate Aspartate Aspartate Glutamic acid 2 ** Valine Valine Valine Leucine 7 Serine Threonine Serine Threonine 14 Threonine Threonine Threonine Asparagine 17 Glutamine Glutamine Glutamine Aspartic acid 18 Proline Proline Proline Glutamine 36*** Phenylalanine Phenylalanine Tyrosine Tyrosine 37* Glutamine Glutamine Leucine Leucine 39* Arginine Arginine Arginine Urine 45 Arginine Arginine Arginine Urine 81 Glutamic acid Glutamic acid Glutamic acid Glutamic acid 83 Valine Valine Valine Leucine **Indicates the position of the Vernier zone* *Indicates that this position is within the 4 amino acids of our modified RDA As mentioned previously, the framework residues that affect antigen binding include the Vernier region and the VL / VH interface residues. When comparing humanized constructs, the mAbs described here retain most of the Vernier region and the VL / VH interface residues that may be important for binding site conformation; however, some changes were made to create less immunogenic humanized variants. The amino acids that were changed—those located in the Vernier region or the VH / VL interface—and the amino acid changes within the four amino acids (sequentially) of the CDRs are indicated by asterisks. There are four positions in the Vernier zone of the heavy chain sequence that differ among the three constructs: positions 48, 67, 69, and 73. H2 retains mouse residues but differs from constructs H1 and H3 in eight positions: 5, 11, 69, 73, 75, 82b, 83, and 85.Of these eight residues, two are Vernier zone residues, at positions 69 and 73. H2 and H1 have substitutions at two Vernier zone positions, specifically residues 48 and 67, which are retained as mouse residues in H3. Because there is no difference in affinity between H1L1 and H3L3, this suggests that the Vernier zone positions at 48 and 67 are unlikely to contribute to the affinity difference, as they are retained in H3 but changed in H1. H2, however, retains the mouse Vernier zone residues at positions 69 and 73, unlike H1 and H3, suggesting that changes at these two Vernier zone positions may contribute to the affinity differences. These two Vernier zone residues underlying CDR2 may have enhanced the binding of H2 compared to H1 and H3. Of the different amino acids among the humanized constructs, positions 69 and 108 are the closest to the binding site.The amino acid in the. RChnnn / 1 ζπζ / ε / υιλι position 69 in H2 (as in the mouse antibody) is leucine, while both of the other humanized constructs have methionine. Of the differences between H2 and the mouse antibody, the changes at amino acids 38, 48, 66, 67, and 109 are closest to the CDRs and may also affect affinity. Because H2L2 has a higher affinity than H2L3, comparing L2 and L3 can further help to delineate the reason for this difference. There were no changes in VL / VH residues, and most of the Vernier zone residues in the light chain remained consistent across the three humanized constructs, except for Vernier positions 2 and 36. Unlike L3, but similar to the mouse antibody, the higher-affinity L2 has a phenylalanine instead of a tyrosine at Vernier position 36. The amino acids at positions 36 and 37 may favorably influence the conformation, as they are located within 5 Å of the binding site and are part of the amino acids at positions 24–35, which were found to be important for maintaining the alpha-bond requirement at the reducing end of GalNAc. Many normal tissues have structures that are not identical, but very similar to TF-Ag, such as the Gaipi3GalNAc-beta linker structures normally found in the central nervous system, NK cells, and peripheral nervous tissue. Maintaining fine specificity is the key feature of this humanization. The H1L1, H2L2, and H3L3 subunits of hJAA-F11 and the chimeric antibody retain this same fine specificity. RPfrnnn / Lznz / E / YiAi showed improved antigen binding compared to the mouse antibody, including limited binding to Gaip 1-3GalNAc-α linkage structures (TF-Ag) and lack of binding to α2-3 sialylated structures based on the glycan matrix. The specificity of the humanized JAA-F11 constructs appeared to be the same or even improved when tested against more than 600 different saccharides. The mouse JAA-F11 and all humanized constructs reacted with only 5 saccharides: TF-Ag and 3 trisaccharides with an additional 1-6 sugar linked to GIcNAc (NeuAc at position 2, and GIcNAc at position 3), or with one containing two additional 1-6 sugars linked to GalGIcNAc. For example, H2L3 showed less binding to all 4 other saccharides that bind to JAA-F11, while H1L1 and H2L2 have no binding to the tetrasaccharide that binds to JAA-F11, compared to chimeric and mouse antibodies.Although these other four saccharides that bind to JAA-F11 are not known to be expressed in normal tissues, increased specificity for the target structure is always desirable. This demonstrates the likely targeting potential of all these antibodies in human therapy. Because H2L3 has increased binding specificity, as shown by binding to fewer than three of the four saccharides compared to the other constructs, the difference between H2 and H3 may have caused this difference. The differences between the variants discussed earlier in the affinity section may have been the reason for this improved specificity. H2L3 differs from H2L2 in its decreased binding to NeuAcalpha2-6(TF-Ag) and GlcNAcbeta2-6(TFRPfrnnn / Lznz / E / YiAi). Ag). Without intending to be limited by theory, it is believed that the reason H1L1 and H2L2 do not bind to the tetrasaccharide at all may be related to a shared difference between these antibodies compared to H3L3 and mouse antibodies. H1 and H2 both have a methionine at position 48, which is both a Vernier region position and sequentially within 4 amino acids of the CDR. This methionine is in place of the isoleucine found in mouse and H3 antibodies. In this description, whole-cell EIA results highlight the ability of hJAA-F11 and chimeric JAA-F11 antibodies to bind to triple-negative breast cancer cell lines. The data obtained suggest a potential therapeutic effect of TF-Ag in targeting all breast cancers, but especially aggressive triple-negative breast cancers (TNBCs), as there are currently no specific therapies for these cancer types. Some lectins and antibodies that bind to TF-Ag cause increased proliferation of TF-Ag-expressing tumor cells. These are generally thought to be related to whether the lectin or antibody binds to both the alpha and beta anomers at the reducing end (causing proliferation) or only to the alpha anomer (inhibiting). While mouse JAA-F11 binds only to the alpha anomer and did not cause tumor cell proliferation, it is important to determine whether humanized constructs had this effect on RPfrnnn / Lznz / E / YiA tumor cells. Humanized JAA-F1 1 and chimeric antibodies do not cause proliferation of cancer cells in vitro; on the contrary, similar to the effect of mouse JAA-F1 1, a small (~6-11%), but significant inhibition of mouse 4T1 and human cancer cell growth was observed. Since ADCC is one of the main mechanisms by which antibodies eliminate tumor cells, and it is important that an antibody be used for cancer therapy, the ability of humanized JAA-F11 variants to induce ADCC was examined. ADCC activity was seen with some, but not all, of the humanized constructs and was observed with PBMCs from three individuals. Both H2L2 and H3L3 antibodies induced significantly more ADCC than either of the chimeric or mouse antibodies in both 4T1 mouse and human breast cancer cell lines, indicating that H2L2 and H3L3 antibodies may currently be the preferred options for direct passive immunotherapy. Mouse JAA-F11 showed no statistically significant ADCC ability in any of the tests. Another effector function that the antibodies used to kill tumor cells is CDC.None of the humanized, chimeric, and mouse JAA-F11 antibodies induced CDC, but this lack of CDC activity does not preclude its use as an immunotherapeutic. In fact, herceptin was found to induce only a nominal amount of CDC, and this is not one of its modes of action. Data with Rituxan actually show that complement binding inhibits the binding of the APfrnnn / Lznz / E / YiAi NK cells and decreases effectiveness. Antibodies can potentially be used to transport drugs or toxins into cells as antibody-drug conjugates. Therefore, the ability of humanized JAA-F11 variants to internalize after binding to TF-Ag in cancer cells was evaluated. Mouse JAA-F11 was previously shown to be internalized within 1 hour. Using an enzyme immunoassay, the mouse antibodies H2L2, chimeric antibodies H2L3, and H1L1 were shown to be significantly internalized in mouse 4T1 breast cancer cells, while the H3L3 antibody did not show significant internalization. A live-cell fluorescence microscopy method confirmed the results obtained from the enzyme immunoassay.We expected that antibodies showing higher ADCC activity would exhibit lower internalization, and the results of the two internalization assays supported this expectation: the H3L3 antibody showed greater ADCC activity than any chimeric or mouse antibody but was not extensively internalized. However, H2L2, despite performing well in the ADCC assay, was internalized. The mechanism for both of these behaviors occurring with the same antibody is not yet understood, but one possibility is that different production rates in CHO cells may result in different fucosylation rates, such that H2L2 is less fucosylated and may therefore function better in ADCC activity while on the cell surface, even though it is internalized. RChnnn / 1 ζπζ / ε / υιλι internalizes. These results show that the chimeric constructs H2L2 and H2L3 have the potential to be used as antibody-drug conjugates. The mouse antibody 124I-JAA-F11 has been shown to localize TF-Ag-bearing breast tumors in mice. The labeled JAAF11 antibody remained bound to the 4T1 tumor for at least 20 days and 24 days for a human triple-negative breast tumor, implying that JAA-F11 could be used to find metastases and treat tumors that support TF-Ag. To see if the iodine-124-labeled humanized antibody would localize to human breast tumors in mice, the highest-affinity humanized antibody, the H2L2 variant (which internalizes and performs ADCC), was used. Imaging showed tumor uptake preference, with uptake in the thyroid and spleen being blocked in a subsequent cold rabbit immunoglobulin experiment. MicroPET imaging demonstrated that the radiolabeled humanized antibody was taken up by the tumor within 24 hours and could be visualized for up to 96 hours.Such radiolocalization in patients could be used to find metastasis and is also used to determine if there is any binding target in a particular patient prior to passive immunotherapy, whether direct or antibody-drug conjugate. The Thomsen-Friedenreich antigen (TF-Ag) is present in more than 80% of various human carcinomas, including several types of breast cancer. It plays a functional role in the The ability of an antibody to target TF-Ag suggests its potential use as an immunotherapeutic product, such as an antibody-drug conjugate, to kill cancer cells and inhibit metastasis. The JAA-F11 antibody, with its unique high specificity for TF-Ag, has great potential as a passive anti-TF-Ag response for the treatment of breast cancer and other cancers. In our humanization approach, we have to maintain this unique specificity, and we have likely enhanced it with the humanized JAA-F11 variants. Example 2 This example provides a description of the materials and methods used to obtain the results described in this document. The JAA-F11 CDRs were previously predicted. Cloning and sequencing of the mouse JAA-F11 antibody was performed to confirm the amino acid sequences of both heavy and light chain variable regions. To have therapeutic benefits in humans, an antibody must maintain its specificity for its target antigen while simultaneously not generating anti-mouse immune reactions. We selected the CDR grafting approach to retain mouse JAA-F11 CDRs to maintain specificity and affinity for TFAga. The CDRs were selected using methods by Chothia and Kabat, as well as X-ray crystal structure and carbohydrate threading. RPfrnnn / Lznz / E / YiAi digital. To select a human acceptor antibody frame for each light and heavy chain of the JAAF-11 variable region, a Blast® Protein Search (BLASTP) was performed on blast.nebi.nlm.nih.gov / Blast.cgi?PAGE=proteins against the complete, non-redundant human GenBank (Homo sapiens) database to identify the human antibodies most homologous to mouse JAA-F11. All non-Homo sapiens protein sequences, humanized antibodies, and phage presentation sequences were removed from the BLASTP results. The ten resulting sequences most homologous to each of the JAA-F11 heavy and light chain sequences were selected as potential human acceptor antibody frames for humanized JAA-F11 (hJAA-F11). The SeaView sequence alignment program was used to align the ten potential human acceptor heavy and light frame sequences with mouse JAA-F11 heavy or light region sequences. The three heavy variable regions (H1, H2, and H3) and three light variable regions (L1, L2, and L3) were constructed from BLAST protein selection of the 10 most similar human immunoglobulin sequences. The final JAA-F11 mouse CDRs were then grafted into the human framework regions by one of three different methods, creating three different heavy chains and three different light chains. In the first method, (a) the heavy H1 and light L1 Fr sequences were engineered to contain the production of human amino acids in In the second method, (b) the heavy H2 and light L2 sequences were designed to contain the most frequently produced human amino acid among the ten selected human FR sequences at each site, unless an amino acid was present in any of the top ten human FR sequences seen that matched mouse JAA-F11; subsequently, the amino acid was retained as in mouse JAA-F11. In the third method, (c) the heavy H3 or light L3 sequences, the three FR residues before and after the CDR1, CDR2, and CD3 of mouse JAA-F11 were retained, and the remaining residues were the amino acids most frequently seen in human sequences as in the H1 or L1 variants. Any of these designed heavy variable chains can be combined with any of the similarly designed light variable chains, for example. H1 / L1, H1 / L2, H1L3, H2L1, H2L3, etc. A chimeric JAA-F11 was also constructed, in which the entire mouse JAA-F1 region was joined to a human IgG1 constant region. The aim is to provide a baseline that should have the original specificity and affinity of the mouse antibody while also possessing the human constant region, for use as a positive control in the evaluation of humanized variants. Evaluation of hJAA-F11 models Several hJAA-F11 constructs were evaluated for conformational effects. Briefly, the proposed heavy and light variable chain sequences of several hJAA-F11 constructs were APfrnnn / Lznz / E / YiAi align with the JAA-F11 sequence. Any amino acid residues in the frame for each humanized variant that could potentially cause severe steric interaction with surrounding amino acids were removed and replaced with the original mouse JAA-F11 frame residue. Prediction of immunogenicity of hJAA-F11 variants using the T20 score. Although humanized or fully human antibodies are considered non-immunogenic and safe for human use, the immunogenicity of fully human and humanized antibodies has been reported in patients. To determine whether the selected sequences had low immunogenicity, an immunogenicity analysis was performed using the T20 scoring method. The T20 scoring analyzer, developed by Gao et al., calculates the humanity of monoclonal antibody variable region sequences using a database of over 38,000 human antibody sequences. In this method, a BLAST protein search is performed, and the analyzed humanized antibody is first compared against all of these human sequences. The humanized antibody is then compared to the 20 best BLAST matches of human antibodies, and the similarity to these sequences is scored. The T20 score for the humanized antibody is obtained from the average of the percentage similarity to the 20 best human sequences. The highest possible score is 100 (most similar to human). RChnnn / 1 ζοζ / ε / υιλι As a proof of concept for the relationship of this method to immunogenicity in patients, Gao et al. compared the in vivo immunogenicity results in patients of more than 90 antibodies that are approved for clinical use or in various stages of clinical development (mouse, chimeric (mouse), humanized (n = 22), and fully human antibody sequences) with the T20 score. They found that the antibodies were immunogenic, and using a T20 score for FR sequences only, those above 85 were not immunogenic. T20 scores for the humanized JAA-F11 variants were calculated using the Human T20 Database Summary at abanalyzer.lakepharma.com / . Codon optimization and gene synthesis. After designing the VH and VL amino acid sequence variants of humanized JAAF11, the corresponding nucleotide sequences were manually selected for optimal codon usage for Cricetulus griseus (CHO) cell protein production and synthesized and inserted into the pUC57 plasmid, a commonly used cloning vector. Subcloning and sequencing of hJAA-F11 and the chimeric JAA-F11 variable light (VL) and heavy (VH) genes in mammalian expression vectors. The hJAA-F11 VH genes were subcloned and inserted into an expression vector (pAH6307) containing a human IgG1 heavy chain leader / constant region under the control of the human cytomegalovirus (CMV) promoter. RPfrnnn / Lznz / E / YiAi ampicillin (Amp) and histidine dehydrogenase (hisD) cassettes. The VL genes were inserted into an expression vector (pAN 6714) containing a human kappa light chain leader / constant region under the control of the human CMV promoter and neomycin phosphotransferase (neoR) cassette. Similarly, the VH and VL genes of chimeric JAA-F11 were expressed and used as a positive control in subsequent analyses. Expression and production of humanized and chimeric JAA-F11 variants. Co-transfection in CHO-K1 cells. Chinese hamster ovary adherent cells (CHO-K1; ATCC #CCL-61, Manassas, VA) were cultured for 18 hours prior to transfection in Ham F12 medium (Corning Cellgro, Manassas, VA) supplemented with 10% fetal calf serum (FCS; Hyclone) at 37°C and 5% carbon dioxide (CO2) in humidified air and harvested at 50-80% confluence. The expression vectors 6307 pAH (VH) and 6714 pAN (VL) were co-transfected into CHO-K1 cells by electroporation (Gene Pulser System (Bio-Rad, Hercules, CA)). Specifically, each plasmid (VH and VL) was linearized using the restriction enzyme Pvul (Promega, Madison, W1), and 5 pg of each plasmid was added to 5 x 10⁶ CHO-K1 cells in cold Ham F12 medium (Corning Cellgro, Manassas, VA) to a total volume of 500 µL in a 0.4 cm³ electroporation cuvette (Bio-Rad, Hercules, CA). The cuvette containing the mixture was electroporated at 960 pF and 250 mV. Subsequently, the transfection mixture was diluted withRChnnn / Lznz / E / YiAi pre-warmed non-selective medium (Ham F12 supplemented with 10% FCS) was inoculated at a concentration of 1 x 10⁵ cells / mL. Two hundred microliters were seeded into 96-well tissue culture plates (BD Bioscience, San Jose, California) at a density of 1 x 10⁴ cells per well. Transfected cells were incubated at 37°C and 5% CO₂ in a tissue culture incubator. At 72 hours, the non-selective medium in the 96-well tissue culture plate was removed and replaced with 200 µl of Ham's F12 selective medium (plus 10% FCS) containing 700 pg / ml of G418 (Gibco, Life Technologies, Grand Island, NY), 5 mM histidinol (Sigma-Aldrich, St. Louis, MO), and 10 mM HEPES buffer (Corning Cellgro, Manassas, VA), and the colonies were allowed to grow for up to 3 weeks. CHO-K1 cells transfected with plasmids 6307 pAH and 6714 pAN will continue to grow, while non-transfected CHO-K1 cells will not.Every 2-4 days for the next 14-21 days, the selective medium was replaced to remove the remains of dead cells and the resistant cell colonies were allowed to grow in the selective medium. Analysis of culture supernatants by enzyme-linked immunosorbent assay (ELISA) Between 14 and 21 days after selection, CHO-K1 culture supernatants were collected from the 96-well plate(s) and analyzed for antibodies by enzyme-linked immunosorbent assay (ELISA). Briefly, 100 pL of culture supernatants were added to Immulon 1 B binding medium and placed in a microtiter plate. RPfrnnn / Lznz / E / YiAi wells (Thermo Scientific, Milford, MA) that had been coated with 1.25 pg / ml of TF-Ag-BSA conjugate in coating buffer (0.1 M Na2CO3 at pH 9.6) and washed five times with TBS-Brij (pH 7.2). A 1:500 dilution of purified mouse JAA-F11 (1 mg / ml) was also added to sterile PBS in 2-3 wells in each plate to serve as positive controls. After a 2-hour incubation at 37°C, the plates were washed five times and 100 pL of anti-human IgG-alkaline phosphatase conjugate secondary antibody (Sigma-Aldrich, St. Louis, MO) in 1% BSA-PBST buffer (1:10000) was added to each well for the transfected cells, while a 1:1000 dilution of secondary antibody (Sigma-Aldrich, St. Louis, MO) in 1% BSA-PBST buffer was used for the mouse JAA-F11 control well.After 1 hour of incubation at room temperature, the plates were washed five times, and then 100 µL of phosphatase substrate (p-nitrophenyl phosphate pNPP) (Sigma-Aldrich, St. Louis, MO) was added to each well. The plates were read at 405 nm using a plate reader after 1 hour of incubation at room temperature to detect the presence of IgG antibodies to TF-Ag. As a blank, HAM F12 medium (plus 10% FCS) was used during the primary antibody incubation, followed by the secondary antibody incubation and the substrate incubation. Generation and production of stable clones After initial screening for the anti-TF-Ag antibody on days 14-21 after screening began, 10-12 clones with the highest absorbance readings were selected and transferred RPfrnnn / Lznz / E / YiAi were transferred to 24-well tissue culture plates for expansion. The culture supernatants from these clones were subsequently analyzed by ELISA. The four clones that yielded the highest absorbance readings were transferred to T25 tissue culture flasks, expanded, and re-screened for anti-TF-Ag antibody by ELISA to ensure that the clones were still producing TF-Ag-reactive antibodies. These four clones were then transferred to T75 tissue culture flasks, expanded, and re-screened for anti-TF-Ag antibody, and the cells were stored frozen in liquid nitrogen. The clone with the highest anti-TF-Ag result was expanded and passed two to three times to ensure cell line stability. Stable single-cell clones for each hJAA-F11 heavy- and light-chain combination of the best previously obtained clone were generated through limiting dilution subcloning. Briefly, cells were cultured to confluence in a T75 tissue culture flask, harvested, counted, and seeded at a dilution of 0.3 cells / well into each well of 96-well plates in selective medium. The plates were then carefully examined under a microscope for 7–10 days, and wells showing a single focus of cells were labeled and monitored until sufficient growth density was observed. The supernatants from these labeled wells were assayed for anti-TF-Ag antibody by ELISA. The four clones that gave the highest absorbance readings were Rffrnnn / Lznz / E / YiAi were expanded as described above to obtain the parental clones. The sub-clone that gave the highest absorbance was expanded and used for supernatant production in T200 NuncTM Tripleflasks cell culture (Thermo Fisher Scientific, Inc.). For the production of Tripleflasks for each humanized JAA-F1 variant, cells were cultured for 3 weeks before the collection of 1 liter of culture supernatant. The clone producing the chimeric JAA-F11 was processed in the same manner as the humanized JAA-F11 variants. Purification of humanized and chimeric JAA-F11 variants The supernatants of humanized and chimeric JAA-F11 variants were purified using a Protein A-Sepharose® 4B, fast-flow affinity column (Sigma-Aldrich, St. Louis, MO). To prepare the Protein A column, a 1:1 suspension of resin in Buffer A (0.02 M NaH₂PO₄, 0.15 M NaCl, pH 8.0) was poured into the column. After the column was adjusted, it was washed with 20 column volumes (CVs) of Buffer A. One liter of CHO-K1 cell culture supernatant was centrifuged at 3,500 rpm for 30 minutes to remove dead cells or debris and then filtered. The filtered supernatant was loaded onto the Protein A column and allowed to trickle down by gravity at a flow rate of 1 mL / min. Subsequently, the column was washed with 10 CVs of buffer A. The antibody was eluted from the protein A column using 3 CVs of buffer B (0.2 M Na₂HPO₄, 0.1 M citric acid, pH 3.9). The eluate was carefully neutralized with 0.1 M NaOH to minimize the effect The low pH RPfrnnn / Lznz / E / YiAi antibody column was re-equilibrated with 20 to 30 CV of buffer A and stored at 2–8°C. Each column was used up to five times for the same antibody. The purified antibody was dialyzed overnight at 4°C (Slide-A-Lyzer Dialysis cassette; Thermo Scientific) against either phosphate-buffered saline (PBS) or RPMI medium with or without phenol red, as required for downstream assays. After dialysis, the antibody was filtered through a 0.22 µm filter (Corning), and the protein concentration was determined by a Bio-Rad protein assay as detailed below. The antibody was stored at 4°C and tested for TF-Ag binding using ELISA. Bio-Rad protein assay The antibody concentration was determined using the Bio-Rad protein assay, which is based on the Bradford dye-fixation method. In short, the dye reagent was diluted with distilled water in a 1:4 ratio and filtered using Whatman #1 filter paper. Serial dilutions of a known protein standard (1.44 mg / mL gamma globulin, Bio-Rad, Hercules, CA) and the humanized antibody sample were prepared using 1X PBS buffer. Ten microliters of each standard and sample solution were placed in triplicate into wells of a microtiter plate. Two hundred microliters of the diluted dye reagent were then added to all wells using a multichannel pipette and thoroughly mixed by pipetting up and down without creating bubbles. The plate was incubated at room temperature for at least 5 minutes to 1 hour. RChnnn / 1 ζπζ / ε / υιλι The absorbance was then read at 595 nm on the plate reader (Bio-Tek Instruments). Analysis of the chemical specificity and affinity of humanized and chimeric JAA-F1 antibodies Determination of chemical specificity The chemical specificity of the various JAA-F11 and chimeric JAA-F11 constructs was determined using the imprinted glycan array. This was compared with previously obtained data for mouse JAA-F11. The glycan array is an indirect immunofluorescence method, described on the Functional Glycomics Consortium website. Each construct was tested for reactivity with 610 different glycans. In short, the imprinted array was sequentially incubated with the antibody, washed, and then incubated with a secondary antibody labeled with FITC. After washing, the image was read on a Perkin Elmer Microscanarray XL4000 spectrophotometer, and a TIFF file was saved for image analysis using Imagene V.6 image analysis software. The relative binding of each glycan was expressed and normalized to the TF-Ag binding of the original glycan.The comparison and statistical analysis of relative bonding capabilities were performed using ANOVA. Determination of relative affinity The relative affinity binding of hJAA-F11 to TF-Ag was analyzed using a competitive ELISA inhibition with mouse JAAF11 antibody and chimeric JAA-F11 antibody. Briefly, 3 pg / ml Humanized JAA-F11 (hJAA-F11) or chimeric JAA-F11 (chimeric JAA-F11) was incubated in the presence of different concentrations of mouse JAA-F11 (10, 8, 6, 4, 2 µg / mL) in 96-well plates coated with 1.25 µg / mL TF-Ag (Thermo Scientific, Milford, MA). Bound antibody was detected by incubation with anti-human IgG secondary antibody, and the substrate and absorbance readings were obtained as described in the Materials and Methods section. The amount of mouse antibody required to inhibit the binding of 3 pg / mL humanized or chimeric antibodies by 50% was determined and compared. The higher the amount of mouse antibody required for inhibition, the greater the relative affinity of the antibody. The relative affinity of each of the humanized and chimeric antibodies was compared using ANOVA. Analysis of the biological efficacy of hJAA-F11 Evaluation of binding to human breast tumor cell lines The binding of hJAA-F11 to various human breast tumor cell lines was analyzed by whole-cell ELISA. The mouse 4T1 breast tumor cell line, which has TF-Ag, served as a positive control, while the P3-X63-Ag8 myeloma cell line (ATCC number: CRL-1580), which was the fusion partner for JAA-F11 hybridoma production, served as a negative control for TF-Ag. Positive binding was determined by comparing the reactivity of each antibody with each cell line to the antibody's reactivity with the cell line. APfrnnn / Lznz / E / YiAi myeloma using the Student's t-test. To normalize the data, the binding to each cell line was expressed by dividing the absorbance reading of the test cell line by the absorbance reading of the myeloma cells. Cell preparation Myeloma and 4T1 breast tumor cell lines were collected using a non-enzymatic Cellstripper (Mediatech, Inc., VA, USA). To avoid cell agglutination, the media, buffer, and reagents were pre-warmed before use. For adherent cells, the medium was removed from the culture vessels, and the cells were washed with 1X Dulbecco's phosphate-buffered saline (DPBS) without calcium and magnesium (Mediatech Inc., Cellgro). Five milliliters of cell dissociation solution were added to each flask, and the flasks were then incubated at 37°C for 10 minutes. The flasks were gently tapped to deagglutinate the cells. For non-adherent myeloma cells, the flasks were thoroughly tapped, and the culture supernatant was centrifuged at 1,000 x G for 10 minutes. The cell pellets were resuspended in the cell dissociation solution and incubated at 37°C for 10 minutes.After incubation, 20 mL of 1X DPBS were added to the cells and pipetted several times to remove clumps. The cell suspensions were centrifuged at 1,000 x G for 10 minutes. The supernatants were decanted, and the pellets were resuspended in 5 mL of DPBS. The cells were counted using trypan blue staining and a hemocytometer. RChnnn / 1 zoz / e / uli were diluted to obtain 1 x 10⁶ viable cells / ml. Two hundred microliters of the cell suspensions (2 x 10⁵ cells) were placed into 5 ml polystyrene quadruple tubes. Two hundred microliters of 4% formaldehyde solution were added to each tube and incubated for 20 minutes at room temperature before being centrifuged at 1,500 x G for 10 minutes. The supernatant was carefully decanted into a throw, the cells were washed in DPBS, followed by centrifugation and decanting. Two hundred microliters of 1% BSA (w / v) PBS-Tween were added to each tube and stored at 4°C overnight or for up to two weeks. Enzyme immunoassay in cells Two hundred microliters of 50 pg / ml mouse JAA-F11, hJAAF11, or chimeric JAA-F11 antibody were added to tubes containing the different cell lines tested in quadruplicate and incubated at 37°C for two hours. A set of tubes treated with 200 µL of 1X PBS-0.1% Tween 20-1% BSA served as a negative control for each cell line tested. The tubes were washed three times with 3 ml of wash buffer (1X PBS Tween, azide-free), then centrifuged for 10 minutes at 1,500 x G. The supernatant was carefully decanted between each wash. Two hundred microliters of anti-mouse IgG (specific y chain) radish peroxidase secondary antibody (1:1,000, SigmaAldrich, St. Louis, MO) or anti-human IgG radish peroxidase secondary antibody (specific y chain) (2000, Sigma Aldrich, St. Louis, MO 1) were added in PBS-Tween-1% BSA to the respective tubes and then incubated for 1 hour at room temperature (RT).After incubation, the tubes were decanted and washed three times, centrifuged at 1,500 x G between washes. Two hundred microliters of ophenylenediamine dihydrochloride (OPD) substrate solution (Sigma, St. Louis, MO) were then added to each tube and incubated for 1 hour at room temperature. After incubation, the reaction was stopped by adding 100 pL of chelating solution (1N H₂SO₄) and centrifuged for 10 minutes at 1,500 x G. Subsequently, 200 µL of supernatant was removed from each tube and transferred to the respective wells in a microtiter plate. Absorbance was read at 490 nm using a microplate reader (Microplate Autoreader, Model EL31 1, BioTek Instruments, Inc.), and unreacted OPD was used as a blank substrate. For the different cell lines, each respective average blank (tubes with only PBS-Tween-1% BSA) is subtracted from its respective average OD to obtain the final optical reading.Each experiment was repeated 3 times. Effects of hJAA-F1 1 on the proliferation of cancer cells in vitro To examine the effects of humanized and chimeric JAA-F11 antibodies on cancer cell growth, the 3-[4,5-dimethyltetrazolium-2-iodine]-2,5-diphenyltetrazolium bromide (thiazolyl blue, MTT) proliferation assay was performed. Mouse 4T1 mammary tumor cells and human mammary tumor cells were seeded at 1 x 10⁴ cells / well in 10 replicates. RChnnn / 1 ζπζ / ε / υιλι was seeded in 96-well plates in the presence of varying amounts of JAAF11, humanized JAA-F11, and chimeric JAA-F11 (4, 2, and 1 mg / ml). When seeded at this cell density, the cells were in the linear portion of their growth curves at 72 hours. Cells grown in antibody-free culture medium served as normal growth controls. The culture medium was used only as a blank. After 68 hours of cell growth at 37°C, 10 μL of the tetrazolium salt MTT (5 mg / ml) was added to each well, and the plates were returned to the incubator for 4 hours. At the end of the incubation, the formazan product resulting in each well was solubilized by the addition of 120 pL of dimethyl sulfoxide (DMSO, Fisher Scientific) and the absorbance of each well was subsequently measured at 570 nm (Microplate Autoreader, Model EL31 1, BioTek Instruments, Inc.). Antibody-dependent cellular cytotoxicity assay To examine the effector functions of hJAAF11 antibodies, antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) assays were performed. ADCC was determined by the lactate dehydrogenase (LDH) release assay (CytoTox 96 non-radioactive cytotoxicity assay; Promega, Madison, Wl) using human peripheral blood mononuclear cells (PBMCs) as effector cells and human breast tumor cell lines as target cells in an effector to target ratio (E:T) of 100:1. RPfrnnn / Lznz / E / YiAi PBMCs were prepared from whole blood by Ficoll-Paque density gradient centrifugation. Whole blood drawn into EDTA vacuum tubes with a violet top was mixed with an equal volume of pre-warmed sterile DPBS. Twenty milliliters of this diluted blood were then gently layered into 15 ml of Ficoll-Paque Plus (GE Healthcare) in 50 ml conical tubes. The samples were centrifuged at 1,300 rpm for 30–40 minutes at room temperature without standing. The PBMC layer was then collected, and sterile PBS was added to the PBMCs to a total volume of 40 ml. The mixture was subsequently centrifuged at 1,000 rpm for 10 minutes at 18–22°C to remove any contaminating Ficoll and platelet / plasma proteins. The supernatant was discarded, the cells were resuspended in fresh sterile PBS and the centrifugation step was repeated.The cells were resuspended in RPMI 1640 culture medium (10% FCS) and counted using a hemocytometer and trypan blue. Target cells (1 x 10⁴; 30 µL) and PBMCs (1 x 10⁶; 30 µL) were added to 96-well U-bottom plates and incubated with hJAA-F11 or chimeric antibodies (200 pg / ml; 30 pL) for 17 hours at 37°C in a tissue culture incubator. Forty-five minutes before the end of the 17-hour incubation period, 10 g L of lysis solution (x10) was added to the wells containing the target cell maximum LDH release control (target cells and medium) and the volume correction control (medium only). At the end of the incubation period, the plate was centrifuged for 4 minutes at 1,000 rpm. RChnnn / 1 ζπζ / ε / υιλι Fifty-microliter aliquots were then transferred from all wells to a fresh, flat-bottomed, 96-well plate. Fifty milliliters of reconstituted substrate mixture were then added to each of these wells. The plate was covered with aluminum foil and incubated for 30 minutes at room temperature. The reaction was stopped by adding 50 milliliters of chelating solution to each well, and the absorbance was recorded at 490 nm using a microplate reader (Microplate Autoreader, Model EL311, Bio-Tek Instruments, Inc.). Cytotoxicity was calculated using the formula: Cytotoxicity (%) = 100 x [(E - SE)] / [(M - SE)], where the substrate absorbance is measured for each condition. The conditions are as follows: E is the experimental well, SE is the spontaneous release without antibody control (target cells incubated with the voter cells and PBS), M is the maximum release determined by target cells lysed with a 10X lysis solution. Complement-dependent cytotoxicity assay (CDC) CDC was determined by the lactate dehydrogenase (LDH) release assay (CytoTox 96 non-radioactive cytotoxicity assay; Promega, Madison, W1) using HCC 1428 mammary tumor cells (ATCC® CRL-2327, Manassas, VA) as the target cell population. An LDH-positive sample containing bovine heart LDH, provided with the reagent kit, was used as a positive control. After comparing rabbit, baby rabbit, and guinea pig complement samples to background cytotoxicity levels, RChnnn / 1 zoz / e / uli used lyophilized guinea pig serum (CL3112, Cedarlane Laboratories, Burlington, NC) as the complement source and reconstituted according to the manufacturer's instructions. The optimum cell concentration used in the assay was 1 x 10⁴ cells / 50 pL per well. The antibodies used were dialyzed against RPMI-free phenol red medium. For the experimental wells, 50 pL of HCC 1428 cell suspension, 20 pL of complement dilution (final dilution to 1:20), and 30 pL of antibody (final concentration to 100 pg / ml) were mixed into each well in quadruplicate sets in a 96-well round-bottom culture plate. Control wells containing the target cells and complement without antibody were included to monitor for any serum-mediated cytotoxicity used as a complement source and spontaneous LDH release.The plate was centrifuged at 1,000 rpm for 4 minutes and then incubated for 2 hours at 37°C with 5% carbon dioxide. Forty-five minutes before the end of the 2-hour incubation, 10 pL of lysis solution (x10) was added to the wells containing the maximum target cell LDH release control and the volume correction control. At the end of the incubation period, the plate was centrifuged for 4 minutes at 1,000 rpm. Fifty microliter aliquots were then transferred from all wells to a fresh, flat-bottomed, 96-well plate. Fifty milliliters of reconstituted substrate mixture were then added to each well. The plate was covered with filter paper. APfrnnn / Lznz / E / YiAi aluminum was added and incubated for 30 minutes at room temperature. The reaction was stopped by adding 50 pL of chelating solution to each well, and then the absorbance was recorded at 490 nm using a microplate reader (Microplate Autoreader, Model EL311, Bio-Tek Instruments, Inc.). Cytotoxicity was calculated using the formula: Cytotoxicity (%) = 100 x [(E - SE)] / [(M - SE)], where the substrate absorbance is measured for each condition. The conditions are as follows: E is the experimental well, SE is spontaneous release without antibody control (target cells incubated with complement and medium), and M is release from target cells using a 10X lysis solution. Cell internalization assays The internalization of humanized and chimeric JAA-F11 in 4T1 breast tumor cells was analyzed using two methods, an enzyme immunoassay with the binding surface measured at 2 incubation temperatures, 4°C and 37°C, and an immunofluorescence microscopy method. Enzyme immunoassay method of internalization The 4T1 mouse mammary cancer cell line was tested for internalization of JAA-F11, hJAA-F11, or chimeric JAA-F11. Five hundred thousand (5 x 10⁵) cells were seeded into the six wells of a 6-well plate and cultured to confluence. Two plates were prepared for each antibody tested. The medium was removed from each plate, and 1 mL of antibody (200 pg / mL) was added to three wells of each plate, followed by 1 mL of PBS dilution to the three wells. RPfrnnn / Lznz / E / YiAi remaining. One set of plates was incubated at 37°C to allow internalization, and the other plate was incubated at 4°C. After 1 hour of incubation, the medium was removed, and the wells were washed 4 times with 1 mL of RPMI-free phenol red medium. Then, 1 mL of 2% paraformaldehyde was added to each well, and the plates were incubated at room temperature for 20 minutes to fix the cells. The plates were then washed 4 times with RPMI-free phenol red medium. Next, 1 mL of anti-mouse or anti-human IgG alkaline phosphatase secondary antibody (specific y chain) (1:5,000, Sigma, St. Louis, MO) was added in 1% BSA / PBS, and the plates were incubated at 37°C for 1 hour. Next, the plates were washed 4 times with phenol red RPMI-free media and 1 ml of p-nitrof phosphate substrate in i I o (pNPP) was added and incubated for 1 hour in the dark.After a 1 h incubation, 200 µL were transferred from each well to the respective wells of a 96-well plate, and the absorbance was recorded at 405 nm using a microplate reader (Microplate Autoreader, Model EL31 1, Bio-Tek Instruments, Inc.). Unreacted substrate was used as a blank. Wells were averaged in triplicate, and the mean optical density of the blanks containing only medium was subtracted from that of the wells containing antibodies. The percentage of internalization was calculated using the formula: % Internalization = 100* [1- (sample at 37°C - blank at 37°C) / (sample at 4°C - blank at 4°C)] Immunofluorescence microscopy method In this method, 4T1 breast cancer cells were seeded on coverslips in the wells of two 6-well tissue culture plates at a density of 3 x 10⁵ cells in 10% RPMI 1640 FCS medium and incubated at 37°C and 5% CO₂ for 24 hours. The medium was removed from the plates, and 1.5 mL of test antibodies (5 pg / mL) diluted in serum-free RPMI 1640 medium was added to the respective wells. Both plates were incubated at 4°C for 20 minutes to allow surface antibody binding. After the 20-minute incubation, the coverslips were transferred from one of the plates into a new 6-well plate. The antibody dilutions were removed from the second plate and pre-warmed serum-free RPMI 1640 medium was added and the plate was incubated at 37°C for 1 hour in a tissue culture incubator.The coverslips from the first plate were washed twice with ice-cold 5% BSA / PBS, rinsed once with 1X PBS, and then fixed with 4% paraformaldehyde solution (Affymetrix) for 15 minutes at room temperature. After incubation for 1 hour at 37°C, the coverslips from the second plate were transferred to a new 6-well plate, washed, rinsed, and fixed. After three washes in 1X PBS, the cells in both plates were permeabilized with 0.1% Triton X-100, 0.1% sodium deoxycholate in PBS for 10 minutes at room temperature. Subsequently, the cells were washed three times with 1X PBS and then incubated in 5% BSA / PBS for 30 minutes at room temperature. The coverslips were incubated with antibody. Rabbit anti-lysosomal membrane protein 1 (LAMP1; Abcam, 24170) Rffrnnn / Lznz / E / YiAi at 1 pg / mL dilutions in 5% BSA / PBS for 1 hour at room temperature. The coverslips were subsequently rinsed three times with 1X PBS and incubated with Alexa 647 anti-mouse IgG and Alexa 488 anti-rabbit IgG (mouse JAA-F11 coverslips) and Alexa 647 anti-mouse IgG and Alexa 488 anti-rabbit IgG secondary antibodies (chimeric and hJAA-F11) (Molecular Probes, Invitrogen) at a 1:500 dilution in 5% BSA / PBS. The Alexa 488 anti-rabbit IgG secondary antibody was used to detect anti-LAMP 1 antibody. Coverslips were incubated for 1 hour at room temperature in the dark. After incubation, the cells were washed three times with 1X PBS in the dark.The coverslips were placed cell-side down on SlowFade Gold reagent (Molecular Probes, Life Technologies) with DAPI medium on microscope slides and sealed with nail polish. The cells were analyzed using an Axiolmager fluorescence microscope (Zeiss). Images were captured and analyzed using AxioVision Release 4.8.2 software. In vivo studies to test the efficacy of humanized JAA-F11 in detecting TF-Ag-bearing breast tumors Using the H2L2 humanized JAA-F11 antibody obtained from previous in vitro biological studies, immunolocalization studies were performed in mice to test whether the iodine-124-labeled humanized antibody will localize TF-Ag mammary tumors in mice. RChnnn / Lznz / E / YiAi hJAA-F11 (H2L2) antibody labeled with

[124] iodine The

[124] iodine labeling of the humanized JAA-F11 antibody was performed using the Bolton Hunter method. Prior to iodination, the humanized antibody was first modified using a water-soluble Bolton Hunter reagent (Sulfo-SHPP) (Thermo Scientific, Rockford, IL, USA). Briefly, 1.9 mg of hJAA-F11 antibody was dissolved in the modification buffer (200 mM borate buffer, pH 9.0). Five mg of the water-soluble Bolton Hunter reagent were dissolved in 1 mL of modification buffer immediately before use. 100 pL of the water-soluble Bolton Hunter reagent solution was then added to the antibody sample and incubated on ice for 3 hours with periodic mixing. The unreacted water-soluble Bolton Hunter reagent was separated by dialysis against phosphate-buffered saline (PBS: 0.1 M sodium phosphate, 150 mM sodium chloride).In this intermediate step, the humanized antibody contains a linker attached to some of the antibody's spindles and is stable for 2 weeks. The modified hJAA-F11 antibody was subsequently labeled using the Chizzonite indirect titration method

[158] . Briefly, a pre-coated Pierce iodination tube (Thermo Fisher, Rockford, IL, USA) was pre-moistened with 1 mL of HighTris iodination buffer (Tris-HClO, 1.25 M, pH 6.8, 0.15 M NaCl). The buffer was decanted, and then 100 pL of HighTris iodination buffer was added directly to the bottom of the tube, followed RPfrnnn / Lznz / E / YiAi was activated by the addition of 280 pL (4.59 millicuries) of sodium iodide-1241 in 0.02 M NaOH solution (IBA Molecular, Richmond, VA). An initial count was performed using a CRC 12 radioisotope calibrator (Capintec). The pH of the mixture was measured to ensure it was neutral at 7. After activation for 6 minutes at room temperature, the activated iodide was added to the previously modified huJAA-F11 solution. After a 9-minute incubation period, 50 pL of scavenging buffer (10 mg tyrosine / mL in Tris-iodination buffer; 25 mM Tris, pH 7.5, 0.4 M NaCl) and the mixture were incubated for 5 minutes. The purpose of the cleaning buffer is to remove the free iodine that reacts with the tyrosine in the buffer. A 1 mL buffer of Tris / NaCl / EDTA (25 mM Tris-HCl, pH 7.5, 0.4 M NaCl, 5 mM EDTA, 0.05% sodium azide) was subsequently added to the reaction mixture. The sample was then added to a 10 mL desalination column that had been previously equilibrated with Tris / NaCl / EDTA buffer. The sample tube was washed with 0.5 mL of Tris / NaCl / EDTA buffer and washed with the added wash. The sample was eluted in fifteen 500 pL fractions using Tris / NaCl / EDTA buffer and assayed for radioactivity. Radiolabeling efficiency was determined in the fraction with the highest activity by high-performance liquid chromatography (HPLC). The radiolabeled antibody was injected into the animals within 2 hours of labeling. To ensure that the labeled humanized JAA-F11 maintains its reactivity to TF-Ag, a radioimmunoassay was also performed. RPfrnnn / Lznz / E / YiAi Animals and tumor models The animals in this study were housed and used in accordance with the Institutional Animal Care and Use Committee (IACUC) regulations. All protocols were approved by the IACUC at the University of Buffalo. Mouse 4T1 mammary cancer cells were implanted into 7- to 8-week-old BALB / C mice by injecting 5 x 10⁴ cells in 0.1 mL of D-PBS subcutaneously beneath one of the right teats. Mice were divided into two groups and injected with [1,24I]-hJAA-F1 (n = 13) and [1,24I]-f1 (n = 7) 0-14 days post-implantation via the tail vein and then underwent biodistribution studies and micro-PET imaging. One mouse from each of the two groups was photographed and followed throughout the study. All mice received water with 0.2 g / L after injection with labeled antibody and throughout the study as a thyroid blockade regimen. Biodistribution studies Mice were euthanized by intraperitoneal injection of 0.1 mL of sodium pentobarbital (Fatal Plus) at 72, 96, 168, and 192 hours post-injection of the radiolabeled antibody. At each time point, three mice receiving the labeled antibody and two mice receiving free iodine were euthanized. Blood, muscle, spleen, lungs, kidneys, heart, liver, small and large intestines, stomach, brain, skin, tumor tissue, bone, tail, esophagus, thyroid, and ovaries were analyzed. RPfrnnn / Lznz / E / YiAi were collected and placed into pre-weighed 5 ml polypropylene tubes. These tubes were reweighed to obtain the actual weight of each tissue / organ. All tubes were capped, and radioactivity was measured using a gamma counter. The radioactivity absorption for each tissue was calculated as the percentage of the injected dose per gram of tissue (% ID / g) according to the following formula as in Appendix 2: Micro-PET imaging The localization of the labeled antibody in one mouse from each of the two groups was determined by microPET imaging using the Focus 120® microPET camera (Siemens Concorde Microsystems) at 24, 48, 72, 96, 168, and 192 hours post-injection using established techniques. Briefly, prior to digitalization, mice were anesthetized with O2 / isoflurane (1%–3% isoflurane), and images were then acquired in the prone position in the microPET scanner portal. The emission analysis window was set between 350 and 750 keV. The scan was performed for 30 minutes for each mouse. Radioimmunoassay To determine the immunoreactivity of the radiolabeled antibody, a radioimmunoassay (RIA) was performed. A microtiter plate was coated with 100 pL of 1.25 pg / mL TF-Ag-BSA conjugate in coating buffer. The buffer was removed from the wells and washed with 1% BSA / PBS-Tween. One hundred microliters of serial dilutions of radiolabeled hJAA-F1 in 1% BSA / PBS-Tween RChnnn / 1 ζπζ / ε / υιλι were added to the wells and allowed to bind at room temperature for 1 hour. After incubation, unbound antibody was removed by washing the wells three times manually with 1% BSA / PBS-Tween using a multichannel pipette. Bound hJAAF11 was removed from the plate by incubating with 200 pL of 1M acetic acid / 0.15M NaCl buffer (pH 2.4) for 30 minutes at room temperature. After incubation, 100 pL of solution from each well were added to separate polypropylene test tubes, and radioactivity was measured using a gamma counter. Although the invention has been described through specific modalities, routine modifications will be evident to those skilled in the art, and such modifications are intended to be within the scope of the present invention. RPfrnnn / Lznz / E / YiAi Rpfcnnn / Lznz / Em

Claims

1. - A monoclonal antibody (mAb) that binds specifically to Gaipi-3GalNAc-a of TF-Ag, the monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a sequence consisting of: EVQLVESGAEVKKPGASVKVSCKASGYTFTTYWMHWVKQAPGQGLEWIG FISPNTDYTEYNQKFRDKATMTADTSISTAYMELSRLRSDDTAVYYCARSF IGYNFDFWGQGTTLTVSS (SEQ ID NO: 9) (H3); and where the light chain comprises a sequence consisting of: DVVMTQSPLSLPVTLGQPASISCRSSQTIVYSNGNTYLEWYLQRPGQSPR LLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVP FTFGSGTKLEIK (SEQ ID NO: 12) (L3). 2,- The mAb according to claim 1, wherein the mAb comprises a human IgG constant region.

3. The mAb according to claim 1, wherein the mAb is conjugated with an agent selected from the group consisting of chemotherapeutic drugs, toxins, and radioactive isotopes.

4. One or more mAbs according to claim 1, for use in the prophylaxis and / or therapy of cancer in an individual, wherein the cancer comprises cancer cells expressing TF-Ag, the use comprising administering to the individual the one or more mAbs according to claim 1, wherein the growth, or survival, or metastasis, or a combination thereof, of the cancer cells in the individual is inhibited subsequent to administration. 5.- The one or more mAbs to be used according to claim 4, wherein the mAb comprises a human IgG constant region.

6. The one or more mAbs for use according to claim 4, wherein the mAb is conjugated with an agent selected from the group consisting of chemotherapeutic drugs, toxins, and radioactive isotopes. 7.- A pharmaceutical composition, characterized in that it comprises the mAb according to claim 1. 8.- An in vitro cell culture, characterized in that the cells in the cell culture express the mAb according to claim RPfrnnn / ίζηζ / Β / γι 1.