Anti-polysialic acid antibodies and uses thereof

Abstract

The present invention relates generally to immunoglobulin-related compositions (e.g., antibodies or antigen-binding fragments thereof) that can bind to polysialic acids and neutralize their activity, and uses thereof. The antibodies of the present technology are useful in methods of detecting and treating polysialic acid-associated cancers in a subject in need thereof.

Description

This application is a divisional application of the invention application with the application date of March 13, 2019, the Chinese application number of 201980031623.6, and the invention name of “Anti-polysialic acid antibodies and their uses”. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 643,141, filed on March 14, 2018, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0002] The present technology generally relates to the preparation of immunoglobulin-related compositions (e.g., antibodies or antigen-binding fragments thereof) that specifically bind to polysialic acid and the use of the immunoglobulin-related compositions. Specifically, the present technology relates to the preparation of polysialic acid neutralizing antibodies and the use of the antibodies in detecting and treating polysialic acid-related cancers. Background Art

[0003] The following description of the background of the present technology is provided only to assist in understanding the present technology and does not admit that the description describes or constitutes prior art to the present technology.

[0004] Abnormal glycosylation has long been considered a hallmark of cancer (Hakomori, S. (1994) Prog Brain Res 101, 241-250). Tumor-associated carbohydrate antigens have been shown to be associated with tumor proliferation, invasion, angiogenesis, metastasis and immunity. Specifically, sialic acid (also known as N-acetylneuraminic acid) is a carbohydrate unit that has shown particular relevance and is included in several cancer-associated glycolipids, including gangliosides GD2, GD3, GM1, GM2 and GM3 (Daniotti, JL, et al. (2013) Front Oncol 3, 306). Summary of the invention

[0005] In one aspect, the present disclosure provides a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domain (V L ) or its antigen-binding fragment, wherein (a) said V H A V comprising a V selected from DYYIH (SEQ ID NO: 1), RYYIH (SEQ ID NO: 7), GYYIH (SEQ ID NO: 8) and NYYIH (SEQ ID NO: 9) H-CDR1 sequence selected from WIYPGSGNTKYNEKFKG (SEQ ID NO: 2), SIYPGSGNTKYNEKFKG (SEQ ID NO: 10), RIYPGSGNTKYNEKFKG (SEQ ID NO: 11), CIYPGSGNTKYNEKFKG (SEQ ID NO: 12), WIYPGSGNTKYNEKFEG (SEQ ID NO: 12) NO:13), WIYPGSGNTKYNQKFQG (SEQ ID NO:14), WIYPGSGNTKYSQKFQG (SEQ ID NO:15), WIYPGSGNTKYSEKFQG (SEQ ID NO:16) and WIYPGSGNTKYSQKFKG (SEQ ID NO:18) H -CDR2 sequence and V of GGKFAMDY (SEQ ID NO: 3) H -CDR3 sequence; and / or (b) the V L comprising a V selected from RSSQSLVHSNGNTYLY (SEQ ID NO: 4) and RSSQSLVHSNGKTYLY (SEQ ID NO: 20); L - CDR1 sequence, V of RVSNRFS (SEQ ID NO: 5) L - CDR2 sequence and a V selected from FQGTHVPYT (SEQ ID NO: 6), FQGTHVPYI (SEQ ID NO: 21) and FQGTHEPYT (SEQ ID NO: 22) L -CDR3 sequence.

[0006] In some embodiments, the present disclosure provides a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domain (V L ) or an antigen-binding fragment thereof, wherein (a) V H V comprising SEQ ID NO: 1 H -CDR1 sequence, V of SEQ ID NO: 2 H -CDR2 sequence, V of SEQ ID NO: 3 H - CDR3 sequence; and / or (b) V L Contains V selected from the following L -CDR1 sequence, V L -CDR2 sequence and V L- CDR3 sequences: SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6; SEQ ID NO:20, SEQ ID NO:5 and SEQ ID NO:6; SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:22.

[0007] In some embodiments, the present disclosure provides a light chain immunoglobulin variable domain (V L ) and the heavy chain immunoglobulin variable domain (V H ) or an antigen-binding fragment thereof, wherein (a) V L V comprising SEQ ID NO: 4 L -CDR1 sequence, V of SEQ ID NO: 5 L -CDR2 sequence, V of SEQ ID NO:6 L - CDR3 sequence; and / or (b) V H Contains V selected from the following H -CDR1 sequence, V H -CDR2 sequence and V H -CDR3 sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3; SEQ ID NO:1, SEQ ID NO:11 and SEQ ID NO:3; SEQ ID NO:1, SEQ ID NO:12, SEQ ID NO:3; SEQ ID NO:1, SEQ ID NO:13 and SEQ ID NO:3; SEQ ID NO:7, SEQ ID NO:2 and SEQ ID NO:3; SEQ ID SEQ ID NO:7, SEQ ID NO:11, and SEQ ID NO:3; SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:3; SEQ ID NO:8, SEQ ID NO:12, and SEQ ID NO:3; SEQ ID NO:9, SEQ ID NO:11, and SEQ ID NO:3; SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:3; SEQ ID NO:1, SEQ ID NO:14 and SEQ ID NO:3; SEQ ID NO:1, SEQ ID NO:15 and SEQ ID NO:3; SEQ ID NO:1, SEQ ID NO:16 and SEQ ID NO:3; and SEQ ID NO:1, SEQ ID NO:18 and SEQ ID NO:3.

[0008] In some embodiments, the present disclosure provides a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domain (V L ) or an antigen-binding fragment thereof, wherein V H Contains V H -CDR1 sequence, V H -CDR2 sequence and V H -CDR1 sequence, and V L Contains V L -CDR1 sequence, V L -CDR2 sequence and V L - CDR3 sequences selected from the group consisting of: a) SEQ ID NOs: 1, 2, 3, 4, 5 and 6, respectively; b) SEQ ID NOs: 1, 2, 3, 4, 5 and 22, respectively; c) SEQ ID NOs: 1, 2, 3, 20, 5 and 6, respectively; d) SEQ ID NOs: 1, 11, 3, 4, 5 and 6, respectively; e) SEQ ID NOs: 1, 12, 3, 4, 5 and 6, respectively; f) SEQ ID NOs: 1, 13, 3, 4, 5 and 6, respectively; g) SEQ ID NOs: 1, 13, 3, 4, 5 and 21, respectively; h) SEQ ID NOs: 7, 2, 3, 4, 5 and 6, respectively; i) 7, 11, 3, 4, 5 and 6, respectively; j) SEQ ID NOs: 8, 10, 3, 4, 5 and 6, respectively; k) 8, 12, 3, 4, 5 and 6, respectively; l) SEQ ID NOs: NO: 9, 11, 3, 4, 5 and 6; and m) are SEQ ID NO: 9, 12, 3, 4, 5 and 6, respectively.

[0009] The antibody may further comprise an Fc domain of an isotype selected from IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, and IgE. In some embodiments, the antibody comprises an IgG1 constant region comprising one or more amino acid substitutions selected from N297A and K322A. Additionally or alternatively, in some embodiments, the antibody comprises an IgG4 constant region comprising an S228P mutation. In certain embodiments, the antigen-binding fragment is selected from Fab, F(ab')2, Fab', scF v and F v. In some embodiments, the antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, or a bispecific antibody. In certain embodiments, the antibody or antigen-binding fragment binds to poly-Sia (high DP poly-Sia) with a high degree of polymerization (e.g., a DP of about 10-20 Sia units, about 20-30 Sia units, about 30-50 Sia units, about 50-70 Sia units, about 70-100 Sia units, about 100-200 Sia units, or about 200-400 Sia units). In some embodiments, the antibody or antigen-binding fragment binds to, for example, high DP poly-Sia with DP>10, DP>20, DP>50, DP>100, or DP>200. In some embodiments, the epitope is a conformational epitope unique to high DP poly-Sia. In some embodiments, the conformational epitope includes 3 or more Sia units. In some embodiments, the 3 or more Sia units may be continuous units. In some embodiments, the antibody or antigen-binding fragment binds to poly-Sia with DP>10, and has higher affinity for increasing DP. Without wishing to be bound by theory, it is hypothesized that the antibodies or antigen-binding fragments disclosed herein bind cooperatively to a single high-DP poly-Sia with two Fab arms based on the co-crystal structure of mAb735 scFv, which shows two scFvs bound to one octasialic acid, with each scFv contacting 3 of the 8 Sia units (Nagae, M. et al. J Biol Chem. 288(47):33784-96 (2013)).

[0010] In another aspect, the present disclosure provides an antibody comprising a heavy chain (HC) amino acid sequence comprising SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, or a variant thereof having one or more conservative amino acid substitutions, and / or comprising SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:60, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85 The light chain (LC) amino acid sequence of NO:84 or a variant thereof having one or more conservative amino acid substitutions.

[0011] In certain embodiments, the antibody comprises a HC amino acid sequence and a LC amino acid sequence, respectively, selected from the group consisting of: SEQ ID NO: 23 and SEQ ID NO: 24 (chimeric P35); SEQ ID NO: 25 and SEQ ID NO: 28 (humanized P35H1L2); SEQ ID NO: 25 and SEQ ID NO: 27 (humanized P35 H1L1); SEQ ID NO: 26 and SEQ ID NO: 28 (humanized P35 H2L2); SEQ ID NO: 26 and SEQ ID NO: 27 (humanized P35 H2L1); SEQ ID NO: 48 and SEQ ID NO: 49 (BC137); SEQ ID NO: 51 and SEQ ID NO: 52 (BC137 KS2); SEQ ID NO: 53 and SEQ ID NO: 54 (BC137 KS34); SEQ ID NO: 55 and SEQ ID NO: 49 (BC137 DS47); SEQ ID NO: 56 and SEQ ID NO: 57 (BC137 DS47). NO:49 (BC137 DS54); SEQ ID NO:57 and SEQ ID NO:49 (BC137 DS47 D31R); SEQ ID NO:58 and SEQ ID NO:49 (BC137 DS54 D31R); SEQ ID NO:55 and SEQ ID NO:50 (BC137-2 DS47); SEQ ID NO:56 and SEQ ID NO:50 (BC137-2 DS54); SEQ ID NO:59 and SEQ ID NO:60 (BC163); SEQ ID NO:74 and SEQ ID NO:82 (rehumanized P35H1L1); SEQ ID NO:75 and SEQ ID NO:82 (rehumanized P35H2L1); SEQ ID NO:76 and SEQ ID NO:82 (rehumanized P35H3L1); SEQ ID NO:77 and SEQ ID NO:82 (heavy humanized P35H4L1); SEQ ID NO:78 and SEQ ID NO:82 (heavy humanized P35H5L1); SEQ ID NO:79 and SEQ ID NO:82 (heavy humanized P35H6L1); SEQ ID NO:80 and SEQ ID NO:82 (heavy humanized P35H7L1); SEQ ID NO:81 and SEQ ID NO:82 (heavy humanized P35H8L1); SEQ ID NO:74 and SEQ ID NO:83 (heavy humanized P35H1L2); SEQ ID NO:75 and SEQ ID NO:83 (heavy humanized P35H2L2);SEQ ID NO:76 and SEQ ID NO:83 (heavy humanized P35H3L2); SEQ ID NO:77 and SEQ ID NO:83 (heavy humanized P35H4L2); SEQ ID NO:78 and SEQ ID NO:83 (heavy humanized P35H5L2); SEQ ID NO:79 and SEQ ID NO:83 (heavy humanized P35H6L2); SEQ ID NO:80 and SEQ ID NO:83 (heavy humanized P35H7L2); SEQ ID NO:81 and SEQ ID NO:83 (heavy humanized P35H8L2); SEQ ID NO:74 and SEQ ID NO:84 (heavy humanized P35H1L3); SEQ ID NO:75 and SEQ ID NO:84 (heavy humanized P35H2L3); SEQ ID NO:76 and SEQ ID NO:84 (heavy humanized P35H3L3); SEQ ID NO:77 and SEQ ID NO:84 (heavy humanized P35H4L3); SEQ ID NO:78 and SEQ ID NO:84 (heavy humanized P35H5L3); SEQ ID NO:79 and SEQ ID NO:84 (heavy humanized P35H6L3); SEQ ID NO:80 and SEQ ID NO:84 (heavy humanized P35H7L3); and SEQ ID NO:81 and SEQ ID NO:84 (heavy humanized P35H8L3). ;

[0012] In one aspect, the disclosure provides an antibody comprising (a) a light chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a light chain immunoglobulin variable domain sequence present in any one of SEQ ID NO:37, 39, 41, 43, 44, 45, 47, 24, 27, 28, 49, 50, 52, 54, 60, 82, 83, or 84; and / or (b) a light chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a light chain immunoglobulin variable domain sequence present in any one of SEQ ID NO:37, 39, 41, 43, 44, 45, 47, 24, 27, 28, 49, 50, 52, 54, 60, 82, 83, or 84; NO:30, 31, 32, 33, 34, 35, 36, 38, 40, 42, 46, 23, 25, 26, 29, 48, 51, 53, 55, 56, 57, 58, 59, 74, 75, 76, 77, 78, 79, 80 or 81. A heavy chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the heavy chain immunoglobulin variable domain sequence present in any one of NO:30, 31, 32, 33, 34, 35, 36, 38, 40, 42, 46, 23, 25, 26, 29, 48, 51, 53, 55, 56, 57, 58, 59, 74, 75, 76, 77, 78, 79, 80 or 81.

[0013] In another aspect, the present disclosure provides an antibody comprising (a) an LC sequence that is at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the LC sequence present in any one of SEQ ID NOs: 24, 27, 28, 49, 50, 52, 54, 60, 82, 83 or 84; and / or (b) an HC sequence that is at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the HC sequence present in any one of SEQ ID NOs: 23, 25, 26, 29, 48, 51, 53, 55, 56, 57, 58, 59, 74, 75, 76, 77, 78, 79, 80 or 81.

[0014] In any of the above embodiments, the antibody is a chimeric antibody, a humanized antibody or a bispecific antibody. Additionally or alternatively, in some embodiments, the antibody comprises an IgG1 constant region containing one or more amino acid substitutions selected from N297A and K322A. In certain embodiments, the antibody of the present technology comprises an IgG4 constant region containing an S228P mutation. In any of the above embodiments, the antibody is bound to a high DP poly-Sia (e.g., a DP of about 10-20 Sia units, about 20-30 Sia units, about 30-50 Sia units, about 50-70 Sia units, about 70-100 Sia units, about 100-200 Sia units, or about 200-400 Sia units). In some embodiments, the antibody or antigen-binding fragment is bound to a high DP poly-Sia, for example, with DP>10, DP>20, DP>50, DP>100, or DP>200. In some embodiments, the epitope is a conformational epitope unique to high DP poly-Sia. In some embodiments, the conformational epitope comprises 3 or more Sia units. In some embodiments, the 3 or more Sia units may be continuous units. Additionally or alternatively, in some embodiments, the antibodies of the present technology lack α-1,6-fucose modification. In certain embodiments, the antibody or antigen-binding fragment recruits T cells for T cell-dependent cellular cytotoxicity (TDCC) against tumor cells expressing poly-Sia. In some embodiments, the tumor cells expressing poly-Sia are resistant to poly-Sia-specific antibody-dependent cell-mediated cytotoxicity (ADCC).

[0015] In one aspect, the disclosure provides a recombinant nucleic acid sequence encoding any of the antibodies described herein. In some embodiments, the recombinant nucleic acid sequence is selected from SEQ ID NO: 92-108.

[0016] In another aspect, the present disclosure provides a host cell or a vector comprising any of the recombinant nucleic acid sequences disclosed herein.

[0017] In one aspect, the present disclosure provides a composition comprising an antibody or antigen-binding fragment of the present technology and a pharmaceutically acceptable carrier, wherein the antibody or antigen-binding fragment is optionally conjugated to an agent selected from the following: an isotope, a dye, a chromagen, a contrast agent, a drug, a toxin, a cytokine, an enzyme, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA or any combination thereof.

[0018] In some embodiments of the bispecific antibodies of the present technology, the bispecific antibodies bind to T cells, B cells, bone marrow cells, plasma cells or mast cells. Additionally or alternatively, in some embodiments, the bispecific antibodies bind to CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, TCRγ / δ, NKp46, KIR or small molecule DOTA hapten.The small molecule DOTA hapten can be selected from DOTA, DOTA-Bn, DOTA-deferoxamine, DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH2, Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH2, DOTA-D-Asp-D-Lys(HSG)-D-Asp-D-Lys(HSG)-NH2; DOTA-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2, DOTA-D-Tyr-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2, DOTA-D-Ala-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2, DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH2, Ac-D-Phe-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-NH2, Ac-D-Phe-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH2, Ac-D-Phe-D-Lys(Bz-DTPA)-D-Tyr-D-Lys(Bz-DTPA)-NH2, Ac-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH2, DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH2, (Tscg-Cys)-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(DOTA)-NH2, Tscg-D-Cys-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2, (Tscg-Cys)-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2, Ac-D-Cys-D-Lys(DOTA)-D-Tyr-D-Ala-D-Lys(DOTA)-D-Cys-NH2, Ac-D-Cys-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH2, Ac-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-D-Lys(Tscg-Cys)-NH2, and Ac-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-D-Lys(Tscg-Cys)-NH2.

[0019] In another aspect, the disclosure provides a method of treating poly-Sia-associated cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of any one of the antibodies disclosed herein. In certain embodiments, the antibody comprises a HC amino acid sequence and a LC amino acid sequence, respectively, selected from the group consisting of: SEQ ID NO: 23 and SEQ ID NO: 24 (chimeric P35); SEQ ID NO: 25 and SEQ ID NO: 28 (humanized P35 H1L2); SEQ ID NO: 25 and SEQ ID NO: 27 (humanized P35 H1L1); SEQ ID NO: 26 and SEQ ID NO: 28 (humanized P35 H2L2); SEQ ID NO: 26 and SEQ ID NO: 27 (humanized P35 H2L1); SEQ ID NO: 48 and SEQ ID NO: 49 (BC137); SEQ ID NO: 51 and SEQ ID NO: 52 (BC137 KS2); SEQ ID NO: 53 and SEQ ID NO: 54 (BC137 KS34); SEQ ID NO: 55 and SEQ ID NO: 49 (BC137 DS47); SEQ ID NO: 56 and SEQ ID NO: NO:49 (BC137 DS54); SEQ ID NO:57 and SEQ ID NO:49 (BC137 DS47 D31R); SEQ ID NO:58 and SEQ ID NO:49 (BC137 DS54D31R); SEQ ID NO:55 and SEQ ID NO:50 (BC137-2 DS47); SEQ ID NO:56 and SEQ ID NO:50 (BC137-2 DS54); SEQ ID NO:59 and SEQ ID NO:60 (BC163); SEQ ID NO:74 and SEQ ID NO:82 (rehumanized P35H1L1); SEQ ID NO:75 and SEQ ID NO:82 (rehumanized P35H2L1); SEQ ID NO:76 and SEQ ID NO:82 (rehumanized P35H3L1); SEQ ID NO:77 and SEQ ID NO:82 (heavy humanized P35H4L1); SEQ ID NO:78 and SEQ ID NO:82 (heavy humanized P35H5L1); SEQ ID NO:79 and SEQ ID NO:82 (heavy humanized P35H6L1); SEQ ID NO:80 and SEQ ID NO:82 (heavy humanized P35H7L1); SEQ ID NO:81 and SEQ ID NO:82 (heavy humanized P35H8L1);SEQ ID NO:74 and SEQ ID NO:83 (heavy humanized P35H1L2); SEQ ID NO:75 and SEQ ID NO:83 (heavy humanized P35H2L2); SEQ ID NO:76 and SEQ ID NO:83 (heavy humanized P35H3L2); SEQ ID NO:77 and SEQ ID NO:83 (heavy humanized P35H4L2); SEQ ID NO:78 and SEQ ID NO:83 (heavy humanized P35H5L2); SEQ ID NO:79 and SEQ ID NO:83 (heavy humanized P35H6L2); SEQ ID NO:80 and SEQ ID NO:83 (heavy humanized P35H7L2); SEQ ID NO:81 and SEQ ID NO:83 (heavy humanized P35H8L2); SEQ ID NO:74 and SEQ ID NO:84 (heavy humanized P35H1L3); SEQ ID NO:75 and SEQ ID NO:84 (heavy humanized P35H2L3); SEQ ID NO:76 and SEQ ID NO:84 (heavy humanized P35H3L3); SEQ ID NO:77 and SEQ ID NO:84 (heavy humanized P35H4L3); SEQ ID NO:78 and SEQ ID NO:84 (heavy humanized P35H5L3); SEQ ID NO:79 and SEQ ID NO:84 (heavy humanized P35H6L3); SEQ ID NO:80 and SEQ ID NO:84 (heavy humanized P35H7L3); and SEQ ID NO:81 and SEQ ID NO:84 (heavy humanized P35H8L3), wherein the antibody specifically binds poly-Sia.;

[0020] In some embodiments, the poly-Sia-associated cancer is small cell or non-small cell lung cancer, neuroblastoma, pancreatic cancer, pituitary tumor, Wilms' tumor, rhabdomyosarcoma, glioblastoma, breast cancer, or acute myeloid leukemia. The poly-Sia-associated cancer may be a metastatic cancer.

[0021] Additionally or alternatively, in some embodiments of the method, the antibody is administered to the subject separately, sequentially or simultaneously with an additional therapeutic agent. Examples of additional therapeutic agents include one or more of the following: alkylating agents, platinum agents, taxanes, vinca agents, anti-estrogen drugs, aromatase inhibitors, ovarian inhibitors, VEGF / VEGFR inhibitors, EGF / EGFR inhibitors, PARP inhibitors, cytostatic alkaloids, cytotoxic antibiotics, antimetabolites, endocrine / hormonal agents, bisphosphonate therapeutics.

[0022] In another aspect, the present disclosure provides a method for detecting a tumor in a subject in vivo, the method comprising (a) administering to the subject an effective amount of an antibody of the present technology, wherein the antibody is configured to localize to a tumor expressing high-DP polySia and is labeled with a radioisotope; and (b) detecting the presence of a tumor in the subject by detecting a level of radioactivity emitted by the antibody that is higher than a reference value. In some embodiments, the subject is diagnosed with or suspected of having cancer. The level of radioactivity emitted by the antibody can be detected using positron emission tomography or single photon emission computed tomography.

[0023] Additionally or alternatively, in some embodiments, the method further comprises administering to the subject an effective amount of an immunoconjugate comprising an antibody of the present technology conjugated to a radionuclide. In some embodiments, the radionuclide is an alpha particle emitting isotope, a beta particle emitting isotope, an Auger emitter, or any combination thereof. Examples of beta particle emitting isotopes include 86 Y. 90 Y. 89 Sr. 165 Dy, 186 Re, 188 Re, 177 Lu and 67 Cu. In some embodiments of the methods, non-specific FcR-dependent binding in normal tissues is eliminated or reduced (eg, via a N297A mutation in the Fc region, which results in deglycosylation).

[0024] Also disclosed herein is a kit for detecting and / or treating poly-Sia-related cancers, the kit comprising at least one immunoglobulin-related composition of the present technology (e.g., any antibody or antigen-binding fragment described herein) or a functional variant thereof (e.g., a substitution variant) and instructions for use. In certain embodiments, the immunoglobulin-related composition is coupled to one or more detectable labels. In one embodiment, the one or more detectable labels include a radioactive label, a fluorescent label, or a chromogenic label.

[0025] Additionally or alternatively, in some embodiments, the kit further comprises a second antibody that specifically binds to the anti-polySia immunoglobulin-related composition described herein. In some embodiments, the second antibody is coupled to at least one detectable label selected from a radioactive label, a fluorescent label, or a chromogenic label.

[0026] In one aspect, the present disclosure provides a method for detecting a solid tumor in a subject in need thereof, the method comprising (a) administering to the subject an effective amount of a complex comprising a radiolabeled DOTA hapten and a bispecific antibody of the present technology that binds to the radiolabeled DOTA hapten and a polySia antigen, wherein the complex is configured to localize to a solid tumor expressing a polySia antigen recognized by the bispecific antibody of the complex; and (b) detecting the presence of a solid tumor in the subject by detecting a level of radioactivity emitted by the complex that is higher than a reference value.

[0027] In another aspect, the present disclosure provides a method for selecting a subject for pretargeted radioimmunotherapy, the method comprising (a) administering to the subject an effective amount of a complex comprising a radiolabeled DOTA hapten and a bispecific antibody of the present technology that binds to the radiolabeled DOTA hapten and a polySia antigen, wherein the complex is configured to localize to a solid tumor expressing a polySia antigen recognized by the bispecific antibody of the complex; (b) detecting the level of radioactivity emitted by the complex; and (c) selecting the subject for pretargeted radioimmunotherapy when the level of radioactivity emitted by the complex is higher than a reference value.

[0028] In one aspect, the present disclosure provides a method for increasing the sensitivity of a tumor to radiation therapy in a subject diagnosed with a poly-Sia-associated cancer, the method comprising administering to the subject an effective amount of a complex comprising a radiolabeled DOTA hapten and a bispecific antibody of the present technology that recognizes and binds to the radiolabeled DOTA hapten and a poly-Sia antigen target, wherein the complex is configured to localize to a tumor expressing the poly-Sia antigen target recognized by the bispecific antibody of the complex.

[0029] In another aspect, the present disclosure provides a method for treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of a complex comprising a radiolabeled DOTA hapten and a bispecific antibody of the present technology that recognizes and binds to the radiolabeled DOTA hapten and a poly-Sia antigen target, wherein the complex is configured to localize to a tumor expressing the poly-Sia antigen target recognized by the bispecific antibody of the complex.

[0030] In any of the above embodiments of the methods disclosed herein, the complex is administered intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intraorbitally, intradermally, intraperitoneally, transtracheally, subcutaneously, intracerebroventricularly, orally, or intranasally. In some embodiments of the methods disclosed herein, the subject is a human. Additionally or alternatively, in any of the above embodiments of the methods disclosed herein, the radiolabeled DOTA hapten comprises 213 Bi, 211 At 225 Ac, 152 Dy, 212 Bi, 223 Ra, 219 Rn, 215 Po, 211 Bi, 221 Fr. 217 At 255 Fm, 86 Y. 90 Y. 89 Sr. 165 Dy, 186 Re, 188 Re, 177 Lu, 67 Cu, 111 In, 67 Ga, 51 Cr, 58 Co. 99m Tc, 103m Rh, 195m Pt, 119 Sb, 161 Ho, 189m Os, 192 Ir, 201 Tl, 203 Pb, 68 Ga, 227 Th or 64 Cu, and optionally contains an alpha-emitting isotope, a beta-emitting isotope, or an Auger emitter.

[0031] In one aspect, the present disclosure provides a method for increasing the sensitivity of a tumor to radiotherapy in a subject diagnosed with poly-Sia-associated cancer, the method comprising (a) administering to the subject an effective amount of an anti-DOTA bispecific antibody of the present technology, wherein the anti-DOTA bispecific antibody is configured to localize to a tumor expressing a poly-Sia antigen target; and (b) administering to the subject an effective amount of a radiolabeled DOTA hapten, wherein the radiolabeled DOTA hapten is configured to bind to the anti-DOTA bispecific antibody. In another aspect, the present disclosure provides a method for treating cancer in a subject in need thereof, the method comprising (a) administering to the subject an effective amount of an anti-DOTA bispecific antibody of the present technology, wherein the anti-DOTA bispecific antibody is configured to localize to a tumor expressing a poly-Sia antigen target; and (b) administering to the subject an effective amount of a radiolabeled DOTA hapten, wherein the radiolabeled DOTA hapten is configured to bind to the anti-DOTA bispecific antibody. In some embodiments, the method of the present technology further comprises administering to the subject an effective amount of a scavenger prior to administering the radiolabeled DOTA hapten.

[0032] Additionally or alternatively, in any of the above embodiments of the methods disclosed herein, the radiolabeled DOTA hapten comprises 213 Bi, 211 At 225 Ac, 152 Dy, 212 Bi, 223 Ra, 219 Rn, 215 Po, 211 Bi, 221 Fr. 217 At 255 Fm, 86 Y. 90 Y. 89 Sr. 165 Dy, 186 Re, 188 Re, 177 Lu, 67 Cu, 111 In, 67 Ga, 51 Cr, 58 Co. 99m Tc, 103m Rh, 195m Pt, 119 Sb, 161 Ho, 189m Os, 192 Ir, 201Tl, 203 Pb, 68 Ga, 227 Th or 64 Cu, and optionally comprises an alpha particle emitting isotope, a beta particle emitting isotope, or an Auger emitter. In any of the above embodiments of the methods disclosed herein, the subject is a human. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1(A) and 1(B) Shown is the expression of polysialic acid on various cancer cell lines.

[0034] Figure 2 Shown are the results of immunohistochemistry of small cell lung cancer (SCLC) patient-derived xenografts stained with a chimeric P35 antibody.

[0035] Figure 3 Relative staining of the neuroblastoma cell line SK-N-BE(1) with four humanized anti-poly-Sia IgG1 antibodies is shown.

[0036] Figure 4 (A) shows a schematic diagram of a poly-Sia x CD3 bispecific antibody in IgG-scFv format. Figure 4 (B) shows the purity of the poly-Sia x CD3 bispecific antibody BC137 by SEC-HPLC, where the main peak (16.0 minutes) is the fully paired BsAb (molecular weight approximately 210 kDa) and the salt buffer peak (25 minutes).

[0037] Figure 5(A) shows a T cell dependent cytotoxicity assay (TDCC) performed with BC137 on the melanoma cell line M14. Figure 5(B) shows a T cell dependent cytotoxicity assay (TDCC) performed with BC137 on the neuroblastoma cell line IMR-32.

[0038] Figure 6 Shown are tumor volumes in a mouse xenograft study with neuroblastoma IMR-32 tumors treated with BC137 and activated T cells (ATC).

[0039] FIG. 7(A) shows the electrostatic potential map of the binding pocket of mAb735 with bound octasialic acid based on the PDB 3WDB crystal structure with two negatively charged residues VH:D31 and VH:D105. Figure 7(B) shows a side view of a ribbon diagram of the binding pocket of mAb735 with bound octasialic acid based on the PDB 3WDB crystal structure with two negatively charged residues VH:D31 and VH:D105. Figure 7(C) shows the relative binding of the P35 H1L2 IgG1 parent and with mutations designed by rational engineering.

[0040] Figure 8 (A) shows the binding kinetics of affinity matured clones based on rational design or yeast display affinity maturation using a direct screening approach. Figure 8 (B) shows the positions of amino acid changes in affinity matured clones.

[0041] Figure 9 (A) shows the binding kinetics of affinity matured clones based on rational design or yeast display affinity maturation using a kinetic screening approach. Figure 9 (B) shows the positions of amino acid changes in affinity matured clones.

[0042] Figure 10(A) shows the TDCC assay of selected BC137 clones on neuroblastoma IMR-32 cells. Figure 10(B) shows the EC50 values ​​of TDCC of selected BC137 clones against melanoma M14, neuroblastoma IMR-32 and neuroblastoma SKNSH cells.

[0043] Fig.11 Shown are tumor volumes in mouse xenograft studies with neuroblastoma IMR-32 tumors treated with BC137 and selected affinity matured variants.

[0044] Fig.12 A schematic diagram of the poly-Sia x DOTA bispecific antibody in IgG-scFv format is shown.

[0045] Figure 13(A)-13(E) The results show that the poly-Sia x DOTA bispecific antibody BC163 and BC137 (0, 0.25, 0.5 or 1 mg) were used. 177 Results of a mouse xenograft study of neuroblastoma IMR-32 tumors treated with Lu-Bn-DOTA.

[0046] Figure 14(A) and 14(B) The human germline content of the humanized clones is shown.

[0047] Figure 15(A) and 15(B) The amino acid sequences of the heavy and light chains of chimeric P35-IgG1 are shown, corresponding to SEQ ID NO: 23 and SEQ ID NO: 24, respectively. The CDR sequences are underlined.

[0048] Figure 16 (A) shows the amino acid and nucleotide sequences of humanized P35 heavy chain H1, which correspond to SEQ ID NO: 25 and SEQ ID NO: 92, respectively. Figure 16 (B) shows the amino acid and nucleotide sequences of humanized P35 heavy chain H2, which correspond to SEQ ID NO: 26 and SEQ ID NO: 93, respectively. The CDR sequences are underlined.

[0049] Figure 17 (A) shows the amino acid and nucleotide sequences of humanized P35 light chain L1, which correspond to SEQ ID NO: 27 and SEQ ID NO: 94, respectively. Figure 17 (B) shows the amino acid and nucleotide sequences of humanized P35 light chain L2, which correspond to SEQ ID NO: 28 and SEQ ID NO: 95, respectively. CDR sequences are underlined.

[0050] Figure 18 (A) and 18(B) The amino acid sequences of the heavy and light chains of the humanized P35 H1L2 IgG1 antibody are shown and correspond to SEQ ID NO: 25 and SEQ ID NO: 28, respectively. The CDR sequences are underlined.

[0051] Fig.19 The amino acid sequence of the heavy chain of a rationally designed affinity-matured clone of a humanized P35 heavy chain with a D31R mutation is shown. The amino acid sequence corresponds to SEQ ID NO: 29. The CDR sequences are underlined. The modified residues in the variable domains are shown in bold.

[0052] Fig. 20 The amino acid sequence of the heavy chain variable domain of DS45 (an affinity mature clone of humanized P35 H1L2) is shown. The amino acid sequence corresponds to SEQ ID NO: 30. The CDR sequences are underlined. Modified residues in the variable domain are shown in bold.

[0053] Fig.21 The amino acid sequence of the heavy chain variable domain of DS47 (an affinity mature clone of humanized P35 H1L2) is shown. The amino acid sequence corresponds to SEQ ID NO: 31. The CDR sequences are underlined. Modified residues in the variable domain are shown in bold.

[0054] Fig. 22 The amino acid sequence of the heavy chain variable domain of DS51 (an affinity matured clone of humanized P35 H1L2) is shown. The amino acid sequence corresponds to SEQ ID NO: 32. The CDR sequences are underlined. Modified residues in the variable domain are shown in bold.

[0055] Fig.23 The amino acid sequence of the heavy chain variable domain of DS53 (an affinity mature clone of humanized P35 H1L2) is shown. The amino acid sequence corresponds to SEQ ID NO: 33. The CDR sequences are underlined. Modified residues in the variable domain are shown in bold.

[0056] Fig.24 The amino acid sequence of the heavy chain variable domain of DS54 (an affinity matured clone of humanized P35 H1L2) is shown. The amino acid sequence corresponds to SEQ ID NO: 34. The CDR sequences are underlined. The modified residues in the variable domain are shown in bold. The CDR sequences are underlined. The modified residues in the variable domain are shown in bold.

[0057] Fig.25 The amino acid sequence of the heavy chain variable domain of DS55 (an affinity mature clone of humanized P35 H1L2) is shown. The amino acid sequence corresponds to SEQ ID NO: 35. The CDR sequences are underlined. Modified residues in the variable domain are shown in bold.

[0058] Figure 26(A) and 26(B) The amino acid sequences of the heavy and light chain variable domains of KS2 (an affinity matured clone of humanized P35 H1L2) are shown. The amino acid sequences correspond to SEQ ID NO:36 and SEQ ID NO:37, respectively. The CDR sequences are underlined. Modified residues in the variable domains are shown in bold.

[0059] Figure 27(A) and 27(B) The amino acid sequences of the heavy and light chain variable domains of KS10 (an affinity matured clone of humanized P35 H1L2) are shown. The amino acid sequences correspond to SEQ ID NO: 38 and SEQ ID NO: 39, respectively.

[0060] Figure 28(A) and 28(B) The amino acid sequences of the heavy and light chain variable domains of KS21 (an affinity matured clone of humanized P35 H1L2) are shown. The amino acid sequences correspond to SEQ ID NO:40 and SEQ ID NO:41, respectively. The CDR sequences are underlined. Modified residues in the variable domains are shown in bold.

[0061] Figure 29(A) and 29(B)The amino acid sequences of the heavy and light chain variable domains of KS23 (an affinity matured clone of humanized P35 H1L2) are shown. The amino acid sequences correspond to SEQ ID NO:42 and SEQ ID NO:43, respectively. The CDR sequences are underlined. Modified residues in the variable domains are shown in bold.

[0062] Fig.30 The amino acid sequence of the light chain variable domain of KS26 (an affinity matured clone of humanized P35 H1L2) is shown. The amino acid sequence corresponds to SEQ ID NO:44.

[0063] Fig.31 The amino acid sequence of the light chain variable domain of KS30 (an affinity matured clone of humanized P35 H1L2) is shown. The amino acid sequence corresponds to SEQ ID NO: 45. The CDR sequences are underlined. Modified residues in the variable domain are shown in bold.

[0064] Figure 32(A) and 32(B) The amino acid sequences of the heavy and light chain variable domains of KS34 (an affinity matured clone of humanized P35 H1L2) are shown. The amino acid sequences correspond to SEQ ID NO:46 and SEQ ID NO:47, respectively. The CDR sequences are underlined. Modified residues in the variable domains are shown in bold.

[0065] Figure 33(A) and 33(B) The amino acid and nucleotide sequences of the heavy chain of the polySia x CD3 bispecific antibody BC137 are shown, corresponding to SEQ ID NO: 48 and SEQ ID NO: 96, respectively. The N297A and K322A substitutions in the Fc domain are shown in bold. Figures 33(C) and 33(D) show the amino acid and nucleotide sequences of the light chain of BC137, corresponding to SEQ ID NO: 49 and SEQ ID NO: 97, respectively. The CDR sequences and GS linker are underlined.

[0066] Figures 34(A) and 34(B) show the amino acid and nucleotide sequences of the modified light chain (BC137-2 light chain) used in the polySia x CD3 bispecific antibody, which correspond to SEQ ID NO:50 and SEQ ID NO:98, respectively. The sequence has been modified to make two Cys to Gly substitutions in the anti-CD3 huOKT3 scFv portion to alter the stability of the disulfide bond. The CDR sequence and GS linker are underlined. The modified Cys residues are shown in bold.

[0067] Figures 35(A) and 35(B) show the amino acid and nucleotide sequences of the heavy chain of affinity matured clone KS2 in polySia x CD3 BsAb format, corresponding to SEQ ID NO:51 and SEQ ID NO:99, respectively. The modified residues in the variable domain and the N297A and K322A substitutions in the Fc domain are shown in bold. Figures 35(C) and 35(D) show the amino acid and nucleotide sequences of the light chain, corresponding to SEQ ID NO:52 and SEQ ID NO:100, respectively. The CDR sequences and GS linker are underlined.

[0068] Fig.36A Figures 36(A) and 36(B) show the amino acid and nucleotide sequences of the heavy chain of affinity matured clone KS34 in polySia x CD3 BsAb format, which correspond to SEQ ID NO:53 and SEQ ID NO:101, respectively. The modified residues in the variable domain and the N297A and K322A substitutions in the Fc domain are shown in bold. Figures 36(C) and 36(D) show the amino acid and nucleotide sequences of the light chain, which correspond to SEQ ID NO:54 and SEQ ID NO:102, respectively. The underlined sequence corresponds to the GS linker sequence. The CDR sequences and GS linker are underlined.

[0069] Figures 37(A) and 37(B) show the amino acid and nucleotide sequences of the heavy chain of affinity matured clone DS47 in poly-Sia x CD3 BsAb format, corresponding to SEQ ID NO:55 and SEQ ID NO:103, respectively. The CDR sequences are underlined. The modified residues in the variable domain and the N297A and K322A substitutions in the Fc domain are shown in bold.

[0070] Figures 38(A) and 38(B) show the amino acid and nucleotide sequences of the heavy chain of affinity matured clone DS54 in poly-Sia x CD3 BsAb format, corresponding to SEQ ID NO: 56 and SEQ ID NO: 104, respectively. The CDR sequences are underlined. The modified residues in the variable domain and the N297A and K322A substitutions in the Fc domain are shown in bold.

[0071] Figures 39(A) and 39(B) show the amino acid and nucleotide sequences of the heavy chain of affinity-matured clone DS47 with D31R mutation in a poly-Sia x CD3 BsAb format, corresponding to SEQ ID NO:57 and SEQ ID NO:105, respectively. The CDR sequences are underlined. The modified residues in the variable domain and the N297A and K322A substitutions in the Fc domain are shown in bold.

[0072] Figures 40(A) and 40(B) show the amino acid and nucleotide sequences of the heavy chain of affinity-matured clone DS54 with D31R mutation in a poly-Sia x CD3 BsAb format, corresponding to SEQ ID NO:58 and SEQ ID NO:106, respectively. The CDR sequences are underlined. The modified residues in the variable domain and the N297A and K322A substitutions in the Fc domain are shown in bold.

[0073] Figures 41(A) and 41(B) show the amino acid and nucleotide sequences of the heavy chain of BC163 (poly Sia x DOTA bispecific antibody), which correspond to SEQ ID NO: 59 and SEQ ID NO: 107, respectively. The modified residues in the variable domain and the N297A and K322A substitutions in the Fc domain are shown in bold. Figures 41(C) and 41(D) show the amino acid and nucleotide sequences of the light chain, which correspond to SEQ ID NO: 60 and SEQ ID NO: 108, respectively. The CDR sequences and GS linker are underlined.

[0074] Fig.42 The amino acid sequence of affinity matured clone DS45 in scFv format is shown, corresponding to SEQ ID NO: 61. The underlined sequence corresponds to the GS linker sequence. The CDR sequences and GS linker are underlined. Modified residues in the variable domains are shown in bold.

[0075] Fig.43 The amino acid sequence of affinity matured clone DS47 in scFv format is shown corresponding to SEQ ID NO: 62. The underlined sequence corresponds to the GS linker sequence. The CDR sequences and GS linker are underlined. Modified residues in the variable domains are shown in bold.

[0076] Fig.44 The amino acid sequence of affinity matured clone DS51 in scFv format is shown, corresponding to SEQ ID NO: 63. The underlined sequence corresponds to the GS linker sequence. The CDR sequences and GS linker are underlined. Modified residues in the variable domains are shown in bold.

[0077] Fig.45 The amino acid sequence of affinity matured clone DS53 in scFv format is shown, corresponding to SEQ ID NO: 64. The underlined sequence corresponds to the GS linker sequence. The CDR sequences and GS linker are underlined. Modified residues in the variable domains are shown in bold.

[0078] Fig.46The amino acid sequence of affinity matured clone DS54 in scFv format is shown, corresponding to SEQ ID NO: 65. The underlined sequence corresponds to the GS linker sequence. The CDR sequences and GS linker are underlined. Modified residues in the variable domains are shown in bold.

[0079] Fig.47 The amino acid sequence of affinity matured clone DS55 in scFv format is shown, corresponding to SEQ ID NO: 66. The underlined sequence corresponds to the GS linker sequence. The CDR sequences and GS linker are underlined. Modified residues in the variable domains are shown in bold.

[0080] Fig.48 The amino acid sequence of affinity matured clone KS2 in scFv format is shown, corresponding to SEQ ID NO: 67. The underlined sequence corresponds to the GS linker sequence.

[0081] Fig.49 The amino acid sequence of affinity matured clone KS10 in scFv format is shown, corresponding to SEQ ID NO: 68. The CDR sequences and GS linker are underlined. Modified residues in the variable domain are shown in bold.

[0082] Fig.50 The amino acid sequence of affinity matured clone KS21 in scFv format is shown, corresponding to SEQ ID NO: 69. The underlined sequence corresponds to the GS linker sequence. The CDR sequences and GS linker are underlined. Modified residues in the variable domains are shown in bold.

[0083] Fig.51 The amino acid sequence of affinity matured clone KS23 in scFv format is shown, corresponding to SEQ ID NO: 70. The underlined sequence corresponds to the GS linker sequence.

[0084] Fig.52 The amino acid sequence of affinity matured clone KS26 in scFv format is shown, corresponding to SEQ ID NO: 71. The underlined sequence corresponds to the GS linker sequence. The CDR sequences and GS linker are underlined. Modified residues in the variable domains are shown in bold.

[0085] Fig.53 The amino acid sequence of affinity matured clone KS30 in scFv format is shown, corresponding to SEQ ID NO: 72. The underlined sequence corresponds to the GS linker sequence. The CDR sequences and GS linker are underlined. Modified residues in the variable domains are shown in bold.

[0086] Fig.54The amino acid sequence of affinity matured clone KS34 in scFv format is shown, corresponding to SEQ ID NO: 73. The underlined sequence corresponds to the GS linker sequence. The CDR sequences and GS linker are underlined. Modified residues in the variable domains are shown in bold.

[0087] Fig.55 The amino acid sequence of HC1 (re-humanized heavy chain based on P35 H1L2) is shown. The amino acid sequence corresponds to SEQ ID NO: 74. The CDR sequences are underlined.

[0088] Fig.56 The amino acid sequence of HC2 (heavy humanized heavy chain based on P35 H1L2) is shown. The amino acid sequence corresponds to SEQ ID NO: 75. The CDR sequences are underlined.

[0089] Fig.57 The amino acid sequence of HC3 (heavy humanized heavy chain based on P35 H1L2) is shown. The amino acid sequence corresponds to SEQ ID NO: 76. The CDR sequences are underlined.

[0090] Fig.58 The amino acid sequence of HC4 (heavy humanized heavy chain based on P35 H1L2 H1L2) is shown. The amino acid sequence corresponds to SEQ ID NO: 77. The CDR sequences are underlined.

[0091] Fig.59 The amino acid sequence of HC5 (heavy humanized heavy chain based on P35 H1L2) is shown. The amino acid sequence corresponds to SEQ ID NO: 78. The CDR sequences are underlined.

[0092] Fig.60 The amino acid sequence of HC6 (heavy humanized heavy chain based on P35 H1L2) is shown. The amino acid sequence corresponds to SEQ ID NO: 79. The CDR sequences are underlined.

[0093] Fig.61 The amino acid sequence of HC7 (heavy humanized heavy chain based on P35 H1L2) is shown. The amino acid sequence corresponds to SEQ ID NO: 80. The CDR sequences are underlined.

[0094] Fig.62 The amino acid sequence of HC8 (heavy humanized heavy chain based on P35 H1L2) is shown. The amino acid sequence corresponds to SEQ ID NO: 81. The CDR sequences are underlined.

[0095] Fig.63 The amino acid sequence of LC1 (heavy humanized light chain based on P35 H1L2) is shown. The amino acid sequence corresponds to SEQ ID NO: 82. The CDR sequences are underlined.

[0096] Fig.64 The amino acid sequence of LC2 (heavy humanized light chain based on P35 H1L2) is shown. The amino acid sequence corresponds to SEQ ID NO: 83. The CDR sequences are underlined.

[0097] Fig.65 The amino acid sequence of LC3 (heavy humanized light chain based on P35 H1L2) is shown. The amino acid sequence corresponds to SEQ ID NO: 84. The CDR sequences are underlined.

[0098] Fig.66 Shown is an antibody-dependent cell-mediated cytotoxicity (ADCC) assay with humanized P35IgG1 antibody on the neuroblastoma cell line IMR-32Luc.

[0099] Fig.67 Shown is the relative binding of various re-humanized HP35 clones disclosed herein to the M14 melanoma cell line (polysialic acid+) as determined by flow cytometry.

[0100] Fig.68 A heat map comparing the antigen binding properties and stability of various re-humanized anti-polySia antibodies of the present technology relative to the chimeric HP35 polySia antibody is shown. Column 1, MFI, based on binding to the M14 melanoma cell line via flow cytometry at 1 μg / million cells, low binding (white) to high binding (dark gray). Column 2, Binding Affinity, based on K D (M), the K D Column 3, stability, based on purity after three weeks of incubation at 37°C using HPCL, low purity (white) to high purity (dark grey). Clone LC2+HC5 was selected for further characterization in vitro and in vivo due to its high binding affinity to its antigen. DETAILED DESCRIPTION

[0101] It should be appreciated that certain aspects, modes, embodiments, variations and features of the methods of the present invention are described below in varying levels of detail in order to provide a substantial understanding of the present technology.

[0102] The present disclosure generally provides immunoglobulin-related compositions (e.g., antibodies or antigen-binding fragments thereof) that can specifically bind to poly-Sia, particularly high-DP poly-Sia, and can neutralize the biological activity of poly-Sia, particularly high-DP poly-Sia. The immunoglobulin-related compositions of the present technology can be used in methods for detecting or treating poly-Sia-related cancers in subjects in need thereof. Therefore, various aspects of the present method relate to the preparation, characterization and manipulation of anti-poly-Sia antibodies. The immunoglobulin-related compositions of the present technology can be used alone or in combination with additional therapeutic agents for treating cancer. In some embodiments, the immunoglobulin-related composition is a humanized antibody, a chimeric antibody, or a bispecific antibody.

[0103] In practicing the methods of the present invention, many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology, and recombinant DNA are used. See, for example, Sambrook and Russell, eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; Ausubel et al., eds. (2007) Current Protocols in Molecular Biology; Methods in Enzymology (Academic Press, Inc., New York); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane, eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait, ed. (1984) Oligonucleotide Synthesis; U.S. Pat. No. 4,683,195; Hames and Higgins, eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins, eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos, ed. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides, ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker, ed. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al., ed. (1996) Weir's Handbook of Experimental Immunology. Methods for detecting and measuring the level of polypeptide gene expression products (i.e., gene translation levels) are well known in the art and include the use of polypeptide detection methods, such as antibody detection and quantification techniques. (See also, Strachan and Read, Human Molecular Genetics, 2nd ed. (John Wiley and Sons, Inc., New York, 1999)). . definition

[0104] Unless otherwise limited, all technical and scientific terms used herein generally have the same meaning as commonly understood by those of ordinary skill in the art to which the technology belongs. As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural designators unless the context clearly indicates otherwise. For example, reference to "a cell" includes combinations of two or more cells, and so on. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry, and nucleic acid chemistry and hybridization described below are those well known and commonly used in the art.

[0105] Unless otherwise indicated or apparent from the context, as used herein, the term "about" with respect to numbers is generally considered to include numbers that fall within 1%, 5%, or 10% in either direction (greater than or less than) of the stated number (except where such a number is below 0% or above 100% of the possible value).

[0106] As used herein, "administering" an agent or drug to a subject includes any route of introducing or delivering a compound to a subject to perform its intended function. Administration can be by any suitable route, including, but not limited to, oral, intranasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), rectal, intrathecal, intratumoral, or topical. Administration includes self-administration and administration by another person.

[0107] "Adjuvant" refers to one or more substances that cause immune system stimulation. In this case, the adjuvant is used to enhance the immune response to one or more vaccine antigens or antibodies. The adjuvant can be administered to the subject before, in combination with, or after the administration of the vaccine. Examples of chemical compounds used as adjuvants include aluminum compounds, oils, block polymers, immunostimulatory complexes, vitamins and minerals (e.g., vitamin E, vitamin A, selenium, and vitamin B12), Quil A (saponin), bacterial and fungal cell wall components (e.g., lipopolysaccharides, lipoproteins, and glycoproteins), hormones, cytokines, and co-stimulatory factors.

[0108] As used herein, the term "antibody" collectively refers to immunoglobulins or immunoglobulin-like molecules, including, for example, but not limited to, IgA, IgD, IgE, IgG, and IgM, combinations thereof, and similar molecules produced during an immune response in any vertebrate, such as mammals (such as humans, goats, rabbits, and mice), as well as non-mammalian species (such as shark immunoglobulins). As used herein, "antibodies" (including complete immunoglobulins) and "antigen-binding fragments" specifically bind to a target molecule (or a group of highly similar target molecules) while substantially excluding binding to other molecules (e.g., the binding constant for the target molecule is at least 10 greater than the binding constant for other molecules in the biological sample). 3 M -1 , at least 10 4 M -1 or at least 10 5 M -1 The term "antibody" also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (e.g., bispecific antibodies). See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Illinois); Kuby, J., Immunology, 3rd Edition, WH Freeman & Co., New York, 1997.

[0109] More specifically, an antibody refers to a polypeptide ligand that specifically recognizes and binds to an antigen epitope and contains at least a light chain immunoglobulin variable region or a heavy chain immunoglobulin variable region. Antibodies are composed of heavy chains and light chains, each of which has a variable region, called a heavy chain variable region (V H ) region and light chain variable (V L ) area. H Area and V LThe regions are jointly responsible for binding to the antigen recognized by the antibody. Typically, immunoglobulins have heavy (H) chains and light (L) chains interconnected by disulfide bonds. There are two types of light chains, namely λ (lambda) and κ (kappa). There are five main heavy chain categories (or isotypes): IgM, IgD, IgG, IgA and IgE, which determine the functional activity of the antibody molecule. Each heavy chain and light chain contains a constant region and a variable region (the region is also referred to as a "domain"). The combined heavy chain variable region and light chain variable region specifically bind to the antigen. The light chain variable region and the heavy chain variable region contain a "framework" region interrupted by three hypervariable regions (also referred to as "complementarity determining regions" or "CDRs"). The scope of the framework region and CDR has been defined (see Kabat et al., Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991, which is hereby incorporated by reference). The Kabat database is currently maintained online. The sequences of the framework regions of different light or heavy chains are relatively conserved within species. The framework region of an antibody (i.e., the combined framework region of the constituent light and heavy chains) predominantly adopts a β-sheet conformation, and the CDR forms a loop connecting the β-sheet structure, and in some cases the loop forms a part of the β-sheet structure. Therefore, the framework region plays a role in forming a scaffold that positions the CDR in the correct orientation through interchain non-covalent interactions.

[0110] The CDRs are primarily responsible for binding to the antigenic epitope. The CDRs of each chain are usually referred to as CDR1, CDR2, and CDR3, are numbered sequentially starting from the N-terminus, and are usually also identified according to the chain in which a particular CDR is located. H CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, while V L CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found. An antibody that binds poly-Sia will have a specific V H Area and V L Region sequence, thus having a specific CDR sequence. Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. Although CDRs are different between different antibodies, only a limited number of amino acid positions in the CDR are directly involved in antigen binding. These positions in the CDR are called specificity determining residues (SDRs). As used herein, "immunoglobulin-related compositions" refer to antibodies (including monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, multispecific antibodies, bispecific antibodies, etc.) and antibody fragments. Antibodies or their antigen-binding fragments specifically bind to antigens.

[0111] As used herein, the term "antibody-related polypeptide" means an antigen-binding antibody fragment including a single-chain antibody, which may contain one or more variable regions alone or in combination with all or part of the following polypeptide elements: the hinge region, CH1, CH2, and CH3 domains of an antibody molecule. The technology also includes any combination of one or more variable regions and hinge regions, CH1, CH2, and CH3 domains. Antibody-related molecules that can be used in the present method include, for example, but are not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (sdFv), and V L or V H Examples include: (i) Fab fragments, i.e., fragments of V L 、V H , C L and CH1 domains; (ii) F(ab')2 fragments, i.e. bivalent fragments comprising two Fab fragments connected by a disulfide bridge in the hinge region; (iii) H and CH1 domains; (iv) a V fragment consisting of a single arm of the antibody L and V H (v) dAb fragment (Ward et al., Nature 341: 544-546, 1989), which consists of V H domain composition; and (vi) isolated complementarity determining regions (CDRs). Thus, an "antibody fragment" or "antigen binding fragment" may comprise a portion of a full-length antibody, typically its antigen binding or variable region. Examples of antibody fragments or antigen binding fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0112] As used herein, "bispecific antibody" or "BsAb" refers to an antibody that can simultaneously bind to two targets with different structures (e.g., two different target antigens, two different epitopes on the same target antigen, or a hapten and a target antigen or an epitope on a target antigen). A variety of different bispecific antibody structures are known in the art. In some embodiments, each antigen-binding portion in a bispecific antibody includes a V H and / or V L region; in some such embodiments, V H and / or V L The V regions are those found in a particular monoclonal antibody. In some embodiments, the bispecific antibody contains two antigen-binding portions, each of which includes a V region from a different monoclonal antibody. H and / or V LIn some embodiments, the bispecific antibody contains two antigen-binding moieties, wherein one of the two antigen-binding moieties comprises a V H and / or V L region of the immunoglobulin molecule, the V H and / or V L The second antigen binding portion comprises a CDR region from the first monoclonal antibody; and the second antigen binding portion comprises a CDR region from the first monoclonal antibody; H and / or V L The antibody fragment of the V region (e.g., Fab, F(ab'), F(ab')2, Fd, Fv, dAB, scFv, etc.), H and / or V L The region contains the CDRs from the second monoclonal antibody.

[0113] As used herein, a "scavenger" is an agent that binds to excess bispecific antibodies present in the blood compartment of a subject to promote rapid clearance via the kidneys. The use of a scavenger prior to hapten administration (e.g., DOTA) helps to achieve a better tumor to background ratio in a pretargeted radioimmunotherapy (PRIT) system. Examples of scavengers include 500kD-dextran-DOTA-Bn(Y) (Orcutt et al., Mol Cancer Ther. 11(6): 1365–1372 (2012)), 500kD aminodextran-DOTA conjugates, antibodies against pretargeted antibodies, and the like.

[0114] As used herein, the term "conjugated" refers to the association of two molecules by any method known to those skilled in the art. Suitable types of association include chemical bonds and physical bonds. Chemical bonds include, for example, covalent bonds and coordination bonds. Physical bonds include, for example, hydrogen bonds, dipole interactions, van der Waals forces, electrostatic interactions, hydrophobic interactions, and aromatic ring stacking.

[0115] As used herein, the term "diabodies" refers to small antibody fragments with two antigen-binding sites, said fragments comprising two antigen-binding sites bound to the same polypeptide chain (V H -V L ) in the light chain variable domain (V L ) connected to the heavy chain variable domain (V H ). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are more fully described in, for example, EP 404,097; WO 93 / 11161; and 30 Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).

[0116] As used herein, the term "single-chain antibody" or "single-chain Fv (scFv)" refers to the two domains V and Fv fragments. L and V H The single-chain antibody molecule may contain a polymer having multiple individual molecules, such as a dimer, trimer or other polymer. v The two domains of the fragment V L and V H The two proteins are encoded by separate genes, but they can be joined by synthetic linkers using recombinant methods to make them into a single protein chain in which V L and V H The domains pair to form a monovalent molecule (called a single-chain F v (scF v )). Bird et al. (1988) Science 242:423-426 and Huston et al. (1988) Proc. Natl. Acad Sci. USA 85:5879-5883. Such single-chain antibodies can be prepared by recombinant techniques or by enzymatic or chemical cleavage of intact antibodies.

[0117] Fragments of any of the foregoing antibodies are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for binding specificity and neutralization activity in the same manner as are intact antibodies.

[0118] As used herein, "antigen" refers to a molecule to which an antibody (or its antigen-binding fragment) can selectively bind. The target antigen can be a protein, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, the target antigen can be a carbohydrate (e.g., a poly-Sia species). Antigens can also be administered to animals to generate an immune response in the animal.

[0119] The term "antigen binding fragment" refers to a fragment of a complete immunoglobulin structure having a polypeptide portion responsible for binding to an antigen. Examples of antigen binding fragments that can be used in the present technology include, but are not limited to, scFv, (scFv)2, scFvFc, Fab, Fab' and F(ab')2.

[0120] "Binding affinity" refers to the strength of the total non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and the molecule's binding partner (e.g., an antigen or antigenic peptide). The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (K D) is represented by. Affinity can be measured by standard methods known in the art (including those described herein). Low affinity complexes contain antibodies that generally tend to dissociate easily from the antigen, while high affinity complexes contain antibodies that generally tend to remain bound to the antigen for a longer period of time.

[0121] As used herein, the term "biological sample" means a sample material derived from living cells. Biological samples can include tissues, cells, proteins or membrane extracts of cells separated from a subject and biological fluids (e.g., ascites or cerebrospinal fluid (CSF)), as well as tissues, cells and fluids present in a subject. The biological samples of this technology include but are not limited to samples taken from: breast tissue, kidney tissue, cervix, endometrium, head or neck, gallbladder, parotid tissue, prostate, brain, pituitary gland, kidney tissue, muscle, esophagus, stomach, small intestine, colon, liver, spleen, pancreas, thyroid tissue, heart tissue, lung tissue, bladder, adipose tissue, lymph node tissue, uterus, ovarian tissue, adrenal tissue, testicular tissue, tonsil, thymus, blood, hair, cheek, skin, serum, plasma, CSF, sperm, prostatic fluid, semen, urine, feces, sweat, saliva, sputum, mucus, bone marrow, lymph and tears. Biological samples can also be obtained from the biopsy of internal organs or from cancer. Biological samples can be obtained from subjects for diagnosis or research; or can be obtained from individuals who are not ill, as controls or for basic research. Samples can be obtained by standard methods, including, for example, venipuncture and surgical biopsy. In certain embodiments, the biological sample is a breast, lung, pancreas, adrenal gland, brain, kidney, nerve or muscle tissue sample obtained by needle biopsy. Surgical biopsy samples can be derived from fresh or frozen samples obtained during surgery, patient-derived xenografts, or cell lines established for surgical specimens.

[0122] As used herein, the term "CDR-grafted antibody" means an antibody in which at least one CDR of a "recipient" antibody is replaced by a CDR "graft" having the desired antigen specificity from a "donor" antibody.

[0123] As used herein, the term "chimeric antibody" refers to an antibody in which the Fc constant region of a monoclonal antibody from one species (e.g., a mouse Fc constant region) is replaced with the Fc constant region of an antibody from another species (e.g., a human Fc constant region) using recombinant DNA technology. See generally, Robinson et al., PCT / US86 / 02269; Akira et al., European Patent Application 184,187; Taniguchi, European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., WO 86 / 01533; Cabilly et al., U.S. Patent No. 4,816,567; Cabilly et al., European Patent Application 0125,023; Better et al., Science 240:1041-1043, 1988; Liu et al., Proc. Natl. Acad. Sci. USA 84:3439-3443, 1987; Liu et al., J. Immunol 139:3521-3526, 1987; Sun et al., Proc. Natl. Acad. Sci. USA 84:214-218, 1987; Nishimura et al., Cancer Res 47:999-1005, 1987; Wood et al., Nature 314:446-449, 1885; and Shaw et al., J. Natl. Cancer Inst. 80:1553-1559, 1988.

[0124] As used herein, the term "consensus FR" means the framework (FR) antibody region in the consensus immunoglobulin sequence. The FR region of an antibody does not contact the antigen.

[0125] As used herein, a "control" is an alternative sample used in an experiment for comparison purposes. A control can be "positive" or "negative." For example, where the purpose of an experiment is to determine the relevance of the efficacy of a therapeutic agent for the treatment of a particular type of disease, a positive control (a compound or composition known to exhibit the desired therapeutic effect) and a negative control (a subject or sample that does not receive treatment or receives a placebo) are typically used.

[0126] As used herein, the term "effective amount" refers to an amount sufficient to achieve the desired therapeutic and / or preventive effect, such as an amount that results in the prevention or reduction of a disease or condition described herein or one or more signs or symptoms associated with a disease or condition described herein. In the case of therapeutic or preventive applications, the amount of the composition administered to the subject will vary according to the composition, the extent, type and severity of the disease, and according to individual characteristics such as general health, age, sex, body weight and drug tolerance. The technician will be able to determine the appropriate dosage based on these and other factors. The composition may also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, the therapeutic composition may be administered to a subject having one or more signs or symptoms of a disease or condition described herein. As used herein, a "therapeutically effective amount" of a composition refers to a composition level at which the physiological effects of a disease or condition are improved or eliminated. The therapeutically effective amount may be administered in one or more administrations.

[0127] As used herein, the term "effector cell" means an immune cell that participates in the effector phase of an immune response, which is opposite to the cognitive phase and activation phase of an immune response. Exemplary immune cells include cells of bone marrow or lymphoid origin, such as lymphocytes (e.g., B cells and T cells, including cytolytic T cells (CTL)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mast cells, and basophils. Effector cells express specific Fc receptors and perform specific immune functions. Effector cells can induce antibody-dependent cell-mediated cytotoxicity (ADCC), such as neutrophils that can induce ADCC. For example, monocytes, macrophages, neutrophils, eosinophils, and lymphocytes expressing FcαR participate in the specific killing of target cells and present antigens to other components of the immune system, or bind to cells presenting antigens.

[0128] As used herein, the term "epitope" means an antigenic determinant that can specifically bind to an antibody. An epitope is usually composed of chemically active surface groups of a molecule (such as amino acids or sugar side chains) and usually has specific three-dimensional structural characteristics, as well as specific charge characteristics. The difference between a conformational epitope and a non-conformational epitope is that in the presence of a denaturing solvent, the binding to the conformational epitope rather than the non-conformational epitope is lost. In some embodiments, the "epitope" of poly-Sia is a conformational epitope specifically employed by high-DP poly-Sia (e.g., DP>10, DP>20, DP>50, DP>100 or DP>200) to which the anti-poly-Sia antibodies of the present technology specifically bind. In order to screen for anti-poly-Sia antibodies that bind to an epitope, a conventional cross-blocking assay can be performed, such as the assay described in the following document: Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Harlow and David Lane (1988). This assay can be used to determine whether an anti-poly-Sia antibody binds to the same site or epitope as the anti-poly-Sia antibodies of the present technology. Alternatively or additionally, epitope mapping can be performed by methods known in the art. For example, the antibody sequence can be mutagenized, such as by alanine scanning, to identify contact residues. In a different approach, poly-Sia species with different DPs can be used in competition assays with multiple test antibodies, or with one test antibody and one antibody with a characterized or known epitope specificity.

[0129] As used herein, "expression" includes one or more of the following: transcription of a gene into a precursor mRNA; splicing and other processing of the precursor mRNA to produce a mature mRNA; mRNA stability; translation of the mature mRNA into protein (including codon usage and tRNA availability); and glycosylation and / or other modifications of the translation product (if required for proper expression and function).

[0130] As used herein, the term "gene" means a segment of DNA that contains all the information for regulated biosynthesis of an RNA product, including promoters, exons, introns, and other untranslated regions that control expression.

[0131] "Homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing the positions in each sequence, which can be aligned for comparison purposes. When the position in the compared sequence is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences varies with the number of matching or homologous positions shared by the sequences. A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) has a certain percentage (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of "sequence identity" with another sequence, which means that, when aligned, the bases (or amino acids) of that percentage are the same in comparing the two sequences. This alignment and homology or sequence identity percentage can be determined using software programs known in the art. In some embodiments, default parameters are used for alignment. One alignment program is BLAST, using default parameters. Specifically, the programs are BLASTN and BLASTP, using the following default parameters: genetic code = standard; filter = none; chain = two; cutoff = 60; expectation = 10; matrix = BLOSUM62; description = 50 sequences; sorting mode = high score (HIGH SCORE); database = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translation + SwissProtein + SPupdate + PIR. Detailed information on these programs can be found at the National Center for Biotechnology Information. Biologically equivalent polynucleotides are those polynucleotides with a specified homology percentage and encoding polypeptides with the same or similar biological activity. If two sequences share less than 40% identity or less than 25% identity with each other, the sequences are considered "unrelated" or "non-homologous".

[0132] As used herein, the "humanized" form of the term non-human (e.g., mouse) antibody is a chimeric antibody containing a minimal sequence derived from a non-human immunoglobulin. For the most part, a humanized antibody is a human immunoglobulin, in which the hypervariable region residues of the receptor are replaced by the hypervariable region residues (donor antibody) with the desired specificity, affinity and ability from a non-human species (e.g., mouse, rat, rabbit or non-human primate). In some embodiments, the Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, a humanized antibody may be included in residues not found in a receptor antibody or a donor antibody. These modifications are carried out to further improve antibody performance such as binding affinity. Typically, a humanized antibody will comprise substantially all of at least one, usually two variable domains (e.g., Fab, Fab', F(ab')2, or Fv), wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the FR regions are those of the consensus FR sequences of human immunoglobulins, but the FR regions may include one or more amino acid substitutions that improve binding affinity. The number of these amino acid substitutions in the FR is typically no more than 6 in the H chain and no more than 3 in the L chain. The humanized antibody may optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin constant region. For further details, see Jones et al., Nature 321:522-525 (1986); Reichmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See, e.g., Ahmed and Cheung, FEBS Letters 588(2):288-297 (2014).

[0133] As used herein, the term "hypervariable region" refers to the amino acid residues in an antibody that are responsible for antigen binding. A hypervariable region generally includes amino acid residues from a "complementarity determining region" or "CDR" (e.g., V L before and after residues 24-34 (L1), 50-56 (L2), and 89-97 (L3), and V H 31-35B (H1), 50-65 (H2), and 95-102 (H3) in the sequence (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Maryland. (1991)) and / or those residues from the "hypervariable loops" (e.g., V LResidues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in V H 26-32 (H1), 52A-55 (H2) and 96-101 (H3) (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).

[0134] As used herein, when used in the context of two or more nucleic acids or polypeptide sequences, the term "identical" or "identity" percentage refers to two or more sequences or subsequences that are identical or have a specified percentage of identical amino acid residues or nucleotides (i.e., about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity over a specified region (e.g., a nucleotide sequence encoding an antibody described herein or an amino acid sequence of an antibody described herein) when compared and aligned for maximum correspondence over a comparison window or a specific region), as measured using the BLAST or BLAST 2.0 sequence comparison algorithm using the default parameters described below or by manual alignment and visual inspection (e.g., the NCBI website). Such sequences are then referred to as "substantially identical". This term also refers to or can be applied to the complement of a test sequence. The terms also include sequences with deletions and / or additions, as well as those with substitutions.In some embodiments, identity exists over a region of at least about 25 amino acids or nucleotides in length, or 50-100 amino acids or nucleotides in length.

[0135] As used herein, the term "intact antibody" or "intact immunoglobulin" means an antibody having at least two heavy (H) chain polypeptides and two light (L) chain polypeptides interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as HCVR or V H ) and a heavy chain constant region. The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as LCVR or V L ) and the light chain constant region. The light chain constant region consists of a domain C L Composition. H and V L The V region can be further subdivided into regions of high variability, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). H and V LIt is composed of three CDRs and four FRs, and the order of arrangement from the amino terminal to the carboxyl terminal is as follows: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0136] As used herein, the term "individual", "patient" or "subject" can be a single organism, a vertebrate, a mammal or a human. In some embodiments, the individual, patient or subject is a human.

[0137] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, that is, except for a possible naturally occurring mutation that may be present in a small amount, the individual antibodies constituting the population are identical. For example, a monoclonal antibody may be an antibody derived from a single clone (including any eukaryotic, prokaryotic or phage clone) rather than a method for producing it. A monoclonal antibody composition exhibits a single binding specificity and affinity to a specific epitope. Monoclonal antibodies are highly specific for a single antigenic site. In addition, compared to conventional (polyclonal) antibody preparations that typically include different antibodies for different determinants (epitopes), each monoclonal antibody is for a single determinant on an antigen. The modifier "monoclonal" indicates that the antibody is characterized by being obtained from a substantially homogeneous antibody population, and should not be construed as requiring the antibody to be produced by any ad hoc method. Monoclonal antibodies can be prepared using a variety of techniques known in the art, including, for example, but not limited to, hybridoma, recombinant and phage display techniques. For example, the monoclonal antibody to be used according to the method of the invention can be prepared by the hybridoma method originally described by Kohler et al., Nature 256:495 (1975), or can be prepared by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). For example, "monoclonal antibodies" can also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991).

[0138] As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal compounds, isotonic and absorption delaying compounds, etc., that are compatible with pharmaceutical administration. Pharmaceutically acceptable carriers and their formulations are known to those skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences (20th edition, A. Gennaro, 2000, Lippincott ed., Williams & Wilkins, Philadelphia, Pennsylvania).

[0139] As used herein, the term "polyclonal antibody" means a preparation of antibodies derived from at least two (2) different antibody-producing cell lines. The use of this term includes preparations of at least two (2) antibodies containing antibodies that specifically bind to different epitopes or regions of an antigen.

[0140] As used herein, the term "polynucleotide" or "nucleic acid" means any RNA or DNA, which can be unmodified or modified RNA or DNA. Polynucleotides include, but are not limited to, single-stranded and double-stranded DNA, DNA as a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, RNA as a mixture of single-stranded and double-stranded regions, and hybrid molecules comprising DNA and RNA, which can be single-stranded or more generally double-stranded or a mixture of single-stranded and double-stranded regions. In addition, polynucleotides refer to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases, and DNA or RNA to which the backbone is modified for stability or other reasons.

[0141] As used herein, the terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer comprising two or more amino acids linked to each other by peptide bonds or modified peptide bonds (i.e., peptide isosteres). Polypeptides refer to both short chains, commonly referred to as peptides, glycopeptides or oligomers, and longer chains, commonly referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. Polypeptides include amino acid sequences modified by natural processes (such as post-translational processing) or by chemical modification techniques well known in the art. Such modifications are fully described in basic textbooks and more detailed monographs as well as lengthy research literature.

[0142] As used herein, "PRIT" or "Pretargeted Radioimmunotherapy" refers to a multi-step process that addresses the slow blood clearance of tumor-targeting antibodies, which leads to undesirable toxicity to normal tissues such as the bone marrow. In pretargeting, a radionuclide or other diagnostic or therapeutic agent is attached to a small hapten. A pretargeting bispecific antibody with binding sites for the hapten as well as the target antigen is first administered. Unbound antibody is then allowed to clear from the circulation, and the hapten is subsequently administered.

[0143] As used herein, the term "recombinant" when used with respect to, for example, a cell or a nucleic acid, protein or vector, indicates that the cell, nucleic acid, protein or vector has been modified by the introduction of a heterologous nucleic acid or protein or by the alteration of a native nucleic acid or protein, or that the material is derived from a cell so modified. Thus, for example, a recombinant cell expresses genes not found in the native (non-recombinant) form of the cell, or expresses native genes that are otherwise abnormally expressed, under-expressed, or not expressed at all.

[0144] As used herein, the term "separate" therapeutic use refers to the simultaneous or substantially simultaneous administration of at least two active ingredients by different routes.

[0145] As used herein, the term "sequential" therapeutic use refers to administration of at least two active ingredients at different times, with the same or different routes of administration. More specifically, sequential use refers to the entire administration of one active ingredient before the administration of the other one or more active ingredients begins. Thus, the other active ingredient may be administered a few minutes, hours, or days before the administration of one or more active ingredients. In this case, there is no simultaneous treatment.

[0146] As used herein, "specifically binds" refers to a molecule (e.g., an antibody or antigen-binding fragment thereof) that recognizes and binds to another molecule (e.g., an antigen) but does not substantially recognize and bind to other molecules. As used herein, the terms "specifically bind," "specifically binds," or "specific for" a particular molecule (e.g., a carbohydrate antigen or an epitope on a carbohydrate antigen) can be defined, for example, by a molecule having about 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 M of K DThe term "specifically binds" may also refer to binding in which a molecule (e.g., an antibody or antigen-binding fragment thereof) binds to a particular antigen (e.g., high-DP poly-Sia) without substantially binding to any other antigen or antigen form (e.g., low-DP poly-Sia).

[0147] As used herein, the term "simultaneous" therapeutic use refers to the administration of at least two active ingredients by the same route and at the same time or substantially the same time.

[0148] As used herein, the term "therapeutic agent" is intended to mean a compound that, when present in an effective amount, produces a desired therapeutic effect on a subject in need thereof.

[0149] As used herein, "treating" or "treatment" encompasses treating a disease or disorder described herein in a subject, such as a human, and includes: (i) inhibiting the disease or disorder, i.e., preventing its development; (ii) alleviating the disease or disorder, i.e., causing regression of the disorder; (iii) slowing the progression of the disorder; and / or (iv) inhibiting, alleviating, or slowing the progression of one or more symptoms of the disease or disorder. In some embodiments, treatment means that symptoms associated with the disease are, for example, alleviated, reduced, cured, or in a state of remission.

[0150] It is also understood that the various treatment modalities for disorders as described herein are intended to mean "substantially," which includes complete treatment but also less than complete treatment, and in which some biologically or medically relevant result is achieved. Treatment can be continuous, long-term treatment for chronic diseases, or a single or several administrations to treat acute conditions.

[0151] One or more amino acid sequence modifications of the anti-polySia antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of anti-polySia antibodies are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the antibody amino acid sequence. Any combination of deletions, insertions and substitutions may be performed to obtain the desired antibody, as long as the antibody obtained has the desired properties. Modifications also include changes in the glycosylation pattern of the protein. The sites of greatest interest for substitution mutagenesis include the hypervariable regions, but FR changes are also contemplated. "Conservative substitutions" are shown in the table below.

[0152] One type of substitution variant involves replacing one or more hypervariable region residues of a parent antibody. A convenient method for generating such substitution variants involves affinity maturation using phage display. In particular, several hypervariable region sites (e.g., 6-7 sites) are mutated to produce all possible amino acid substitutions at each site. The antibody variants thus produced are displayed as fusions of the gene III product of M13 packaged in each particle in a monovalent manner from filamentous phage particles. The variants displayed by phage display are then screened for their biological activity (e.g., binding affinity), as disclosed herein. In order to identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively or additionally, analyzing the crystal structure of the antigen-antibody complex may be beneficial to identify the contact points between the antibody and the antigen. According to the technology described in detail herein, such contact residues and adjacent residues are candidates for substitution. Once such variants are produced, this group of variants is screened as described herein, and antibodies with similar or better properties in one or more related assays can be selected for further development. Polysialic acid

[0153] It was found that sialic acid itself can also be linked together in long chains of up to 200 residues, called polysialic acid (polySia), which are linked via α2-8 bonds and are mainly found on the outer chains of N-linked oligosaccharides of neural cell adhesion molecules (NCAM) and other proteins (Finne, J., et al. Biochem Biophys Res Commun 112, 482-487 (1983)). NCAM is expressed on cells of neuroectodermal origin and plays a role in neural tissue development and regeneration (Sadoul, R., et al. Nature 304, 347-349 (1983)). PolySia regulates homophilic interactions between NCAM molecules and heterophilic interactions with other adhesion molecules (Schreiber, SC, et al. Gastroenterology 134, 1555-1566 (2008)). The biological properties of poly-Sia depend on the degree of polymerization (DP), which is high in embryonic development but low in adult tissues (Rutishauser, U. Nat Rev Neurosci 9, 26-35 (2008)). High-DP poly-Sia has shown an inhibitory effect on cell adhesion (Muhlenhoff, M., et al. Curr Opin Struct Biol 8, 558-564 (1998); Johnson, CP, et al. J Biol Chem 280, 137-145 (2005)) and is expressed on several metastatic cancers, including small cell and non-small cell lung cancer, neuroblastoma, pancreatic cancer, pituitary tumors, Wilms' tumor, rhabdomyosarcoma (Falconer, RA, et al. Curr Cancer Drug Targets 12, 925-939 (2012)), glioblastoma (Amoureux, MC, et al. BMC Cancer 10, 91 (2010)) and breast cancer (Wang, X., et al. Int J Mol Med 37, 197-206 (2016)). In neuroblastoma, the DP of poly-Sia is greater than 55 Sia units (Livingston, BD, et al. J Biol Chem 263, 9443-9448 (1988)).Since poly-Sia is widely expressed in a variety of tumor types and is almost absent in most adult tissues, poly-Sia is a target for cancer immunotherapy.

[0154] Several monoclonal antibodies have been developed for mono- and disialic acid (1-2 Sia units), oligosialic acid (3-5 Sia units) and poly-Sia (at least 8 Sia units). See Sato, C., and Kitajima, KJ Biochem 154, 115-136 (2013). Among them, only mouse mAb735 (Bitter-Suermann, D., and Roth, J. Immunol Res 6, 225-237 (1987)) is described as having the highest specificity for high DP poly-Sia (requiring at least 11 Sia units) (Sato, C., and Kitajima, KJ Biochem 154, 115-136 (2013)). Further studies of the binding kinetics of mAb735 showed that it has a high affinity (subnanomolar dissociation constant K) for poly-Sia with high DP (about 200 Sia units). D ), and the affinity for lower degrees of polymerization gradually decreases (Hayrinen, J., et al. Mol Immunol 39, 399-411 (2002)). Immunoglobulin-related compositions of the present technology

[0155] The present technology describes methods and compositions for producing and using anti-polySia immunoglobulin-related compositions (e.g., anti-polySia antibodies or antigen-binding fragments thereof). The anti-polySia immunoglobulin-related compositions of the present disclosure can be used to diagnose or treat polySia-related cancers. Anti-polySia immunoglobulin-related compositions within the scope of the present technology include, for example, but not limited to, monoclonal antibodies, chimeric antibodies, humanized antibodies, and diabodies that specifically bind to polySia, its homologs, derivatives, or fragments. Anti-polySia immunoglobulin-related compositions within the scope of the present technology also include compositions in the form of bispecific antibodies to enhance anti-tumor efficacy (e.g., via T cell recruitment or payload delivery). The present disclosure also provides an antigen-binding fragment of any anti-polySia antibody disclosed herein, wherein the antigen-binding fragment is selected from Fab, F(ab)'2, Fab', scF v and F v The amino acid sequences of the CDR regions of the immunoglobulin-related compositions disclosed herein are defined according to the Kabat system.

[0156] In one aspect, the present technology provides a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domain (V L ) or its antigen-binding fragment, wherein (a) said V HA V comprising a V selected from DYYIH (SEQ ID NO: 1), RYYIH (SEQ ID NO: 7), GYYIH (SEQ ID NO: 8) and NYYIH (SEQ ID NO: 9) H -CDR1 sequence selected from WIYPGSGNTKYNEKFKG (SEQ ID NO: 2), SIYPGSGNTKYNEKFKG (SEQ ID NO: 10), RIYPGSGNTKYNEKFKG (SEQ ID NO: 11), CIYPGSGNTKYNEKFKG (SEQ ID NO: 12), WIYPGSGNTKYNEKFEG (SEQ ID NO: 12) NO:13), WIYPGSGNTKYNQKFQG (SEQ ID NO:14), WIYPGSGNTKYSQKFQG (SEQ ID NO:15), WIYPGSGNTKYSEKFQG (SEQ ID NO:16) and WIYPGSGNTKYSQKFKG (SEQ ID NO:18) H -CDR2 sequence and V of GGKFAMDY (SEQ ID NO: 3) H -CDR3 sequence; and / or (b) the V L comprising a V selected from RSSQSLVHSNGNTYLY (SEQ ID NO: 4) and RSSQSLVHSNGKTYLY (SEQ ID NO: 20); L - CDR1 sequence, V of RVSNRFS (SEQ ID NO: 5) L - CDR2 sequence and a V selected from FQGTHVPYT (SEQ ID NO: 6), FQGTHVPYI (SEQ ID NO: 21) and FQGTHEPYT (SEQ ID NO: 22) L -CDR3 sequence.

[0157] In some embodiments, the present disclosure provides a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domain (V L ) or an antigen-binding fragment thereof, wherein (a) V H V comprising SEQ ID NO: 1 H -CDR1 sequence, V of SEQ ID NO: 2 H -CDR2 sequence, V of SEQ ID NO: 3 H - CDR3 sequence; and / or (b) V L Contains V selected from the following L -CDR1 sequence, VL -CDR2 sequence and V L - CDR3 sequences: SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6; SEQ ID NO:20, SEQ ID NO:5 and SEQ ID NO:6; SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:22.

[0158] In some embodiments, the present disclosure provides a light chain immunoglobulin variable domain (V L ) and the heavy chain immunoglobulin variable domain (V H ) or an antigen-binding fragment thereof, wherein (a) V L V comprising SEQ ID NO: 4 L -CDR1 sequence, V of SEQ ID NO: 5 L -CDR2 sequence, V of SEQ ID NO:6 L - CDR3 sequence; and / or (b) V H Contains V selected from the following H -CDR1 sequence, V H -CDR2 sequence and V H -CDR3 sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3; SEQ ID NO:1, SEQ ID NO:11 and SEQ ID NO:3; SEQ ID NO:1, SEQ ID NO:12, SEQ ID NO:3; SEQ ID NO:1, SEQ ID NO:13 and SEQ ID NO:3; SEQ ID NO:7, SEQ ID NO:2 and SEQ ID NO:3; SEQ ID SEQ ID NO:7, SEQ ID NO:11, and SEQ ID NO:3; SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:3; SEQ ID NO:8, SEQ ID NO:12, and SEQ ID NO:3; SEQ ID NO:9, SEQ ID NO:11, and SEQ ID NO:3; SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:3; SEQ ID NO:1, SEQ ID NO:14 and SEQ ID NO:3; SEQ ID NO:1, SEQ ID NO:15 and SEQ ID NO:3; SEQ ID NO:1, SEQ ID NO:16 and SEQ ID NO:3; and SEQ ID NO:1, SEQ ID NO:18 and SEQ ID NO:3.

[0159] In some embodiments, the present disclosure provides a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domain (V L ) or an antigen-binding fragment thereof, wherein V H Contains V H -CDR1 sequence, V H -CDR2 sequence and V H -CDR1 sequence, and V L Contains V L -CDR1 sequence, V L -CDR2 sequence and V L - CDR3 sequences selected from the group consisting of: a) SEQ ID NOs: 1, 2, 3, 4, 5 and 6, respectively; b) SEQ ID NOs: 1, 2, 3, 4, 5 and 22, respectively; c) SEQ ID NOs: 1, 2, 3, 20, 5 and 6, respectively; d) SEQ ID NOs: 1, 11, 3, 4, 5 and 6, respectively; e) SEQ ID NOs: 1, 12, 3, 4, 5 and 6, respectively; f) SEQ ID NOs: 1, 13, 3, 4, 5 and 6, respectively; g) SEQ ID NOs: 1, 13, 3, 4, 5 and 21, respectively; h) SEQ ID NOs: 7, 2, 3, 4, 5 and 6, respectively; i) 7, 11, 3, 4, 5 and 6, respectively; j) SEQ ID NOs: 8, 10, 3, 4, 5 and 6, respectively; k) 8, 12, 3, 4, 5 and 6, respectively; l) SEQ ID NOs: NO: 9, 11, 3, 4, 5 and 6; and m) are SEQ ID NO: 9, 12, 3, 4, 5 and 6, respectively.

[0160] In some embodiments, the antibody further comprises an Fc domain of any isotype, such as, but not limited to, IgG (including IgG1, IgG2, IgG3, and IgG4), IgA (including IgA1 and IgA2), IgD, IgE, or IgM and IgY. Non-limiting examples of constant region sequences include:

[0161] Human IgD constant region, Uniprot: P01880 (SEQ ID NO: 85) APTKAPDVFPIISGCRHPKDNSPVVLACLITGYHPTSVTVTWYMGTQSQPQRTFPEIQRRDSYYMTSSQLSTPLQQWRQGEYKCVVQHTASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDHGPMK

[0162] Human IgG1 constant region, Uniprot: P01857 (SEQ ID NO:86) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0163] Human IgG2 constant region, Uniprot: P01859 (SEQ ID NO:87) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0164] Human IgG3 constant region, Uniprot: P01860 (SEQ ID NO:88) ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK

[0165] Human IgM constant region, Uniprot: P01871 (SEQ ID NO:89) GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITLSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY

[0166] Human IgG4 constant region, Uniprot: P01861 (SEQ ID NO:90) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0167] Human IgA1 constant region, Uniprot: P01876 (SEQ ID NO:91) ASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQDASGDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY

[0168] Human IgA2 constant region, Uniprot: P01877 (SEQ ID NO:17) ASPTSPKVFPLSLDSTPQDGNVVVACLVQGFFPQEPLSVTWSESGQNVTARNFPPSQDASGDLYTTSSQLTLPATQCPDGKSVTCHVKHYTNPSQDVTVPCPVPPPPPCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGATFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAQPWNHGETFTCTAAHPELKTPLTANITKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRMAGKPTHVNVSVVMAEVDGTCY

[0169] Human Igκ constant region, Uniprot: P01834 (SEQ ID NO:19) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0170] In some embodiments, the immunoglobulin-related compositions of the present technology comprise a heavy chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ ID NO: 17 or 85-91. Additionally or alternatively, in some embodiments, the immunoglobulin-related compositions of the present technology comprise a light chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ ID NO: 19. In some embodiments, the immunoglobulin-related compositions of the present technology bind to high DP poly-Sia (e.g., a DP of about 10-20 Sia units, about 20-30 Sia units, about 30-50 Sia units, about 50-70 Sia units, about 70-100 Sia units, about 100-200 Sia units, or about 200-400 Sia units). In some embodiments, the antibody or antigen-binding fragment binds to high DP poly-Sia, for example, with DP>10, DP>20, DP>50, DP>100, or DP>200. In some embodiments, the epitope is a conformational epitope unique to high DP poly-Sia. In some embodiments, the conformational epitope includes 3 or more Sia units. In some embodiments, the 3 or more Sia units can be continuous units.

[0171] In another aspect, the disclosure provides an isolated immunoglobulin-related composition (e.g., an antibody or an antigen-binding fragment thereof) comprising a heavy chain (HC) amino acid sequence comprising SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, or a variant thereof having one or more conservative amino acid substitutions.

[0172] Additionally or alternatively, in some embodiments, the immunoglobulin-related compositions of the present technology comprise a light chain (LC) amino acid sequence comprising SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:60, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, or variants thereof having one or more conservative amino acid substitutions.

[0173] In some embodiments, the immunoglobulin-related compositions of the present technology comprise HC amino acid sequences and LC amino acid sequences selected from the following: SEQ ID NO:23 and SEQ ID NO:24 (chimeric P35); SEQ ID NO:25 and SEQ ID NO:28 (humanized P35 H1L2); SEQ ID NO:25 and SEQ ID NO:27 (humanized P35 H1L1); SEQ ID NO:26 and SEQ ID NO:28 (humanized P35 H2L2); SEQ ID NO:26 and SEQ ID NO:27 (humanized P35 H2L1); SEQ ID NO:48 and SEQ ID NO:49 (BC137); SEQ ID NO:51 and SEQ ID NO:52 (BC137 KS2); SEQ ID NO:53 and SEQ ID NO:54 (BC137 KS34); SEQ ID NO:55 and SEQ ID NO:49 (BC137 DS47); SEQ ID NO:56 and SEQ ID NO:57 (BC137 DS47); SEQ ID NO:56 and SEQ ID NO:49 (BC137 DS54); SEQ ID NO:57 and SEQ ID NO:49 (BC137 DS47 D31R); SEQ ID NO:58 and SEQ ID NO:49 (BC137 DS54 D31R); SEQ ID NO:55 and SEQ ID NO:50 (BC137-2 DS47); SEQ ID NO:56 and SEQ ID NO:50 (BC137-2 DS54); SEQ ID NO:59 and SEQ ID NO:60 (BC163); SEQ ID NO:74 and SEQ ID NO:82 (rehumanized P35H1L1); SEQ ID NO:75 and SEQ ID NO:82 (rehumanized P35H2L1); SEQ ID NO:76 and SEQ ID NO:82 (rehumanized P35H3L1); SEQ ID NO:77 and SEQ ID NO:82 (heavy humanized P35H4L1); SEQ ID NO:78 and SEQ ID NO:82 (heavy humanized P35H5L1); SEQ ID NO:79 and SEQ ID NO:82 (heavy humanized P35H6L1); SEQ ID NO:80 and SEQ ID NO:82 (heavy humanized P35H7L1); SEQ ID NO:81 and SEQ ID NO:82 (heavy humanized P35H8L1); SEQ ID NO:74 and SEQ ID NO:83 (heavy humanized P35H1L2);SEQ ID NO:75 and SEQ ID NO:83 (heavy humanized P35H2L2); SEQ ID NO:76 and SEQ ID NO:83 (heavy humanized P35H3L2); SEQ ID NO:77 and SEQ ID NO:83 (heavy humanized P35H4L2); SEQ ID NO:78 and SEQ ID NO:83 (heavy humanized P35H5L2); SEQ ID NO:79 and SEQ ID NO:83 (heavy humanized P35H6L2); SEQ ID NO:80 and SEQ ID NO:83 (heavy humanized P35H7L2); SEQ ID NO:81 and SEQ ID NO:83 (heavy humanized P35H8L2); SEQ ID NO:74 and SEQ ID NO:84 (heavy humanized P35H1L3); SEQ ID NO:75 and SEQ ID NO:84 (heavy humanized P35H2L3); SEQ ID NO:76 and SEQ ID NO:84 (heavy humanized P35H3L3); SEQ ID NO:77 and SEQ ID NO:84 (heavy humanized P35H4L3); SEQ ID NO:78 and SEQ ID NO:84 (heavy humanized P35H5L3); SEQ ID NO:79 and SEQ ID NO:84 (heavy humanized P35H6L3); SEQ ID NO:80 and SEQ ID NO:84 (heavy humanized P35H7L3); and SEQ ID NO:81 and SEQ ID NO:84 (heavy humanized P35H8L3). ;

[0174] In any of the above embodiments of the immunoglobulin-related compositions, the HC and LC immunoglobulin variable domain sequences form an antigen binding site that binds to high DP poly-Sia (e.g., a DP of about 10-20 Sia units, about 20-30 Sia units, about 30-50 Sia units, about 50-70 Sia units, about 70-100 Sia units, about 100-200 Sia units, or about 200-400 Sia units). In some embodiments, the antibody or antigen binding fragment binds to high DP poly-Sia, for example, with DP>10, DP>20, DP>50, DP>100, or DP>200. In some embodiments, the epitope is a conformational epitope unique to high DP poly-Sia. In some embodiments, the conformational epitope includes 3 or more Sia units. In some embodiments, the 3 or more Sia units may be continuous units.

[0175] In some embodiments, the HC and LC immunoglobulin variable domain sequences are components of the same polypeptide chain. In other embodiments, the HC and LC immunoglobulin variable domain sequences are components of different polypeptide chains. In certain embodiments, the antibody is a full-length antibody.

[0176] In some embodiments, the immunoglobulin-related compositions of the present technology specifically bind to poly-Sia. In some embodiments, the immunoglobulin-related compositions of the present technology are at about 10 -3 M, 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 The dissociation constant (K D ) binds to high DP poly Sia. In certain embodiments, the immunoglobulin-related composition is a monoclonal antibody, a chimeric antibody, a humanized antibody, or a bispecific antibody. In some embodiments, the antibody comprises a human antibody framework region.

[0177] In certain embodiments, the immunoglobulin-related compositions include one or more of the following features: (a) a light chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the light chain immunoglobulin variable domain sequence present in any one of SEQ ID NO: 37, 39, 41, 43, 44, 45, 47, 24, 27, 28, 49, 50, 52, 54, 60, 82, 83, or 84; and / or (b) a light chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the light chain immunoglobulin variable domain sequence present in any one of SEQ ID NO: 37, 39, 41, 43, 44, 45, 47, 24, 27, 28, 49, 50, 52, 54, 60, 82, 83, or 84; NO:30,31,32,33,34,35,36,38,40,42,46,23,25,26,29,48,51,53,55,56,57,58,59,74,75,76,77,78,79,80 or 81 in any one of the heavy chain immunoglobulin variable domain sequence present at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical heavy chain immunoglobulin variable domain sequence. In another aspect, one or more amino acid residues in the immunoglobulin-related compositions provided herein are substituted by another amino acid. The substitution can be a "conservative substitution" as defined herein.

[0178] In some embodiments, the immunoglobulin-related composition comprises a scFv having an amino acid sequence of any one of SEQ ID NOs: 61-73.

[0179] In some embodiments, the immunoglobulin-related composition comprises (a) an LC sequence that is at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the LC sequence present in any one of SEQ ID NO:24, 27, 28, 49, 50, 52, 54, 60, 82, 83 or 84; and / or (b) an HC sequence that is at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the HC sequence present in any one of SEQ ID NO:23, 25, 26, 29, 48, 51, 53, 55, 56, 57, 58, 59, 74, 75, 76, 77, 78, 79, 80 or 81.

[0180] In certain embodiments, the immunoglobulin-related composition comprises an IgG1 constant region comprising one or more amino acid substitutions selected from N297A and K322A. Additionally or alternatively, in some embodiments, the immunoglobulin-related composition comprises an IgG4 constant region comprising an S228P mutation.

[0181] The amino acid and nucleotide sequences of exemplary anti-polySia immunoglobulin-related compositions are shown, for example, in Figures 15-65.

[0182] In some aspects, the anti-polySia immunoglobulin-related compositions described herein contain structural modifications to promote rapid binding and cellular uptake and / or slow release. In some aspects, the anti-polySia immunoglobulin-related compositions (e.g., antibodies) of the present technology may contain deletions in the CH2 constant heavy chain region to promote rapid binding and cellular uptake and / or slow release. In some aspects, Fab fragments are used to promote rapid binding and cellular uptake and / or slow release. In some aspects, F(ab)'2 fragments are used to promote rapid binding and cellular uptake and / or slow release.

[0183] In one aspect, the present technology provides a nucleic acid sequence encoding a heavy chain or light chain of an immunoglobulin-related composition described herein. Also disclosed herein is a recombinant nucleic acid sequence encoding any antibody described herein. In some embodiments, the nucleic acid sequence is selected from SEQ ID NO: 92-108. In another aspect, the present technology provides a host cell that expresses any nucleic acid sequence encoding a heavy chain or light chain of an immunoglobulin-related composition described herein.

[0184] The immunoglobulin-related compositions (e.g., anti-poly-Sia antibodies) of the present technology can be monospecific, bispecific, trispecific, or have greater multispecificity. Multispecific antibodies can be specific for poly-Sia with different degrees of polymerization, or can be specific for both poly-Sia and heterologous compositions (e.g., heterologous polypeptides or solid support materials). See, e.g., WO 93 / 17715; WO 92 / 08802; WO 91 / 00360; WO 92 / 05793; Tutt et al., J. Immunol. 147: 60-69 (1991); U.S. Pat. Nos. 5,573,920, 4,474,893, 5,601,819, 4,714,681, 4,925,648; 6,106,835; Kostelny et al., J. Immunol. 148: 1547-1553 (1992). In some embodiments, the immunoglobulin-related compositions are chimeric. In certain embodiments, the immunoglobulin-related compositions are humanized.

[0185] The immunoglobulin-related compositions of the present technology can be further recombinantly fused to a heterologous polypeptide at the N-terminus or C-terminus, or chemically conjugated (including covalent and non-covalent conjugation) to a polypeptide or other composition. For example, the immunoglobulin-related compositions of the present technology can be recombinantly fused or conjugated to molecules and effector molecules (such as heterologous polypeptides, drugs or toxins) that can be used as markers in detection assays. See, for example, WO 92 / 08495; WO 91 / 14438; WO 89 / 12624; U.S. Patent No. 5,314,995; and EP 0 396 387.

[0186] In any of the above embodiments of the immunoglobulin-related compositions of the present technology, the antibody or antigen-binding fragment can be optionally conjugated to an agent selected from the following: an isotope, a dye, a chromogen, a contrast agent, a drug, a toxin, a cytokine, an enzyme, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA, or any combination thereof. For chemical bonds or physical binding, the functional groups on the immunoglobulin-related compositions are typically associated with the functional groups on the agent. Alternatively, the functional groups on the agent are associated with the functional groups on the immunoglobulin-related compositions.

[0187] Functional groups on the medicament and functional groups on the immunoglobulin-related composition can be directly associated. For example, functional groups (e.g., sulfhydryl groups) on the medicament can be associated with functional groups (e.g., sulfhydryl groups) on the immunoglobulin-related composition to form disulfide bonds. Alternatively, the functional groups can be associated through a cross-linking agent (i.e., a linker). Some examples of cross-linking agents are described below. The cross-linking agent can be attached to the medicament or the immunoglobulin-related composition. The number of medicaments or immunoglobulin-related compositions in the conjugate is also limited by the number of functional groups present on the other party. For example, the maximum number of medicaments associated with the conjugate depends on the number of functional groups present on the immunoglobulin-related composition. Alternatively, the maximum number of immunoglobulin-related compositions associated with the medicament depends on the number of functional groups present on the medicament.

[0188] In another embodiment, the conjugate comprises an immunoglobulin-related composition associated with an agent. In one embodiment, the conjugate comprises at least one agent chemically bonded (e.g., conjugated) to at least one immunoglobulin-related composition. The agent can be chemically bonded to the immunoglobulin-related composition by any method known to those skilled in the art. For example, the functional group on the agent can be directly attached to the functional group on the immunoglobulin-related composition. Some examples of suitable functional groups include, for example, amino, carboxyl, sulfhydryl, maleimide, isocyanate, isothiocyanate, and hydroxyl.

[0189] The agent may also be chemically bonded to the immunoglobulin-related composition by a cross-linking agent such as a dialdehyde, carbodiimide, dimaleimide, etc. Cross-linking agents may be obtained, for example, from Pierce Biotechnology, Inc. of Rockford, IL. The Pierce Biotechnology, Inc. website may provide assistance. Additional cross-linking agents include platinum cross-linking agents described in the following documents: U.S. Pat. Nos. 5,580,990; 5,985,566; and 6,133,038 to Kreatech Biotechnology, BV of Amsterdam, The Netherlands.

[0190] Alternatively, the functional groups on the agent and the immunoglobulin-related composition can be the same. Homobifunctional crosslinkers are generally used to crosslink the same functional groups. Examples of homobifunctional crosslinkers include EGS (i.e., ethylene glycol bis[succinimidyl succinate]), DSS (i.e., disuccinimidyl suberate), DMA (i.e., dimethyl adipimidate.2HCl), DTSSP (i.e., 3,3'-dithiobis[sulfosuccinimidyl propionate]), DPDPB (i.e., 1,4-bis-[3'-(2'-pyridyldithio)-propionamido] butane) and BMH (i.e., bismaleimidohexane). Such homobifunctional crosslinkers can also be obtained from Pierce Biotechnology, Inc.

[0191] In other cases, it may be beneficial to cleave the agent from the immunoglobulin-related composition. The website of Pierce Biotechnology, Inc. can also provide assistance to those skilled in the art in selecting a suitable cross-linking agent, which can be cleaved by, for example, an enzyme in the cell. Thus, the agent can be separated from the immunoglobulin-related composition. Examples of cleavable linkers include SMPT (i.e., 4-succinimidyloxycarbonyl-methyl-α-[2-pyridyldithio]toluene), sulfo-LC-SPDP (i.e., sulfosuccinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), LC-SPDP (i.e., succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), sulfo-LC-SPDP (e.g., sulfosuccinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), SPDP (i.e., N-succinimidyl 3-[2-pyridyldithio]-propionamidohexanoate), and AEDP (i.e., 3-[(2-aminoethyl)dithio]propionic acid HCl).

[0192] In another embodiment, the conjugate comprises at least one agent physically bound to at least one immunoglobulin-related composition. The agent can be physically bound to the immunoglobulin-related composition by any method known to those skilled in the art. For example, the immunoglobulin-related composition and the agent can be mixed by any method known to those skilled in the art. The order of mixing is not important. For example, the agent can be physically mixed with the immunoglobulin-related composition by any method known to those skilled in the art. For example, the immunoglobulin-related composition and the agent can be placed in a container and stirred by, for example, shaking the container to mix the immunoglobulin-related composition and the agent.

[0193] The immunoglobulin-related compositions can be modified by any method known to those skilled in the art. For example, as described above, the immunoglobulin-related compositions can be modified by cross-linking agents or functional groups. A. Methods for preparing anti-polySia antibodies of the present technology

[0194] Overview. First, a target poly-Sia species is selected against which antibodies of the present technology can be generated. For example, antibodies can be generated against high DP poly-Sia, such as having DP>10, DP>20, DP>50, DP>100, or DP>200. Techniques for generating antibodies against such target antigens are well known to those skilled in the art. Examples of such techniques include, but are not limited to, techniques involving display libraries, xenogeneic or human mice, hybridomas, and the like. The preparation of antibodies specific for poly-Sia is described herein.

[0195] It will be appreciated that recombinantly engineered antibodies and antibody fragments (eg, antibody-related polypeptides) directed against poly-Sia are suitable for use in accordance with the present disclosure.

[0196] Anti-polySia antibodies that can be subjected to the techniques described herein include monoclonal antibodies and polyclonal antibodies, as well as antibody fragments (e.g., Fab, Fab', F(ab')2, Fd, scFv), diabodies, antibody light chains, antibody heavy chains, and / or antibody fragments. Methods that can be used to produce high yields of antibody Fv-containing polypeptides such as Fab' and F(ab')2 antibody fragments have been described. See U.S. Patent No. 5,648,237.

[0197] Typically, antibodies are obtained from a species of origin. More specifically, nucleic acid or amino acid sequences of the variable portions of the light chain, heavy chain, or both of an antibody of the species of origin that is specific for the target antigen are obtained. The species of origin is any species that can be used to generate antibodies or antibody libraries of the present technology, such as rats, mice, rabbits, chickens, monkeys, humans, etc.

[0198] Phage or phagemid display technology is a technology that can be used to derive antibodies of the present technology. The technology used to produce and clone monoclonal antibodies is well known to those skilled in the art. The expression of sequences encoding antibodies of the present technology can be carried out in Escherichia coli.

[0199] Due to the degeneracy of nucleic acid coding sequences, other sequences encoding amino acid sequences substantially identical to those of naturally occurring proteins can be used in the practice of the present technology. These sequences include, but are not limited to, nucleic acid sequences including all or part of the nucleic acid sequences encoding the above-mentioned polypeptides, which are altered by substitution of different codons for functionally equivalent amino acid residues within the coding sequence, thereby producing silent changes. It should be understood that the nucleotide sequences of immunoglobulins according to the present technology allow for up to 25% sequence homology changes as calculated by standard methods ("Current Methods in Sequence Comparison and Analysis," Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pages 127-149, 1998, Alan R. Liss, Inc.), as long as such variants form effective antibodies that recognize poly-Sia species. For example, one or more amino acid residues within a polypeptide sequence can be substituted by another amino acid of similar polarity, which acts as a functional equivalent, resulting in silent changes. Substituents for amino acids within a sequence can be selected from other members of the class to which the amino acids belong. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. Positively charged (basic) amino acids include arginine, lysine and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Also included within the scope of the present technology are proteins or fragments or derivatives thereof, which are differentially modified during or after translation, such as by glycosylation, proteolytic cleavage, connection to antibody molecules or other cellular ligands, etc. In addition, the nucleic acid sequence encoding the immunoglobulin can be mutated in vitro or in vivo to generate and / or destroy the translation, initiation and / or termination of the sequence, or to produce variations in the coding region and / or form new restriction endonuclease sites or destroy the previously existing sites to facilitate further in vitro modification. Any mutagenesis technique known in the art may be used, including but not limited to in vitro site-directed mutagenesis (J. Biol. Chem. 253:6551), use of Tab linkers (Pharmacia), and the like.

[0200] Preparation of polyclonal antisera and immunogens. Methods for producing antibodies or antibody fragments of the present technology generally include immunizing a subject (typically a non-human subject, such as a mouse or rabbit) with a purified poly-Sia target (e.g., a high-DP poly-Sia having DP>10, DP>20, DP>50, DP>100, or DP>200) or with a poly-Sia(+) cell line expressing high-DP poly-Sia. Suitable immunogenic preparations can contain, for example, recombinantly expressed poly-Sia species or chemically synthesized poly-Sia species.

[0201] If desired, the immunogenicity of the poly-Sia target can be increased by fusion or conjugation with a hapten, such as keyhole limpet hemocyanin (KLH) or ovalbumin (OVA). Many such haptens are known in the art. The poly-Sia target can also be combined with a conventional adjuvant, such as Freund's complete or incomplete adjuvant, to enhance the subject's immune response to the polypeptide. Various adjuvants used to enhance the immune response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surfactants (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), human adjuvants (e.g., Bacille Calmette-Guerin and Corynebacterium parvum), or similar immunostimulatory compounds. These techniques are standard in the art.

[0202] When describing the present technology, the immune response may be described as a "primary" or "secondary" immune response. A primary immune response, also referred to as a "protective" immune response, refers to an immune response generated in an individual as a result of initial exposure (e.g., initial "immunization") to a specific antigen (e.g., high DP poly-Sia). In some embodiments, immunization can be performed by vaccinating an individual with a vaccine containing the antigen. For example, the vaccine can be a poly-Sia vaccine comprising one or more high DP poly-Sia species. Over time, the primary immune response may weaken or diminish, and may even disappear or at least become so weakened that it cannot be detected. Therefore, the present technology also relates to a "secondary" immune response, also referred to herein as a "memory immune response." The term secondary immune response refers to an immune response elicited in an individual after a primary immune response has been generated.

[0203] Thus, a secondary immune response may be elicited, for example, to enhance an existing immune response that has been weakened or attenuated, or to regenerate a previous immune response that has disappeared or can no longer be detected. A secondary or memory immune response may be a humoral (antibody) response or a cellular response. A secondary or memory humoral response occurs following stimulation of memory B cells generated upon initial presentation of an antigen. A delayed type hypersensitivity (DTH) reaction is a CD4 +A type of cellular secondary or memory immune response mediated by T cells. The first exposure to an antigen primes the immune system, and one or more additional exposures result in DTH.

[0204] After appropriate immunization, anti-polySia antibodies can be prepared from the subject's serum. If desired, antibody molecules directed against polySia targets can be isolated from mammals (eg, from blood) and further purified by well-known techniques such as polypeptide A chromatography to obtain IgG fractions.

[0205] Monoclonal antibodies. In one embodiment of the present technology, the antibody is an anti-polySia monoclonal antibody. For example, in some embodiments, the anti-polySia monoclonal antibody can be a human or mouse anti-polySia monoclonal antibody. In order to prepare monoclonal antibodies or derivatives, fragments, analogs or homologs thereof against high DP polySia proteins, any technique for producing antibody molecules by continuous cell line culture can be used. Such techniques include, but are not limited to, hybridoma technology (see, e.g., Kohler and Milstein, 1975. Nature 256: 495-497); tri-source hybridoma technology; human B cell hybridoma technology (see, e.g., Kozbor et al., 1983. Immunol. Today 4: 72) and EBV hybridoma technology to produce human monoclonal antibodies (see, e.g., Cole et al., 1985. In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies can be used in the practice of the present technology, and can be produced by using human hybridomas (see, for example, Cote et al., 1983. Proc. Natl. Acad. Sci. USA 80: 2026-2030) or by transforming human B cells with Epstein Barr virus in vitro (see, for example, Cole et al., 1985. In: MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). For example, a nucleic acid group encoding an antibody region can be isolated. PCR using primers derived from sequences encoding antibody conserved regions is used to amplify sequences encoding antibody portions from the population, and then DNA encoding antibodies or fragments thereof (such as variable domains) is reconstructed from the amplified sequences. Such amplified sequences can also be fused with DNA encoding other proteins, such as phage coats or bacterial cell surface proteins, to express and display fusion polypeptides on phages or bacteria. The amplified sequences can then be expressed and further selected or isolated based on, for example, the affinity of the expressed antibodies or fragments thereof for high-DP poly-Sia or its derivatives, fragments, analogs or homologs. Alternatively, hybridomas expressing anti-poly-Sia monoclonal antibodies can be prepared by immunizing a subject and then isolating the hybridomas from the spleen of the subject using conventional methods. See, for example, Milstein et al. (Galfre and Milstein, Methods Enzymol (1981) 73: 3-46). Screening of hybridomas using standard methods will produce monoclonal antibodies with different specificities (i.e., against different epitopes) and affinities.A selected monoclonal antibody having a desired property (e.g., high DP poly-Sia binding) as expressed by a hybridoma can be used, can be conjugated to a molecule (e.g., polyethylene glycol (PEG)) to alter its properties, or the cDNA encoding the monoclonal antibody can be isolated, sequenced, and manipulated in various ways. Other manipulations include substitution or deletion of specific aminoacyl residues that contribute to the instability of the antibody during storage or after administration to a subject, and affinity maturation techniques to improve the affinity of the anti-poly-Sia antibody for high DP poly-Sia.

[0206] Hybridoma technology. In some embodiments, the antibody of the present technology is an anti-polySia monoclonal antibody produced by a hybridoma, wherein the hybridoma includes a B cell obtained from a transgenic non-human animal (e.g., a transgenic mouse) having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell. Hybridoma technology includes those known in the art and taught in the following documents: Harlow et al., Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 349 (1988); Hammerling et al., Monoclonal Antibodies And T-Cell Hybridomas, 563-681 (1981). Other methods for producing hybridomas and monoclonal antibodies are well known to those skilled in the art.

[0207] Phage display technology. As described above, antibodies of the present technology can be produced by applying recombinant DNA and phage display technology. For example, various phage display methods known in the art can be used to prepare anti-polySia antibodies. In the phage display method, functional antibody domains are displayed on the surface of phage particles, and the phage particles carry polynucleotide sequences encoding the functional antibody domains. By directly selecting with antigens (usually antigens bound to or captured on solid surfaces or beads), phages with desired binding properties are selected from libraries or combinatorial antibody libraries (e.g., humans or mice). The phages used in these methods are typically filamentous phages, which include fd and M13 with Fab, Fv or disulfide-stabilized Fv antibody domains, and the domains are recombinantly fused to phage gene III or gene VIII proteins. Furthermore, the methods can be adapted for the construction of Fab expression libraries (see, e.g., Huse et al., Science 246: 1275-1281, 1989) to allow rapid and efficient identification of monoclonal Fab fragments with the desired specificity for high-DP poly-Sia or a derivative, fragment, analog or homolog thereof.Other examples of phage display methods that can be used to make antibodies of the present technology include those disclosed in the following references: Huston et al., Proc. Natl. Acad. Sci USA, 85:5879-5883, 1988; Chaudhary et al., Proc. Natl. Acad. Sci USA, 87:1066-1070, 1990; Brinkman et al., J. Immunol. Methods 182:41-50, 1995; Ames et al., J. Immunol. Methods 184:177-186, 1995; Kettleborough et al., Eur. J. Immunol. 24:952-958, 1994; Persic et al., Gene 187:9-18, 1997; Burton et al., Advances in Immunology 57:191-280,1994; PCT / GB91 / 01134; WO 90 / 02809; WO 91 / 10737; WO 92 / 01047; WO 92 / 18619; WO 93 / 11236; WO 95 / 15982; WO 95 / 20401; WO 96 / 06213; WO 92 / 01047 (Medical Research Council et al); WO 97 / 08320 (Morphosys); WO92 / 01047 (CAT / MRC); WO 91 / 17271 (Affymax) and U.S. Patent Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727 and 5,733,743. U.S. Patent No. 6,753,136 to Lohning has described methods that can be used to display polypeptides on the surface of phage particles by attaching the polypeptides via disulfide bonds. As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to produce whole antibodies (including human antibodies) or any other desired antigen-binding fragments and expressed in any desired host (including mammalian cells, insect cells, plant cells, yeast and bacteria).For example, techniques for recombinantly producing Fab, Fab' and F(ab')2 fragments can also be utilized using methods known in the art, such as those disclosed in WO 92 / 22324; Mullinax et al., BioTechniques 12:864-869, 1992; and Sawai et al., AJRI 34:26-34, 1995; and Better et al., Science 240:1041-1043, 1988.

[0208] Typically, hybrid antibodies or hybrid antibody fragments cloned into display vectors can be selected for the appropriate antigen to identify variants that retain good binding activity because the antibody or antibody fragment will be present on the surface of the phage or phagemid particle. See, for example, Barbas III et al., Phage Display, A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001). However, other vector formats can be used for this process, such as cloning antibody fragment libraries into lytic phage vectors (modified T7 or Lambda Zap systems) for selection and / or screening.

[0209] Expression of recombinant anti-poly-Sia antibodies. As described above, antibodies of the present technology can be produced by applying recombinant DNA technology. Recombinant polynucleotide constructs encoding anti-poly-Sia antibodies of the present technology generally include expression control sequences operably linked to the coding sequence of the anti-poly-Sia antibody chain, and the expression control sequences include naturally associated or heterologous promoter regions. Therefore, another aspect of the present technology includes vectors containing one or more nucleic acid sequences encoding anti-poly-Sia antibodies of the present technology. For the recombinant expression of one or more polypeptides of the present technology, a nucleic acid containing all or a portion of a nucleotide sequence encoding an anti-poly-Sia antibody is inserted into an appropriate cloning vector or expression vector (i.e., a vector containing the necessary elements for transcription and translation of the inserted polypeptide coding sequence) by recombinant DNA technology well known in the art and described in detail below. Methods for generating a variety of vector populations have been described in U.S. Patent Nos. 6,291,160 and 6,680,192 to Lerner et al.

[0210] In general, expression vectors that can be used in recombinant DNA technology are usually in the form of plasmids. In this disclosure, "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used vector form. However, the present technology is intended to include such other forms of expression vectors that are not technically plasmids and perform equivalent functions, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses). Such viral vectors allow infection of a subject and expression of the construct in the subject. In some embodiments, the expression control sequence is a eukaryotic promoter system in a vector capable of transforming or transfecting a eukaryotic host cell. Once the vector is incorporated into an appropriate host, the host is maintained under conditions suitable for high-level expression of nucleotide sequences encoding anti-poly-Sia antibodies and collection and purification of anti-poly-Sia antibodies (e.g., cross-reactive anti-poly-Sia antibodies). See generally, US2002 / 0199213. These expression vectors can generally be replicated in a host organism as an episome or as a component of the host chromosomal DNA. Typically, the expression vector contains a selection marker, such as ampicillin resistance or hygromycin resistance, to allow detection of cells transformed with the desired DNA sequence. The vector may also encode a signal peptide that can be used to direct secretion of the extracellular antibody fragment, such as pectin lyase. See U.S. Patent No. 5,576,195.

[0211] The recombinant expression vector of the present technology comprises a nucleic acid encoding a protein having polySia binding properties, and the recombinant expression vector is in a form suitable for the expression of the nucleic acid in a host cell, which means that the recombinant expression vector includes one or more regulatory sequences selected according to the host cell for expression, and the one or more regulatory sequences are operably connected to the nucleic acid sequence to be expressed. In the recombinant expression vector, "operably connected" is intended to mean that the target nucleotide sequence is connected to one or more regulatory sequences in a manner that allows the nucleotide sequence to be expressed (for example, in an in vitro transcription / translation system or in a host cell when the vector is introduced into a host cell). The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (for example, polyadenylation signals). Such regulatory sequences are described in, for example, the following documents: Goeddel, GENE EXPRESSION TECHNOL OGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, California (1990). Regulatory sequences include those that guide the constitutive expression of nucleotide sequences in many types of host cells and those that guide the expression of nucleotide sequences only in certain host cells (for example, tissue-specific regulatory sequences). Those skilled in the art will appreciate that the design of the expression vector may depend on factors such as the choice of the host cell to be transformed, the expression level of the desired polypeptide, etc. Typical regulatory sequences that can be used as promoters for expression of recombinant polypeptides (e.g., anti-poly Sia antibodies) include, for example, but not limited to, promoters of 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, in particular, promoters from: alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization. In one embodiment, the polynucleotide encoding the anti-poly Sia antibody of the present technology is operably linked to the ara B promoter and is expressible in the host cell. See U.S. Patent No. 5,028,530. The expression vector of the present technology can be introduced into a host cell to produce a polypeptide or peptide encoded by the nucleic acid described herein, including a fusion polypeptide (e.g., anti-poly Sia antibodies, etc.).

[0212] Another aspect of the present technology relates to host cells expressing anti-poly-Sia antibodies, which contain nucleic acids encoding one or more anti-poly-Sia antibodies. The recombinant expression vectors of the present technology can be designed to express anti-poly-Sia antibodies in prokaryotic or eukaryotic cells. For example, anti-poly-Sia antibodies can be expressed in bacterial cells (such as Escherichia coli), insect cells (using baculovirus expression vectors), fungal cells (such as yeast, yeast cells) or mammalian cells. Suitable host cells are further discussed in the following literature: Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS INENZYMOLOGY 185, Academic Press, San Diego, California (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example, using T7 promoter regulatory sequences and T7 polymerase. Methods that can be used to prepare and screen polypeptides (such as anti-poly-Sia antibodies) with predetermined properties via expression of randomly generated polynucleotide sequences have been previously described. See U.S. Patent Nos. 5,763,192; 5,723,323; 5,814,476; 5,817,483; 5,824,514; 5,976,862; 6,492,107; 6,569,641.

[0213] The expression of polypeptides in prokaryotes is most often carried out in Escherichia coli using vectors containing constitutive or inducible promoters that direct the expression of fusion or non-fusion polypeptides. Fusion vectors add many amino acids to the polypeptide encoded therein, usually to the amino terminus of the recombinant polypeptide. Such fusion vectors usually have three purposes: (i) increase the expression of the recombinant polypeptide; (ii) increase the solubility of the recombinant polypeptide; and (iii) assist in the purification of the recombinant polypeptide by acting as a ligand in affinity purification. Typically, in a fusion expression vector, a proteolytic cleavage site is introduced at the junction of the fusion part and the recombinant polypeptide so that the recombinant polypeptide can be separated from the fusion part after the fusion polypeptide is purified. Such enzymes and their homologous recognition sequences include factor Xa, thrombin, and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67:31-40), pMAL (New England Biolabs, Beverly, Massachusetts) and pRIT5 (Pharmacia, Piscataway, New Jersey), which fuse glutathione S-transferase (GST), maltose E binding polypeptide or polypeptide A to the target recombinant polypeptide, respectively.

[0214] Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS INENZYMOLOGY 185, Academic Press, San Diego, CA (1990) 60-89). Pack et al., U.S. Pat. Nos. 6,294,353; 6,692,935, have described methods for targeted assembly of different active peptides or protein domains via polypeptide fusion to produce multifunctional polypeptides. One strategy to maximize the expression of recombinant polypeptides (e.g., anti-polySia antibodies) in E. coli is to express the polypeptide in a host bacterium that has an impaired ability to proteolytically cleave the recombinant polypeptide. See, for example, Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, California (1990) 119-128. Another strategy is to change the nucleic acid sequence of the nucleic acid to be inserted into the expression vector so that the individual codons for each amino acid are those codons that are preferentially used in the expression host (e.g., E. coli) (see, for example, Wada et al., 1992. Nucl. Acids Res. 20: 2111-2118). This change in the nucleic acid sequence of the present technology can be performed by standard DNA synthesis techniques.

[0215] In another embodiment, the anti-polySia antibody expression vector is a yeast expression vector. Examples of vectors for expression in Saccharomyces cerevisiae include pYepSec1 (Baldari et al., 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, Cell 30: 933-943, 1982), pJRY88 (Schultz et al., Gene 54: 113-123, 1987), pYES2 (Invitrogen Corporation, San Diego, California) and picZ (Invitrogen Corp, San Diego, California). Alternatively, baculovirus expression vectors can be used to express anti-polySia antibodies in insect cells. Baculovirus vectors that can be used to express polypeptides (eg, anti-polySia antibodies) in cultured insect cells (eg, SF9 cells) include the pAc series (Smith et al., Mol. Cell. Biol. 3:2156-2165, 1983) and the pVL series (Lucklow and Summers, 1989. Virology 170:31-39).

[0216] In yet another embodiment, a nucleic acid encoding an anti-polySia antibody of the present technology is expressed in a mammalian cell using a mammalian expression vector. Examples of mammalian expression vectors include, for example, but not limited to, pCDM8 (Seed, Nature 329: 840, 1987) and pMT2PC (Kaufman et al., EMBO J. 6: 187-195, 1987). When used in mammalian cells, the control functions of the expression vector are generally provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma virus, adenovirus 2, cytomegalovirus, and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells that can be used to express the anti-polySia antibodies of the present technology, see, for example, Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, Chapters 16 and 17.

[0217] In another embodiment, the recombinant mammalian expression vector is capable of directing the expression of the nucleic acid in a specific cell type (e.g., tissue-specific regulatory elements). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al., Genes Dev. 1: 268-277, 1987), lymphoid-specific promoters (Calame and Eaton, Adv. Immunol. 43: 235-275, 1988), T cell receptors (Winoto and Baltimore, EMBO J. 8: 729-733, 1989) and immunoglobulins (Banerji et al., 1983. Cell 33: 729-740; Queen and Baltimore, Cell 33: 741-748, 1983.), neuron-specific promoters (e.g., neurofilament promoters; Byrne and Ruddle, Proc. Natl. Acad. Sci. USA 86:5473-5477, 1989), pancreas-specific promoters (Edlund et al., 1985. Science 230:912-916) and mammary gland-specific promoters (e.g., whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally regulated promoters are also contemplated, such as the mouse hox promoter (Kessel and Gruss, Science 249:374-379, 1990) and the alpha-fetoprotein promoter (Campes and Tilghman, Genes Dev. 3:537-546, 1989).

[0218] Another aspect of the inventive method relates to a host cell into which a recombinant expression vector of the present technology has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It should be understood that such terms refer not only to a specific subject cell, but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in the offspring due to mutations or environmental influences, such progeny may in fact be different from the parental cell, but are still included in the scope of the term as used herein.

[0219] The host cell can be any prokaryotic or eukaryotic cell. For example, anti-polySia antibodies can be expressed in bacterial cells (such as Escherichia coli), insect cells, yeast or mammalian cells. Mammalian cells are hosts suitable for expressing nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes To Clones, (VCH Publishers, New York, 1987). Many suitable host cell lines capable of secreting complete heterologous proteins have been developed in the art, and the suitable host cell lines include Chinese hamster ovary (CHO) cell lines, various COS cell lines, HeLa cells, L cells and myeloma cell lines. In some embodiments, the cells are non-human. The expression vectors of these cells can include expression control sequences, such as replication origins, promoters, enhancers, and necessary processing information sites, such as ribosome binding sites, RNA splicing sites, polyadenylation sites and transcription terminator sequences. Queen et al., Immunol. Rev. 89: 49, 1986. Illustrative expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papilloma virus, etc. Co et al., J Immunol. 148: 1149, 1992. Other suitable host cells are known to those skilled in the art.

[0220] Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional conversion or transfection techniques. As used herein, the terms "conversion" and "transfection" are intended to refer to various art-recognized techniques for introducing exogenous nucleic acid (e.g., DNA) into host cells, including calcium phosphate or calcium chloride coprecipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, gene guns, or transfection based on viruses. Other methods for transforming mammalian cells include using polybrene, protoplast fusion, liposomes, electroporation, and microinjection (generally referring to Sambrook et al., Molecular Cloning). Suitable methods for transforming or transfecting host cells can be found in the following documents: Sambrook et al. (MOLECULAR CLONING: ALABORATORY MANUAL. 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989) and other laboratory manuals. Depending on the type of cell host, the vector containing the target DNA segment can be transferred to the host cell by a well-known method.

[0221] For stable transfection of mammalian cells, it is known that only a small portion of cells can integrate foreign DNA into their genomes, depending on the expression vector and transfection technique used. In order to identify and select these integrants, genes encoding selectable markers (e.g., resistance to antibiotics) are usually introduced into host cells together with the target gene. Various selectable markers include those that confer resistance to drugs (such as G418, hygromycin and methotrexate). The nucleic acid encoding the selectable marker can be introduced into the host cell on the same vector as the vector encoding the anti-polySia antibody, or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells incorporating the selectable marker gene will survive, while other cells die).

[0222] Host cells (such as prokaryotic or eukaryotic host cells in culture) comprising anti-polySia antibodies of the present technology can be used to produce (i.e., express) recombinant anti-polySia antibodies. In one embodiment, the method comprises culturing host cells (into which a recombinant expression vector encoding an anti-polySia antibody has been introduced) in a suitable culture medium, thereby producing anti-polySia antibodies. In another embodiment, the method further comprises the step of isolating the anti-polySia antibody from the culture medium or the host cell. Once expressed, the anti-polySia antibody, such as a collection of anti-polySia antibodies or anti-polySia antibody-related polypeptides, is purified from the culture medium and the host cell. Anti-polySia antibodies can be purified according to standard procedures in the art (including HPLC purification, column chromatography, gel electrophoresis, etc.). In one embodiment, anti-polySia antibodies are produced in a host organism by the method of Boss et al., U.S. Patent No. 4,816,397. Typically, anti-polySia antibody chains are expressed together with signal sequences and are thereby released into the culture medium. However, if the anti-polySia antibody chains are not naturally secreted by the host cells, the anti-polySia antibody chains can be released by treatment with a mild detergent. Purification of recombinant polypeptides is well known in the art and includes ammonium sulfate precipitation, affinity chromatography purification techniques, column chromatography, ion exchange purification techniques, gel electrophoresis, and the like (see generally Scopes, Protein Purification (Springer-Verlag, New York, 1982)).

[0223] Polynucleotides encoding anti-polySia antibodies, such as the coding sequence of anti-polySia antibodies, can be incorporated into a transgene for introduction into the genome of a transgenic animal and subsequently expressed in the milk of the transgenic animal. See, for example, U.S. Patent Nos. 5,741,957, 5,304,489, and 5,849,992. Suitable transgenes include coding sequences for light and / or heavy chains operably linked to promoters and enhancers from mammary gland-specific genes, such as casein or β-lactoglobulin. For the production of transgenic animals, the transgene can be microinjected into fertilized oocytes, or the transgene can be incorporated into the genome of embryonic stem cells and the nuclei of such cells transferred into enucleated oocytes.

[0224] Single-chain antibodies. In one embodiment, the anti-polySia antibody of the present technology is a single-chain anti-polySia antibody. According to the present technology, the technology can be applied to produce single-chain antibodies specific for polySia (see, e.g., U.S. Patent No. 4,946,778). Examples of technologies that can be used to produce single-chain Fvs and antibodies of the present technology include those described in the following documents: U.S. Patent Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology, 203:46-88, 1991; Shu, L. et al., Proc. Natl. Acad. Sci. USA, 90:7995-7999, 1993; and Skerra et al., Science 240:1038-1040, 1988.

[0225] Chimeric and humanized antibodies. In one embodiment, the anti-poly-Sia antibody of the present technology is a chimeric anti-poly-Sia antibody. In one embodiment, the anti-poly-Sia antibody of the present technology is a humanized anti-poly-Sia antibody. In one embodiment of the present technology, the donor antibody and the recipient antibody are monoclonal antibodies from different species. For example, the recipient antibody is a human antibody (to minimize its antigenicity in humans), in which case the resulting CDR-grafted antibody is referred to as a "humanized" antibody.

[0226] Recombinant anti-polySia antibodies (such as chimeric monoclonal antibodies and humanized monoclonal antibodies) comprising human and non-human portions can be prepared using standard recombinant DNA techniques and are within the scope of the present technology. For certain uses, including in vivo uses of the anti-polySia antibodies of the present technology in humans and uses of these agents in in vitro detection assays, chimeric or humanized anti-polySia antibodies can be used. Such chimeric monoclonal antibodies and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art. Such useful methods include, for example, but are not limited to, methods described in the following documents: International Application No. PCT / US86 / 02269; U.S. Patent No. 5,225,539; European Patent No. 184187; European Patent No. 171496; European Patent No. 173494; PCT International Publication No. WO 86 / 01533; U.S. Patent Nos. 4,816,567; 5,225,539; European Patent No. 125023; Better et al., 1988. Science 240:1041-1043; Liu et al., 1987. Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al., 1987. J. Immunol. 139:3521-3526; Sun et al., 1987. Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al., 1987. Cancer Res. 47:999-1005; Wood et al., 1985. Nature 314:446-449; Shaw et al., 1988. J. Natl. Cancer Inst. 80:1553-1559; Morrison (1985) Science 229:1202-1207; Oi et al. (1986) BioTechniques 4:214; Jones et al., 1986. Nature 321:552-525; Verhoeyan et al., 1988. Science 239:1534; Morrison, Science 229:1202, 1985; Oi et al., BioTechniques 4:214, 1986; Gillies et al., J. Immunol. Methods, 125:191-202, 1989; U.S. Patent No. 5,807,715; and Beidler et al., 1988. J. Immunol. 141:4053-4060.For example, antibodies can be humanized using a variety of techniques including CDR grafting (EP 0 239 400; WO 91 / 09967; U.S. Pat. Nos. 5,530,101; 5,585,089; 5,859,205; 6,248,516; EP 460 167), veneering or resurfacing (EP 0 592 106; EP 0 519 596; Padlan E.A., Molecular Immunology, 28:489-498, 1991; Studnicka et al., Protein Engineering 7:805-814, 1994; Roguska et al., PNAS 91:969-973, 1994), and chain shuffling (U.S. Pat. No. 5,565,332). In one embodiment, the cDNA encoding the murine anti-polySia monoclonal antibody is digested with a specifically selected restriction endonuclease to remove the sequence encoding the Fc constant region and replace it with the equivalent portion of the cDNA encoding the human Fc constant region (see Robinson et al., PCT / US86 / 02269; Akira et al., European Patent Application 184,187; Taniguchi, European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., WO 86 / 01533; Cabilly et al., U.S. Patent No. 4,816,567; Cabilly et al., European Patent Application 125,023; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J Immunol 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al. (1987) Cancer Res 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559; U.S. Pat. No. 6,180,370; U.S. Pat. Nos. 6,300,064; 6,696,248; 6,706,484; 6,828,422.

[0227] In one embodiment, the present technology provides the construction of humanized anti-poly-Sia antibodies that are less likely to induce a human anti-mouse antibody (hereinafter referred to as "HAMA") response while still having effective antibody effector function. As used herein, the terms "human" and "humanized" with respect to antibodies refer to any antibody that is expected to elicit a weak immunogenic response that is therapeutically tolerable in human subjects. In one embodiment, the present technology provides humanized anti-poly-Sia antibodies, heavy chain and light chain immunoglobulins.

[0228] CDR antibodies. In some embodiments, the anti-polySia antibodies of the present technology are anti-polySia CDR antibodies. Typically, the donor and recipient antibodies used to generate anti-polySia CDR antibodies are monoclonal antibodies from different species; typically, the recipient antibody is a human antibody (to minimize its antigenicity in humans), in which case the resulting CDR-grafted antibody is called a "humanized" antibody. The graft may have a single V of the recipient antibody. H or V L A single CDR (or even a portion of a single CDR) within a H and V L In general, all three CDRs in all variable domains of the recipient antibody will be replaced by the corresponding donor CDRs, but only as many replacements are required so that the resulting CDR-grafted antibody fully binds to polySia. Methods for producing CDR-grafted and humanized antibodies are taught in the following literature: U.S. Pat. No. 5,585,089 to Queen et al.; U.S. Pat. No. 5,693,761; U.S. Pat. No. 5,693,762; and U.S. Pat. No. 5,225,539 to Winter; and EP 0682040. It can be used to prepare V H and V L Polypeptide methods are taught in Winter et al., US Patent Nos. 4,816,397; 6,291,158; 6,291,159; 6,291,161; 6,545,142; EP0368684; EP0451216; and EP0120694.

[0229] After selecting suitable framework region candidates from the same family and / or same family member, one or both of the heavy chain and light chain variable regions are produced by transplanting the CDR from the species of origin into the hybrid framework region. Conventional methods known to those skilled in the art can be used to complete the assembly of hybrid antibodies or hybrid antibody fragments with hybrid variable chain regions in any of the above aspects. For example, DNA sequences encoding hybrid variable domains as described herein (i.e., frameworks based on target species and CDRs from species of origin) can be produced by oligonucleotide synthesis and / or PCR. Suitable restriction enzymes can also be used to separate the nucleic acids encoding CDR regions from species of origin antibodies, and connected to the target species framework by connecting with suitable ligases. Alternatively, the framework regions of the variable chains of species of origin antibodies can be changed by site-directed mutagenesis.

[0230] Since hybrids are constructed from selections between multiple candidates corresponding to each framework region, there are many sequence combinations suitable for construction according to the principles described herein. Thus, libraries of hybrids can be assembled whose members have different combinations of individual framework regions. Such libraries can be electronic database collections of sequences or physical collections of hybrids.

[0231] This process does not usually change the FRs of the recipient antibody flanking the transplanted CDRs. However, one skilled in the art can sometimes improve the antigen binding affinity of the resulting anti-polySia CDR-grafted antibody by replacing certain residues of a given FR to make the FR more similar to the corresponding FR of the donor antibody. Suitable substitution positions include amino acid residues adjacent to the CDR, or amino acid residues that can interact with the CDR (see, for example, US 5,585,089, especially columns 12-16). Alternatively, one skilled in the art can start with the donor FR and modify it to make it more similar to the recipient FR or human common FR. The techniques for making these modifications are known in the art. In particular, if the resulting FR conforms to the human common FR at that position or is at least 90% or more identical to such a common FR, this may not significantly increase the antigenicity of the resulting modified anti-polySia CDR-grafted antibody compared to the same antibody with fully human FRs.

[0232] Bispecific antibodies (BsAb). Bispecific antibodies are antibodies that can simultaneously bind to two targets with different structures (e.g., two different target antigens, two different epitopes on the same target antigen, or a hapten and a target antigen or an epitope on a target antigen). BsAb can be prepared, for example, by combining heavy chains and / or light chains that recognize different epitopes of the same or different antigens. In some embodiments, through molecular function, a bispecific binder binds to one antigen (or epitope) on one of its two binding arms (a VH / VL pair) and binds to a different antigen (or epitope) on its second arm (a different VH / VL pair). According to this definition, a bispecific binder has two different antigen binding arms (both specificity and CDR sequences are different) and is monovalent for each antigen it binds.

[0233] The bispecific antibodies (BsAb) and bispecific antibody fragments (BsFab) of the present technology have at least one arm that specifically binds to, for example, poly-Sia and at least one other arm that specifically binds to a second target antigen. In some embodiments, the second target antigen is an antigen or epitope of a B cell, a T cell, a bone marrow cell, a plasma cell, or a mast cell. Additionally or alternatively, in certain embodiments, the second target antigen is selected from CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, TCRγ / δ, NKp46, and KIR. In certain embodiments, BsAb is capable of binding to tumor cells that express poly-Sia on the cell surface. In some embodiments, BsAb has been engineered to promote killing of tumor cells by directing (or recruiting) cytotoxic T cells to the tumor site. Other exemplary BsAbs include those having a first antigen binding site specific for poly-Sia and a second antigen binding site specific for a small molecule hapten (e.g., DTP A, IMP288, DOTA, DOTA-Bn, DOTA-deferoxamine, other DOTA chelates described herein, biotin, fluorescein, or those disclosed in Goodwin, D A. et al., 1994, Cancer Res. 54(22):5937-5946).

[0234] A variety of bispecific fusion proteins can be produced using molecular engineering. For example, BsAbs utilizing complete immunoglobulin frameworks (e.g., IgG), single-chain variable fragments (scFv) or combinations thereof have been constructed. In some embodiments, bispecific fusion proteins are divalent, comprising, for example, scFvs with a single binding site for an antigen and Fab fragments with a single binding site for a second antigen. In other embodiments, bispecific fusion proteins are tetravalent, comprising, for example, immunoglobulins (e.g., IgGs) with two binding sites for an antigen and two identical scFvs for a second antigen. BsAbs consisting of two scFv units in series have been shown to be clinically successful bispecific antibody forms. In some embodiments, BsAbs comprise two single-chain variable fragments (scFvs) in series, which are designed to connect the scFvs in conjunction with tumor antigens (e.g., poly-Sia) to the scFvs of engaging T cells (e.g., by combining CD3). In this way, T cells are recruited to the tumor site so that they can mediate the cytotoxic killing of tumor cells. See, eg, Dreier et al., J. Immunol. 170:4397-4402 (2003); Bargou et al., Science 321:974-977 (2008).

[0235] The latest methods for producing BsAbs include engineered recombinant monoclonal antibodies with additional cysteine ​​residues that make them more firmly cross-linked than more common immunoglobulin isotypes. See, for example, FitzGerald et al., Protein Eng. 10 (10): 1221-1225 (1997). Another method is an engineered recombinant fusion protein that connects two or more different single-chain antibodies or antibody fragment segments with the desired dual specificity. See, for example, Coloma et al., Nature Biotec h. 15: 159-163 (1997). A variety of bispecific fusion proteins can be produced using molecular engineering.

[0236] Bispecific fusion proteins connecting two or more different single-chain antibodies or antibody fragments are produced in a similar manner. Recombinant methods can be used to produce a variety of fusion proteins. In some specific embodiments, the BsAb according to the present technology comprises an immunoglobulin containing a heavy chain and a light chain and an scFv. In some specific embodiments, the scFv is connected to the C-terminus of the heavy chain of any anti-polySia immunoglobulin disclosed herein. In some specific embodiments, the scFv is connected to the C-terminus of the light chain of any anti-polySia immunoglobulin disclosed herein. In various embodiments, the scFv is connected to the heavy chain or light chain via a linker sequence. By PCR reaction, the appropriate linker sequence necessary for in-frame connection of the heavy chain Fd with the scFv is introduced into the VL and V κ The DNA fragment encoding the scFv was then ligated into a staging vector containing a DNA sequence encoding the CH1 domain. The resulting scFv-CH1 construct was excised and ligated to a V vector containing a DNA sequence encoding an anti-polySia antibody. H The obtained vector can be used to transfect appropriate host cells, such as mammalian cells, to express the bispecific fusion protein.

[0237] In some embodiments, the linker length is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids. In some embodiments, the linker is characterized in that it tends not to adopt a rigid three-dimensional structure, but to provide flexibility (e.g., the first and / or second antigen binding site) for the polypeptide. In some embodiments, based on the specific characteristics conferred on the BsAb, such as increased stability, a linker is used in the BsAb described herein. In some embodiments, the BsAb of the present technology comprises a G4S linker. In some specific embodiments, the BsAb of the present technology comprises (G4S) n A linker wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more.

[0238] Fc modification. In some embodiments, the anti-polySia antibody of the present technology comprises a variant Fc region, wherein the variant Fc region comprises at least one amino acid modification relative to the wild-type Fc region (or parent Fc region), such that the affinity of the molecule for an Fc receptor (e.g., FcγR) is altered, provided that the variant Fc region does not have a substitution at a position that directly contacts the Fc receptor based on crystallographic and structural analyses of Fc-Fc receptor interactions (such as those disclosed by Sondermann et al., Nature, 406:267-273 (2000)). Examples of positions within the Fc region that directly contact an Fc receptor (such as FcγR) include amino acids 234-239 (hinge region), amino acids 265-269 (B / C loop), amino acids 297-299 (C7E loop), and amino acids 327-332 (F / G) loop.

[0239] In some embodiments, the anti-polySia antibodies of the present technology have altered affinity for activating and / or inhibitory receptors, wherein the variant Fc region has one or more amino acid modifications, wherein the one or more amino acid modifications are N297 substituted for alanine or K322 substituted for alanine.

[0240] Glycosylation modification. In some embodiments, the anti-polySia antibodies of the present technology have an Fc region that contains variant glycosylation compared to a parent Fc region. In some embodiments, the variant glycosylation includes the absence of fucose; in some embodiments, expression in GnT1-deficient CHO cells results in variant glycosylation.

[0241] In some embodiments, the antibodies of the present technology may have modified glycosylation sites relative to an appropriate reference antibody that binds to an antigen of interest (e.g., poly-Sia) without altering the functionality of the antibody, such as binding activity to the antigen. As used herein, "glycosylation site" includes any specific amino acid sequence in an antibody to which an oligosaccharide (i.e., a carbohydrate containing two or more monosaccharides linked together) will be specifically and covalently attached.

[0242] Oligosaccharide side chains are usually connected to the backbone of the antibody via N-linked or O-linked. N-linked glycosylation refers to the attachment of the oligosaccharide moiety to the side chain of an asparagine residue. O-linked glycosylation refers to the attachment of the oligosaccharide moiety to a hydroxyamino acid, such as serine, threonine. For example, Fc-glycoforms lacking certain oligosaccharides (including fucose) and terminal N-acetylglucosamine can be produced in special CHO cells and exhibit enhanced ADCC effector function.

[0243] In some embodiments, the carbohydrate content of the immunoglobulin-related compositions disclosed herein is modified by adding or deleting glycosylation sites. Methods for modifying the carbohydrate content of antibodies are well known in the art and are included in the present technology, see, for example, U.S. Patent No. 6,218,149; EP 0359096B1; U.S. Patent Publication No. US2002 / 0028486; International Patent Application Publication WO 03 / 035835; U.S. Patent Publication No. 2003 / 0115614; U.S. Patent No. 6,218,149; U.S. Patent No. 6,472,511; All of these references are incorporated herein by reference in their entirety. In some embodiments, the carbohydrate content of an antibody (or its related part or component) is modified by deleting one or more endogenous carbohydrate parts of the antibody. In some specific embodiments, the present technology includes deleting the glycosylation site of the Fc region of an antibody by modifying the asparagine at position 297 to alanine.

[0244] Engineered glycoforms can be used for a variety of purposes, including but not limited to enhancing or reducing effector functions. Engineered glycoforms can be produced by any method known to those skilled in the art, such as by using engineered or variant expression strains, by coexpression with one or more enzymes (e.g., DI N-acetylglucosamine transferase III (GnTIII)), by expressing molecules comprising the Fc region in various organisms or cell lines from various organisms, or by modifying one or more carbohydrates after having expressed a molecule comprising the Fc region. Methods for generating engineered glycoforms are known in the art and include, but are not limited to, those described in Umana et al., 1999, Nat. Biotechnol. 17:176-180; Davies et al., 2001, Biotechnol. Bioeng. 74:288-294; Shields et al., 2002, J. Biol. Chem. 277:26733-26740; Shinkawa et al., 2003, J. Biol. Chem. 278:3466-3473; U.S. Patent No. 6,602,684; U.S. Patent Application Serial No. 10 / 277,370; U.S. Patent Application Serial No. 10 / 113,929; International Patent Application Publication Nos. WO 00 / 61739A1; WO 01 / 292246A1; WO 02 / 311140A1; WO 02 / 30954A1; POTILLEGENT TM Technology (Biowa, Inc., Princeton, NJ); GLYCOMAB TM Glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland); each of these documents is incorporated herein by reference in its entirety. See, for example, International Patent Application Publication No. WO 00 / 061739; U.S. Patent Application Publication No. 2003 / 0115614; Okazaki et al., 2004, JMB, 336: 1239-49.

[0245] Fusion protein. In one embodiment, the anti-polySia antibody of the present technology is a fusion protein. When fused with a second protein, the anti-polySia antibody of the present technology can be used as an antigen tag. Examples of domains that can be fused to polypeptides include not only heterologous signal sequences, but also other heterologous functional regions. The fusion is not necessarily direct, but can be performed through a linker sequence. Moreover, the fusion protein of the present technology can also be engineered to improve the characteristics of the anti-polySia antibody. For example, a region of additional amino acids (particularly charged amino acids) can be added to the N-terminus of the anti-polySia antibody to improve stability and durability during purification from host cells or subsequent processing and storage. In addition, a peptide portion can be added to the anti-polySia antibody to facilitate purification. Such regions can be removed before the final preparation of the anti-polySia antibody. Adding a peptide portion to facilitate the processing of polypeptides is a conventional technique well known in the art. The anti-polySia antibody of the present technology can be fused to a marker sequence (such as a peptide that promotes the purification of the fusion polypeptide). In selected embodiments, the marker amino acid sequence is a hexa-histidine peptide, particularly as provided in the pQE vector (QIAGEN, Inc., Chatsworth, Calif.), many of which are commercially available. As described in Gentz ​​et al., Proc. Natl. Acad. Sci. USA 86:821-824, 1989, for example, hexa-histidine allows for convenient purification of the fusion protein. Another peptide tag that can be used for purification, the "HA" tag, corresponds to an epitope derived from the influenza hemagglutinin protein. Wilson et al., Cell 37:767, 1984.

[0246] Thus, any of these above-described fusion proteins can be engineered using the polynucleotides or polypeptides of the present technology.Additionally, in some embodiments, the fusion proteins described herein exhibit increased half-life in vivo.

[0247] Fusion proteins with a disulfide-linked dimeric structure (due to IgG) can bind and neutralize other molecules more efficiently than individual monomeric secreted proteins or protein fragments. Fountoulakis et al., J. Biochem. 270:3958-3964, 1995.

[0248] Similarly, EP-AO 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various parts of the constant region of an immunoglobulin molecule and another human protein or a fragment thereof. In many cases, the Fc portion in the fusion protein is beneficial in therapy and diagnosis, and can therefore lead to, for example, improved pharmacokinetic properties. See EP-A 0232262. Alternatively, it may be necessary to delete or modify the Fc portion after expression, detection and purification of the fusion protein. For example, if the fusion protein is used as an antigen for immunization, the Fc portion may hinder therapy and diagnosis. In drug discovery, for example, human proteins (such as hIL-5) have been fused to the Fc portion for the purpose of high-throughput screening assays to identify antagonists of hIL-5. Bennett et al., J.Molecular Recognition 8:52-58, 1995; Johanson et al., J.Biol.Chem., 270:9459-9471, 1995.

[0249] Labeled anti-poly-Sia antibodies. In one embodiment, the anti-poly-Sia antibodies of the present technology are coupled to a labeling portion, i.e., a detectable group. The specific label or detectable group conjugated to the anti-poly-Sia antibody is not a key aspect of the present technology, as long as it does not significantly interfere with the specific binding of the anti-poly-Sia antibody of the present technology to poly-Sia. The detectable group can be any material with detectable physical or chemical properties. Such detectable labels have been well developed in the fields of immunoassays and imaging. In general, almost any label that can be used in such methods can be applied to the present technology. Therefore, a label is any composition that can be detected by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Labels useful in the practice of the present technology include magnetic beads (e.g., Dynabeads TM ), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas Red, rhodamine, etc.), radioactive labels (e.g., 3 H. 14 C. 35 S. 125 I. 121 I. 131 I. 112 In, 99 mTc), other imaging agents such as microbubbles (for ultrasound imaging), 18 F. 11 C. 15 O (for positron emission tomography), 99m TC, 111In (for single photon emission tomography), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, and other enzymes commonly used in ELISA), and calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. Patents describing the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241, each of which is incorporated herein by reference in its entirety and for all purposes. See also Handbook of Fluorescent Probes and Research Chemicals (6th Edition, Molecular Probes, Inc., Eugene, Oregon).

[0250] The label can be coupled directly or indirectly to the desired component of the assay according to methods well known in the art. As described above, a variety of labels can be used, and the choice of label depends on factors such as the sensitivity required, ease of conjugation to the compound, stability requirements, available instrumentation, and disposal regulations.

[0251] Non-radioactive labels are usually attached by indirect means. Typically, a ligand molecule (e.g., biotin) is covalently bound to a molecule. The ligand is then bound to an anti-ligand (e.g., streptavidin) molecule, which is inherently detectable or covalently bound to a signaling system such as a detectable enzyme, fluorescent compound, or chemiluminescent compound. Many ligands and anti-ligands can be used. In the case where a ligand (e.g., biotin, thyroxine, and cortisol) has a natural anti-ligand, the ligand can be used in combination with a labeled naturally occurring anti-ligand. Alternatively, any hapten or antigenic compound can be used in combination with an antibody such as an anti-poly-Sia antibody.

[0252] The molecule can also be directly conjugated to the compound that produces the signal, for example, by conjugating with an enzyme or a fluorophore. The target enzyme as a label will mainly be a hydrolase, particularly a phosphatase, an esterase and a glycosidase, or an oxidoreductase, particularly a peroxidase. Fluorescent compounds that can be used as labeling parts include, but are not limited to, for example, fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc. Chemiluminescent compounds that can be used as labeling parts include, but are not limited to, for example, fluorescein and 2,3-dihydrophthalazinediones, such as luminol. For a review of various labels or signal generating systems that can be used, see U.S. Patent No. 4,391,904.

[0253] The means of detecting the label is well known to those skilled in the art. Therefore, for example, in the case where the label is a radioactive label, the detection means includes a scintillation counter or film, such as in radioautography. In the case where the label is a fluorescent label, it can be detected by exciting the fluorescent dye with light of appropriate wavelength and detecting the fluorescence produced. Fluorescence can be detected in a visual form, by means of film, by using an electronic detector such as a charge coupled device (CCD) or a photomultiplier tube, etc. Similarly, enzyme labels can be detected by providing an appropriate substrate for the enzyme and detecting the resulting reaction product. Finally, simple colorimetric labels can be simply detected by observing the color associated with the label. Therefore, in various dipstick assays, conjugated gold often appears pink, and various conjugated beads present the color of beads.

[0254] Some assay formats do not require the use of labeled components. For example, an agglutination assay can be used to detect the presence of a target antibody, such as an anti-polySia antibody. In this case, antigen-coated particles are agglutinated by a sample containing the target antibody. In this format, no components need to be labeled, and the presence of the target antibody is detected by simple visual inspection. B. Identification and Characterization of Anti-Poly-Sia Antibodies of the Present Technology

[0255] Methods for identifying and / or screening anti-poly-Sia antibodies of the present technology. Methods for identifying and screening antibodies against poly-Sia species for antibodies with desired specificity for poly-Sia include any immune-mediated techniques known in the art. Components of the immune response can be detected in vitro by various methods well known to those of ordinary skill in the art. For example, (1) cytotoxic T lymphocytes can be incubated with radiolabeled target cells and the lysis of these target cells detected by the release of radioactivity; (2) helper T lymphocytes can be incubated with antigen and antigen-presenting cells, and cytokine synthesis and secretion measured by standard methods (Windhagen A et al., Immunity, 2:373-80, 1995); (3) antigen-presenting cells can be incubated with whole protein antigen and the presentation of the antigen on MHC detected by T lymphocyte activation assay or biophysical methods (Harding et al., Proc. Natl. Acad. Sci., 86:4230-4, 1989); (4) mast cells can be incubated with agents that cross-link their Fc-ε receptors and histamine release measured by enzyme immunoassay (Siraganian et al., TIPS, 4:432-437, 1983); and (5) enzyme-linked immunosorbent assay (ELISA).

[0256] Similarly, the products of immune responses in model organisms (e.g., mice) or human subjects can also be detected by various methods well known to those of ordinary skill in the art. For example, (1) the production of antibodies in response to vaccination can be easily detected by standard methods currently used in clinical laboratories, such as ELISA; (2) the migration of immune cells to the site of inflammation can be detected by scratching the skin surface and placing a sterile container to capture the migrating cells at the scratch site (Peters et al., Blood, 72:1310-5, 1988); (3) the use of 3 H-thymidine measures the proliferation of peripheral blood mononuclear cells (PBMCs) in response to mitogens or mixed lymphocyte reactions; (4) the phagocytic capacity of granulocytes, macrophages, and other phagocytes in PBMCs can be measured by placing PBMCs in wells with labeled particles (Peters et al., Blood, 72:1310-5, 1988); and (5) the differentiation of immune system cells can be measured by labeling PBMCs with antibodies to CD molecules (such as CD4 and CD8) and measuring the fraction of PBMCs expressing these markers.

[0257] In one embodiment, the display of high DP polySia on the surface of a replicable genetic package is used to select for anti-polySia antibodies of the present technology. See, e.g., U.S. Patent Nos. 5,514,548; 5,837,500; 5,871,907; 5,885,793; 5,969,108; 6,225,447; 6,291,650; 6,492,160; EP 585 287; EP 605522; EP 616640; EP1024191; EP 589 877; EP 774 511; EP 844 306. Methods useful for producing / selecting filamentous phage particles containing a phagemid genome encoding a binding molecule with a desired specificity have been described. See, for example, EP 774 511; US ​​5871907; US 5969108; US 6225447; US 6291650; US 6492160.

[0258] In some embodiments, the display of high DP polySia on the surface of yeast host cells is used to select anti-polySia antibodies of the present technology. Methods that can be used to isolate scFv polypeptides by display on the surface of yeast have been described by Kieke et al., Protein Eng. 1997 Nov; 10(11): 1303-10.

[0259] In some embodiments, anti-polySia antibodies of the present technology are selected using ribosome display. Methods that can be used to identify ligands in peptide libraries using ribosome display have been described by Mattheakis et al., Proc. Natl. Acad. Sci. USA 91:9022-26, 1994; and Hanes et al., Proc. Natl. Acad. Sci. USA 94:4937-42, 1997.

[0260] After selecting the desired anti-polySia antibody, it is expected that the antibody can be produced in large quantities by any technique known to those skilled in the art (e.g., prokaryotic or eukaryotic cell expression, etc.). Anti-polySia antibodies (which are, for example, but not limited to, anti-polySia hybrid antibodies or fragments) can be produced by constructing an expression vector encoding an antibody heavy chain using conventional techniques, in which the CDRs and (if necessary) the minimum portion of the variable region framework required to retain the binding specificity of the origin species antibody and (if necessary) are derived from the origin species antibody, and the remainder of the antibody is derived from the target species immunoglobulin that can be manipulated as described herein, thereby generating a vector for expressing the hybrid antibody heavy chain.

[0261] Measurement of poly-Sia binding. In some embodiments, a poly-Sia binding assay refers to an assay format in which high-DP poly-Sia and anti-poly-Sia antibodies are mixed under conditions suitable for binding between high-DP poly-Sia and anti-poly-Sia antibodies and assessing the amount of binding between high-DP poly-Sia and anti-poly-Sia antibodies. The amount of binding is compared with a suitable control, which can be the amount of binding in the absence of high-DP poly-Sia, the amount of binding in the presence of a non-specific immunoglobulin composition, or both. The amount of binding can be assessed by any suitable method. Binding assays include, for example, ELISA, radioimmunoassay, proximity scintillation assay, fluorescence energy transfer assay, liquid chromatography, membrane filtration assay, and the like. Biophysical assays for directly measuring the binding of high-DP poly-Sia to anti-poly-Sia antibodies are, for example, nuclear magnetic resonance, fluorescence, fluorescence polarization, surface plasmon resonance (BIACORE chip), and the like. Specific binding is determined by standard assays known in the art, such as radioligand binding assays, ELISA, FRET, immunoprecipitation, SPR, NMR (2D-NMR), mass spectrometry, and the like. If the specific binding of a candidate anti-poly-Sia antibody is at least 1% greater than the binding observed in the absence of the candidate anti-poly-Sia antibody, the candidate anti-poly-Sia antibody can be used as an anti-poly-Sia antibody in accordance with the present technology.

[0262] Measurement of poly-Sia neutralization. As used herein, "poly-Sia neutralization" refers to the reduction of the activity and / or expression of poly-Sia, or the reduction of the activity and / or expression of a protein modified with a poly-Sia moiety, by the binding of an anti-poly-Sia antibody. The ability of the anti-poly-Sia antibodies of the present technology to neutralize poly-Sia activity / expression can be assessed in vitro or in vivo using methods known in the art. Uses of anti-poly-Sia antibodies of the present technology

[0263] Overview. The anti-poly-Sia antibodies of the present technology can be used in methods known in the art related to the localization and / or quantification of poly-Sia (e.g., for measuring the level of high-DP poly-Sia in appropriate physiological samples, for diagnostic methods, for polypeptide imaging, etc.). The antibodies of the present technology can be used to separate high-DP poly-Sia by standard techniques such as affinity chromatography or immunoprecipitation. The anti-poly-Sia antibodies of the present technology can facilitate the purification of immunoreactive high-DP poly-Sia species from biological samples such as mammalian serum or cells, as well as the purification of recombinantly produced immunoreactive high-DP poly-Sia expressed in a host system. In addition, anti-poly-Sia antibodies can be used to detect immunoreactive high-DP poly-Sia species (e.g., in plasma, cell lysates, or cell supernatants) to evaluate the expression abundance and pattern of immunoreactive high-DP poly-Sia. The anti-poly-Sia antibodies of the present technology can be used to diagnostically monitor immunoreactive high-DP poly-Sia levels in tissues as part of a clinical testing procedure, for example, to determine the efficacy of a given treatment regimen. As described above, detection can be facilitated by coupling (i.e., physically linking) the anti-poly-Sia antibodies of the present technology to a detectable substance.

[0264] Detection of poly-Sia. An exemplary method for detecting the presence of immunoreactive high-DP poly-Sia in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with an anti-poly-Sia antibody of the present technology capable of detecting immunoreactive high-DP poly-Sia, thereby detecting the presence of immunoreactive high-DP poly-Sia in the biological sample. Detection can be accomplished by a detectable label attached to the antibody.

[0265] The term "labeled" with respect to anti-polySia antibodies is intended to encompass direct labeling of the antibody by coupling (i.e., physically linking) a detectable substance to the antibody, as well as indirect labeling of the antibody by reactivity with another compound (e.g., a secondary antibody) that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin so that it can be detected with fluorescently labeled streptavidin.

[0266] In some embodiments, the anti-poly-Sia antibodies disclosed herein are conjugated to one or more detectable labels. For such uses, the anti-poly-Sia antibodies can be detectably labeled by covalent or non-covalent attachment of chromogenic agents, enzymatic agents, radioisotopic agents, isotopic agents, fluorescent agents, toxic agents, chemiluminescent agents, nuclear magnetic resonance contrast agents, or other labels.

[0267] Examples of suitable chromogenic labels include diaminobenzidine and 4-hydroxyazo-benzene-2-carboxylic acid. Examples of suitable enzyme labels include malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triosephosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.

[0268] Examples of suitable radioisotope labels include 3 H. 111 In, 125 I. 131 I. 32 P. 35 S. 14 C. 51 Cr, 57 To, 58 Co. 59 Fe, 75 Se, 152 Eu, 90 Y. 67 Cu, 217 Ci, 211 At 212 Pb, 47 Sc, 109 Pd etc. 111 In is an exemplary isotope when using in vivo imaging because it avoids 125 I or 131 I-labeled polySia-conjugated antibodies are dehalogenated by the liver. In addition, this isotope has a gamma emission energy that is more favorable for imaging (Perkins et al., Eur. J. Nucl. Med. 70:296-301 (1985); Carasquillo et al., J. Nucl. Med. 25:281-287 (1987)). For example, monoclonal antibodies conjugated with 1-(P-isothiocyanatobenzyl)-DPTA 111In exhibits little uptake in non-tumor tissues (particularly the liver) and enhances the specificity of tumor localization (Esteban et al., J. Nucl. Med. 28:861-870 (1987)). Examples of suitable non-radioactive isotope labels include 157 Gd, 55 Mn, 162 Dy, 52 Tr and 56 Fe.

[0269] Examples of suitable fluorescent labels include 152 Eu labeling, fluorescein labeling, isothiocyanate labeling, rhodamine labeling, phycoerythrin labeling, phycocyanin labeling, allophycocyanin labeling, green fluorescent protein (GFP) labeling, o-phthalaldehyde labeling and fluorescamine labeling.Examples of suitable toxin labels include diphtheria toxin, ricin toxin and cholera toxin.

[0270] Examples of chemiluminescent labels include luminol labels, isoluminol labels, aromatic acridinium ester labels, imidazole labels, acridinium salt labels, oxalate labels, luciferin labels, luciferase labels, and aequorin labels. Examples of nuclear magnetic resonance contrast agents include heavy metal nuclei such as Gd, Mn, and iron.

[0271] The detection methods of the present technology can be used to detect immunoreactive high-DP poly-Sia in biological samples in vitro and in vivo. In vitro techniques for detecting immunoreactive high-DP poly-Sia include enzyme-linked immunosorbent assay (ELISA), protein blotting, immunoprecipitation, radioimmunoassay, and immunofluorescence. In addition, in vivo techniques for detecting immunoreactive high-DP poly-Sia include introducing labeled anti-poly-Sia antibodies into a subject. For example, an anti-poly-Sia antibody can be labeled with a radioactive marker, the presence and location of which in a subject can be detected by standard imaging techniques. In one embodiment, the biological sample contains high-DP poly-Sia from a test subject.

[0272] Immunoassays and imaging. The anti-poly-Sia antibodies of the present technology can be used to determine the levels of immunoreactive high-DP poly-Sia in biological samples (e.g., human plasma) using antibody-based techniques. For example, high-DP poly-Sia expression in tissues can be studied using classical immunohistological methods. Jalkanen, M. et al., J. Cell. Biol. 101:976-985, 1985; Jalkanen, M. et al., J. Cell. Biol. 105:3087-3096, 1987. Other antibody-based methods that can be used to detect poly-Sia expression include immunoassays such as enzyme-linked immunosorbent assays (ELISA) and radioimmunoassays (RIA). Suitable antibody assay labels are known in the art and include enzyme labels (such as glucose oxidase) and radioisotopes or other radioactive agents (such as iodine ( 125 I. 121 I. 131 I), carbon ( 14 C) Sulfur ( 35 S), tritium ( 3 H), Indium ( 112 In) and technetium ( 99 mTc)) and fluorescent labels such as fluorescein, rhodamine, and green fluorescent protein (GFP), as well as biotin.

[0273] In addition to determining the level of immunoreactive high-DP poly-Sia in biological samples, the anti-poly-Sia antibodies of the present technology can also be used for in vivo imaging of high-DP poly-Sia. Antibodies that can be used in this method include those that can be detected by X-ray radiography, NMR or ESR. For X-ray radiography, suitable labels include radioisotopes, such as barium or cesium, which emit detectable radiation but are not significantly harmful to the subject. Labels suitable for NMR and ESR include those with detectable characteristic spins, such as deuterium, which can be incorporated into anti-poly-Sia antibodies by labeling nutrients that are only used for the relevant scFv clone.

[0274] The cells have been treated with an appropriate detectable imaging moiety such as a radioisotope (e.g. 131 I. 112 In, 99 The anti-polySia antibody labeled with mTc, a radiopaque substance, or a material detectable by nuclear magnetic resonance is introduced (e.g., parenterally, subcutaneously, or intraperitoneally) into a subject. It will be understood in the art that the size of the subject and the imaging system used will determine the amount of imaging moiety required to produce a diagnostic image. In the case of a radioisotope moiety, for a human subject, the amount of radioactivity injected is generally between about 5 and 20 millicuries. 99The labeled anti-poly-Sia antibody will then accumulate in the cell locations containing the specific target antigen. For example, the labeled anti-poly-Sia antibody of the present technology will accumulate in cells and tissues in the subject where high-DP poly-Sia has been located.

[0275] Therefore, the present technology provides a method for diagnosing a medical condition, which involves: (a) determining the expression of immunoreactive high-DP poly-Sia by measuring the binding of anti-poly-Sia antibodies of the present technology in cells or body fluids of an individual; (b) comparing the amount of immunoreactive high-DP poly-Sia present in the sample with a standard reference, wherein an increase or decrease in the level of immunoreactive high-DP poly-Sia compared to the standard indicates a medical condition.

[0276] Affinity purification. The anti-polySia antibodies of the present technology can be used to purify immunoreactive high-DP polySia from a sample. In some embodiments, the antibody is immobilized on a solid support. Examples of such solid supports include plastics (such as polycarbonates), complex carbohydrates (such as agarose and sepharose), acrylic resins, and beads such as polyacrylamide and latex. The technology for coupling antibodies to such solid supports is well known in the art (Weir et al., "Handbook of Experimental Immunology" 4th edition, Blackwell Scientific Publications, Oxford, UK, Chapter 10 (1986); Jacoby et al., Meth. Enzym. 34 Academic Press, New York (1974)).

[0277] The simplest method of combining antigen with antibody-support matrix is ​​to collect beads in a column and pass the antigen solution downward through the column. The efficiency of this method depends on the contact time between the immobilized antibody and the antigen, and the contact time can be extended by using a low flow rate. The immobilized antibody captures the antigen when the antigen flows through. Alternatively, the antigen can be contacted with the antibody support matrix in the following manner: the antigen solution is mixed with a support (e.g., beads) and rotated or shaken to achieve maximum contact between the antigen and the immobilized antibody. After the binding reaction is completed, the slurry is passed into the column to collect beads. The beads are washed with a suitable washing buffer solution, and then the pure or substantially pure antigen is eluted.

[0278] The antibody polypeptide of interest can be conjugated to a solid support (e.g., beads). In addition, if desired, a first solid support such as beads can also be conjugated to a second solid support (which can be a second bead or other support) by any suitable means (including those disclosed herein for conjugating an antibody polypeptide to a support). Therefore, any conjugation methods and means disclosed herein for conjugating an antibody polypeptide to a solid support can also be used to conjugate a first support to a second support, wherein the first and second solid supports can be the same or different.

[0279] Suitable linkers (which may be crosslinkers) for conjugating antibody polypeptides to solid supports include a variety of agents that can react with functional groups present on the support surface or with antibody polypeptides or both. Reagents that can be used as crosslinkers include homobifunctional agents and particularly heterobifunctional agents. Useful bifunctional crosslinkers include, but are not limited to, N-SIAB, dimaleimide, DTNB, N-SATA, N-SPDP, SMCC, and 6-HYNIC. Crosslinkers can be selected to provide selectively cleavable bonds between antibody polypeptides and solid supports. For example, photolabile crosslinkers such as 3-amino-(2-nitrophenyl) propionic acid can be used as a means of cutting antibody polypeptides from solid supports. (Brown et al., Mol. Divers, pp. 4-12 (1995); Rothschild et al., Nucl. Acids Res., 24: 351-66 (1996); and U.S. Pat. No. 5,643,722). Other crosslinking agents are well known in the art. (See, e.g., Wong (1991), supra; and Hermanson (1996), supra).

[0280] The antibody polypeptide can be fixed on a solid support (such as beads) by a covalent amide bond formed between the carboxyl-functionalized beads and the amino terminus of the antibody polypeptide, or conversely by a covalent amide bond formed between the amino-functionalized beads and the carboxyl terminus of the antibody polypeptide. In addition, a bifunctional trityl linker can be attached to a support via an amino resin through an amino group or a carboxyl group on the resin, such as a 4-nitrophenyl active ester attached to a resin (such as Wang resin). When using the bifunctional trityl method, the solid support may need to be treated with a volatile acid (such as formic acid or trifluoroacetic acid) to ensure that the antibody polypeptide is cut and can be removed. In this case, the antibody polypeptide can be deposited on the bottom of the hole of the solid support or on the flat surface of the solid support as a plaque without beads. After adding the matrix solution, the antibody polypeptide can be desorbed into the MS.

[0281] Hydrophobic trityl linkers can also be used as acid-labile linkers by cleaving the amino-linked trityl group from the antibody polypeptide using a volatile acid or an appropriate matrix solution (e.g., a matrix solution containing 3-HPA). Acid instability can also be changed. For example, a trityl group, a monomethoxytrityl group, a dimethoxytrityl group, or a trimethoxytrityl group can be changed to a tritylamine derivative that is appropriately para-substituted or more acid-labile to the antibody polypeptide, i.e., a trityl ether bond and a tritylamine bond can be formed with the antibody polypeptide. Therefore, the antibody polypeptide can be removed from the hydrophobic linker, for example, by destroying the hydrophobic attraction under acidic conditions or by cleaving the trityl ether bond or the tritylamine bond, the acidic conditions including (if necessary) under typical MS conditions, where a matrix such as 3-HPA is used as an acid.

[0282] Orthogonal cleavable linkers can also be used to bind a first solid support (e.g., beads) to a second solid support, or can be used to bind a polypeptide of interest (e.g., an antibody polypeptide) to a solid support. Using such linkers, the first solid support (e.g., beads) can be selectively cleaved from the second solid support without cleaving the antibody polypeptide from the support; the antibody polypeptide can then be cleaved from the beads at a later time. For example, a disulfide linker that can be cleaved using a reducing agent such as DTT can be used to bind beads to a second solid support, and an acid-cleavable bifunctional trityl group can be used to fix the antibody polypeptide to the support. As needed, the connection between the antibody polypeptide and the solid support can be cleaved first, for example, to keep the connection between the first and second supports intact. The trityl linker can provide covalent or hydrophobic conjugation, and regardless of the nature of the conjugation, the trityl group is easily cleaved under acidic conditions.

[0283] For example, beads can be combined with a second support through a linking group, and a linking group with the following length and chemical properties can be selected, which promotes high-density binding of beads to solid supports or high-density binding of polypeptides to beads. Such linking groups can have, for example, a "tree-like" structure, thereby providing multiple functional groups for each attachment site on the solid support. Examples of such linking groups include polylysine, polyglutamic acid, penta-erythrole, and trihydroxyaminomethane.

[0284] Non-covalent binding association. Through non-covalent interactions, the antibody polypeptide can be conjugated to a solid support, or the first solid support can also be conjugated to a second solid support. For example, magnetic beads made of a ferromagnetic material that can be magnetized can be attracted to a magnetic solid support and can be released from the support by removing the magnetic field. Alternatively, the solid support can have an ionic or hydrophobic portion, which can allow the ionic or hydrophobic portion to interact with the antibody polypeptide (e.g., an antibody polypeptide containing an attached trityl group) or with a second solid support having a hydrophobic characteristic, respectively.

[0285] The solid support may also have members of a specific binding pair and thus may be conjugated to an antibody polypeptide or a second solid support containing a complementary binding moiety. For example, beads coated with avidin or with streptavidin may be bound to an antibody polypeptide into which a biotin moiety has been incorporated, or to a second solid support coated with biotin or a biotin derivative such as iminobiotin.

[0286] It should be appreciated that any binding member disclosed herein or otherwise known in the art can be reversed. Thus, for example, biotin can be incorporated into an antibody polypeptide or a solid support, and conversely, avidin or other biotin binding moiety can be incorporated into a support or an antibody polypeptide, respectively. Other specific binding pairs contemplated for use herein include, but are not limited to, hormones and their receptors, enzymes and their substrates, nucleotide sequences and their complementary sequences, antibodies and their specifically interacting antigens, and other such pairs known to those skilled in the art. A. Diagnostic Uses of Anti-Poly-Sia Antibodies of the Present Technology

[0287] Overview. The anti-poly-Sia antibodies of the present technology can be used in diagnostic methods. Therefore, the present technology provides methods for diagnosing high DP poly-Sia activity in a subject using the antibodies. The anti-poly-Sia antibodies of the present technology can be selected so that they have any level of epitope binding specificity and very high binding affinity for high DP poly-Sia. In general, the higher the binding affinity of the antibody, the more stringent washing conditions can be performed in the immunoassay to remove non-specifically bound material without removing the target antigen. Therefore, the anti-poly-Sia antibodies of the present technology that can be used in diagnostic assays typically have about 10 8 M -1 , 10 9 M -1 , 10 10 M -1 , 10 11 M -1 or 10 12 M -1Furthermore, it is desirable that anti-polySia antibodies for use as diagnostic reagents have sufficient kinetic association rates to reach equilibrium under standard conditions within at least 12 h, at least five (5) h, or at least one (1) hour.

[0288] Anti-poly-Sia antibodies can be used to detect immunoreactive high-DP poly-Sia in a variety of standard assay formats. Such formats include immunoprecipitation, Western blot, ELISA, radioimmunoassay, and immunometric assays. See Harlow and Lane, Antibodies, A Laboratory Manual (Cold Spring Harbor Publications, New York, 1988); U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,879,262; 4,034,074; 3,791,932; 3,817,837; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; and 4,098,876. A biological sample may be obtained from any tissue or fluid of a subject. In certain embodiments, the subject is in the early stages of cancer. In one embodiment, the early stages of cancer are determined by the level or expression pattern of high DP poly-Sia in a sample obtained from the subject. In certain embodiments, the sample is selected from urine, blood, serum, plasma, saliva, amniotic fluid, cerebrospinal fluid (CSF), and biopsy body tissue.

[0289] Immunoassay or sandwich assay is a form of diagnostic method of the present technology. See U.S. Patent Nos. 4,376,110, 4,486,530, 5,914,241 and 5,965,375. Such assays use an antibody (e.g., an anti-polySia antibody or anti-polySia antibody population) fixed to a solid phase and another anti-polySia antibody or anti-polySia antibody population in a solution. Typically, the solution anti-polySia antibody or anti-polySia antibody population is labeled. If an antibody population is used, the population may contain antibodies that bind to polySia with different polymerization degrees. Therefore, the same population can be used for both solid phase and solution antibodies. If an anti-polySia monoclonal antibody is used, a first and a second polySia monoclonal antibody with different binding specificities is used for the solid phase and the solution phase. The solid phase (also referred to as "capture") and solution (also referred to as "detection") antibodies can be contacted with the target antigen in any order or simultaneously. If the solid phase antibody is contacted first, the assay is called a forward assay. On the contrary, if the solution antibody is contacted first, the assay is called a reverse assay. If the target is contacted with two antibodies at the same time, the assay is called a simultaneous assay. After contacting poly-Sia with the anti-poly-Sia antibody, the sample is incubated for a period of time, which generally varies from about 10 minutes to about 24 hours, and is generally about 1 hour. A washing step is then performed to remove components in the sample that are not specifically bound to the anti-poly-Sia antibody used as a diagnostic reagent. When the solid phase antibody and the solution antibody are combined in a separate step, washing can be performed after either or both of the binding steps. After washing, the binding is quantified, usually by detecting the label attached to the solid phase through the binding of the labeled solution antibody. Usually for a given antibody pair or antibody population and a given reaction condition, a calibration curve is prepared from a sample containing a known concentration of the target antigen. The concentration of immunoreactive high DP poly-Sia in the sample being tested is then read by interpolation from the calibration curve (i.e., the standard curve). The analyte can be measured from the amount of labeled solution antibody bound at equilibrium or by kinetic measurements of the bound labeled solution antibody at a series of time points before reaching equilibrium. The slope of this curve is a measure of the high DP poly-Sia concentration in the sample.

[0290] Suitable supports for use in the above methods include, for example, nitrocellulose membranes, nylon membranes and derivatized nylon membranes, and also particles such as agarose, dextran-based gels, dipsticks, microparticles, microspheres, magnetic particles, test tubes, microtiter wells, SEPHADEX TM (Amersham Pharmacia Biotech, Piscataway, NJ) etc. Immobilization can be achieved by absorption or by covalent attachment. Optionally, the anti-polySia antibody can be linked to a linker molecule (such as biotin) to attach to a surface-bound linker (such as avidin).

[0291] In some embodiments, the present disclosure provides an anti-polySia antibody of the present technology conjugated to a diagnostic agent. The diagnostic agent may comprise a radioactive or non-radioactive label, a contrast agent (such as for magnetic resonance imaging, computed tomography, or ultrasound), and the radioactive label may be a gamma, beta, alpha, Auger electron, or positron emitting isotope. The diagnostic agent is a molecule administered by conjugation to an antibody portion, i.e., an antibody or antibody fragment or subfragment, and can be used to diagnose or detect disease by locating cells containing the antigen.

[0292] Useful diagnostic agents include, but are not limited to, radioisotopes, dyes (such as using biotin-streptavidin complexes), contrast agents, fluorescent compounds or molecules, and enhancers (such as paramagnetic ions) for magnetic resonance imaging (MRI). U.S. Patent No. 6,331,175 describes MRI technology and the preparation of antibodies conjugated to MRI enhancers, and is incorporated by reference in its entirety. In some embodiments, the diagnostic agent is selected from radioisotopes, enhancers for magnetic resonance imaging, and fluorescent compounds. In order to load radioactive metals or paramagnetic ions on the antibody component, it may be necessary to react it with a reagent having a long tail, to which a variety of chelating groups for binding ions are attached. This tail can be a polymer, such as polylysine, polysaccharides or other derivatized or derivatizable chains, and the polymer has a side group that can be combined with a chelating group, and the chelating group is, for example, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrin, polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and similar groups known to be useful for this purpose. Chelates can be coupled to the antibodies of the present technology using standard chemical methods. Chelates are typically linked to antibodies via a group that is capable of forming a bond with the molecule with minimal loss of immunoreactivity and minimal aggregation and / or internal crosslinking. Other methods and reagents for conjugating chelates to antibodies are disclosed in U.S. Pat. No. 4,824,659. Particularly useful metal-chelate combinations include 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs for radioimaging with diagnostic isotopes. When complexed with non-radioactive metals such as manganese, iron, and gadolinium, the same chelates can be used for MRI when used with the anti-polySia antibodies of the present technology.

[0293] Macrocyclic chelates such as NOTA (1,4,7-triaza-cyclononane-N,N',N"-triacetic acid), DOTA, and TETA (p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid) are used with a variety of metals and radioactive metals (e.g., radionuclides of gallium, yttrium, and copper), respectively. Such metal-chelate complexes can be stabilized by tailoring the ring size to the metal of interest. Other examples of DOTA chelates include (i) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH2; (ii) Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH2; (iii) DOTA-D- Asp-D-Lys(HSG)-D-Asp-D-Lys(HSG)-NH2; (iv)DOTA-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (v)DOTA-D-Tyr-D-Lys(HSG)-D-Glu-D-Lys(H SG)-NH2; (vi)DOTA-D-Ala-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (vii)DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH2; (viii)Ac-D-Phe-D-L ys(DOTA)-D-Tyr-D-Lys(DOTA)-NH2; (ix)Ac-D-Phe-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH2; (x)Ac-D-Phe-D-Lys(Bz-DTPA)-D-Tyr-D-Lys(Bz- DTPA)-NH2; (xi)Ac-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH2; (xii)DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-C ys)-NH2; (xiii) (Tscg-Cys)-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(DOTA)-NH2; (xiv)Tscg-D-Cys-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(H SG)-NH2; (xv) (Tscg-Cys)-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (xvi)Ac-D-Cys-D-Lys(DOTA)-D-Tyr-D-Ala-D-Lys(DOTA)-D-Cys-NH2;(xvii)Ac-D-Cys-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH2; (xviii)Ac-D-Lys(DTPA)-D-Tyr-D-Lys(D TPA)-D-Lys(Tscg-Cys)-NH2; and (xix)Ac-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-D-Lys(Tscg-Cys)-NH2. ;

[0294] Also considered are the stable binding nuclides (such as those used in RAIT). 223 Other cyclic chelates of interest include macrocyclic polyethers. B. Therapeutic Uses of Anti-Poly-Sia Antibodies of the Present Technology

[0295] The immunoglobulin-related compositions (e.g., antibodies or antigen-binding fragments thereof) of the present technology can be used to treat poly-Sia-related cancers. Such treatment can be used for patients identified as having pathologically high levels of high-DP poly-Sia (e.g., those diagnosed by the methods described herein) or patients diagnosed with diseases known to be associated with such pathological levels. In one aspect, the present disclosure provides a method for treating poly-Sia-related cancers in a subject in need thereof, the method comprising administering to the subject an effective amount of an antibody (or antigen-binding fragment thereof) of the present technology. Examples of cancers that can be treated by antibodies of the present technology include, but are not limited to: small cell or non-small cell lung cancer, neuroblastoma, pancreatic cancer, pituitary tumor, Wilms' tumor, rhabdomyosarcoma, glioblastoma, breast cancer, or acute myeloid leukemia.

[0296] The compositions of the present technology can be used in combination with other therapeutic agents that can be used to treat poly-Sia-related cancers. For example, the antibodies of the present technology can be administered separately, sequentially or simultaneously with at least one additional therapeutic agent selected from the following: alkylating agents, platinum agents, taxanes, vinca agents, anti-estrogen drugs, aromatase inhibitors, ovarian inhibitors, VEGF / VEGFR inhibitors, EGF / EGFR inhibitors, PARP inhibitors, cell-inhibiting alkaloids, cytotoxic antibiotics, antimetabolites, endocrine / hormonal agents, bisphosphonate therapeutic agents, and targeted biological therapeutic agents (e.g., therapeutic peptides described in US 6306832, WO 2012007137, WO2005000889, WO 2010096603, etc.). In some embodiments, the at least one additional therapeutic agent is a chemotherapeutic agent. Specific chemotherapeutic agents include, but are not limited to, cyclophosphamide, fluorouracil (or 5-fluorouracil or 5-FU), methotrexate, edatrexate (10-ethyl-10-deaza-aminopterin), thiotepa, carboplatin, cisplatin, taxanes, paclitaxel, protein-bound paclitaxel, docetaxel, vinorelbine, tamoxifen, raloxifene, toremifene, fulvestrant, gemcitabine, irinotecan, ixabepilone, temozolomide, topotecan, vincristine, vinblastine, eribulin, mutamycin, capecitabine, a Nastrazole, exemestane, letrozole, leuprolide, abarelix, buserelin, goserelin, megestrol acetate, risedronate, pamidronate, ibandronate, alendronate, denosumab, zoledronate, trastuzumab, lapatinib, anthracyclines (e.g., daunorubicin and doxorubicin), bevacizumab, oxaliplatin, melphalan, etoposide, nitrogen mustard, bleomycin, microtubule poison, annonaceous acetone, or a combination thereof.

[0297] The compositions of the present technology can optionally be administered to a subject in need thereof in the form of a single bolus injection. Alternatively, the dosing regimen can include multiple administrations at different times after the appearance of a tumor.

[0298] Administration can be by any suitable route, including oral, intranasal, parenteral (intravenous, intramuscular, intraperitoneal or subcutaneous), rectal, intracranial, intrathecal or topical. Administration includes self-administration and administration by another person. It is also understood that the various treatments of medical conditions as described herein are intended to mean "substantially," which includes complete treatment but also less than complete treatment, and in which some biologically or medically relevant results are achieved.

[0299] In some embodiments, the antibodies of the present technology include pharmaceutical formulations that can be administered to a subject in need thereof in one or more doses. The dosage regimen can be adjusted to provide a desired response (e.g., a therapeutic response).

[0300] Typically, the effective amount of the antibody composition of the present technology sufficient to achieve a therapeutic effect is within the range of about 0.000001 mg per kilogram of body weight per day to about 10,000 mg per kilogram of body weight per day. Typically, the dosage range is about 0.0001 mg per kilogram of body weight per day to about 100 mg per kilogram of body weight per day. For administration of anti-poly-Sia antibodies, the dosage range is 0.0001 to 100 mg / kg of subject body weight per week, every two weeks or every three weeks, and more typically 0.01 to 5 mg / kg of subject body weight. For example, the dosage can be 1 mg / kg body weight or 10 mg / kg body weight per week, every two weeks or every three weeks, or within the range of 1-10 mg / kg per week, every two weeks or every three weeks. In one embodiment, the single dose range of the antibody is 0.1-10,000 micrograms per kilogram of body weight. In one embodiment, the antibody concentration in the carrier ranges from 0.2 to 2000 micrograms per milliliter delivered. Exemplary treatment regimens require administration once every two weeks or once a month or once every 3 to 6 months. Anti-polySia antibodies can be administered at multiple times. The intervals between individual doses can be hourly, daily, weekly, monthly or annual. Intervals can also be irregular as indicated by measuring the blood level of the antibody in the subject. In some methods, the dose is adjusted to achieve the following serum antibody concentration in the subject: about 75 μg / mL to about 125 μg / mL, 100 μg / mL to about 150 μg / mL, about 125 μg / mL to about 175 μg / mL or about 150 μg / mL to about 200 μg / mL. Alternatively, anti-polySia antibodies can be administered as sustained release formulations, in which case less frequent administration is required. The dose and frequency vary depending on the half-life of the antibody in the subject. The dose and frequency of administration can vary depending on whether the treatment is preventive or therapeutic. In preventive applications, relatively low doses are administered at relatively infrequent intervals over a long period of time. In therapeutic applications, relatively high doses are sometimes required to be administered at relatively short intervals until the progression of the disease is reduced or terminated, or until the subject shows partial or complete improvement in symptoms of the disease. Thereafter, the patient may be given a preventive regimen.

[0301] In another aspect, the present disclosure provides a method for detecting a tumor in a subject in vivo, the method comprising (a) administering to the subject an effective amount of an antibody (or an antigen-binding fragment thereof) of the present technology, wherein the antibody is configured to localize to a tumor expressing poly-Sia and is labeled with a radioactive isotope; and (b) detecting the presence of a tumor in the subject by detecting a level of radioactivity emitted by the antibody that is higher than a reference value. In some embodiments, the reference value is expressed as injected dose per gram (%ID / g). The reference value can be calculated by measuring the level of radioactivity present in non-tumor (normal) tissues and calculating the average radioactivity level ± standard deviation present in non-tumor (normal) tissues. In some embodiments, the ratio of radioactivity levels between tumor and normal tissue is about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, or 100:1.

[0302] In some embodiments, the subject is diagnosed with or suspected of having cancer.The level of radioactivity emitted by the antibody can be detected using positron emission tomography or single photon emission computed tomography.

[0303] Additionally or alternatively, in some embodiments, the method further comprises administering to the subject an effective amount of an immunoconjugate comprising an antibody of the present technology conjugated to a radionuclide. In some embodiments, the radionuclide is an alpha particle emitting isotope, a beta particle emitting isotope, an Auger emitter, or any combination thereof. Examples of beta particle emitting isotopes include 86 Y. 90 Y. 89 Sr. 165 Dy, 186 Re, 188 Re, 177 Lu and 67 Cu. Examples of alpha-emitting isotopes include 213 Bi, 211 At 225 Ac, 152 Dy, 212 Bi, 223 Ra, 219 Rn, 215 Po, 211 Bi, 221 Fr. 217 At and 255Fm. Examples of Auger emitters include 111 In, 67 Ga, 51 Cr, 58 Co. 99m Tc, 103m Rh, 195m Pt, 119 Sb, 161 Ho, 189m Os, 192 Ir, 201 Tland 203 Pb. In some embodiments of the method, non-specific FcR-dependent binding in normal tissues is eliminated or reduced (e.g., via N297A mutations in the Fc region, which result in deglycosylation). The therapeutic effectiveness of such an immunoconjugate can be determined by calculating the area under the curve (AUC) tumor: AUC normal tissue ratio. In some embodiments, the AUC tumor: AUC normal tissue ratio of the immunoconjugate is about 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 15: 1, 20: 1, 25: 1, 30: 1, 35: 1, 40: 1, 45: 1, 50: 1, 55: 1, 60: 1, 65: 1, 70: 1, 75: 1, 80: 1, 85: 1, 90: 1, 95: 1 or 100: 1.

[0304] PRIT. In one aspect, the present disclosure provides a method for detecting a solid tumor in a subject in need thereof, the method comprising (a) administering to the subject an effective amount of a complex comprising a radiolabeled DOTA hapten and a bispecific antibody of the present technology that binds to the radiolabeled DOTA hapten and a high-DP poly-Sia antigen, wherein the complex is configured to localize to a solid tumor expressing a high-DP poly-Sia antigen recognized by the bispecific antibody of the complex; and (b) detecting the presence of a solid tumor in the subject by detecting a level of radioactivity emitted by the complex that is above a reference value. In some embodiments, the subject is a human.

[0305] In another aspect, the present disclosure provides a method for selecting a subject for pretargeted radioimmunotherapy, the method comprising (a) administering to the subject an effective amount of a complex comprising a radiolabeled DOTA hapten and a bispecific antibody of the present technology that binds to the radiolabeled DOTA hapten and a high-DP poly-Sia antigen, wherein the complex is configured to localize to a solid tumor expressing a high-DP poly-Sia antigen recognized by the bispecific antibody of the complex; (b) detecting the level of radioactivity emitted by the complex; and (c) selecting the subject for pretargeted radioimmunotherapy when the level of radioactivity emitted by the complex is higher than a reference value. In some embodiments, the subject is a human.

[0306] Examples of DOTA haptens include (i) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH2; (ii) Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH2; (iii) DOTA-D-Asp-D-Lys(HSG)-D-Asp-D-Lys(HSG)-NH2; (iv) DOTA-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (v) DOTA-D-Tyr-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (vi) DOTA-D-Ala-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (vii) DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH2; (viii) Ac-D-Phe-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-NH2; (ix) Ac-D-Phe-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH2; (x) Ac-D-Phe-D-Lys(Bz-DTPA)-D-Tyr-D-Lys(Bz-DTPA)-NH2; (xi) Ac-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH2; (xii) DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH2; (xiii) (Tscg-Cys)-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(DOTA)-NH2; (xiv) Tscg-D-Cys-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (xv) (Tscg-Cys)-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (xvi) Ac-D-Cys-D-Lys(DOTA)-D-Tyr-D-Ala-D-Lys(DOTA)-D-Cys-NH2; (xvii) Ac-D-Cys-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH2; (xviii) Ac-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-D-Lys(Tscg-Cys)-NH2;(xix) Ac-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-D-Lys(Tscg-Cys)-NH2 and (xx) DOTA. The radioactive label may be an alpha particle emitting isotope, a beta particle emitting isotope or an Auger emitter. Examples of radioactive labels include; 213 Bi, 211 At 225 Ac, 152 Dy, 212 Bi, 223 Ra, 219 Rn, 215 Po, 211 Bi, 221 Fr. 217 At 255 Fm, 86 Y. 90 Y. 89 Sr. 165 Dy, 186 Re, 188 Re, 177 Lu, 67 Cu, 111 In, 67 Ga, 51 Cr, 58 Co. 99m Tc, 103m Rh, 195m Pt, 119 Sb, 161 Ho, 189m Os, 192 Ir, 201 Tl, 203 Pb, 68 Ga, 227 Th or 64 Cu.

[0307] In some embodiments of the methods disclosed herein, the level of radioactivity emitted by the complex is detected using positron emission tomography or single photon emission computed tomography. Additionally or alternatively, in some embodiments of the methods disclosed herein, the subject is diagnosed with or suspected of having a poly-Sia-associated cancer, such as small cell or non-small cell lung cancer, neuroblastoma, pancreatic cancer, pituitary tumor, Wilms' tumor, rhabdomyosarcoma, glioblastoma, breast cancer, or acute myeloid leukemia.

[0308] Additionally or alternatively, in some embodiments of the methods disclosed herein, the complex is administered intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intraorbitally, intradermally, intraperitoneally, transtracheally, subcutaneously, intracerebroventricularly, orally, or intranasally. In certain embodiments, the complex is administered to the subject's cerebrospinal fluid or blood.

[0309] In some embodiments of the methods disclosed herein, the radioactivity level emitted by the complex is detected between 2 and 120 hours after administration of the complex. In certain embodiments of the methods disclosed herein, the radioactivity level emitted by the complex is expressed as a percentage of injected dose per gram of tissue (% ID / g). The reference value can be calculated by measuring the radioactivity level present in non-tumor (normal) tissues and calculating the average radioactivity level ± standard deviation present in non-tumor (normal) tissues. In some embodiments, the reference value is a standard uptake value (SUV). See Thie JA, J Nucl Med. 45 (9): 1431-4 (2004). In some embodiments, the ratio of radioactivity levels between tumor and normal tissue is about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, or 100:1.

[0310] In another aspect, the present disclosure provides a method for increasing the sensitivity of a tumor to radiotherapy in a subject diagnosed with poly-Sia-associated cancer, the method comprising (a) administering to the subject an effective amount of an anti-DOTA bispecific antibody of the present technology, wherein the anti-DOTA bispecific antibody is configured to localize to a tumor expressing high-DP poly-Sia; and (b) administering to the subject an effective amount of a radiolabeled DOTA hapten, wherein the radiolabeled DOTA hapten is configured to bind to the anti-DOTA bispecific antibody. In some embodiments, the subject is a human.

[0311] The anti-DOTA bispecific antibody is administered under conditions and for a period of time (e.g., according to a dosing regimen) sufficient to allow saturation of the tumor cells. In some embodiments, after administration of the anti-DOTA bispecific antibody, unbound anti-DOTA bispecific antibody is removed from the bloodstream. In some embodiments, a radiolabeled DOTA hapten is administered after a period of time sufficient to allow clearance of unbound anti-DOTA bispecific antibody.

[0312] The radiolabeled DOTA hapten can be administered anywhere from 1 minute to 4 or more days after administration of the anti-DOTA bispecific antibody. For example, in some embodiments, the radiolabeled DOTA hapten is administered 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 1.25 hours, 1.5 hours, 1.75 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, or any range therein, after administration of the anti-DOTA bispecific antibody. Alternatively, the radiolabeled DOTA hapten can be administered at any time after 4 or more days following administration of the anti-DOTA bispecific antibody.

[0313] Additionally or alternatively, in some embodiments, the method further comprises administering to the subject an effective amount of a scavenger prior to administering the radiolabeled DOTA hapten. The scavenger can be any molecule (glucan or dendrimer or polymer) that can be conjugated to the C825 hapten. In some embodiments, the scavenger is no more than 2000 kD, 1500 kD, 1000 kD, 900 kD, 800 kD, 700 kD, 600 kD, 500 kD, 400 kD, 300 kD, 200 kD, 100 kD, 90 kD, 80 kD, 70 kD, 60 kD, 50 kD, 40 kD, 30 kD, 20 kD, 10 kD or 5 kD. In some embodiments, the scavenging agent is a 500 kD aminodextran-DOTA conjugate (eg, 500 kD dextran-DOTA-Bn(Y), 500 kD dextran-DOTA-Bn(Lu), or 500 kD dextran-DOTA-Bn(In), etc.).

[0314] In some embodiments, a scavenger and a radiolabeled DOTA hapten are administered without further administration of an anti-DOTA bispecific antibody of the present technology. For example, in some embodiments, an anti-DOTA bispecific antibody of the present technology is administered according to a regimen comprising at least one of the following cycles: (i) administration of an anti-DOTA bispecific antibody of the present technology (optionally, to saturate the relevant tumor cells); (ii) administration of a radiolabeled DOTA hapten and an optional scavenger; (iii) optional additional administration of a radiolabeled DOTA hapten and / or scavenger without additional administration of an anti-DOTA bispecific antibody. In some embodiments, the method may include multiple such cycles (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more cycles).

[0315] Additionally or alternatively, in some embodiments of the methods, the anti-DOTA bispecific antibody and / or the radiolabeled DOTA hapten is administered intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intraorbitally, intradermally, intraperitoneally, transtracheally, subcutaneously, intracerebroventricularly, orally, or intranasally.

[0316] In one aspect, the present disclosure provides a method for increasing the sensitivity of a tumor to radiotherapy in a subject diagnosed with a poly-Sia-associated cancer, the method comprising administering to the subject an effective amount of a complex comprising a radiolabeled DOTA hapten and a bispecific antibody of the present technology that recognizes and binds to the radiolabeled DOTA hapten and a high-DP poly-Sia antigen target, wherein the complex is configured to localize to a tumor expressing a high-DP poly-Sia antigen target recognized by the bispecific antibody of the complex. The complex can be administered intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intraorbitally, intradermally, intraperitoneally, transtracheally, subcutaneously, intracerebroventricularly, orally, or intranasally. In some embodiments, the subject is a human.

[0317] In another aspect, the present disclosure provides a method for treating cancer in a subject in need thereof, the method comprising (a) administering to the subject an effective amount of an anti-DOTA bispecific antibody of the present technology, wherein the anti-DOTA bispecific antibody is configured to localize to a tumor expressing a high-DP poly-Sia antigen target; and (b) administering to the subject an effective amount of a radiolabeled DOTA hapten, wherein the radiolabeled DOTA hapten is configured to bind to the anti-DOTA bispecific antibody. The anti-DOTA bispecific antibody is administered under certain conditions and for a period of time (e.g., according to a dosing regimen), and the conditions and time period are sufficient to saturate it in tumor cells. In some embodiments, after administration of the anti-DOTA bispecific antibody, unbound anti-DOTA bispecific antibodies are removed from the bloodstream. In some embodiments, the radiolabeled DOTA hapten is administered after a period of time sufficient to allow clearance of unbound anti-DOTA bispecific antibodies. In some embodiments, the subject is a human.

[0318] Thus, in some embodiments, the method further comprises administering to the subject an effective amount of a clearing agent prior to administering the radiolabeled DOTA hapten. The radiolabeled DOTA hapten can be administered at any time between 1 minute to 4 or more days after administration of the anti-DOTA bispecific antibody. For example, in some embodiments, the radiolabeled DOTA hapten is administered 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 1.25 hours, 1.5 hours, 1.75 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, or any range therein, after administration of the anti-DOTA bispecific antibody. Alternatively, the radiolabeled DOTA hapten can be administered at any time after 4 or more days following administration of the anti-DOTA bispecific antibody.

[0319] The scavenger can be a 500kD aminodextran-DOTA conjugate (e.g., 500kD dextran-DOTA-Bn(Y), 500kD dextran-DOTA-Bn(Lu) or 500kD dextran-DOTA-Bn(In), etc.). In some embodiments, the scavenger and the radiolabeled DOTA hapten are administered without further administration of the anti-DOTA bispecific antibody. For example, in some embodiments, the anti-DOTA bispecific antibody is administered according to a regimen comprising at least one of the following cycles: (i) administering an anti-DOTA bispecific antibody of the present technology (optionally, to saturate the relevant tumor cells); (ii) administering a radiolabeled DOTA hapten and an optional scavenger; (iii) optionally administering a radiolabeled DOTA hapten and / or a scavenger in addition without additional administration of the anti-DOTA bispecific antibody. In some embodiments, the method can include multiple such cycles (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more cycles).

[0320] Also provided herein is a method for treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of a complex comprising a radiolabeled DOTA hapten and a bispecific antibody of the present technology that recognizes and binds to the radiolabeled DOTA hapten and a high-DP poly-Sia antigen target, wherein the complex is configured to localize to a tumor expressing a high-DP poly-Sia antigen target recognized by the bispecific antibody of the complex. The therapeutic effectiveness of such a complex can be determined by calculating the area under the curve (AUC) tumor: AUC normal tissue ratio. In some embodiments, the AUC tumor:AUC normal tissue ratio of the complex is about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, or 100:1.

[0321] Toxicity. Optimally, an effective amount (e.g., dose) of an anti-polySia antibody described herein will provide therapeutic benefit without causing substantial toxicity to a subject. The toxicity of an anti-polySia antibody described herein can be determined by standard pharmaceutical procedures in cell culture or experimental animals, such as by measuring LD 50 (dose lethal to 50% of the population) or LD 100The therapeutic index is determined by the dose ratio between toxicity and therapeutic effect. The data obtained from these cell culture assays and animal studies can be used to formulate a dosage range that is nontoxic to humans. The dosage of the anti-polySia antibodies described herein is within a range of circulating concentrations that includes an effective dose with little or no toxicity. The dosage can vary within this range depending on the dosage form employed and the route of administration utilized. The exact formulation, route of administration, and dosage can be selected by an individual physician based on the circumstances of the subject. See, e.g., Fingl et al., In: The Pharmacological Basis of Therapeutics, Chapter 1 (1975).

[0322] Formulation of pharmaceutical compositions. According to the methods of the present technology, anti-polySia antibodies can be incorporated into pharmaceutical compositions suitable for administration. Pharmaceutical compositions typically comprise recombinant or substantially purified antibodies and a pharmaceutically acceptable carrier in a form suitable for administration to a subject. The pharmaceutically acceptable carrier depends in part on the specific composition being administered, as well as on the specific method used to administer the composition. Thus, there are a variety of suitable formulations of pharmaceutical compositions for administering antibody compositions (see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 18th edition, 1990). Pharmaceutical compositions are typically formulated to be sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0323] The terms "pharmaceutically acceptable", "physiologically tolerable" and grammatical variants thereof, when they refer to compositions, carriers, diluents and agents, are used interchangeably and indicate that the material can be administered to or onto a subject without producing undesirable physiological effects to the extent that would prohibit administration of the composition. For example, "pharmaceutically acceptable excipient" means an excipient that can be used to prepare a pharmaceutical composition that is generally safe, non-toxic and desirable, and includes excipients that are acceptable for veterinary and human pharmaceutical use. Such excipients can be solid, liquid, semisolid, or, in the case of aerosol compositions, gas. "Pharmaceutically acceptable salts and esters" means salts and esters that are pharmaceutically acceptable and have the desired pharmacological properties. Such salts include salts that can be formed when acidic protons present in the composition are able to react with inorganic or organic bases. Suitable inorganic salts include those formed with alkali metals such as sodium and potassium, magnesium, calcium and aluminum. Suitable organic salts include those formed with organic bases, such as amine bases, for example, ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. Such salts also include acid addition salts formed with inorganic acids such as hydrochloric acid and hydrobromic acid and organic acids such as acetic acid, citric acid, maleic acid, and alkanesulfonic and aromatic sulfonic acids such as methanesulfonic acid and benzenesulfonic acid. Pharmaceutically acceptable esters include esters formed with carboxyl, sulfonyloxy, and phosphonooxy groups present in the anti-polySia antibody, such as C 1-6 Alkyl esters. When two acidic groups are present, the pharmaceutically acceptable salt or ester may be a monoacid monosalt or ester or a disalt or ester; and similarly, when more than two acidic groups are present, some or all of such groups may be salified or esterified. The anti-polySia antibodies named in this technology may exist in an unsalted or unesterified form, or in a salified and / or esterified form, and the naming of such anti-polySia antibodies is intended to include the original (unsalted and unesterified) compound and its pharmaceutically acceptable salts and esters. In addition, certain embodiments of the present technology may exist in more than one stereoisomer form, and the naming of such anti-polySia antibodies is intended to include all single stereoisomers and all mixtures of such stereoisomers (whether racemic or otherwise). It is not difficult for a person of ordinary skill in the art to determine the appropriate timing, sequence, and dosage for administering a particular drug and composition of the present technology.

[0324] Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles, such as fixed oils, may also be used. The use of such media and compounds for pharmaceutically active substances is well known in the art. Unless any conventional media or compounds are incompatible with the anti-poly Sia antibody, their use in the composition is contemplated. Supplementary active compounds may also be incorporated into the composition.

[0325] The pharmaceutical compositions of the present technology are formulated to be compatible with their intended route of administration. The anti-poly-Sia antibody compositions of the present technology can be administered parenterally, topically, intravenously, orally, subcutaneously, intraarterially, intradermally, transdermally, rectally, intracranially, intrathecally, intraperitoneally, intranasally; or intramuscularly, or as an inhalant. The anti-poly-Sia antibodies can be administered optionally in combination with other agents that are at least partially effective in treating various poly-Sia-related cancers.

[0326] Solutions or suspensions for parenteral, intradermal or subcutaneous administration may include the following components: a sterile diluent, such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents; antibacterial compounds, such as benzyl alcohol or methyl paraben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating compounds, such as ethylenediaminetetraacetic acid (EDTA); buffers (such as acetates, citrates or phosphates), and compounds for adjusting tonicity (such as sodium chloride or dextrose). pH can be adjusted with acids or bases (such as hydrochloric acid or sodium hydroxide). Parenteral preparations can be packaged in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0327] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL TM (BASF, Percival, New Jersey) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that it is easy to inject. The composition must be stable under manufacturing and storage conditions, and its preservation must be resistant to the contaminating effects of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium, which contains, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. For example, by using a coating such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using a surfactant, appropriate fluidity can be maintained. Preventing the effects of microorganisms can be achieved by a variety of antibacterial and antifungal compounds (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.). In many cases, it is desirable to include isotonic compounds in the composition, such as sugars, polyols (such as mannitol, sorbitol), sodium chloride. Extended absorption of injectable compositions can be achieved by including compounds that delay absorption in the composition, such as aluminum monostearate and gelatin.

[0328] Sterile injectable solutions can be prepared by incorporating the anti-polySia antibodies of the present technology in the desired amount into an appropriate solvent optionally having one or a combination of the ingredients listed above, followed by filtration sterilization. Typically, dispersions are prepared by incorporating the anti-polySia antibodies into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for preparing sterile injectable solutions, the preparation methods are vacuum drying and freeze drying, which produce powders of the active ingredient and any additional required ingredients from a previously sterile filtered solution. The antibodies of the present technology can be administered in the form of a depot injection or implant formulation, which can be formulated in a manner that allows for sustained or pulsatile release of the active ingredient.

[0329] Oral compositions typically include an inert diluent or edible carrier. They can be encapsulated in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, anti-polySia antibodies can be incorporated with excipients and used in the form of tablets, lozenges or capsules. Oral compositions can also be prepared using a fluid carrier used as a mouthwash, where the compound in the fluid carrier is administered orally and rinsed and spit out or swallowed. Pharmaceutically compatible binding compounds and / or auxiliary materials can be included as part of the composition. Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients or compounds of similar properties: binders, such as microcrystalline cellulose, tragacanth or gelatin; excipients, such as starch or lactose, disintegrating compounds, such as alginic acid, Primogel or corn starch; lubricants, such as magnesium stearate or Sterotes; glidants, such as colloidal silica; sweetening compounds, such as sucrose or saccharin; or flavoring compounds, such as mint, methyl salicylate or orange flavor.

[0330] For administration by inhalation, the anti-poly Sia antibody can be delivered in the form of an aerosol spray from a pressured container or dispenser which contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

[0331] Systemic administration can also be performed by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants suitable for the barrier to be penetrated are used in the formulation. Such penetrants are generally known in the art and include (for example, for transmucosal administration) detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished by using nasal sprays or suppositories. For transdermal administration, the anti-polySia antibody is formulated into an ointment, salves, gel, or cream as generally known in the art.

[0332] Anti-poly Sia antibodies can also be prepared as pharmaceutical compositions for rectal delivery in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas.

[0333] In one embodiment, the anti-polySia antibody is prepared with a carrier that prevents the rapid clearance of the anti-polySia antibody from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be clear to those skilled in the art. The materials can also be commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, such as those described in U.S. Patent No. 4,522,811. C. Kit

[0334] The present technology provides a kit for detecting and / or treating poly-Sia-related cancers, the kit comprising at least one immunoglobulin-related composition of the present technology (e.g., any antibody or antigen-binding fragment described herein) or a functional variant thereof (e.g., a substitution variant). Optionally, the above components of the kit of the present technology are packaged in a suitable container and labeled for diagnosis and / or treatment of poly-Sia-related cancers. The above components can be stored in a unit container or a multi-dose container (e.g., a sealed ampoule, vial, bottle, syringe, and test tube) as an aqueous solution (preferably a sterile solution) or as a lyophilized (preferably sterile) preparation for reconstitution. The kit may also include a second container containing a diluent suitable for diluting the pharmaceutical composition to a larger volume. Suitable diluents include, but are not limited to, pharmaceutically acceptable excipients and saline solutions of the pharmaceutical composition. In addition, the kit may include instructions for diluting the pharmaceutical composition and / or instructions for administering the diluted or undiluted pharmaceutical composition. The container can be made of a variety of materials (such as glass or plastic) and can have a sterile access port (for example, the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). The kit can further include more containers containing pharmaceutically acceptable buffers, such as phosphate-buffered saline, Ringer's solution, and dextrose solution. The kit can further include other materials that are desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, syringes, culture media for one or more suitable hosts. The kit can optionally include instructions that are typically included in the commercial packaging of the treatment or diagnostic product, the instructions containing information about, for example, the indications, usage, dosage, manufacture, administration, contraindications, and / or warnings for using such treatment or diagnostic products.

[0335] The kit can be used to detect the presence of immunoreactive high-DP poly-Sia in a biological sample, such as any body fluid, including but not limited to, for example, serum, plasma, lymph, cystic fluid, urine, feces, cerebrospinal fluid, ascites or blood, and including biopsy samples of body tissue. For example, the kit may include: one or more humanized, chimeric or bispecific anti-poly-Sia antibodies (or antigen-binding fragments thereof) of the present technology that are capable of binding to high-DP poly-Sia in a biological sample; a device for determining the amount of high-DP poly-Sia in a sample; and a device for comparing the amount of immunoreactive high-DP poly-Sia in a sample with a standard. One or more anti-poly-Sia antibodies may be labeled. Kit components (e.g., reagents) may be packaged in suitable containers. The kit may further include instructions for using the kit to detect immunoreactive high-DP poly-Sia.

[0336] For antibody-based kits, the kit can include, for example, 1) a first antibody attached to a solid support, such as a humanized, chimeric or bispecific anti-poly-Sia antibody (or antigen-binding fragment thereof) of the present technology, which first antibody binds to high-DP poly-Sia; and, optionally; 2) a second different antibody that binds to high-DP poly-Sia or the first antibody and is conjugated to a detectable label.

[0337] The kit may also include, for example, a buffer, a preservative, or a protein stabilizer. The kit may further include other components required for detecting the detectable label, such as an enzyme or a substrate. The kit may also contain a control sample or a series of control samples, which can be assayed and compared with the test sample. Each component of the kit can be encapsulated in a separate container, and all the different containers can be placed in a single package together with instructions for explaining the results of the assay using the kit. The kit of the present technology may contain a written product on or in the kit container. The written product describes how to use the reagents contained in the kit, for example, for detecting high DP poly-Sia in vitro or in vivo, or for treating poly-Sia-related cancers in subjects in need. In certain embodiments, the reagents can be used according to the methods of the present technology. Example

[0338] The present technology is further illustrated by the following examples, which should not be construed as limiting in any way. The following examples demonstrate the preparation, characterization, and use of illustrative anti-polySia antibodies of the present technology. The following examples demonstrate the generation of chimeric, humanized, and bispecific antibodies of the present technology, and the characterization of their binding specificity and in vivo biological activity. Example 1: Materials and methods used to generate and characterize polysialic acid antibodies of the present technology

[0339] Generation of polySia x CD3 bispecific antibodies. PolySia x CD3 BsAbs were constructed by fusing humanized OKT3 scFv to the C-terminus of the P35 H1L2 antibody light chain via a (G4S)3 linker, as previously described in: Xu H et al., Cancer Immunology Research 3:266-277 (2015) and Lopez-Albaitero A et al., OncoImmunology 6:e1267891 (2017). N297A and K322A mutations were introduced in the Fc region of the antibody to abolish FcR and complement binding activities, respectively (Shields RL et al., Journal of Biological Chemistry 276:6591-6604 (2001); Idusogie EE et al., Journal of Immunology 164:4178-4184 (2000)). The DNA construct was then transfected into CHO-S cells and stable clones were selected for high antibody production levels. For larger scale antibody purification, the selected stable clones were expanded in shake flasks. The bispecific antibodies were purified from the supernatant using one-step protein A affinity chromatography.

[0340] SEC-HPLC analysis. BC137 size and purity were analyzed using an HPLC system (Shimadzu Scientific Instruments Inc., Columbia, Maryland). Monomer species were identified using molecular weight standards (Bio-Rad Laboratories, Hercules, California), and monomer percentages were calculated based on the relative area under the curve (AUC) of different non-buffered peaks.

[0341] T cell-dependent cytotoxicity (TDCC) assay. 51 Cytotoxicity assays were performed using either the Cr release assay or the Pierce LDH release assay (Thermo Fisher Scientific, Cambridge, MA). For both assays, T cells activated by exposure to anti-CD3 / anti-CD28 Dynaband for 14 days were then used as effector cells, except for sorted cells from PBMCs, which were used for TDCC assays without prior stimulation. As previously described in Cheng M et al., International Journal of Cancer 136:476-486 (2015) 51Cr assay. The LDH assay was performed according to the manufacturer's instructions with the following modifications. Briefly, for each assay well of a 96-well round-bottom plate, 1.5 × 10 4 Target cells were incubated with variable numbers of effector cells. Antibodies were then added at different dilutions and the plates were incubated at 37 °C for 16 h. Each condition was performed in triplicate. The supernatant was then transferred to a flat-bottom plate with reaction substrate and incubated for 30 min before reading at 490 nm with 680 nm as reference wavelength. EC was calculated by fitting the curves to a 4-parameter nonlinear regression model using GraphPad Prism 50 value.

[0342] In vivo tumor therapy. To test the antitumor efficacy of BC137, immunodeficient mice were used for human tumor xenografts. Neuroblastoma IMR-32 tumors were implanted subcutaneously, where tumor cells were mixed with matrigel and implanted in Balb / cRag2 - / - IL2Rγ - / - The flank of a DKO mouse (now commercially available as CIEA BRG mouse from Taconic (Rensselaer, NY) was obtained. Tumor growth was monitored by measuring tumor volume weekly using either a caliper or a digital device, the Peira TM900 scanner (Peira Scientific Instruments, Turnhout, Belgium).

[0343] Treatment was started on day 7 after tumor implantation. 100 μg of BsAb was injected into each mouse (iv, twice a week for five weeks), and the next day 10 million CD3 / CD28 bead-activated T cells were injected (once a week for five weeks).

[0344] Yeast display was used for affinity maturation. Parent P35 H1L2 was converted into scFv form with 20 amino acid (G4S) 3 joints and cloned into yeast display vector. P35 H1L2 scFv was randomly mutated using GeneMorph II mutagenesis kit (Agilent Technologies, Santa Clara, California). PCR product was electroporated into yeast together with linearized vector, and the library was subjected to 4 rounds of sorting using biotinylated poly-Sia. PCR amplification and sequencing were performed on the last round of individual clones to analyze mutation patterns. The selected scFv clone was converted into BsAb form using a one-step 4-fragment connection method, wherein the mol ratio of the vector to the insert of 50ng linearized vector and other 3 components was 1:3. Connected at room temperature for 1 hour with a rapid DNA connection kit (Thermo Fisher Scientific, Cambridge, Massachusetts). Type II restriction enzyme SapI (New England Biolabs, Ipswich, Massachusetts) was used to ensure seamless connection between different components. The selected clones were transiently expressed using the Expi293 expression system (Thermo Fisher Scientific, Cambridge, Mass.) according to the manufacturer's instructions. After 4-5 days of culture in shake flasks, supernatants from Expi293 cells were used to purify antibodies using MabSelect SuRe (GE Healthcare, Chicago, Ill.) and dialyzed against pH 8.0 citrate buffer in a dialysis membrane (Spectrum Laboratories, Inc., Rancho Dominguez, Calif.).

[0345] Surface plasmon resonance (SPR) analysis. Polyacetylneuraminic acid (polySia with about 100 Sia units) was immobilized on a CM5 chip. Using Biacore TM Five concentrations of 2-fold serial dilutions of antibody IgG or BsAb were flowed over the chip using a T100 system (GE Healthcare, Chicago, IL). Binding kinetics were measured at 25° C. Sensorgrams were fitted with a 1:1 binding model for both to derive kinetic parameters. Example 2: Generation of chimeric and humanized P35 antibodies

[0346] Chimeric and humanized anti-poly-Sia antibodies were generated using a CDR grafting approach using closely homologous human germline sequences. Two different humanized VH (P35 H1, P35 H2) and humanized VL (P35 L1, P35 L2) sequences were combined to generate 4 different humanized anti-poly-Sia IgG1 antibodies. Figure 3Differences in antigen binding were demonstrated by flow cytometry of the four combinations H1L1, H1L2, H2L1, and H2L2. P35 H1L2 exhibited the highest binding ( Figure 3 ) and was further studied.

[0347] These results demonstrate that the antibodies or antigen-binding fragments thereof of the present technology specifically bind to polysialic acid antigens with high binding affinity. Therefore, the immunoglobulin-related compositions disclosed herein can be used for methods of detecting polysialic acid (high DP poly-Sia) with a high degree of polymerization in a sample and for methods of detecting tumors in a subject. Example 3: Expression of Poly-Sia in Various Cancer Cell Lines

[0348] The expression of poly-Sia was tested on a number of cancer cell lines using chimeric P35 IgG1. An isotype-matched control antibody (anti-RSV palivizumab) was used as a control and the mean fluorescence intensity was expressed as Figure 1(A) and 1(B) High levels of poly-Sia were observed for neuroblastoma, small cell lung cancer (SCLC), and acute myeloid leukemia (AML). The detection of poly-Sia in AML cells was unexpected, considering that high levels of poly-Sia have not been reported for AML previously.

[0349] These results demonstrate that the antibodies or antigen-binding fragments thereof of the present technology specifically bind to polysialic acid antigens with high binding affinity. Therefore, the immunoglobulin-related compositions disclosed herein can be used for methods of detecting polysialic acid (high DP poly-Sia) with a high degree of polymerization in a sample and for methods of detecting tumors in a subject. Example 4: Expression of Poly-Sia on SCLC Patient-Derived Xenografts (PDX)

[0350] The expression of poly-Sia was tested by immunohistochemistry on 14 different SCLC PDX samples using a chimeric p35 antibody. The samples were rated using a grading scale of 1-4, and 11 of the 14 samples showed areas of high-level staining (grade 3 or 4). Figure 2 .

[0351] These results demonstrate that the antibodies or antigen-binding fragments thereof of the present technology specifically bind to polysialic acid antigens with high binding affinity. Therefore, the immunoglobulin-related compositions disclosed herein can be used for methods of detecting polysialic acid (high DP poly-Sia) with a high degree of polymerization in a sample and for methods of detecting tumors in a subject. Example 5: Generation of poly-Sia x CD3 bispecific antibodies

[0352] When tested for antibody-dependent cell-mediated cytotoxicity (ADCC) against polySia(+) cell lines, the humanized P35 IgG1 antibody exhibited no antitumor activity ( Fig.66 ). In order to confer anti-tumor properties to the humanized P35 antibody, a bispecific antibody targeting polySia x CD3 was generated, thereby redirecting T cells to kill tumors. FIG. 4(A) shows a schematic diagram of a polySia x CD3 BsAb. The first BsAb generated utilized the P35 H1L2 sequence to make Biclone 137 (BC137). FIG. 4(B) shows the purity of the polySia x CD3 bispecific antibody BC137 by SEC-HPLC, wherein the main peak of BC137 (absorbance at 280 nm of 95%) had a MW of approximately 210 kDa. After multiple freeze and thaw cycles, the BC137 BsAb remained stable by SEC-HPLC. Example 6: Poly-Sia-BsAb redirected T cell cytotoxicity against human cancer cell lines

[0353] The polySia(+) cell lines melanoma M14 and neuroblastoma IMR-32 were tested in a 24-hour lactate dehydrogenase (LDH) cytotoxicity assay using activated human T cells (effector / target ratio: 10:1) to test BC137 redirected T cell cytotoxicity.

[0354] Figure 5(A)-5(B) BC137 showed an EC of nearly 100 ng / ml 50 The high T cell-dependent cell-mediated cytotoxicity (TDCC) exhibited by BC137 was unexpected, considering that the humanized P35 H1L2 antibody exhibited a lack of ADCC.

[0355] These results demonstrate that the antibodies or antigen-binding fragments thereof of the present technology specifically bind to polysialic acid antigens with high binding affinity. Therefore, the immunoglobulin-related compositions disclosed herein can be used for methods of detecting polysialic acid (high DP poly-Sia) with a high degree of polymerization in a sample and for methods of detecting tumors in a subject. Example 7: Poly-Sia-BsAb in Mouse Xenograft Studies with Neuroblastoma IMR-32 Tumors Shows anti-tumor efficacy

[0356] To test the antitumor efficacy of BC137, immunodeficient mice were used for human tumor xenografts. Neuroblastoma IMR-32 tumors were implanted subcutaneously in Balb / c Rag2 - / - IL2Rγ - / - The flank of a (DKO) mouse. Figure 6As shown, in the absence of antibody treatment, both the tumor group and the tumor + activated T cell group showed rapid tumor growth. In contrast, 10 doses of BC137 in 5 weeks effectively cured mice, and the mice remained tumor-free for at least 50 days. No clinical toxicity was observed.

[0357] In summary, these results demonstrate that the antibodies or antigen-binding fragments of the present technology can detect tumors and inhibit the progression of tumor growth and / or metastasis. Therefore, the immunoglobulin-related compositions disclosed herein can be used in methods for detecting and / or treating poly-Sia-related cancers in subjects in need thereof. Example 8: Affinity maturation of anti-polySia antibodies by computer modeling and rational design

[0358] In the attempt to further improve the effectiveness of BC137, affinity maturation was attempted by both reasonable engineering and random mutagenesis / yeast display. In order to carry out reasonable engineering, based on the previously resolved crystal structure (PDB 3WDB) of the scFv combined with eight sialic acid ligands, the binding pocket of mAb735 was subjected to electrostatic analysis. The DelPhi algorithm was used to generate electrostatic surface diagram (Fig. 7 (A)) using BioviaDiscovery Studio modeling software (Dassault System, San Diego, California). The electrostatic potential diagram shows 2 negatively charged residues (VH:D31 on the pocket surface, and VH:D105 in the depths of the binding pocket). Fig. 7 (B) shows a cross-sectional ribbon diagram of the binding pocket of mAb735. It is not desirable to be bound by theory, and it is believed that the charge change at D31 and D105 to positively charged residues enhances poly-Sia combination. At these sites, mutations are introduced into P35 H1L2 IgG1, and then tested for combination with poly-Sia (+) neuroblastoma BE (1) n cells. Surprisingly, D105H showed weaker binding than the parent P35 H1L2, whereas the positively charged amino acid at D31 showed enhanced binding; while D31R showed the most enhanced binding ( FIG. 7(C) ).

[0359] These results demonstrate that the antibodies or antigen-binding fragments thereof of the present technology specifically bind to polysialic acid antigens with high binding affinity. Therefore, the immunoglobulin-related compositions disclosed herein can be used for methods of detecting polysialic acid (high DP poly-Sia) with a high degree of polymerization in a sample and for methods of detecting tumors in a subject. Example 9: Affinity maturation of poly-Sia-BsAb by yeast display

[0360] In an attempt to further improve the effectiveness of poly-Sia-BsAb, the scFv affinity maturation derived from P35 H1L2 was performed using yeast display. Two screening methods, direct method and kinetic method (see Boder, ET and Wittrup, KD, Biotechnol Prog., 1998, 14: 55-62) were used. In the direct method, yeast cells were incubated with antigen to equilibrium before sorting. In the kinetic screening, yeast cells were stained with saturated amounts of labeled antigen, and then non-labeled antigen was added as a competitor. Based on sequence analysis, 6 clones were selected from the direct screening method, 8 clones were selected from the kinetic screening method, and one clone (D31R) was selected from the computer design. Surface plasma resonance (SPR) was performed on the selected clones.

[0361] The clones selected from the direct screen all contained mutations in D31 and W50, and two of them had mutations in K38. The clones were then compared by dissociation constant (K D , Figure 8(A)-8(B) ) were sorted. Clone DS54 showed the lowest K compared to the wild-type clone. D , a greater than 20-fold improvement in affinity. Clone DS47 had the smallest K off , 15 times slower than the wild-type clone. D31R had the fastest association rate, nearly 250 times faster than the wild-type clone. Two additional clones were generated, DS47+D31R and DS54+D31R.

[0362] Regarding clones selected from the kinetic screen ( Figure 9(A)-9(B) ), KS34 had a 3-fold improvement in affinity relative to the wild-type clone. Clone KS2 had the slowest off rate, more than 6-fold slower than the wild-type clone. Even though most of the clones from the kinetic screen shared the same D65C mutation, the clones from the kinetic screen also had lower conservation in terms of common amino acids. Six clones were then converted back to the bispecific format.

[0363] The three cell lines were used to compare the efficacy of selected clones in poly-Sia x CD3 BsAb-redirected T cell cytotoxicity. Figure 10(A)-10(B) The selected clones were shown to be effective in polySia xCD3 BsAB-redirected T cell cytotoxicity. 50 The values ​​demonstrated that clones DS47 and DS54 were more potent than wild-type BsAb.

[0364] In the in vivo therapy study, DKO mice were subcutaneously implanted with IMR-32 cells mixed with PBMC (inactivated from buffy coat) (1:1). Treatment with BsAB (100 μg, intravenous injection, twice a week for five weeks) was started on day 4, and tumor volume was measured weekly. Poly-Sia-BsAB and clone 47 exhibited antitumor effects compared to controls (isotype control and deglycosylated huOKT3) and clone 54 ( Fig.11 On day 38, the average tumor size in the control group reached greater than 1600 mm 3 , while the average tumor size of BC137-2 DS47 was less than 280 mm 3 (83% reduction, p=0.004), and the mean tumor size of BC137 was 310 mm 3 (81% reduction, p=0.005).

[0365] These results demonstrate that the antibodies or antigen-binding fragments thereof of the present technology specifically bind to polysialic acid antigens with high binding affinity. Therefore, the immunoglobulin-related compositions disclosed herein can be used for methods of detecting polysialic acid (high DP poly-Sia) with a high degree of polymerization in a sample and for methods of detecting tumors in a subject. Example 10: Pretargeted radioimmunotherapy using poly-Sia x DOTA bispecific antibodies

[0366] To determine whether humanized P35 antibodies could be used for pretargeted radioimmunotherapy, polySia x DOTABsAbs were generated using an IgG-scFv format, in which a humanized anti-DOTA scFv (huC825) was fused to the C-terminus of the light chain of a humanized P35 IgG1 with a silent Fc ( Fig.12 Using P35 H1L2, a poly-Sia x DOTA BsAb (BC163) was generated. A therapeutic pre-targeted radioimmunotherapy (PRIT) experiment was designed to test the efficacy of the poly-Sia x DOTA BsAb.

[0367] Different doses of poly-Sia x DOTA BsAb (0, 0.25 mg, 0.5 mg, and 1 mg) were injected into the 3 Tumor burden of IMR-32 nude mice. Antibody was injected once a week for three weeks (except for mice given 1 mg BsAb, which was only injected for two weeks). 177 Lu-DOTA-Bn and scavengers. No treatment or 177 The Lu-DOTA-Bn mice group showed rapid tumor growth ( Figure 13(A)-13(B) Groups of mice receiving 0.25, 0.5 or 1.0 mg BC163 showed almost complete tumor ablation with no signs of toxicity ( Figure 13(C)-13(E)).

[0368] To further optimize and reduce potential immunogenicity in patients, eight additional heavy chain variants (87%-88% human germline content) and three additional light chain variants (92% human germline content) were generated ( Figure 14(A)-14(B) ).

[0369] In summary, these results demonstrate that the antibodies or antigen-binding fragments of the present technology can detect tumors, increase the sensitivity of tumors to radiotherapy, and inhibit the progression of tumor growth and / or metastasis, and can be used to select patients for pre-targeted immunotherapy. Therefore, the immunoglobulin-related compositions disclosed herein can be used in methods for detecting and treating poly-Sia-positive cancers in subjects in need thereof. Example 11: Characterization of the therapeutic effect of the rehumanized anti-poly-Sia antibody

[0370] The affinity values ​​of the re-humanized anti-polySia antibodies to polyacetylneuraminic acid were measured via surface plasmon resonance. Re-humanized HP35 heavy chain (HC) variants 1-8 correspond to SEQ ID NOs: 74-81, respectively. See Figure 55-62 Heavy humanized HP35 light chain (LC) variants 1-3 correspond to SEQ ID NOs: 82-84, respectively. Figure 63-65 The results of these determinations are shown in Table 2: Table 2 sample ka1(1lMs) kd1(1 / s) ka2(1 / s) kd2(1 / s) KD(M) HP35_LC2+HC5 2.46E+06 3.89E-01 7.30E-03 1.37E-03 2.50E-08 HP35_LC3+HC7 3.72E+05 2.46E-01 3.01E-02 1.84E-03 3.80E-08 HP35-chimeric 1.01E+06 5.26E-01 2.47E-02 1.97E-03 3.86E-08 HP35_LC2+HC6 1.12E+06 6.76E-01 2.30E-02 1.58E-03 3.90E-08 HP35_LC2+HC8 1.23E+06 6.63E-01 2.34E-02 1.88E-03 4.01E-08 HP35_LC2+HC1 1.08E+06 5.90E-01 2.45E-02 1.96E-03 4.04E-08 HP35_LC2+HC7 1.49E+06 7.21E-01 1.86E-02 1.73E-03 4.12E-08 HP35_LC3+HC1 7.32E+05 8.59E-01 2.72E-02 1.04E-03 4.33E-08 HP35_LC2+HC3 1.44E+06 7.01E-01 1.74E-02 1.70E-03 4.33E-08 HP35_LC3+HC4 2.46E+05 2.04E-01 3.22E-02 1.86E-03 4.55E-08 HP35_LC2+HC4 1.32E+06 7.13E-01 1.91E-02 1.75E-03 4.56E-08 HP35_LC3+HC5 1.93E+06 7.37E-01 1.53E-02 2.10E-03 4.61E-08 HP35_LC3+HC8 2.35E+05 1.86E-01 3.22E-02 2.01E-03 4.65E-08 HP35_LC3+HC6 2.48E+05 1.97E-01 3.07E-02 2.03E-03 4.92E-08 HP35_LC3+HC3 2.78E+05 2.56E-01 3.23E-02 1.96E-03 5.28E-08 HP35_LC2+HC2 1.18E+06 7.23E-01 1.98E-02 1.90E-03 5.36E-08 HP35_LC3+HC2 2.42E+05 2.14E-01 2.95E-02 2.03E-03 5.68E-08 HP35_LC1+HC1 7.39E+05 4.67E-01 2.55E-02 2.58E-03 5.81E-08 HP35_LC1+HC5 2.16E+06 3.30E-01 3.68E-03 2.69E-03 6.46E-08 HP35_LC1+HC7 1.45E+06 7.26E-01 1.55E-02 2.69E-03 7.37E-08 HP35_LC1+HC3 1.41E+06 6.89E-01 1.40E-02 2.62E-03 7.69E-08 HP35_LC1+HC2 1.30E+06 6.64E-01 1.51E-02 2.88E-03 8.15E-08 HP35_LC1+HC6 1.33E+06 7.09E-01 1.52E-02 2.74E-03 8.16E-08 HP35_LC1+HC8 9.10E+05 5.87E-01 1.75E-02 3.01E-03 9.50E-08 HP35_LC1+HC4 1.28E+06 6.77E-01 1.41E-02 3.08E-03 9.51E-08

[0371] To evaluate the long-term stability of the rehumanized anti-polySia antibodies under accelerated storage conditions, the antibody solutions were stored in a temperature-controlled incubator at 37°C. Samples were taken out at different times and analyzed for integrity by HPLC. As shown in Table 3, after 3 weeks at 37°C, most of the rehumanized polySia antibodies were 76% to 95% intact. Table 3 sample start Finish HP35_LC3+HC4 99.3 95.3 HP35_LC3+HC6 99.2 94.7 HP35_LC3+HC7 99.3 94.7 HP35_LC3+HC5 99.3 94.7 HP35_LC3+HC8 98.6 94.2 HP35_LC3+HC3 99.3 83.5 HP35_LC2+HC7 99.1 83.0 HP35_LC2+HC6 99.1 82.9 HP35_LC3+HC2 99.3 82.5 HP35_LC3+HC1 98.7 81.9 HP35_LC2+HC8 98.4 81.8 HP35_LC1+HC7 99.3 81.5 HP35_LC2+HC2 99.2 81.5 HP35_LC1+HC8 99.0 81.4 HP35_LC2+HC1 98.4 81.4 HP35_LC1+HC5 99.5 81.0 HP35_LC1+HC3 99.4 80.9 HP35_LC1+HC6 99.3 80.9 HP35_LC1+HC4 99.3 80.4 HP35_LC2+HC5 99.2 79.6 HP35_LC2+HC4 99.0 79.3 HP35_LC2+HC3 99.0 79.2 HP35_LC1+HC2 99.3 78.7 HP35 Chimeric 98.8 78.5 HP35_LC1+HC1 97.8 76.0

[0372] The binding of various rehumanized anti-polySia antibodies of the present technology to M14 melanoma cells was determined by flow cytometry. Fig.67 Shown are the mean fluorescence intensities (MFI) of the re-humanized anti-poly-Sia antibodies of the present technology. Fig.68 Shown is a heat map comparing the antigen binding properties and stability of various re-humanized anti-poly-Sia antibodies of the present technology relative to the chimeric HP35 poly-Sia antibody.

[0373] These results demonstrate that the anti-poly-Sia antibodies or antigen-binding fragments of the present technology can be used in methods for detecting polysialic acid with a high degree of polymerization (high-DP poly-Sia) in biological samples and methods for detecting poly-Sia-positive cancers in subjects in need thereof. Example 12: Evaluation of the therapeutic effect of rehumanized anti-poly-Sia antibodies

[0374] The same PRIT protocol described in this paper will be used to test Figure 14(A) and 14(B) Eight additional heavy chain variants (87%-88% human germline content) and three additional light chain variants (92% human germline content) as shown in were tested for the efficacy of the polySia x DOTA BsAb. It is expected that tumor-bearing animals receiving the variant anti-polySia antibodies disclosed herein will exhibit a reduction in tumor volume.

[0375] These results will demonstrate that the immunoglobulin-related compositions disclosed herein can be used in methods for detecting and treating poly-Sia-positive cancers in subjects in need thereof. Equivalent

[0376] The present technology is not limited to the specific embodiments described in the application, and the embodiments are intended to be a single description of the individual aspects of the present technology. It is clear to those skilled in the art that various modifications and changes can be made to the present technology without departing from the spirit and scope of the present technology. It will be clear to those skilled in the art according to the foregoing description that, in addition to the methods and devices listed herein, functionally equivalent methods and devices within the scope of the present technology. Such modifications and changes are intended to fall within the scope of the present technology. It should be understood that the present technology is not limited to specific methods, reagents, compounds, compositions or biological systems, and of course they can change. It should also be understood that the terms used herein are only used for the purpose of describing specific embodiments, and are not intended to be restrictive.

[0377] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0378] It will be understood by those skilled in the art that for any and all purposes, particularly with respect to providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily identified as fully describing the same range and enabling the same range to be decomposed into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be easily decomposed into a lower third, a middle third, and an upper third, etc. As will be understood by those skilled in the art, all such as "up to", "at least", "greater than", "less than", etc., include the numbers and relate to the ranges that can subsequently be decomposed into sub-ranges as described above. Finally, it will be understood by those skilled in the art that a range includes each individual member. Thus, for example, a group having 1-3 cells refers to a group having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to a group having 1, 2, 3, 4, or 5 cells, etc.

[0379] All patents, patent applications, pending applications, and publications mentioned or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are consistent with the explicit teachings of this specification.

[0380] The present disclosure relates to the following embodiments. 1. A method comprising a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domain (V L ) or an antigen-binding fragment thereof, wherein: (a) V H A V comprising a V selected from DYYIH (SEQ ID NO: 1), RYYIH (SEQ ID NO: 7), GYYIH (SEQ ID NO: 8) and NYYIH (SEQ ID NO: 9) H -CDR1 sequence selected from WIYPGSGNTKYNEKFKG (SEQ ID NO: 2), SIYPGSGNTKYNEKFKG (SEQ ID NO: 10), RIYPGSGNTKYNEKFKG (SEQ ID NO: 11), CIYPGSGNTKYNEKFKG (SEQ ID NO: 12), WIYPGSGNTKYNEKFEG (SEQ ID NO: 12) NO:13), WIYPGSGNTKYNQKFQG (SEQ ID NO:14), WIYPGSGNTKYSQKFQG (SEQ ID NO:15), WIYPGSGNTKYSEKFQG (SEQ ID NO:16) and WIYPGSGNTKYSQKFKG (SEQ ID NO:18) H-CDR2 sequence and V of GGKFAMDY (SEQ ID NO: 3) H - CDR3; and / or (b) V L A V comprising a sequence selected from RSSQSLVHSNGNTYLY (SEQ ID NO: 4) and RSSQSLVHSNGKTYLY (SEQ ID NO: 20) L - CDR1 sequence, V of RVSNRFS (SEQ ID NO: 5) L - CDR2 sequence and a V selected from FQGTHVPYT (SEQ ID NO: 6), FQGTHVPYI (SEQ ID NO: 21) and FQGTHEPYT (SEQ ID NO: 22) L -CDR3 sequence. 2. The antibody or antigen-binding fragment according to claim 1, further comprising an Fc domain of an isotype selected from IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD and IgE. 3. The antibody according to item 2, comprising an IgG1 constant region comprising one or more amino acid substitutions selected from N297A and K322A. 4. The antibody according to item 2, comprising an IgG4 constant region comprising an S228P mutation. 5. The antigen-binding fragment according to item 1, wherein the antigen-binding fragment is selected from Fab, F(ab')2, Fab', scF v and F v . 6. The antibody or antigen-binding fragment according to any one of items 1 to 5, wherein the antibody or antigen-binding fragment is bound to polysialic acid having a high degree of polymerization (high-DP poly-Sia). 7. The antibody according to any one of items 1-4 or 6, wherein the antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody or a bispecific antibody. 8. An antibody comprising a heavy chain (HC) amino acid sequence comprising SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, or a variant thereof having one or more conservative amino acid substitutions, and / or a heavy chain (HC) amino acid sequence comprising SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:60, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85 The light chain (LC) amino acid sequence of NO:84 or a variant thereof having one or more conservative amino acid substitutions. 9. The antibody according to item 8, comprising a HC amino acid sequence and a LC amino acid sequence respectively selected from the following: a) SEQ ID NO: 23 and SEQ ID NO: 24 (chimeric P35); b) SEQ ID NO: 25 and SEQ ID NO: 28 (humanized P35 H1L2); c) SEQ ID NO: 25 and SEQ ID NO: 27 (humanized P35 H1L1); d) SEQ ID NO: 26 and SEQ ID NO: 28 (humanized P35 H2L2); e) SEQ ID NO: 26 and SEQ ID NO: 27 (humanized P35 H2L1); f) SEQ ID NO:48 and SEQ ID NO:49 (BC137); g) SEQ ID NO:51 and SEQ ID NO:52 (BC137 KS2); h) SEQ ID NO:53 and SEQ ID NO:54 (BC137 KS34); i) SEQ ID NO:55 and SEQ ID NO:49 (BC137 DS47); j) SEQ ID NO:56 and SEQ ID NO:49 (BC137 DS54); k) SEQ ID NO:57 and SEQ ID NO:49 (BC137 DS47 D31R); l) SEQ ID NO:58 and SEQ ID NO:49 (BC137 DS54 D31R); m) SEQ ID NO:55 and SEQ ID NO:50 (BC137-2 DS47); n) SEQ ID NO:56 and SEQ ID NO:50 (BC137-2 DS54); o) SEQ ID NO: 59 and SEQ ID NO: 60 (BC163), p) SEQ ID NO: 74 and SEQ ID NO: 82 (rehumanized P35H1L1); q) SEQ ID NO: 75 and SEQ ID NO: 82 (rehumanized P35H2L1); r) SEQ ID NO:76 and SEQ ID NO:82 (rehumanized P35H3L1); s) SEQ ID NO: 77 and SEQ ID NO: 82 (rehumanized P35H4L1); t) SEQ ID NO: 78 and SEQ ID NO: 82 (rehumanized P35H5L1); u) SEQ ID NO: 79 and SEQ ID NO: 82 (rehumanized P35H6L1); v) SEQ ID NO: 80 and SEQ ID NO: 82 (rehumanized P35H7L1); w) SEQ ID NO:81 and SEQ ID NO:82 (rehumanized P35H8L1); x) SEQ ID NO: 74 and SEQ ID NO: 83 (rehumanized P35H1L2); y) SEQ ID NO: 75 and SEQ ID NO: 83 (rehumanized P35H2L2); z) SEQ ID NO: 76 and SEQ ID NO: 83 (rehumanized P35H3L2); aa) SEQ ID NO:77 and SEQ ID NO:83 (rehumanized P35H4L2); bb) SEQ ID NO:78 and SEQ ID NO:83 (rehumanized P35H5L2); cc) SEQ ID NO:79 and SEQ ID NO:83 (rehumanized P35H6L2); dd) SEQ ID NO: 80 and SEQ ID NO: 83 (rehumanized P35H7L2); ee) SEQ ID NO:81 and SEQ ID NO:83 (rehumanized P35H8L2); ff) SEQ ID NO: 74 and SEQ ID NO: 84 (rehumanized P35H1L3); gg) SEQ ID NO: 75 and SEQ ID NO: 84 (rehumanized P35H2L3); hh) SEQ ID NO:76 and SEQ ID NO:84 (rehumanized P35H3L3); ii) SEQ ID NO: 77 and SEQ ID NO: 84 (rehumanized P35H4L3); jj) SEQ ID NO:78 and SEQ ID NO:84 (rehumanized P35H5L3); kk) SEQ ID NO: 79 and SEQ ID NO: 84 (rehumanized P35H6L3); 11) SEQ ID NO:80 and SEQ ID NO:84 (rehumanized P35H7L3); and mm) SEQ ID NO:81 and SEQ ID NO:84 (rehumanized P35H8L3). 10. An antibody comprising (a) a light chain immunoglobulin variable domain sequence that is at least 95% identical to the light chain immunoglobulin variable domain sequence present in any one of SEQ ID NO:37, 39, 41, 43, 44, 45, 47, 24, 27, 28, 49, 50, 52, 54, 60, 82, 83 or 84; and / or (b) a heavy chain immunoglobulin variable domain sequence that is at least 95% identical to the heavy chain immunoglobulin variable domain sequence present in any one of SEQ ID NO:30, 31, 32, 33, 34, 35, 36, 38, 40, 42, 46, 23, 25, 26, 29, 48, 51, 53, 55, 56, 57, 58, 59, 74, 75, 76, 77, 78, 79, 80 or 81. 11. An antibody comprising (a) an LC sequence that is at least 95% identical to the LC sequence present in any one of SEQ ID NO: 24, 27, 28, 49, 50, 52, 54, 60, 82, 83 or 84; and / or (b) a HC sequence that is at least 95% identical to the HC sequence present in any one of SEQ ID NO: 23, 25, 26, 29, 48, 51, 53, 55, 56, 57, 58, 59, 74, 75, 76, 77, 78, 79, 80 or 81. 12. The antibody according to any one of items 8 to 11, wherein the antibody is a chimeric antibody, a humanized antibody or a bispecific antibody. 13. An antibody according to any one of items 8-12, wherein the antibody binds to a high DP epitope comprising at least three consecutive sialic acid residues. 14. The antibody according to any one of items 8 to 13, wherein the antibody comprises an IgG1 constant region comprising one or more amino acid substitutions selected from N297A and K322A. 15. An antibody according to any one of items 8-13, wherein the antibody comprises an IgG4 constant region comprising an S228P mutation. 16. A recombinant nucleic acid sequence encoding the antibody according to any one of items 8 to 15. 17. A recombinant nucleic acid sequence selected from SEQ ID NO: 92-108. 18. A host cell or vector comprising the recombinant nucleic acid sequence according to item 16 or item 17. 19. A composition comprising an antibody or antigen-binding fragment according to any one of items 1-7 and a pharmaceutically acceptable carrier, wherein the antibody or antigen-binding fragment is optionally conjugated to an agent selected from the following: an isotope, a dye, a chromogen, a contrast agent, a drug, a toxin, a cytokine, an enzyme, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA or any combination thereof. 20. A composition comprising an antibody according to any one of items 8-15 and a pharmaceutically acceptable carrier, wherein the antibody is optionally conjugated to an agent selected from the following: an isotope, a dye, a chromogen, a contrast agent, a drug, a toxin, a cytokine, an enzyme, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA or any combination thereof. 21. The antibody according to any one of items 1-4, 6 or 7, wherein the antibody lacks α-1,6-fucose modification. 22. The antibody according to any one of items 8-15, wherein the antibody lacks α-1,6-fucose modification. 23. The bispecific antibody of item 7 or 12, wherein the bispecific antibody binds to T cells, B cells, bone marrow cells, plasma cells or mast cells. 24. The bispecific antibody of claim 7 or 12, wherein the bispecific antibody binds to CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, TCRγ / δ, NKp46, KIR or a small molecule DOTA hapten. 25. A method for treating polysialic acid (polySia)-related cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of an antibody comprising an HC amino acid sequence and an LC amino acid sequence, wherein the HC amino acid sequence and the LC amino acid sequence each comprise a sequence selected from the group consisting of: a) SEQ ID NO: 23 and SEQ ID NO: 24 (chimeric P35); b) SEQ ID NO: 25 and SEQ ID NO: 28 (humanized P35 H1L2); c) SEQ ID NO: 25 and SEQ ID NO: 27 (humanized P35 H1L1); d) SEQ ID NO: 26 and SEQ ID NO: 28 (humanized P35 H2L2); e) SEQ ID NO: 26 and SEQ ID NO: 27 (humanized P35 H2L1); f) SEQ ID NO:48 and SEQ ID NO:49 (BC137); g) SEQ ID NO:51 and SEQ ID NO:52 (BC137 KS2); h) SEQ ID NO:53 and SEQ ID NO:54 (BC137 KS34); i) SEQ ID NO:55 and SEQ ID NO:49 (BC137 DS47); j) SEQ ID NO:56 and SEQ ID NO:49 (BC137 DS54); k) SEQ ID NO:57 and SEQ ID NO:49 (BC137 DS47 D31R); l) SEQ ID NO:58 and SEQ ID NO:49 (BC137 DS54 D31R); m) SEQ ID NO:55 and SEQ ID NO:50 (BC137-2 DS47); n) SEQ ID NO:56 and SEQ ID NO:50 (BC137-2 DS54); o) SEQ ID NO: 59 and SEQ ID NO: 60 (BC163); p) SEQ ID NO: 74 and SEQ ID NO: 82 (rehumanized P35H1L1); q) SEQ ID NO: 75 and SEQ ID NO: 82 (rehumanized P35H2L1); r) SEQ ID NO:76 and SEQ ID NO:82 (rehumanized P35H3L1); s) SEQ ID NO: 77 and SEQ ID NO: 82 (rehumanized P35H4L1); t) SEQ ID NO: 78 and SEQ ID NO: 82 (rehumanized P35H5L1); u) SEQ ID NO: 79 and SEQ ID NO: 82 (rehumanized P35H6L1); v) SEQ ID NO: 80 and SEQ ID NO: 82 (rehumanized P35H7L1); w) SEQ ID NO:81 and SEQ ID NO:82 (rehumanized P35H8L1); x) SEQ ID NO: 74 and SEQ ID NO: 83 (rehumanized P35H1L2); y) SEQ ID NO: 75 and SEQ ID NO: 83 (rehumanized P35H2L2); z) SEQ ID NO: 76 and SEQ ID NO: 83 (rehumanized P35H3L2); aa) SEQ ID NO:77 and SEQ ID NO:83 (rehumanized P35H4L2); bb) SEQ ID NO:78 and SEQ ID NO:83 (rehumanized P35H5L2); cc) SEQ ID NO:79 and SEQ ID NO:83 (rehumanized P35H6L2); dd) SEQ ID NO: 80 and SEQ ID NO: 83 (rehumanized P35H7L2); ee) SEQ ID NO:81 and SEQ ID NO:83 (rehumanized P35H8L2); ff) SEQ ID NO: 74 and SEQ ID NO: 84 (rehumanized P35H1L3); gg) SEQ ID NO: 75 and SEQ ID NO: 84 (rehumanized P35H2L3); hh) SEQ ID NO:76 and SEQ ID NO:84 (rehumanized P35H3L3); ii) SEQ ID NO: 77 and SEQ ID NO: 84 (rehumanized P35H4L3); jj) SEQ ID NO:78 and SEQ ID NO:84 (rehumanized P35H5L3); kk) SEQ ID NO: 79 and SEQ ID NO: 84 (rehumanized P35H6L3); 11) SEQ ID NO:80 and SEQ ID NO:84 (rehumanized P35H7L3); and mm) SEQ ID NO: 81 and SEQ ID NO: 84 (heavy humanized P35H8L3), wherein the antibody specifically binds to poly-Sia. 26. The method of claim 25, wherein the poly-Sia-associated cancer is small cell or non-small cell lung cancer, neuroblastoma, pancreatic cancer, pituitary tumor, Wilms' tumor, rhabdomyosarcoma, glioblastoma, breast cancer or acute myeloid leukemia. 27. A method according to item 25 or 26, wherein the antibody and the additional therapeutic agent are administered to the subject separately, sequentially or simultaneously. 28. The method of claim 27, wherein the additional therapeutic agent is one or more of the following: an alkylating agent, a platinum agent, a taxane, a vinca agent, an anti-estrogen drug, an aromatase inhibitor, an ovarian inhibitor, a VEGF / VEGFR inhibitor, an EGF / EGFR inhibitor, a PARP inhibitor, a cytostatic alkaloid, a cytotoxic antibiotic, an antimetabolite, an endocrine / hormonal agent, a bisphosphonate therapeutic agent. 29. A method for detecting a tumor in a subject in vivo, the method comprising (a) administering to the subject an effective amount of an antibody according to any one of items 8 to 15, wherein The antibody is configured to localize to tumors expressing polysialic acid with a high degree of polymerization (high-DP poly-Sia) and is labeled with a radioisotope; and (b) detecting the presence of a tumor in the subject by detecting a level of radioactivity emitted by the antibody above a reference value. 30. A method according to claim 29, wherein the subject is diagnosed with or suspected of having cancer. 31. A method according to item 29 or 30, wherein the level of radioactivity emitted by the antibody is detected using positron emission tomography or single photon emission computed tomography. 32. The method according to any one of items 29-31, further comprising administering to the subject an effective amount of an immunoconjugate comprising the antibody according to any one of items 8-15 conjugated to a radionuclide. 33. A method according to claim 32, wherein the radionuclide is an alpha particle emitting isotope, a beta particle emitting isotope, an Auger emitter or any combination thereof. 34. A method according to claim 33, wherein the isotope emitting beta particles is selected from 86 Y. 90 Y. 89 Sr. 165 Dy, 186 Re, 188 Re, 177 Lu and 67 Cu. 35. A kit comprising the antibody according to any one of items 1-15 and instructions for use. 36. A kit according to item 35, wherein the antibody according to any one of items 1-15 is coupled to at least one detectable label selected from a radioactive label, a fluorescent label and a chromogenic label. 37. The kit according to item 35 or 36, further comprising a secondary antibody that specifically binds to the antibody according to any one of items 1-15. 38. The bispecific antibody of claim 7 or 12, wh...

Claims

1. An anti-high DP poly-Sia antibody or an antigen-binding fragment thereof, comprising a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domain (V L ),in: (a) V H V comprising RYYIH (SEQ ID NO: 7) H -CDR1 sequence, V of WIYPGSGNTKYNEKFKG (SEQ ID NO: 2) H -CDR2 sequence and V of GGKFAMDY (SEQ ID NO: 3) H -CDR3 sequence, and the V L V comprising RSSQSLVHSNGNTYLY (SEQ ID NO: 4) L -CDR1 sequence, V of RVSNRFS (SEQ ID NO: 5) L -CDR2 sequence and V of FQGTHVPYT (SEQ ID NO: 6) L - CDR3 sequence, or (b) V H V comprising GYYIH (SEQ ID NO: 8) H -CDR1 sequence, V of SIYPGSGNTKYNEKFKG (SEQ ID NO: 10) H -CDR2 sequence and V of GGKFAMDY (SEQ ID NO: 3) H -CDR3 sequence, and the V L V comprising RSSQSLVHSNGNTYLY (SEQ ID NO: 4) L -CDR1 sequence, V of RVSNRFS (SEQ ID NO: 5) L -CDR2 sequence and V of FQGTHVPYT (SEQ ID NO: 6) L - CDR3 sequence, or (c) V H V comprising NYYIH (SEQ ID NO: 9) H -CDR1 sequence, V of RIYPGSGNTKYNEKFKG (SEQ ID NO: 11) H -CDR2 sequence and V of GGKFAMDY (SEQ ID NO: 3) H -CDR3 sequence, and the V L V comprising RSSQSLVHSNGNTYLY (SEQ ID NO: 4) L -CDR1 sequence, V of RVSNRFS (SEQ ID NO: 5) L -CDR2 sequence and V of FQGTHVPYT (SEQ ID NO: 6) L - CDR3 sequence, or (d) V H V comprising GYYIH (SEQ ID NO: 8) H -CDR1 sequence, V of CIYPGSGNTKYNEKFKG (SEQ ID NO: 12) H -CDR2 sequence and V of GGKFAMDY (SEQ ID NO: 3) H -CDR3 sequence, and the V L V comprising RSSQSLVHSNGNTYLY (SEQ ID NO: 4) L -CDR1 sequence, V of RVSNRFS (SEQ ID NO: 5) L -CDR2 sequence and V of FQGTHVPYT (SEQ ID NO: 6) L - CDR3 sequence, or (e) V H V comprising DYYIH (SEQ ID NO: 1) H -CDR1 sequence, V of RIYPGSGNTKYNEKFKG (SEQ ID NO: 11) H -CDR2 sequence and V of GGKFAMDY (SEQ ID NO: 3) H -CDR3 sequence, and the V L V comprising RSSQSLVHSNGNTYLY (SEQ ID NO: 4) L -CDR1 sequence, V of RVSNRFS (SEQ ID NO: 5) L -CDR2 sequence and V of FQGTHVPYT (SEQ ID NO: 6) L - CDR3 sequence, or (f) V H V comprising NYYIH (SEQ ID NO: 9) H -CDR1 sequence, V of CIYPGSGNTKYNEKFKG (SEQ ID NO: 12) H -CDR2 sequence and V of GGKFAMDY (SEQ ID NO: 3) H -CDR3 sequence, and the V L V comprising RSSQSLVHSNGNTYLY (SEQ ID NO: 4) L -CDR1 sequence, V of RVSNRFS (SEQ ID NO: 5) L -CDR2 sequence and V of FQGTHVPYT (SEQ ID NO: 6) L - CDR3 sequence, or (g) V H V comprising DYYIH (SEQ ID NO: 1) H -CDR1 sequence, V of WIYPGSGNTKYNEKFEG (SEQ ID NO: 13) H -CDR2 sequence and V of GGKFAMDY (SEQ ID NO: 3) H -CDR3 sequence, and the V L V comprising RSSQSLVHSNGNTYLY (SEQ ID NO: 4) L -CDR1 sequence, V of RVSNRFS (SEQ ID NO: 5) L -CDR2 sequence and V of FQGTHVPYI (SEQ ID NO: 21) L - CDR3 sequence, or (h) V H V comprising DYYIH (SEQ ID NO: 1) H -CDR1 sequence, V of CIYPGSGNTKYNEKFKG (SEQ ID NO: 12) H -CDR2 sequence and V of GGKFAMDY (SEQ ID NO: 3) H -CDR3 sequence, and the V L V comprising RSSQSLVHSNGNTYLY (SEQ ID NO: 4) L -CDR1 sequence, V of RVSNRFS (SEQ ID NO: 5) L -CDR2 sequence and V of FQGTHVPYT (SEQ ID NO: 6) L - CDR3 sequence, or (i) V H V comprising DYYIH (SEQ ID NO: 1) H -CDR1 sequence, V of WIYPGSGNTKYNEKFKG (SEQ ID NO: 2) H -CDR2 sequence and V of GGKFAMDY (SEQ ID NO: 3) H -CDR3 sequence, and the V L V comprising RSSQSLVHSNGKTYLY (SEQ ID NO: 20) L -CDR1 sequence, V of RVSNRFS (SEQ ID NO: 5) L -CDR2 sequence and V of FQGTHVPYT (SEQ ID NO: 6) L - CDR3 sequence, or (j) V H V comprising DYYIH (SEQ ID NO: 1) H -CDR1 sequence, V of WIYPGSGNTKYNEKFKG (SEQ ID NO: 2) H -CDR2 sequence and V of GGKFAMDY (SEQ ID NO: 3) H -CDR3 sequence, and the V L V comprising RSSQSLVHSNGNTYLY (SEQ ID NO: 4) L -CDR1 sequence, V of RVSNRFS (SEQ ID NO: 5) L -CDR2 sequence and V of FQGTHEPYT (SEQ ID NO: 22) L - CDR3 sequence, or (k) V H V comprising RYYIH (SEQ ID NO: 7) H -CDR1 sequence, V of RIYPGSGNTKYNEKFKG (SEQ ID NO: 11) H -CDR2 sequence and V of GGKFAMDY (SEQ ID NO: 3) H -CDR3 sequence, and the V L V comprising RSSQSLVHSNGNTYLY (SEQ ID NO: 4) L -CDR1 sequence, V of RVSNRFS (SEQ ID NO: 5) L -CDR2 sequence and V of FQGTHVPYT (SEQ ID NO: 6) L - CDR3 sequence, or (l) V H V comprising DYYIH (SEQ ID NO: 1) H -CDR1 sequence, V of WIYPGSGNTKYNQKFQG (SEQ ID NO: 14) H -CDR2 sequence and V of GGKFAMDY (SEQ ID NO: 3) H -CDR3 sequence, and the V L V comprising RSSQSLVHSNGNTYLY (SEQ ID NO: 4) L -CDR1 sequence, V of RVSNRFS (SEQ ID NO: 5) L -CDR2 sequence and V of FQGTHVPYT (SEQ ID NO: 6) L - CDR3 sequence, or (m) V H V comprising DYYIH (SEQ ID NO: 1) H -CDR1 sequence, V of WIYPGSGNTKYSQKFQG (SEQ ID NO: 15) H -CDR2 sequence and V of GGKFAMDY (SEQ ID NO: 3) H -CDR3 sequence, and the V L V comprising RSSQSLVHSNGNTYLY (SEQ ID NO: 4) L -CDR1 sequence, V of RVSNRFS (SEQ ID NO: 5) L -CDR2 sequence and V of FQGTHVPYT (SEQ ID NO: 6) L - CDR3 sequence, or (n) V H V comprising DYYIH (SEQ ID NO: 1) H -CDR1 sequence, V of WIYPGSGNTKYSEKFQG (SEQ ID NO: 16) H -CDR2 sequence and V of GGKFAMDY (SEQ ID NO: 3) H -CDR3 sequence, and the V L V comprising RSSQSLVHSNGNTYLY (SEQ ID NO: 4) L -CDR1 sequence, V of RVSNRFS (SEQ ID NO: 5) L -CDR2 sequence and V of FQGTHVPYT (SEQ ID NO: 6) L - CDR3 sequence, or (o) V H V comprising DYYIH (SEQ ID NO: 1) H -CDR1 sequence, V of WIYPGSGNTKYSQKFKG (SEQ ID NO: 18) H -CDR2 sequence and V of GGKFAMDY (SEQ ID NO: 3) H -CDR3 sequence, and the V L V comprising RSSQSLVHSNGNTYLY (SEQ ID NO: 4) L -CDR1 sequence, V of RVSNRFS (SEQ ID NO: 5) L -CDR2 sequence and V of FQGTHVPYT (SEQ ID NO: 6) L - CDR3 sequence; or (p) V H V comprising NYYIH (SEQ ID NO: 9) H -CDR1 sequence, V of RIYPGSGNTKYNEKFKG (SEQ ID NO: 11) H -CDR2 sequence and V of GGKFAMDY (SEQ ID NO: 3) H -CDR3 sequence, and the V L V comprising RSSQSLVHSNGNTYLY (SEQ ID NO: 4) L -CDR1 sequence, V of RVSNRFS (SEQ ID NO: 5) L -CDR2 sequence and V of FQGTHVPYT (SEQ ID NO: 6) L - CDR3 sequence, The antibody or antigen-binding fragment thereof is bound to polysialic acid with a high degree of polymerization (high DP poly-Sia).

2. The anti-high-DP poly-Sia antibody or antigen-binding fragment thereof according to claim 1, further comprising an Fc domain of an isotype selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD and IgE, wherein: IgG1 comprises one or more amino acid substitutions selected from the group consisting of N297A and K322A; or IgG4 contains the S228P mutation, or The antibody lacks α-1,6-fucose modification.

3. The anti-high DP poly-Sia antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is a monoclonal antibody, a chimeric antibody or a humanized antibody, or the antigen-binding fragment is selected from Fab, F(ab')2, Fab', scF v and F v Composed of groups.

4. An anti-high-DP poly-Sia antibody or an antigen-binding fragment thereof, comprising: A heavy chain immunoglobulin variable domain and a light chain immunoglobulin variable domain that are 100% identical to a high DP poly-Sia heavy chain immunoglobulin variable domain sequence and a high DP poly-Sia light chain immunoglobulin variable domain sequence, wherein the high DP poly-Sia heavy chain immunoglobulin variable domain sequence and the high DP poly-Sia light chain immunoglobulin variable domain sequence are respectively present in: a) SEQ ID NO:81 and SEQ ID NO:84 (rehumanized P35H8L3); b) SEQ ID NO: 25 and SEQ ID NO: 28 (humanized P35 H1L2); c) SEQ ID NO: 25 and SEQ ID NO: 27 (humanized P35 H1L1); d) SEQ ID NO: 26 and SEQ ID NO: 28 (humanized P35 H2L2); e) SEQ ID NO: 26 and SEQ ID NO: 27 (humanized P35 H2L1); f) SEQ ID NO:48 and SEQ ID NO:49 (BC137); g) SEQ ID NO:51 and SEQ ID NO:52 (BC137 KS2); h) SEQ ID NO:53 and SEQ ID NO:54 (BC137 KS34); i) SEQ ID NO:55 and SEQ ID NO:49 (BC137 DS47); j) SEQ ID NO:56 and SEQ ID NO:49 (BC137 DS54); k) SEQ ID NO:57 and SEQ ID NO:49 (BC137 DS47 D31R); l) SEQ ID NO:58 and SEQ ID NO:49 (BC137 DS54 D31R); m) SEQ ID NO:55 and SEQ ID NO:50 (BC137-2 DS47); n) SEQ ID NO:56 and SEQ ID NO:50 (BC137-2 DS54); o) SEQ ID NO: 59 and SEQ ID NO: 60 (BC163), p) SEQ ID NO: 74 and SEQ ID NO: 82 (rehumanized P35H1L1); q) SEQ ID NO: 75 and SEQ ID NO: 82 (rehumanized P35H2L1); r) SEQ ID NO:76 and SEQ ID NO:82 (rehumanized P35H3L1); s) SEQ ID NO: 77 and SEQ ID NO: 82 (rehumanized P35H4L1); t) SEQ ID NO: 78 and SEQ ID NO: 82 (rehumanized P35H5L1); u) SEQ ID NO: 79 and SEQ ID NO: 82 (rehumanized P35H6L1); v) SEQ ID NO: 80 and SEQ ID NO: 82 (rehumanized P35H7L1); w) SEQ ID NO:81 and SEQ ID NO:82 (rehumanized P35H8L1); x) SEQ ID NO: 74 and SEQ ID NO: 83 (rehumanized P35H1L2); y) SEQ ID NO: 75 and SEQ ID NO: 83 (rehumanized P35H2L2); z) SEQ ID NO: 76 and SEQ ID NO: 83 (rehumanized P35H3L2); aa) SEQ ID NO:77 and SEQ ID NO:83 (rehumanized P35H4L2); bb) SEQ ID NO:78 and SEQ ID NO:83 (rehumanized P35H5L2); cc) SEQ ID NO:79 and SEQ ID NO:83 (rehumanized P35H6L2); dd) SEQ ID NO: 80 and SEQ ID NO: 83 (rehumanized P35H7L2); ee) SEQ ID NO:81 and SEQ ID NO:83 (rehumanized P35H8L2); ff) SEQ ID NO: 74 and SEQ ID NO: 84 (rehumanized P35H1L3); gg) SEQ ID NO: 75 and SEQ ID NO: 84 (rehumanized P35H2L3); hh) SEQ ID NO:76 and SEQ ID NO:84 (rehumanized P35H3L3); ii) SEQ ID NO: 77 and SEQ ID NO: 84 (rehumanized P35H4L3); jj) SEQ ID NO:78 and SEQ ID NO:84 (rehumanized P35H5L3); kk) SEQ ID NO:79 and SEQ ID NO:84 (rehumanized P35H6L3); and 11) SEQ ID NO:80 and SEQ ID NO:84 (rehumanized P35H7L3).

5. The anti-high-DP poly-Sia antibody or antigen-binding fragment thereof according to claim 4, further comprising an Fc domain of an isotype selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD and IgE, wherein: IgG1 comprises one or more amino acid substitutions selected from the group consisting of N297A and K322A; or IgG4 contains the S228P mutation, or The antibody lacks α-1,6-fucose modification.

6. The anti-high DP poly-Sia antibody or antigen-binding fragment thereof according to claim 4, wherein the antibody is a monoclonal antibody, a chimeric antibody or a humanized antibody, or the antigen-binding fragment is selected from Fab, F(ab')2, Fab', scF v and F v The group composed of.

7. A recombinant nucleic acid sequence encoding the anti-high-DP poly-Sia antibody or its antigen-binding fragment according to claim 1 or 4.

8. A host cell or vector comprising the recombinant nucleic acid according to claim 7.

9. A composition comprising the anti-high-DP poly-Sia antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier.

10. The composition of claim 9, wherein the anti-high-DP poly-Sia antibody or antigen-binding fragment thereof is optionally conjugated to an agent selected from the group consisting of an isotope, a dye, a chromogen, a contrast agent, a drug, a toxin, a cytokine, an enzyme, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA, or any combination thereof.

11. A method for detecting a tumor in a subject in vivo, comprising (a) administering to the subject an effective amount of an anti-high-DP poly-Sia antibody or an antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the antibody is configured to localize to a tumor expressing polysialic acid with a high degree of polymerization (high-DP poly-Sia) and is labeled with a radioactive isotope; and (b) detecting the presence of a tumor in the subject by detecting a level of radioactivity emitted by the antibody that is above a reference value, wherein The subject is diagnosed with or suspected of having cancer, or The level of radioactivity emitted by the antibody is detected using positron emission tomography or single photon emission computed tomography.

12. The method of claim 11, further comprising administering to the subject an effective amount of an immunoconjugate comprising the anti-high-DP poly-Sia antibody or antigen-binding fragment thereof according to any one of claims 1-6 conjugated to a radionuclide.

13. The method of claim 12, wherein the radionuclide is an alpha-emitting isotope, a beta-emitting isotope, an Auger emitter, or any combination thereof, wherein the beta-emitting isotope is selected from 86 Y. 90 Y. 89 Sr. 165 Dy, 186 Re, 188 Re, 177 Lu and 67 Cu.

14. A kit comprising the anti-high-DP poly-Sia antibody or antigen-binding fragment thereof according to any one of claims 1-6 and instructions for use.

15. The kit according to claim 14, wherein the antibody is coupled to at least one detectable label selected from the group consisting of a radioactive label, a fluorescent label and a chromogenic label. The kit according to claim 14 or 15 , further comprising a secondary antibody that specifically binds to the antibody.

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