De-N-acetylated polysialic acid (dPSA) binding agents and methods of use thereof

Abstract

dPSA binding agents, immunoglobulin heavy and light chain polypeptides thereof, and methods of using the dPSA binding agents to treat cancer and kill cancer cells.

Description

[Background technology]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims the benefit of U.S. Provisional Patent Application No. 63 / 299,841, filed January 14, 2022, and U.S. Provisional Patent Application No. 63 / 299,843, filed January 14, 2022, which are incorporated by reference in their entireties.

[0002] Incorporation by Reference of Electronically Submitted Materials Incorporated herein by reference in its entirety is a computer readable nucleotide / amino acid sequence listing, which was submitted contemporaneously herewith and is identified as follows: one 56,304 byte XML file entitled "514163.xml", created on January 13, 2023.

[0003] 2. Background of the Invention In general, the goal of anti-cancer immunotherapy is to identify stable antigens that are not excreted or secreted by tumor cells but are highly expressed, and this antigen can be used as the basis of immunotherapy, for example, as antigens for cancer vaccines or as targets for antibody-based cancer therapy.Optimally, such tumor antigens are tolerably specific to cancer target cells, so as to reduce the harmful side effects that may result from cross-reactivity with non-cancerous cells of the subject being treated.If cross-reactivity affects repopulating cells, the specificity requirements of immunotherapy may be relaxed.

[0004] Altered glycosylation patterns of cell surface proteins occur in almost all types of cancer. Hypersialylation of glycoproteins and glycolipids is central to the aberrant regulation of cell adhesion in metastatic cancers, which in turn arises from the re-expression and / or overexpression of genes that are not expressed in cells of adult normal tissues, but are normally expressed during development. In particular, poly-α2→8 N-acetylneuraminic acid or polysialic acid (polySia) is expressed primarily during fetal development and is restricted to very few regenerating tissues after development. The de-N-acetylated form of polysialic acid (dPSA), present on the surface of cancer cells but not on the surface of human cells after development, serves as a tumor antigen for cancer identification and treatment.

[0005] Therefore, new agents capable of binding to cells expressing dPSA are needed. Summary of the Invention

[0006] Provided herein are dPSA binding agents that include immunoglobulin heavy and light chain polypeptides. Also provided herein are methods of using the dPSA binding agents to treat cancer, kill cancer cells, and deliver payloads to cells that express dPSA.

[0007] As will become apparent from the detailed description that follows, related compositions and methods are also provided. [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 is a graph comparing the mean fluorescence intensity (MFI) of the SAC-1 antibody with the concentration of the SEAM3 reference antibody, demonstrating that the test antibody does not bind to the same epitope as the reference antibody. [Diagram 2] FIG. 2 is a graph comparing the mean fluorescence intensity (MFI) of the SAC-2 antibody with the concentration of the SEAM3 reference antibody, demonstrating that the test antibody does not bind to the same epitope as the reference antibody. [Diagram 3]3A and 3B show the proteins co-precipitated by SAC-1, SAC-2 and a control IgG1 antibody on SDS-PAGE. [Figure 4] Figures 4A, 4B and 4C show SAC-3 staining of normal human breast tissue (Figure 4A) and breast tumor (Figure 4C) compared to staining of the tumor with a control IgG2a antibody (Figure 4B). [Diagram 5] Figures 5A, 5B, and 5C are graphs showing the amount of SAC-1 antibody (Figure 5A) or SAC-2 antibody (Figure 5B) versus the relative light units (RLU) indicating the ADCC of each antibody against various cell lines, and the quantification values ​​(Figure 5C). [Figure 6] Figures 6A and 6B are graphs plotting cytotoxicity versus concentration of afucosylated SAC-2.1C and SAC-2.1D (referred to as "SAC-2.1CaFUC" and "SAC-2.1DaFUC," respectively) showing the effect of afucosylated antibodies on ADCC activity against human A375 melanoma (Figure 6A) and MDA-MB-231 breast cancer (Figure 6B) cell lines. [Figure 7] 7A and 7B show the dose-dependence of SAC-1.1 (FIG. 7A) and SAC-2 (FIG. 7B) treatment on tumor growth in the A375 xenograft mouse model of human melanoma. [Figure 8] Figures 8A and 8B show the effects of SAC-1.1, murine SAC-2, and SAC-2.1C on tumor growth compared to vehicle control and cyclophosphamide treatment in the MDA-MB-231 xenograft mouse model of human breast cancer (Figure 8A), as well as the effect of the addition of human PBMCs in combination with SAC-2.1C on tumor growth (Figure 8B). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Detailed Description of the Invention dPSA is a de-N-acetylated form of polysialic acid (dPSA) that is found on the surface of cancer cells but not other non-cancerous post-emergence human cells (Granoff et al., J. Immunol.160(1):5028-36 (1998); Moe et al., J. Immunol.182(10):6610-7 (2009); Moe et al., Infect Immunol.73(4):2123-8 (2005); Moe et al., J. Exp.Clin.Cancer Res.40(1):293 (2021); Steirer et al., PLoS ONE 6:e27249 (2011)). Because cell surface dPSA is unique to cancer cells and is widely expressed across different cancers, agents that preferentially bind to cells expressing dPSA can be used to target cancer cells for both diagnostics and therapeutics.

[0010] Humans have two genes, ST8SIA2 and ST8SIA4, that code for enzymes that synthesize polysialic acid (polysialyltransferases ST8SIA2 and ST8SIA4, respectively). Both genes are highly expressed during fetal development in humans (Angata et al., J. Biol.Chem., 272(11): 7182-90 (1997)), but ST8SIA4 is expressed primarily in lymphoid tissues and lymphocytes (Drake et al., PNAS 106(29):11995-2000 (2009)); ST8SIA2 does not appear to be present at significant levels in any adult normal tissues based on Northern blots (Angata et al., J. Biol.Chem., 272(11): 7182-90 (1997)).

[0011] Several proteins have been identified as polysialylated in humans (e.g., Curreli et al., J. Biol.Chem., 282(42):30346-56 (2007); Finne et al., Biochem.Biophys.Res. Commun.112(2):482-7 (1983); Simon et al., J. Biol.Chem., 288(26):18825-33 (2013); Werneburg et al., Glia, 64(8):1314-30 (2016); Werneburg et al., Glia, 63(7):1240-55 (2015); Yabe et al., J. Biol.Chem., 278(16):13875-80 (2003)). Neural cell adhesion molecules (NCAMs) are the most abundant and most thoroughly studied, especially during fetal development (Rutishauser, U., Nat'l Rev. Neurosci., 9(1):26-35 (2008)). Many human cancers have been reported to aberrantly express polysialic acid NCAM (Amoureaux et al., BMC Cancer, 10: 91 (2010); Gluer et al., Pediatr.Res. 43(1):145-7 (1998); Roth et al., Am.J. Pathol.133(2):227-40 (1988); Tanaka et al., Cancer Res. 60(11): 3072-80 (2000)), and its role in mediating cell-cell and cell-extracellular matrix interactions has been associated with metastasis and poor clinical prognosis (Amoureaux et al., BMC Cancer, 10: 91 (2010); Tanaka et al., Cancer Res. 60(11): 3072-80 (2000)). Recently, we identified nucleolin as a protein that modifies or associates with dPSA and showed that cell surface dPSA depends on ST8SIA2 expression (Moe et al., J. Exp. Clin. Cancer Res. 40(1):293 (2021)).

[0012] Provided herein are binding agents (e.g., antibodies or antibody fragments) that selectively bind to cells expressing dPSA, particularly cancer cells. In some embodiments, the dPSA binding agent binds to nucleolin modified with dPSA. Without wishing to be bound by any particular theory or mechanism of action, it is believed that the binding agent binds to an antigen (e.g., nucleolin) that comprises an epitope that is at least partially defined by one or more dPSA residues. Thus, the binding agent is hereinafter referred to as a "dPSA binding agent."

[0013] The dPSA binding agents provided herein comprise Ig heavy and light chain polypeptides, each polypeptide comprising at least an Ig heavy chain variable region and an Ig light chain variable region, respectively. Each of the Ig heavy chain variable region and the Ig light chain variable region comprises three complementarity determining regions (CDRs), which are usually referred to as CDR1, CDR2, or CDR3, in order. The CDR regions can also be referred to using "H" or "L" in the nomenclature to indicate heavy or light chain, respectively, i.e., CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3. The CDRs of a given Ig sequence can be determined by any of several conventional numbering schemes, such as Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo (these are commonly used names for numbering schemes that are widely known in the art and described in published literature, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH (1991), describing the "Kabat" numbering scheme; Chothia et al., Canonical Structures for the Hypervariable Regions of Immunoglobulins, J. Mol.Biol., 196:901-917 (1987) and Al-Lazikani et al., Standard Conformations for the Canonical Structures of Immunoglobulins, J. Mol.Biol., 273:927 - 948 (1997), describing the "Chothia" numbering scheme; The "Chothia" numbering scheme is described in Abhinandan et al., Analysis and Improvements to Kabat and Structurally Correct Numbering of Antibody Variable Domains, Mol. Immunol., 45: 3832 - 3839 (2008); see Lefranc et al., The IMGT unique numbering for immunoglobulins, T cell Receptors and Ig-like domains, The Immunologist, 7: 132-136 (1999) and Lefranc et al., IMGT unique numbering for immunoglobulin and T cell receptor variable domains and I superfamily V-like domains, Dev.Comp. Immunol., 27:55-77 (2003), describing the "IMGT" numbering scheme; and Honegger et al., Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool, J. Mol.Biol. 309: 657 - 670 (2001), describing the "AHo" numbering scheme. Identification of CDRs can also be performed through relevant empirical binding data, such as crystallographic studies of the interaction of a binding agent with its target (e.g., an antigen or portion thereof that contains the binding epitope), optionally in combination with any of the aforementioned numbering systems.

[0014] The dPSA binding agents provided herein are artificial and do not exist in nature.They are produced by laboratory techniques and therefore are properly considered to be recombinant or synthetic molecules that contain recombinant or synthetic amino acid sequences.Ig heavy and light chain polypeptides can be "isolated" in the sense that they are removed from the environment in which they are produced (e.g., cell culture) and purified to any degree.

[0015] In some embodiments, the dPSA binding agent comprises an Ig heavy chain variable region comprising any of SEQ ID NOs: 1-4 or at least the CDRs thereof; and an Ig light chain variable region comprising SEQ ID NO: 5 or at least the CDRs thereof. The CDRs may be determined using any known numbering scheme, such as Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo. In some embodiments, the antibody comprises a heavy chain variable region of any of SEQ ID NOs: 1-4 and a light chain variable region of SEQ ID NO: 5, or at least the CDRs thereof as determined by Kabat. In some embodiments, the antibody comprises a heavy chain variable region of any of SEQ ID NOs: 1-4 and a light chain variable region of SEQ ID NO: 5, or at least the CDRs thereof as determined by Chothia. In some embodiments, the antibody comprises a heavy chain variable region of any of SEQ ID NOs: 1-4 and a light chain variable region of SEQ ID NO: 5, or at least the CDRs thereof as determined by Martin. In some embodiments, the antibody comprises a heavy chain variable region of any of SEQ ID NOs: 1-4 and a light chain variable region of SEQ ID NO: 5, or at least the CDRs thereof as determined by IGMT. In some embodiments, the antibody comprises a heavy chain variable region of any of SEQ ID NOs: 1-4 and a light chain variable region of SEQ ID NO: 5, or at least the CDRs thereof as determined by AHo. In some embodiments, the dPSA binding agent comprises any of the following combinations of Ig heavy chain variable regions and light chain variable regions, or at least the CDRs thereof as determined by any of Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo:

[0016] JPEG2025502369000001.jpg31140

[0017] In some embodiments, the dPSA binding agent comprises an Ig heavy chain variable region comprising any of SEQ ID NOs: 17-20 or at least the CDRs thereof; and an Ig light chain variable region comprising SEQ ID NO: 21 or at least the CDRs thereof. The CDRs may be determined using any known numbering scheme, such as Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo. In some embodiments, the antibody comprises a heavy chain variable region of any of SEQ ID NOs: 17-20 and a light chain variable region of SEQ ID NO: 21, or at least the CDRs thereof as determined by Kabat. In some embodiments, the antibody comprises a heavy chain variable region of any of SEQ ID NOs: 17-20 and a light chain variable region of SEQ ID NO: 21, or at least the CDRs thereof as determined by Chothia. In some embodiments, the antibody comprises a heavy chain variable region of any of SEQ ID NOs: 17-20 and a light chain variable region of SEQ ID NO: 21, or at least the CDRs thereof as determined by Martin. In some embodiments, the antibody comprises a heavy chain variable region of any of SEQ ID NOs:17-20 and a light chain variable region of SEQ ID NO:21, or at least the CDRs thereof as determined by IGMT. In some embodiments, the antibody comprises a heavy chain variable region of any of SEQ ID NOs:17-20 and a light chain variable region of SEQ ID NO:21, or at least the CDRs thereof as determined by AHo. In some embodiments, the dPSA binding agent comprises any of the following combinations of Ig heavy chain variable regions and light chain variable regions, or at least the CDRs thereof as determined by any of Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo:

[0018] JPEG2025502369000002.jpg52135

[0019] In some embodiments, provided herein is a dPSA binding agent comprising an Ig heavy chain variable region and an Ig light chain variable region, wherein the Ig heavy chain variable region comprises a CDR1 comprising any one of SEQ ID NOs:6-9 or 24-27, a CDR2 comprising SEQ ID NO:10 or 28, and a CDR3 comprising SEQ ID NO:11 or 29; and the Ig light chain variable region comprises a CDR1 comprising SEQ ID NO:12 or 30, a CDR2 comprising SEQ ID NO:13(RMS) or 31, and a CDR3 comprising SEQ ID NO:14 or 32.

[0020] In some embodiments, provided herein is a dPSA binding agent comprising an Ig heavy chain variable region and an Ig light chain variable region, wherein the Ig heavy chain variable region comprises a CDR1 comprising any one of SEQ ID NOs:6-9, a CDR2 comprising SEQ ID NO:10, and a CDR3 comprising SEQ ID NO:11; and the Ig light chain variable region comprises a CDR1 comprising SEQ ID NO:12, a CDR2 comprising SEQ ID NO:13, and a CDR3 comprising SEQ ID NO:14.

[0021] JPEG2025502369000003.jpg46160

[0022] In some embodiments, provided herein is a dPSA binding agent comprising an Ig heavy chain variable region and an Ig light chain variable region, wherein the Ig heavy chain variable region comprises a CDR1 comprising any one of SEQ ID NOs:24-27, a CDR2 comprising SEQ ID NO:28, and a CDR3 comprising SEQ ID NO:29; and the Ig light chain variable region comprises a CDR1 comprising SEQ ID NO:30, a CDR2 comprising SEQ ID NO:31, and a CDR3 comprising SEQ ID NO:32.

[0023] JPEG2025502369000004.jpg83160

[0024] According to yet another embodiment of the present disclosure, the dPSA binding agent comprises an Ig heavy chain variable region comprising an amino acid sequence having at least 80% sequence identity (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to any of SEQ ID NOs. 1-4; and The Ig light chain variable region comprises an amino acid sequence having at least 80% sequence identity to NO:5 (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%).In some embodiments, the dPSA binding agent is an Ig light chain polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO:15 (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%); and and an Ig light chain polypeptide comprising an amino acid sequence having at least 80% sequence identity (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to SEQ ID NO:16. In any of the foregoing embodiments, the Ig heavy chain variable region and the Ig light chain variable region can include (retain) the CDRs of the heavy and light chain variable regions of the sequence, which may be determined using any known numbering scheme, such as Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo, or as otherwise defined herein (e.g., SEQ ID NOs:6-14, above). In some embodiments, the Ig heavy chain variable region comprises one of SEQ ID NOs: 1-4, and the Ig light chain variable region comprises SEQ ID NO: 5. In some embodiments, the dPSA binding agent comprises Ig heavy and light chain polypeptides comprising SEQ ID NO: 15 and SEQ ID NO: 16, respectively.

[0025] According to yet another embodiment of the present disclosure, the dPSA binding agent comprises an Ig heavy chain variable region comprising an amino acid sequence having at least 80% sequence identity (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to any of SEQ ID NOs. 17-20; and and an Ig light chain variable region comprising an amino acid sequence having at least 80% sequence identity to NO:21 (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%).In some embodiments, the dPSA binding agent is an Ig heavy chain polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO:22 (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%); and SEQ ID NO: 23 having at least 80% sequence identity (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%). In any of the foregoing embodiments, the Ig heavy chain variable region and the Ig light chain variable region can include (retain) the CDRs of the heavy chain variable region and the light chain variable region, which CDRs may be determined using any known numbering scheme, such as Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo, or as otherwise defined herein (e.g., SEQ ID NOs:6-14 or 24-32, above). In some embodiments, the Ig heavy and light chain variable regions each comprise one of SEQ ID NOs:17-20, and the Ig light chain variable region each comprises SEQ ID NO:21. In some embodiments, the dPSA binding agent comprises an Ig heavy and light chain polypeptide comprising SEQ ID NO:22 and SEQ ID NO:23, respectively.

[0026] In some embodiments, the dPSA binding agent comprises Ig heavy and light chain polypeptides comprising SEQ ID NOs: 33 and 34, respectively.

[0027] In some embodiments, the dPSA binding agent comprises an Ig heavy chain variable region comprising SEQ ID NO: 35 or at least its CDRs; and an Ig light chain variable region comprising SEQ ID NO: 36 or at least its CDRs. The CDRs may be determined using any known numbering scheme, such as Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo. In some embodiments, the antibody comprises a heavy chain variable region of SEQ ID NO: 35 and a light chain variable region of SEQ ID NO: 36, or at least its CDRs as determined by Kabat. In some embodiments, the antibody comprises a heavy chain variable region of SEQ ID NO: 35 and a light chain variable region of SEQ ID NO: 36, or at least its CDRs as determined by Chothia. In some embodiments, the antibody comprises a heavy chain variable region of SEQ ID NO: 35 and a light chain variable region of SEQ ID NO: 36, or at least its CDRs as determined by Martin. In some embodiments, the antibody comprises a heavy chain variable region of SEQ ID NO: 35 and a light chain variable region of SEQ ID NO: 36, or at least the CDRs thereof as determined by IGMT. In some embodiments, the antibody comprises a heavy chain variable region of SEQ ID NO: 35 and a light chain variable region of SEQ ID NO: 36, or at least the CDRs thereof as determined by AHo.

[0028] In some embodiments, the dPSA binding agent comprises an Ig heavy chain variable region comprising SEQ ID NO:51 or at least the CDRs thereof; and an Ig light chain variable region comprising SEQ ID NO:52 or 53 or at least the CDRs thereof. The CDRs may be determined using any known numbering scheme, such as Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo. In some embodiments, the antibody comprises a heavy chain variable region of SEQ ID NO:51 and a light chain variable region of SEQ ID NO:52 or 53, or at least the CDRs thereof as determined by Kabat. In some embodiments, the antibody comprises a heavy chain variable region of SEQ ID NO:51 and a light chain variable region of SEQ ID NO:52 or 53, or at least the CDRs thereof as determined by Chothia. In some embodiments, the antibody comprises a heavy chain variable region of SEQ ID NO:51 and a light chain variable region of SEQ ID NO:52 or 53, or at least the CDRs thereof as determined by Martin. In some embodiments, the antibody comprises a heavy chain variable region of SEQ ID NO:51 and a light chain variable region of SEQ ID NO:52 or 53, or at least the CDRs thereof as determined by IGMT. In some embodiments, the antibody comprises a heavy chain variable region of SEQ ID NO:51 and a light chain variable region of SEQ ID NO:52 or 53, or at least the CDRs thereof as determined by AHo.

[0029] In some embodiments, provided herein is a dPSA binding agent comprising an Ig heavy chain variable region and an Ig light chain variable region, wherein the Ig heavy chain variable region comprises a CDR1 comprising SEQ ID NO: 39 or 45, a CDR2 comprising SEQ ID NO: 40 or 46, and a CDR3 comprising SEQ ID NO: 41 or 47; and the Ig light chain variable region comprises a CDR1 comprising SEQ ID NO: 42 or 48, a CDR2 comprising SEQ ID NO: 43(GTN), 49, or 56, and a CDR3 comprising SEQ ID NO: 44 or 50. In some embodiments, the dPSA binding agent comprises a CDR1 comprising SEQ ID NO: 39, a CDR2 comprising SEQ ID NO: 40, and a CDR3 comprising SEQ ID NO: 41 or 47; and the Ig light chain variable region comprises a CDR1 comprising SEQ ID NO: 42, a CDR2 comprising SEQ ID NO: 43 or 56, and a CDR3 comprising SEQ ID NO: 44.

[0030] In some embodiments, provided herein is a dPSA binding agent comprising an Ig heavy chain variable region and an Ig light chain variable region, wherein the Ig heavy chain variable region comprises a CDR1 comprising SEQ ID NO: 45, a CDR2 comprising SEQ ID NO: 46, and a CDR3 comprising SEQ ID NO: 41 or 47; and the Ig light chain variable region comprises a CDR1 comprising SEQ ID NO: 48, a CDR2 comprising SEQ ID NO: 49 or 56, and a CDR3 comprising SEQ ID NO: 50.

[0031] JPEG2025502369000005.jpg103160

[0032] In some embodiments, the dPSA binding agent comprises an Ig heavy chain variable region comprising an amino acid sequence having at least 80% sequence identity (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to SEQ ID NO:35; and and an Ig light chain variable region comprising an amino acid sequence having at least 80% sequence identity to NO:36 (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%).In some embodiments, the dPSA binding agent is an Ig heavy chain polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO:37 (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%); and and an Ig light chain polypeptide comprising an amino acid sequence having at least 80% sequence identity (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to SEQ ID NO:38. In any of the foregoing embodiments, the Ig heavy chain variable region and the Ig light chain variable region can comprise (retain) the CDRs of the heavy chain variable region and light chain variable region of SEQ ID NO:35 and 36, respectively, which CDRs may be determined using any known numbering scheme, such as Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo, or as otherwise defined herein (e.g., SEQ ID NO:39-44). In some embodiments, the Ig heavy and light chain variable regions comprise SEQ ID NO: 35 and SEQ ID NO: 36, respectively. In some embodiments, the dPSA binding agent comprises Ig heavy and light chain polypeptides comprising SEQ ID NO: 37 and SEQ ID NO: 38, respectively.

[0033] In some embodiments, the dPSA binding agent comprises an Ig heavy chain variable region comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO:51 (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%); and and an Ig light chain variable region comprising an amino acid sequence having at least 80% sequence identity (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to NO:52 or 53.In some embodiments, the dPSA binding agent is an Ig heavy chain polypeptide having at least 80% sequence identity to SEQ ID NO:51 (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%); and and an Ig light chain polypeptide comprising an amino acid sequence having at least 80% sequence identity (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to SEQ ID NO:52 or 53. In any of the foregoing embodiments, the Ig heavy chain variable region and the Ig light chain variable region can comprise (retain) the CDRs of the heavy chain variable region and light chain variable region of SEQ ID NO:51 and 52 or 53, respectively, which CDRs can be determined using any known numbering scheme, such as Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo, or as otherwise defined herein. In some embodiments, the Ig heavy and light chain variable regions comprise SEQ ID NO:51 and SEQ ID NO:52 or 53, respectively.

[0034] In some embodiments, the dPSA binding agent comprises an Ig heavy chain variable region comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO:57 (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%); and and an Ig light chain variable region comprising an amino acid sequence having at least 80% sequence identity (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to NO:58 or 59.In some embodiments, the dPSA binding agent is a heavy chain polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO:57 (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%); and and an Ig light chain polypeptide comprising an amino acid sequence having at least 80% sequence identity (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to SEQ ID NO:58 or 59. In any of the foregoing embodiments, the Ig heavy chain variable region and the Ig light chain variable region can comprise (retain) the CDRs of the heavy chain variable region and light chain variable region of SEQ ID NO:57 and 58 or 59, respectively, which CDRs can be determined using any known numbering scheme, such as Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo, or as otherwise defined herein. In some embodiments, the Ig heavy and light chain variable regions comprise SEQ ID NO:57 and SEQ ID NO:58 or 59, respectively.

[0035] In some embodiments, the dPSA binding agent has an Ig heavy and light chain variable region comprising SEQ ID NOs:33 and 34, or at least the CDRs thereof as determined using any known numbering scheme, such as Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo; and / or at least 80% sequence identity (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to SEQ ID NOs:33 or 34, and optionally retains the CDRs thereof. In some embodiments, the dPSA binding agent comprises an Ig heavy chain variable region and a light chain variable region comprising at least the CDRs thereof as determined using SEQ ID NOs:54 and 55, or any known numbering scheme such as Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo; and / or has at least 80% sequence identity (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to SEQ ID NOs:54 or 55, and optionally retains the CDRs thereof.

[0036] The "identity" of a sequence, as used in reference to a nucleic acid or amino acid sequence, can be determined by comparing a nucleic acid or amino acid sequence of interest to a reference nucleic acid or amino acid sequence. The percent identity is the percentage of nucleotides or amino acid residues that are the same (i.e., identical) between the sequence of interest and the reference sequence when optimally aligned. Many mathematical algorithms are known and publicly available for obtaining optimal alignment and calculating the identity between two or more sequences. Examples of such programs include CLUSTAL-W, T-Coffee, and ALIGN (for aligning nucleic acid and amino acid sequences), BLAST programs (e.g., BLAST 2.1, BL2SEQ, and later versions thereof operated by the National Center for Biotechnology Information, Bethesda, MD), and FASTA programs (e.g., FASTA3x, FASTM, and SSEARCH) (for sequence alignment and sequence similarity searching). Sequence alignment algorithms are also disclosed, for example, in Altschul et al., J. Molecular Biol:215(3):403-410(1990); Beigert et al., Proc.Natl. Acad.Sci. USA, 106(10):3770-3775 (2009); Durbin et al., eds., Biological Sequence Analysis:Probalistic Models of Proteins and Nucleic Acids, Cambridge University Press, Cambridge, UK (2009); Soding, Bioinformatics, 21(7):951-960 (2005); Altschul et al., Nucleic Acids Res., 25(17):3389-3402 (1997); Gusfield, Algorithms on Strings, Trees and Sequences, Cambridge University Press, Cambridge UK (1997).

[0037] For sequences with less than 100% identity to the heavy and light chain sequences specifically defined above, one or more amino acids of the aforementioned immunoglobulin heavy and / or light chain polypeptides can be replaced or substituted with a different amino acid, and / or one or more amino acids can be deleted from or inserted into the disclosed amino acid sequences, provided that the biological activity of the polypeptide (e.g., the ability of the dPSA binder to bind to dPSA) is substantially retained. The biological activity of a dPSA binder can be measured, for example, by binding affinity to a particular dPSA epitope and / or cross-reactivity with targets other than dPSA. The aforementioned properties or characteristics can be observed, measured, and / or assessed using standard techniques, including, but not limited to, ELISA, competitive ELISA, surface plasmon resonance analysis (BIACORE™), or solution phase competition (KINEXA™), as well as other in vitro or in vivo neutralization assays, binding assays, fluorescence activated cell binding (FACS), or other suitable assays.

[0038] A dPSA binding agent may be part of a multispecific (e.g., bispecific or "dual reactive") construct (e.g., a multispecific antibody such as a bispecific or dual reactive antibody) that binds dPSA and another antigen. Such a construct may include a combination of immunoglobulin heavy and light chain polypeptides that bind dPSA as described herein with immunoglobulin heavy and light chains from an immunoglobulin that binds an antigen other than dPSA.

[0039] The dPSA binding agent can be part of a conjugate.For example, the dPSA binding agent can be a conjugate of (1) an anti-dPSA antibody or a fragment thereof, and (2) a second protein or non-protein moiety.To further illustrate, the dPSA binding agent can include an anti-dPSA antibody or a fragment thereof conjugated with another peptide, a fluorescent molecule, or a chemotherapeutic (e.g., cytotoxic) agent.

[0040] In some embodiments, the dPSA binding agent can be a "whole" immunoglobulin or an antigen-binding immunoglobulin "fragment." A "whole" immunoglobulin typically consists of four polypeptides: two heavy (H) chain polypeptides and two light (L) chain polypeptides. Each heavy chain contains one N-terminal variable region (V H ) and three C-terminal constant regions (C H 1, C H 2, C H 3), each light chain containing one N-terminal variable region (V L ) and one C-terminal constant region (C L ). The light chain of an antibody can be assigned to one of two different types, kappa (κ) or lambda (λ), based on the amino acid sequence of the constant domain. In a typical immunoglobulin, each light chain is linked to a heavy chain by a disulfide bond, and the two heavy chains are linked to each other by disulfide bonds. In this configuration, the variable region of the light chain is generally aligned with the variable region of the heavy chain, and the constant region of the light chain is generally aligned with the first constant region of the heavy chain. The remaining constant regions of the heavy chains are generally aligned with each other.

[0041] The variable or hypervariable regions of each pair of light and heavy chains form the antigen-binding site of an antibody. H and V LThe regions have the same general structure, and each region contains four framework (FW or FR) regions. As used herein, the term "framework region" refers to a relatively conserved amino acid sequence in the variable region located between the hypervariable or complementarity determining regions (CDRs). There are four framework regions in each variable domain, which are designated FR1, FR2, FR3, and FR4. The framework regions form a β-sheet that provides a structural framework for the variable region (see, for example, CA Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, NY (2001)). The framework regions are linked by three complementarity determining regions (CDRs). The three CDRs, known as CDR1, CDR2, and CDR3, form the "hypervariable region" of an antibody and are generally believed to be involved in antigen binding.

[0042] As used herein, the term "antibody fragment" and similar terms (e.g., "fragment of an antibody," "antibody fragment," "functional fragment of an antibody") are used interchangeably to mean one or more fragments or portions of an antibody that retain the ability to specifically bind to an antigen (see generally, Holliger et al., Nat. Biotech:23(9):1126-1129 (2005)). Antibody "fragment," as used herein and routinely in the art, includes not only fragments or portions of whole antibodies in the literal sense, but also other known engineered antibody-like constructs that may contain linkers or other elements not naturally occurring in "whole antibodies." Examples of antibody fragments include: (i) V L , V H , C L (ii) a Fab fragment, which is a monovalent fragment consisting of the CH1 domain; (iii) a F(ab')2 fragment, which is a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; and (iv) a V fragment consisting of a single arm of an antibody. L and V HExamples of single chain antibody fragments include, but are not limited to, Fv fragments consisting of two domains (i.e., V and VFv), (iv) Fab' fragments resulting from cleavage of the disulfide bridges of the F(ab')2 fragment using mild reducing conditions, and (v) disulfide stabilized Fv fragments (dsFv). The dPSA binders can also be single chain antibody fragments. Examples of single chain antibody fragments include (i) single chain Fv (scFv), which consists of two domains (i.e., V and VFv) of the Fv fragment. L and V H ) are monovalent molecules consisting of two domains joined by a synthetic linker that allows the two domains to be synthesized as a single polypeptide chain (e.g., Bird et al., Science, 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA, 85: 5879-5883 (1988); and Osbourn et al., Nat. Biotechnol, 16: 778 (1998)), and (ii) diabodies, which are dimers of polypeptide chains, each of which is composed of a V H and V L V is bound by a peptide linker that is too short to allow pairing with L V connected to H , which allows for different V H -V L These include, but are not limited to, diabodies, which drive pairing between complementary domains on polypeptide chains to generate dimeric molecules with two functional antigen-binding sites.Any other antigen-binding antibody-like constructs known in the art, including Ig heavy and light chain CDRs or variable regions, can also be used, and for the purposes of this disclosure, are antibody fragments.In some embodiments, the dPSA binding agent is a chimeric antigen receptor (or a part thereof).

[0043] In some embodiments, the dPSA binder is fragment crystallizable (F c) region or a portion thereof. The Fc region can be of any Ig class / subclass, including IgA (IgA1, IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3, and IgG4), IgM, and variants thereof. In some embodiments, the dPSA binding agent is a "whole" or "intact" Ig (i.e., antibody); and in other embodiments, the binding agent is an antibody fragment conjugated or linked to an Fc region. In some embodiments, the dPSA binding agent comprises an IgG Fc region, such as an IgG1 or IgG4. For example, the dPSA binding agent can be an IgG1 antibody or an IgG4 antibody. In some embodiments, the dPSA binding agent comprises an Fc region that can mediate complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the dPSA binding agent comprises an Fc region that activates natural killer (NK) cells.

[0044] The dPSA binder may be a human or humanized antibody, a non-human antibody, or a chimeric antibody. By "chimeric" is meant an antibody or fragment thereof that contains both human and non-human regions. Preferably, the dPSA binder is a humanized antibody. A "humanized" antibody is a monoclonal antibody that contains a human antibody scaffold and at least one CDR obtained or derived from a non-human antibody. Non-human antibodies include antibodies isolated from non-human animals, such as rodents (e.g., mice and rats). A humanized antibody may contain one, two, or three CDRs obtained or derived from a non-human antibody.

[0045] Human, non-human, chimeric, and humanized antibodies can be obtained by any means, including through in vitro sources (e.g., hybridomas and cell lines that recombinantly produce antibodies) and in vivo sources (e.g., rodents). Methods for producing antibodies are known in the art, and are described, for example, in Koehler and Milstein, Eur.J. Immunol., 5: 511-519 (1976); Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988); and Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, NY (2001)). In certain embodiments, human or chimeric antibodies can be made using transgenic animals (e.g., mice) in which one or more endogenous immunoglobulin genes have been replaced with one or more human immunoglobulin genes (see, e.g., Lonberg, Nat. Biotechnol., 23(9):1117-25 (2005), and Lonberg, Handb. Exp. Pharmacol., 181: 69-97 (2008)). Humanized antibodies can be made using any suitable method known in the art (see, e.g., An, Z. (ed.), Therapeutic Monoclonal Antibodies: From Bench to Clinic, John Wiley & Sons, Inc., Hoboken, New Jersey (2009)), such as grafting non-human CDRs onto a human antibody scaffold (e.g., Kashmiri et al., Methods, 36(1):25-34(2005); and Hou et al., J. Biochem., 144(1):115-120 (2008)).

[0046] The dPSA binding agent provided herein can be used for any purpose.For example, the dPSA binding agent can be used to target or kill cancer cells that express dPSA (e.g., contain dPSA on the cell surface).Therefore, provided herein is a method for targeting or killing cancer cells in vitro or in vivo, comprising administering the dPSA binding agent described herein to cancer cells.When the method is used to target or kill cancer cells in vivo, the dPSA binding agent can be administered to cancer cells by administering the dPSA binding agent to a subject that contains cancer cells.

[0047] The expressing cancer cell can be any cancer cell that expresses dPSA on the cell surface. For example, the cancer cell can contain a protein such as nucleolin to which dPSA is linked or otherwise associated on the cell surface. In some embodiments, the cancer cell expresses ST8SIA2.

[0048] The method of targeting or killing cancer cells can be used for diagnostic or therapeutic purposes and can be used in vitro, ex vivo, or in vivo. For example, the dPSA binding agent can be attached to a detectable label or support (e.g., radioactive label, fluorescent label, bead, scaffold, etc.) to analyze dPSA expression levels in a biological sample (biological fluid or tissue sample) from a subject, or to facilitate detection of cancer cells expressing dPSA. For example, the detectable moiety can be a radioisotope (e.g., 3 H, 14 C. 32 P, 35 S, or 125I), fluorescent or chemiluminescent compounds (e.g., fluorescein isothiocyanate, rhodamine, luciferin, etc.), enzymes (alkaline phosphatase, β-galactosidase, horseradish peroxidase, etc.), or supports (beads, scaffolds, biosensor surfaces, etc.). Any method known in the art for separately binding antigen-binding agents (e.g., antibodies) to such sites can be employed in the context of the present invention (e.g., Hunter et al., Nature, 194:495-496 (1962); David et al., Biochemistry, 13:1014-1021 (1974); Pain et al., J. Immunol.Meth., 40:219-230 (1981); and Nygren, J. Histochem. and Cytochem., 30:407-412 (1982)).

[0049] Alternatively, dPSA binding agents can be used to kill cancer cells. For example, dPSA binding agents can be conjugated to chemotherapeutic agents (e.g., cytotoxic agents) and used to target and deliver chemotherapeutic agents to cancer cells in expressing dPSA, thereby killing cancer cells in a subject. In other embodiments, dPSA binding agents exhibit antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) and are used to kill cancer cells expressing dPSA by binding to the cancer cells and causing ADCC or CDC-mediated cell death. In some embodiments, dPSA binding agents are afucosylated. Afucosylated binding agents (e.g., antibodies) can be prepared by any suitable technique, such as by expressing nucleic acids encoding the Ig heavy and light chains of dPSA binding agents in cell lines with disrupted or deleted FUT8 gene (e.g., FUT8-deleted CHO cells).

[0050] Thus, the dPSA binding agents provided herein and methods of using same can be used to treat cancers characterized by dPSA surface expression. As used herein, the terms "treatment", "treat" and the like refer to obtaining a desired pharmacological and / or physiological effect, such as reducing the severity or inhibiting the progression of a disease and / or adverse symptoms caused by the disease. To this end, the method of the present invention includes administering a "therapeutically effective amount" of a dPSA binding agent. A "therapeutically effective amount" refers to an amount effective at the dosage and for the period necessary to achieve a desired therapeutic result. A therapeutically effective amount can vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the dPSA binding agent to elicit a desired response in the individual.

[0051] The methods and compositions provided herein are useful in the context of treating or preventing a wide variety of cancers, including carcinomas, sarcomas, leukemias, myelomas, and lymphomas.

[0052] Carcinomas treatable by the methods disclosed herein include, but are not limited to, esophageal carcinoma, hepatocellular carcinoma, basal cell carcinoma (a type of skin cancer), squamous cell carcinoma (various tissues), bladder cancer (including transitional cell carcinoma (malignant neoplasm of the bladder)), bronchial carcinoma, colon carcinoma, colorectal carcinoma, gastric carcinoma, lung cancer (including small cell and non-small cell carcinoma of the lung), adrenal cortical carcinoma, thyroid carcinoma, pancreatic carcinoma, breast cancer, ovarian cancer, prostate cancer, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical carcinoma, uterine carcinoma, testicular carcinoma, osteogenic carcinoma, epithelial carcinoma, and nasopharyngeal carcinoma.

[0053] Sarcomas that may be suitable for treatment with the methods disclosed herein include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovium, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas.

[0054] Other solid tumors that may be suitable for treatment with the methods disclosed herein include, but are not limited to, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, hemangioma, melanoma, neuroblastoma, and retinoblastoma.

[0055] Other cancers include leukemia, lymphoma, and myeloma (including multiple myeloma). Leukemias treatable by the methods disclosed herein include, but are not limited to, a) chronic myeloproliferative syndromes (neoplastic disorders of pluripotent hematopoietic stem cells); b) acute myeloid leukemia (neoplastic transformation of pluripotent hematopoietic stem cells or lineage-restricted hematopoietic cells); c) chronic lymphocytic leukemia (CLL; clonal proliferation of immunologically immature and dysfunctional small lymphocytes, including B-cell CLL, T-cell CLL prolymphocytic leukemia, hairy cell leukemia; and d) acute lymphoblastic leukemia (characterized by accumulation of lymphoblasts). Lymphomas treatable using the subject methods include, but are not limited to, B-cell lymphomas (e.g., Burkitt's lymphoma); Hodgkin's lymphoma; non-Hodgkin's lymphoma, and the like.

[0056] Other cancers treatable according to the methods disclosed herein include atypical meningioma (brain), islet cell carcinoma (pancreas), medullary carcinoma (thyroid), mesenchymal (intestine), hepatocellular carcinoma (liver), hepatoblastoma (liver), clear cell carcinoma (kidney), and neurofibroma mediastinalis.

[0057] Further exemplary cancers that may be suitable for treatment using the methods disclosed herein include, but are not limited to, cancers of neuroectodermal origin and cancers of epithelial origin. Examples of cancers of neuroectodermal origin include, but are not limited to, Ewing's sarcoma, spinal cord tumor, brain tumor, upper ventricular primitive neuroectodermal tumor of childhood, tubulocystic carcinoma, mucinous tubular carcinoma and spindle cell carcinoma, renal tumor, mediastinal tumor, glioma, neuroblastoma, and sarcoma of adolescents and young adults. Examples of epithelial origin include, but are not limited to, small cell lung cancer, breast cancer, eye lens cancer, colon cancer, pancreatic cancer, renal cancer, liver cancer, ovarian cancer, and bronchial epithelial carcinoma. In some embodiments, the subject method does not include treatment of melanoma (i.e., the cancer is other than melanoma). In other embodiments, the subject method does not include treatment of lymphoma (i.e., the cancer is other than lymphoma).

[0058] The dPSA binding agent may be part of a composition suitable for administration to a mammal. Preferably, the composition is a pharma- ceutically acceptable (e.g., physiologically acceptable) composition, which includes a carrier, preferably a pharma-ceutically acceptable (e.g., physiologically acceptable) carrier, and the amino acid sequence, antigen binding agent, or vector of the present invention. In the context of the present invention, any suitable carrier may be used, and such carriers are well known in the art. The choice of carrier is determined, in part, by the particular site to which the composition may be administered and the particular method used to administer the composition. The composition may also include any other excipients used in the formulation of therapeutic molecules (e.g., proteins or antibodies), particularly parenteral formulations, such as buffers, tonicity adjusters, stabilizers, surfactants, and the like. The composition may be sterile. The composition may be frozen or lyophilized for storage and reconstituted in a suitable sterile carrier prior to use. The composition may be produced according to conventional techniques, such as those described in, for example, Remington: The Science and Practice of Pharmacy, 21st Edition, Lippincott Williams & Wilkins, Philadelphia, PA (2001).

[0059] Administration can be performed using any standard administration technique, including oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. Preferably, the composition is suitable for parenteral administration. The term "parenteral" as used herein includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. More preferably, the composition is administered to the mammal using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.

[0060] The dPSA binding agent of the present invention can be administered alone or in combination with other drugs.For example, the dPSA binding agent can be administered in combination with other drugs for the treatment or prevention of diseases disclosed herein, such as other anti-cancer drugs.In this regard, for example, the dPSA binding agent can be used in combination with at least one other drug, including, for example, chemotherapeutic agents, vaccines, biological therapies (e.g., other monoclonal antibodies), radiation therapy, bone marrow transplantation, chemotherapy treatment, biological response modifier treatment, and / or surgery.

[0061] Further provided herein are nucleic acids encoding dPSA binding agents (ie, encoding the immunoglobulin heavy chain polypeptides and / or immunoglobulin light chain polypeptides of a dPSA binding agent).

[0062] The nucleic acid can be a polymer of DNA or RNA (or both, such as hybrid DNA / RNA), can be single-stranded or double-stranded, and can include non-natural or altered nucleotides. The nucleic acid can be part of a vector. The vector can be, for example, a plasmid, an episome, a cosmid, a viral vector (e.g., a retrovirus or an adenovirus), or a phage. Suitable vectors and methods for vector preparation are well known in the art (see, for example, Sambrook et al., Molecular Cloning, a Laboratory Manual, 3rd edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (2001), and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, NY (1994)).

[0063] A vector typically contains expression control sequences, such as promoters, enhancers, polyadenylation signals, transcription terminators, signal peptides (e.g., osteonectin signal peptide), internal ribosome entry sites (IRES), etc., that provide for expression of a coding sequence in a host cell. Exemplary expression control sequences are known in the art and described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, Calif. (1990).

[0064] Numerous promoters, including constitutive, inducible and repressible promoters, are well known in the art from a variety of different sources. Representative sources of promoters include, for example, viruses, mammals, insects, plants, yeast and bacteria, and suitable promoters from these sources are readily available or can be synthetically produced based on publicly available sequences, for example, from depositories such as ATCC and other commercial or individual sources. Promoters can be unidirectional (i.e., initiate transcription in one direction) or bidirectional (i.e., initiate transcription in either 3' or 5' direction). Non-limiting examples of promoters include, for example, T7 bacterial expression system, pBAD (araA) bacterial expression system, cytomegalovirus (CMV) promoter, SV40 promoter, RSV promoter. Inducible promoters include, for example, the Tet system (U.S. Pat. Nos. 5,464,758 and 5,814,618), the Ecdysone-inducible system (No et al., Proc. Natl. Acad. Sci., 93:3346-3351 (1996)), the T-REX™ system (Invitrogen, Carlsbad, Calif.), the LACSWITCH™ system (Stratagene, San Diego, Calif.), and the Cre-ERT tamoxifen-inducible recombinase system (Indra et al., Nuc. Acid. Res., 27: 4324-4327 (1999); Nuc. Acid. Res., 28: e99 (2000); U.S. Pat. No. 7,112,715; and Kramer & Fussenegger, Methods Mol. Biol., 308:123-144(2005)).

[0065] The term "enhancer" as used herein refers to a DNA sequence that, for example, increases the transcription of a nucleic acid sequence to which it is operably linked. Enhancers can be located many kilobases away from the coding region of a nucleic acid sequence and can mediate the binding of regulatory factors, patterns of DNA methylation, or changes in DNA structure. Many enhancers from a variety of different sources are well known in the art and are available as or within cloned polynucleotides (e.g., from depositories such as ATCC, and other commercial or individual sources). Many polynucleotides that contain a promoter (such as the commonly used CMV promoter) also contain enhancer sequences. Enhancers can be located upstream of the coding sequence, within the coding sequence, or downstream of the coding sequence.

[0066] Vectors may also contain a "selectable marker gene." The term "selectable marker gene," as used herein, refers to a nucleic acid sequence that allows cells expressing the nucleic acid sequence to be specifically selected or not selected in the presence of a corresponding selection agent. Suitable selectable marker genes are known in the art and are described, for example, in International Patent Application Publication Nos. WO 1992 / 008796 and WO 1994 / 028143; Wigler et al., Proc. Natl. Acad. Sci. USA, 77:3567-3570 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA, 78:1527-1531 (1981); Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78:2072-2076 (1981); Colberre-Garapin et al., J. Mol. Biol., 150:1-14 (1981); Santerre et al., Gene, 30:147-156 (1984); Kent et al., Science, 237:901-903 (1985). (1987); Wigler et al., Cell, 11: 223-232 (1977); Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA, 48: 2026-2034 (1962); Lowy et al., Cell, 22: 817-823 (1980); and U.S. Patents 5,122,464 and 5,770,359.

[0067] In some embodiments, the vector is an "episomal expression vector" or "episome" and is capable of replicating in a host cell and persisting as an extrachromosomal segment of DNA in the host cell in the presence of appropriate selective pressure (see, e.g., Conese et al., Gene Therapy, 11:1735-1742 (2004)). Representative commercially available episomal expression vectors include, but are not limited to, episomal plasmids that utilize the Epstein-Barr nuclear antigen 1 (EBNA1) and Epstein-Barr virus (EBV) origin of replication (oriP). The vectors pREP4, pCEP4, pREP7, and pcDNA3.1 from Invitrogen (Carlsbad, CA), and pBK-CMV from Stratagene (La Jolla, CA) represent non-limiting examples of episomal vectors that use T-antigen and the SV40 origin of replication in place of EBNA1 and oriP.

[0068] Other suitable vectors include integrative expression vectors that are randomly integrated into the DNA of a host cell or contain recombination sites that allow specific recombination between the expression vector and the host cell chromosome. Such integrative expression vectors can utilize endogenous expression control sequences of the host cell chromosome to provide expression of the desired protein. Examples of site-specifically integrating vectors include, for example, components of the flp-in system (e.g., pcDNA™5 / FRT) from Invitrogen (Carlsbad, CA), or the cre-lox system as found in the pExchange-6 core vector from Stratagene (La Jolla, CA). Examples of vectors that are randomly integrated into the host cell chromosome include, for example, pcDNA3.1 (when introduced in the absence of T-antigen) from Life Technologies (Carlsbad, CA), UCOE from Millipore (Billerica, MA), pCI or pFN10A(ACT)FLEXI™ from Promega (Madison, WI).

[0069] Viral vectors can also be used. Exemplary commercially available viral expression vectors include, but are not limited to, the adenovirus-based Per.C6 system available from Crucell (Leiden, The Netherlands), the lentivirus-based pLP1 available from Invitrogen (Carlsbad, Calif.), and the retroviral vectors pFB-ERV and pCFB-EGSH available from Stratagene (La Jolla, Calif.).

[0070] Nucleic acid sequences encoding the amino acid sequences of the present invention can be provided to the cells on the same vector (i.e., in cis). A unidirectional promoter can be used to control the expression of each nucleic acid sequence. In another embodiment, a combination of bidirectional and unidirectional promoters can be used to control the expression of multiple nucleic acid sequences. Nucleic acid sequences encoding the amino acid sequences of the present invention can alternatively be provided to a population of cells on separate vectors (i.e., in trans). Each nucleic acid sequence of the separate vectors can contain the same or different expression control sequences. The separate vectors can be provided to the cells simultaneously or sequentially.

[0071] A vector containing a nucleic acid encoding the amino acid sequence of the present invention can be introduced into a host cell (including any suitable prokaryotic or eukaryotic cell) capable of expressing the polypeptide encoded thereby.Thus, the present invention provides an isolated cell containing the vector of the present invention.Preferred host cells are cells that can be easily and reliably grown, have a reasonably fast growth rate, have a well-characterized expression system, and can be easily and efficiently transformed or transfected.

[0072] Examples of suitable prokaryotic cells include, but are not limited to, cells of the genera Bacillus (such as Bacillus subtilis and Bacillus brevis), Escherichia (such as E. coli), Pseudomonas, Streptomyces, Salmonella, and Erwinia. Particularly useful prokaryotic cells include various strains of E. coli, such as K12, HB101 (ATCC No. 33694), DH5α, DH10, MC1061 (ATCC No. 53338), and CC102.

[0073] In some embodiments, the vector is introduced into a eukaryotic cell. Suitable eukaryotic cells are known in the art, and include, for example, yeast cells, insect cells, and mammalian cells. Examples of suitable yeast cells include those of the genera Kluyveromyces, Pichia, Rhino-sporidium, Saccharomyces, and Schizosaccharomyces. Preferred yeast cells include, for example, Saccharomyces cerivisae and Pichia pastoris.

[0074] Suitable insect cells are described, for example, in Kitts et al., Biotechniques, 14: 810-817 (1993); Lucklow, Curr. Opin. Biotechnol., 4: 564-572 (1993); and Lucklow et al., J. Virol., 67: 4566-4579 (1993). Preferred insect cells include Sf-9 and HI5 (Invitrogen, Carlsbad, Calif.).

[0075] In some embodiments, mammalian cells are utilized in the present invention. Numerous suitable mammalian host cells are known in the art, and many are available from the American Type Culture Collection (ATCC, Manassas, VA). Examples of suitable mammalian cells include, but are not limited to, Chinese hamster ovary cells (CHO) (e.g., CHO-K1 cells, ATCC No. CCL61), CHO DHFR- cells (Urlaub et al., Proc. Natl. Acad. Sci. USA, 97:4216-4220 (1980)), human embryonic kidney (HEK) 293 or 293T cells (ATCC No. CRL1573), and 3T3 cells (ATCC No. CCL92). Other suitable mammalian cell lines are the monkey COS-1 cell line (ATCC No. CRL1650) and COS-7 cell line (ATCC No. CRL1651), as well as the CV-1 cell line (ATCC No. CCL70). Further exemplary mammalian host cells include primate and rodent cell lines, including transformed cell lines. Normal diploid cells, cell lines derived from in vitro culture of primary tissues, and primary explants are also suitable. Other suitable mammalian cell lines include, but are not limited to, mouse neuroblastoma N2A cells, HeLa, mouse L-929 cells, and BHK or HaK hamster cell lines, all of which are available from ATCC. Methods for selecting suitable mammalian host cells, and for transforming, culturing, amplifying, screening, and purifying cells are known in the art.

[0076] In one embodiment, the mammalian cell is a human cell.For example, the mammalian cell can be a human lymphocyte or lymphocyte-derived cell line, such as a pre-B lymphocyte-derived cell line.Examples of human lymphoid cell lines include, but are not limited to, RAMOS (CRL-1596), Daudi (CCL-213), EB-3 (CCL-85), DT40 (CRL-2111), 18-81 (Jack et al., Proc.Natl. Acad.Sci. USA, 85: 1581-1585 (1988)), Raji cells (CCL-86), PER.C6 cells (Crucell Holland BV, Leiden, The Netherlands), and their derivatives.

[0077] The nucleic acid sequence encoding the amino acid sequence of the present invention can be introduced into a cell by any suitable method, such as "transfection", "transformation" or "transduction". "Transfection", "transformation" or "transduction" as used herein refers to the introduction of one or more exogenous polynucleotides into a host cell using physical or chemical methods. Many suitable techniques are known in the art, such as calcium phosphate DNA co-precipitation (see, for example, Murray EJ (ed.), Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols, Humana Press (1991)); DEAE-dextran; electroporation; cationic liposome-mediated transfection; tungsten particle-facilitated microparticle bombardment (Johnston, Nature, 346:776-777 (1990)); and strontium phosphate DNA co-precipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)). Phage or viral vectors can be introduced into host cells after propagation of the infectious particles in appropriate packaging cells, many of which are commercially available.

[0078] The nucleic acids and cells can be used for any purpose, such as the manufacture of dPSA binders as described herein. In this regard, the present invention provides a method for preparing a dPSA binder, comprising culturing a cell comprising a nucleic acid or nucleic acid sequence encoding the heavy and / or light immunoglobulin polypeptide of the dPSA binder. In other words, the method comprises expressing a nucleic acid encoding the immunoglobulin heavy and / or light chain of the dPSA binder in a cell (e.g., an in vitro cell, such as any of the cell lines discussed herein, including CHO cells and CHO-K1 cells). It will be understood that the immunoglobulin heavy and light chains can be expressed from a single nucleic acid in a given cell, or the immunoglobulin heavy and light chains can be expressed from separate nucleic acids in the same cell. The method may further comprise harvesting and / or purifying the dPSA binder from the cell or cell culture medium using known techniques. EXAMPLES

[0079] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

[0080] Example 1 The following examples demonstrate the selective binding of the dPSA binding agents provided herein to the dPSA antigen.

[0081] Antibodies designated SAC-1 and SAC-2, having the sequences shown below, were expressed as recombinant chimeras with human IgG1 Fc in a CHO cell line that does not express dPSA. The antibodies were tested for specific binding to dPSA by ELISA and cell-based assays. Humanized antibodies were prepared with the same CDRs as the SAC-1 and SAC-2 antibodies. The antibodies were designated SAC-1.1, SAC-2.1 (also called "SAC-2 humanized D"), and SAC-2.2 (also called "SAC-2 humanized C"), and have the sequences shown below.

[0082] JPEG2025502369000006.jpg66161

[0083] Preparation of dPSA antigen The dPSA antigen used in the ELISA was prepared by combining 100 milligrams of colominic acid (MilliporeSigma), 10 milligrams of sodium borohydride (MilliporeSigma) in 10 milliliters of 2 molar sodium and heating at 100°C for 40 minutes. The resulting dPSA was neutralized with 2 M hydrochloric acid, dialyzed twice against 4 L of water, and lyophilized. dPSA (20 milligrams) in 0.75 milliliters of 0.1 M sodium acetate (pH 6.5) was first oxidized with sodium periodate (0.25 milliliters of 10 millimolar periodic acid) in the dark at ambient temperature for 30 minutes. After the addition of 100 microliters of 10% (volume / volume) ethylene glycol, the reaction mixture was dialyzed and lyophilized as above. Twenty milligrams of oxidized dPSA and 10 milligrams of ovalbumin (IMJECT™ OVALBUMIN, Pierce Chemical Co.) were combined in phosphate-buffered saline (PBS) containing approximately 5 milligrams of sodium cyanoborohydride. The solution was stirred overnight at ambient temperature in the dark. The dPSA-ovalbumin conjugate was purified by size-exclusion chromatography using a ToyoPearl HW-65F column in 0.9% (wt / vol) sodium chloride, 10 mM potassium phosphate, pH 7.1. Fractions containing the dPSA-KLH conjugate were concentrated (SpinX, Corning) to 2 milligrams / milliliter of protein and 4 milligrams / milliliter of dPSA with approximately 30% de-N-acetylated residues as measured by a modified resorcinol assay.

[0084] Binding to dPSA antigen The ELISA was performed starting with an antibody concentration of 10 micrograms per ml with eight serial 3-fold dilutions as described in Moe et al., J. Exp. & Clin. Can. Res. 40(1):293 (2021). Specificity for dPSA was determined by performing the same ELISA in the presence of 100 micrograms of polysialic acid (i.e., colominic acid, MilliporeSigma) in the buffer. The results (OD405 of ≥ 0.5 mean fluorescence intensity (MFI) after 30 min) confirmed that the antibody specifically bound to dPSA.

[0085] The antibody was also tested for binding to the human neuroblastoma cell line CHP-134 and the human myeloma cell line NCI-H929 at a fixed concentration of 10 μg / ml by flow cytometry as described by Moe et al., J of Exp & Clin Can Res 2021, 40:293. Briefly, adherent cells were suspended in RPMI 1640 cell culture medium containing 10% (vol / vol) fetal bovine serum (FBS, ThermoFisher Scientific) by pipetting or treatment with Accutase™ (Innovative Cell Technologies). Cells were centrifuged (200xg, 8 min) and the cell pellet was suspended in medium to a concentration of 0.5-2x10^6 cells per ml. Cells and antibodies were placed in 1.5 milliliter Eppendorf tubes and mixed by end over end rotation at ambient temperature for 1 h. The tubes were centrifuged (200xg, 2 min), washed once with fresh medium, and the cells were suspended in medium containing AlexaFluor 488-conjugated goat anti-human F(ab')2 secondary antibody. After 30 min of rotational mixing, the cells were washed once with fresh medium and suspended in PBS buffer containing 0.5% (vol / vol) formaldehyde. Cell fluorescence was measured by flow cytometry (Acea NovoCyte). The results, expressed as mean fluorescence intensity (MFI) relative to an irrelevant human IgG1 negative control antibody (BioXCell), are summarized in Table 1.

[0086] [Table 1]

[0087] Example 2 The following examples show binding of the antibodies provided herein to additional diverse cancer cell lines, but not to CHP-134 neuroblastoma cells in which the polysialyltransferase genes ST8SIA2 and ST8SIA4 have been knocked out (CHP-134 KO). dPSA on the surface of CHP-134 cells is dependent on the expression of ST8SIA2, as described in Moe et al., J of Exp & Clin Can Res 2021, 40:293.

[0088] The antibodies provided in Example 1 were further tested for binding to several additional cell lines following procedures similar to those described in Example 1 for CHP-134 and NCI-H929 cells. Table 2 shows the EC 50 The maximum MFI and maximum MFI values ​​are shown, indicating that the antibody binds to multiple diverse types of cancer cells, and the antibody exhibits significantly enhanced binding compared to the reference anti-dPSA antibody, SEAM 3 (Steirer and Moe et al., PLoS One 6(11): e27249 (2011)).

[0089] [Table 2]

[0090] Example 3 The following examples demonstrate that the antibodies provided herein bind to different epitopes than a reference antibody.

[0091] To determine whether the epitope recognized by the antibody of Example 1 is different from the epitope recognized by the reference anti-dPSA antibody, SEAM 3 (Steirer and Moe et al., PLoS One 6(11): e27249 (2011)), a constant concentration of the antibody 10-fold higher than the EC50 for binding to CHP-134 cells was combined with serial 2-fold dilutions of SEAM 3. As shown in Figures 1 and 2, the antibody of Example 1 did not inhibit binding of SEAM 3, indicating that the antibody recognizes a different epitope.

[0092] Example 4 This example demonstrates that the antibodies provided herein bind to multiple human cancer cell lines.

[0093] Cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and routinely tested for mycoplasma contamination (MycoStripTM, InvivoGen, San Diego, CA). Cell lines were grown in ATCC-recommended media in a humidified chamber with a 5% CO2 atmosphere. Adherent cell lines were suspended by treatment with StemProTM AccutaseTM cell dissociation reagent (Thermo Fisher Scientific, Carlsbad, CA). Suspension cells were diluted 1:5 in cell culture medium, centrifuged (200xg, 10 min), and suspended in fresh medium to a viable cell density of 1–10 million cells / mL. Viability was determined by trypan blue staining (Thermo Fisher Scientific, Carlsbad, CA), and cells were counted using SKC, Inc. C-ChipTM Disposable Hemacytometers (Fisher Scientific, Pittsburgh, PA). Cells and antibodies (SAC-1, SAC-1 humanized (SAC-1.1), SAC-2, SAC-2 mouse (SEQ ID NO: 54 and SEQ ID NO: 55), SAC-2 humanized C (SAC-2.2), and SAC-2 humanized D (SAC-2.1)) were combined in a tube and incubated for 1 hour at ambient temperature with end and end continuous mixing. Cells were centrifuged (200xg, 2 minutes), the supernatant was aspirated, and cells were suspended in medium containing the appropriate (i.e., anti-mouse or anti-human IgG H+L) secondary antibody labeled with Alexa Fluor 488™ (AffiniPure F(ab')2 Fragment Goat Anti-Mouse IgG (H+L), Jackson ImmunoResearch, West Grove, PA). The cells and secondary antibody were incubated for 30 min at ambient temperature with end and end continuous mixing, then centrifuged (200 x g, 2 min), the supernatant was aspirated, and the cells were suspended in phosphate-buffered saline containing 0.5% formaldehyde.

[0094] Finally, cells were analyzed by flow cytometry (Acea NovoCyte, Agilent, Santa Clara, CA or similar). Binding curves of antibody concentration dependence of mean fluorescence intensity (MFI) were analyzed with curve fitting software (GraphPad Prism, San Diego, CA) to determine the binding constant (K D The MFI and maximum MFI (MFImax) were calculated. The results are shown in Table 3 (SAC-1 antibody) and Table 4 (SAC-2 antibody). Neg. means negative, and NA means not applicable. As shown in Tables 3 and 4, the tested antibodies showed excellent activity against multiple cancer cell lines and nanomolar K D The MFImax values ​​vary depending on the epitope density of each cell line. The data indicate that the antigen recognized by the antibody is present in a wide range of human cancers and is dependent on the expression of the polysialyltransferase STSIA2, as knockout of the gene encoding the enzyme in CHP-134 cells abolishes binding.

[0095] [Table 3]

[0096] [Table 4]

[0097] Example 5 This example demonstrates that the antibodies provided herein recognize derivatives of nucleolin, including dPSA.

[0098] Preparation of subcellular fractions: Human melanoma A375 cells (80% confluent in a T-175 flask) were extracted using the ProteoExtract® Subcellular Proteome Extraction Kit (MilliporeSigma). Briefly, the differential detergent extraction method used four extraction buffers in sequence, along with a protease inhibitor cocktail to prevent protein degradation during extraction, and Benzonase® nuclease (Sigma-Aldrich, St. Louis, MO) to degrade contaminating nucleic acids. Extraction was performed according to the manufacturer's instructions, and the cell extract was divided into four fractions: F1 (cytoplasmic fraction), F2 (cell membrane fraction), F3 (nuclear protein fraction), and F4 (cytoskeleton fraction).

[0099] Co-immunoprecipitation: Dynabeads M-270 epoxy magnetic beads (Thermo Fisher Scientific, Carlsbad, CA) covalently coupled with SAC-1, SAC-2, or irrelevant human IgG1 antibodies (BioXCell, Lebanon, NH) were prepared according to the manufacturer's protocol. Antigens reacting with the antibodies were purified by co-immunoprecipitation as follows: F2 membrane fractions were incubated separately with SAC-1, SAC-2, or irrelevant IgG1-conjugated magnetic beads. The beads were separated by a magnet and washed with the respective extraction buffer alone, and then with a buffer containing polysialic acid (polySia) (50 μg / mL; Sigma-Aldrich, Colominic Acid, St. Louis, MO) to remove nonspecifically bound antigens. Finally, antigens were eluted from the beads using SDS-PAGE sample buffer without reducing agent (NuPAGE, Thermo Fisher Scientific) by heating at 80 °C for 10 min. Proteins eluted from the beads were separated by 4%-12% SDS-PAGE (NuPAGE, Thermo Fisher Scientific) and either stained with SimplyBlue® Coomassie stain (Thermo Fisher Scientific) or transferred to PVDF membranes (Immobilon®-FL, Millipore, Waltham, MA) using a NuPAGE transfer cell (Thermo Fisher Scientific) for Western blotting. After blocking the PVDF membranes with 5% (wt / vol) dry whole milk in phosphate-buffered saline (PBS) buffer overnight, the immunoprecipitates were stained with anti-nucleolin antibody MS-3 (Santa Cruz Biotechnology, Santa Cruz, CA) in blocking buffer for 2 h at ambient temperature. After washing three times with PBS buffer, bound antibodies were detected with IRDye® 800CW-conjugated donkey anti-mouse IgG (H+L) secondary antibody (LI-COR, Lincoln, NE). Images of the gel and blots were recorded with an Odyssey® Fc Imaging System (LI-COR), and the results are shown in Figures 3A and 3B.

[0100] Figure 3A shows proteins co-precipitated by each antibody separated by SDS-PAGE. There are multiple bands with similar apparent mass distributions and strong staining co-precipitated by SAC-1 and SAC-2, but not by an irrelevant IgG1 antibody. We previously showed that an antibody that binds de-N-acetylpolysialic acid (dPSA) co-immunoprecipitates dPSA-modified nucleolin from the F2 fraction of cancer cells (J Exp Clin Cancer Res. 2021 Sep 20; 40(1): 293). Cell surface nucleolin is unique to cancer cells and has a range of apparent masses when resolved on SDS-PAGE gels. This is due to multiple post-translational modifications and heterogeneity in the length and charge of dPSA.

[0101] Figure 3B shows Western blot staining of the co-precipitated proteins with anti-nucleolin antibody MS-3. The major band in the SAC-1 and SAC-2 samples (i.e., the band with an apparent mass of 77 kDa) is nucleolin based on its reactivity with MS-3. Post-translational modifications of nucleolin that affect its migration in SDS-PAGE gels may not be detectable by MS-3 made to unmodified recombinant nucleolin. To confirm that other bands with apparent masses different from the major bands detected by MS-3 are also derivatives of nucleolin, slices of the gel indicated in brackets in Figure 5A were excised for each co-immunoprecipitated sample and processed as previously described for analysis by LC-MS / MS mass spectrometry for protein identification (J Exp Clin Cancer Res. 2021 Sep 20; 40(1): 293). LC-MS / MS mass spectrometry confirmed that the portions of the gel analyzed also contained nucleolin.

[0102] Quantification of nucleolin for each sample is also shown in Figure 5A, corresponding to the relative amount of Coomassie staining in that portion of the SDS-PAGE gel for each sample. In summary, the data show that SAC-1 and SAC-2 bind to the same modified form of nucleolin located in the membrane fraction of A375 human melanoma cells, and that that derivative of nucleolin has the characteristics (i.e., the range of apparent masses on SDS-PAGE) that are modified with dPSA as described above.

[0103] Example 6 This example demonstrates that the antibodies provided herein are reactive with human tumor and normal human tissues by immunohistochemistry.

[0104] To examine the specificity of the SAC antibody, which binds to cancer cells but not to normal developmental human tissues, we used SAC-2 to immunohistochemically stain tissue microarrays containing tissue specimens from normal human tissues and tumors.

[0105] SAC-2 mouse monoclonal antibodies (SEQ ID NO: 54 and SEQ ID NO: 55) were used at 2.5 micrograms / milliliter using Tris-based pH 9.5 Heat-Induced Epitope Retrieval; isotype control (mouse IgG2a) was used under the same conditions. Formalin-fixed paraffin-embedded (FFPE) sections were stained on a Biocare intelliPATH automated staining platform (Biocare Medical, Pacheco, CA) using the manufacturer's recommended settings. Sections were incubated with Biocare Peroxidase Blocker (Biocare, Cat. #PX968) and Background Punisher (Biocare, Cat. #BP974M) to block nonspecific background. Mouse primary antibodies were detected using the MACH4 HRP-polymer Detection System (Biocare, Cat. #MRH534). Chromogenic detection and counterstaining kits IntelliPATH FLX DAB chromogen (Biocare, Cat. #IPK5010) and IntelliPATH Hematoxylin (Biocare Medical, Cat. #XMF963) were used. Tissue microarrays (TMA) were from Pantomics (Fairfield, CA). The TMA contained an FDA-recommended panel of normal human tissues (MNO961) from three different donors. Human tumor TMAs included a multi-tumor array (MTU481) and arrays of breast cancer (BRC1022), colon cancer (COC1021), lung cancer (primary and metastatic, LUM961), metastatic cancer (MET961), ovarian cancer (OVC1021), pancreatic cancer (PAN1021), and lymphoma (LYM1021). TMA slides were stained with a mouse version of the SAC-2 antibody to eliminate background from secondary antibodies that bind human IgG in the samples.Stained slides were digitized at 20x magnification using a TissueScope LE whole slide scanner (Huron Digital Pathology, St. Jacobs, Ontario, Canada). For verification, glass slides were viewed under an upright bright-field microscope.

[0106] Figure 4 shows examples of SAC-2 staining of normal human breast tissue (Figure 4A) and breast tumors (Figure 4C) compared to staining of the tumor with an irrelevant mouse IgG2a antibody (Figure 4B). Staining of the tumor is indicated by rust-brown membrane staining of the tumor cells, whereas normal breast tissue does not stain with SAC-2 and tumors do not stain with an irrelevant IgG2a antibody. SAC-2 staining of breast tumor cells was uniform throughout the specimen. The intensity of SAC-2 staining of tumor cells varied in different specimens, but the homogeneous staining characteristic was seen in all specimens that were positive for SAC-2 binding.

[0107] Table 5 summarizes the staining results for normal human tissues. Positive staining was observed in only one of three prostate specimens in normal human tissue. The stained prostate specimen was described as benign prostatic hyperplasia, but may have also contained early stage prostate cancer cells. Many of the tumor specimens were stained, especially metastatic tumors. Table 6 summarizes the results of staining tumor tissue microarrays with SAC-2. In summary, SAC-2 was shown not to bind to normal human developmental tissues but to recognize antigens expressed on cells of several different human primary and metastatic tumors.

[0108] [Table 5-1]

[0109] [Table 5-2]

[0110] [Table 6]

[0111] Example 7 This example demonstrates that the antibodies provided herein have antibody-dependent cellular cytotoxicity activity.

[0112] Five adherent (CHP-134 and Kelly neuroblastoma, SK-MEL-28 melanoma, SK-OV-3 ovarian, and AsPC-1 pancreatic) and two non-adherent (NCI-H020 myeloma, Jurkat leukemia) cancer cell lines were tested with SAC-1 and SAC-2 for their ability to mediate ADCC activity in vitro. Cell lines were obtained and prepared for use in the assay as described in Example 4 above. ADCC activity was measured using the InvivoGen Jurkat-Lucia™ NFAT-CD16 Reporter Assay Kit according to the manufacturer's instructions. This assay measures the cell killing CD-16 Fcγ receptor-mediated signaling pathway, which is activated by binding of an antibody to an antigen on the cancer surface and binding of the Fc portion of the antibody to a receptor on the reporter cell that results in the expression of luciferase and the production of luminescence in the presence of luciferin. The output signal was measured as relative luminescence units (RLU) using a luminescence plate reader (Synergy HTX Multimode Reader, Agilent, Santa Clara, CA or similar).

[0113] Results for the mouse-human Fc IgG1 chimeric versions of SAC-1 and SAC-2 are shown in the graphs in Figures 5A-5C. In general, SAC-1 gave less signal than SAC-2, reflecting a lower epitope density as reflected by the relative MFI determined in the antibody binding assay (Table 3), but was able to activate ADCC activity against most cell lines tested. SAC-2 had higher ADCC activity against most cell lines tested, consistent with the higher epitope density observed in the binding assay (Table 4), although SAC-2 lacked activity against some cell lines. In summary, the data show that both antibodies are able to mediate ADCC activity against multiple different human cancer cell lines.

[0114] Example 8 This example shows that the antibody-dependent cellular cytotoxicity (ADCC) activity of humanized SAC-2.1C and D antibodies is enhanced by reducing or eliminating fucosylation (eg, afucosylated antibodies).

[0115] The ADCC activity of reduced fucosylated antibodies (aFUC) was compared using the DELFIA® cell cytotoxicity assay kit (PerkinElmer, Billerica, MA). The target cells used were human A375 melanoma cells and MDA-MB-231 breast cancer cells. The assay was performed according to the manufacturer's instructions. Briefly, target cells were harvested and suspended in complete medium. Target cells (1x10^6) were labeled with 2uL of fluorescence-enhancing ligand (DELFIA® BATDA Reagent) for 20 min at 37°C. After washing the cells four times with phosphate-buffered saline (PBS), the cell density was adjusted to 1x10^5 / mL and seeded at 100mL / well in a 96-well assay plate. For the ADCC assay, 50μL of serially diluted antibody solution was added to the wells and incubated at 37°C, 5% CO2 for 15 min. Natural killer effector cells (NK92 / CD16a) were added to a 96-well plate at 50 μL / well at a ratio of 8 effector cells to 1 target cell. The assay plate containing effector cells, antibody, and target cells was incubated at 37°C, 5% CO2 for 3 hours.

[0116] When cells are intact, the BATDA ligand remains intracellular. When europium solution is added to the supernatant of an intact cell sample, BATDA is not released into the supernatant and therefore europium cannot form a fluorescent chelate with BATDA. The europium solution is not fluorescent in its natural state. When cells are lysed by effector cells, BATDA is released outside the cells into the supernatant. When europium solution is added to the supernatant, europium can form a highly fluorescent and stable chelate with the released BATDA (EuTDA). The measured fluorescent signal directly correlates with the amount of lysed cells in a cytotoxicity assay.

[0117] To measure the maximum release of BATDA, 10 μL of Lysis Buffer was added to the control wells, and 20 μL of the supernatant from all wells was transferred to a flat-bottom detection plate. Europium solution (200 μL) was added to the supernatant in the flat-bottom well plate, the plate was shaken at room temperature for 15 min, and finally the fluorescence was measured within 5 h in a time-resolved fluorometer. The killing rate was calculated as follows: ADCC = (fluorescence of sample - autofluorescence of target and effector cell mixture) / (maximum fluorescence of target cells - autofluorescence of target and effector cell mixture) × 100%. The killing rate curves were analyzed to determine the EC50 using GraphPad Prism 6 (GraphPad, San Diego, CA). The results are shown in Figures 6A and 6B and summarized in Table 7. Depending on the antibody and target cells tested, the ADCC activity was increased 4- to 10-fold.

[0118] [Table 7]

[0119] Example 9 This example demonstrates that the antibodies provided herein have in vivo tumor inhibitory activity in xenograft mouse models of human cancer.

[0120] The ability of the SAC antibody to inhibit tumor growth in xenograft mouse models of human cancer was tested in athymic BALB / c nu / nu mice and NSG mice supplemented with human peripheral blood mononuclear cells (PBMCs) using two models: A375 human melanoma and MDA-MB-231 human breast cancer. Mice were inoculated subcutaneously in the hind limb with 1x10^6 cells and Matrigel. Tumors were measured with digital calipers and averaged 100-200 mm. 3Mice were grouped into treatment groups (10 per group) based on the size of the tumor. Mice were treated with SAC antibody at 2 mg / kg, 6 mg / kg, or 20 mg / kg twice weekly, standard of care (paclitaxel 7.5 mg / kg for the A375 model and cyclophosphamide 30 mg / kg for the MDA-MB-231 model ip daily) or vehicle alone in the control group. Mice receiving human PBMCs were treated once with PBMCs at the beginning of the treatment period. Comparisons of significance were performed by repeated measures ANOVA with Dunnett's multiple comparison test using GraphPad Prism (San Diego, CA) software. The dose-dependence of SAC antibody treatment compared to standard of care paclitaxel is shown in Figures 7A and 7B. Data for SAC-1.1, murine SAC-2, and SAC-2.1C in the MDA-MB-231 human breast cancer xenograft mouse model are shown in Figure 8A, and the effect of adding human PBMCs with SAC-2.1C in Figure 8B. The data show that SAC-1 and SAC-2 can inhibit tumor growth in human melanoma and breast cancer xenograft mouse models in an antibody concentration-dependent manner.

[0121] All references cited in this specification, including publications, patent applications, and patents, are incorporated by reference herein to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein.

[0122] The use of "a," "an," "the," "at least one," and similar reference words in the context of describing the present invention (particularly in the context of the claims below) are to be construed as covering both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term "at least one" followed by a list of one or more items (e.g., "at least one of A and B") is to be construed as meaning one item selected from the listed items (A or B), or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to"), unless otherwise indicated. The description of ranges of values ​​herein is intended to serve merely as a shorthand method for individually referring to each separate value falling within the range, and each separate value is incorporated herein as if it were individually described herein, unless otherwise indicated herein. All methods described herein can be performed in any suitable order, unless otherwise indicated herein or clearly contradicted by context. Any examples provided herein, or the use of exemplary language (e.g., "such as"), are intended merely to facilitate a better understanding of the invention, and do not limit the scope of the invention unless otherwise asserted. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0123] Preferred embodiments of the invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of these preferred embodiments will become apparent to those skilled in the art upon reading the foregoing description. The inventors expect that such variations will be adopted by those skilled in the art as appropriate, and the inventors do not intend to practice the invention otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

1. dPSA binders including the following: (a) The immunoglobulin heavy chain variable region including SEQ ID NO: 51 or at least its complementarity-determining region (CDR); and the immunoglobulin light chain variable region including SEQ ID NO: 52 or 53 or at least its CDR; (b) Immunoglobulin heavy chain variable region including one or at least one of SEQ ID NO: 17-20 or its complementarity-determining region (CDR); and immunoglobulin light chain variable region including SEQ ID NO: 21 or at least its CDR; (c) Immunoglobulin heavy chain variable region including one or at least one of SEQ ID NO: 1 to 4 or its complementarity-determining region (CDR); and immunoglobulin light chain variable region including SEQ ID NO: 5 or at least its CDR; or (d) an immunoglobulin heavy chain variable region including SEQ ID NO: 35 or at least its complementarity-determining region (CDR); and an immunoglobulin light chain variable region including SEQ ID NO: 36 or at least its CDR.

2. dPSA binders including the following: (a） SEQ ID NO: CDRH1 containing one of 6-9; SEQ ID NO: CDRH2 including 10; CDRH3 containing SEQ ID NO: 11; This includes the immunoglobulin heavy chain variable region, and CDRL1 containing SEQ ID NO: 12; CDRL2 including SEQ ID NO: 13; and CDRL3 containing SEQ ID NO: 14; The immunoglobulin light chain variable region, (b） SEQ ID NO: CDRH1 containing one of 24-27; CDRH2 containing SEQ ID NO: 28; CDRH3 containing SEQ ID NO: 29; The immunoglobulin heavy chain variable region including and CDRL1 containing SEQ ID NO: 30; CDRL2 including SEQ ID NO: 31; and CDRL3 containing SEQ ID NO: 32; The immunoglobulin light chain variable region, (c） CDRH1 containing SEQ ID NO: 39; SEQ ID NO: CDRH2 including 40; and CDRH3 containing SEQ ID NO: 41 or 47; The immunoglobulin heavy chain variable region including and CDRL1 containing SEQ ID NO: 42; SEQ ID NO: CDRL2 including 43 or 56; and CDRL3 containing SEQ ID NO: 44; The immunoglobulin light chain variable region, or （d） CDRH1 containing SEQ ID NO: 45; CDRH2 including SEQ ID NO: 46; and CDRH3 containing SEQ ID NO: 41 or 47; The immunoglobulin heavy chain variable region, and CDRL1 containing SEQ ID NO: 48; SEQ ID NO: CDRL2 including 49 or 56; and CDRL3 containing SEQ ID NO: 50; The immunoglobulin light chain variable region, including the immunoglobulin light chain variable region.

3. The dPSA conjugate according to claim 1, wherein the dPSA conjugate is an antibody, an antigen-binding antibody fragment, a conjugate thereof, or a chimeric antigen receptor.

4. The dPSA binder is F(ab') 2 The dPSA binder according to claim 1, which is a fragment, a Fab' fragment, a Fab fragment, an Fv fragment, an scFv fragment, a dsFv fragment, or a dAb fragment.

5. The dPSA conjugate according to claim 1, wherein the dPSA conjugate comprises an Fc region capable of mediating complement-dependent cell injury (CDC) or antibody-dependent cell injury (ADCC).

6. The dPSA conjugate according to claim 1, wherein the dPSA conjugate is an IgG1 or IgG4 antibody.

7. The dPSA binder according to claim 1, wherein the dPSA binder is afucosylated.

8. (a) a dPSA binder according to any one of claims 1 to 7 or a nucleic acid encoding the same, and (b) a pharmaceutically acceptable carrier.

9. A nucleic acid encoding the immunoglobulin heavy chain variable region and / or light chain variable region of a dPSA binder according to any one of claims 1 to 7, wherein the nucleic acid is optionally present in the vector.

10. The nucleic acid according to claim 9, further encoding a leader sequence of the immunoglobulin heavy chain variable region and / or light chain variable region.

11. A cell containing the nucleic acid described in claim 9.

12. A cell line expressing the dPSA binding agent according to any one of claims 1 to 7.

13. A method for preparing a dPSA binder according to any one of claims 1 to 7, comprising expressing in vitro in cells a nucleotide sequence encoding an immunoglobulin heavy chain polypeptide and a nucleic acid sequence encoding an immunoglobulin light chain polypeptide.

14. A method for delivering a payload to cells expressing dPSA in vitro, comprising contacting the cells with a dPSA conjugate according to any one of claims 1 to 7, which is bound to the payload.

15. The method according to claim 14, wherein the payload is a cytotoxic molecule or a detectable label.

16. A method for detecting soluble dPSA in vitro in a biological fluid or tissue sample, comprising contacting the biological fluid or tissue sample with a dPSA binder according to any one of claims 1 to 7, which is optionally conjugated to a detectable label.

17. A dPSA conjugate according to any one of claims 1 to 7 for treating cancer.

18. The composition according to claim 8 for treating cancer.

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