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

Abstract

A dPSA binding agent and immunoglobulin heavy chain and light chain polypeptides of the binding agent, and methods of treating cancer and killing cancer cells using the dPSA binding agent.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This patent application claims the benefit of U.S. Provisional Patent Application No. 63 / 299,841 filed on January 14, 2022 and U.S. Provisional Patent Application No. 63 / 299,843 filed on January 14, 2022, which are incorporated herein by reference in their entirety.

[0003] Incorporation by Reference of Electronically Submitted Materials

[0004] Incorporated herein by reference in its entirety is the computer-readable nucleotide / amino acid sequence listing filed concurrently with this document, which is identified as follows: a 56,304-byte XML file named "514163.xml" created on January 13, 2023. Background Art

[0005] In general, the goal of anti-cancer immunotherapy is to identify stable antigens that are highly expressed but not shed or secreted from tumor cells, which can then be used as the basis for immunotherapy, for example, as antigens in cancer vaccines or as targets for antibody-based cancer therapy. Optimally, such tumor antigens have acceptable specificity for cancerous target cells in order to reduce harmful side effects that can be caused by cross-reactivity with non-cancerous cells of the treated subject. In cases where cross-reactivity affects cells that can be repopulated, it may be acceptable to relax this requirement for immunotherapy specificity.

[0006] Altered glycosylation patterns of cell surface proteins occur in almost all types of cancer. Excessive sialylation of glycoproteins and glycolipids is at the core of abnormal regulation of cell adhesion in metastatic cancer, which in turn can be caused by re-expression and / or over-expression of genes that are normally expressed during development but not in cells of adult normal tissues. In particular, poly-α2->8N-acetylneuraminic acid or polysialic acid (polySia) is primarily expressed during fetal development and is highly restricted to only a few regenerative tissues after development. The de-N-acetylated form of polySia (dPSA) on the surface of cancer but not post-developmental human cells can be used as a tumor antigen for cancer identification and treatment.

[0007] Therefore, there is a need for new agents that can bind to cells expressing dPSA. Summary of the invention

[0008] Provided herein is a dPSA-binding agent comprising an immunoglobulin heavy chain and light chain polypeptide. Also provided herein is a method of using a dPSA-binding agent to treat cancer, kill cancer cells, and deliver a payload to cells expressing dPSA.

[0009] Related compositions and methods are also provided, as will be apparent from the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 is a graph depicting the mean fluorescence intensity (MFI) of the SAC-1 antibody vs. the concentration of the SEAM 3 reference antibody, indicating that the test antibody does not bind to the same epitope as the reference antibody.

[0011] Figure 2 is a graph depicting the mean fluorescence intensity (MFI) of the SAC-2 antibody vs. the concentration of the SEAM 3 reference antibody, indicating that the test antibody does not bind to the same epitope as the reference antibody.

[0012] Figure 3A and 3B Shown are proteins co-immunoprecipitated by SAC-1, SAC-2 and control IgG1 antibodies on SDS-PAGE.

[0013] Figure 4A , 4B and 4C show normal human breast tissue ( Figure 4A ) and breast tumors ( Figure 4C ) of SAC-3 staining, compared with the control IgG2a antibody staining of tumors ( Figure 4B ) compared.

[0014] Figure 5A , 5B and 5C show a depiction of SAC-1 ( Figure 5A ) or SAC-2 antibody ( Figure 5B ) vs. relative luminescence units (RLU), indicating the ADCC of each antibody against various cell lines, and the quantitative values ​​( Figure 5C ).

[0015] Fig. 6A and 6B is a graph plotting cell killing against concentrations of non-fucosylated SAC-2.1C and SAC-2.1D (referred to as "SAC-2.1CaFUC" and SAC-2.1DaFUC", respectively), illustrating the effects of non-fucosylated antibodies against human A375 melanoma ( Fig. 6A ) and MDA-MB-231 breast cancer ( Figure 6B ) cell lines.

[0016] Fig. 7A and 7B SAC-1.1 ( Fig. 7A ) and SAC-2( Figure 7B ) treatment on the dose-dependency of tumor growth in the A375 xenograft mouse model of human melanoma.

[0017] Fig. 8A and8B Shown are the effects of SAC-1.1, mouse 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 ( Fig. 8A ), and the effect of adding human PBMCs and SAC-2.1C on tumor growth ( Figure 8B ). DETAILED DESCRIPTION

[0018] dPSA is a de-N-acetylated form of polySia (dPSA) that is present on the surface of cancer cells but not other non-cancerous post-developmental 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)). Since cell surface dPSA is unique to cancer cells and widely expressed in different cancers, agents that preferentially bind to cells expressing dPSA can be used to target cancer cells for diagnosis and treatment.

[0019] Humans have two genes, ST8SIA2 and ST8SIA4, which encode enzymes that synthesize polySia (polysialyltransferases ST8SIA2 and ST8SIA4, respectively). Although both genes are highly expressed in humans during fetal development (Angata et al., J. Biol. Chem., 272 (11): 7182-90 (1997)), ST8SIA4 is mainly expressed in lymphoid tissues and lymphocytes (Drake et al., PNAS 106 (29): 11995-2000 (2009)); based on Northern blotting, ST8SIA2 does not appear to be present at significant levels in any adult normal tissue (Angata et al., J. Biol. Chem., 272 (11): 7182-90 (1997)).

[0020] Several proteins have been shown to be polysialylated in humans (see, 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 molecule (NCAM) is the most abundant, especially during fetal development, and the most thoroughly studied (Rutishauser, U., Nat'l Rev. Neurosci., 9(1):26-35 (2008)). Many human cancers have been reported to aberrantly express polySia-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)), where its role in mediating interactions between cells and between cells and the extracellular matrix is ​​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, the present inventors identified nucleolin as a dPSA-modified or dPSA-associated protein and showed that cell surface dPSA is dependent on ST8SIA2 expression (Moe et al., J. Exp. Clin. Cancer Res. 40(1):293 (2021)).

[0021] Provided herein is a binding agent (e.g., an antibody or antibody fragment) that selectively binds 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, the binding agent is believed to bind to an antigen (e.g., nucleolin) that comprises an epitope that is at least partially defined by one or more dPSA residues. Therefore, the binding agent is hereinafter referred to as a "dPSA binding agent".

[0022] The dPSA binders provided herein include Ig heavy and light chain polypeptides, each of which includes at least an Ig heavy chain variable region and an Ig light chain variable region, respectively. The Ig heavy and light chain variable regions each include three complementary determining regions (CDRs), commonly referred to as CDR1, CDR2, or CDR3. The CDR regions can also be referred to in the nomenclature using "H" or "L" to represent a heavy chain or a light chain, i.e., CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3, respectively. The CDRs of a given Ig sequence can be identified by any of several conventional numbering schemes, such as Kabat, Chothia, Martin (enhanced Chothia), IGMT, or AHo (these are common names for numbering schemes that are widely known in the art and described in the published literature, see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, USDepartment of Health and Human Services, NIH (1991), which describes 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), which describes the "Chothia" numbering scheme; Abhinandan et al., Analysis and Improvements to Kabat and StructurallyCorrect Numbering of Antibody Variable Domains,Mol.Immunol., 45:3832–3839 (2008), which describes the “Martin” or “enhanced Chothia” numbering scheme; 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), which describes 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), which describes the “AHo” numbering scheme). Identification of CDRs may also be performed by correlation with empirical binding data, such as crystallographic studies of the interaction of a binding agent with its target (e.g., an antigen or portion thereof comprising a binding epitope), optionally in combination with any of the foregoing numbering systems.

[0023] The dPSA binders provided herein are artificial and non-naturally occurring. They have been generated by laboratory techniques and are therefore appropriately considered to be recombinant or synthetic molecules comprising recombinant or synthetic amino acid sequences. The Ig heavy and light chain polypeptides can be "isolated", i.e., they are removed from the environment in which they were produced (e.g., cell culture) and purified to any degree.

[0024] In some embodiments, the dPSA binder comprises an Ig heavy chain variable region comprising any one of SEQ ID NOs: 1-4 or at least its CDRs; and an Ig light chain variable region comprising SEQ ID NO: 5 or at least its CDRs. The CDRs can 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 one of SEQ ID NOs: 1-4 and a light chain variable region of SEQ ID NO: 5, or at least its CDRs determined by Kabat. In some embodiments, the antibody comprises a heavy chain variable region of any one of SEQ ID NOs: 1-4 and a light chain variable region of SEQ ID NO: 5, or at least its CDRs determined by Chothia. In some embodiments, the antibody comprises a heavy chain variable region of any one of SEQ ID NOs: 1-4 and a light chain variable region of SEQ ID NO: 5, or at least its CDRs determined by Martin. In some embodiments, the antibody comprises a heavy chain variable region of any one of SEQ ID NOs: 1-4 and a light chain variable region of SEQ ID NO: 5, or at least its CDRs as determined by IGMT. In some embodiments, the antibody comprises a heavy chain variable region of any one of SEQ ID NOs: 1-4 and a light chain variable region of SEQ ID NO: 5, or at least its CDRs as determined by AHo. In some embodiments, the dPSA binder comprises one of the following combinations of Ig heavy and light chain variable regions, or at least its CDRs as determined by any one of Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo:

[0025] Heavy chain variable region Light chain variable region 1 SEQ ID NO: 1 or its CDRs SEQ ID NO: 5 or its CDRs 2 SEQ ID NO: 2 or its CDRs SEQ ID NO: 5 or its CDRs 3 SEQ ID NO: 3 or its CDRs SEQ ID NO: 5 or its CDRs 4 SEQ ID NO:4 or its CDRs SEQ ID NO: 5 or its CDRs

[0026] In some embodiments, the dPSA binder comprises an Ig heavy chain variable region comprising any one of SEQ ID NOs: 17-20 or at least its CDRs; and an Ig light chain variable region comprising SEQ ID NO: 21 or at least its CDRs. The CDRs can 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 one of SEQ ID NOs: 17-20 and a light chain variable region of SEQ ID NO: 21, or at least its CDRs determined by Kabat. In some embodiments, the antibody comprises a heavy chain variable region of any one of SEQ ID NOs: 17-20 and a light chain variable region of SEQ ID NO: 21, or at least its CDRs determined by Chothia. In some embodiments, the antibody comprises a heavy chain variable region of any one of SEQ ID NOs: 17-20 and a light chain variable region of SEQ ID NO: 21, or at least its CDRs determined by Martin. In some embodiments, the antibody comprises a heavy chain variable region of any one of SEQ ID NOs: 17-20 and a light chain variable region of SEQ ID NO: 21, or at least its CDRs as determined by IGMT. In some embodiments, the antibody comprises a heavy chain variable region of any one of SEQ ID NOs: 17-20 and a light chain variable region of SEQ ID NO: 21, or at least its CDRs as determined by AHo. In some embodiments, the dPSA binder comprises one of the following combinations of Ig heavy and light chain variable regions, or at least its CDRs as determined by any one of Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo:

[0027] Heavy chain variable region Light chain variable region 1 SEQ ID NO: 17 or its CDRs SEQ ID NO:21 or its CDRs 2 SEQ ID NO: 18 or its CDRs SEQ ID NO:21 or its CDRs 3 SEQ ID NO: 19 or its CDRs SEQ ID NO:21 or its CDRs 4 SEQ ID NO: 20 or its CDRs SEQ ID NO:21 or its CDRs

[0028] 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 NOs: 10 or 28, and a CDR3 comprising SEQ ID NOs: 11 or 29; and the Ig light chain variable region comprises: a CDR1 comprising SEQ ID NOs: 12 or 30, a CDR2 comprising SEQ ID NOs: 13 (RMS) or 31, and a CDR3 comprising SEQ ID NOs: 14 or 32.

[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 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.

[0030] CDRH1 CDRH2 CDRH3 CDRL1 CDRL2 CDRL3 1 SEQ ID NO:6 SEQ ID NO:10 SEQ ID NO:11 SEQ ID NO:12 SEQ ID NO:13 SEQ ID NO:14 2 SEQ ID NO:7 SEQ ID NO:10 SEQ ID NO:11 SEQ ID NO:12 SEQ ID NO:13 SEQ ID NO:14 3 SEQ ID NO:8 SEQ ID NO:10 SEQ ID NO:11 SEQ ID NO:12 SEQ ID NO:13 SEQ ID NO:14 4 SEQ ID NO:9 SEQ ID NO:10 SEQ ID NO:11 SEQ ID NO:12 SEQ ID NO:13 SEQ ID NO:14

[0031] 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.

[0032]

[0033] According to yet another aspect of the present disclosure, a 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 one of SEQ ID NOs: 1-4; 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 any one of SEQ ID NOs: 1-4; NO:5 has an amino acid sequence with 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 some embodiments, the dPSA-binding agent comprises an Ig heavy 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:15; 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:15; NO: 16 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%) of the amino acid sequence. In any of the foregoing embodiments, the Ig heavy chain variable region and the Ig light chain variable region can comprise (preserve) the CDRs of the heavy chain and light chain variable regions of the sequence, which can be determined using any known numbering scheme, such as Kabat, Chothia, Martin (enhanced Chothia), IGMT or AHo, or as otherwise described 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, a dPSA-binding agent comprises Ig heavy and light chain polypeptides comprising SEQ ID NO: 15 and SEQ ID NO: 16, respectively.

[0034] According to yet another aspect of the present disclosure, a 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 one of SEQ ID NOs: 17-20; 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 any one of SEQ ID NOs: 17-20; NO:21 has an amino acid sequence with 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 some embodiments, a dPSA-binding agent comprises an Ig heavy 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:22; 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:22; NO:23 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%) of the amino acid sequence. 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 and light chain variable regions, which can be determined using any known numbering scheme, such as Kabat, Chothia, Martin (enhanced Chothia), IGMT or AHo, or as otherwise described herein (e.g., SEQ ID NO:6-14 or 24-32 above). In some embodiments, the Ig heavy and light chain variable regions comprise one of SEQ ID NOs: 17-20, respectively, and the Ig light chain variable region comprises SEQ ID NO: 21.In some embodiments, a dPSA-binding agent comprises Ig heavy and light chain polypeptides comprising SEQ ID NO:22 and SEQ ID NO:23, respectively.

[0035] In some embodiments, a dPSA-binding agent comprises Ig heavy and light chain polypeptides comprising SEQ ID NOs: 33 and 34, respectively.

[0036] In some embodiments, the dPSA binder 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 can 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 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 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 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 its CDRs 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 its CDRs as determined by AHo.

[0037] In some embodiments, the dPSA binder comprises an Ig heavy chain variable region comprising SEQ ID NO: 51 or at least its CDRs; and an Ig light chain variable region comprising SEQ ID NO: 52 or 53 or at least its CDRs. The CDRs can 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 its CDRs 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 its CDRs 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 its CDRs 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 its CDRs 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 its CDRs as determined by AHo.

[0038] 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.

[0039] 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.

[0040]

[0041] In some embodiments, a 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 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 SEQ ID NO: 35; NO:36 has an amino acid sequence with 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 some embodiments, the dPSA-binding agent comprises an Ig heavy 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:37; 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:37; NO:38 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%) of the amino acid sequence. 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 and light chain variable regions of SEQ ID NOs:35 and 36, respectively, which CDRs can be identified using any known numbering scheme, such as Kabat, Chothia, Martin (enhanced Chothia), IGMT or AHo, or as otherwise described herein (e.g., SEQ ID NOs: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.

[0042] In some embodiments, a 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:51; 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 SEQ ID NO:51; NO:52 or 53 has an amino acid sequence with 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 some embodiments, a dPSA-binding agent comprises an Ig heavy 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:51; 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:51; IDNO: 52 or 53 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%) amino acid sequence. 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 and light chain variable regions of SEQ IDNO: 51 and 52 or 53, respectively, which can be determined using any known numbering scheme, such as Kabat, Chothia, Martin (enhanced Chothia), IGMT or AHo, or as otherwise described 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.

[0043] In some embodiments, a 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:57; 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 SEQ ID NO:57; NO:58 or 59 has an amino acid sequence with 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 some embodiments, a dPSA-binding agent comprises an Ig heavy 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:57; 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:57; IDNO: 58 or 59 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%) amino acid sequence. 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 and light chain variable regions of SEQ ID NO: 57 and 58 or 59, respectively, which can be determined using any known numbering scheme, such as Kabat, Chothia, Martin (enhanced Chothia), IGMT or AHo, or as otherwise described 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.

[0044] In some embodiments, a dPSA-binding agent comprises Ig heavy and light chain variable regions 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 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 NOs:33 or 34, optionally while retaining the CDRs thereof. In some embodiments, a dPSA-binding agent comprises Ig heavy and light chain variable regions comprising SEQ ID NOs:54 and 55, 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 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 NOs:54 or 55, optionally while retaining the CDRs thereof.

[0045] Sequence "identity" used for nucleic acid or amino acid sequence can be determined by comparing the nucleic acid or amino acid sequence of interest with a reference nucleic acid or amino acid sequence. Percent identity is the percentage of nucleotides or amino acid residues that are identical (i.e., identical) between the sequence of interest and the reference sequence when the best comparison is made. Many mathematical algorithms for obtaining the best comparison and calculating the identity between two or more sequences are known and publicly available. Examples of such programs include CLUSTAL-W, T-Coffee and ALIGN (for comparison of nucleic acid and amino acid sequences), BLAST programs (e.g., BLAST 2.1, BL2SEQ and subsequent 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 searches). Sequence alignment algorithms are also disclosed in, for example, 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); and Gusfield, Algorithms on Strings, Trees and Sequences, Cambridge University Press, Cambridge UK (1997).

[0046] With respect to sequences having less than 100% identity to the heavy and light chain sequences specifically described above, one or more amino acids of the aforementioned immunoglobulin heavy chain polypeptides and / or light chain polypeptides may be replaced or substituted with different amino acids, and / or one or more amino acids may 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 dPSA) is substantially retained. The biological activity of the dPSA binder can be measured, for example, by binding affinity to a specific dPSA epitope and / or cross-reactivity with targets other than dPSA. The aforementioned properties or characteristics can be observed, measured and / or evaluated using standard techniques, including but not limited to ELISA, competitive ELISA, surface plasmon resonance analysis (BIACORE TM ) or solution phase competition (KINEXA TM ), as well as other in vitro or in vivo neutralization assays, binding assays, fluorescence activated cell binding (FACS), or other suitable assays.

[0047] The dPSA binder can 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 can 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.

[0048] The dPSA binder can be part of a conjugate. For example, the dPSA binder can be a conjugate of (1) an anti-dPSA antibody or fragment thereof and (2) a secondary protein or non-protein moiety. As a further illustration, the dPSA binder can comprise an anti-dPSA antibody or fragment thereof conjugated to another peptide, a fluorescent molecule, or a chemotherapeutic (e.g., cytotoxic) agent.

[0049] In some embodiments, a dPSA binder can be a "whole" immunoglobulin or an antigen-binding immunoglobulin "fragment". A "whole" immunoglobulin is typically composed of four polypeptides: two heavy (H) chain polypeptides and two light (L) chain polypeptides. Each heavy chain contains an N-terminal variable (V H ) region and three C-terminal constant (C H 1. C H 2 and C H 3) region, and each light chain contains an N-terminal variable (V L ) region and a C-terminal constant (C L) region. Based on the amino acid sequence of the constant domain, the light chain of an antibody can be classified into one of two different types, kappa (κ) or lambda (λ). 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 light chain variable region is usually aligned with the variable region of the heavy chain, and the light chain constant region is usually aligned with the first constant region of the heavy chain. The remaining constant regions of the heavy chains are usually aligned with each other.

[0050] The variable or hypervariable regions of each pair of light and heavy chains form the antigen-binding site of the antibody. H and V L The regions have the same general structure, each region comprising four framework (FW or FR) regions. As used herein, the term "framework region" refers to a relatively conservative amino acid sequence within the variable region, which is located between the hypervariable region or the complementary determining region (CDR). There are four framework regions in each variable domain, named FR1, FR2, FR3 and FR4. The framework region forms a β sheet that provides a structural framework for the variable region (see, for example, CA Janeway et al. (eds.), Immunobiology, 5th edition, Garland Publishing, New York, NY (2001)). The framework region is connected by three complementary determining regions (CDRs). These three CDRs are called CDR1, CDR2 and CDR3, forming the "hypervariable region" of the antibody, which is generally considered to be responsible for antigen binding.

[0051] The term "antibody fragment" and similar terms (e.g., "fragment of an antibody", "antibody fragment", "functional fragment of an antibody") are used interchangeably herein to refer to 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)). As used herein and in the art, antibody "fragments" include not only fragments or pieces of a literal intact antibody, but also other known engineered antibody-like constructs that may include linkers or other elements that do not naturally occur in "intact" antibodies. Examples of antibody fragments include, but are not limited to, (i) a Fab fragment, which is a fragment of V L 、V H , C L and CH1 domains, (ii) F(ab')2 fragments, which are bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region, and (iii) V fragments consisting of a single arm of an antibody. L and V H(iv) Fab' fragments, which are generated by breaking the disulfide bridges of the F(ab')2 fragment using mild reducing conditions, and (v) disulfide-stabilized Fv fragments (dsFv). dPSA binders can also be single-chain antibody fragments. Examples of single-chain antibody fragments include, but are not limited to, (i) single-chain Fv (scFv), which is composed of two domains of the Fv fragment (i.e., V and V) connected by a synthetic linker. L and V H ), which synthetic linker enables the two domains to be synthesized as a single polypeptide chain (see, 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 comprises a V domain connected to a V domain by a peptide linker. L V H , the peptide linker is too short to allow V on the same polypeptide chain H and V L pairing to drive different V H -V L Pairing between complementary domains on polypeptide chains to generate a dimeric molecule with two functional antigen binding sites. Any other antigen-binding antibody-like constructs known in the art comprising Ig heavy and light chain CDRs or variable regions may also be used and are antibody fragments for the purposes of the present disclosure. In some embodiments, the dPSA binder is a chimeric antigen receptor (or is part of a chimeric antigen receptor).

[0052] In some embodiments, a dPSA-binding agent comprises a heavy chain constant region, such as a fragment crystallizable (F c ) region or a portion thereof. The Fc region can be any Ig class / subclass (IgA (IgA1, IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3, and IgG4), IgM, including variants thereof. In some embodiments, the dPSA binder is a "complete" or "complete" Ig (i.e., an antibody); and in other embodiments, the binder is an antibody fragment conjugated or linked to an Fc region. In some embodiments, the dPSA binder comprises an IgG Fc region, such as IgG1 or IgG4. For example, the dPSA binder can be an IgG1 or IgG4 antibody. In some embodiments, the dPSA binder comprises an Fc region that can mediate complement dependent cytotoxicity (CDC) or antibody dependent cellular cytotoxicity (ADCC). In some embodiments, the dPSA binder comprises an Fc region that activates natural killer (NK) cells.

[0053] The dPSA binder can be a human or humanized antibody, a non-human antibody, or a chimeric antibody. "Chimeric" means an antibody or fragment thereof comprising human and non-human regions. Preferably, the dPSA binder is a humanized antibody. A "humanized" antibody is a monoclonal antibody comprising a human antibody scaffold and at least one CDR obtained or derived from a non-human antibody. Non-human antibodies include antibodies isolated from any non-human animal, such as, for example, a rodent (e.g., a mouse or rat). A humanized antibody may comprise one, two, or three CDRs obtained or derived from a non-human antibody.

[0054] Human antibodies, non-human antibodies, chimeric antibodies, or humanized antibodies can be obtained by any means, including via in vitro sources (e.g., hybridomas or recombinant antibody-producing cell lines) and in vivo sources (e.g., rodents). Methods for generating antibodies are known in the art and are described, e.g., 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 antibodies or chimeric antibodies can be produced using transgenic animals (e.g., mice) in which one or more endogenous immunoglobulin genes are replaced by 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 generated 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)), including, for example, grafting non-human CDRs onto human antibody scaffolds (see, e.g., Kashmiri et al., Methods, 36(1):25-34 (2005); and Hou et al., J. Biochem., 144(1):115-120 (2008)).

[0055] The dPSA binders provided herein can be used for any purpose. For example, dPSA binders can be used to target or kill cancer cells that express dPSA (e.g., comprising dPSA on the cell surface). Therefore, a method for targeting or killing cancer cells in vitro or in vivo is provided herein, comprising administering a dPSA binder as described herein to the cancer cells. When using this method to target or kill cancer cells in vivo, the dPSA binder can be administered to the cancer cells by administering the dPSA binder to a subject comprising the cancer cells.

[0056] The cancer cell can be any cancer cell that expresses dPSA on the cell surface. For example, the cancer cell can comprise a protein, such as nucleolin, wherein dPSA is linked to or otherwise associated with the protein on the cell surface. In some embodiments, the cancer cell expresses ST8SIA2.

[0057] Methods 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, a dPSA binder can be conjugated to a detectable marker or support (e.g., a radioactive marker, a fluorescent marker, a bead, a scaffold, etc.) to analyze dPSA expression levels or facilitate detection of cancer cells expressing dPSA in a biological sample (biological fluid or tissue sample) from a subject. For example, the detectable moiety can be a radioactive isotope (e.g., 3 H. 14 C. 32 P. 35 S or 125 I), fluorescent or chemiluminescent compounds (e.g., fluorescein isothiocyanate, rhodamine or luciferin), enzymes (e.g., alkaline phosphatase, β-galactosidase or horseradish peroxidase) or supports (beads, scaffolds, biosensor surfaces, etc.). In the context of the present invention, any method known in the art for conjugating antigen-binding agents (e.g., antibodies) to such moieties, respectively, can be used (see, 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)).

[0058] Alternatively, dPSA binders can be used to kill cancer cells. For example, dPSA binders can be conjugated with chemotherapeutic agents (e.g., cytotoxic agents) and used to target and deliver chemotherapeutic agents to cancer cells expressing dPSA, thereby killing cancer cells in subjects. In other embodiments, dPSA binders exhibit antibody-dependent cellular toxicity (ADCC) or complement-dependent cytotoxicity (CDC), and are used to kill cancer cells expressing dPSA by binding to cancer cells and achieving ADCC or CDC-mediated cell death. In some embodiments, dPSA binders are non-fucosylated. Non-fucosylated binders (e.g., antibodies) can be prepared by any suitable technique, such as by expressing nucleic acids encoding Ig heavy and light chains of dPSA binders in cell lines (e.g., FUT8-deficient CHO cells) in which the FUT8 gene has been destroyed or deleted.

[0059] Therefore, the dPSA binders provided herein and methods of use thereof can be used to treat cancers characterized by surface expression of dPSA. As used herein, the term "treatment (treatment / treating, etc.)" refers to obtaining the desired pharmacological and / or physiological effects, for example, to reduce the severity of the disease and / or adverse symptoms attributable to the disease or inhibit the progression of the disease and / or adverse symptoms attributable to the disease. To this end, the methods of the present invention include administering a "therapeutically effective amount" of a dPSA binder. "Therapeutically effective amount" refers to an amount that is effective to achieve the desired therapeutic outcome within the necessary dosage and time period. The therapeutically effective amount can vary according to factors such as the individual's disease state, age, sex, and weight, and the ability of the dPSA binder to elicit the desired response in the individual.

[0060] The methods and compositions provided herein can be used to treat or prevent a variety of cancers, including carcinomas, sarcomas, leukemias, myelomas, and lymphomas.

[0061] Cancers that may be treated by the methods disclosed herein include, but are not limited to, esophageal cancer, hepatocellular carcinoma, basal cell carcinoma (a form of skin cancer), squamous cell carcinoma (of various tissues), bladder cancer, including transitional cell carcinoma (a malignant tumor of the bladder), bronchial cancer, colon cancer, colorectal cancer, gastric cancer, lung cancer, including small cell carcinoma and non-small cell carcinoma of the lung, adrenocortical carcinoma, thyroid cancer, pancreatic cancer, 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, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, osteoblastic carcinoma, epithelial carcinoma, and nasopharyngeal carcinoma.

[0062] Sarcomas that may be treated by the methods disclosed herein include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas.

[0063] Other solid tumors that can be treated by the methods disclosed herein include, but are not limited to, gliomas, astrocytomas, medulloblastomas, craniopharyngiomas, ependymomas, pinealomas, hemangioblastomas, acoustic neuromas, oligodendrogliomas, menangiomas, melanomas, neuroblastomas, and retinoblastomas.

[0064] Other cancers include leukemia, lymphoma and myeloma (including multiple myeloma). Leukemias that can be treated by the methods disclosed herein include, but are not limited to, a) chronic myeloproliferative syndrome (neoplastic disorder of pluripotent hematopoietic stem cells); b) acute myeloid leukemia (neoplastic transformation of pluripotent hematopoietic stem cells or hematopoietic cells of restricted lineage potential); c) chronic lymphocytic leukemia (CLL; clonal proliferation of immunologically immature and incomplete small lymphocytes), including B cell CLL, T cell CLL, prolymphocytic leukemia and hairy cell leukemia; and d) acute lymphoblastic leukemia (characterized by the accumulation of lymphoblasts). Lymphomas that can be treated 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, etc.

[0065] Other cancers that may be treated according to the methods disclosed herein include atypical meningioma (brain), islet cell carcinoma (pancreas), medullary carcinoma (thyroid), mesenchymal carcinoma (intestine), hepatocellular carcinoma (liver), hepatoblastoma (liver), clear cell carcinoma (kidney), and mediastinal neurofibroma.

[0066] Other exemplary cancers that can be treated using the methods disclosed herein include, but are not limited to, cancers of neuroectodermal and epithelial origin. Examples of cancers of neuroectodermal origin include, but are not limited to, Ewing's sarcoma, spinal tumors, brain tumors, primitive neuroectodermal tumors on the tendons of infancy, tubular cystic carcinomas, mucinous tubular and spindle cell carcinomas, renal tumors, mediastinal tumors, gliomas, neuroblastomas, and adolescent and youth sarcomas. Examples of epithelial origin include, but are not limited to, small cell lung cancer, breast cancer, eye lens cancer, colon cancer, pancreatic cancer, kidney cancer, liver cancer, ovarian cancer, and bronchial epithelial carcinoma. In some embodiments, the subject method does not include the treatment of melanoma (that is, cancer is not melanoma). In other embodiments, the subject method does not include the treatment of lymphoma (that is, cancer is not lymphoma).

[0067] The dPSA binder may be part of a composition suitable for administration to a mammal. Preferably, the composition is a pharmaceutically acceptable (e.g., physiologically acceptable) composition comprising a carrier, preferably a pharmaceutically acceptable (e.g., physiologically acceptable) carrier, and an amino acid sequence, antigen binding agent or carrier 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 will be determined in part by the specific site where the composition can be administered and the specific method for administering the composition. The composition may also include any other excipients for preparations of therapeutic molecules (e.g., proteins or antibodies), particularly parenteral preparations, including, for example, buffers, tension regulators, stabilizers, surfactants, etc. The composition may be sterile. The composition may be frozen or lyophilized for storage and reconstituted in a suitable sterile carrier before use. The composition may be generated according to conventional techniques described in, for example, Remington: The Science and Practice of Pharmacy, 21st edition, Lippincott Williams & Wilkins, Philadelphia, PA (2001).

[0068] Administration can be achieved using any standard administration technique, including oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual or suppository administration. The composition is preferably suitable for parenteral administration. As used herein, the term "parenteral" includes intravenous, intramuscular, subcutaneous, rectal, vaginal and intraperitoneal administration. More preferably, the composition is administered to a mammal using peripheral systemic delivery by intravenous, intraperitoneal or subcutaneous injection.

[0069] The dPSA binders of the present invention can be administered alone or in combination with other drugs. For example, the dPSA binders can be administered in combination with other agents for treating or preventing the diseases disclosed herein, such as other anticancer agents. In this regard, for example, the dPSA binders can be used in combination with at least one other agent, including, for example, chemotherapeutic agents, vaccines, biological therapies (e.g., other monoclonal antibodies), radiation therapy, bone marrow transplantation, chemotherapy, biological response modifier therapy and / or surgery.

[0070] Also provided herein is a nucleic acid encoding a dPSA-binding agent (ie, encoding an immunoglobulin heavy chain polypeptide and / or an immunoglobulin light chain polypeptide of a dPSA-binding agent).

[0071] Nucleic acid can be a polymer of DNA or RNA (or both, such as hybrid DNA / RNA), which can be single-stranded or double-stranded, and can contain non-natural or altered nucleotides. Nucleic acid can be a part of a vector. The vector can be, for example, a plasmid, an episome, a cosmid, a viral vector (e.g., a retrovirus or 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)).

[0072] Vectors typically include expression control sequences, such as promoters, enhancers, polyadenylation signals, transcription terminators, signal peptides (e.g., osteonectin signal peptides), internal ribosome entry sites (IRES), etc., which provide for expression of the coding sequence in a host cell. Exemplary expression control sequences are known in the art and are described in, for example, Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, Calif. (1990).

[0073] A large number of promoters from a variety of different sources, including constitutive promoters, inducible promoters and repressible promoters, are well known in the art. Representative sources of promoters include, for example, viruses, mammals, insects, plants, yeasts and bacteria, and suitable promoters from these sources are easily available, or can be prepared based on, for example, sequences publicly available from depository institutions such as ATCC and other commercial or personal sources. The promoter can be unidirectional (i.e., initiating transcription in one direction) or bidirectional (i.e., initiating transcription in the 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, Tet system (U.S. Patents 5,464,758 and 5,814,618), ecdysone inducible system (No et al., Proc. Natl. Acad. Sci., 93:3346-3351 (1996)), T-REX TMSystem (Invitrogen, Carlsbad, CA), LACSWITCH TM system (Stratagene, San Diego, CA) 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. Patent 7,112,715; and Kramer & Fussenegger, Methods Mol. Biol., 308:123-144 (2005)).

[0074] As used herein, the term "enhancer" refers to a DNA sequence that increases, for example, the transcription of a nucleic acid sequence that is operably connected thereto. The enhancer can be located thousands of bases away from the coding region of the nucleic acid sequence, and can mediate the binding of regulatory factors, the pattern of DNA methylation, or the change of DNA structure. A large number of enhancers from various sources are well known in the art, and can be obtained as cloned polynucleotides or in cloned polynucleotides (from, for example, depository institutions such as ATCC and other commercial or personal sources). Many polynucleotides comprising promoters (such as the commonly used CMV promoter) also include enhancer sequences. The enhancer can be located upstream, internal, or downstream of the coding sequence.

[0075] The vector may also contain a "selectable marker gene". As used herein, the term "selectable marker gene" refers to a nucleic acid sequence that allows cells expressing the nucleic acid sequence to be specifically selected or not specifically selected in the presence of a corresponding selection agent. Suitable selectable marker genes are known in the art and are described in, for example, International Patent Application Publications 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 (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.

[0076] In some embodiments, the vector is an "episomal expression vector" or "episome" that is capable of replicating in a host cell and persisting as an extrachromosomal segment of DNA within 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 utilizing Epstein-Barr nuclear antigen 1 (EBNA1) and Epstein-Barr virus (EBV) replication origin (oriP). 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 SV40 replication origin instead of EBNA1 and oriP.

[0077] Other suitable vectors include integrative expression vectors that can be randomly integrated into the DNA of the host cell, or can include recombination sites to achieve 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 achieve expression of the desired protein. Examples of vectors that integrate in a site-specific manner include, for example, components of the flp-in system from Invitrogen (Carlsbad, CA) (e.g., pcDNA TM 5 / FRT), or a cre-lox system, such as can be found in the pExchange-6 core vector from Stratagene (La Jolla, CA). Examples of vectors that randomly integrate into the host cell chromosome include, for example, pcDNA3.1 from Life Technologies (Carlsbad, CA) (when introduced in the absence of T antigen), UCOE from Millipore (Billerica, MA), and pCI or pFN10A (ACT) FLEXI from Promega (Madison, WI). TM .

[0078] Viral vectors may also be used. Representative commercially available viral expression vectors include, but are not limited to, the adenovirus-based Per.C6 system available from Crucell, Inc. (Leiden, The Netherlands), the lentivirus-based pLP1 from Invitrogen (Carlsbad, CA), and the retroviral vector pFB-ERV plus pCFB-EGSH from Stratagene (La Jolla, CA).

[0079] The nucleic acid sequence encoding the amino acid sequence of the present invention can be provided to cells on the same vector (i.e., cis). A unidirectional promoter can be used to control the expression of each nucleic acid sequence. In another embodiment, a combination of a bidirectional promoter and a unidirectional promoter can be used to control the expression of multiple nucleic acid sequences. Alternatively, the nucleic acid sequence encoding the amino acid sequence of the present invention can be provided to a cell population on a separate vector (i.e., trans). Each nucleic acid sequence in each separate vector can contain the same or different expression control sequences. A separate vector can be provided to cells simultaneously or sequentially.

[0080] The vector comprising the nucleic acid encoding the amino acid sequence of the present invention can be introduced into a host cell capable of expressing the polypeptide encoded therein, including any suitable prokaryotic or eukaryotic cell. Therefore, the present invention provides a cell comprising an isolated vector of the present invention. Preferred host cells are those that can be easily and reliably grown, have a fairly fast growth rate, have a well-characterized expression system, and can be easily and effectively transformed or transfected.

[0081] Examples of suitable prokaryotic cells include, but are not limited to, cells from 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 (e.g., K12, HB101 (ATCC No. 33694), DH5α, DH10, MC1061 (ATCC No. 53338), and CC102).

[0082] 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 from Kluyveromyces, Pichia, Rhino-sporidium, Saccharomyces, and Schizosaccharomyces. Preferred yeast cells include, for example, Saccharomyces cerivisae and Pichia pastoris.

[0083] 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, CA).

[0084] In some embodiments, the present invention utilizes mammalian cells.Many suitable mammalian host cells are known in the art, and many can be obtained from the American Type Culture Collection (ATCC, Manassas, VA).The example of suitable mammalian cells includes but is not limited to Chinese hamster ovary cells (CHO) (e.g., CHO-K1 cells, ATCC numbering 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 numbering CRL1573) and 3T3 cells (ATCC numbering CCL92).Other suitable mammalian cell lines are monkey COS-1 (ATCC numbering CRL1650) and COS-7 cell lines (ATCC numbering CRL1651), and CV-1 cell lines (ATCC numbering CCL70).Other exemplary mammalian host cells include primate cell lines and rodent cell lines, including transformed cell lines. Normal diploid cells, cell lines derived from primary tissue in vitro cultures, 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 methods for transforming, culturing, amplifying, screening, and purifying cells are known in the art.

[0085] In one embodiment, the mammalian cell is a human cell. For example, the mammalian cell can be a human lymphoid or lymphoid-derived cell line, such as a cell line derived from a pre-B lymphocyte. 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 derivatives thereof.

[0086] The nucleic acid sequence encoding the amino acid sequence of the invention can be introduced into the cell by any suitable method, such as by "transfection", "transformation" or "transduction". As used herein, "transfection", "transformation" or "transduction" refers to the introduction of one or more exogenous polynucleotides into a host cell by using physical or chemical methods. Many suitable techniques are known in the art and include, for example, calcium phosphate DNA coprecipitation (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-promoted microprojectile bombardment (Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNA coprecipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)). After the infectious particles are grown in suitable packaging cells, the phage or viral vector can be introduced into the host cell, and many packaging cells are commercially available.

[0087] Nucleic acids and cells can be used for any purpose, such as for making the dPSA binders 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 a heavy chain and / or light chain immunoglobulin polypeptide of a dPSA binder. In other words, the method includes expressing a nucleic acid encoding an immunoglobulin heavy chain and / or light chain of a dPSA binder in a cell (e.g., an in vitro cell, such as any cell line discussed herein, including CHO and CHO-K1 cells). It should be understood that the immunoglobulin heavy and light chains can be expressed by a single nucleic acid in a given cell, or that the immunoglobulin heavy and light chains can be expressed by separate nucleic acids from the same cell. The method may also include harvesting and / or purifying the dPSA binder from cells or cell culture media using known techniques.

[0088] The following examples further illustrate the invention but, of course, should not be construed as limiting its scope in any way.

[0089] Example 1

[0090] The following examples illustrate the selective binding of dPSA binding agents provided herein to dPSA antigen.

[0091] Antibodies named SAC-1 and SAC-2 having the following sequences 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 named SAC-1.1, SAC-2.1 (also referred to as "SAC-2 humanized D") and SAC-2.2 (also referred to as "SAC-2 humanized C") and had the sequences described below.

[0092]

[0093] dPSA antigen preparation

[0094] The dPSA antigen for ELISA was prepared by combining 100 mg of polyacetylneuraminic acid (MilliporeSigma), 10 mg of sodium borohydride (MilliporeSigma) in 10 mL of 2 molar sodium and heating to 100°C for 40 minutes. The resulting dPSA was neutralized with 2M hydrochloric acid, dialyzed twice against 4 L of water, and lyophilized. dPSA (20 mg) in 0.75 mL of 0.1 M sodium acetate (pH 6.5) was first oxidized with sodium periodate (0.25 mL of 10 mM periodate) for 30 minutes at ambient temperature in the dark. After adding 100 μL of 10% (vol / vol) ethylene glycol, the reaction mixture was dialyzed and lyophilized as described above. 20 mg of oxidized dPSA and 10 mg of ovalbumin (Imject TM Ovalbumin, Pierce Chemical) was combined in phosphate buffered saline (PBS) containing about 5 mg of sodium cyanoborohydride. The solution was stirred overnight in the dark at ambient temperature. The dPSA-ovalbumin conjugate was purified by size exclusion chromatography on a ToyoPearl HW-65F column in 0.9% (weight / volume) sodium chloride, 10 mM potassium phosphate, pH 7.1. The fractions containing the dPSA-KLH conjugate were concentrated (SpinX, Corning) to 2 mg / mL protein and 4 mg / mL dPSA containing about 30% de-N-acetylated residues, which was determined by a modified resorcinol assay.

[0095] Binding to dPSA antigen

[0096] As described in Moe et al., J. Exp. & Clin. Can. Res. 40(1): 293 (2021), the ELISA was performed starting with an antibody concentration of 10 μg / ml and 8 serial 3-fold dilutions. The specificity for dPSA was determined by performing the same ELISA in buffer in the presence of 100 μg of polysialic acid (i.e., polyacetylneuraminic acid, MilliporeSigma). The results (OD405>0.5 mean fluorescence intensity (MFI) within 30 minutes) confirmed that the antibody specifically bound to dPSA.

[0097] The antibodies were also tested for binding to the human neuroblastoma cell line CHP-134 and the human myeloma cell line NCI-H929 by flow cytometry at a fixed concentration of 10 μg / ml as described in Moe et al., J of Exp & Clin Can Res 2021, 40:293. Briefly, the antibodies were removed by pipetting or with Accutase TM (Innovative Cell Technologies), adherent cells were suspended in RPMI 1640 cell culture medium containing 10% (volume / volume) fetal bovine serum (FBS, ThermoFisher Scientific). The cells were centrifuged (200xg, 8 minutes), and the cell pellet was suspended in culture medium to a concentration of 0.5-2×10^6 cells / ml. The cells and antibodies were combined in 1.5 ml Eppendorf tubes and mixed by inversion rotation for 1 hour at ambient temperature. The tubes were centrifuged (200xg, 2 minutes), washed once in fresh culture medium, and the cells were suspended in culture medium containing AlexaFluor 488-conjugated goat anti-human F(ab')2 secondary antibodies. After inversion mixing for 30 minutes, the cells were washed once with fresh culture medium, and the cells were suspended in PBS buffer containing 0.5% (volume / volume) formaldehyde. The fluorescence of the cells was measured by flow cytometry (Acea NovoCyte). The results are summarized in Table 1 , expressed as mean fluorescence intensity (MFI) relative to an irrelevant human IgG1 negative control antibody (BioXCell).

[0098] Table 1

[0099] Antibody CHP-134MFI NCI-H929 MFI SAC-1 155,241 66,816 SAC-2 59,426 67,142

[0100] Example 2

[0101] The following examples illustrate the binding of antibodies provided herein to other different cancer cell lines, but not to CHP-134 neuroblastoma cells (CHP-134KO) in which polysialyltransferase genes ST8SIA2 and ST8SIA4 have been knocked out. dPSA on the surface of CHP-134 cells depends on the expression of ST8SIA2, as described in Moe et al., J of Exp & Clin Can Res 2021, 40: 293.

[0102] The antibodies provided in Example 1 were further tested for binding to a number of additional cell lines according to procedures similar to those described in Example 1 for CHP-134 and NCI-H929 cells. Table 2 lists the EC values ​​for each cell line. 50 and maximum MFI values, and showed that the antibody binds to a variety of different types of cancer cells. In addition, the antibody showed significantly enhanced binding compared to the reference anti-dPSA antibody SEAM 3 (Steirer and Moe et al., PLoS One 6(11):e27249(2011)).

[0103] Table 2

[0104]

[0105] *ND, not determined.

[0106] Example 3

[0107] The following examples illustrate that the antibodies provided herein bind to a different epitope than a reference antibody.

[0108] 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 fixed concentration of antibody 10-fold greater than the EC50 for binding to CHP-134 cells was combined with serial 2-fold dilutions of SEAM 3. Figure 1 and Figure 2 As shown, the antibody of Example 1 did not inhibit SEAM 3 binding, indicating that the antibody recognized a different epitope.

[0109] Example 4

[0110] This example demonstrates that the antibodies provided herein bind to a variety of human cancer cell lines.

[0111] Cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and routinely tested for mycoplasma contamination (MycoStrip™, InvivoGen, San Diego, CA). Cell lines were grown in a humidified chamber under a 5% CO2 atmosphere in ATCC recommended medium. Adherent cell lines were suspended by treatment with StemPro™ Accutase™ Cell Dissociation Reagent (Thermo Fisher Scientific, Carlsbad, CA). The suspended cells were diluted 1:5 with cell culture medium, centrifuged (200 x g, 10 minutes), and suspended in fresh medium to a density of 1-10 million viable cells / mL. Viability was determined by trypan blue staining (Thermo Fisher Scientific, Carlsbad, CA) and quantified using SKC, Inc. C-Chip TM Cells were counted using a disposable hemocytometer (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 continuous mixing by inversion. Cells were centrifuged (200 x g, 2 minutes), the supernatant was removed by aspiration, and the cells were suspended in a 4% paraformaldehyde solution containing AlexaFluor 488 TM The cells were incubated with the appropriate (i.e., anti-mouse or anti-human IgG H+L) labeled secondary antibody (AffiniPure F(ab')2 fragment goat anti-mouse IgG (H+L), Jackson ImmunoResearch, West Grove, PA) in a culture medium. The cells and secondary antibody were mixed and incubated for 30 minutes at ambient temperature by inversion, then centrifuged (200 x g, 2 minutes), the supernatant was removed by aspiration, and the cells were suspended in phosphate-buffered saline containing 0.5% formaldehyde.

[0112] Finally, cells were analyzed by flow cytometry (Acea NovoCyte, Agilent, Santa Clara, CA or similar). Binding curves of the dependence of mean fluorescence intensity (MFI) on antibody concentration were analyzed by curve fitting software (GraphPad Prism, San Diego, CA) to determine the binding constant (K D) and maximum MFI (MFImax). The results are presented in Table 3 (SAC-1 antibody) and Table 4 (SAC-2 antibody), where Neg. means negative and NA means not applicable. As shown in Tables 3 and 4, the test antibodies were tested with K in the nanomolar range. D Binds to cell lines from a variety of cancers with variable MFImax values, depending on the epitope density of each cell line. The data show that the antigen recognized by the antibody is present in a broad 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 abolished binding.

[0113] Table 3

[0114]

[0115] Table 4

[0116]

[0117]

[0118] Example 5

[0119] This example demonstrates that the antibodies provided herein recognize derivatives of nucleolin that contain dPSA.

[0120] Preparation of subcellular fractions: Use Human melanoma A375 cells (80% confluent in T-175 flasks) were extracted using the Subcellular Proteome Extraction Kit (MilliporeSigma). Briefly, a differential detergent extraction procedure used four extraction buffers in sequence, along with a protease inhibitor cocktail to prevent protein degradation during extraction, and a Nuclease (Sigma-Aldrich, St. Louis, MO) degrades contaminating nucleic acids. Following the manufacturer's extraction instructions, the cell extract was separated into four fractions: F1 (cytosolic fraction), F2 (cell membrane fraction), F3 (nucleoprotein fraction), and F4 (cytoskeleton fraction).

[0121] Immunoprecipitation: Dynabeads M-270 epoxy magnetic beads (Thermo Fisher Scientific, Carlsbad, CA) covalently linked to SAC-1, SAC-2 or unrelated human IgG1 antibodies (BioXCell, Lebanon, NH) were prepared following the manufacturer's protocol. Antigens reacting with the antibodies were purified by immunoprecipitation as follows. The F2 membrane fraction was incubated with SAC-1, SAC-2 or unrelated IgG1-linked magnetic beads, respectively. The beads were separated using a magnet, washed with the corresponding extraction buffer alone, and then washed with a buffer containing polysialic acid (polySia) (50 μg / mL; polyacetylneuraminic acid from Sigma-Aldrich, St. Louis, MO) to remove nonspecifically bound antigens. Finally, the antigens were eluted from the beads by heating to 80°C for 10 minutes using a reducing agent-free SDS-PAGE sample buffer (NuPAGE, Thermo Fisher Scientific). The proteins eluted from the beads were resolved on 4%-12% SDS-PAGE (NuPAGE, Thermo Fisher Scientific) and purified by The cells were stained with Coomassie Brilliant Blue (Thermo Fisher Scientific) or transferred to PVDF membrane using NuPAGE transfer (Thermo Fisher Scientific). -FL, Millipore, Waltham, MA). The PVDF membrane was blocked with 5% (weight / volume) whole milk powder in phosphate-buffered saline (PBS) buffer overnight, and the immunoprecipitates were then stained with anti-nucleolin antibody MS-3 (Santa Cruz Biotechnology, Santa Cruz, CA) in blocking buffer at ambient temperature for 2 h. After washing three times with PBS buffer, the membranes were stained with Bound antibodies were detected with 800CW-conjugated donkey anti-mouse IgG (H+L) secondary antibody (LI-COR, Lincoln, NE). Images of gels and blots were recorded on an Fc imaging system (LI-COR). Results are presented in Figure 3A and Figure 3B middle.

[0122] Figure 3AThe proteins co-immunoprecipitated by each antibody resolved on SDS-PAGE are shown. There are multiple strongly stained bands with the same apparent mass distribution, which are co-immunoprecipitated by SAC-1 and SAC-2, but not by unrelated IgG1 antibodies. Previously, we showed that antibodies bound to de-N-acetylated polysialic acid (dPSA) co-immunoprecipitated dPSA-modified nucleolin from cancer cell F2 fractions (J Exp Clin Cancer Res. 2021 Sep 20; 40 (1): 293). Cell surface nucleolin is unique to cancer cells and has a certain apparent mass range when resolved on SDS-PAGE gels, because of multiple post-translational modifications of dPSA and length and charge heterogeneity.

[0123] Figure 3B Staining of proteins co-immunoprecipitated with the anti-nucleolin antibody MS-3 on a Western blot is shown. The major band for 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 migration in SDS-PAGE gels may not be detected by MS-3, which is used for unmodified recombinant nucleolin. To confirm that other bands with an apparent mass different from the major band detected with MS-3 are also derivatives of nucleolin, for each co-immunoprecipitated sample, a Figure 5A The gel slices indicated in brackets were processed as previously described (J Exp Clin Cancer Res. 2021 Sep 20; 40(1): 293) for analysis by LC-MS / MS mass spectrometry for protein identification. LC-MS / MS mass spectrometry confirmed that the analyzed gel slices also contained nucleolin.

[0124] The quantification of nucleolin for each sample is also shown in Figure 5A In the Figure 1, and corresponding to the relative amount of Coomassie blue staining in the SDS-PAGE gel slice of 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 the derivatives of nucleolin have the properties of being modified by dPSA as described above (i.e., the apparent mass range on SDS-PAGE).

[0125] Example 6

[0126] This example demonstrates by immunohistochemistry that the antibodies provided herein exhibit reactivity with human tumors and normal human tissues.

[0127] To determine the specificity of SAC antibody binding to cancer cells and not normal developmental human tissues, tissue microarrays containing tissue samples from normal human tissues and tumors were stained by immunohistochemistry with SAC-2.

[0128] Using 2.5 μg / ml of SAC-2 mouse monoclonal antibody (SEQ ID NO:54 and SEQ ID NO:55), 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 the Biocare intelliPATH automated staining platform (Biocare Medical, Pacheco, CA) using the manufacturer's recommended settings. Sections were incubated with Biocare Peroxidase Blocker (Biocare, Cat. No. PX968) and Background Punisher (Biocare, Cat. No. BP974M) to block nonspecific background. For detection of mouse primary antibodies, the MACH4 HRP-polymer detection system (Biocare, Cat. No. MRH534) was used. The color detection and counterstaining kits IntelliPATH FLX DAB chromogen (Biocare, Cat. No. #IPK5010) and IntelliPATH hematoxylin (Biocare Medical, Cat. No. #XMF963) were used. Tissue microarrays (TMAs) were from Pantomics (Fairfield, CA). TMAs included normal human tissue panels (MNO961) from three different donors recommended by the FDA. TMAs of human tumors included arrays of multiple tumor arrays (MTU481) and breast cancer (BRC1022), colorectal 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 mouse-formed SAC-2 antibodies to eliminate the background generated by the combination of secondary antibodies with human IgG in the sample. The stained slides were digitized with a TissueScope LE full slide scanner (Huron Digital Pathology, St. Jacobs, Ontario, Canada) at 20x magnification. For the purpose of confirmation, slides were examined on a vertical bright field microscope.

[0129] Figure 4 shows normal human breast tissue ( Figure 4A ) and breast tumors ( Figure 4C ) and an example of SAC-2 staining of a tumor with an unrelated mouse IgG2a antibody ( Figure 4B) compared to the rust-brown membrane staining of the tumor cells. The tumor staining was shown by the rust-brown membrane staining of the tumor cells, while normal breast tissue was not stained by SAC-2, and the tumor was not stained by the unrelated IgG2a antibody. The SAC-2 staining of breast tumor cells was uniform throughout the sample. Although the intensity of SAC-2 staining of tumor cells was variable, for different samples, the uniform staining characteristic was present in all samples that were positive for SAC-2 binding.

[0130] Table 5 summarizes the staining results of normal human tissue. The only positive staining observed for normal human tissue was one of three prostate samples. The stained prostate sample was described as benign prostatic hyperplasia, which may also contain early prostate cancer cells. Many tumor specimens, especially metastatic tumors, were stained. Table 6 summarizes the results of staining tumor tissue microarrays with SAC-2. In short, the results show that SAC-2 does not bind to normal post-developmental tissues, but does recognize antigens expressed on several different human primary and metastatic tumor cells.

[0131] Table 5. Summary of SAC-2 staining of normal human tissue (Pantomics MNO96) by immunohistochemistry.

[0132]

[0133]

[0134] *The stained specimen is one of three specimens described as benign prostatic hyperplasia.

[0135] Table 6. Summary of SAC-2 immunohistochemical staining of tumor samples on tissue microarray.

[0136] Patient tumor set (Pantomics catalog number) Positive percentage (number of positive samples / total number) 15 different cancer types (MTU481) 46％(22 / 48) Breast cancer (BRC1022) 78％(62 / 80) Lung cancer (LUM961) 73％(73 / 100) Colorectal cancer (COCR1021) 20％(20 / 100) Lymphoma (LYM1021) 17％(15 / 88) Pancreatic cancer (PAN1021) 21％(19 / 88) Ovarian cancer (OVC1021) 0％(0 / 96) Metastatic cancer (MET961) 81％(73 / 90)

[0137] Example 7

[0138] This example demonstrates that the antibodies provided herein have antibody-dependent cellular cytotoxicity activity.

[0139] The ability of in vitro mediation ADCC activity of five kinds of adherent cell lines (CHP-134 and Kelly neuroblastoma, SK-MEL-28 melanoma, SK-OV-3 ovary and AsPC-1 pancreas) and two kinds of non-adherent (NCI-H020 myeloma, Jurkat leukemia) cancer cell lines was tested with SAC-1 and SAC-2. Cell lines are obtained and prepared for the determination described in Example 4 above. Following the manufacturer's instructions, ADCC activity is measured using InvivoGen Jurkat-LuciaTM NFAT-CD16 reporter gene assay kit. The assay measures the signal transduction pathway mediated by cell killing CD-16Fcγ receptors, which is activated by binding of antibodies to antigens on cancer surfaces and the Fc part of antibodies to receptors on reporter cells, resulting in luciferase expression and luminescence in the presence of luciferin. Output signals as relative luminescence units (RLU) are measured using a luminescent plate reader (Synergy HTX multifunctional microplate reader, Agilent, Santa Clara, CA or similar).

[0140] The results of mouse-human Fc IgG1 chimera forms SAC-1 and SAC-2 are shown in Figures 5A-5C In the figure. In general, SAC-1 has less signal than SAC-2, reflecting the lower epitope density reflected in the relative MFI determined in the binding study of the antibody (Table 3), but can activate ADCC activity against all tested cell lines. SAC-2 has higher ADCC activity against most tested cell lines, which is also consistent with the higher epitope density observed in the binding study (Table 4), but SAC-2 lacks activity against some cell lines. In summary, the data show that both antibodies can mediate ADCC activity against a variety of different human cancer cell lines.

[0141] Example 8

[0142] This example demonstrates that by reducing or eliminating fucosylation (eg, afucosylated antibodies), the antibody-dependent cellular cytotoxicity (ADCC) activity of humanized SAC-2.1C and D antibodies is enhanced.

[0143] use The ADCC activity of antibodies with reduced fucosylation (aFUC) was compared using a cytotoxicity assay kit (PerkinElmer, Billerica, MA). The target cells used were human A375 melanoma and MDA-MB-231 breast cancer cells. The assay was performed using the manufacturer's instructions. In brief, target cells were harvested and suspended in complete medium. Target cells (1×10^6) were labeled with 2uL of fluorescence enhancing ligand (BATDA reagent) at 37°C for 20 min. The cells were washed 4 times with phosphate buffered saline (PBS), and then the cell density was adjusted to 1×10^5 / mL and seeded into a 96-well assay plate at 100mL / well. In the ADCC assay, 50μL of serially diluted antibody solution was added to the wells and incubated at 37°C, 5% CO2 for 15min. Natural killer effector cells (NK92 / CD16a) were added to 50μL / well of a 96-well plate at a ratio of 8 effector cells to 1 target cell. The assay plate with effector cells, antibodies and target cells was incubated at 37°C, 5% CO2 for 3 hours.

[0144] When cells are intact, the BATDA ligand remains in the cells. When a europium solution is added to the supernatant from an intact cell sample, europium cannot form a fluorescent chelate with BATDA because no BATDA is released into the supernatant. Europium solution does not fluoresce in its unchanged state. If cells are lysed by effector cells, BATDA is released outside the cells into the supernatant. Upon addition of the europium solution to the supernatant, europium can form a highly fluorescent and stable chelate with the released BATDA (EuTDA). The measured fluorescence signal is directly related to the amount of cells lysed in the cytotoxicity assay.

[0145] To determine the maximum release of BATDA, 10 μL of lysis buffer was added to the control wells, and 20 μL of supernatant from all wells was transferred to a flat-bottomed detection plate. Europium solution (200 μL) was added to the supernatant of the flat-well plate, shaken at room temperature for 15 min, and finally the fluorescence was measured using a time-resolved fluorimeter within 5 hours. The percentage of killing was calculated as follows: ADCC = (sample fluorescence - autofluorescence of the mixture of target cells and effector cells) / (maximum target cell fluorescence - autofluorescence of the mixture of target cells and effector cells) × 100%. The percentage of killing curve was analyzed using GraphPad Prism 6 (GraphPad, San Diego, CA) to determine the EC50. The results are shown in Fig. 6A and Figure 6B The ADCC activity was increased 4- to 10-fold depending on the tested antibody and target cells.

[0146] Table 7

[0147]

[0148] Example 9

[0149] This example demonstrates that the antibodies provided herein have in vivo tumor suppressor activity in a human cancer xenograft mouse model.

[0150] The ability of SAC antibodies to inhibit tumor growth in human cancer xenograft mouse models 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 with 1×10^6 cells plus Matrigel injected subcutaneously in the hind legs. Tumors were measured by caliper (digital caliper) and grouped into treatment groups (10 mice per group), with an average size of 100-200mm 3 . Mice were treated with SAC antibody at 2 mg / kg, 6 mg / kg, and 20 mg / kg twice weekly, or with standard of care drugs (7.5 mg / kg paclitaxel for the A375 model; 30 mg / kg cyclophosphamide ip daily for the MDA-MB-231 model), or a control group received vehicle alone. Mice given human PBMCs received a single PBMC treatment at the beginning of the treatment period. Significance comparisons were performed by repeated measures ANOVA with Dunnett's multiple comparison test using GraphPad Prism (San Diego, CA) software. The dose-dependency of SAC antibody treatment compared to standard of care paclitaxel is shown in Fig. 7A and Figure 7B Data on SAC-1.1, mouse SAC-2, and SAC-2.1C in the MDA-MB-231 human breast cancer xenograft mouse model are shown in Fig. 8A The effect of adding human PBMCs with SAC-2.1C was shown in Figure 8B Data show that SAC-1 and SAC-2 can inhibit tumor growth in xenograft mouse models of human melanoma and breast cancer, depending on the concentration of the antibody.

[0151] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0152] Unless otherwise specified herein or clearly contradictory to the context, in the context of describing the present invention (especially in the context of the following claims), the use of the terms "one", "a kind of", "the", "at least one" and similar indicators should be interpreted as covering the singular and plural. Unless otherwise specified herein or clearly contradictory to the context, the use of a list of one or more items (e.g., "at least one of A and B") following the term "at least one" should be interpreted as meaning an 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 specified, the terms "comprising", "having", "including" and "containing" should be interpreted as open terms (i.e., meaning "including but not limited to"). Unless otherwise specified herein, the enumeration of the range of values ​​herein is intended only to be used as a shorthand method for individually referring to each individual value falling within the range, and each individual value is incorporated into this specification as if it were individually listed herein. Unless otherwise specified herein or clearly contradictory to the context, all methods described herein can be performed in any suitable order. Unless otherwise claimed, the use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the invention and does not limit the scope of the invention. Language in this specification should not be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0153] Preferred embodiments of the present invention are described herein, including the best mode known to the inventor for implementing the present invention. After reading the foregoing description, changes in those preferred embodiments may become apparent to those of ordinary skill in the art. The inventors expect that skilled technicians will appropriately adopt such changes, and the inventors wish to practice the present invention in a manner different from that specifically described herein. Therefore, the present invention includes all modifications and equivalents of the subject matter listed in the appended claims as allowed by applicable law. In addition, unless otherwise stated herein or clearly contradictory to the context, any combination of the above-mentioned elements in all possible variations thereof is encompassed by the present invention.

Claims

1. A dPSA binder comprising (a) an immunoglobulin heavy chain variable region comprising SEQ ID NO: 51 or at least its complementarity determining region (CDR); and an immunoglobulin light chain variable region comprising SEQ ID NO: 52 or 53 or at least its CDR; (b) an immunoglobulin heavy chain variable region comprising any one of SEQ ID NOs: 17-20 or at least the complementarity determining regions (CDRs) thereof; and an immunoglobulin light chain variable region comprising SEQ ID NO: 21 or at least the CDRs thereof; (c) an immunoglobulin heavy chain variable region comprising any one of SEQ ID NOs: 1-4 or at least a complementarity determining region (CDR) thereof; and an immunoglobulin light chain variable region comprising SEQ ID NO: 5 or at least a CDR thereof; or (d) an immunoglobulin heavy chain variable region comprising SEQ ID NO:35 or at least its complementarity determining region (CDR); and an immunoglobulin light chain variable region comprising SEQ ID NO:36 or at least its CDR.

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

50.

3. The dPSA binder of claim 1 or 2, wherein the dPSA binder is an antibody, an antigen-binding antibody fragment, a conjugate thereof, or a chimeric antigen receptor.

4. The dPSA-binding agent of any one of claims 1-3, wherein the dPSA-binding agent is a F(ab')2 fragment, a Fab' fragment, a Fab fragment, a Fv fragment, a scFv fragment, a dsFv fragment, or a dAb fragment.

5. The dPSA-binding agent of any one of claims 1-3, wherein the dPSA-binding agent comprises an Fc region capable of mediating complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC).

6. The dPSA-binding agent of any one of claims 1-3, wherein the dPSA-binding agent is an IgG1 or IgG4 antibody.

7. The dPSA binder of any one of claims 1-6, wherein the dPSA binder is non-fucosylated.

8. A composition comprising (a) the dPSA-binding agent or a nucleic acid encoding the same according to any one of claims 1 to 7 and (b) a pharmaceutically acceptable carrier.

9. A nucleic acid encoding the immunoglobulin heavy chain and / or light chain variable region of the dPSA-binding agent of any one of claims 1-7, optionally in a vector.

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

11. A cell comprising the nucleic acid according to claim 9 or 10.

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

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

14. A method of killing a cancer cell expressing dPSA, the method comprising contacting the cancer cell with a dPSA-binding agent according to any one of claims 1-7 or a composition according to claim 8.

15. A method of treating a cancer characterized by dPSA expression in a subject, the method comprising administering to the subject a dPSA-binding agent according to any one of claims 1-7 or a composition according to claim 8.

16. A method of delivering a payload to a cell expressing dPSA, the method comprising contacting the cell with a dPSA binder according to any one of claims 1-7 conjugated to the payload.

17. The method of claim 16, wherein the payload is a cytotoxic molecule or a detectable label.

18. The method of claim 16 or 17, wherein the cell is a cancer cell.

19. A method of detecting soluble dPSA in a biological fluid or tissue sample, comprising contacting the biological fluid or tissue sample with a dPSA binding agent according to any one of claims 1 to 7, optionally conjugated to a detectable label.

20. The method of claim 19, wherein the method is used to detect cancer or select patients for treatment with a dPSA binding agent.

21. The dPSA binding agent of any one of claims 1-7 or the composition of claim 8 for use in treating cancer.

22. Use of a dPSA binder according to any one of claims 1 to 7 or a composition according to claim 8 in the manufacture of a medicament for treating cancer.

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