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

Abstract

The invention relates to dPSA binding agent conjugates, dPSA binding agents, and methods for treating cancer and killing cancer cells.

Description

Cross-reference to related applications

[0001] This patent application claims priority to U.S. Provisional Patent Application 63 / 513,221, filed July 12, 2023, the entire disclosure of which is incorporated herein by reference.

[0002] Merging of sequence lists The computer-readable nucleotide / amino acid sequence listing submitted concurrently with this application is incorporated herein by reference in its entirety by reference as follows: a 57,102-byte ASCII (text) file named "514961ST.26.xml", created on July 11, 2024. Background Technology

[0003] Typically, the goal of anticancer immunotherapy is to identify stable antigens that are highly expressed in tumor cells but not shed or secreted. These antigens can then be used as the basis for immunotherapy, for example, as antigens in cancer vaccines or as targets for antibody-based cancer therapies. Ideally, such tumor antigens have acceptable specificity for cancer cell targets to reduce potentially harmful side effects from cross-reactions with non-cancerous cells in the treated subject. This requirement for immunotherapy specificity can be relaxed if cross-reactions affect regenerative cells.

[0004] Altered glycosylation patterns of cell surface proteins occur in almost all types of cancer. Excessive sialylation of glycoproteins and glycolipids is central to the aberrant regulation of cell adhesion in metastatic cancers, which may stem from the re-expression and / or overexpression of genes normally expressed during development but not in normal adult tissue cells. In particular, poly(α2→8) N-acetylneuraminic acid, or polysialic acid (polySia), is primarily expressed during fetal development and is highly confined to a few regenerating tissues post-development. A de-N-acetylated form of polySia (dPSA) exists on the surface of cancer cells but not on the surface of post-developmental human cells and could serve as a tumor antigen for cancer recognition and treatment.

[0005] Therefore, there is a need for new formulations that can target cells expressing dPSA and deliver payloads (e.g., cytotoxic fractions). Summary of the Invention

[0006] This article provides a dPSA binding conjugate comprising an immunoglobulin heavy chain and a light chain polypeptide, and a payload conjugated to a binding agent. This article also provides a method for delivering the payload to dPSA-expressing cells using the dPSA binding agent, and for killing cancer cells or otherwise treating cancer.

[0007] As will be apparent in the following detailed description, related compositions and methods are also provided. Attached Figure Description

[0008] Figure 1 The graph depicts the average fluorescence intensity (MFI) of the SAC-1 antibody relative to the concentration of the SEAM 3 reference antibody, indicating that the test antibody does not bind to the same epitope as the reference antibody.

[0009] Figure 2 The graph depicts the average fluorescence intensity (MFI) of the SAC-2 antibody relative to the concentration of the SEAM-3 reference antibody, indicating that the test antibody does not bind to the same epitope as the reference antibody.

[0010] Figure 3A and 3B The protein was shown to be co-immunoprecipitated on SDS-PAGE by SAC-1, SAC-2 and control IgG1 antibodies.

[0011] Figure 4A , 4B 4C showed that SAC-3 had an effect on normal human breast tissue ( Figure 4A ) and breast tumors ( Figure 4C The staining of tumors with IgG2a antibody was compared with that of control IgG2a antibody. Figure 4B (Compare)

[0012] Figure 5A , 5B 5C indicates the amount of SAC-1 antibody ( Figure 5A ) or SAC-2 antibody level ( Figure 5B The curves relative to relative luminescent units (RLU) demonstrate the antibody-dependent cytotoxicity (ADCC) of each antibody against various cell lines, as well as the quantitative values ​​( Figure 5C ).

[0013] Figure 6A and 6B The graph plots show the cell-killing effect of defucosylated SAC-2.1C and SAC-2.1D (referred to as "SAC-2.1CaFUC" and "SAC-2.1DaFUC" respectively) against the concentrations of these antibodies, illustrating the effect of defucosylated antibodies on human A375 melanoma (…). Figure 6A ) and MDA-MB-231 breast cancer ( Figure 6B The effect of ADCC activity on cell lines.

[0014] Figure 7A and 7B The results show that SAC-1.1 (SAC-1.1) was effective in the A375 xenograft mouse model of human melanoma. Figure 7A ) and SAC-2 ( Figure 7BThe dose-dependent effect of treatment on tumor growth.

[0015] Figure 8A and 8B The study demonstrated the effects of SAC-1.1, mouse SAC-2, and SAC-2.1C on tumor growth in an MDA-MB-231 xenograft mouse model of human breast cancer, compared to the vector control and cyclophosphamide treatment. Figure 8A ), and the effect of adding human PBMCs in combination with SAC-2.1C on tumor growth ( Figure 8B ).

[0016] Figure 9 The structure of the vc-MMAE linker-payload combination for antibody-drug conjugates (ADCs) for SAC-1 and SAC-2D is depicted.

[0017] Figure 10A and 10B This plots a curve showing the response (mAU) versus retention time (min), displaying the purity of the SAC-2D-vc-MMAE ADC determined by size exclusion chromatography (SEC). Figure 10A The drug-antibody ratio (DAR) of the ADC was determined by high-performance ion exchange chromatography (HIC).

[0018] Figure 11A and 11B In the A375 human melanoma mouse model treated with SAC-2D-vc-MMAE, tumor volume relative to the number of days after treatment initiation ( Figure 11A ) and relative change in weight (%) relative to the number of days after treatment started ( Figure 11B The curve of SAC-2D-vc-MMAE shows that it inhibits tumor growth or causes complete tumor regression in a dose-dependent manner, and the treatment did not result in weight loss.

[0019] Figure 12A and 12B In the HCC1954 human breast cancer mouse model treated with SAC-2D-vc-MMAE, the tumor volume relative to the number of days after treatment initiation ( Figure 12A ) and relative change in weight (%) relative to the number of days after treatment started ( Figure 12B The curve of SAC-2D-vc-MMAE shows that it inhibits tumor growth or causes complete tumor regression in a dose-dependent manner, and the treatment did not result in weight loss. Detailed Implementation

[0020] dPSA is a de-N-acetylated polySia (dPSA) form present on the surface of cancer cells but not on the surface of 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 ONE6:e27249 (2011)). Since cell surface dPSA is specific to cancer cells and is widely expressed in various cancers, formulations that preferentially bind to cells expressing dPSA can be used to target cancer cells for diagnosis and treatment.

[0021] Humans have two genes, ST8SIA2 and ST8SIA4, which encode enzymes that synthesize polysia (polysialidases ST8SIA2 and ST8SIA4, respectively). Although both genes are highly expressed during human 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 normal tissue in adults (Angata et al., J. Biol. Chem., 272(11): 7182-90 (1997)).

[0022] Several proteins have been shown to be polysialylated in humans (see, for example, Curreli et al., J. Biol. Chem., 282(42): 30346-56 (2007); Finne et al., Biochem. Biophys. Res. Commun. 112(2): 482-7 (1983); Simon et al., J. Biol. Chem., 288(26):18825-33 (2013); Werneburg et al., Glia, 64(8):1314-30 (2016); Werneburg et al., Glia, 63(7):1240-55 (2015); Yabe et al., J. Biol. Chem., 278(16): 13875-80 (2003)). Neural cell adhesion molecules (NCAMs) are the most abundant, especially during fetal development, and are also the most thoroughly studied (Rutishauser, U., Nat'l Rev. Neurosci., 9(1): 26-35 (2008)). It has been reported that many human cancers 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)), and its role in mediating cell-cell and cell-extracellular matrix interactions 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 inventors identified nucleolin as a protein modified by or associated with dPSA and showed that cell surface dPSA depends on the expression of ST8SIA2 (Moe et al., J. Exp. Clin. Cancer Res. 40(1): 293 (2021)).

[0023] This document provides a binding conjugate comprising a dPSA conjugate (e.g., an antibody or antibody fragment) conjugated to a payload (e.g., a cytotoxic portion). The dPSA conjugate selectively binds to cells expressing dPSA, particularly cancer cells. In some embodiments, the dPSA conjugate binds to nucleolin modified with dPSA. It is not intended to be limited to any particular theory or mechanism of action, but it is considered that the conjugate binds to antigens (e.g., nucleolin) containing epitopes at least partially defined by one or more dPSA residues.

[0024] dPSA binders can contain immunoglobulin heavy and light chain polypeptides, each polypeptide containing at least one immunoglobulin heavy chain variable region and one immunoglobulin light chain variable region. Each immunoglobulin heavy and light chain variable region contains three complementarity-determining regions (CDRs), commonly referred to as CDR1, CDR2, or CDR3. CDR regions can also be designated using "H" or "L" in nomenclature to represent the heavy or light chain, i.e., CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3. The CDR of a given immunoglobulin sequence can be determined using any of several conventional numbering schemes, such as Kabat, Chothia, Martin (enhanced Chothia), IGMT, or AHo (these are common names for numbering schemes widely used in this field and described in published literature; see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, NIH (1991) describing the "Kabat" numbering scheme; Chothia et al., Canonical Structures for the Hypervariable Regions of Immunoglobulins, J. Mol. Biol., 196:901-917 (1987) and Al-Lazikani et al., Standard Conformations for the Canonical Structures of Immunoglobulins, J. Mol. Biol., 273:927–948 (1997) describing the "Chothia" numbering scheme; Abhinandan et al., Analysis and Improvements to Kabatand Structurally Correct Numbering of Antibody Variable Domains, Mol.Immunol., 45: 3832 – 3839 (2008) 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 Isuperfamily V-like domains, Dev. Comp. Immunol., 27: 55 - 77 (2003) describe 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) describe the "AHo" numbering scheme. The identification of CDRs can also be performed using relevant empirical data, such as crystallographic studies of the interaction between the binder and its target (e.g., an antigen containing a binding epitope or a portion thereof), optionally in conjunction with any of the aforementioned numbering systems.

[0025] The dPSA binders described herein are artificial and not naturally occurring. They are produced using laboratory techniques and are therefore appropriately considered as recombinant or synthetic molecules containing recombinant or synthetic amino acid sequences. Immunoglobulin heavy and light chain polypeptides can be "isolated," meaning they are removed from their environment of origin (e.g., cell culture) and purified to any extent.

[0026] In some embodiments, the dPSA binder comprises an immunoglobulin heavy chain variable region comprising any one of SEQ ID NOs: 1-4 or at least its CDR; and an immunoglobulin light chain variable region comprising SEQ ID NO: 5 or at least its CDR. The CDR can be determined according to any known numbering scheme, such as Kabat, Chothia, Martin (enhanced Chothia), IGMT, or AHo. In some embodiments, the antibody comprises the heavy chain variable region of any one of SEQ ID NOs: 1-4 and the light chain variable region of SEQ ID NO: 5, or at least its CDR as determined by Kabat. In some embodiments, the antibody comprises the heavy chain variable region of any one of SEQ ID NOs: 1-4 and the light chain variable region of SEQ ID NO: 5, or at least its CDR as determined by Chothia. In some embodiments, the antibody comprises the heavy chain variable region of any one of SEQ ID NOs: 1-4 and the light chain variable region of SEQ ID NO: 5, or at least its CDR as 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 CDR 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 CDR as determined by AHo. In some embodiments, the dPSA binder comprises one of the following combinations of immunoglobulin heavy and light chain variable regions, or at least its CDR as determined by any one of Kabat, Chothia, Martin (enhanced Chothia), IGMT, or AHo: Heavy chain variable region Light chain variable region 1 SEQ ID NO: 1 or its CDR SEQ ID NO: 5 or its CDR 2 SEQ ID NO: 2 or its CDR SEQ ID NO: 5 or its CDR 3 SEQ ID NO: 3 or its CDR SEQ ID NO: 5 or its CDR 4 SEQ ID NO: 4 or its CDR SEQ ID NO: 5 or its CDR In some embodiments, the dPSA binder comprises an immunoglobulin heavy chain variable region comprising any one or at least one of the CDRs of SEQ ID NOs:17-20; and an immunoglobulin light chain variable region comprising SEQ ID NO:21 or at least one of its CDRs. The CDR can be determined according to any known numbering scheme, such as Kabat, Chothia, Martin (enhanced Chothia), IGMT, or AHo. In some embodiments, the antibody comprises the heavy chain variable region of any one of SEQ ID NOs:17-20 and the light chain variable region of SEQ ID NO:21, or at least its CDR as determined by Kabat. In some embodiments, the antibody comprises the heavy chain variable region of any one of SEQ ID NOs:17-20 and the light chain variable region of SEQ ID NO:21, or at least its CDR as 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 CDR as 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 CDR 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 CDR as determined by AHo. In some embodiments, the dPSA binder comprises one of the following combinations of immunoglobulin heavy and light chain variable regions, or at least its CDR as determined by any one of Kabat, Chothia, Martin (enhanced Chothia), IGMT, or AHo: Heavy chain variable region Light chain variable region 1 SEQ ID NO: 17 or its CDR SEQ ID NO: 21 or its CDR 2 SEQ ID NO: 18 or its CDR SEQ ID NO: 21 or its CDR 3 SEQ ID NO: 19 or its CDR SEQ ID NO: 21 or its CDR 4 SEQ ID NO: 20 or its CDR SEQ ID NO: 21 or its CDR In some embodiments, this document provides a dPSA binder comprising an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, wherein the immunoglobulin heavy chain variable region comprises: CDR1, comprising any one of SEQ ID NOs: 6-9 or 24-27; CDR2, comprising SEQ ID NO: 10 or 28; and CDR3, comprising SEQ ID NO: 11 or 29; and the immunoglobulin light chain variable region comprises CDR1, comprising SEQ ID NO: 12 or 30; CDR2, comprising SEQ ID NO: 13 (RMS) or 31; and CDR3, comprising SEQ ID NO: 14 or 32.

[0027] In some embodiments, this document provides a dPSA binder comprising an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, wherein the immunoglobulin heavy chain variable region comprises: CDR1, comprising any one of SEQ ID NOs: 6-9; CDR2, comprising SEQ ID NO: 10; and CDR3, comprising SEQ ID NO: 11; and the immunoglobulin light chain variable region comprises CDR1, comprising SEQ ID NO: 12; CDR2, comprising SEQ ID NO: 13; and CDR3, comprising SEQ ID NO: 14.

[0028] 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 In some embodiments, this document provides a dPSA binder comprising an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, wherein the immunoglobulin heavy chain variable region comprises: CDR1, comprising any one of SEQ ID NOs:24-27; CDR2, comprising SEQ ID NO: 28; and CDR3, comprising SEQ ID NO: 29; and the immunoglobulin light chain variable region comprises CDR1, comprising SEQ ID NO: 30; CDR2, comprising SEQ ID NO: 31; and CDR3, comprising SEQ ID NO: 32.

[0029] CDRH1 CDRH2 CDRH3 CDRL1 CDRL2 CDRL3 1 SEQ ID NO: 24 SEQ ID NO: 28 SEQ ID NO: 29 SEQ ID NO: 30 SEQ ID NO: 31 SEQ ID NO: 32 2 SEQ ID NO: 25 SEQ ID NO: 28 SEQ ID NO: 29 SEQ ID NO: 30 SEQ ID NO: 31 SEQ ID NO: 32 3 SEQ ID NO: 26 SEQ ID NO: 28 SEQ ID NO: 29 SEQ ID NO: 30 SEQ ID NO: 31 SEQ ID NO: 32 4 SEQ ID NO: 27 SEQ ID NO: 28 SEQ ID NO: 29 SEQ ID NO: 30 SEQ ID NO: 31 SEQ ID NO: 32 5 SEQ ID NO: 24 SEQ ID NO: 10 SEQ ID NO: 11 SEQ ID NO: 12 SEQ ID NO: 13 SEQ ID NO: 14 6 SEQ ID NO: 25 SEQ ID NO: 10 SEQ ID NO: 11 SEQ ID NO: 12 SEQ ID NO: 13 SEQ ID NO: 14 7 SEQ ID NO: 26 SEQ ID NO: 10 SEQ ID NO: 11 SEQ ID NO: 12 SEQ ID NO: 13 SEQ ID NO: 14 8 SEQ ID NO: 27 SEQ ID NO: 10 SEQ ID NO: 11 SEQ ID NO: 12 SEQ ID NO: 13 SEQ ID NO: 14 According to another aspect of this disclosure, the dPSA binder comprises an immunoglobulin heavy chain variable region comprising an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 1-4 (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%); and an immunoglobulin light chain variable region comprising an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 1-4. 5. 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%). In some embodiments, the dPSA binder comprises an immunoglobulin heavy chain polypeptide containing an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 15 (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%); and an immunoglobulin light chain polypeptide containing an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 16 (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%). In any of the foregoing embodiments, the immunoglobulin heavy chain variable region and the immunoglobulin light chain variable region may comprise (reserved) sequences of the CDRs of the heavy chain and light chain variable regions, which may be determined using any known numbering scheme, such as Kabat, Chothia, Martin (enhanced Chothia), IGMT, or AHo, or as described elsewhere herein (e.g., SEQ ID NOs: 6-14 above). In some embodiments, the immunoglobulin heavy chain variable region comprises one of SEQ ID NOs: 1-4, and the immunoglobulin light chain variable region comprises SEQ ID NO: 5.In some embodiments, the dPSA binder comprises immunoglobulin heavy chain and light chain polypeptides, comprising SEQ ID NO: 15 and SEQ ID NO: 16, respectively.

[0030] According to another aspect of this disclosure, the dPSA binder comprises an immunoglobulin heavy chain variable region containing an amino acid sequence having at least 80% sequence identity with any of SEQ ID NOs: 17-20 (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%); and an immunoglobulin light chain variable region containing an amino acid sequence having at least 80% sequence identity with any of SEQ ID NOs: 17-20. 21. 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%). In some embodiments, the dPSA binder comprises an immunoglobulin heavy chain polypeptide containing an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 22 (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%); and an immunoglobulin light chain polypeptide containing an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 23 (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%). In any of the foregoing embodiments, the immunoglobulin heavy chain variable region and the immunoglobulin light chain variable region may include (retain) the CDRs of the heavy chain and light chain variable regions, which may be identified using any known numbering scheme, such as Kabat, Chothia, Martin (enhanced Chothia), IGMT, or AHo, or as described elsewhere herein (e.g., SEQ ID NOs: 6-14 or 24-32 above). In some embodiments, the immunoglobulin heavy chain and light chain variable regions respectively include one of SEQ ID NOs: 17-20 and SEQ ID NO: 21.In some embodiments, the dPSA binder comprises immunoglobulin heavy chain and light chain polypeptides, comprising SEQ ID NO: 22 and SEQ ID NO: 23, respectively.

[0031] In some embodiments, the dPSA binder comprises immunoglobulin heavy chain and light chain polypeptides, comprising SEQ ID NOs: 33 and 34, respectively.

[0032] In some embodiments, the dPSA binder comprises an immunoglobulin heavy chain variable region comprising SEQ ID NO: 35 or at least its CDR; and an immunoglobulin light chain variable region comprising SEQ ID NO: 36 or at least its CDR. The CDR can be determined according to any known numbering scheme, such as Kabat, Chothia, Martin (enhanced Chothia), IGMT, or AHo. In some embodiments, the antibody comprises the heavy chain variable region of SEQ ID NO: 35 and the light chain variable region of SEQ ID NO: 36, or at least its CDR as determined by Kabat. In some embodiments, the antibody comprises the heavy chain variable region of SEQ ID NO: 35 and the light chain variable region of SEQ ID NO: 36, or at least its CDR as determined by Chothia. In some embodiments, the antibody comprises the heavy chain variable region of SEQ ID NO: 35 and the light chain variable region of SEQ ID NO: 36, or at least its CDR as determined by Martin. In some embodiments, the antibody comprises the heavy chain variable region of SEQ ID NO: 35 and the light chain variable region of SEQ ID NO: 36, or at least its CDR as determined by IGMT. In some embodiments, the antibody comprises the heavy chain variable region of SEQ ID NO: 35 and the light chain variable region of SEQ ID NO: 36, or at least its CDR as determined by AHo.

[0033] In some embodiments, the dPSA binder comprises an immunoglobulin heavy chain variable region comprising SEQ ID NO: 51 or at least its CDR; and an immunoglobulin light chain variable region comprising SEQ ID NO: 52 or 53 or at least its CDR. The CDR can be determined according to any known numbering scheme, such as Kabat, Chothia, Martin (enhanced Chothia), IGMT, or AHo. In some embodiments, the antibody comprises the heavy chain variable region of SEQ ID NO: 51 and the light chain variable region of SEQ ID NO: 52 or 53, or at least its CDR as determined by Kabat. In some embodiments, the antibody comprises the heavy chain variable region of SEQ ID NO: 51 and the light chain variable region of SEQ ID NO: 52 or 53, or at least its CDR as determined by Chothia. In some embodiments, the antibody comprises the heavy chain variable region of SEQ ID NO: 51 and the light chain variable region of SEQ ID NO: 52 or 53, or at least its CDR as determined by Martin. In some embodiments, the antibody comprises the heavy chain variable region of SEQ ID NO: 51 and the light chain variable region of SEQ ID NO: 52 or 53, or at least its CDR as determined by IGMT. In some embodiments, the antibody comprises the heavy chain variable region of SEQ ID NO: 51 and the light chain variable region of SEQ ID NO: 52 or 53, or at least its CDR as determined by AHo.

[0034] In some embodiments, this document provides a dPSA binder comprising an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, wherein the immunoglobulin heavy chain variable region comprises: CDR1, comprising SEQ ID NO: 39 or 45; CDR2, comprising SEQ ID NO: 40 or 46; and CDR3, comprising SEQ ID NO: 41 or 47; and the immunoglobulin light chain variable region comprises CDR1, comprising SEQ ID NO: 42 or 48; CDR2, comprising SEQ ID NO: 43 (GTN), 49 or 56; and CDR3, comprising SEQ ID NO: 44 or 50. In some embodiments, the dPSA binder comprises CDR1, comprising SEQ ID NO: 39; CDR2, comprising SEQ ID NO: 40; and CDR3, comprising SEQ ID NO: 41 or 47; and the immunoglobulin light chain variable region comprises CDR1, comprising SEQ ID NO: 42; CDR2, comprising SEQ ID NO: 43 or 56; and CDR3, comprising SEQ ID NO: 44.

[0035] In some embodiments, this document provides a dPSA binder comprising an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, wherein the immunoglobulin heavy chain variable region comprises: CDR1, comprising SEQ ID NO: 45; CDR2, comprising SEQ ID NO: 46; and CDR3, comprising SEQ ID NO: 41 or 47; and the immunoglobulin light chain variable region comprises CDR1, comprising SEQ ID NO: 48; CDR2, comprising SEQ ID NO: 49 or 56; and CDR3, comprising SEQ ID NO: 50.

[0036] CDRH1 CDRH2 CDRH3 CDRL1 CDRL2 CDRL3 1 SEQ ID NO: 39 SEQ ID NO: 40 SEQ ID NO: 41 or 47 SEQ ID NO: 42 SEQ ID NO: 43 or 56 SEQ ID NO: 44 2 SEQ ID NO: 39 SEQ ID NO: 40 SEQ ID NO: 47 SEQ ID NO: 42 SEQ ID NO: 43 SEQ ID NO: 44 3 SEQ ID NO: 39 SEQ ID NO: 40 SEQ ID NO: 41 SEQ ID NO: 42 SEQ ID NO: 43 SEQ ID NO: 44 4 SEQ ID NO: 39 SEQ ID NO: 40 SEQ ID NO: 47 SEQ ID NO: 42 SEQ ID NO: 56 SEQ ID NO: 44 5 SEQ ID NO: 39 SEQ ID NO: 40 SEQ ID NO: 41 SEQ ID NO: 42 SEQ ID NO: 56 SEQ ID NO: 44 6 SEQ ID NO: 45 SEQ ID NO: 46 SEQ ID NO: 41 or 47 SEQ ID NO: 48 SEQ ID NO: 49 or 56 SEQ ID NO: 50 7 SEQ ID NO: 45 SEQ ID NO: 46 SEQ ID NO: 47 SEQ ID NO: 48 SEQ ID NO: 49 SEQ ID NO: 50 8 SEQ ID NO: 45 SEQ ID NO: 46 SEQ ID NO: 41 SEQ ID NO: 48 SEQ ID NO: 49 SEQ ID NO: 50 9 SEQ ID NO: 45 SEQ ID NO: 46 SEQ ID NO: 47 SEQ ID NO: 48 SEQ ID NO: 56 SEQ ID NO: 50 10 SEQ ID NO: 45 SEQ ID NO: 46 SEQ ID NO: 41 SEQ ID NO: 48 SEQ ID NO: 56 SEQ ID NO: 50 In some embodiments, the dPSA binder comprises an immunoglobulin heavy chain variable region containing an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 35 (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%); and an immunoglobulin light chain variable region containing an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 36 (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%). In some embodiments, the dPSA binder comprises an immunoglobulin heavy chain polypeptide comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO:37 (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%); and an immunoglobulin light chain polypeptide comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO:38 (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%). In any of the foregoing embodiments, the immunoglobulin heavy chain variable region and the immunoglobulin light chain variable region may include (retain) the CDRs of the heavy chain and light chain variable regions, respectively, of SEQ ID NO: 35 and 36. These CDRs may be determined using any known numbering scheme, such as Kabat, Chothia, Martin (enhanced Chothia), IGMT, or AHo, or as described elsewhere herein (e.g., SEQ ID NOs: 39-44). In some embodiments, the immunoglobulin heavy chain and light chain variable regions comprise SEQ ID NO: 35 and SEQ ID NO: 36, respectively.In some embodiments, the dPSA binder comprises immunoglobulin heavy chain and light chain polypeptides, comprising SEQ ID NO: 37 and SEQ ID NO: 38, respectively.

[0037] In some embodiments, the dPSA binder comprises an immunoglobulin heavy chain variable region containing an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 51 (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%); and an immunoglobulin light chain variable region containing an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 52 or 53 (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 binder comprises an immunoglobulin heavy chain polypeptide comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 51 (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%); and an immunoglobulin light chain polypeptide comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 52 or 53 (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%). In any of the foregoing embodiments, the immunoglobulin heavy chain variable region and the immunoglobulin light chain variable region may include (retain) the CDRs of the heavy chain and light chain variable regions, respectively, of SEQ ID NO: 51 and 52 or 53. These CDRs may be determined using any known numbering scheme, such as Kabat, Chothia, Martin (enhanced Chothia), IGMT, or AHo, or as described elsewhere herein. In some embodiments, the immunoglobulin heavy chain and light chain variable regions respectively include SEQ ID NO: 51 and SEQ ID NO: 52 or 53.

[0038] In some embodiments, the dPSA binder comprises an immunoglobulin heavy chain variable region containing an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 57 or 60 (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%); and an immunoglobulin light chain variable region containing an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 57 or 60. 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, the dPSA binder comprises an immunoglobulin heavy chain polypeptide containing an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 57 (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%); and an immunoglobulin light chain polypeptide containing an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 58 or 59 (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%). In any of the foregoing embodiments, the immunoglobulin heavy chain variable region and the immunoglobulin light chain variable region may include (retain) the CDRs of the heavy chain and light chain variable regions, respectively, of SEQ ID NO: 57 and 58 or 59. These CDRs may be determined using any known numbering scheme, such as Kabat, Chothia, Martin (enhanced Chothia), IGMT, or AHo, or as described elsewhere herein. In some embodiments, the immunoglobulin heavy chain and light chain variable regions respectively include SEQ ID NO: 57 and SEQ ID NO: 58 or 59.In some embodiments, the variable regions of the immunoglobulin heavy chain and light chain respectively comprise SEQ ID NO: 60 and SEQ ID NO: 58 or 59.

[0039] In some embodiments, the dPSA binder comprises variable regions of the immunoglobulin heavy and light chains, which contain SEQ ID NO: 33 and 34, or at least their CDRs determined using any known numbering scheme (e.g., Kabat, Chothia, Martin (enhanced Chothia), IGMT, or AHo); and / or have at least 80% sequence identity with SEQ ID NO: 33 or 34 (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%), while optionally retaining its CDR. In some embodiments, the dPSA binder comprises variable regions of the immunoglobulin heavy and light chains, which contain SEQ ID NO: 54 and 55, or at least their CDRs determined using any known numbering scheme (e.g., Kabat, Chothia, Martin (enhanced Chothia), IGMT, or AHo); and / or have at least 80% sequence identity with SEQ ID NO: 54 or 55 (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%), while optionally retaining its CDR.

[0040] Sequence "identity," used when dealing with nucleic acid or amino acid sequences, is determined by comparing the nucleic acid or amino acid sequence of interest with a reference nucleic acid or amino acid sequence. When the alignment is optimal, the sequence identity percentage is the percentage of identical (i.e., consistent) nucleotide or amino acid residues between the sequence of interest and the reference sequence. Many mathematical algorithms for obtaining optimal alignments and calculating identity between two or more sequences are known and publicly available. Examples of such programs include CLUSTAL-W, T-Coffee, and ALIGN (for nucleic acid and amino acid sequence alignment), BLAST programs (e.g., BLAST 2.1, BL2SEQ, and later versions 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)).

[0041] For sequences having less than 100% identity with the aforementioned specific heavy and light chain sequences, one or more amino acids of the aforementioned immunoglobulin heavy chain polypeptide and / or light chain polypeptide may be replaced or substituted with different amino acids, and / or one or more amino acids may be deleted or inserted from the published amino acid sequence, provided that the bioactivity of the polypeptide (e.g., the ability of the dPSA binder to bind dPSA) is substantially preserved. The bioactivity 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, and surface plasmon resonance analysis (BIACORE). TM ) or solution phase competition (KINEXA) TM (and other in vitro or in vivo neutralization assays, binding assays, fluorescence-activated cell binding assays (FACS) or other suitable assays.)

[0042] dPSA binders 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 may comprise heavy and light chain polypeptides of immunoglobulins that bind dPSA as described herein, combined with heavy and light chains of immunoglobulins that bind antigens other than dPSA.

[0043] A dPSA binder may be part of a conjugate. For example, a dPSA binder may be a conjugate of (1) an anti-dPSA antibody or a fragment thereof, and (2) a secondary protein or non-protein portion. By further explanation, a dPSA binder may comprise an anti-dPSA antibody or a fragment thereof conjugated to another peptide, fluorescent molecule, or chemotherapeutic (e.g., cytotoxic) agent.

[0044] In some implementations, the dPSA binder can be a "whole" immunoglobulin or a "fragment" of an antigen-binding immunoglobulin. A "whole" immunoglobulin typically consists of four polypeptides: two heavy (H) chains and two light (L) chains. Each heavy chain contains an N-terminal variable (VH) region and three C-terminal constant (CH1, CH2, and CH3) regions, and each light chain contains an N-terminal variable (VL) region and a C-terminal constant (CL) region. The light chains of an antibody can be classified into one of two different types based on the amino acid sequence of their constant domains: κ (kappa) or λ (lambda). In typical immunoglobulins, each light chain is linked to one heavy chain by a disulfide bond, and two heavy chains are linked to each other by disulfide bonds. In this configuration, the variable region of the light chain is typically aligned with the variable region of the heavy chain, and the constant region of the light chain is typically aligned with the first constant region of the heavy chain. The remaining constant regions of the heavy chains are typically aligned with each other.

[0045] The variable or hypervariable regions of each pair of light and heavy chains form the antigen-binding site of the antibody. The VH and VL regions have the same overall structure, each containing four frame (FW or FR) regions. As used herein, the term "frame region" refers to a relatively conserved amino acid sequence located within a variable region between a hypervariable or complementarity-determining region (CDR). Each variable domain has four frame regions, designated FR1, FR2, FR3, and FR4. The frame regions form a β-sheet that provides the structural framework for the variable region (see, e.g., CA Janeway et al. (ed.), Immunobiology, 5th Ed., Garland Publishing, New York, NY (2001)). The frame regions are connected by three complementarity-determining regions (CDRs). These three CDRs, CDR1, CDR2, and CDR3, form the "hypervariable region" of the antibody, which is generally considered to be the region responsible for antigen binding.

[0046] The term "antibody fragment" and similar terms (e.g., "antibody fragment," "functional antibody fragment") are used interchangeably herein to refer to one or more antibody fragments or portions that retain the ability to specifically bind to antigens (see, generally, Holliger et al., Nat. Biotech., 23(9): 1126-1129 (2005)). As is commonly used herein and in the field, the term "antibody fragment" includes not only fragments or segments of a complete antibody in the literal sense, but also other known engineered antibody-like constructs that may contain linkers or other elements not naturally present in a "complete" antibody. Examples of antibody fragments include, but are not limited to, (i) Fab fragments, which are monovalent fragments containing VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, which are bivalent fragments containing two Fab fragments connected by a disulfide bridge in the hinge region; (iii) Fv fragments, which consist of the VL and VH domains of an antibody single arm; (iv) Fab' fragments, which are generated by cleaving the disulfide bridge of the F(ab')2 fragment under 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 a monovalent molecule composed of two domains (i.e., VL and VH) of an Fv fragment linked by a synthetic linker that allows these two domains to be synthesized as a single polypeptide chain (see, for example, 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) biantibodies, which are dimers of polypeptide chains, wherein each polypeptide chain contains VH and VL linked by a peptide linker that is too short to allow VH and VL on the same polypeptide chain to pair, thereby driving pairing between complementary domains on different VH-VL polypeptide chains to generate a dimer molecule with two functional antigen-binding sites. Any other antigen-binding antibody-like constructs known in the art that contain CDRs or variable regions of immunoglobulin heavy and light chains may also be used and are considered antibody fragments for the purposes of this disclosure. In some embodiments, the dPSA binder is (or a portion thereof) a chimeric antigen receptor.

[0047] In some embodiments, the dPSA binder comprises a heavy chain constant region, such as a fragment crystallizable (Fc) region or a portion thereof. The Fc region can be any immunoglobulin class / subclass (IgA (IgA1, IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3, and IgG4), IgM, including its variants). In some embodiments, the dPSA binder is a "complete" or "full" immunoglobulin (i.e., an antibody); in other embodiments, the binder is an antibody fragment conjugated to 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.

[0048] In some embodiments, the dPSA binder includes an Fc region that binds FcγR and can mediate complement-dependent cytotoxicity (CDC) or antibody-dependent cytotoxicity (ADCC). In some embodiments, the dPSA binder includes an Fc region that activates natural killer (NK) cells. In some embodiments, the Fc region includes modifications that increase FcγR binding and / or CDC or ADCC compared to the same binder without such modifications. Examples of such modifications include, for example, defucosylation, S298A / E333A / K334A; S239D / I332E (DE); S239D / A330L / I332E (DLE); G236A; G236A / S239D / I332E (ADE); G236A / A330L / I332E (GAALIE); G236A / S239D / A330L / I332E (GASDALIE); F243L / R292P / Y300L / V305I / P396L (LPLIL); L235V / F243L / R292P / Y300L / P396L (VLPLL); or other modifications known in the art.

[0049] In some embodiments, the dPSA binder has reduced FcγR binding and / or reduced complement-dependent cytotoxicity (CDC) or antibody-dependent cytotoxicity (ADCC). This reduced effector function is considered particularly advantageous when the binder is part of an antibody-drug conjugate. Thus, for example, the dPSA binder may comprise an Fc region modified to have reduced FcγR binding and / or reduced complement-dependent cytotoxicity (CDC) or antibody-dependent cytotoxicity (ADCC) compared to the same binder without such modification. Examples of such modifications include, for example, deglycosylation (N297A / Q / G); L235A / G237A / E318A; L234A / L235A (LALA); L234A / L235A / P329G (LALA-PG); S228P / L235E (PE); G236R / L328R (RR); S298G / T299A (GA); L234F / L235E / P331S (FES); H268Q / V309L / A330S / P331S; L234F / L235E / D265A (FEA); or V234A / G237A / P238S / H268A / V309L / A330S / P331S. The dPSA binder can be a human or humanized antibody, a non-human antibody, or a chimeric antibody. "Chimeric" refers to an antibody or fragment thereof containing both 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 derived from a non-human antibody. Non-human antibodies include antibodies isolated from any non-human animal (e.g., rodents, such as mice or rats). Humanized antibodies may contain one, two, or three CDRs derived from non-human antibodies.

[0050] Human antibodies, non-human antibodies, chimeric antibodies, or humanized antibodies can be obtained by any means, including from in vitro sources (e.g., hybridomas or recombinant antibody-producing cell lines) and in vivo sources (e.g., rodents). Methods for producing antibodies are known in the field and described, for example, Köhler 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 some implementations, transgenic animals (e.g., mice) may be used to generate human antibodies or chimeric antibodies, wherein one or more endogenous immunoglobulin genes are replaced with one or more human immunoglobulin genes (see, for example, 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 field (see, for example, An, Z. (ed.), Therapeutic Monoclonal Antibodies: From Benchto Clinic, John Wiley & Sons, Inc., Hoboken, New Jersey (2009)), including, for example, grafting nonhuman CDRs onto human antibody scaffolds (see, for example, Kashmiri et al., Methods, 36(1): 25-34 (2005); and Hou et al., J. Biochem., 144(1): 115-120 (2008)).

[0051] The payload can be any molecule intended to be delivered to cells expressing dPSA. In some embodiments, the payload is a cytotoxic fraction or molecule. In some embodiments, the payload is an antibiotic or an anticancer or antitumor agent. For example, in some embodiments, the payload is a tubulin inhibitor, a DNA topoisomerase I inhibitor, or a DNA topoisomerase II inhibitor. In some embodiments, the payload is monomethylolpropamine E / Dolastatin-10 (MMAE), monomethylolpropamine F (MMAF), maytansine (DM1), maytansine alkaloid derivatives, maytansine (DM1), N2'-deacetyl-N2'-(4-mercapto-4-methyl-1-oxopentyl)matansine (DM4), 12-ethyl-9-hydroxycamptothecin (7-ethyl-10-hydroxycamptothecin (SN-38)), essanotecan mesylate (DX-8951f), PNU-1596821 (a metabolite of the anthracycline drug nemorubicin), pyrrolobenzodiazepines (PBD or SGD-1882), essanotecan derivatives (such as DXD), camptothecin derivatives (such as Camp 98), or taxanes (e.g., paclitaxel). In some embodiments, the payload is essanotecan or an essanotecan derivative (such as DXD).

[0052] The payload can be conjugated to the dPSA binder directly or via a connector. Any suitable connector can be used to attach the payload to the dPSA binder. In some embodiments, connectors used to conjugate the binder to the payload are maleimide hexanoyl-L-valine-L-citrulline-p-aminobenzyl alcohol p-nitrophenyl carbonate (MC-VC-PAB-PNP), glutamic acid-valine-citrulline-p-aminobenzyl alcohol (Glu-VC-PAB), N-hydroxysuccinimide-glutamic acid-valine-citrulline-p-aminobenzyl alcohol (NHS-Glu-VC-PAB), and azido-glutamic acid-valine-citrulline-p-aminobenzyl alcohol (Azido-Glu-V C-PAB), hydrazone, succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester (SMCC), fluorenemethoxycarbonyl-valine-alanine-p-aminobenzyl alcohol p-nitrophenyl carbonate (Fmoc-Val-Ala-PAB-PNP), maleimide hexanoyl-L-valine-L-citrulline-p-aminobenzyl alcohol pentafluorophenyl carbonate (MC-VC-PAB-PFP), maleimide hexanoyl-valine-lysine (PEG)-p-aminobenzyl alcohol (MC-Val-Lys) (PEG)-PAB), fluorenylmethoxycarbonyl-valine-alanine-p-aminobenzyl alcohol pentafluorophenyl carbonate (Fmoc-Val-Ala-PAB-PFP), Fmoc-L-valine-L-citrulline-p-aminobenzyl alcohol (Fmoc-VC-PAB), maleimide hexanoyl-L-valine-L-citrulline-p-aminobenzyl alcohol (MC-VC-PAB), L-valine-L-citrulline-p-aminobenzyl alcohol (VC-P) AB), maleimide hexanoyl-glycine-glycine-L-phenylalanine-glycine (MC-GGFG), succinimide-(N-maleimidepropamido-N-maleimidepropamido-diethylene glycol)-glycine-glycine-L-phenylalanine-glycine-4-aminohexanoate (SM-GGFG-4AH), or maleimide hexanoyl-β-glucuronide (MC-beta-glucuronide). In some embodiments, the binder comprises a linker conjugated to a lysine residue of the antibody (e.g., as described above) and a payload attached to the linker. In some embodiments, the binder comprises a linker conjugated to a cysteine ​​residue of the antibody (e.g., as described above) and a payload attached to the linker.

[0053] In some embodiments, the binder is conjugated with a load and linker selected from the following: valine-citrulline-monomethylaurestatin E (vcMMAE), maleimide hexanoylmonomethylaurestatin F (mcMMAF), N-((S)-7-benzyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazatridecane-13-yl)-6-(2,5-dioxo) -2,5-Dihydro-1H-pyrrolo-1-yl)hexanoamide (MC-GGFG-Dxd), 6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-N-(2-((2-(((S)-1-((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)hexanoamide (MC- GGFG-Essanotecan), 4-((S)-2-((S)-2-(2-(aminooxy)acetamido)-3-methylbutamido)-5-ureidopentamido)benzyl((S)-1-(((S)-1-(((3R,4S,5R)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (HA- VC-PAB-MMAE), 4-(2-(4-aminobutyl)-35-(4-((4-((2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)methyl)cyclohexane-1-carboxamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxa-3,9-diazapentadecanoamide)benzyl((S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolazino[1,2-b]quinoline-4-yl) carbonate (CL2A-SN38), or 2,5-Dioxopyrrolidine-1-yl(1S,4r)-4-((3-((3-(((S)-1-(((14S,16S,32S,33S,2R,4S,10E,12E,14R)-86-chloro-14-hydroxy-85,14-dimethoxy-33,2,7,10-tetramethyl-12,6-dioxo-7-aza-1(6,4)-oxazine-3(2,3)-ethylene oxide-8(1,3)-phenylcyclotetradecane-10,12-dioxopyrrolidine-1-yl(1S,4)-oxazine-3(2,3)-ethylene oxide-8(1,3)-phenylcyclotetradecane-10,12-dioxopyrrolidine-1-yl(1S,4r)-4-((3-((3-(((S)-1-(((14S,16S,32S,33S,2R,4S,10E,12E,14R)-86-chloro-14-hydroxy-85,14-dimethoxy-33,2,7,10-tetramethyl-12,6-dioxo-7-aza-1(6 ... MC-4-yl)oxy)-1-oxopropane-2-yl)methyl)amino)-3-oxopropyl)thio)-2,5-dioxopyrrolidone-1-yl)methyl)cyclohexane-1-carboxylic acid ester (DM1-SMCC); MC-β-glucuronide-MMAE; MC-Val-Lys(PEG)-PAB-paclitaxel; MC-Val-Cit-PAB-matansine; MC-GGFG-exatecan; or MC-Val-Cit-PAB-exatecan.

[0054] The dPSA binders or conjugates provided herein can be used for any purpose. For example, dPSA binders or conjugates can be used to target or kill cancer cells expressing dPSA (e.g., cancer cells containing dPSA on their cell surface). Therefore, this document provides a method for targeting or killing cancer cells, in vitro or in vivo, comprising administering a dPSA binder or conjugate as described herein to cancer cells. When the method is used to target or kill cancer cells in vivo, the dPSA binder or conjugate can be administered to the cancer cells by administering the dPSA binder or conjugate to a subject containing the cancer cells.

[0055] Cancer cells can be any cancer cell that expresses dPSA on its cell surface. For example, cancer cells may contain a protein, such as a nucleolin linked to or associated with dPSA on its surface. In some embodiments, cancer cells express ST8SIA2.

[0056] Binders can also be used for diagnostic or therapeutic purposes and can be used in vitro, ex vivo, or in vivo. For example, dPSA binders can be conjugated to detectable markers or supports (e.g., radiolabeled, fluorescently labeled, beads, scaffolds, etc.) to analyze dPSA expression levels or facilitate the detection of cancer cells expressing dPSA in biological samples (biological fluids or tissue samples) from a subject. For example, the detectable portion can be a radioisotope (e.g., 3 H, 14 C 32 P, 35 S or 1251) 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.). Within the scope of this invention, any method known in the art for conjugating antigen-binding agents (e.g., antibodies) to such portions may be used (see, for example, 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)).

[0057] In some embodiments, the dPSA binder exhibits antibody-dependent cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) and kills dPSA-expressing cancer cells by binding to cancer cells and mediating ADCC or CDC-mediated cell death. In some embodiments, the dPSA binder is defucosylated. The defucosylated binder (e.g., an antibody) can be prepared by any suitable technique, such as by expressing nucleic acids encoding the heavy and light chains of the immunoglobulin that encode the dPSA binder in a cell line where the FUT8 gene is disrupted or missing (e.g., FUT8-deficient CHO cells).

[0058] Therefore, the dPSA binders and their methods of use provided herein can be used to treat cancers characterized by dPSA surface expression. As used herein, the terms "treatment," "treating," etc., refer to achieving the desired pharmacological and / or physiological effects, such as reducing the severity of disease or inhibiting the progression of disease and / or adverse symptoms attributable to disease. To this end, the method of the present invention comprises administering a "therapeuticly effective amount" of a dPSA binder or its conjugate. A "therapeuticly effective amount" refers to an amount that effectively achieves the desired therapeutic outcome within the necessary dose and time period. Therapeuticly effective amounts can vary depending on factors such as disease state, individual age, sex, weight, and the ability of the dPSA binder or conjugate to elicit the desired response in the individual.

[0059] The methods and compositions provided herein are useful in the treatment or prevention of a variety of cancers, including carcinoma, sarcoma, leukemia, myeloma, and lymphoma.

[0060] Cancers that can be treated using the methods disclosed herein include, but are not limited to: esophageal cancer, hepatocellular carcinoma, basal cell carcinoma (a type of skin cancer), squamous cell carcinoma (various tissues), bladder cancer (including transitional cell carcinoma (a malignant bladder tumor)), bronchogenic carcinoma, colon cancer, colorectal cancer, gastric cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), adrenocortical carcinoma, thyroid cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary carcinoma, papillary adenocarcinoma, cystic adenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, nephroblastoma, cervical cancer, uterine cancer, testicular cancer, bone-derived cancer, epithelial carcinoma, and nasopharyngeal carcinoma.

[0061] Sarcomas that can be treated using the methods disclosed herein include, but are not limited to: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteosarcoma, osteosarcoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovoma, mesothelioma, Ewing sarcoma, leiomyosarcoma, rhabdomyosarcoma and other soft tissue sarcomas.

[0062] Other solid tumors that can be treated using the methods disclosed herein include, but are not limited to: glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0063] Other cancers include leukemia, lymphoma, and myeloma (including multiple myeloma). Leukemias that can be treated with the methods disclosed herein include, but are not limited to: a) chronic myeloproliferative syndromes (neoplastic diseases of pluripotent hematopoietic stem cells); b) acute myeloid leukemia (neoplastic transformation of pluripotent hematopoietic stem cells or lineage-restricted hematopoietic cells); c) chronic lymphocytic leukemia (CLL; clonal proliferation of immunologically immature and dysfunctional small lymphocytes), including B-cell CLL, T-cell CLL, prolymphocytic leukemia, and hairy cell leukemia; and d) acute lymphoblastic leukemia (characterized by lymphoblastic accumulation). Lymphomas that can be treated with the methods described herein include, but are not limited to, B-cell lymphomas (e.g., Burkitt lymphoma); Hodgkin lymphoma; non-Hodgkin lymphoma, etc.

[0064] Other cancers that can be treated using the methods disclosed in this article include atypical meningioma (brain), islet cell carcinoma (pancreas), medullary carcinoma (thyroid), mesenchymal carcinoma (intestine), hepatocellular carcinoma (liver), hepatoblastoma (liver), clear cell carcinoma (kidney), and neurofibromatosis mediastinum.

[0065] Further exemplary cancers that can be treated according to the methods disclosed herein include, but are not limited to, cancers of neuroectodermal and epithelial origin. Examples of neuroectodermal cancers include, but are not limited to, Ewing sarcoma, spinal cord tumors, brain tumors, infantile supratentorial primitive neuroectodermal tumors, tubular cystic carcinomas, mucinous tubular and spindle cell carcinomas, renal tumors, mediastinal tumors, gliomas, neuroblastomas, and sarcomas in adolescents and young adults. Examples of epithelial cancers include, but are not limited to, small cell lung cancer, cancers of the breast, lens, colon, pancreas, kidney, liver, ovary, and bronchial epithelium. In some embodiments, the methods described herein do not include treatment of melanoma (i.e., the cancer is not melanoma). In other embodiments, the methods described herein do not include treatment of lymphoma (i.e., the cancer is not lymphoma).

[0066] The dPSA binder or conjugate may be part of a composition suitable for administration to mammals. 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 the amino acid sequence, antigen binder, conjugate, or carrier of the present invention. Any suitable carrier may be used within the scope of the invention, and such carriers are well known in the art. The choice of carrier will depend in part on the specific site on which the composition may be administered and the specific method of administration. The composition may also contain any other excipients for formulation of therapeutic molecules (e.g., proteins or antibodies), particularly parenteral preparations, including, for example, buffers, osmotic regulators, stabilizers, surfactants, etc. The composition may be sterile. The composition may be freeze-dried or lyophilized and reconstituted in a suitable sterile carrier prior to use. The composition may be produced according to conventional techniques, for example, as described in Remington: The Science and Practice of Pharmacy, 21st Edition, Lippincott Williams & Wilkins, Philadelphia, PA (2001).

[0067] The composition can be administered using any standard application 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 via intravenous, intraperitoneal, or subcutaneous injection into the peripheral system of mammals.

[0068] dPSA binders or binder conjugates can be administered alone or in combination with other drugs. For example, dPSA binders or binder conjugates can be administered in combination with other agents used to treat or prevent the diseases disclosed herein, such as other anticancer agents. In this regard, for example, dPSA binders or binder conjugates can be used in combination with at least one other agent, including, for example, chemotherapeutic agents, vaccines, biological therapies (e.g., other monoclonal antibodies), radiotherapy, bone marrow transplantation, chemotherapy, biological response modifier therapy, and / or surgery.

[0069] This article also provides nucleic acids encoding dPSA binders (i.e., immunoglobulin heavy chain peptides and / or immunoglobulin light chain peptides encoding dPSA binders).

[0070] The nucleic acid may be a polymer of DNA or RNA (or both, e.g., hybrid DNA / RNA), may be single-stranded or double-stranded, and may contain non-natural or modified nucleotides. The nucleic acid may be part of a vector. The vector may be, for example, a plasmid, episome, granule, viral vector (e.g., retrovirus or adenovirus), or bacteriophage. Suitable vectors and methods of vector preparation are well known in the field (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)).

[0071] The vector typically contains expression control sequences, such as promoters, enhancers, polyadenylation signals, transcription terminators, signal peptides (e.g., osteonectin signal peptide), internal ribosome entry sites (IRES), etc., which provide for the expression of the coding sequence in the host cell. Exemplary expression control sequences are known in the art and described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, Calif. (1990).

[0072] A wide variety of promoters from diverse sources, including constitutive, inducible, and repressible promoters, are well-known in this field. Representative sources of promoters include, for example, viruses, mammals, insects, plants, yeast, and bacteria, from which suitable promoters are readily available or can be synthesized based on publicly available sequences (e.g., from repositories such as ATCC and commercial or personal sources). Promoters 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, the T7 bacterial expression system, the pBAD(araA) bacterial expression system, the cytomegalovirus (CMV) promoter, the SV40 promoter, and the RSV promoter. Inducible promoters include, for example, the Tet system (US Patents 5,464,758 and 5,814,618), the ecdysone-inducible system (No. et al., Proc. Natl. Acad. Sci., 93: 3346-3351 (1996)), and T-REX. TM Systems (Invitrogen, Carlsbad, CA), LACSWITCH TM The system (Stratagene, San Diego, CA) and the Cre-ERT tamoxifen-induced recombinase system (Indra et al., Nuc. Acid. Res., 27: 4324-4327 (1999); Nuc. Acid. Res., 28:e99 (2000); US Patent 7,112,715; and Kramer & Fussenegger, Methods Mol. Biol., 308:123-144 (2005)).

[0073] As used herein, the term "enhancer" refers to a DNA sequence that adds, for example, an operationally linked nucleic acid sequence to the transcription. Enhancers can be located many kilobases outside the coding region of a nucleic acid sequence and can mediate the binding of regulatory factors, DNA methylation patterns, or alterations in DNA structure. A large number of enhancers from a wide variety of sources are well-known in the field and are available as cloned polynucleotides or contained therein (from, for example, repositories such as ATCC, as well as commercial or personal sources). Many polynucleotides containing promoters (such as the commonly used CMV promoter) also contain enhancer sequences. Enhancers can be located upstream, inside, or downstream of the coding sequence.

[0074] The vector may also contain a "selective marker gene". As used herein, the term "selective marker gene" refers to a nucleic acid sequence that allows cells expressing that nucleic acid sequence to be specifically selected or eliminated in the presence of a suitable selector. Suitable selective marker genes are known in the field and described, for example, in international patent applications published 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.

[0075] In some embodiments, the vector is an "attaposome expression vector" or "attaposome" that is capable of replicating in a host cell and persists as an extrachromosomal DNA fragment within the host cell under appropriate selection pressure (see, for example, Conese et al., Gene Therapy, 11: 1735-1742 (2004)). Representative commercially available attachment expression vectors include, but are not limited to, attachment plasmids utilizing Epstein Barr nuclear antigen 1 (EBNA1) and the Epstein Barr virus (EBV) origin of replication (oriP). The vectors pREP4, pCEP4, pREP7, and pcDNA3.1 from Invitrogen (Carlsbad, CA) and pBK-CMV from Stratagene (La Jolla, CA) are non-limiting examples of attachment vectors that use T antigen and SV40 origin of replication instead of EBNA1 and oriP.

[0076] Other suitable vectors include integrative expression vectors, which can be randomly integrated into the host cell's DNA or contain recombination sites to achieve specific recombination between the expression vector and the host cell's chromosome. Such integrative expression vectors can utilize endogenous expression control sequences of the host cell's chromosome to influence the expression of the desired protein. Examples of site-specific integrative vectors include, for example, components of the flp-in system from Invitrogen (Carlsbad, CA) (e.g., pcDNA). TM 5 / FRT), or cre-lox systems, such as those found in the pExchange-6 core vector from Stratagene (La Jolla, CA). Examples of vectors that integrate randomly 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 .

[0077] Viral vectors may also be used. Representative commercially available viral expression vectors include, but are not limited to, the adenovirus-based Per.C6 system 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).

[0078] The nucleic acid sequences encoding the amino acid sequences 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 bidirectional and unidirectional promoters can be used to control the expression of multiple nucleic acid sequences. Alternatively, the nucleic acid sequences encoding the amino acid sequences of the present invention can be provided to a population of cells on separate vectors (i.e., trans). Each nucleic acid sequence in each separate vector can contain the same or different expression control sequences. The separate vectors can be provided to cells simultaneously or sequentially.

[0079] Vectors containing nucleic acids encoding the amino acid sequences of the present invention can be introduced into host cells capable of expressing polypeptides encoded therefrom, including any suitable prokaryotic or eukaryotic cells. Therefore, the present invention provides isolated cells containing the vectors of the present invention. Preferred host cells are those that are readily and reliably grown, have a reasonably rapid growth rate, possess a well-characterized expression system, and are readily and efficiently transformed or transfected.

[0080] Examples of suitable prokaryotic cells include, but are not limited to, cells from the following genera: Bacillus (e.g., Bacillus subtilis and Bacillus brevis), Escherichia (e.g., Escherichia coli), Pseudomonas, Streptomyces, Salmonella, and Erwinia. Particularly useful prokaryotic cells include various strains of Escherichia coli (e.g., K12, HB101 (ATCC number 33694), DH5α, DH10, MC1061 (ATCC number 53338), and CC102).

[0081] In some embodiments, the vector is introduced into eukaryotic cells. 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 cells from the genera *Kluyveromyces*, *Pichia*, *Rhinospordium*, *Saccharomyces*, and *Schizosaccharomyces*. Preferred yeast cells include, for example, *Saccharomyces cerevisiae* and *Pichia pastoris*.

[0082] 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).

[0083] In some implementations, mammalian cells are utilized. Many suitable mammalian host cells are known in the art, and many are available from the United States Type Culture Collection (ATCC, Manassas, VA). Examples of suitable mammalian cells include, but are not limited to, Chinese hamster ovary cells (CHO) (e.g., CHO-K1 cells, ATCC number 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 number CRL1573), and 3T3 cells (ATCC number CCL92). Other suitable mammalian cell lines are monkey COS-1 (ATCC number CRL1650) and COS-7 (ATCC number CRL1651), and the CV-1 cell line (ATCC number CCL70). Further exemplary mammalian host cells include primate and rodent cell lines, including transformed cell lines. Normal diploid cells, cell lines cultured in vitro from primary tissues, and primary explants are also suitable. Other suitable mammalian cell lines include, but are not limited to, mouse neuroblastoma N2A cells, HeLa, mouse L-929 cells, and BHK or HaK hamster cell lines, all of which are available from ATCC. The methods for selecting suitable mammalian host cells and for transforming, culturing, expanding, screening, and purifying cells are known in the field.

[0084] In one embodiment, the mammalian cell is a human cell. For example, the mammalian cell may be a human lymphocyte or a lymphocyte-derived cell line, such as a pre-B lymphocyte-derived cell line. Examples of human lymphocytes 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.

[0085] The nucleic acid sequence encoding the amino acid sequence of the present invention can be introduced into cells by any suitable method, such as by "transfection", "transformation" or "transduction". As used herein, "transfection", "transformation" or "transduction" means the introduction of one or more exogenous polynucleotides into a host cell using physical or chemical methods. Many suitable techniques are known in the art, including, 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-glucan; electroporation; cationic liposome-mediated transfection; tungsten particle-promoted microparticle bombardment (Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNA coprecipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)). Infectious particles can be grown in suitable packaging cells, after which phages or viral vectors can be introduced into the host cells. Many packaging cells are commercially available.

[0086] The nucleic acids and cells can be used for any purpose, such as for manufacturing the dPSA binders described herein. In this regard, the present invention provides a method for preparing dPSA binders, comprising culturing cells containing nucleic acid or nucleic acid sequences encoding heavy and / or light immunoglobulin polypeptides encoding dPSA binders. In other words, the method comprises expressing nucleic acids encoding the heavy and / or light chains of immunoglobulins encoding dPSA binders in cells (e.g., in vitro cells, such as any cell lines discussed herein, including CHO and CHO-K1 cells). It should be understood that the heavy and light chains of immunoglobulins can be expressed from a single nucleic acid in a given cell, or the heavy and light chains can be expressed from separate nucleic acids in the same cell. The method may also include harvesting and / or purifying the dPSA binders from the cells or cell culture medium using known techniques.

[0087] The following embodiments further illustrate the invention, but should not be construed as limiting its scope in any way.

[0088] Example 1 The following example illustrates the selective binding of the dPSA binder provided in this article to the dPSA antigen.

[0089] Antibodies SAC-1 and SAC-2, having the sequences shown below, were recombinantly expressed as chimeras with human IgG1 Fc in a CHO cell line that does not express dPSA. The specific binding of the antibodies to dPSA was tested by ELISA and cell-based assays. Humanized antibodies were prepared using the same CDR as the SAC-1 and SAC-2 antibodies. These antibodies were named SAC-1.1, SAC-2.1 (also known as "SAC-2 Humanized D" or "SAC-2D"), and SAC-2.2 (also known as "SAC-2 Humanized C" or "SAC-2C"), and their sequences are shown below.

[0090] <![CDATA[ Antibody ]]> <![CDATA[ Heavy chain variable region ]]> <![CDATA[ Light chain variable region ]]> <![CDATA[ Heavy chain total length ]]> <![CDATA[ Light chain full length ]]> SAC-1 SEQ ID NO: 1 SEQ ID NO: 5 SEQ ID NO: 15 SEQ ID NO: 16 SAC-2 SEQ ID NO: 35 SEQ ID NO: 36 SEQ ID NO: 37 SEQ ID NO: 38 SAC-1.1 SEQ ID NO: 17 SEQ ID NO: 21 SEQ ID NO: 22 SEQ ID NO: 23 SAC-1.1 M1 SEQ ID NO: 18 SEQ ID NO: 21 SAC-1.1 M2 SEQ ID NO: 19 SEQ ID NO: 21 SAC-1.1 M3 SEQ ID NO: 20 SEQ ID NO: 21 SAC-2.1 (SAC-2D) SEQ ID NO: 51 SEQ ID NO: 52 SEQ ID NO: 57 SEQ ID NO: 58 SAC-2.2 (SAC-2C) SEQ ID NO: 51 SEQ ID NO: 53 SEQ ID NO: 57 SEQ ID NO: 59 Preparation of dPSA antigen The preparation method of dPSA antigen for ELISA is as follows: 100 mg of polysialic acid (MilliporeSigma), 10 mg of sodium borohydride (MilliporeSigma), and 10 mL of 2 mol sodium hydroxide were mixed and heated to 100°C for 40 minutes. The resulting dPSA was neutralized with 2 M hydrochloric acid, dialyzed twice with 4 L of water, and then lyophilized. 20 mg of dPSA was dissolved in 0.75 mL of 0.1 M sodium acetate, pH 6.5, and first oxidized with sodium periodate (0.25 mL of 10 mmol periodate) at room temperature in the dark for 30 minutes. After adding 100 μL of 10% (v / v) ethylene glycol, the reaction mixture was dialyzed and lyophilized as described above. 20 mg of oxidized dPSA and 10 mg of ovalbumin (Imject) were then mixed. TM Ovalbumin (Pierce Chemical Company) was mixed in phosphate-buffered saline (PBS) containing approximately 5 mg of sodium cyanoborohydride. The solution was stirred overnight at room temperature in the dark. The dPSA-ovalbumin conjugate was purified by size exclusion chromatography using a ToyoPearl HW-65F column in 0.9% (w / v) sodium chloride and 10 mM potassium phosphate (pH 7.1). The fraction containing the dPSA-ovalbumin conjugate was concentrated (SpinX, Corning) to a protein concentration of 2 mg / mL and a dPSA concentration of 4 mg / mL, containing approximately 30% de-N-acetyl residues, as determined by a modified resorcinol method.

[0091] Binding with dPSA antigen ELISA was performed as described by Moe et al. (J. Exp. & Clin. Can. Res. 40(1):293 (2021)), with an initial antibody concentration of 10 μg / mL and eight consecutive 3-fold dilutions. Specificity for dPSA was determined by adding 100 μg of polysialic acid (i.e., polysialic acid, MilliporeSigma) to the same ELISA buffer as described above. The results (OD405 > 0.5 mean fluorescence intensity (MFI) over 30 minutes) confirmed the antibody's specific binding to dPSA.

[0092] As described by Moe et al. (J of Exp & Clin Can Res 2021, 40:293), the binding of antibodies to the human neuroblastoma cell line CHP-134 and the human myeloma cell line NCI-H929 at a fixed concentration of 10 μg / mL was tested by flow cytometry. In short, adherent cells were obtained by pipetting or using Accutase. TM (Innovative Cell Technologies) treated cells and suspended them in RPMI 1640 cell culture medium containing 10% (v / v) fetal bovine serum (FBS, Thermo Fisher Scientific). Cells were centrifuged (200xg, 8 min), and the cell pellet was resuspended in culture medium to a concentration of 0.5-2x10⁻⁶. 6 Cells / mL. Cells and antibodies were mixed in 1.5 mL Eppendorf tubes and incubated end-to-end at room temperature for 1 hour. After centrifugation (200 x g, 2 min), the cells were washed once with fresh medium and resuspended in medium containing Alexa Fluor 488-labeled goat anti-human F(ab')2 secondary antibody. After end-to-end mixing for 30 min, the cells were washed once with fresh medium and resuspended in PBS buffer containing 0.5% (v / v) formaldehyde. Cell fluorescence was measured by flow cytometry (Acea NovoCyte). Results are expressed as mean fluorescence intensity (MFI) relative to the irrelevant human IgG1 negative control antibody (BioXCell) and summarized in Table 1.

[0093] Table 1 Antibody CHP-134 MFI NCI-H929 MFI SAC-1 155,241 66,816 SAC-2 59,426 67,142 Example 2 The following examples illustrate the binding of the antibodies presented in this article to a variety of other cancer cell lines, but not to CHP-134 neuroblastoma cells (CHP-134 KO) in which the polysialotyltransferase genes ST8SIA2 and ST8SIA4 are knocked out. As described by Moe et al. (J of Exp & Clin Can Res 2021, 40:293), dPSA on the surface of CHP-134 cells depends on the expression of ST8SIA2.

[0094] Following a similar procedure to that described in Example 1 for CHP-134 and NCI-H929 cells, the binding of the antibody provided in Example 1 to a variety of other cell lines was further tested. Table 2 lists the EC50 and maximum MFI values ​​for each cell line, indicating that the antibody binds to a variety of different types of cancer cells. Furthermore, this 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)).

[0095] Table 2 *ND, not measured.

[0096] Example 3 The following example illustrates that the epitopes bound by the antibodies presented in this article differ from those of the reference antibodies.

[0097] To determine whether the epitope recognized by the antibody in Example 1 differed from that recognized by the reference anti-dPSA antibody SEAM 3 (Steirer and Moe et al., PLoS One 6(11): e27249 (2011)), an antibody at a fixed concentration of 10 times the EC50 value binding to CHP-134 cells was mixed with sequentially 2-fold dilutions of SEAM 3. Figure 1 and Figure 2 As shown, the antibody in Example 1 does not inhibit the binding of SEAM 3, which demonstrates that these antibodies recognize different epitopes.

[0098] Example 4 This embodiment demonstrates that the antibody provided herein binds to a variety of human cancer cell lines.

[0099] The cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and were regularly tested for mycoplasma contamination (MycoStrip). TM (InvivoGen, San Diego, CA). Cell lines were grown in ATCC-recommended medium in a humidified incubator with a 5% CO2 atmosphere. Adherent cell lines were grown using StemPro.TM Accutase TM Cell dissociation reagent (ThermoFisher Scientific, Carlsbad, CA) was used to treat the suspension. The suspended cells were diluted 1:5 with cell culture medium, centrifuged (200xg, 10 min), and then resuspended in fresh culture medium at a density of 1-10 million viable cells / mL. Viability was determined by trypan blue staining (ThermoFisher Scientific, Carlsbad, CA) and analyzed using SKC, Inc. C-Chip. TM Cells were counted using a disposable hematology counter (Fisher Scientific, Pittsburgh, PA). Cells were mixed with antibodies (SAC-1, SAC-1 humanized antibody (SAC-1.1), SAC-2, SAC-2 mouse antibody (SEQ ID NO: 54 and SEQ ID NO: 55), SAC-2 humanized C (SAC-2.2), and SAC-2 humanized D (SAC-2.1)) in tubes and incubated end-to-end for 1 hour at room temperature. Cells were centrifuged (200xg, 2 min), the supernatant was aspirated, and the cells were resuspended in Alexa Fluor 488 containing the corresponding (i.e., anti-mouse or anti-human IgG H+L) antibodies. TM Cells were incubated with the labeled secondary antibody (AffiniPure F(ab')2 Fragment GoatAnti-Mouse IgG (H+L), Jackson ImmunoResearch, West Grove, PA) in medium. Cells and secondary antibody were incubated end-to-end at room temperature for 30 minutes, followed by centrifugation (200xg, 2 minutes), aspirating the supernatant, and resuspending the cells in phosphate-buffered saline containing 0.5% formaldehyde.

[0100] Finally, cells were analyzed by flow cytometry (Acea NovoCyte, Agilent, Santa Clara, CA, or similar instruments). Binding curves dependent on antibody concentration for mean fluorescence intensity (MFI) were analyzed using curve fitting software (GraphPad Prism, San Diego, CA) to determine the binding constant (KD), half-maximal effector concentration (EC50), and maximum MFI (MFImax). Results are shown in Tables 3 (SAC-1 antibody) and 4 (SAC-2 antibody), where Neg. indicates negative and NA indicates not applicable. As shown in Tables 3 and 4, the tested antibodies bound to cell lines of various cancers with KDs in the nanomolar range, and exhibited different MFImax values ​​depending on the epitope density of each cell line. These data indicate that the antigens recognized by the antibodies are present in a variety of human cancers, and that this binding is dependent on the expression of the polysialic acid transferase STSIA2, as the knockout of the gene encoding STSIA2 in CHP-134 cells eliminates the binding.

[0101] Table 3

[0102] Table 4

[0103] Example 5 This embodiment demonstrates that the antibody provided herein recognizes a nucleolar protein derivative containing dPSA.

[0104] Preparation of subcellular components: Human melanoma A375 cells (80% density in T-175 culture flasks) were extracted using the ProteoExtract® Subcellular Proteome Extraction Kit (MilliporeSigma). In short, this differential detergent extraction procedure sequentially used four extraction buffers, with the addition of a protease inhibitor mixture to prevent protein degradation during extraction, and the addition of Benzobonase® nuclease (Sigma-Aldrich, St. Louis, MO) to degrade contaminating nucleic acids. Following the manufacturer's extraction instructions, the cell extract was separated into four components: F1 (cytoplasmic component), F2 (cell membrane component), F3 (nucleoprotein component), and F4 (cytoskeleton component).

[0105] Co-immunoprecipitation: Dynabeads M-270 epoxy magnetic beads (Thermo Fisher Scientific, Carlsbad, CA) covalently linked to SAC-1, SAC-2, or irrelevant human IgG1 antibodies (BioXCell, Lebanon, NH) were prepared according to the manufacturer's protocol. Antigens were purified by co-immunoprecipitation based on their reactivity with the antibodies, as follows: F2 membrane fractions were incubated with SAC-1, SAC-2, or irrelevant IgG1-linked magnetic beads, respectively. The beads were separated using a magnet, washed first with the appropriate extraction buffer, and then with a buffer containing polysialic acid (50 µg / mL; Sigma-Aldrich, St. Louis, MO) to remove non-specifically bound antigens. Finally, the antigens were eluted from the beads by heating to 80°C for 10 minutes using SDS-PAGE loading buffer (NuPAGE, Thermo Fisher Scientific) without reducing agents. Proteins eluted from magnetic beads were separated on 4%–12% SDS-PAGE (NuPAGE, Thermo Fisher Scientific) and stained with SimplyBlue® Coomassie stain (Thermo Fisher Scientific), or transferred to PVDF membranes (Immobilon®-FL, Millipore, Waltham, MA) using a NuPAGE transfer system (Thermo Fisher Scientific) for Western blotting. PVDF membranes were blocked overnight in phosphate-buffered saline (PBS) buffer containing 5% (w / v) skim milk powder, and then stained with anti-nucleolar protein antibody MS-3 (Santa Cruz Biotechnology, Santa Cruz, CA) in blocking buffer at room temperature for 2 hours. After washing three times with PBS buffer, antibody binding was detected using IRDye® 800CW-labeled donkey anti-mouse IgG (H+L) secondary antibody (LI-COR, Lincoln, NE). Images of the gel and blot were recorded on an Odyssey® Fc imaging system (LI-COR). Results are as follows. Figure 3A and 3B As shown.

[0106] Figure 3AThe separation of proteins co-immunoprecipitated by each antibody on SDS-PAGE is shown. Multiple strongly stained bands with similar molecular weight distributions were observed, co-immunoprecipitated by SAC-1 and SAC-2, but not by unrelated IgG1 antibodies. We have previously shown that antibodies binding to de-N-acetylated polysialic acid (dPSA) can co-immunoprecipitate dPSA-modified nucleolar proteins from the F2 fraction of cancer cells (J Exp Clin Cancer Res. 2021 Sep 20;40(1):293). Cell surface nucleolar proteins are cancer cell-specific and exhibit a wide molecular weight range when separated on SDS-PAGE gels due to various post-translational modifications and the length and charge heterogeneity of dPSA.

[0107] Figure 3B The results of co-immunoprecipitated proteins were shown by Western blot staining with anti-nucleolar protein antibody MS-3. The major bands (i.e., the bands with an apparent molecular weight of 77 kDa) in SAC-1 and SAC-2 samples were confirmed as nucleolar proteins based on their reactivity with MS-3. Post-translational modifications affecting nucleolar protein migration in SDS-PAGE gels may not be detected by MS-3 because MS-3 was prepared for unmodified recombinant nucleolar proteins. To confirm that other bands with apparent molecular weights different from the major bands detected by MS-3 are also derivatives of nucleolar proteins, [further details needed]. Figure 5A The gel fractions indicated in square brackets were cut from each co-immunoprecipitation sample and processed according to the previously described method (J Exp Clin Cancer Res. 2021Sep 20;40(1):293), and protein identification analysis was performed by LC-MS / MS mass spectrometry. LC-MS / MS mass spectrometry confirmed that the analyzed gel fractions also contained nucleolar proteins.

[0108] Nucleolar protein quantification for each sample was also shown. Figure 5A The values ​​in the table correspond to the relative amount of Coomassie staining in the corresponding fraction of the SDS-PAGE gel for each sample. In summary, the data indicate that SAC-1 and SAC-2 bind to the same modified form of nucleolar protein located in the membrane fraction of A375 human melanoma cells, and that the nucleolar protein derivative has (i.e., expressed as a molecular weight range on SDS-PAGE) the characteristics of being modified with dPSA as described above.

[0109] Example 6 This embodiment demonstrates that the antibody provided herein exhibits reactivity with human tumors and normal human tissues via immunohistochemistry.

[0110] To determine the specificity of SAC antibodies in binding to cancer cells but not to normally developed human tissues, tissue microarrays containing samples of normal human tissue and tumor tissue were stained using SAC-2 via immunohistochemistry.

[0111] SAC-2 mouse monoclonal antibodies (SEQ ID NO: 54 and SEQ ID NO: 55) were administered at a concentration of 2.5 μg / mL using Tris-based pH 9.5 heat-induced epitope retrieval; isotype controls (mouse IgG2a) were 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 manufacturer-recommended settings. Sections were incubated with Biocare peroxidase inhibitor (Biocare, catalog #PX968) and background remover (Biocare, catalog #BP974M) to block nonspecific background. For detection of the mouse primary antibody, the MACH4 HRP-polymer detection system (Biocare, catalog #MRH534) was used. The IntelliPATH FLX DAB chromogenic reagent (Biocare, catalog number #IPK5010) and IntelliPATH hematoxylin (Biocare Medical, catalog number #XMF963) were used for colorimetric detection and counterstaining. Tissue microarrays (TMAs) were purchased from Pantomics (Fairfield, CA). These TMAs included the FDA-recommended combination of normal human tissues from three different donors (MNO961). TMAs for human tumors included multitumor arrays (MTU481) and arrays for 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 sections were stained with a mouse version of the SAC-2 antibody to eliminate background caused by the binding of the secondary antibody to human IgG in the sample. The stained sections were digitized at 20x magnification using a TissueScope LE whole-slide scanner (Huron Digital Pathology, St. Jacobs, Ontario, Canada). For confirmation, the glass sections were also examined under an upright bright-field microscope.

[0112] Figure 4 shows SAC-2 stained normal human breast tissue ( Figure 4A ) and breast tumors ( Figure 4C Examples of tumor staining with unrelated mouse IgG2a antibodies ( ) Figure 4B The staining was compared. The rust-brown membrane staining of tumor cells revealed the tumor's staining, while normal breast tissue was not stained by SAC-2, and the tumor was not stained by irrelevant IgG2a antibodies. SAC-2 staining of breast tumor cells was uniform throughout the sample. Although the staining intensity of SAC-2 on tumor cells varied between samples, this uniform staining characteristic was consistent in all SAC-2-binding positive samples.

[0113] Table 5 summarizes the staining results of normal human tissues. The only positive staining observed in normal human tissue was in one of the three prostate samples. This stained prostate sample was described as benign prostatic hyperplasia, which may also contain early prostate cancer cells. Many tumor samples, particularly metastatic tumors, were stained. Table 6 summarizes the results of staining tumor tissue microarrays with SAC-2. In summary, the results indicate that SAC-2 does not bind to normal post-developmental human tissues but can recognize antigens expressed on several different human primary and metastatic tumor cells.

[0114] Table 5. Summary of SAC-2 immunohistochemical staining results on normal human tissue (Pantomics MNO96).

[0115] organize positive staining adrenal glands 0, sample size 3 bladder 0, sample size 3 marrow 0, sample size 1 Eye 0, sample size 2 breast 0, sample size 3 Brain, cerebellum 0, sample size 3 Brain, cerebral cortex 0, sample size 3 oviduct 0, sample size 3 esophagus 0, sample size 3 Stomach 0, sample size 3 Small intestine 0, sample size 3 colon 0, sample size 3 rectum 0, sample size 3 heart 0, sample size 3 kidney 0, sample size 5 Kidneys, Cortex 0, sample size 1 liver 0, sample size 3 lung 0, sample size 3 ovaries 0, sample size 3 pancreas 0, sample size 3 parathyroid glands 0, sample size 1 pituitary 0, sample size 2 placenta 0, sample size 3 prostate 1, sample size 3* skin 0, sample size 2 spinal cord 0, sample size 2 spleen 0, sample size 2 skeletal muscle 0, sample size 3 testis 0, sample size 3 thymus 0, sample size 3 thyroid 0, sample size 3 tonsil 0, sample size 3 ureter 0, sample size 3 uterus, cervix 0, sample size 3 Uterus, endometrium 0, sample size 3 *One-third of the stained samples were described as having benign prostatic hyperplasia.

[0116] Table 6. Summary of SAC-2 immunohistochemical staining results of tumor samples on tissue microarray.

[0117] Patient Tumor Combination (Pantomics catalog number) Positive percentage (number of positive samples / total number of samples) 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) Example 7 This embodiment demonstrates that the antibody provided herein possesses antibody-dependent cell-mediated cytotoxic activity.

[0118] The ability of five adherent cell lines (CHP-134 and Kelly neuroblastoma, SK-MEL-28 melanoma, SK-OV-3 ovarian cancer, and AsPC-1 pancreatic cancer) and two non-adherent cancer cell lines (NCI-H020 myeloma and Jurkat leukemia) to mediate ADCC activity with SAC-1 and SAC-2 was tested. Cell lines for assays were obtained and prepared as described in Example 4. InvivoGen Jurkat-Lucia was used. TMThe NFAT-CD16 reporter gene assay kit measures ADCC activity according to the manufacturer's instructions. This assay detects activation of the CD-16 Fcγ receptor-mediated signaling pathway, which is activated by the binding of the antibody to cancer cell surface antigens and the binding of the antibody Fc moiety to the receptor on reporter cells, leading to the expression of luciferase and luminescence in the presence of luciferin. The output signal is a relative luminescent unit (RLU), measured using a luminescent microplate reader (Synergy HTX MultimodeReader, Agilent, Santa Clara, CA, or similar instrument).

[0119] Results from mouse-human Fc IgG1 chimeric versions of SAC-1 and SAC-2 showed Figures 5A-5C The charts show that SAC-1 typically shows a lower signal than SAC-2, reflecting a lower epitope density relative to the MFI identified in the binding studies, but still activating ADCC activity against all tested cell lines. SAC-2 exhibits higher ADCC activity against most tested cell lines, consistent with the higher epitope density observed in the binding studies, but lacks activity against some cell lines. In summary, the data indicate that both antibodies can mediate ADCC activity against a variety of different human cancer cell lines.

[0120] Example 8 This embodiment demonstrates that the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of humanized SAC-2C and SAC-2D antibodies is enhanced by reducing or eliminating fucosylation (e.g., defucosylated antibodies).

[0121] The defucosylation antibody was produced using a commercially available method. ADCC activity with reduced fucosylation (aFUC) was compared using the DELFIA® Cytotoxicity Assay Kit (PerkinElmer, Billerica, MA). Human A375 melanoma and MDA-MB-231 breast cancer cells were used as target cells. Assays were performed according to the manufacturer's instructions. In short, target cells were harvested and suspended in complete culture medium. Target cells (1 x 10⁻⁶) were... 6 Cells were labeled with 2 µL of fluorescence-enhancing ligand (DELFIA® BATDA reagent) at 37°C for 20 min. Cells were washed four times with phosphate-buffered saline (PBS), and then the cell density was adjusted to 1 × 10⁻⁶ cells / day. 5 / mL, seeded at 100 µL / well in a 96-well assay plate. For ADCC assay, 50 µL of serially diluted antibody solution was added to each well and incubated at 37°C and 5% CO2 for 15 minutes. Natural killer effector cells (NK92 / CD16a) were added at a ratio of 8 effector cells to 1 target cell, 50 µL per well. The assay plate containing effector cells, antibody, and target cells was incubated at 37°C and 5% CO2 for 3 hours.

[0122] When cells are intact, the BATDA ligand remains intracellular. When europium solution is added to the supernatant from intact cell samples, europium cannot form a fluorescent chelate with BATDA because no BATDA is released into the supernatant. The europium solution does not glow in its unchanged state. If cells are lysed by effector cells, BATDA is released extracellularly into the supernatant. Upon addition of europium solution to the supernatant, europium forms a highly fluorescent and stable chelate with the released BATDA (EuTDA). The measured fluorescence signal is directly correlated with the number of lysed cells in the cytotoxicity assay.

[0123] 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 plate, and the plate was shaken at room temperature for 15 minutes. Fluorescence was measured using a time-resolved fluorometer over 5 hours. The percentage kill was calculated using the formula: ADCC = (sample fluorescence - autofluorescence of the target and effector cell mixture) / (maximum fluorescence of target cells - autofluorescence of the target and effector cell mixture) × 100%. The percentage kill curve was analyzed using a GraphPad Prism 6 (GraphPad, San Diego, CA) to determine the EC50. Results are as follows: Figure 6A and 6B As shown, and summarized in Table 7. Depending on the antibody and target cells tested, ADCC activity increased by 4 to 10 times.

[0124] Table 7

[0125] Example 9 This embodiment demonstrates that the antibody provided herein has in vivo tumor-suppressive activity in a human cancer xenograft mouse model.

[0126] The ability of SAC antibodies to inhibit tumor growth in human cancer xenograft mouse models was tested using two models: A375 human melanoma and MDA-MB-231 human breast cancer, in thymic-agnostic BALB / c nu / nu mice and NSG mice supplemented with human peripheral blood mononuclear cells (PBMCs), respectively. The assay was performed by subcutaneous injection of 1x10n cells into the hind leg. 6 Mice were inoculated with individual cells and matrix gel. Tumor size was measured using digital calipers, and mice were divided into treatment cohorts (n=10 per group). The average tumor size was 100-200 mm. 3 Mice were treated twice weekly with SAC antibodies at doses of 2 mg / kg, 6 mg / kg, and 20 mg / kg, or with standard treatment (paclitaxel 7.5 mg / kg for the A375 model; cyclophosphamide 30 mg / kg intraperitoneally daily for the MDA-MB-231 model) or as a control group using only the carrier. Mice given human PBMCs received a single dose of PBMCs at the start of treatment. Repeated measures ANOVA and Dunnett's multiple comparison test were performed using GraphPad Prism (San Diego, CA) software to assess significance. The dose-dependent effect of SAC antibody treatment compared to standard paclitaxel treatment was as follows: Figure 7A and 7B As shown. Data on SAC-1.1, mouse SAC-2, and SAC-2C in the MDA-MB-231 human breast cancer xenograft mouse model are as follows. Figure 8A As shown, the effects of adding human PBMC and SAC-2C are as follows: Figure 8B As shown, data indicate that SAC-1 and SAC-2 can inhibit tumor growth in human melanoma and breast cancer xenograft mouse models, depending on the antibody concentration.

[0127] Example 10 This example illustrates the preparation of antibody-drug conjugates.

[0128] The SAC-2D-vc-MMAE antibody-drug conjugate (ADC) is prepared by linking the MC-Val-Cit-PAB-MMAE linker / load to cysteine ​​residues on the antibody. The structure of the linker-MMAE load linked to the antibody cysteine ​​is as follows... Figure 9 As shown. Figure 10A and 10B The results showed that size exclusion chromatography analysis revealed that over 98% of the SAC-2D-vc-MMAE ADC was monomeric; high-performance ion exchange chromatography analysis showed that 76% of the product contained four MMAE molecules per antibody molecule. Endotoxin levels in the SAC-2D-vd-MMAE product were measured using the Limulus Amebocyte Lysate (LAL) assay and found to be below 0.092 EU / mg.

[0129] Example 11 This embodiment demonstrates that the SAC-2D-vc-MMAE antibody-drug conjugate binds to cancer cell lines representing various human cancers and exhibits cytotoxicity due to binding to the dPSA-nucleolar protein antigen on the cell surface, being taken up by the cell, and releasing the MMAE drug after the antibody-drug linker lyses within the cell.

[0130] The binding assay of the SAC-2D-vc-MMAE antibody-drug conjugate (ADC) was performed as described in Example 4. In vitro cytotoxicity assays were performed as follows: Depending on the specific cell line, a number of cells (90 µL volume) were seeded into 96-well flat-bottomed, transparent black polystyrene tissue culture dishes on day 1 and incubated overnight in a tissue incubator. On day 2, cells were treated with the test compound at nine concentration points. Control wells included untreated cells and wells containing only culture medium. ADC was prepared from the stock solution to obtain a 10x solution of 200 µg / mL. Serial dilutions of 1:5 were performed in culture medium to obtain nine concentration points. The dilutions were then added to the cells (10 µL of diluted compound + 90 µL of culture medium) to achieve final ADC concentrations of 20000, 4000, 800, 160, 32, 6.4, 1.28, and 0.0512 ng / mL on the cells. Cells were incubated with the compound for 96 hours, and then on day 4, the number of viable cells was measured using Celltiter-Glo® according to the manufacturer’s (Promega, Inc., Madison, WS) recommended protocol.

[0131] As summarized in Table 8, the SAC-2D-vc-MMAE ADC is cytotoxic to cancer cell lines expressing dPSA-nucleolar antigen. The sensitivity of cell lines to ADC treatment (expressed as the half-maximal inhibitory concentration, IC50) does not necessarily correspond to the amount of antigen present on the cell surface. This discrepancy between antigen level and ADC effect may stem from differences in the ability of specific cell lines to resist drug effects. For example, cell lines may overexpress multidrug efflux pumps, preventing sufficient intracellular accumulation of drugs to achieve cytotoxic levels.

[0132] Table 8.

[0133] Example 12 This embodiment demonstrates that the SAC-2D-vc-MMAE ADC inhibits or completely ablates tumor growth in a human cancer mouse model in a dose-dependent manner. The ability of this ADC antibody to inhibit tumor growth in human cancer xenograft mouse models was tested using two models established in nude mice (BALB / c nu / nu): the A375 human melanoma model and the HCC1954 human breast cancer model. In the experiment, tumor cells were first implanted into mice and allowed to form a volume exceeding approximately 150 mm². 3 The tumor will be evaluated again later.

[0134] In the A375 melanoma model, mice were treated with the vector alone, 20 mg / kg SAC-2D or its defucosylated variant, 5 mg / kg or 20 mg / kg SAC-2D-vc-MMAE ADC, or the positive control standard treatment (vemurafenib) 50 mg / kg. Mice treated with the vector or antibody received intraperitoneal injections twice weekly. Mice treated with vemurafenib received oral administration four times weekly. Figure 11A As shown, only mice treated with vemurafenib or 5 mg / kg SAC-2D-vc-MMAE had tumors that remained quiescent during the test, while mice treated with 20 mg / kg SAC-2D-vc-MMAE showed complete tumor regression. SAC-2D ADC treatment was well tolerated, as evidenced by the mice maintaining or increasing their body weight during treatment. Figure 11B ).

[0135] Mice in the HCC1954 human breast cancer mouse model were treated twice weekly with either the carrier alone, 20 mg / kg SAC-2D or its defucosylated variant, 1 mg / kg, 3 mg / kg, or 10 mg / kg SAC-2D-vc-MMAE ADC, or the positive control standard treatment Buparlisib (BMK-120) orally at a dose of 35 mg / kg, four days a week. Although HCC1954 cells are relatively low expressers of dPSA-nucleolar protein, treatment with each of the three doses of SAC-2D-vc-MMAE resulted in tumor regression, compared to no such effect in the control group treated with the carrier alone. Figure 12A In contrast, SAC-2D or its defucosylated variants had no effect on tumor growth inhibition. Standard treatment with Buparlisib achieved tumor arrest but not regression. Similarly, SAC-2D-vc-MMAE was well tolerated at all tested doses, as evidenced by a steady increase in mouse body weight. Figure 12B Overall, the results indicate that SAC-2D-vc-MMAE is superior to SAC-2D or its defucosylated variants in inhibiting or eliminating tumor growth.

[0136] Example 13 This example illustrates the preparation of the following antibody-drug conjugates: LP-001: MC-Val-Cit-PAB-Exanotecan HOBt (62.1 mg, 459 μmol) and DIEA (59.4 mg, 459 μmol) were added to a solution of compound 1 (152 mg, 206 μmol) and compound 2 (100 mg, 229 μmol) in pyridine (2 mL). The mixture was stirred at 25 °C for 2 hours. LC-MS showed that the reaction was complete. The reaction mixture was concentrated to obtain a residue, which was purified by preparative HPLC (column: Waters Atlantis T3 150 30 mm 5 μm; mobile phase: [water (TFA)-acetonitrile]; gradient: phase B from 20% to 60% over 20 minutes) to give LP-001 as a yellow solid.

[0137] LP-003: MC-GGFG-Exanotecan DIEA (444 mg, 3.44 mmol, 598 μL) was added to a solution of compound 1 (500 mg, 1.15 mmol, MeSO3H), compound 2 (609 mg, 1.15 mmol, MeSO3H), and HATU (653 mg, 1.72 mmol) in DMF (15.0 mL). The mixture was stirred at 25 °C for 1 hour. LC-MS showed that compound 1 had been consumed, and 88.6% of the target compound was detected. The reaction mixture was concentrated, and then acetonitrile (6.00 mL) and methyl tert-butyl ether (24.0 mL) were added. The solid precipitated from the solution was filtered, and the filter cake was dried under vacuum. Compound 3 (1.40 g, crude product) was given as a dark brown solid.

[0138] Compound 3 (1.4 g, 1.64 mmol) was added to a solution of trifluoroacetic acid (7.68 g, 67.3 mmol, 5.00 mL) in dichloromethane (30 mL). The mixture was stirred at 25 °C for 0.5 h. LC-MS showed that compound 3 had been consumed, with 87% of the target compound detected. The reaction mixture was concentrated, and the residue was purified by preparative HPLC (TFA conditions; column: CD05-Phenomenex luna C18 150*40*10 μm; mobile phase: [water (TFA)-acetonitrile]; gradient: phase B from 16% to 46% over 10 min). Compound 4 (350 mg, 28.0% yield, 99.6% purity) was given as a pale yellow solid.

[0139] To a solution of compound 4 (120 mg, 159 μmol) in DMF (5 mL), DIEA (41.2 mg, 318 μmol, 55.5 μL) and compound 5 (68.7 mg, 223 μmol) were added. The mixture was stirred at 25 °C for 5 hours. LC-MS showed that compound 4 had been consumed, with 71.3% of the target compound detected. The reaction mixture was concentrated, and then acetonitrile (6.00 mL) and methyl tert-butyl ether (24.0 mL) were added. The solid precipitated from the solution was filtered, and the filter cake was dried under vacuum. The crude product was purified by preparative HPLC (TFA conditions; column: CD01-Phenomenex luna C18 150*25*10 μm; mobile phase: [water (TFA)-acetonitrile]; gradient: phase B from 26% to 56% over 10 minutes). LP-003 was given as a yellow solid.

[0140] LP-004: MC-Val-Cit-PAB-Maytansine Compound 1 (100 mg, 181 μmol) was dissolved in anhydrous DMF (0.3 mL) to obtain a clear, colorless solution, which was transferred to a dry, double-necked round-bottom flask under nitrogen protection. Anhydrous THF (1.2 mL) was added, followed by DIEA (141 mg, 1.09 mmol). A solution of compound 2 (158 mg, 1.22 mmol) was added, resulting in a clear, colorless solution. Dry, finely ground Zn(OTf)₂ (264 mg, 726 μmol) was added to the stirred solution, and the reaction mixture was stirred at 20 °C for 48 hours. LCMS showed 19.7% of compound 1 remaining and 51.8% of the target compound detected. The reaction mixture was quenched at 25 °C by adding NaHCO3 (1.2 M, 2 mL), extracted with ethyl acetate (20 mL), washed with brine (10 mL x 1), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was then analyzed by preparative HPLC (column: Welch Xtimate C18 40*200 mm 7 μm; mobile phase: [water (NH4HCO3)- [Methanol]; gradient: phase B increased from 40% to 80% within 25 minutes) purification yielded (14S,16S,32S,33S,2R,4S,10E,12E,14R)-86-chloro-14-hydroxy-85,14-dimethoxy-33,2,7,10-tetramethyl-12,6-dioxo-7-aza-1(6,4)-oxazacyclobutane-3(2,3)-ethylene oxide-8(1,3)-benzocyclotetradecane-10,12-diene-4-yl D-alanine methyl ester (29.0 mg, 16.9% yield, 94.9% purity).

[0141] The mixture of compound 3 (25.0 mg, 38.5 μmol), compound 4 (22.7 mg, 30.8 μmol), DIEA (9.94 mg, 76.9 μmol, 13.4 μL), and HOBt (10.4 mg, 76.9 μmol) in pyridine (1 mL) was degassed and purged three times with nitrogen, then stirred at 25 °C for 2 h under a nitrogen atmosphere. LCMS (EB12738-44-P1B) showed that the reaction was complete. The reaction mixture was filtered and concentrated under reduced pressure to give a residue, which was purified by preparative HPLC (Welch Xtimate C18 40 * 200 mm 7 μm; mobile phase: [water (formic acid) - acetonitrile]; gradient: phase B from 24% to 64% over 25 min) to give LP004 as a white solid.

[0142] LP-005: MC-Val-Lys (PEG)-PAB-Taxol t-BuOK (1 M, 7.73 mL) was added to a solution of compound 1 (6.60 g, 7.73 mmol) in THF (150 mL) at 40 °C. The mixture was stirred and heated to -20 °C (possibly 0 °C) for 20 min, then compound 2 (1.77 g, 7.73 mmol, 1.21 mL) was added. After the addition was complete, the mixture was heated to 20 °C and stirred for 2 h. LC-MS showed detection of 31.9% of the target compound (retention time = 2.170 min). TLC (petroleum ether / ethyl acetate = 2 / 1, Rf = 0.80) showed the formation of a major new spot with low polarity. The reaction mixture was quenched by adding NH4Cl (50 mL) at 25 °C, diluted with ethyl acetate (100 mL), and extracted with ethyl acetate (200 mL, 100 mL x 2). The combined organic layers were dried over Na₂SO₄, filtered, and concentrated under reduced pressure to obtain a residue, which was purified by column chromatography (petroleum ether / ethyl acetate = 10 / 1 to 1 / 1). Compound 3 (2.88 g, 27.4% yield, 73.7% purity) was given as a white solid.

[0143] Pd / C (10%, 0.5 g) was added to a solution of compound 3 (2.88 g, 2.87 mmol) in ethyl acetate (40 mL) and THF (10 mL) under a nitrogen atmosphere. The suspension was degassed and purged five times with hydrogen. The mixture was stirred at 25 °C for 0.5 h under hydrogen (15 Psi). LC-MS showed detection of 64.7% of the target compound (retention time = 1.896 min). The reaction mixture was filtered and concentrated under reduced pressure to give the residue. The residue was purified by column chromatography (petroleum ether / ethyl acetate = 100 / 1 to 10 / 1) to give compound 4 (1.80 g, 65.6% yield, 95.5% purity) as a white solid.

[0144] To a solution of compound 6-1 (9.41 g, 21.6 mmol) in DME (150 mL), NaHCO3 (3.15 g, 37.5 mmol) was added. The mixture was stirred at 25 °C for 2 h. LC-MS showed detection of 89.7% of the target compound (retention time = 1.865 min). The pH of the mixture was adjusted to 3.0 with 0.5 N HCl solution. After removing DME under vacuum, a large amount of white solid precipitated and was suspended in water. The mixture was filtered, and the solid was washed twice with water. The solid was then dried under vacuum below 40 °C to give compound 6-3 (4.64 g, 44.5% yield, 99.1% purity) as a white solid.

[0145] A mixture of compound 6-3 (4.64 g, 8.41 mmol), (4-aminophenyl)methanol (2.07 g, 16.8 mmol), and EEDQ (4.16 g, 16.8 mmol) in methanol (30 mL) and dichloromethane (100 mL) was degassed and purged three times with nitrogen, then stirred at 20 °C for 8 hours under a nitrogen atmosphere. LC-MS showed detection of 65.1% of the target compound (retention time = 1.890 min). The reaction mixture was concentrated under reduced pressure to give a residue, which was ground with acetonitrile (100 mL) and methyl tert-butyl ether (150 mL). Compound 6 (3.05 g, 51.0% yield, 92.4% purity) was given as a white solid.

[0146] A mixture of compound 4 (300 mg, 329 μmol), compound 6 (325 mg, 494 μmol), diphenyl azidophosphate (272 mg, 987 μmol, 213 μL), and DIEA (128 mg, 987 μmol, 172 μL) in toluene (15 mL) was degassed and purged three times with nitrogen. The mixture was then stirred at 80 °C for 2 h under nitrogen atmosphere, followed by stirring at 100 °C for 2 h. LC-MS showed detection of 28.3% of the target compound (retention time = 2.279 min). The three batches of reaction mixture were combined and concentrated under reduced pressure to obtain the residue, which was purified by column chromatography (dichloromethane:ethanol = 100 / 1 to 20 / 1). Compound 7 (514 mg, 29.6% yield, 88.9% purity) was given as a yellow foam.

[0147] A mixture of compound 7 (300 mg, 192 μmol), 1,3-dimethylhexahydropyrimidine-2,4,6-trione (120 mg, 766192 μmol), and Pd(PPh3)4 (22.1 mg, 19.2 μmol) in THF (6 mL) was degassed and purged three times with nitrogen. The reaction mixture was then stirred at 25 °C for 1.5 h under a nitrogen atmosphere. LC-MS analysis showed a yield of 75.0% for the target compound (retention time Rt = 2.043 min). The reaction mixture was concentrated under reduced pressure to give crude compound 8 (283 mg, crude product) as an orange solid.

[0148] LP-006: MC-β-glucuronide-MMAE A solution of EEDQ (2.17 g, 8.78 mmol) in dichloromethane (20.0 mL) and methanol (40.0 mL) was added to a solution of compound 1 (2.00 g, 4.39 mmol) and compound 2 (1.37 g, 4.39 mmol). The mixture was stirred at 25 °C for 1 hour. LC-MS showed detection of 65% of the target compound. TLC (dichloromethane / methanol = 5 / 1, Rf = 0.4) showed that the reaction was complete. The reaction mixture was diluted with water (80.0 mL) and extracted with dichloromethane (50.0 mL x 3). The combined organic layers were dried over Na2SO4, filtered, and the filtrate was concentrated to give the residue. The residue was purified by column chromatography (silica gel, petroleum ether / ethyl acetate = 100 / 1 to 3 / 1) to give compound 3 (2.25 g, 2.76 mmol) as a yellow solid.

[0149] Pyridine (528 mg, 6.68 mmol, 539 μL) was added to a solution of compound 3 (1.00 g, 1.34 mmol) in dichloromethane (15.0 mL) at 0 °C. A solution of (4-nitrophenyl)chloroformate (403 mg, 2.00 mmol) in dichloromethane (0.5 mL) was added dropwise. The mixture was stirred at 0 °C for 2 hours. LC-MS showed detection of 71.1% of the target compound. TLC (petroleum ether / ethyl acetate = 5 / 1, Rf = 0.5) indicated that the reaction was complete. The reaction mixture was diluted with water (100 mL) and extracted with dichloromethane (80 mL x 3). The combined organic layers were dried over Na2SO4, filtered, and the filtrate was concentrated to give the residue. The residue was purified by column chromatography (silica gel, petroleum ether / ethyl acetate = 100 / 1 to 3 / 1) to give compound 5 (1.02 g, 72.5% yield, 86.8% purity) as a yellow solid.

[0150] HOBt (88.7 mg, 656 μmol) and DIEA (84.8 mg, 656 μmol, 114 μL) were added to a solution of compound 5 (235 mg, 328 μmol) in DMF (4.00 mL) and pyridine (1.00 mL). The mixture was stirred at 30 °C for 4 hours. LC-MS showed detection of 42.2% of the target compound. TLC (dichloromethane / methanol = 20 / 1, Rf = 0.43) showed that the reaction was complete. The reaction mixture was concentrated under vacuum at 30 °C to give the residue. The residue was purified by column chromatography (silica gel, dichloromethane / methanol = 20 / 1 to 10 / 1) to give compound 7 (390 mg, 223 μmol) as a yellow solid.

[0151] LiOH·H₂O (33.7 mg, 804 μmol) was added to a solution of compound 7 (200 mg, 134 μmol) in THF (8.00 mL) and water (2.00 mL). The mixture was stirred at 25 °C for 4 hours. LC-MS showed a detection of 46.9% of the target compound. The pH of the reaction mixture was adjusted to 4 with AcOH and concentrated using a vacuum pump. The residue was purified by preparative HPLC (column: CD04-Welch Ultimate C18 150*25*7 μm; mobile phase: [water (TFA)-acetonitrile]; gradient: phase B from 24% to 54% over 10 minutes) to give compound 8 (71.0 mg, 45.9% yield) as a white solid.

[0152] DIEA (22.3 mg, 172 μmol, 30.0 μL) was added to a solution of compound 8 (65 mg, 57.5 μmol) and compound 9 (24.8 mg, 80.5 μmol) in DMF (2.00 mL). The mixture was stirred at 20 °C for 1 hour. LC-MS showed detection of 68.4% of the target compounds. DMF was removed from the reaction mixture at 35 °C. Acetonitrile (0.50 mL) and methyl tert-butyl ether (6.00 mL) were added to the mixture. The combined organic layers were filtered, and the filter cake was concentrated to give the residue, which was purified by preparative HPLC (TFA conditions; column: CD04-Welch Ultimate C18 150*25*7 μm; mobile phase: [water (TFA)-acetonitrile]; gradient: phase B from 30% to 60% over 10 minutes). LP006 was given as a grayish-white solid.

[0153] Linker - Conjugation of payload molecules to antibodies: Conjugation was performed to prepare a drug-antibody ratio (DAR) of 4.0. SAC-2D antibody (1106.19 µL, 9.04 mg / mL) was pipetted into a 50 mL tube in buffer (PBS, pH 7.4, 0.02% PS80), and then 69.73 µL of 2 mM TCEP dissolved in water (TCEP / mAb molar ratio set at 2.00) was added for reduction. Reaction buffer (49.07 µL, 50 mM phosphate buffer, pH 6.5) was added to the tube to bring the final mAb concentration to 4.0 mg / mL. The tube was placed in a constant-temperature shaker at 22°C and gently shaken at 60 rpm. After 18 hours of reduction, 10 mM (10.53 mg / mL) of the linker-loaded polymer in DMA (55.78 µL) was added to the sample to bring the drug-antibody molar equivalent ratio to 8.0. PG (propylene glycol) solvent (1194.22 µL) was added to the reduced sample to ensure an organic solvent percentage of 50%. The reaction sample was then incubated for 1 hour at 22°C with gentle shaking at 60 rpm. After 1 hour, the sample was purified using a Zeba desalting column (40 kDa, 5 mL, Thermo Scientific-87770) and Amicon ultrafiltration centrifuge tubes (50 kDa, 15 mL, Millipore-UFC905024) to obtain the SAC-LP-002 product. The product was characterized by LC-MS, SEC-HPLC, HIC-HPLC, RP-HPLC, and KTA. All conjugates were processed using similar procedures.

[0154] Example 14 This example demonstrates the effects of the conjugate provided in Example 13 on various cell lines.

[0155] The SAC-2D conjugates prepared in Example 13 were >98% monomers as determined by size exclusion chromatography, and >70% of the product, as determined by high-performance ion exchange chromatography, contained four linker-loador molecules per antibody molecule. Endotoxin levels in the SAC-2D linker-loador product were measured using the Limulus Amebocyte Lysate (LAL) assay and found to be below 0.1 EU / mg.

[0156] These conjugates were screened for various cell lines. Cell lines were tested as described in Example 11, and the results are shown in Tables 9 and 10. Entries marked "na" indicate that no cytotoxic activity was observed.

[0157] Table 9

[0158] Table 10

[0159] All references cited in this article, including publications, patent applications and patents, are incorporated herein by reference to the same extent that each reference is individually and specifically indicated to be incorporated by reference and listed in full.

[0160] When the terms "a," "an," "the," and "at least one," and similar indicators are used in the context of describing the invention (particularly in the context of the following claims), they should be interpreted to cover both the singular and the plural, unless otherwise stated herein or obviously contradicted by the context. When the term "at least one" is followed by a list of one or more items (e.g., "at least one of A and B"), it should be interpreted as selecting one item (A or B) from the list or any combination of two or more of the listed items (A and B), unless otherwise stated herein or obviously contradicted by the context. The terms "comprising," "having," "including," and "containing" should be interpreted as open-ended terms (i.e., meaning "including but not limited to"), unless otherwise stated. The enumeration of numerical ranges herein is intended only as a shorthand method for individually referring to each individual value falling within that range, unless otherwise stated herein, and each individual value is incorporated into the specification as if it were individually enumerated herein. All methods described herein may be performed in any suitable order, unless otherwise stated herein or obviously contradicted by the context. 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 constitute a limitation on the scope of the invention unless otherwise required. No language in the specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0161] Preferred embodiments of the invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of these preferred embodiments may become apparent to those skilled in the art upon reading the foregoing description. The inventors expect those skilled in the art to employ such variations as appropriate, and the inventors desire that the invention be practiced in ways different from those specifically described herein. Therefore, the invention includes all modifications and equivalents of the subject matter as set forth in the appended claims, provided they comply with applicable law. Furthermore, unless otherwise stated herein or clearly contradicted by the context, any combination of all possible variations of the foregoing elements is included in the invention.

Claims

1. A dPSA binding conjugate comprising a dPSA binding agent and a cytotoxic payload conjugated thereto, wherein the cytotoxic payload is monomethylolpropamine E / Dolastatin-10 (MMAE), monomethylolpropamine F (MMAF), maytansin (DM1), N2'-deacetyl-N2'-(4-mercapto-4-methyl-1-oxopentyl)matansin (DM4), 12-ethyl-9-hydroxycamptothecin (7-ethyl-10-hydroxycamptothecin (SN-38)), ethatecan mesylate (DX-8951f), PNU-1596821, pyrrolobenzodiazepine (PBD or SGD-1882), an ethatecan derivative, or a camptothecin derivative.

2. The dPSA binding conjugate according to claim 1, wherein the cytotoxic payload is MMAE, ethatecan, or an ethatecan derivative.

3. The dPSA binder conjugate according to claim 1 or 2, wherein the dPSA binder comprises: (a) The immunoglobulin heavy chain variable region, which includes SEQ ID NO: 51 or at least its complementarity-determining region (CDR); and the immunoglobulin light chain variable region, which includes SEQ ID NO: 52 or 53 or at least its CDR; (b) The immunoglobulin heavy chain variable region, comprising any one or at least one of SEQ ID NO: 17-20 and its complementarity-determining region (CDR); and the immunoglobulin light chain variable region, comprising SEQ ID NO: 21 or at least its CDR; (c) An immunoglobulin heavy chain variable region comprising any one or at least one of SEQ ID NO: 1-4 and its complementarity-determining region (CDR); and an immunoglobulin light chain variable region comprising SEQ ID NO: 5 or at least its CDR; or (d) The immunoglobulin heavy chain variable region, which includes SEQ ID NO: 35 or at least its complementarity-determining region (CDR); and the immunoglobulin light chain variable region, which includes SEQ ID NO: 36 or at least its CDR.

4. The dPSA binder conjugate according to claim 1 or 2, wherein the dPSA binder comprises: (a) The variable region of the immunoglobulin heavy chain, which includes: CDRH1 contains any one of SEQ ID NO: 6-9; CDRH2 contains SEQ ID NO: 10; CDRH3, containing SEQ ID NO: 11; and The variable region of the immunoglobulin light chain includes: CDRL1 contains SEQ ID NO: 12; CDRL2, containing SEQ ID NO: 13; and CDRL3 contains SEQ ID NO: 14; (b) The variable region of the immunoglobulin heavy chain, which includes: CDRH1 contains any one of SEQ ID NO: 24-27; CDRH2 contains SEQ ID NO: 28; CDRH3, containing SEQ ID NO: 29; and The variable region of the immunoglobulin light chain includes: CDRL1 contains SEQ ID NO: 30; CDRL2, containing SEQ ID NO: 31; and CDRL3 contains SEQ ID NO: 32; (c) The variable region of the immunoglobulin heavy chain, which includes: CDRH1 contains SEQ ID NO: 39; CDRH2, containing SEQ ID NO: 40; and CDRH3, containing SEQ ID NO: 41 or 47; and The variable region of the immunoglobulin light chain includes: CDRL1 contains SEQ ID NO: 42; CDRL2, containing SEQ ID NO: 43 or 56; and CDRL3, containing SEQ ID NO: 44; or (d) The variable region of the immunoglobulin heavy chain, which includes: CDRH1 contains SEQ ID NO: 45; CDRH2, containing SEQ ID NO: 46; and CDRH3, containing SEQ ID NO: 41 or 47; and The variable region of the immunoglobulin light chain includes: CDRL1 contains SEQ ID NO: 48; CDRL2, containing SEQ ID NO: 49 or 56; and CDRL3 contains SEQ ID NO:

50.

5. The dPSA binding conjugate according to claim 1 or 2, wherein the dPSA binding agent comprises a heavy chain immunoglobulin polypeptide comprising SEQ ID NO: 57 or SEQ ID NO: 60, and a light chain immunoglobulin polypeptide comprising SEQ ID NO: 58 or SEQ ID NO:

59.

6. The dPSA conjugate according to any one of claims 1-4, wherein the dPSA conjugate is an antibody or an antigen-binding antibody fragment.

7. The dPSA conjugate according to any one of claims 1-4, wherein the dPSA conjugate is an IgG1 or IgG4 antibody.

8. The dPSA binder conjugate according to any one of claims 1-4, wherein the dPSA binder is an F(ab')2 fragment, a Fab' fragment, a Fab fragment, an Fv fragment, a scFv fragment, a dsFv fragment, or a dAb fragment.

9. The dPSA binding conjugate according to any one of claims 1-8, wherein the dPSA binding agent comprises an Fc region, and the Fc region comprises modifications that reduce FcγR binding and / or reduce complement-dependent cytotoxicity (CDC) or antibody-dependent cytotoxicity (ADCC).

10. The dPSA binder conjugate according to any one of claims 1-9, wherein the cytotoxic payload is conjugated to the dPSA binder via a linker.

11. A dPSA binder comprising an immunoglobulin heavy chain comprising SEQ ID NO: 57 and an immunoglobulin light chain comprising SEQ ID NO: 58 or SEQ ID NO:

59.

12. A dPSA binder comprising an immunoglobulin heavy chain comprising SEQ ID NO: 60 and an immunoglobulin light chain comprising SEQ ID NO: 58 or SEQ ID NO:

59.

13. The dPSA binder according to claim 11 or 12, wherein the dPSA binder is defucosylated.

14. The dPSA binder of claim 11, wherein the dPSA binder comprises an immunoglobulin heavy chain comprising SEQ ID NO:57, the heavy chain having modifications to reduce FcγR binding and / or reduce complement-dependent cytotoxicity (CDC) or antibody-dependent cytotoxicity (ADCC).

15. A dPSA binding conjugate comprising the dPSA binding agent of any one of claims 11-14 and a cytotoxic payload.

16. The dPSA binding conjugate according to claim 15, wherein the cytotoxic payload is monomethylolpropamine E / Dolastatin-10 (MMAE), monomethylolpropamine F (MMAF), maytansin (DM1), N2'-deacetyl-N2'-(4-mercapto-4-methyl-1-oxopentyl)matansin (DM4), 12-ethyl-9-hydroxycamptothecin (7-ethyl-10-hydroxycamptothecin (SN-38)), ethatecan mesylate (DX-8951f), PNU-1596821, pyrrolobenzodiazepine (PBD or SGD-1882), ethatecan derivatives, or camptothecin derivatives.

17. The dPSA binding conjugate according to claim 15 or 16, wherein the cytotoxic payload is MMAE, ethatecan mesylate, or an ethatecan derivative.

18. A nucleic acid encoding the immunoglobulin heavy chain and / or light chain variable region of the dPSA binder of any one of claims 11-14, optionally in a vector.

19. The nucleic acid of claim 18, further encoding a guide sequence for the variable region of the immunoglobulin heavy chain and / or light chain.

20. A cell comprising the nucleic acid of claim 18 or 19.

21. A cell line expressing the dPSA binder of any one of claims 11-14.

22. A method for preparing the dPSA binder according to any one of claims 11-14, the method comprising expressing in cells a nucleotide sequence encoding the immunoglobulin heavy chain polypeptide and a nucleic acid sequence encoding the immunoglobulin light chain polypeptide.

23. A composition comprising (a) a dPSA conjugate according to any one of claims 1-10 or 15-17, a dPSA conjugate or a nucleic acid encoding the conjugate according to any one of claims 11-14, and (b) a pharmaceutically acceptable carrier.

24. A method for killing cancer cells expressing dPSA, the method comprising contacting the cancer cells with a dPSA binding conjugate according to any one of claims 1-10 or 15-17, a dPSA binding agent according to any one of claims 11-14, or a composition according to claim 23.

25. A method of treating a subject with cancer characterized by dPSA expression, the method comprising administering to the subject a dPSA binding conjugate according to any one of claims 1-10 or 15-17, a dPSA binding agent according to any one of claims 11-14, or a composition according to claim 23.

26. A method for delivering a cytotoxic payload to cells expressing dPSA, the method comprising contacting the cells with a dPSA binding conjugate as described in any one of claims 1-10 or 15-17.

27. The method of claim 26, wherein the cell is a cancer cell.

28. The dPSA binder conjugate of any one of claims 1-10 or 15-17, the dPSA binder of any one of claims 11-14, or the composition of claim 23, for the treatment of cancer.

29. Use of the dPSA binder conjugate of any one of claims 1-10 or 15-17, the dPSA binder of any one of claims 11-14, or the composition of claim 23 in the preparation of a medicament for treating cancer.

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