De-n-acetylated polysialic acid (DPSA) binding agent conjugates and mel'hod of using same

Abstract

A dPSA-binding agent conjugate and methods of use to treat cancer and kill cancer cells.

Description

Leydig 515590 1 DE-N-ACETYLATED POLYSIALIC ACID (dPSA) BINDING AGENT CONJUGATES AND METHOD OF USING SAME CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims priority to U.S. provisional patent application 63 / 670,443, filed on July 12, 2024, the entire disclosure of which is hereby incorporated by reference. INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0002] Incorporated by reference in its entirety herein is a computer-readable nucleotide / amino acid sequence listing submitted concurrently herewith and identified as follows: One 57,10 Byte ASCII (Text) file named "515590ST26.xml," created on July 14, 2025. BACKGROUND OF THE INVENTION

[0003] In general, the goal of anti-cancer immunotherapy has been to identify stable antigens that are highly expressed but not shed or secreted from tumor cells, which antigens can then be used as the basis of immunotherapy, e.g., as the antigen in a cancer vaccine or as a target for antibody-based cancer therapy. Optimally, such tumor antigens are acceptably specific for the cancerous target cells, so as to reduce deleterious side effects that can result from cross-reactivity with non-cancerous cells of the subject being treated. Where cross-reactivity affects cells that can be repopulated, it may be acceptable to relax this requirement for the specificity of immunotherapy.

[0004] Altered glycosylation patterns of cell surface proteins occur in nearly all types of cancer. Excessive sialylation of glycoproteins and glycolipids is central to the aberrant regulation of cell adhesion in metastatic cancer, which in turn can result from re-expression and / or overexpression of genes normally expressed during development but not in cells of adult normal tissues. In particular, poly alpha 2->8 N-acetyl neuraminic acid or polysialic acid (polySia) is expressed mainly during fetal development and is highly restricted to just a few regenerative tissues post development. A de-N-acetylated form of polySia (dPSA) on the surface of cancerLeydig 515590 2 but not post-development human cells, and can serve as a tumor antigen for cancer identification and therapy.

[0005] Thus, new agents capable of targeting cells expressing dPSA and delivering cargo, such as cytotoxic moieties, are needed. BRIEF SUMMARY OF THE INVENTION

[0006] Provided herein is a dPSA-binding agent conjugate comprising immunoglobulin heavy and light chain polypeptides, a linker attached to the dPSA binding agent, and a payload attached to the linker, wherein the linker is resistant to degradation in human serum and / or by one or more neutrophil serine proteases.

[0007] Related compositions and methods also are provided, as will be apparent from the following detailed description. DETAILED DESCRIPTION OF THE INVENTION

[0008] Provided herein is a binding agent conjugate comprising a dPSA binding agent (e.g., antibody or antibody fragment), a linker attached to the dPSA binding agent, and a payload (e.g., a cytotoxic moiety) attached to the linker, wherein the linker is resistant to degradation in human serum and / or resistant to degradation (cleavage) by one or more neutrophil serine proteases. In some embodiments, the linker is resistant to degradation by elastase, proteinase 3, cathepsin G, and / or neutrophil serine protease 4 (NSP 4), which proteases are secreted by neutrophils undergoing development in bone marrow and when stimulated the blood stream. In some embodiments, the linker when exposed to one or more (or all) of the neutrophil serine proteases degrades at a rate that is less than the rate of degradation of one or more of an MC-Val-Cit-PAB linker (MC-VC-PAB), an MC-Gly-Gly-Phe-Gly linker (MC-GGFG), an MC-Val-Lys (PEG)- PAB linker, and / or an MC-beta-glucuronide linker. In some embodiments, the linker when exposed to one or more (or all) of the neutrophil serine proteases degrades at a rate that is less than the rate of degradation of a MC-VC-PAB linker. In some embodiments, the linker when exposed to one or more (or all) of the neutrophil serine proteases degrades at a rate that is less than the rate of degradation of a MC-GGFG linker. In some embodiments, the linker when exposed to one or more (or all) of the neutrophil serine proteases degrades at a rate that is lessLeydig 515590 3 than the rate of degradation of a MC-Val-Lys (PEG)-PAB linker. In some embodiments, the linker when exposed to one or more (or all) of the neutrophil serine proteases degrades at a rate that is less than the rate of degradation of a MC-beta-glucuronide linker. In some embodiments, the linker comprises a dipeptide moiety comprising phenylalanine and lysine (FK). In some embodiments, the linker can comprise a glutamic acid-glycine-citrulline (EGCit) tripeptide moiety. In some embodiments, the linker can further comprise a para-aminobenzyloxycarbonyl (PAB) moiety, and a functional group (e.g., maleimidocaprolyl or “MC”) to facilitate conjugation with a free amine on a lysine residue or thiol group of a cysteine residue on the antibody. By way of further illustration, the linker can be a maleimidocaprolyl-phe-lys-PAB linker (MC-FK-PAB), or a maleimidocaprolyl-glutamic acid-glycine-citrulline-PAB linker (MC- EGCit-PAB).

[0009] The dPSA binding agent selectively binds to cells, particularly cancer cells, that express dPSA. In some embodiments, the dPSA binding agent binds nucleolin modified with dPSA Without wishing to be bound by any particular theory or mechanism of action, the binding agents are believed to bind an antigen (e.g., nucleolin) comprising an epitope defined at least in part by one or more dPSA residues.

[0010] The dPSA-binding agent can comprise Ig heavy chain and light chain polypeptides, each of which comprise at least an Ig heavy chain variable region and an Ig light chain variable region, respectively. The Ig heavy and light chain variable regions, in turn, each comprise three complementarity determining regions (CDRs), usually referred to as CDR1, CDR2, or CDR3. The CDR regions also can be referred to using an “H” or “L” in the nomenclature to denote the heavy or light chain, respectively, i.e., CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3. The CDRs of a given Ig sequence can be determined by any of several conventional numbering schemes, such as Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo (these are commonly used names for numbering schemes widely known in the field and described in published literature see, e.g., 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 – 948Leydig 515590 4 (1997) describing the “Chothia” numbering scheme; Abhinandan et al., Analysis and Improvements to Kabat and Structurally Correct Numbering of Antibody Variable Domains, Mol. Immunol., 45: 3832 – 3839 (2008) describing the “Martin” or “Enhanced Chothia” numbering scheme; Lefranc et al., The IMGT unique numbering for immunoglobulins, T cell Receptors and Ig-like domains, The Immunologist, 7: 132-136 (1999) and Lefranc et al., IMGT unique numbering for immunoglobulin and T cell receptor variable domains and I superfamily V-like domains, Dev. Comp. Immunol., 27: 55 – 77 (2003) describing the “IMGT” numbering scheme; and Honegger et al., Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool, J. Mol. Biol.309: 657 – 670 (2001) describing the “AHo” numbering scheme). The identification of CDRs also can be made through relevant empirical binding data, such as the crystallography studies of the binding agent interactions with its target (e.g., antigen or portion thereof comprising the binding epitope), optionally in conjunction with any of the foregoing numbering systems.

[0011] The dPSA-binding agents described herein are man-made and non-naturally occurring. They have been generated by laboratory techniques and, thus, are properly considered recombinant or synthetic molecules comprising recombinant or synthetic amino acid sequences. The Ig heavy and light chain polypeptides can be “isolated” in the sense that they are removed from the environment in which they are produced (e.g., cell culture) and purified to any degree.

[0012] In some embodiments, the dPSA-binding agent comprises an Ig heavy chain variable region comprising any of SEQ ID NOs: 1-4 or at least the CDRs thereof; and an Ig light chain variable region comprising SEQ ID NO: 5 or at least the CDRs thereof. The CDRs can be as determined using any known numbering scheme, such as Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo. In some embodiments, the antibody comprises a heavy chain variable region of any of SEQ ID NOs: 1-4 and light chain variable region of SEQ ID NO: 5, or at least the CDRs thereof as determined by Kabat. In some embodiments, the antibody comprises a heavy chain variable region of any of SEQ ID NOs: 1-4 and light chain variable region of SEQ ID NO: 5, or at least the CDRs thereof as determined by Chothia. In some embodiments, the antibody comprises a heavy chain variable region of any of SEQ ID NOs: 1-4 and light chain variable region of SEQ ID NO: 5, or at least the CDRs thereof as determined by Martin. In some embodiments, the antibody comprises a heavy chain variable region of any of SEQ ID NOs: 1-4Leydig 515590 5 and light chain variable region of SEQ ID NO: 5, or at least the CDRs thereof as determined by IGMT. In some embodiments, the antibody comprises a heavy chain variable region of any of SEQ ID NOs: 1-4 and light chain variable region of SEQ ID NO: 5, or at least the CDRs thereof as determined by AHo. In some embodiments, the dPSA binding agent comprises one of the following combination of Ig heavy and light chain variable regions, or at least the CDRs thereof as determined by any of Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo: Heavy Chain Variable Region Light Chain Variable Region 1 SEQ ID NO: 1 or CDRs thereof SEQ ID NO: 5 or CDRs thereof, variable region comprising any of SEQ ID NOs: 17-20 or at least the CDRs thereof; and an Ig light chain variable region comprising SEQ ID NO: 21 or at least the CDRs thereof. The CDRs can be as determined using any known numbering scheme, such as Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo. In some embodiments, the antibody comprises a heavy chain variable region of any of SEQ ID NOs: 17-20 and light chain variable region of SEQ ID NO: 21, or at least the CDRs thereof as determined by Kabat. In some embodiments, the antibody comprises a heavy chain variable region of any of SEQ ID NOs: 17-20 and light chain variable region of SEQ ID NO: 21, or at least the CDRs thereof as determined by Chothia. In some embodiments, the antibody comprises a heavy chain variable region of any of SEQ ID NOs: 17-20 and light chain variable region of SEQ ID NO: 21, or at least the CDRs thereof as determined by Martin. In some embodiments, the antibody comprises a heavy chain variable region of any of SEQ ID NOs: 17-20 and light chain variable region of SEQ ID NO: 21, or at least the CDRs thereof as determined by IGMT. In some embodiments, the antibody comprises a heavy chain variable region of any of SEQ ID NOs: 17-20 and light chain variable region of SEQ ID NO: 21, or at least the CDRs thereof as determined by AHo. In some embodiments, the dPSA binding agent comprises one of the following combination of Ig heavy and light chain variable regions, or at least the CDRs thereof as determined by any of Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo: Heavy Chain Variable Region Light Chain Variable RegionLeydig 515590 6 1 SEQ ID NO: 17 or CDRs thereof SEQ ID NO: 21 or CDRs thereof 2 SEQ ID NO: 18 or CDRs thereof SEQ ID NO: 21 or CDRs thereof f ft comprising an Ig heavy chain variable region and an Ig light chain variable region, wherein the Ig heavy chain variable region comprises: a CDR1 comprising any one of SEQ ID NOs: 6-9 or 24-27, CDR2 comprising SEQ ID NO: 10 or 28, and CDR3 comprising SEQ ID NO: 11 or 29; and the Ig light chain variable region comprises a CDR1 comprising SEQ ID NO: 12 or 30, CDR2 comprising SEQ ID NO: 13 (RMS) or 31, and CDR3 comprising SEQ ID NO: 14 or 32.

[0015] In some embodiments, provided herein is a dPSA binding agent comprising an Ig heavy chain variable region and an Ig light chain variable region, wherein the Ig heavy chain variable region comprises: a CDR1 comprising any one of SEQ ID NOs: 6-9, CDR2 comprising SEQ ID NO: 10, and CDR3 comprising SEQ ID NO: 11; and the Ig light chain variable region comprises a CDR1 comprising SEQ ID NO: 12, CDR2 comprising SEQ ID NO: 13, and CDR3 comprising SEQ ID NO: 14. CDRH1 CDRH2 CDRH3 CDRL1 CDRL2 CDRL3 1 SEQ ID SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: : : :

[0016] In some embodiments, provided herein is a dPSA binding agent comprising an Ig heavy chain variable region and an Ig light chain variable region, wherein the Ig heavy chain variable region comprises: a CDR1 comprising any one of SEQ ID NOs: 24-27, CDR2 comprising SEQ ID NO: 28, and CDR3 comprising SEQ ID NO: 29; and the Ig light chain variable region comprises a CDR1 comprising SEQ ID NO: 30, CDR2 comprising SEQ ID NO: 31, and CDR3 comprising SEQ ID NO: 32. CDRH1 CDRH2 CDRH3 CDRL1 CDRL2 CDRL3Leydig 515590 7 1 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: 24 28 29 30 31 32 O: : : : : : :cco g o ye a o e aspec o e sc osu e, e - g age co p ses an Ig heavy chain variable region comprising 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%) to any of SEQ ID NOs: 1-4; and an Ig light chain variable region comprising 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%) to SEQ ID NO: 5. In some embodiments, the dPSA- binding agent comprises an Ig heavy chain polypeptide comprising 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%) to SEQ ID NO: 15; and an Ig light chain polypeptide comprising 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%) to SEQ ID NO: 16. In any of the foregoingLeydig 515590 8 embodiments, the Ig heavy chain variable region and Ig light chain variable region can comprise (retain) the CDRs of the heavy and light chain variable regions of sequences, which CDRs can be determined using any known numbering scheme, such as Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo, or as otherwise set forth herein (e.g., SEQ ID NOs: 6-14 above). In some embodiments, the Ig heavy chain variable region comprises one of SEQ ID NOs: 1-4 and the Ig light chain variable region comprises SEQ ID NO: 5. In some embodiments, the dPSA binding agent comprises Ig heavy and light chain polypeptides comprising SEQ ID NO: 15 and SEQ ID NO: 16, respectively.

[0018] According to still another aspect of the disclosure, the dPSA-binding agent comprises an Ig heavy chain variable region comprising 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%) to any of SEQ ID NOs: 17-20; and an Ig light chain variable region comprising 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%) to SEQ ID NO: 21. In some embodiments, the dPSA- binding agent comprises an Ig heavy chain polypeptide comprising 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%) to SEQ ID NO: 22; and an Ig light chain polypeptide comprising 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%) to SEQ ID NO: 23. In any of the foregoing embodiments, the Ig heavy chain variable region and Ig light chain variable region can comprise (retain) the CDRs of the heavy and light chain variable regions, which CDRs can be determined using any known numbering scheme, such as Kabat, Chothia, Martin (Enhanced Chothia),Leydig 515590 9 IGMT, or AHo, or as otherwise set forth herein (e.g., SEQ ID NOs: 6-14 or 24-32 above). In some embodiments, the Ig heavy and light chain variable regions comprises one of SEQ ID NOs: 17-20 and the Ig light chain variable region comprises SEQ ID NO: 21, respectively. In some embodiments, the dPSA binding agent comprises Ig heavy and light chain polypeptides comprising SEQ ID NO: 22 and SEQ ID NO: 23, respectively.

[0019] In some embodiments, the dPSA binding agent comprises Ig heavy and light chain polypeptides comprising SEQ ID NOs: 33 and 34, respectively.

[0020] In some embodiments, the dPSA-binding agent comprises an Ig heavy chain variable region comprising SEQ ID NO: 35 or at least the CDRs thereof; and an Ig light chain variable region comprising SEQ ID NO: 36 or at least the CDRs thereof. The CDRs can be as determined using any known numbering scheme, such as Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo. In some embodiments, the antibody comprises a heavy chain variable region of SEQ ID NO: 35 and light chain variable region of SEQ ID NO: 36, or at least the CDRs thereof as determined by Kabat. In some embodiments, the antibody comprises a heavy chain variable region of SEQ ID NO: 35 and light chain variable region of SEQ ID NO: 36, or at least the CDRs thereof as determined by Chothia. In some embodiments, the antibody comprises a heavy chain variable region of SEQ ID NO: 35 and light chain variable region of SEQ ID NO: 36, or at least the CDRs thereof as determined by Martin. In some embodiments, the antibody comprises a heavy chain variable region of SEQ ID NO: 35 and light chain variable region of SEQ ID NO: 36, or at least the CDRs thereof as determined by IGMT. In some embodiments, the antibody comprises a heavy chain variable region of SEQ ID NO: 35 and light chain variable region of SEQ ID NO: 36, or at least the CDRs thereof as determined by AHo.

[0021] In some embodiments, the dPSA-binding agent comprises an Ig heavy chain variable region comprising SEQ ID NO: 51 or at least the CDRs thereof; and an Ig light chain variable region comprising SEQ ID NO: 52 or 53 or at least the CDRs thereof. The CDRs can be as determined using any known numbering scheme, such as Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo. In some embodiments, the antibody comprises a heavy chain variable region of SEQ ID NO: 51 and light chain variable region of SEQ ID NO: 52 or 53, or at least the CDRs thereof as determined by Kabat. In some embodiments, the antibody comprises a heavy chain variable region of SEQ ID NO: 51 and light chain variable region of SEQ ID NO: 52 or 53,Leydig 515590 10 or at least the CDRs thereof as determined by Chothia. In some embodiments, the antibody comprises a heavy chain variable region of SEQ ID NO: 51 and light chain variable region of SEQ ID NO: 52 or 53, or at least the CDRs thereof as determined by Martin. In some embodiments, the antibody comprises a heavy chain variable region of SEQ ID NO: 51 and light chain variable region of SEQ ID NO: 52 or 53, or at least the CDRs thereof as determined by IGMT. In some embodiments, the antibody comprises a heavy chain variable region of SEQ ID NO: 51 and light chain variable region of SEQ ID NO: 52 or 53, or at least the CDRs thereof as determined by AHo.

[0022] In some embodiments, the dPSA binding agent comprises an Ig heavy chain variable region and an Ig light chain variable region, wherein the Ig heavy chain variable region comprises: a CDR1 comprising SEQ ID NO: 39 or 45, CDR2 comprising SEQ ID NO: 40 or 46, and CDR3 comprising SEQ ID NO: 41 or 47; and the Ig light chain variable region comprises a CDR1 comprising SEQ ID NO: 42 or 48, CDR2 comprising SEQ ID NO: 43 (GTN), 49, or 56, and CDR3 comprising SEQ ID NO: 44 or 50. In some embodiments, the dPSA binding agent comprises a CDR1 comprising SEQ ID NO: 39, CDR2 comprising SEQ ID NO: 40, and CDR3 comprising SEQ ID NO: 41 or 47; and the Ig light chain variable region comprises a CDR1 comprising SEQ ID NO: 42, CDR2 comprising SEQ ID NO: 43 or 56, and CDR3 comprising SEQ ID NO: 44.

[0023] In some embodiments, the dPSA binding agent comprises an Ig heavy chain variable region and an Ig light chain variable region, wherein the Ig heavy chain variable region comprises: a CDR1 comprising SEQ ID NO: 45, CDR2 comprising SEQ ID NO: 46, and CDR3 comprising SEQ ID NO: 41 or 47; and the Ig light chain variable region comprises a CDR1 comprising SEQ ID NO: 48, CDR2 comprising SEQ ID NO: 49 or 56, and CDR3 comprising SEQ ID NO: 50. CDRH1 CDRH2 CDRH3 CDRL1 CDRL2 CDRL3Leydig 515590 11 5 SEQ ID NO: SEQ ID SEQ ID NO: SEQ ID SEQ ID NO: SEQ ID 39 NO: 40 41 NO: 42 56 NO: 44, g g p g y le region comprising 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%) to SEQ ID NO: 35; and an Ig light chain variable region comprising 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%) to SEQ ID NO: 36. In some embodiments, the dPSA-binding agent comprises an Ig heavy chain polypeptide comprising 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%) to SEQ ID NO: 37; and an Ig light chain polypeptide comprising 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%) to SEQ ID NO: 38. In any of the foregoing embodiments, the Ig heavy chain variable region and Ig light chain variable region can comprise (retain) the CDRs of the heavy and light chain variable regions of SEQ ID NO: 35 and 36, respectively, which CDRs can be determinedLeydig 515590 12 using any known numbering scheme, such as Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo, or as otherwise set forth herein (e.g., SEQ ID NOs: 39-44). In some embodiments, the Ig heavy and light chain variable regions comprise SEQ ID NO: 35 and SEQ ID NO: 36, respectively. In some embodiments, the dPSA binding agent comprises Ig heavy and light chain polypeptides comprising SEQ ID NO: 37 and SEQ ID NO: 38, respectively.

[0025] In some embodiments, the dPSA-binding agent comprises an Ig heavy chain variable region comprising 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%) to SEQ ID NO: 51; and an Ig light chain variable region comprising 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%) to SEQ ID NO: 52 or 53. In some embodiments, the dPSA-binding agent comprises an Ig heavy chain polypeptide comprising 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%) to SEQ ID NO: 51; and an Ig light chain polypeptide comprising 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%) to SEQ ID NO: 52 or 53. In any of the foregoing embodiments, the Ig heavy chain variable region and Ig light chain variable region can comprise (retain) the CDRs of the heavy and light chain variable regions of SEQ ID NO: 51 and 52 or 53, respectively, which CDRs can be determined using any known numbering scheme, such as Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo, or as otherwise set forth herein. In some embodiments, the Ig heavy and light chain variable regions comprise SEQ ID NO: 51 and SEQ ID NO: 52 or 53, respectively.Leydig 515590 13

[0026] In some embodiments, the dPSA-binding agent comprises an Ig heavy chain comprising 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%) to SEQ ID NO: 60; and an Ig light chain comprising 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%) to SEQ ID NO: 58 or 59. In some embodiments, the dPSA-binding agent comprises an Ig heavy chain polypeptide comprising 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%) to SEQ ID NO: 57; and an Ig light chain polypeptide comprising 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%) to SEQ ID NO: 58 or 59. In any of the foregoing embodiments, the Ig heavy chain variable region and Ig light chain variable region can comprise (retain) the CDRs of the heavy and light chain variable regions of SEQ ID NO: 57 or 60 and 58 or 59, respectively, which CDRs can be determined using any known numbering scheme, such as Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo, or as otherwise set forth herein. In some embodiments, the Ig heavy and light chain regions comprise SEQ ID NO: 57 and SEQ ID NO: 58 or 59, respectively. In some embodiments, the Ig heavy and light chain regions comprise SEQ ID NO: 60 and SEQ ID NO: 58 or 59, respectively.

[0027] In some embodiments, the dPSA binding agent comprises an Ig heavy and light chain variable region comprising SEQ ID NO: 33 and 34, or at least the CDRs thereof as determined using any known numbering scheme, such as Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo; and / or having at least 80% sequence identity (e.g., at least 80%, at least 81%, atLeydig 515590 14 least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to SEQ ID NO: 33 or 34, optionally while retaining the CDRs thereof. In some embodiments, the dPSA binding agent comprises an Ig heavy and light chain variable region comprising SEQ ID NO: 54 and 55, or at least the CDRs thereof as determined using any known numbering scheme, such as Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo; and / or having at least 80% sequence identity (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to SEQ ID NO: 54 or 55, optionally while retaining the CDRs thereof.

[0028] Sequence “identity” as used in reference to nucleic acid or amino acid sequences can be determined by comparing a nucleic acid or amino acid sequence of interest to a reference nucleic acid or amino acid sequence. The percent identity is the percentage of nucleotides or amino acid residues that are the same (i.e., that are identical) as between the sequence of interest and the reference sequence when optimally aligned. A number of mathematical algorithms for obtaining the optimal alignment 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 alignment of nucleic acid and amino acid sequences), BLAST programs (e.g., BLAST 2.1, BL2SEQ, and later versions thereof operated by the National Center for Biotechnology Information, Bethesda, MD) and FASTA programs (e.g., FASTA3x, FASTM, and SSEARCH) (for sequence alignment and sequence similarity searches). Sequence alignment algorithms also are 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)).

[0029] With respect to sequences having less than 100% identity to the heavy and light chain sequences specifically set forth above, one or more amino acids of the aforementionedLeydig 515590 15 immunoglobulin heavy chain polypeptides and / or light chain polypeptides can be replaced or substituted with a different amino acid, and / or one of more amino acids can be deleted from or inserted into the disclosed amino acid sequences, provided the biological activity of the polypeptide (e.g., the ability of the dPSA binding agent to bind dPSA) is substantially retained. The biological activity of a dPSA-binding agent can be measured, for example, by the binding affinity for a particular dPSA epitope and / or cross-reactivity with targets other than dPSA. The aforementioned properties or characteristics can be observed, measured, and / or assessed using standard techniques including, but not limited to, ELISA, competitive ELISA, surface plasmon resonance analysis (BIACORE™), or solution phase competition (KINEXA™), as well as other in vitro or in vivo neutralization assays, binding assays, fluorescence-activated cell binding (FACS), or other suitable assays.

[0030] The dPSA-binding agent can be part of a multispecific (e.g., bispecific or “dual reactive”) construct (e.g., a multispecific antibody, such as a bispecific or dual reactive antibody) that binds dPSA and another antigen. Such a construct can comprise immunoglobulin heavy and light chain polypeptides that bind dPSA as described herein in combination with immunoglobulin heavy chains and light chains from an immunoglobulin that binds an antigen other than dPSA.

[0031] In some embodiments, the dPSA-binding agent can be a “whole” immunoglobulin or an antigen-binding immunoglobulin “fragment.” A “whole” immunoglobulin typically consists of four polypeptides: two heavy (H) chain polypeptides and two light (L) chain polypeptides. Each of the heavy chains contains one N-terminal variable (VH) region and three C-terminal constant (CH1, CH2, and CH3) regions, and each light chain contains one N-terminal variable (VL) region and one C-terminal constant (CL) region. The light chains of antibodies can beassigned to one of two distinct types, either kappa ( ) or lambda ( ), based upon the amino acidsequences of their constant domains. In a typical immunoglobulin, each light chain is linked to a heavy chain by disulfide bonds, and the two heavy chains are linked to each other by disulfide bonds. In this configuration, the light chain variable region is generally aligned with the variable region of the heavy chain, and the light chain constant region is generally aligned with the first constant region of the heavy chain. The remaining constant regions of the heavy chains are generally aligned with each other.Leydig 515590 16

[0032] The variable regions or hypervariable regions of each pair of light and heavy chains form the antigen binding site of an antibody. The VHand VLregions have the same general structure, with each region comprising four framework (FW or FR) regions. The term “framework region,” as used herein, refers to the relatively conserved amino acid sequences within the variable region, which are located between the hypervariable or complementary determining regions (CDRs). There are four framework regions in each variable domain, which are designated FR1, FR2, FR3, and FR4. The framework regions form the sheets that provide the structural framework of the variable region (see, e.g., C.A. Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, NY (2001)). The framework regions are connected by three complementarity determining regions (CDRs). The three CDRs, known as CDR1, CDR2, and CDR3, form the “hypervariable region” of an antibody, which is generally considered to be responsible for antigen binding.

[0033] The term “antibody fragment” and like terms (e.g., “fragment of an antibody,” “antibody fragment,” “functional fragment of an antibody”) are used interchangeably herein to mean one or more fragments or portions of an antibody that retain the ability to specifically bind to an antigen (see, generally, Holliger et al., Nat. Biotech., 23(9): 1126-1129 (2005)). Antibody “fragments,” as used herein and routinely in the art, include not only fragments or pieces of a whole antibody in the literal sense, but also other known engineered antibody-like constructs, which might include linkers or other elements that do not naturally occur in a “whole” antibody. Examples of antibody fragments include, but are not limited to, (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1domains, (ii) a F(ab’)2fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, (iii) a Fv fragment consisting of the VLand VHdomains of a single arm of an antibody, (iv) a Fab’ fragment, which results from breaking the disulfide bridge of an F(ab’)2fragment using mild reducing conditions, and (v) a disulfide-stabilized Fv fragment (dsFv). The dPSA-binding agent also can be a single chain antibody fragment. Examples of single chain antibody fragments include, but are not limited to, (i) a single chain Fv (scFv), which is a monovalent molecule consisting of the two domains of the Fv fragment (i.e., VLand VH) joined by a synthetic linker which enables the two domains to be synthesized as a single polypeptide chain (see, e.g., Bird et al., Science, 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci.Leydig 515590 17 USA, 85: 5879-5883 (1988); and Osbourn et al., Nat. Biotechnol., 16: 778 (1998)) and (ii) a diabody, which is a dimer of polypeptide chains, wherein each polypeptide chain comprises a VHconnected to a VLby a peptide linker that is too short to allow pairing between the VHand VLon the same polypeptide chain, thereby driving the pairing between the complementary domains on different VH-VLpolypeptide chains to generate a dimeric molecule having two functional antigen binding sites. Any other antigen-binding antibody-like constructs known in the art that comprise Ig heavy and light chain CDRs or variable regions can also be used and are antibody fragments for the purposes of this disclosure. In some embodiments, the dPSA binding agent is (or is part of) a chimeric antigen receptor.

[0034] In some embodiments, the dPSA-binding agent comprises a heavy chain constant region, such as a fragment crystallizable (Fc) region or portion thereof. The Fc region can be of any Ig class / subclass (IgA (IgA1, IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3 and IgG4), IgM, including variants thereof. In some embodiments, the dPSA-binding agent is a “whole” or “complete” Ig (i.e., an antibody); and in other embodiments, the binding agent is an antibody fragment conjugated or linked to an Fc region. In some embodiments, the dPSA binding agent comprises an IgG Fc region, such as IgG1 or IgG4. For instance, the dPSA binding agent can be an IgG1 or IgG4 antibody.

[0035] In some embodiments, the dPSA-binding agent comprises an Fc region that binds FcγR and can mediate complement dependent cytotoxicity (CDC) or antibody-dependent cytotoxicity (ADCC). In some embodiments, the dPSA-binding agent comprises an Fc region that activates natural killer (NK) cells. In some embodiments, the Fc region comprises a modification that increases FcγR binding and / or CDC or ADCC as compared to the same binding agent without such modifications. Examples of such modifications include, for instance, afucosylation, 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.

[0036] In some embodiments, the dPSA-binding agent has reduced FcγR binding and / or reduced complement dependent cytotoxicity (CDC) or antibody-dependent cytotoxicity (ADCC). Such reduced effector function is believed to be particularly advantageous when the bindingLeydig 515590 18 agent is part of an antibody-drug conjugate. Thus, for instance, the dPSA-binding agent can comprises an Fc region that is modified to have reduced FcγR binding and / or reduced complement dependent cytotoxicity (CDC) or antibody-dependent cytotoxicity (ADCC) as compared to the same binding agent without such modification. Examples of such modifications include, for instance, aglycosylation (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-binding agent can be a human or humanized antibody, a non-human antibody, or a chimeric antibody. By “chimeric” is meant an antibody or fragment thereof comprising both human and non-human regions. Preferably, the dPSA-binding agent is a humanized antibody. A “humanized” antibody is a monoclonal antibody comprising a human antibody scaffold and at least one CDR obtained or derived from a non-human antibody. Non-human antibodies include antibodies isolated from any non-human animal, such as, for example, a rodent (e.g., a mouse or rat). A humanized antibody can comprise, one, two, or three CDRs obtained or derived from a non-human antibody.

[0037] A human antibody, a non-human antibody, a chimeric antibody, or a humanized antibody can be obtained by any means, including via in vitro sources (e.g., a hybridoma or a cell line producing an antibody recombinantly) and in vivo sources (e.g., rodents). Methods for generating antibodies are known in the art and are described in, 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 certain embodiments, a human antibody or a chimeric antibody can be generated using a transgenic animal (e.g., a mouse) wherein one or more endogenous immunoglobulin genes are replaced with one or more human immunoglobulin genes (see, e.g., Lonberg, Nat. Biotechnol., 23(9): 1117-25 (2005), and Lonberg, Handb. Exp. Pharmacol., 181: 69-97 (2008)). A humanized antibody can be generated using any suitable method known in the art (see, e.g., An, Z. (ed.), Therapeutic Monoclonal Antibodies: From Bench to Clinic, John Wiley & Sons, Inc., Hoboken, New Jersey (2009)), including, e.g., graftingLeydig 515590 19 of non-human CDRs onto a human antibody scaffold (see, e.g., Kashmiri et al., Methods, 36(1): 25-34 (2005); and Hou et al., J. Biochem., 144(1): 115-120 (2008)).

[0038] The payload can be any molecule that is desired to be delivered to a cell that expresses dPSA. Illustrative examples of payloads include a peptide or protein moiety, a fluorescent molecule, a radioactive molecule, an MRI imaging agent, a targeting molecule (e.g., a molecule that binds a biological target, such as another antibody or antibody fragment, a ligand, a receptor, or a small molecule), or a chemotherapeutic (e.g., cytotoxic) agent.

[0039] In some embodiments, the payload is a cytotoxic moiety or molecule. In some embodiments, the payload is an antibiotic or anti-cancer or antineoplastic agent. For example, in some embodiments, the payload is a tubulin inhibitor, a DNA Topoisomerase I inhibitor, or DNA Topoisomerase II inhibitor. In some embodiments, the payload is monomethyl auristatin E / Dolastatin-10 (MMAE), Monomethylauristatin F (MMAF), Mertansine, a maytansoid derivative, maytansine (DM1), N2’-deaetyl-N2’-(4-mercapto-4-methyl-1-oxopentyl) maytansine (DM4), 12-Ethyl-9-hydroxycamptothecin (7-ethyl-10-hydroxycamptothecin (SN-38), Exatecan mesylate (DX-8951f), PNU-1596821, which is a metabolite of the anthracycline nemorubicin, Pyrrolobenzodiazepine (PBD or SGD-1882), Exatecan derivatives (such as DXD), Camptothecin derivatives (such as Camp 98), or taxane (e.g., paclitaxel). In some embodiments, the payload is Exatecan or an Exatecan derivative (such as DXD).

[0040] The payload can be conjugated to the dPSA binding agent through the linker that is resistant to degradation by neutrophil proteases as described herein. In one embodiment, the linker comprises a dipeptide moiety comprising phenylalanine and lysine (FK). In another embodiment, the linker can comprise a glutamic acid-glycine-citrulline (EGCit) tripeptide moiety. The linker can further comprise a para-aminobenzyloxycarbonyl (PAB) moiety, and a functional group to facilitate conjugation with a free amine on a lysine residue or thiol group of a cysteine residue on the antibody. For example, the linker can be a maleimidocaprolyl-phe-lys- PAB linker (MC-FK-PAB), or a maleimidocaprolyl-glutamic acid-glycine-citrulline-PAB linker (MC-EGCit-PAB). Any cytotoxic payload can be used. However, in a specific embodiment, the conjugate further comprises Exatecan or an Exatecan derivative (e.g., DXD). Any method known in the art for conjugating an antigen-binding agent (e.g., an antibody) to a linker and cytotoxic moieties may be employed in the context of the invention (see, e.g., Hunter et al.,Leydig 515590 20 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)).

[0041] The dPSA binding agent conjugate can comprise any suitable ratio of payload molecules to binding agent (e.g., drug-to-antibody ratio, or DAR). For instance, the DAR can be 1 or more, such as 2 or more or 3 or more (e.g., 4 or more, or 5 or more). The DAR will typically be 15 or less, such as 12 or less or 10 or less (e.g., 8 or less, 6 or less, or 4 or less). Any of the foregoing can be combined to provide DAR ranges, e.g., a DAR of 1 to 15, 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 2 to 15, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 4, 3 to 15, 3 to 12, 3 to 10, 3 to 8, 3 to 6, 3 to 4, 4 to 15, 4 to 12, 4 to 10, 4 to 8, 4 to 6, 5 to 15, 5 to 12, 5 to 10, 5 to 8, or 5 to 6.

[0042] The dPSA-binding agent conjugate provided herein can be used for any purpose. For example, the dPSA-binding agent can be used for targeting or killing a cancer cell that expresses dPSA (e.g., that comprises dPSA on the cell surface). Thus, provided herein is a method of targeting or killing a cancer cell, in vitro or in vivo, comprising administering to the cancer cell a dPSA binding agent conjugate as described herein. When the method is used to target or kill a cancer cell in vivo, the dPSA binding agent conjugate can be administered to the cancer cell by administering the dPSA binding agent conjugate to the subject comprising the cancer cell.

[0043] The cancer cell expresses can be any cancer cell that expresses dPSA on the cell surface. For instance, the cancer cell can comprise a protein such as nucleolin with dPSA linked to or otherwise associated with the protein on the cell surface). In some embodiments, the cancer cell expresses ST8SIA2.

[0044] Thus, the dPSA binding agent provided herein, and methods of using same, can be used to treat cancer characterized by dPSA surface expression. As used herein, the terms “treatment,” “treating,” and the like refer to obtaining a desired pharmacologic and / or physiologic effect, e.g., to reduce the severity, or inhibit the progress, of a disease and / or adverse symptom attributable to the disease. To this end, the inventive method comprises administering a “therapeutically effective amount” of the dPSA-binding agent. A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. The therapeutically effective amount may vary according to factorsLeydig 515590 21 such as the disease state, age, sex, and weight of the individual, and the ability of the dPSA binding agent to elicit a desired response in the individual.

[0045] The methods and compositions provided herein are useful in the context of treating or preventing a wide variety of cancers, including carcinomas, sarcomas, leukemias, myelomas, and lymphomas.

[0046] Carcinomas that can be amenable to therapy by a method disclosed herein include, but are not limited to, esophageal carcinoma, hepatocellular carcinoma, basal cell carcinoma (a form of skin cancer), squamous cell carcinoma (various tissues), bladder carcinoma, including transitional cell carcinoma (a malignant neoplasm of the bladder), bronchogenic carcinoma, colon carcinoma, colorectal carcinoma, gastric carcinoma, lung carcinoma, including small cell carcinoma and non-small cell carcinoma of the lung, adrenocortical carcinoma, thyroid carcinoma, pancreatic carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical carcinoma, uterine carcinoma, testicular carcinoma, osteogenic carcinoma, epithelial carcinoma, and nasopharyngeal carcinoma.

[0047] Sarcomas that can be amenable to therapy by a method disclosed herein include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas.

[0048] Other solid tumors that can be amenable to therapy by a method disclosed herein include, but are not limited to, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.

[0049] Other cancers include leukemias, lymphomas, and myelomas (including multiple myeloma). Leukemias that can be amenable to therapy by a method disclosed herein include, but are not limited to, a) chronic myeloproliferative syndromes (neoplastic disorders of multipotential hematopoietic stem cells); b) acute myelogenous leukemias (neoplasticLeydig 515590 22 transformation of a multipotential hematopoietic stem cell or a hematopoietic cell of restricted lineage potential); c) chronic lymphocytic leukemias (CLL; clonal proliferation of immunologically immature and functionally incompetent small lymphocytes), including B-cell CLL, T-cell CLL prolymphocytic leukemia, and hairy cell leukemia; and d) acute lymphoblastic leukemias (characterized by accumulation of lymphoblasts). Lymphomas that can be treated using a subject method include, but are not limited to, B-cell lymphomas (e.g., Burkitt's lymphoma); Hodgkin's lymphoma; non-Hodgkin's lymphoma, and the like.

[0050] Other cancers that can be amenable to treatment according to the methods disclosed herein include atypical meningioma (brain), islet cell carcinoma (pancreas), medullary carcinoma (thyroid), mesenchymal (intestine), hepatocellular carcinoma (liver), hepatoblastoma (liver), clear cell carcinoma (kidney), and neurofibroma mediastinum.

[0051] Further exemplary cancers that can be amenable to treatment using a methods disclosed herein include, but art not limited to, cancers of neuroectodermal and epithelial origin. Examples of cancers of neuroectodermal origin include, but are not limited to, Ewing's sarcoma, spinal tumors, brain tumors, supratenbrial primitive neuroectodermal tumors of infancy, tubulocystic carcinoma, mucinous tubular and spindle cell carcinoma, renal tumors, mediastinum tumors, neurogliomas, neuroblastomas, and sarcomas in adolescents and young adults. Examples of epithelial origin include, but are not limited to, small cell lung cancer, cancers of the breast, eye lens, colon, pancreas, kidney, liver, ovary, and bronchial epithelium. In some embodiments, the subject methods do not include treatment of melanoma (i.e., the cancer is other than melanoma). In other embodiments, the subject methods do not include treatment of lymphoma (i.e., the cancer is other than lymphoma).

[0052] In some embodiments, the cancer to be treated is breast, lung, prostate, melanoma, or colorectal cancer. In another embodiment, the cancer to be treated is pancreatic, lung, liver, or brain cancer.

[0053] The dPSA-binding agent conjugate can be part of a composition suitable for administration to a mammal. Preferably, the composition is a pharmaceutically acceptable (e.g., physiologically acceptable) composition, which comprises a carrier, preferably a pharmaceutically acceptable (e.g., physiologically acceptable) carrier, and the inventive amino acid sequences, antigen-binding agent, or vector. Any suitable carrier can be used within theLeydig 515590 23 context of the invention, and such carriers are well known in the art. The choice of carrier will be determined, in part, by the particular site to which the composition may be administered and the particular method used to administer the composition. The composition also can comprise any other excipient used in the formulation of therapeutic molecules (e.g., proteins or antibodies), particularly parenteral formulations, including, for instance, buffers, tonicity modifiers, stabilizers, surfactants and the like. The composition can be sterile. The composition can be frozen or lyophilized for storage and reconstituted in a suitable sterile carrier prior to use. The compositions can be generated in accordance with conventional techniques described in, e.g., Remington: The Science and Practice of Pharmacy, 21st Edition, Lippincott Williams & Wilkins, Philadelphia, PA (2001).

[0054] Administration may be effected using any standard administration techniques, including oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. The composition preferably is suitable for parenteral administration. The term “parenteral,” as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. More preferably, the composition is administered to a mammal using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.

[0055] The dPSA-binding agent conjugate of the invention may be administered alone or in combination with other drugs. For example, the dPSA-binding agent can be administered in combination with other agents for the treatment or prevention of the diseases disclosed herein, such as other anti-cancer agents. In this respect, for example, the dPSA-binding agent conjugate can be used in combination with at least one other agent including, for example, chemotherapeutic agents, vaccines, biological therapies (e.g., other monoclonal antibodies), radiation therapy, bone marrow transplantation, chemotherapeutic treatment, biological response modifier treatment and / or surgery.

[0100] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope. EXAMPLES

[0101] The following dPSA binding agents are referenced in these Examples:Leydig 515590 24

[0102] Antibodies designated SAC-1 and SAC-2 having the sequences set forth below were expressed as a recombinant chimera with a human IgG1 Fc in a CHO cell line that does not express dPSA. The antibodies were tested for specific binding to dPSA by ELISA and cell based assay. Humanized antibodies were prepared with the same CDRs as the SAC-1 and SAC-2 antibodies. The antibodies were designated SAC-1.1, SAC-2.1 (also referred to as “SAC-2 humanized D” or “SAC-2D”), and SAC-2.2 (also referred to as “SAC-2 humanized C” or “SAC- 2C), and have sequences as set forth below. Antibody Heavy Chain Light Chain Full Heavy Full Light V ri bl R i n V ri bl R i n Ch in Ch in 6 8 3 8 9 8 9EXAMPLE 1

[0103] This example illustrates the preparation of an antibody-drug conjugate comprising SAC-2D antibody and a MC-Phe-Lys-PAB-Exatecan linker:Leydig 515590 25

[0104] Preparation of LP-002: MC-Phe-Lys-PAB-Exatecan, ,EEDQ (2.38 g, 9.60 mmol) in DCM (60 mL) and MeOH (20 mL) was degassed and purged with N2for 3 times, and then the mixture was stirred at 25 °C for 2 hrs under N2atmosphere. LC-MS showed 84.5% of desired compound (Rt = 1.920 min) was detected. The reaction mixture was concentrated under reduced pressure to give a residue which was triturated with CH3CN (50 mL)and MTBE (50 mL). Compound 3 (3.50 g, 79.7% yield) was obtained as a white solid.

[0106] To a solution of compound 3 (2.98 g, 5.19 mmol) in DMF (60 mL) was added Et2NH(11.40 g, 156 mmol, 16.1 mL). The mixture was stirred at 25 °C for 0.5 hr. LC-MS showed 15.4% of desired compound (Rt = 1.168 min) was detected. The reaction mixture wasLeydig 515590 26 concentrated under reduced pressure to give a residue. Compound 4 (1.83 g, crude) wasobtained as a brown oil.

[0107] To a solution of compound 4 (1.80 g, 5.12 mmol) and compound 5 (2.36 g, 4.87mmol) in DMF (60 mL) was added DIEA (993 mg, 7.68 mmol, 1.34 mL). The mixture was stirred at 20 °C for 4 hrs. LC-MS showed 64.0% of desired compound (Rt = 2.020 min) was detected. The reaction mixture was concentrated under reduced pressure to give a residue which was triturated with CH3CN (50 mL) and MTBE (50 mL). Compound 6 (2.53 g, 63.8% yield,93.2% purity) was obtained as a white solid.added compound 6 (400 mg, 451 μmol), HOBt (101 mg, 752 μmol), pyridine (8 mL) and DIEA(243 mg,1.88 mmol, 327 μL). The mixture was stirred at 25 °C for 2 hrs. LC-MS showed the reaction was complete. The reaction mixture was concentrated under reduced pressure to give a residue which was triturated with MTBE (30 mL) at 25 oC for 20 mins to give compound 8 (450mg, 88.9% yield) was obtained as a brown solid.Leydig 515590 27

[0109] To a solution of compound 8 (450 mg, 380 μmol) in DMF (10 mL) was added Et2NH(1.42 g, 19.4 mmol, 2 mL). The mixture was stirred at 25 °C for 1 hr. LC-MS showed the reaction was complete. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: CD01-Phenomenex luna C18 150*25*10um;mobile phase: [water(TFA)-ACN];gradient:29%-59% B over 10 min) to giveCompound 9 (230 mg, 62.6% yield) was obtained as a yellow solid.71.3 μL) and compound 10 (88.4 mg, 286 μmol) .The mixture was stirred at 25 °C for 1 hr. LC-MS showed the reaction was complete. The reaction mixture was concentrated under reduced pressure to give a residue. The crude product was triturated with MTBE (20 mL ) at 25oC for 10mins to give compound 11 (230 mg, 72.1% yield) was obtained as a white solid.

[0111] To a solution of compound 11 (230 mg, 199 μmol) in DCM (3 mL) was added TFA(0.46 mL). The mixture was stirred at 25 °C for 0.5 hr. LC-MS showed the reaction was complete. The reaction mixture was concentrated under reduced pressure to give a residue whichwas purified by prep-HPLC (column: Welch Ultimate C18150*25mm *5um; mobileLeydig 515590 28 phase:[water(TFA)-ACN]; gradient:26%-56% B over 10 min) to give the compound LP002 as a yellow solid.

[0112] A similar procedure can be used to prepare an MC-EGCit-PAB-Exatecan linker.

[0113] Conjugation of Linker-Payload molecules to Antibody:

[0114] Conjugation was performed to prepare the conjugate with a target drug antibody ratio of 4.0. SAC-2D antibody (1106.19 μL, 9.04 mg / mL) in buffer (PBS, pH 7.4, 0.02% PS80) was pipetted into 50 mL tube, and then 69.73 μL of 2 mM TCEP dissolved in water (TCEP / mAb molar ratio was set as 2.00) was added into the antibody solution to reduce. Reaction buffer (49.07 μL, 50 mM phosphate buffer, pH 6.5) was added to the tube to make the final mAb concentration at 4.0 mg / mL. The tube was placed in an incubator-shaker under 22oC with gentle mixing at speed of 60 rpm. After 18 hours of reduction, 10 mM (10.53 mg / mL) MC-FK-PAB- Exatecan linker-payload (55.78 μL) in DMA was added to sample to make the drug to antibody molar eq. at 8.0. PG (Propylene glycol) solvent (1194.22 μL) was added into above reduced sample to make sure the percentage of organic solvent at 50%, and then the reaction sample was incubated in an incubator-shaker under 22 oC with gentle mixing at speed of 60 rpm for another 1 hour. After 1 hour, the sample was purified by zeba spin desalting column (40 kDa, 5 mL, Thermo Scientific-87770) and Amicon (50 kDa, 15 mL, Millipore-UFC905024) to give the product of SAC2D-LP-002. The product was characterized by LC-MS, SEC-HPLC, HIC-HPLC,Leydig 515590 29 RP_HPLC and KTA. A similar procedure was used with an MC-VC-PAB-Exatecan linker / payload to provide SAC2D-LP-001, and with an MC-GGFG-PAB-Exatecan linker / payload to provide SAC2D-LP-003. As used herein in connection with the linkers “VC” refers to Valine-Citruline or ValCit.

[0115] In addition, a conjugate comprising a version of the SAC-2D antibody with a silent FC region and the MC-EGCit-PAB-Exatecan linker was prepared (SAC2D-LP-004). The silent FC region of SAC2D-LP-004 was constructed by mutating the DNA sequence encoding the SAC-2D heavy chain (SEQ_ID_NO: 57) to encode an amino acid sequence having L234A, L235A, and P329G mutations (SEQ_ID_NO: 60). The amino acid sequence changes result in an Fc sequence that has reduced binding to Fc receptors or complement proteins as compared to SAC-2D without the mutations. The light chain amino acid sequence of the construct is the same as that of SAC-2D (SEQ_ID_NO: 58). The silent Fc antibody was produced and purified by Protein A / G affinity chromatography and conjugated through the MC-EGCit-PAB linker to Exatecan as described above. The prepared linkers and drug-to-antibody ratio (DAR) are as set forth in Table 1: Table 1 Antibody / ADC Silent Linker Payload DAR FCEXAMPLE 2

[0116] This Example demonstrates the effect of conjugates provided in Example 1 on various cell lines.

[0117] SAC2D-LP-002 was screened against various cell lines and compared to two other SAC-2D conjugates: LP-001 comprising an MC-VC-PAB-Exatecan linker, LP-003 comprising an MC-GGFG-Exatecan linker / payload, and a SAC-2D conjugate comprising an MC-VC-PAB- MMAE linker / payload (“MMAE”).Leydig 515590 30

[0118] The SAC-2D MC-FK-PAB-Exatecan conjugate (SAC2D-LP-002) prepared in Example 1 was >98% monomeric by size exclusion chromatography and >70% of the product contained 4 molecules of linker-payload per molecule of antibody as measured by high performance ion exchange chromatography. Endotoxin in the SAC-2D-linker-payload product was measured using a standard limulus amebocyte lysate (LAL) assay and found to be less than 0.1 EU / mg.

[0119] The conjugates were screened against various cell lines using an in vitro cellular cytotoxicity assay. A construct comprising SAC-2D conjugated to MC-VC-PAB-MMAE linker / payload (“MMAE” or “SAC2D-MMAE”) provided another comparison.

[0120] A standardized method for measuring the cytotoxicity of each cell line with the indicated ADCs was performed as follows: The cell lines were obtained from ATCC and cultured as recommended by ATCC. The cells (the number of cells depending on the particular cell line) were added to the wells of a 96-well flat clear-bottom white polystyrene tissue culture dishes on day 1 (80μL volume) and placed in the tissue culture incubator overnight. On day 2, the cells were treated with test compounds over a 9-point dose curve. Control wells include untreated cells and wells with media only. The ADC was prepared from a stock solution to give a 5x solution of 100μg / mL. The dilutions were added to the cells (20 μL diluted compound + 80 μL media) to give the final concentrations of the ADC on the cells of 20000, 4000, 800, 160, 32, 6.4, 0.0256, 1.28 and 0.0512ng / mL. The cells were incubated with the compounds for 144 hours before they being processed with CelltiterGlo (Promega) according to the manufacturer’s directions. Survival % = ((sample signal-blank signal) / untreated control signal) x100.

[0121] The results provided in Table 2 show that the construct with the Phe-Lys linker sequence (LP-002) and EGCit linker (LP-003) resulted in increased cytotoxic activity as compared to the VC linker (LP-001) in the cell lines where LP-001 was tested. LP-002 showed superior cytotoxic activity as compared to the other linkers in several cell lines tested.Leydig 515590 31 Table 2 SAC-2D ADC A375 NCI-H1395 A549 Melanoma NSCLC NSCLC y3

[0122] This example demonstrates that the SAC-2D antibody drug conjugates bind to cancer cell lines representing multiple diverse human cancers and is cytotoxic as a result of binding to dPSA-nucleolin antigen on the cell surface, being taken up by the cells, and the Exatecan drug being released inside the cell after cleavage of the antibody-drug linker.

[0123] In vitro cytotoxicity was measured as described in Example 2. An MMAE conjugate SAC-2D-MMAE provided further comparison. As summarized in Table 3, below, both the SAC2D-LP-002 and SAC2D-LP-004 conjugates exhibited cytotoxic activity in several tested cell lines. The SAC-2D-MC-FK-PAB-Exatecan conjugate (SAC2D-LP-002) results in cytotoxicity to multiple cancer cell lines including several lung cancer cell lines that were resistant to killing by SAC2D-MMAE and SAC2D-LP-004, which has the same Exatecan payload but a different linker (MC-EGCit-PAB). In addition, the maximum killing percent achieved by SAC2D-LP-002 was consistently ~90% to 100% compared to SAC-2D-VCit- MMAE, which either had no cytotoxic activity or was less than 90% for 9 of the 19 cell lines tested. SAC2D-LP-004 had activity against 8 of 16 cell lines tested and greater than 90% killing against 1 of the 8 cell lines. The results show that SAC2D-LP-002 with the MC-FK-PAB linker and Exatecan payload had superior in vitro cytotoxic activity compared to SAC2D-MMAE withLeydig 515590 32 the MC-VCit-PAB linker or SAC2D-LP-004 having the same Exatecan payload as SAC-LP-002 but a greater DAR and the MC-EGCit-PAB linker. Table 3 Cancer Cell line SAC-2D-MMAE SAC2D-LP-002 SAC-2D-LP-004 ) 7EXAMPLE 4

[0124] The following is a protocol used for testing ADH conjugates for the ability to inhibit or cause regression of human cancers in xenograft mouse models of human cancer using MiaLeydig 515590 33 PaCa-2 pancreatic cancer cells as an example. All cell lines were obtained from the American Type Culture Collection (ATCC) and were cultured according to instructions provided with the cell line.

[0125] The MiaPaCa-2 cells (pancreatic, ATCC® CRL-1420) were maintained in vitro as a monolayer culture in Dulbecco’s Modified Eagle’s Medium (DMEM) a supplemented with 10% fetal bovine serum (FBS), 100U / ml penicillin and 100ug / ml streptomycin, maintained at 37 °C in an atmosphere containing 5% CO2 in air. The tumor cells were routinely subcultured at a ratio of 1:2 to 1:3 every 3- 4 days by Trypsin-EDTA treatment. The cells growing in an exponential growth phase were harvested and counted for tumor inoculation. Each mouse was inoculated subcutaneously at the right flank with 3 x 10^6 MiaPaCa-2 cells in 0.1ml for tumor development (day 0). All the procedures related to animal handling, care and treatment in this study were performed according to the protocol approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Chempartner following the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).

[0126] At the time of routine monitoring, the animals were checked for any effects of tumor growth on normal behavior such as mobility, food and water consumption (by observation), body weight gain / loss (body weights were measured once a day), eye / hair matting and any other abnormal effects. Death and observed clinical signs were recorded on the basis of the numbers of animals within each subset. Animals that were observed to be in a continuing deteriorating condition were euthanized. Tumor sizes were measured in two dimensions using a caliper, andthe volumes were expressed in mm3using the formula: V = 0.5 a × b2where a and b are thelongest and shortest diameters of the tumor respectively. Body weights were also measured.Dosing was started when the average tumor size reached 130.11 mm3for the efficacy study. 56tumor-bearing mice were randomized into 7 groups with 8 mice in each group. Animals were weighed and tumor volume was measured before administration.

[0127] The major endpoint was tumor growth inhibition. The tumor size was used for calculation of Tumor growth inhibition (TGI) values according to the following equation: TGI (%) = (1 - (TVTreatment / Dn - TVTreatment / D0) / (TVControl / Dn– TVControl / D0)) × 100%. For comparison between two groups, an independent sample t-test was used. For comparison among three or more groups, a one-way ANOVA was performed. If a significant F-statistics (a ratio ofLeydig 515590 34 treatment variance to the error variance) was obtained, multiple comparison procedures was applied after ANOVA. All data were analyzed using SPSS 17.0. P < 0.05 is considered to be statistically significant.

[0128] In vivo ability of SAC-2D ADC conjugates to inhibit growth or cause regression of initial tumor volume is summarized in Table 4, below. The results demonstrate that the SAC- 2D-FK-Exatecan antibody drug conjugate (SAC2D-LP-002) and SAC-2D-EGCit-Exatecan (SAC2D-LP-004) binds to cancer cell lines representing multiple diverse human cancers and is cytotoxic as a result of binding to dPSA-nucleolin antigen on the cell surface, being taken up by the cells, and the Exatecan drug being released inside the cell after cleavage of the antibody-drug linker. Table 4 Cancer ADC Construct Best result Pancreatic SAC2D-MMAE Regression: 3mg / kg Q4D 4x x x x x

[0129] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0130] The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein areLeydig 515590 35 merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0131] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

Leydig 515590 36 CLAIMS:

1. A dPSA binding agent conjugate comprising a dPSA binding agent, a linker resistant to a neutrophil protease attached to the dPSA binding agent, and a cytotoxic payload attached to the linker.

2. The dPSA binding agent of claim 1, wherein the linker comprises a dipeptide Phe- Lys (FK) moiety.

3. The dPSA binding agent of claim 1, wherein the linker comprises a tripeptide glutamic acid-glycine-citrulline (EGCit) moiety.

4. The dPSA binding agent of any of claims 2 or 3, wherein the linker further comprises a para-aminobenzyloxycarbonyl (PAB) moiety.

5. The dPSA binding agent of claim 1, wherein the linker is a maleimidocaprolyl- phe-lys-PAB linker (MC-FK-PAB).

6. The dPSA binding agent of claim 1, wherein the linker is a maleimidocaprolyl- glutamic acid-glycine-citrulline-PAB linker (MC-EGCit-PAB).

7. The dPSA binding agent of any of claims 1-6, wherein the cytotoxic payload is MMAE, Exatecan or an Exatecan derivative.

8. The dPSA-binding agent conjugate of any of claims 1-7, wherein the dPSA binding agent comprises: (a) an immunoglobulin heavy chain variable region comprising SEQ ID NO: 51 or at least the complementarity determining regions (CDRs) thereof; and an immunoglobulin light chain variable region comprising SEQ ID NO: 52 or 53 or at least the CDRs thereof; (b) an immunoglobulin heavy chain variable region comprising any of SEQ ID NOs: 17-Leydig 515590 37 20 or at least the complementarity determining regions (CDRs) thereof; and an immunoglobulin light chain variable region comprising SEQ ID NO: 21 or at least the CDRs thereof; (c) an immunoglobulin heavy chain variable region comprising any of SEQ ID NOs: 1-4 or at least the complementarity determining regions (CDRs) thereof; and an immunoglobulin light chain variable region comprising SEQ ID NO: 5 or at least the CDRs thereof; or (d) an immunoglobulin heavy chain variable region comprising SEQ ID NO: 35 or at least the complementarity determining regions (CDRs) thereof; and an immunoglobulin light chain variable region comprising SEQ ID NO: 36 or at least the CDRs thereof.

9. The dPSA binding agent conjugate of any of claims 1-7, wherein the dPSA binding agent comprises: (a) an immunoglobulin heavy chain variable region comprising: CDRH1 comprising any one of SEQ ID NOs: 6-9; CDRH2 comprising SEQ ID NO: 10; CDRH3 comprising SEQ ID NO: 11; and an immunoglobulin light chain variable region comprising CDRL1 comprising SEQ ID NO: 12; CDRL2 comprising SEQ ID NO: 13; and CDRL3 comprising SEQ ID NO: 14; (b) an immunoglobulin heavy chain variable region comprising: CDRH1 comprising any one of SEQ ID NOs: 24-27; CDRH2 comprising SEQ ID NO: 28; CDRH3 comprising SEQ ID NO: 29; and an immunoglobulin light chain variable region comprising CDRL1 comprising SEQ ID NO: 30; CDRL2 comprising SEQ ID NO: 31; and CDRL3 comprising SEQ ID NO: 32; (c) an immunoglobulin heavy chain variable region comprising: CDRH1 comprising SEQ ID NO: 39; CDRH2 comprising SEQ ID NO: 40; andLeydig 515590 38 CDRH3 comprising SEQ ID NO: 41 or 47; and an immunoglobulin light chain variable region comprising CDRL1 comprising SEQ ID NO: 42; CDRL2 comprising SEQ ID NO: 43 or 56; and CDRL3 comprising SEQ ID NO: 44; or (d) an immunoglobulin heavy chain variable region comprising: CDRH1 comprising SEQ ID NO: 45; CDRH2 comprising SEQ ID NO: 46; and CDRH3 comprising SEQ ID NO: 41 or 47; and an immunoglobulin light chain variable region comprising CDRL1 comprising SEQ ID NO: 48; CDRL2 comprising SEQ ID NO: 49 or 56; and CDRL3 comprising SEQ ID NO:

50.

10. The dPSA binding agent conjugate of any of claims 1-7, 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.

11. The dPSA-binding agent conjugate of any of claims 1-10, wherein the dPSA- binding agent is an antibody or an antigen-binding antibody fragment.

12. The dPSA binding agent conjugate of any of claims 1-10, wherein the dPSA- binding agent is an IgG1 or IgG4 antibody.

13. The dPSA-binding agent conjugate of any of claims 1-10, wherein the dPSA- binding agent is a F(ab’)2fragment, a Fab’ fragment, a Fab fragment, a Fv fragment, a scFv fragment, a dsFv fragment, or a dAb fragment.

14. The dPSA binding agent conjugate of any of claims 1-7, wherein the dPSA- binding agent comprises an Fc region, and the Fc region comprises a modification that reduces FcγR binding and / or reduces complement dependent cytotoxicity (CDC) or antibody-dependent cytotoxicity (ADCC).Leydig 515590 39 15. The dPSA binding agent conjugate of claim 1, wherein the dPSA-binding agent conjugate comprises (a) a payload comprising Exatecan, (b) a linker comprising MC-Phe-Lys- PAB, and (c) a dPSA binding agent comprising an immunoglobulin heavy chain comprising SEQ ID NO: 57 and an immunoglobulin light chain comprising SEQ ID NO:

58.

16. The dPSA binding agent conjugate of claim 1, wherein the dPSA-binding agent conjugate comprises (a) a payload comprising Exatecan, (b) a linker comprising MC-Phe-Lys- PAB, and (c) a dPSA binding agent comprising an immunoglobulin heavy chain comprising SEQ ID NO: 57 and an immunoglobulin light chain comprising SEQ ID NO:

59.

17. The dPSA binding agent conjugate of claim 1, wherein the dPSA-binding agent conjugate comprises (a) a payload comprising Exatecan, (b) a linker comprising MC-FK-PAB, and (c) a dPSA binding agent comprising an immunoglobulin heavy chain comprising SEQ ID NO: 60 and an immunoglobulin light chain comprising SEQ ID NO:

58.

18. The dPSA binding agent conjugate of claim 1, wherein the dPSA-binding agent conjugate comprises (a) a payload comprising Exatecan, (b) a linker comprising MC-FK-PAB, and (c) a dPSA binding agent comprising an immunoglobulin heavy chain comprising SEQ ID NO: 60 and an immunoglobulin light chain comprising SEQ ID NO:

59.

19. The dPSA binding agent conjugate of claim 1, wherein the dPSA-binding agent conjugate comprises (a) a payload comprising Exatecan, (b) a linker comprising MC-EGCit- PAB, and (c) a dPSA binding agent comprising an immunoglobulin heavy chain comprising SEQ ID NO: 57 and an immunoglobulin light chain comprising SEQ ID NO:

58.

20. The dPSA binding agent conjugate of claim 1, wherein the dPSA-binding agent conjugate comprises (a) a payload comprising Exatecan, (b) a linker comprising MC-EGCit- PAB, and (c) a dPSA binding agent comprising an immunoglobulin heavy chain comprising SEQ ID NO: 57 and an immunoglobulin light chain comprising SEQ ID NO:

59.

21. The dPSA binding agent conjugate of claim 1, wherein the dPSA-binding agent conjugate comprises (a) a payload comprising Exatecan, (b) a linker comprising MC-EGCit-Leydig 515590 40 PAB, and (c) a dPSA binding agent comprising an immunoglobulin heavy chain comprising SEQ ID NO: 60 and an immunoglobulin light chain comprising SEQ ID NO:

58.

22. The dPSA binding agent conjugate of claim 1, wherein the dPSA-binding agent conjugate comprises (a) a payload comprising Exatecan, (b) a linker comprising MC-EGCit- PAB, and (c) a dPSA binding agent comprising an immunoglobulin heavy chain comprising SEQ ID NO: 60 and an immunoglobulin light chain comprising SEQ ID NO:

59.

23. A composition comprising (a) the dPSA-binding agent conjugate of any one of claims 1-22, and (b) a pharmaceutically acceptable carrier.

24. A method of killing a cancer cell that expresses dPSA, the method comprising contacting the cancer cell with a dPSA binding agent conjugate of any of claims 1-22 or composition of claim 23.

25. A method of treating cancer characterized by dPSA expression in a subject, the method comprising administering to the subject a dPSA binding agent conjugate of any of claims 1-22 or composition of claim 23.

26. A method of delivering a cytotoxic payload to a cell that expresses dPSA, the method comprising contacting the cell with a dPSA binding agent conjugate of any of claims 1- 22 or composition of claim 23.

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

28. A dPSA binding agent conjugate of any of claims 1-22 or composition of claim 23 for treating cancer.

29. Use of a dPSA binding agent conjugate of any of claims 1-22 or composition of claim 23 for the manufacture of a medicament for treating cancer.

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