ENPP3 × CD3 bispecific antibodies and use thereof

Abstract

The present disclosure provides ENPP3 binding agents, including bispecific ENPP3 × CD3 binding proteins, and related compositions and methods of use.

Description

ENPP3 x CD3 Bispecific Antibodies and Use Thereof1. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 609,152, filed on 12 December 2023, which is incorporated herein by reference in its entirety.2. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing, which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on December 6, 2024, is named JBI6865WOPCTl_SL.xml, and is 62,319 bytes in size.3. FIELD

[0003] The present disclosure generally relates to a T cell engager that binds anti- ectonucleotide pyrophosphatase / phosphodiesterase family member 3 (ENPP3) and cluster of differentiation 3 (CD3), compositions comprising same, and methods of using same.4. BACKGROUND

[0004] Solid tumors have been a challenge for antibody-mediated T cell redirection therapy, with very limited, if any, drugs yet approved by the US Food and Drug Administration (FDA). Limitations of the approach may be related to the lack of cancer specificity of targets leading to toxicity occurring at sub-efficacious doses. Unmet need remains across solid tumors to prolong and improve patient outcomes and overall treatment duration.

[0005] There exists a need for innovative approaches for effective targeting of solid tumors and minimizing treatment toxicities. The present disclosure meets this and other needs.5. SUMMARY

[0006] In some embodiments, the invention provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a bispecific antibody comprising an ENPP3-binding domain and a CD3-binding domain, wherein: A) the ENPP3-binding domain comprises the heavy chain complementarity determining region 1 (HCDR1 ), the HCDR2, andthe HCDR3 of a heavy chain variable region (VH) of SEQ ID NO:22, and the light chain complementarity determining region 1 (LCDR1 ), the LCDR2, and the LCDR3 of a light chain variable region (VL) of SEQ ID NO:23; and B) the CD3-binding domain comprises the HCDR1, the HCDR2, and the HCDR3 of a VH or SEQ ID NO: 47, and the LCDR1, the LCDR2, and the LCDR3 of a VL of SEQ ID NO: 48; and wherein the cancer is a solid tumor. In some embodiments: A) the ENPP3 -binding domain comprises a HCDR1, a HCDR2, a HCDR3, a LCDR1, a LCDR2, and a LCDR3 comprising the amino acid sequences of: i) SEQ ID NO:1, 2, 3, 4, 5, and 6, respectively; ii) SEQ ID NO:7, 8, 3, 4, 5, and 6, respectively; iii) SEQ ID NOV, 10, 3, 4, 5, and 6, respectively; iv) SEQ ID NO: 11, 12, 13, 14, 15, and 16, respectively; or v) SEQ ID NO: 17, 18, 19, 20, 21, and 6, respectively; and B) the CD3-binding domain comprises a HCDR1, a HCDR2, a HCDR3, a LCDR1, a LCDR2, and a LCDR3 comprising the amino acid sequences of: i) SEQ ID NO:26, 27, 28, 29, 30, and 31, respectively; ii) SEQ ID NO:32, 33, 28, 29, 30, and 31, respectively; iii) SEQ ID NO:34, 35, 28, 29, 30, and 31, respectively; iv) SEQ ID NO:36, 37, 38, 39, 40, and 41, respectively; or v) SEQ ID NO:42, 43, 44, 45, 46, and 31, respectively.

[0007] In some embodiments, the cancer is selected from the group consisting of renal cell cancer (RCC), lung adenocarcinoma, endometrioid ovarian cancer, endometrioid uterine carcinoma, and colorectal adenocarcinoma (CRC).

[0008] In some embodiments, the ENPP3 -binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO:22 and a VL comprising the amino acid sequence of SEQ ID NO 23.

[0009] In some embodiments, the CD3 -binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO:47 and a VL comprising the amino acid sequence of SEQ ID NO:48. In some embodiments, the CD3-binding domain comprises an spFv comprising the amino acid sequence of SEQ ID NO:49.

[0010] In some embodiments, the ENPP3 -binding domain comprises a first heavy chain (HC) peptide comprising the amino acid sequence of SEQ ID NO:24 and a first light chain (LC) peptide comprising the amino acid sequence of SEQ ID NO:25.

[0011] In some embodiments, the CD3 -binding domain comprises an spFv-Fc fusion peptide comprising an amino acid sequence of SEQ ID NO: 50.

[0012] In some embodiments, the subject has measurable or evaluable cancer. In some embodiments, the subject has a measurable lesion per Response Evaluation Criteria in Solid Tumors (RECIST) vl.l. In some embodiments, = the cancer is ovarian cancer and the subject has disease evaluable per RECIST vl.l or a cancer antigen (CA) at least 125 greater than twice the upper limit of normal (ULN).

[0013] In some embodiments, the bispecific antibody is administered at a dose selected from the group consisting of: 0.017 mg, 0.05 mg, 0.15 mg, 0.5 mg, 0.65 mg, 0.8 mg, 1.75 mg, 2 mg, 4 mg, 6 mg, 21 mg, 70 mg, and 100 mg. In some embodiments, the bispecific antibody is administered at a dose of 0.017 mg. In some embodiments, the bispecific antibody is administered at a dose of 0.05 mg. In some embodiments, the bispecific antibody is administered at a dose of 0.15 mg. In some embodiments, the bispecific antibody is administered at a dose of 0.5 mg. In some embodiments, the bispecific antibody is administered at a dose of 0.65 mg. In some embodiments, the bispecific antibody is administered at a dose of 0.8 mg. In some embodiments, the bispecific antibody is administered at a dose of 1.75 mg. In some embodiments, the bispecific antibody is administered at a dose of 2 mg. In some embodiments, the bispecific antibody is administered at a dose of 4 mg. In some embodiments, the bispecific antibody is administered at a dose of 6 mg. In some embodiments, the bispecific antibody is administered at a dose of 21 mg. In some embodiments, the bispecific antibody is administered at a dose of 70 mg. In some embodiments, the bispecific antibody is administered at a dose of 100 mg. In some embodiments, the dose comprises a step-up dose and a target dose. In some embodiments, the step-up dose comprises one or more step-up doses.

[0014] In some embodiments, the bispecific antibody is administered once every week. In some embodiments, the bispecific antibody is administered once every two weeks. In some embodiments, the bispecific antibody is administered once every three weeks.

[0015] In some embodiments, the bispecific antibody is administered subcutaneously.6. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 shows a schematic of NPP3B815 (NPP3B56 x CD3B2030-N106A), a bispecific antibody targeting CD3 and ENPP3. AAS, L234A, L235A, D265S; CD, cluster of differentiation; Fab, fragment antigen-binding; hole, T366S, L368A, Y407V; knob, T366W;ENPP3, ectonucleotide pyrophosphatase / phosphodi esterase family member 3; Ig, immunoglobulin; spFv, stapled single-chain fragment variable.

[0017] Figure 2 shows ENPP3 expression (receptor density) in different cancer cell lines with variable expression levels. Flow-cytometry-based membrane ENPP3 detection and receptor occupancy quantification measured using a commercial ENPP3 antibody (i.e., clone NP4D6) on a panel of endogenous cancer cell lines. Representative histogram showing ENPP3 expression in a high, medium, and negative cell line. Abbreviations: ABC, antibody binding capacity; ENPP3, ectonucleotide pyrophosphatase / phosphodi esterase family member 3; HCC, hepatocellular carcinoma; LD, Live Dead; LLOD, lower limit of detection; Med, medium; Ne.g., negative; RCC, renal cell carcinoma; ULOD, upper limit of detection.

[0018] Figure 3 shows ENPP3 expression in different in vivo CDX and PDX model systems from ex vivo tumors. Flow-cytometry (using commercial antibody clone NP4D6) and IHC-based evaluation (using commercial antibody clone E5M2W) of ENPP3 expression on 2 CDX models, i.e., VMRCRCW (RCC) and HepG2 (HCC), and a RCC PDX model, i.e., RXF488.Magnification is 30X for all images. Abbreviations: ENPP3, ectonucleotide pyrophosphatase / phosphodiesterase family member 3; CDX, cell-line-derived xenograft; HCC, hepatocellular carcinoma; IHC, immunohistochemistry; LD, Live Dead; PDX, patient-derived xenograft; RCC, renal cell carcinoma.

[0019] Figure 4A and Figure 4B show the binding of NPP3B815 to endogenous ENPP3- expressing tumor cell lines and isolated T cells. Figure 4A shows the binding of ENPP3 x CD3 (NPP3B815) and Null x CD3 (CD3B2533) on A704 (ENPP3-high), VMRCRCW (ENPP3- medium), and HepG2 ENPP3KO (ENPP3 -negative) cell lines was evaluated by flow cytometry. Figure 4B shows the binding of ENPP3 x CD3 (NPP3B815, NPP3B815) and isotype control (79C3B613) antibodies evaluated by flow cytometry on T cells isolated from 6 different healthy human donors. Solid lines denote NPP3B815 and dotted lines (bottom of graph) denote 79C3B613 binding. Abbreviations: CD, cluster of differentiation; ENPP3, ectonucleotide pyrophosphatase / phosphodiesterase family member 3; geomean, geometric mean.

[0020] Figure 5 shows binding of NPP3B815 to ENPP1, ENPP2, and ENPP3 overexpressing cell lines. Binding of ENPP3 x CD3 (NPP3B815) and isotype control (79C3B613) on CHO parental cell line or CHO cell lines overexpressing ENPP1, ENPP2 or ENPP3 was evaluated by flow cytometry. Abbreviations: CD, cluster of differentiation; ENPP1, ectonucleotidepyrophosphatase / phosphodiesterase family member 1; ENPP2, ectonucleotide pyrophosphatase / phosphodiesterase family member 2; ENPP3, ectonucleotide pyrophosphatase / phosphodi esterase family member 3.

[0021] Figure 6 shows NPP3B815-induced tumor cell killing of a panel of tumor cell lines with endogenous ENPP3 expression. Incucyte- based assessment of tumor cell killing (i.e., loss of Nuclight-red-positive cells) was measured upon treatment with ENPP3 x CD3 (NPP3B815, NPP3B815), ENPP3 x Null (NPP3B812), and Null x CD3 (79C3B615) antibodies in the presence of isolated T cells from Donor 888668965 at E:T ratio of 3:1 on a panel of cell lines. Data plotted at 72 hours post treatment. Error bars are SEM. Abbreviations: CD, cluster of differentiation; ENPP3, ectonucleotide pyrophosphatase / phosphodiesterase family member 3; E:T ratio, effector-to-target cell ratio; SEM, standard error of the mean.

[0022] Figure 7 shows NPP3B815-induced T cell activation in a panel of tumor cell lines with endogenous ENPP3 expression. T cell activation (i.e., CD25+ T cells) was measured by flow cytometry upon treatment with ENPP3 x CD3 (NPP3B815, NPP3B815), ENPP3 x Null (NPP3B812), and Null x CD3 (79C3B615) antibodies for 48 hours in the presence of isolated T cells from Donor 888668965 at E:T ratio of 3:1 with A704 (ENPP3-high), VMRCRCW (ENPP3 -medium), HepG2 (ENPP3 -medium), and HepG2 ENPP3 KO (ENPP3 -negative) cell lines. Error bars are SEM. Abbreviations: CD, cluster of differentiation; ENPP3, ectonucleotide pyrophosphatase / phosphodiesterase family member 3; E:T ratio, effector-to-target cell ratio.

[0023] Figure 8A and Figure 8B show NPP3B815-induced tumor cell killing and T cell activation in the presence of T cells isolated from multiple donors in ENPP3 -positive and ENPP3 -negative cell lines. (Figure 8A) Incucyte-based assessment of tumor cell killing (i.e., loss of Nuclight-red-positive cells) measured upon treatment with ENPP3 x CD3 (NPP3B815) antibody in the presence of T cells (E:T ratio of 3: 1) isolated from 6 different healthy human donors tested with A704 (ENPP3-high), VMRCRCW (ENPP3 -medium), and HepG2 ENPP3 KO (ENPP3 -negative) cell lines. Data plotted at 72 hours post treatment. Error bars are SEM. (Figure 8B) T cell activation (i.e., CD25+ T cells) was measured by flow cytometry upon treatment with ENPP3 x CD3 (NPP3B815) for 48 hours in the presence of T cells (E:T ratio of 3: 1) isolated from 6 different healthy human donors tested with A704 (ENPP3-high), VMRCRCW (ENPP3- medium), and HepG2 ENPP3 KO (ENPP3 -negative) cell lines. Error bars are SEM. Abbreviations: CD, cluster of differentiation; ENPP3, ectonucleotidepyrophosphatase / phosphodiesterase family member 3; E:T ratio, effector-to-target cell ratio; SEM, standard error of the mean.

[0024] Figure 9A and Figure 9B show NPP3B815-induced tumor cell killing in the presence of T cells isolated from multiple donors at different E:T ratios. (Figure 9A) Incucyte-based assessment of tumor cell killing (i.e., loss of Nuclight-red-positive cells) measured upon treatment with ENPP3 x CD3 (NPP3B815) in the presence of T cells isolated from 6 different healthy human donors and added at 2 different E:T ratios of 1 : 1 and 1 :3 to the assay with the ENPP3-high cell line, A704. Data plotted at 72- and 120-hours post-treatment. Error Bars are SEM. (Figure 9B) Incucyte-based assessment of tumor cell killing (i.e., loss of Nuclight-red- positive cells) measured upon treatment with ENPP3 x CD3 (NPP3B815) in the presence of T cells isolated from 6 different healthy human donors and added at 2 different E:T ratios of 1 : 1 and 1:3 to the assay with VMRCRCW (ENPP3 -medium) cells. Data plotted at 72 and 120 hours post treatment. Error Bars are SEM. Abbreviations: CD, cluster of differentiation; Cone., concentration; D, donor; ENPP3, ectonucleotide pyrophosphatase / phosphodiesterase family member 3; E:T ratio, effector-to-target cell ratio; IFN, interferon; IL, interleukin; SEM, standard error of the mean; TNF, tumor necrosis factor.

[0025] Figure 10 shows NPP3B815-induced cytokine release in the presence of T cells isolated from multiple donors in ENPP3-high cancer cell line A704. Cytokine release was measured by flow cytometry upon treatment with ENPP3 x CD3 (NPP3B815) for 48 hours in the presence of T cells (E:T ratio of 3: 1) isolated from 6 different healthy human donors tested with A704 (ENPP3-high) cells. Error bars are SEM. Abbreviations: CD, cluster of differentiation; Cone., concentration; D, donor; ENPP3, ectonucleotide pyrophosphatase / phosphodi esterase family member 3; E:T ratio, effector-to-target cell ratio; IFN, interferon; IL, interleukin; SEM, standard error of the mean; TNF, tumor necrosis factor.

[0026] Figure 11 shows NPP3B815-induced tumor cell killing with PBMC donors at different E:T ratios. Incucyte-based assessment of tumor cell killing (i.e., loss of Nuclight-red- positive cells) measured upon treatment with ENPP3 x CD3 (NPP3B815) and Null x CD3 (79C3B615) antibodies in the presence of PBMCs (NPP3B815: E:T ratio of 5: 1, 3:1 or 1 :1; 79C3B615: E:T ratio of 5: 1) isolated from 6 different healthy human donors tested with A704 (ENPP3-high) cell line. Data plotted at 68 hours or 72 hours post treatment. Error bars are SEM. Abbreviations: CD, cluster of differentiation; Cone., concentration; D, donor; ENPP3,ectonucleotide pyrophosphatase / phosphodiesterase family member 3; E:T ratio, effector-to-target cell ratio; PBMC, peripheral blood mononuclear cell; SEM, standard error of the mean.

[0027] Figure 12 shows human and cynomolgus monkey T-cell activation and T-cell mediated cytotoxicity. Tumor cell killing and T-cell activation (i.e., CD25+T cells) was measured by flow cytometry upon treatment with lead ENPP3 x CD3 (NPP3B815), matched Null x CD3 (79C3B615; with CD3B2030-N106A arm), ENPP3 x CD3 tool (NPP3B847; lead ENPP3-binding domain NPP3B56 with cyno cross reactive CD3B219) and matched Null x CD3 (NPP3B41; with CD3B219 arm) antibodies for 48 hours in the presence of human and cyno T cells (E:T ratio of 3:1) tested with HepG2-huENPP3-KO cell line with cyno ENPP3-OE. Error Bars are SEM. Abbreviations: CD, cluster of differentiation; cyno, cynomolgus monkey; ENPP3, ectonucleotide pyrophosphatase / phosphodiesterase family member 3; E:T ratio, effector-to-target cell ratio; SEM, standard error of the mean.

[0028] Figure 13 A and Figure 13B show the effect of NPP3B815 on VMRCRCW established xenografts in mice (Study ONC2022-035). NSG mice bearing established VMRCRCW xenografts were IP dosed with NPP3B815 or Null x CD3 control antibody at the indicated doses. (Figure 13 A) Group tumor volumes are graphed as mean ± SEM. Tumor cells were implanted on Day 0 and T cells were implanted on Day 10. Treatment with NPP3B815 or Null x CD3 control antibodies was on Days 11, 14, 18, 21, 25, 28, 31, and 34 (represented by line underneath X axis). (Figure 13B) Individual tumor graphs for CD3xNull control antibody and NPP3B815 1 mg / kg treated groups. * Denotes significant difference of NPP3B815-treated groups on Day 39 (n=10 / group) versus the control group. Abbreviations: CD, cluster of differentiation; IP, intraperitoneal; NSG, non-obese diabetic (NOD) severe combined immunodeficiency (scid) gamma or NOD.Cg-Prkdcscid I12rgtmlWjl / SzJ; SEM, standard error of the mean.7. DETAILED DESCRIPTION

[0029] Solid tumors have been a challenge for antibody-mediated T-cell redirection therapy, with very limited, if any, drugs yet approved by the US Food and Drug Administration (FDA). Limitation of the approach may be related to the lack of cancer specificity of targets leading to toxicity occurring at subefficacious doses. To mitigate this liability, the work described hereinhas focused on the identification of cancer-specific antigens with potentially greater therapeutic index based on apically expressed targets. Apical protein targets are unreachable by directed antibody through the blood stream in normal tissues, however in tumors characterized by disorganized expression, these proteins are reachable through the blood stream. Using this approach, ENPP3 was identified as a target with mainly apical expression in normal tissues and depolarized expression in cancer.

[0030] The invention provides bispecific ENPP3 x CD3 binding agents and methods of use thereof. In one embodiment, provided are compositions comprising the bispecific binding agents. The described compositions can be used to carry out the methods of using the binding agents provided herein, or other methods known to those skilled in the art. Accordingly, the described compositions can include one or more of the binding agents (e.g., antibodies or proteins), or an antigen binding region(s) thereof, described herein. In one embodiment, provided is a bispecific binding antibody comprising a first binding region specifically binding ENPP3 (an ENPP3 -binding domain) and a second binding region specifically binding CD3s (a CD3s- binding domain).

[0031] In some embodiments, the invention provides for the use of the compositions or bispecific binding agents of the invention for the diagnosis or treatment of a disease or disorder. In one embodiment, the disease or disorder is cancer.

[0032] In some embodiments, the invention provides for the use of the compositions or bispecific binding agents of the invention to target T cells to an ENPP3 expressing cell. In one embodiment, the ENPP3 expressing cell is a cancer cell.7.1 General Techniques

[0033] Techniques and procedures described or referenced herein include those that are generally well understood and / or commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual (3d ed. 2001); Current Protocols in Molecular Biology (Ausubel et al. eds., 2003); Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed. 2009); Monoclonal Antibodies: Methods and Protocols (Albitar ed. 2010); and Antibody Engineering Vols 1 and 2 (Kontermann and Dtibel eds., 2d ed. 2010).7.2 Terminology

[0034] Unless described otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. For purposes of interpreting this specification, the following description of terms will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. All patents, applications, published applications, and other publications are incorporated by reference in their entirety. In the event that any description of terms set forth conflicts with any document incorporated herein by reference, the description of term set forth below shall control.

[0035] Unless otherwise defined herein, technical and scientific terms used in the present description have the meanings that are commonly understood by those of ordinary skill in the art. Whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any description of a term set forth conflicts with any document incorporated herein by reference, the description of the term set forth below shall control.

[0036] The term “binding agent” as used herein refers to a molecule that binds a specific antigen or target (e.g., ENPP3 and / or CD3). A binding agent may comprise a protein, peptide, nucleic acid, carbohydrate, lipid, or small molecular weight compound. In some embodiments, a binding agent comprises a full-length antibody. In some embodiments, a binding agent is an antigen binding fragment of an antibody. In some embodiments, a binding agent comprises an alternative protein scaffold or artificial scaffold (e.g., a non-immunoglobulin backbone). In some embodiments, a binding agent is a fusion protein comprising an antigen-binding site. In some embodiments, a binding agent is a bispecific molecule comprising at least two antigenbinding sites. In some embodiments, a binding agent is a multispecific molecule comprising at least three antigen-binding sites.

[0037] The terms “ENPP3” “ENPP3 protein” and “ENPP3 polypeptide” encompass a polypeptide (“polypeptide” and “protein” are used interchangeably herein), including any native polypeptide, from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys (cynomolgus)), dogs, and rodents (e.g., mice and rats), unless otherwise indicated. In certain embodiments, the terms include “related ENPP3 polypeptides,” including SNP variants thereof. The term “ENPP3” also encompasses “full-length,” unprocessed ENPP3 as well as any form that results from processing. The amino acid sequence of a full length ENPP3 is shown in SEQ ID NO: 51.Human ENPP3 :MESTLTLATEQPVKKNTLKKYKIACIVLLALLVIMSLGLGLGLGLRKLEKQGSCR KKCFDASFRGLENCRCDVACKDRGDCCWDFEDTCVESTRIWMCNKFRCGETRLEASLC SCSDDCLQRKDCCADYKSVCQGETSWLEENCDTAQQSQCPEGFDLPPVILFSMDGFRAE YLYTWDTLMPNINKLKTCGIHSKYMRAMYPTKTFPNHYHVTGLYPESHGIIDNNMYDV NLNI<NFSLSSI<EQNNPAWWHGQPMWLTAMYQGLI<AATYFWPGSEVAINGSFPSIYMP YNGSVPFEERISTLLKWLDLPKAERPRFYTMYFEEPDSSGHAGGPVSARVIKALQVVDH AFGMLMEGLKQRNLHNCVNIILLADHGMDQTYCNKMEYMTDYFPRINFFYMYEGPAP RIRAHNIPHDFFSFNSEEIVRNLSCRKPDQHFKPYLTPDLPKRLHYAKNVRIDKVHLFVD QQWLAVRSKSNTNCGGGNHGYNNEFRSMEAIFLAHGPSFKEKTEVEPFENIEVYNLMC DLLRIQPAPNNGTHGSLNHLLKVPFYEPSHAEEVSKFSVCGFANPLPTESLDCFCPHLQN STQLEQVNQMLNLTQEEITATVKVNLPFGRPRVLQKNVDHCLLYHREYVSGFGKAMR MPMWS S YTVPQLGDTSPLPPTVPDCLRADVRVPPSES QKCSF YE ADKNITHGFLYPPAS NRTSDSQYDALITSNLVPMYEEFRKMWDYFHSVLLIKHATERNGVNVVSGPIFDYNYD GHFDAPDEITKHLANTDVPIPTHYFWLTSCKNKSHTPENCPGWLDVLPFIIPHRPTNVES CPEGKPEALWVEERFTAHIARVRDVELLTGLDFYQDKVQPVSEILQLKTYLPTFETTI (SEQ ID NO:51)

[0038] The term “Cluster of Differentiation 3 e” or “CD3s” refers to a known protein which is also called “T-cell surface glycoprotein CD3 epsilon chain,” or “T3E.” CD3s, together with CD3-gamma, -delta and -zeta, and the T-cell receptor alpha / beta and gamma / delta heterodimers, forms the T-cell receptor-CD3 complex. This complex plays an important role in coupling antigen recognition to several intracellular signal-transduction pathways. The CD3 complex mediates signal transduction, resulting in T cell activation and proliferation. CD3 is required for the immune response. The amino acid sequence of a full length CD3s is shown in SEQ ID NO:60. The amino acid sequence of the extracellular domain (ECD) of CD3s is shown in SEQ ID NO:61. Throughout the specification, “CD3s-specific” or “specifically binds CD3s” or “anti- CD3s antibody” refers to antibodies that bind specifically to the CD3s polypeptide (SEQ ID NO:60), including antibodies that bind specifically to the CD3s extracellular domain (ECD) (SEQ ID NO:61).Human CD3 epsilon:MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPG SEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLY LRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI (SEQ ID NQ:60)Human CD3 epsilon extracellular domain:DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGS DEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMD (SEQ ID NO:61)

[0039] The terms “binds” or “binding” refer to an interaction between molecules including, for example, to form a complex. Interactions can be, for example, non-covalent interactions including hydrogen bonds, ionic bonds, hydrophobic interactions, and / or van der Waals interactions. A complex can also include the binding of two or more molecules held together by covalent or non-covalent bonds, interactions, or forces. The strength of the total non-covalent interactions between a single antigen-binding site on an antibody and a single epitope of a target molecule, such as an antigen, is the affinity of the antibody or functional fragment for that epitope. The ratio of dissociation rate (kOff) to association rate (kon) of a binding molecule (e.g., an antibody) to a monovalent antigen (kOff / kOn) is the dissociation constant KD, which is inversely related to affinity. The lower the KD value, the higher the affinity of the antibody. The value of KD varies for different complexes of antibody and antigen and depends on both konand kOff. The dissociation constant KD for an antibody provided herein can be determined using any method provided herein or any other method well known to those skilled in the art. The affinity at one binding site does not always reflect the true strength of the interaction between an antibody and an antigen. When complex antigens containing multiple, repeating antigenic determinants, such as a polyvalent antigen, come in contact with antibodies containing multiple binding sites, the interaction of antibody with antigen at one site will increase the probability of a reaction at a second site. The strength of such multiple interactions between a multivalent antibody and antigen is called the avidity.

[0040] In connection with the binding molecules described herein terms such as “bind to,” “that specifically bind to,” and analogous terms are also used interchangeably herein and refer to binding molecules of antigen binding domains that specifically bind to an antigen, such as a polypeptide. A binding molecule or antigen binding domain that binds to or specifically binds to an antigen can be identified, for example, by immunoassays, Octet®, Biacore®, or other techniques known to those of skill in the art. In some embodiments, a binding molecule orantigen binding domain binds to or specifically binds to an antigen when it binds to an antigen with higher affinity than to any cross-reactive antigen as determined using experimental techniques, such as enzyme linked immunosorbent assay (ELISA). Typically, a specific or selective reaction will be at least twice background signal or noise and may be more than 10 times background. See, e.g., Fundamental Immunology 332-36 (Paul ed., 2d ed. 1989) for a discussion regarding binding specificity. In certain embodiments, the extent of binding of a binding molecule or antigen binding domain to a “non-targef ’ protein is less than about 10% of the binding of the binding molecule or antigen binding domain to its particular target antigen, for example, as determined by fluorescence activated cell sorting (FACS) analysis. A binding molecule or antigen binding domain that binds to an antigen includes one that is capable of binding the antigen with sufficient affinity such that the binding molecule is useful, for example, as a therapeutic and / or diagnostic agent in targeting the antigen. In certain embodiments, a binding molecule or antigen binding domain that binds to an antigen has a dissociation constant (KD) of less than or equal to IpM, 800 nM, 600 nM, 550 nM, 500 nM, 300 nM, 250 nM, 100 nM, 50 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM. In certain embodiments, a binding molecule or antigen binding domain binds to an epitope of an antigen that is conserved among the antigen from different species.

[0041] The term “antibody,” “immunoglobulin,” or “Ig” is used interchangeably herein, and is used in the broadest sense and specifically covers, for example polyclonal antibodies, monoclonal antibodies (including agonist, antagonist, neutralizing antibodies, full-length monoclonal antibodies), antibody compositions with polyepitopic or monoepitopic specificity, recombinantly produced antibodies, single domain (e.g., VHH) antibodies, multispecific antibodies, including bispecific antibodies, synthetic antibodies, chimeric antibodies, humanized antibodies, or human versions of antibodies having full-length heavy and / or light chains. Antibodies also include antibody fragments (and / or polypeptides that comprise antibody fragments) that retain binding characteristics of their parental antibodies. Non-limiting examples of antibody fragments include antigen-binding regions and / or effector regions of the antibody, e.g., Fab, Fab’, F(ab’)2, Fv, scFv, (scFv)2, spFv, single chain antibody molecule, dual variable domain antibody, single variable domain, linear antibody, V region, a multispecific antibody formed from antibody fragments, F(ab)2, Fd, Fc, diabody, di-diabody, disulfide-linked Fvs(dsFv), single-domain antibody (e.g., nanobody) or other fragments (e.g., fragments consisting of the variable regions of the heavy and light chains that are non-covalently coupled). In some embodiments, an antibody comprises a fusion protein comprising an antigen-binding site. In some embodiments, an antibody comprises an spFv-Fc fusion protein. In some embodiments, an antibody comprises a full-length antibody arm, comprising a heavy chain and a light chain. In some embodiments, an antibody comprises an spFv-Fc fusion protein and a full-length antibody arm. In general terms, a variable (V) region domain may be any suitable arrangement of immunoglobulin heavy (VH) and / or light (VL) variable domains. For example, antibodies also include tetrameric antibodies comprising two heavy chain and two light chain molecules, an antibody light chain monomer, and an antibody heavy chain monomer. Thus, for example, the V region domain may be dimeric and contain VHH-VHH, VH-VH, VH-VL, or VL-VL dimers that bind ENPP3. If desired, the VH and VL may be covalently coupled either directly or through a linker to form a single chain Fv (scFv). For ease of reference, scFv proteins are referred to herein as included in the category “antibody fragments.” Another form of an antibody fragment is a peptide comprising one or more complementarity determining regions (CDRs) of an antibody. CDRs (also termed “minimal recognition units” or “hypervariable regions”) can be obtained by constructing polynucleotides that encode one or more CDRs of interest. Such polynucleotides are prepared, for example, by using the polymerase chain reaction to synthesize the variable region using mRNA of antibody-producing cells as a template (see, for example, Larrick et al., Methods: A Companion to Methods in Enzymology, 2: 106 (1991); Courtenay- Luck, “Genetic Manipulation of Monoclonal Antibodies,” in Monoclonal Antibodies Production, Engineering and Clinical Application, Ritter et al. (eds.), page 166, Cambridge University Press (1995); and Ward et al., “Genetic Manipulation and Expression of Antibodies,” in Monoclonal Antibodies: Principles and Applications, Birch et al., (eds.), page 137, Wiley-Liss, Inc. (1995)). Antibody fragments may be incorporated, for example, into single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, variable domains of new antigen receptors (v-NAR), and bis-single chain Fv regions (see, e.g., Hollinger and Hudson, Nature Biotechnology, 23(9): 1126-1136, 2005). In some embodiments, antibodies comprising a VH and / or VL contain a light chain and / or a heavy chain constant region, such as one or more constant regions, including one or more IgGl, IgG2, IgG3 and / or IgG4 constant regions. In some embodiments, antibodies can include epitope-binding fragments of any of the above. Theantibodies described herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2) of immunoglobulin molecule.

[0042] An “intact” antibody is one comprising an antigen-binding site as well as a CL and at least heavy chain constant regions, CHI, CH2 and CH3. The constant regions may include human constant regions or amino acid sequence variants thereof. In certain embodiments, an intact antibody has one or more effector functions. A “functional fragment,” “binding fragment,” or “antigen binding fragment” of a therapeutic antibody will exhibit at least one if not some or all of the biological functions attributed to the intact antibody, the function comprising at least binding to the target antigens (e.g., a bispecific ENPP3 x CD3 binding fragment or fragment that binds to ENPP3 and CD3).

[0043] A typical 4-chain antibody unit is a heterotetrametric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. In the case of IgGs, the 4-chain unit is generally about 150,000 Daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the a and y chains and four CH domains for p and s isotypes. Each L chain has at the N-terminus, a variable domain (VL) followed by a constant domain (CL) at its other end. The VL is aligned with the VH, and the CL is aligned with the first constant domain of the heavy chain (CHI). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of a VH and VL together forms a single antigen-binding site. For the structure and properties of the different classes of antibodies, see, for example, Basic and Clinical Immunology 71 (Stites et al. eds., 8th ed. 1994); and Immunobiology (Janeway et al. eds., 5thed. 2001).

[0044] The term “variable region,” “variable domain,” “V region,” or “V domain” refers to a portion of the light or heavy chains of an antibody that is generally located at the amino-terminal of the light or heavy chain and has a length of about 120 to 130 amino acids in the heavy chain and about 100 to 110 amino acids in the light chain, and are used in the binding and specificity of each particular antibody for its particular antigen. The variable region of the heavy chain may be referred to as “VH.” The variable region of the light chain may be referred to as “VL.” The term “variable” refers to the fact that certain segments of the variable regions differ extensivelyin sequence among antibodies. The V region mediates antigen binding and defines specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the 110-amino acid span of the variable regions. Instead, the V regions consist of less variable (e.g., relatively invariant) stretches called framework regions (FRs) of about 15-30 amino acids separated by shorter regions of greater variability (e.g., extreme variability) called “hypervariable regions” that are each about 9-12 amino acids long. The variable regions of heavy and light chains each comprise four FRs, largely adopting a 0 sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases form part of, the 0 sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest (5th ed. 1991)). The constant regions are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). The variable regions differ extensively in sequence between different antibodies. In specific embodiments, the variable region is a human variable region.

[0045] The term “variable region residue numbering according to Kabat” or “amino acid position numbering as in Kabat,” and variations thereof, refer to the numbering system used for heavy chain variable regions or light chain variable regions of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, an FR or CDR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 and three inserted residues (e.g., residues 82a, 82b, and 82c, etc. according to Kabat) after residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence. The Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., supra . The “EU numbering system” or “EU index” is generally used when referring to a residue in an immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). The “EU index as in Kabat” refers to the residue numbering ofthe human IgG 1 EU antibody. Other numbering systems have been described, for example, by AbM, Chothia, Contact, IMGT, and AHon.

[0046] The term “heavy chain” when used in reference to an antibody refers to a polypeptide chain of about 50-70 kDa, wherein the amino-terminal portion includes a variable region of about 120 to 130 or more amino acids, and a carboxy -terminal portion includes a constant region. The constant region can be one of five distinct types, (e.g., isotypes) referred to as alpha (a), delta (5), epsilon (s), gamma (y), and mu (p), based on the amino acid sequence of the heavy chain constant region. The distinct heavy chains differ in size: a, 5, and y contain approximately 450 amino acids, while p and s contain approximately 550 amino acids. When combined with a light chain, these distinct types of heavy chains give rise to five well known classes (e.g., isotypes) of antibodies, IgA, IgD, IgE, IgG, and IgM, respectively, including four subclasses of IgG, namely IgGl, IgG2, IgG3, and IgG4.

[0047] The term “light chain” when used in reference to an antibody refers to a polypeptide chain of about 25 kDa, wherein the amino-terminal portion includes a variable region of about 100 to about 110 or more amino acids, and a carboxy -terminal portion includes a constant region. The approximate length of a light chain is 211 to 217 amino acids. There are two distinct types, referred to as kappa (K) or lambda (X) based on the amino acid sequence of the constant domains.

[0048] As used herein, the terms “hypervariable region,” “HVR,” “Complementarity Determining Region,” and “CDR” are used interchangeably. A “CDR” refers to one of three hypervariable regions (Hl, H2 or H3) within the non-framework region of the immunoglobulin (Ig or antibody) VH P-sheet framework, or one of three hypervariable regions (LI, L2 or L3) within the non-framework region of the antibody VL P-sheet framework. CDR1 , CDR2 and CDR3 in VH domain are also referred to as HCDR1, HCDR2 and HCDR3, respectively. CDR1, CDR2 and CDR3 in VL domain are also referred to as LCDR1, LCDR2 and LCDR3, respectively. Accordingly, CDRs are variable region sequences interspersed within the framework region sequences.

[0049] CDR regions are well known to those skilled in the art and have been defined by well-known numbering systems. For example, the Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (see, e.g., Kabat et al., supra,' Nick Deschacht et al., J Immunol 2010; 184:5696-5704). Chothia refers instead to thelocation of the structural loops (see, e.g., Chothia and Lesk, J. Mol. Biol. 196:901-17 (1987)). The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35 A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular’s AbM antibody modeling software (see, e.g., Antibody Engineering Vol. 2 (Kontermann and Diibel eds., 2d ed. 2010)). The “contact” hypervariable regions are based on an analysis of the available complex crystal structures. Another universal numbering system that has been developed and widely adopted is ImMunoGeneTics (IMGT) Information System® (Lafranc et al., Dev. Comp. Immunol. 27(l):55-77 (2003)). IMGT is an integrated information system specializing in immunoglobulins (IG), T-cell receptors (TCR), and major histocompatibility complex (MHC) of human and other vertebrates. Herein, the CDRs are referred to in terms of both the amino acid sequence and the location within the light or heavy chain. As the “location” of the CDRs within the structure of the immunoglobulin variable domain is conserved between species and present in structures called loops, by using numbering systems that align variable domain sequences according to structural features, CDR and framework residues are readily identified. This information can be used in grafting and replacement of CDR residues from immunoglobulins of one species into an acceptor framework from, typically, a human antibody. An additional numbering system (AHon) has been developed by Honegger and Pliickthun, J. Mol. Biol. 309: 657-70 (2001). Correspondence between the numbering system, including, for example, the Kabat numbering and the IMGT unique numbering system, is well known to one skilled in the art (see, e.g, Kabat, supra,' Chothia and Lesk, supra,' Martin, supra,' Lefranc et al., supra). The residues from each of these hypervariable regions or CDRs are exemplified in the table below.Exemplary CDRs According to Various Numbering Systems

[0050] The boundaries of a given CDR may vary depending on the scheme used for identification. Thus, unless otherwise specified, the terms “CDR” and “complementary determining region” of a given antibody or region thereof, such as a variable region, as well as individual CDRs (e.g., CDR-H1, CDR-H2) of the antibody or region thereof, should be understood to encompass the complementary determining region as defined by any of the known schemes described herein above. In some instances, the scheme for identification of a particular CDR or CDRs is specified, such as the CDR as defined by the IMGT, Kabat, Chothia, or Contact method. In other cases, the particular amino acid sequence of a CDR is given. It should be noted CDR regions may also be defined by a combination of various numbering systems, e.g., a combination of Kabat and Chothia numbering systems, or a combination of Kabat and IMGT numbering systems. Therefore, the term such as “a CDR1 as set forth in a specific VH” includes any CDR1 as defined by the exemplary CDR numbering systems described above, but is not limited thereby. Once a variable region (e.g., a VH or VL) is given, those skilled in the art would understand that CDRs within the region can be defined by different numbering systems or combinations thereof.

[0051] Hypervariable regions may comprise “extended hypervariable regions” as follows: 24-36 or 24-34 (LI), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in the VL, and 26-35 or 26- 35A (Hl), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in the VH.

[0052] The term “framework” or “FR” refers to those variable region residues flanking the CDRs. FR residues are present, for example, in chimeric, humanized, human, domain antibodies, diabodies, linear antibodies, and bispecific antibodies. FR residues are those variable domain residues other than the hypervariable region residues or CDR residues.

[0053] The term “constant region” or “constant domain” refers to a carboxy terminal portion of the light and heavy chain which is not directly involved in binding of the antibody to antigen but exhibits various effector function, such as interaction with the Fc receptor. The term refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable region, which contains the antigen binding site. The constant region may contain the CHI, CH2, and CH3 regions of the heavy chain and the CL region of the light chain.

[0054] The term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain, including, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is often defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxylterminus thereof. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Accordingly, a composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue. A “functional Fc region” possesses an “effector function” of a native sequence Fc region. Exemplary “effector functions” include Clq binding; CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor), etc. Such effector functions generally require the Fc region to be combined with a binding region or binding domain (e.g., an antibody variable region or domain) and can be assessed using various assays known to those skilled in the art. A “variant Fc region” comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification (e.g., substituting, addition, or deletion). In certain embodiments, the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide, for example, from about one to about ten amino acid substitutions, or from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of a parent polypeptide. The variant Fc region herein can possess at least about 80% homology witha native sequence Fc region and / or with an Fc region of a parent polypeptide, or at least about 90% homology therewith, for example, at least about 95% homology therewith.

[0055] The term “antigen binding domain” or “antigen binding region” refers to a binding agent or a portion of a binding agent as described herein (such as a protein or an antibody or fragment thereof) that binds an antigen. In some embodiments, an antigen binding region can comprise one or more fragments or portions of an intact antibody as described herein. The term “antigen binding domain” or “antigen binding region” can be an antibody fragment as described above.

[0056] The term “Fab” or “Fab region” refers to an antibody region that binds to antigens. A conventional IgG usually comprises two Fab regions, each residing on one of the two arms of the Y-shaped IgG structure. Each Fab region is typically composed of one variable region and one constant region of each of the heavy and the light chain. More specifically, the variable region and the constant region of the heavy chain in a Fab region are VH and CHI regions, and the variable region and the constant region of the light chain in a Fab region are VL and CL regions. The VH, CHI, VL, and CL in a Fab region can be arranged in various ways to confer an antigen binding capability according to the present disclosure. For example, VH and CHI regions can be on one polypeptide, and VL and CL regions can be on a separate polypeptide, similarly to a Fab region of a conventional IgG. Alternatively, VH, CHI, VL and CL regions can all be on the same polypeptide and oriented in different orders as described in more detail the sections below.

[0057] The term “single chain Fv” or “scFv” refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region (VL) and at least one antibody fragment comprising a heavy chain variable region (VH), wherein the VL and the VH are contiguously linked via a polypeptide linker, and capable of being expressed as a single chain polypeptide. Unless specified, as used herein, a scFv may have the VL and VH variable regions in either order, e.g., with respect to the N- terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.

[0058] “Stapled single chain Fv” or “spFv” refers to a scFv that comprises one or more disulfide bonds between the VH and the linker or the VL and the linker. Typically the spFv may comprise one disulfide bond between the VH and the linker, one disulfide bond between the VL and the linker, or two disulfide bonds between the VH and the linker and the VL and the linker.scFv molecules which comprise disulfide bonds between the VH and the VL are excluded from the term “spFv”.

[0059] ‘ ‘Anchor point” refers to a scFv VH or a VL framework Cysteine (Cys) residue that can be mutagenized to Cys without adverse effect to the overall scFv structure and is capable of forming a disulfide bond with a Cys residing in the scFv linker.

[0060] “Staple” refers to the scFv linker that contains one or two Cys residues which are capable of forming a disulfide bond with the anchor point Cys.

[0061] The term “multispecific” refers to a molecule, such as an antibody that specifically binds two or more distinct antigens or two or more distinct epitopes within the same antigen. Multispecific molecule may have cross-reactivity to other related antigens, for example to the same antigen from other species (homologs), such as human or monkey, for example Macaca fascicularis (cynomolgus, cyno) or Pan troglodytes, or may bind an epitope that is shared between two or more distinct antigens.

[0062] The term “bispecific” refers to a molecule (such as a protein or an antibody) that specifically binds two distinct antigens or two distinct epitopes within the same antigen. The bispecific molecule may have cross-reactivity to other related antigens, for example to the same antigen from other species (homologs), such as human or monkey, for example Macaca cynomolgus (cynomolgus, cyno) or Pan troglodytes, or may bind an epitope that is shared between two or more distinct antigens.

[0063] The terms “bispecific anti-ENPP3 / anti-CD3 antibody,” “ENPP3 / CD3 antibody,” “ENPP3 x CD3 antibody,” “anti-ENPP3 / anti-CD3 protein,” and the like refer to an antibody that binds ENPP3 and CD3, i.e., comprising at least one binding domain specifically binding ENPP3 (ENPP3 -binding domain) and at least one binding domain specifically binding CD3 (CD3- binding domain). The domains specifically binding ENPP3 and CD3 are typically VH / VL pairs. The bispecific ENPP3 x CD3 antibody may be monovalent in terms of its binding to either ENPP3 or CD3.

[0064] The term “monoclonal antibody” as used herein refers to a substantially homogenous antibody population involved in the highly specific recognition and binding of a single antigenic determinant or epitope. The term “monoclonal antibody” encompasses intact and full-length antibodies as well as antibody fragments (e.g., Fab, Fab', F(ab')2, Fv), single chain antibodies, scFv, fusion proteins comprising an antigen-binding antibody fragment, and any other modifiedimmunoglobulin molecule comprising at least one antigen-binding site. Furthermore, “monoclonal antibody” refers to such antibodies made by any number of techniques, including but not limited to, hybridoma production, phage library display, recombinant expression, and transgenic animals.

[0065] The terms “epitope” and “antigenic determinant” are used interchangeably herein and refer to that portion of an antigen or target capable of being recognized and bound by a particular antibody. When the antigen or target is a polypeptide, epitopes can be formed both from contiguous amino acids and noncontiguous amino acids juxtaposed by tertiary folding of the protein. Epitopes formed from contiguous amino acids (also referred to as linear epitopes) are typically retained upon protein denaturing, whereas epitopes formed by tertiary folding (also referred to as conformational epitopes) are typically lost upon protein denaturing. An epitope typically includes at least 3, and more usually, at least 5, 6, 7, or 8-10 amino acids in a unique spatial conformation. Epitopes can be predicted using any one of a large number of publicly available bioinformatic software tools. X-ray crystallography may be used to characterize an epitope on a target protein by analyzing the amino acid residue interactions of an antigen / antibody complex.

[0066] The term “chimeric antibody” refers to an antibody in which a portion of the heavy and / or light chain is derived from a first source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0067] The term “humanized antibody” as used herein refers to an antibody that comprises a human heavy chain variable region and a light chain variable region wherein the native CDR amino acid residues are replaced by residues from corresponding CDRs from a non-human antibody (e.g., mouse, rat, rabbit, or non-human primate), wherein the non-human antibody has the desired specificity, affinity, and / or activity. In some embodiments, one or more framework region amino acid residues of the human heavy chain or light chain variable regions are replaced by corresponding residues from the non-human antibody. Furthermore, humanized antibodies can comprise amino acid residues that are not found in the human antibody or in the non-human antibody. In some embodiments, these modifications are made to further refine and / or optimize antibody characteristics. In some embodiments, the humanized antibody comprises at least a portion of a human immunoglobulin constant region ( .g, CHI, CH2, CH3, Fc, and / or hinge region). 1

[0068] The term “human antibody” as used herein refers to an antibody that possesses an amino acid sequence that corresponds to an antibody produced by a human and / or an antibody that has been made using any of the techniques that are known to those of skill in the art for making human antibodies. These techniques include, but not limited to, phage display libraries, yeast display libraries, transgenic animals, recombinant protein production, and B-cell hybridoma technology.

[0069] The terms “polypeptide” and “peptide” and “protein” are used interchangeably herein and refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid, including but not limited to, unnatural amino acids, as well as other modifications known in the art. It is understood that, because the polypeptides of this disclosure may be based upon antibodies, the term “polypeptide” encompasses polypeptides as a single chain and polypeptides of two or more associated chains.

[0070] The terms “polynucleotide” and “nucleic acid” and “nucleic acid molecule” are used interchangeably herein and refer to polymers of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase.

[0071] The terms “identical” or percent “identity” in the context of two or more nucleic acids or polypeptides, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity may be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that may be used to obtain alignments of amino acid or nucleotide sequences are well-known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variants thereof. In some embodiments, twonucleic acids or polypeptides of the disclosure are substantially identical, meaning they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. In some embodiments, identity exists over a region of the sequences that is at least about 10, at least about 20, at least about 20-40, at least about 40-60, at least about 60-80 nucleotides or amino acids in length, or any integral value there between. In some embodiments, identity exists over a longer region than 60-80 nucleotides or amino acids, such as at least about 80-100 nucleotides or amino acids, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, for example, (i) the coding region of a nucleotide sequence or (ii) an amino acid sequence.

[0072] The term “vector” as used herein means a construct that is capable of delivering, and usually expressing, one or more gene(s) or sequence(s) of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid, or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, and DNA or RNA expression vectors encapsulated in liposomes.

[0073] The term “isolated” as used herein refers to a polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition that is in a form not found in nature. An “isolated” antibody is substantially free of material from the cellular source from which it is derived. In some embodiments, isolated polypeptides, soluble proteins, antibodies, polynucleotides, vectors, cells, or compositions are those that have been purified to a degree that they are no longer in a form in which they are found in nature. In some embodiments, a polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition that is isolated is substantially pure. A polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition can be isolated from a natural source (e.g., tissue) or from a source such as an engineered cell line.

[0074] The term “substantially pure” as used herein refers to material that is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0075] The term “subject” refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, canines, felines, rabbits, rodents, and the like.

[0076] The term “pharmaceutical composition” or “pharmaceutical formulation” as used herein refers to a preparation that is in such form as to permit the biological activity of the binding agent to be effective. A pharmaceutical formulation or composition generally comprises additional components, such as a pharmaceutically acceptable excipient, carrier, adjuvant, buffers, etc.

[0077] The term “effective amount” or “therapeutically effective amount” as used herein refers to the amount of an agent that is sufficient to reduce and / or ameliorate the severity and / or duration of (i) a disease, disorder or condition in a subject, and / or (ii) a symptom in a subject. The term also encompasses an amount of an agent necessary for the (i) reduction or amelioration of the advancement or progression of a given disease, disorder, or condition, (ii) reduction or amelioration of the recurrence, development, or onset of a given disease, disorder, or condition, and / or (iii) the improvement or enhancement of the prophylactic or therapeutic effect(s) of another agent or therapy (e.g., an agent other than the binding agents provided herein).

[0078] The term “treat” or “treatment” or “treating” or “to treat” or “alleviate” or alleviation” or “alleviating” or “to alleviate” as used herein refers to therapeutic measures that aim to cure, slow down, lessen symptoms of, and / or halt progression of a pathologic condition or disorder. Thus, those in need of treatment include those already with the disorder.

[0079] The term “step-up dose” as used herein refers to incrementally increasing the dose of the ENPP3xCD3 bispecific antibody administered to a patient prior to reaching the target dose level, in order to lower toxicity. The step-up dosing may include one or more doses that are lower than the target dose, administered prior to the target dose. In some embodiments, the one or more step-up doses are administered one week prior to the target dose. In some embodiments, the one or more step-up doses are administered less than one week prior to the target dose. In some embodiments, the one or more step-up doses are administered 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day prior to the target dose.

[0080] The term “immune response” as used herein includes responses from both the innate immune system and the adaptive immune system. It includes both cell-mediated and / or humoral immune responses. It includes both T-cell and B-cell responses, as well as responses from other cells of the immune system such as natural killer (NK) cells, monocytes, macrophages, dendritic cells, etc.

[0081] As used herein, reference to “about” or “approximately” a value or parameter includes (and describes) embodiments that are directed to that value or parameter. For example, a description referring to “about X” includes description of “X.”

[0082] As used in the present disclosure and claims, the singular forms “a,” “an” and “the” include plural forms unless the context clearly dictates otherwise.

[0083] It is understood that wherever embodiments are described herein with the term “comprising” otherwise analogous embodiments described in terms of “consisting of’ and / or “consisting essentially of’ are also provided. It is also understood that wherever embodiments are described herein with the phrase “consisting essentially of’ otherwise analogous embodiments described in terms of “consisting of’ are also provided.

[0084] The term “and / or” as used in a phrase such as “A and / or B” herein is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).7.3 Bispecific Binding Molecules

[0082] In one aspect, provided herein is a bispecific binding molecule that binds ENPP3 and CD3. In some embodiments, the bispecific binding molecule is a bispecific antibody (ENPP3 x CD3 bispecific antibody). In some embodiments, bispecific antibodies described herein are monoclonal antibodies that have binding specificities for at least two different antigens. In certain embodiments, bispecific antibodies are human or humanized antibodies. In certain embodiments, one of the binding specificities is for ENPP3 (an ENPP3 -binding domain) and the other is for CD3 (a CD3-binding domain). Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab’)2 bispecific antibodies).7.4 ENPP3 Binding Domains

[0083] In one aspect, provided herein is a bispecific antibody comprising at least one antigen binding region that binds ENPP3 (e.g., human ENPP3; an ENPP3-binding domain) and a second binding region that binds CD3 (e.g., human CD3s; a CD3-binding domain).

[0084] In some embodiments, the ENPP3 -binding domain binds an ENPP3 protein or a fragment thereof of a mammalian origin. In some embodiments, the ENPP3 -binding domain binds human ENPP3 protein or a fragment thereof. In some embodiments, the ENPP3 -binding domain binds an ENPP3 protein or a fragment thereof originated from a non-human mammalian species. In some embodiments, the non-human mammalian species is a rodent (e.g., mice and rats). In some embodiments, the non-human mammalian species is a dog. In some embodiments, the non-human mammalian species is a cynomolgus monkeys (cynomolgus).

[0085] In some embodiments, the ENPP3 -binding domain is chimeric. In some embodiments, the ENPP3 -binding domain is humanized. In some embodiments, the ENPP3- binding domain is human. In some embodiments, the ENPP3 -binding domain is an IgG isotype. In some embodiments, the ENPP3 -binding domain is an IgGl isotype. In some embodiments, the ENPP3 -binding domain comprises an IgG heavy chain. In some embodiments, the ENPP3- binding domain comprises an IgGl heavy chain. In some embodiments, the ENPP3 -binding domain comprises a kappa light chain. In some embodiments, the ENPP3 -binding domain comprises a kappa light chain constant region.

[0086] In some embodiments, the ENPP3-binding domain is any one of those in Table 1.

[0087] In some embodiments, the ENPP3 -binding domain provided herein comprises one or more CDR sequences of the VH or VL having the amino acid sequence set forth in any one of Tables 1.

[0088] In one embodiment, the ENPP3 -binding domain comprises a VH comprising a HCDR1 , a HCDR2, and a HCDR3 having an amino acid sequence of a HCDR1 , a HCDR2, and a HCDR3, respectively, of a VH having an amino acid sequence of SEQ ID NO:22. In another embodiment, the ENPP3 -binding domain comprises a VL comprising a LCDR1, a LCDR2, and a LCDR3 having an amino acid sequence of a LCDR1, a LCDR2, and a LCDR3, respectively, of a VL having an amino acid sequence of SEQ ID NO:23. In yet another embodiment, the ENPP3- binding domain comprises: (i) a VH comprising a HCDR1, a HCDR2, and a HCDR3 having an amino acid sequence of a HCDR1, a HCDR2, and a HCDR3, respectively, of a VH having an amino acid sequence of SEQ ID NO:22, and (ii) a VL comprising a LCDR1, a LCDR2, and a LCDR3 having an amino acid sequence of a LCDR1, a LCDR2, and a LCDR3, respectively, of a VL having an amino acid sequence of SEQ ID NO:23. CDR sequences can be determined according to well-known numbering systems or a combination thereof. In some embodiments,the CDRs are according to IMGT numbering. In some embodiments, the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering.

[0089] In some specific embodiments, the ENPP3 -binding domain provided herein comprises one or more CDRs in Table 1.

[0090] In some embodiments, the ENPP3 -binding domain provided herein comprises anHCDR1 comprising an amino acid sequence of any of SEQ ID NOs: 1, 7, 9, 11, and 17; (ii) an HCDR2 comprising an amino acid sequence of any of SEQ ID NOs:2, 8, 10, 12, and 18, (iii) an HCDR3 comprising an amino acid sequence of SEQ ID NOs: 3, 13, and 19; (iv) a LCDR1 comprising an amino acid sequence of SEQ ID NOs:4, 14, and 20; (v) a LCDR2 comprising an amino acid sequence of SEQ ID NOs: 5, 15, and 21; and / or (vi) a LCDR3 comprising an amino acid sequence of SEQ ID NOs: 6 and 16.

[0091] In some specific embodiments, in the ENPP3 -binding domain provided herein, theHCDR1 comprises the amino acid sequence of SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence of SEQ ID NO:2, the HCDR3 comprises the amino acid sequence of SEQ ID NO:3, the LCDR1 comprises the amino acid sequence of SEQ ID NO:4, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 5, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:6.

[0092] In some specific embodiments, in the ENPP3 -binding domain provided herein, theHCDR1 comprises the amino acid sequence of SEQ ID NO: 7, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 8, the HCDR3 comprises the amino acid sequence of SEQ ID NO:3, the LCDR1 comprises the amino acid sequence of SEQ ID NO:4, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 5, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:6.

[0093] In some specific embodiments, in the ENPP3 -binding domain provided herein, theHCDR1 comprises the amino acid sequence of SEQ ID NO: 9, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 10, the HCDR3 comprises the amino acid sequence of SEQ ID NO:3, the LCDR1 comprises the amino acid sequence of SEQ ID NO:4, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 5, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:6.

[0094] In some specific embodiments, in the ENPP3 -binding domain provided herein, the HCDR1 comprises the amino acid sequence of SEQ ID NO: 11, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 12, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 13, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 14, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 15, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 16.

[0095] In some specific embodiments, in the ENPP3 -binding domain provided herein, the HCDR1 comprises the amino acid sequence of SEQ ID NO: 17, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 18, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 19, the LCDR1 comprises the amino acid sequence of SEQ ID NO:20, the LCDR2 comprises the amino acid sequence of GAS , and the LCDR3 comprises the amino acid sequence of SEQ ID NO:6.

[0096] In one embodiment, provided herein is an ENPP3 -binding domain , comprising a VH comprising a HCDR1, a HCDR2, and a HCDR3 having an amino acid sequence of SEQ ID NOs:l, 2, and 3, respectively. In another embodiment, provided herein is a binding region that binds ENPP3, comprising a VL comprising a LCDR1, a LCDR2, and a LCDR3 having an amino acid sequence of SEQ ID NOs:4, 5, and 6, respectively. In another embodiment, provided herein is a binding region that binds ENPP3, comprising: (i) a VH comprising a HCDR1, a HCDR2, and a HCDR3 having an amino acid sequence of SEQ ID NOs:l, 2, and 3, respectively, and (ii) a VL comprising a LCDR1, a LCDR2, and a LCDR3 having an amino acid sequence of SEQ ID NOs:4, 5, and 6, respectively.

[0097] In one embodiment, provided herein is an ENPP3 -binding domain , comprising a VH comprising a HCDR1, a HCDR2, and a HCDR3 having an amino acid sequence of SEQ ID NOs:7, 8, and 3, respectively. In another embodiment, provided herein is a binding region that binds ENPP3, comprising a VL comprising a LCDR1, a LCDR2, and a LCDR3 having an amino acid sequence of SEQ ID NOs:4, 5, and 6, respectively. In another embodiment, provided herein is a binding region that binds ENPP3, comprising: (i) a VH comprising a HCDR1, a HCDR2, and a HCDR3 having an amino acid sequence of SEQ ID NOs:7, 8, and 3, respectively, and (ii) a VL comprising a LCDR1, a LCDR2, and a LCDR3 having an amino acid sequence of SEQ ID NOs:4, 5, and 6, respectively.

[0098] In one embodiment, provided herein is an ENPP3 -binding domain , comprising a VH comprising a HCDR1, a HCDR2, and a HCDR3 having an amino acid sequence of SEQ ID NOs:9, 10, and 3, respectively. In another embodiment, provided herein is a binding region that binds ENPP3, comprising a VL comprising a LCDR1, a LCDR2, and a LCDR3 having an amino acid sequence of SEQ ID NOs:4, 5, and 6, respectively. In another embodiment, provided herein is a binding region that binds ENPP3, comprising: (i) a VH comprising a HCDR1, a HCDR2, and a HCDR3 having an amino acid sequence of SEQ ID NOs:9, 10, and 3, respectively, and (ii) a VL comprising a LCDR1, a LCDR2, and a LCDR3 having an amino acid sequence of SEQ ID NOs:4, 5, and 6, respectively.

[0099] In one embodiment, provided herein is an ENPP3 -binding domain , comprising a VH comprising a HCDR1, a HCDR2, and a HCDR3 having an amino acid sequence of SEQ ID NOs:l 1, 12, and 13, respectively. In another embodiment, provided herein is a binding region that binds ENPP3, comprising a VL comprising a LCDR1, a LCDR2, and a LCDR3 having an amino acid sequence of SEQ ID NOs:14, 15, and 16, respectively. In another embodiment, provided herein is a binding region that binds ENPP3, comprising: (i) a VH comprising a HCDR1, a HCDR2, and a HCDR3 having an amino acid sequence of SEQ ID NOs: 11, 12, and 13, respectively, and (ii) a VL comprising a LCDR1, a LCDR2, and a LCDR3 having an amino acid sequence of SEQ ID NOs: 14, 15, and 16, respectively.[000100] In one embodiment, provided herein is an ENPP3 -binding domain, comprising a VH comprising a HCDR1, a HCDR2, and a HCDR3 having an amino acid sequence of SEQ ID NOs: 17, 18, and 19, respectively. In another embodiment, provided herein is a binding region that binds ENPP3, comprising a VL comprising a LCDR1, a LCDR2, and a LCDR3 having an amino acid sequence of SEQ ID NOs:20, 21, and 6, respectively. In another embodiment, provided herein is a binding region that binds ENPP3, comprising: (i) a VH comprising a HCDR1, a HCDR2, and a HCDR3 having an amino acid sequence of SEQ ID NOs: 17, 18, and 19, respectively, and (ii) a VL comprising a LCDR1, a LCDR2, and a LCDR3 having an amino acid sequence of SEQ ID NOs:20, 21, and 6, respectively.[000101] In some embodiments, the ENPP3 -binding domain further comprises one or more framework regions of the VH or VL having the amino acid sequence of any one of SEQ ID NO:22 and SEQ ID NO:23. Framework regions described herein are determined based upon the boundaries of the CDR numbering system. In other words, if the CDRs are determined by, e.g.,Kabat, IMGT, or Chothia, then the framework regions are the amino acid residues surrounding the CDRs in the variable region in the format, from the N-terminus to C-terminus: FR1-CDR1- FR2-CDR2-FR3-CDR3-FR4. For example, FR1 is defined as the amino acid residues N- terminal to the CDR1 amino acid residues as defined by, e.g., the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system, FR2 is defined as the amino acid residues between CDR1 and CDR2 amino acid residues as defined by, e.g., the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system, FR3 is defined as the amino acid residues between CDR2 and CDR3 amino acid residues as defined by, e.g., the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system, and FR4 is defined as the amino acid residues C-terminal to the CDR3 amino acid residues as defined by, e.g., the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system.[000102] In one embodiment, provided herein is an ENPP3 -binding domain , comprising a VH having an amino acid sequence of SEQ ID NO:22. In another embodiment, provided herein is a binding region that binds ENPP3, comprising a VL having an amino acid sequence of SEQ ID NO:23. In another embodiment, provided herein is a binding region that binds ENPP3, comprising: (i) a VH having an amino acid sequence of SEQ ID NO:22, and (ii) a VL having an amino acid sequence of SEQ ID NO:23.Table 1: NPP3B56 Binding Region Sequences[000103] CDR sequences can be determined according to well-known numbering systems or a combination thereof. In some embodiments, the CDRs are according to IMGT numbering. In some embodiments, the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering. [000104] In some embodiments, upon binding to the ENPP3 molecule, the present ENPP3- binding molecule binds to the cell expressing the ENPP3 protein. In some embodiments, the ENPP3 -expressing cell is a cancer cell. In some embodiments, the cancer is a renal cell cancer (RCC), a clear cell RCC (CCRCC), a papillary renal cancer, endometrioid ovarian cancer, endometrioid uterine carcinoma, colorectal adenocarcinoma (CRC), and lung adenocarcinoma.7.5 CD3-Binding Domains

[0105] In particular embodiments, the CD3s-binding domain of the bispecific binding protein comprises one or more binding sequences set forth in Table 2. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are according to IMGT numbering. In some embodiments, the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering. In some embodiments, the CD3s-binding domain ishumanized. In some embodiments, the CD3s-binding domain comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.

[0106] In some embodiments, the CD3s-binding domain provided herein comprises a VH comprising an HCDR1, HCDR2, and HCDR3 of a VH as set forth in SEQ ID NO:47. In some embodiments, the CD3s-binding domain provided herein comprises a VL comprising an LCDR1, LCDR2, and LCDR3 of a VL as set forth in SEQ ID NO:48. In some embodiments, the CD3s-binding domain provided herein comprises a VH comprising an HCDR1, HCDR2, and HCDR3 of a VH as set forth in SEQ ID NO: 47, and a VL comprising an LCDR1, LCDR2, and LCDR3 of a VL as set forth in SEQ ID NO:48. CDR sequences can be determined according to well-known numbering systems or a combination thereof. In some embodiments, the CDRs are according to IMGT numbering. In some embodiments, the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering.

[0107] In some specific embodiments, the CD3 binding region provided herein comprises one or more CDRs in Table 2.

[0108] In some embodiments, the CD3s-binding domain provided herein comprises an HCDR1 comprising an amino acid sequence of any of SEQ ID NO:26, 32, 34, 36, or 42; (ii) an HCDR2 comprising an amino acid sequence of any of SEQ ID NO:27, 33, 35, 37, or 43; (iii) an HCDR3 comprising an amino acid sequence SEQ ID NO:28, 38, or 44; (iv) a LCDR1 comprising an amino acid sequence of SEQ ID NO:29, 39, or 45; (v) a LCDR2 comprising an amino acid sequence of SEQ ID NO: 30, 40, or 46; and / or (vi) a LCDR3 comprising an amino acid sequence of SEQ ID NO:31 or 41.

[0109] In some specific embodiments, in the CD3s-binding domain provided herein, the HCDR1 comprises the amino acid sequence of SEQ ID NO:26, the HCDR2 comprises the amino acid sequence of SEQ ID NO:27, the HCDR3 comprises the amino acid sequence of SEQ ID NO:28, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 29, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 30, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:31.[HO] In some specific embodiments, in the CD3s-binding domain provided herein, the HCDR1 comprises the amino acid sequence of SEQ ID NO: 32, the HCDR2 comprises the amino acidsequence of SEQ ID NO: 33, the HCDR3 comprises the amino acid sequence of SEQ ID NO:28, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 29 the LCDR2 comprises the amino acid sequence of SEQ ID NO: 30, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:31.

[0111] In some specific embodiments, in the CD3s-binding domain provided herein, the HCDR1 comprises the amino acid sequence of SEQ ID NO: 34, the HCDR2 comprises the amino acid sequence of SEQ ID NO:35, the HCDR3 comprises the amino acid sequence of SEQ ID NO:28, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 29, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 30, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:31.

[0112] In some specific embodiments, in the CD3s-binding domain provided herein, the HCDR1 comprises the amino acid sequence of SEQ ID NO: 36, the HCDR2 comprises the amino acid sequence of SEQ ID NO:37, the HCDR3 comprises the amino acid sequence of SEQ ID NO:38, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 39, the LCDR2 comprises the amino acid sequence of SEQ ID NO:40, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:41.

[0113] In some specific embodiments, in the CD3s-binding domain provided herein, the HCDR1 comprises the amino acid sequence of SEQ ID NO:42, the HCDR2 comprises the amino acid sequence of SEQ ID NO:43, the HCDR3 comprises the amino acid sequence of SEQ ID NO:44, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 45, the LCDR2 comprises the amino acid sequence of DSS and the LCDR3 comprises the amino acid sequence of SEQ ID NO:31.

[0114] In one embodiment, provided herein is a binding region that binds CD3s, comprising a VH comprising a HCDR1, a HCDR2, and a HCDR3 having an amino acid sequence of SEQ ID NOs:26, 27, and 28, respectively. In another embodiment, provided herein is a binding region that binds CD3s, comprising a VL comprising a LCDR1, a LCDR2, and a LCDR3 having an amino acid sequence of SEQ ID NOs:29, 30, and 31, respectively. In another embodiment, provided herein is a binding region that binds CD3s, comprising: (i) a VH comprising a HCDR1, a HCDR2, and a HCDR3 having an amino acid sequence of SEQ ID NOs:26, 27, and 28, respectively, and (ii) a VL comprising a LCDR1 , a LCDR2, and a LCDR3 having an amino acid sequence of SEQ ID NOs:29, 30, 31, respectively.

[0115] In one embodiment, provided herein is a binding region that binds CD3s, comprising a VH comprising a HCDR1, a HCDR2, and a HCDR3 having an amino acid sequence of SEQ ID NOs:32, 33, and 28, respectively. In another embodiment, provided herein is a binding region that binds CD3s, comprising a VL comprising a LCDR1, a LCDR2, and a LCDR3 having an amino acid sequence of SEQ ID NOs:29, 30, and 31, respectively. In another embodiment, provided herein is a binding region that binds CD3s, comprising: (i) a VH comprising a HCDR1, a HCDR2, and a HCDR3 having an amino acid sequence of SEQ ID NOs:32, 33, and 28, respectively, and (ii) a VL comprising a LCDR1 , a LCDR2, and a LCDR3 having an amino acid sequence of SEQ ID NOs:29, 30, and 31, respectively.

[0116] In one embodiment, provided herein is a binding region that binds CD3s, comprising a VH comprising a HCDR1, a HCDR2, and a HCDR3 having an amino acid sequence of SEQ ID NOs:34, 35, and 28, respectively. In another embodiment, provided herein is a binding region that binds CD3s, comprising a VL comprising a LCDR1, a LCDR2, and a LCDR3 having an amino acid sequence of SEQ ID NOs:29, 30, and 31, respectively. In another embodiment, provided herein is a binding region that binds CD3s, comprising: (i) a VH comprising a HCDR1, a HCDR2, and a HCDR3 having an amino acid sequence of SEQ ID NOs:34, 35, and 28, respectively, and (ii) a VL comprising a LCDR1 , a LCDR2, and a LCDR3 having an amino acid sequence of SEQ ID NOs:29, 30, and 31, respectively.

[0117] In one embodiment, provided herein is a binding region that binds CD3s, comprising a VH comprising a HCDR1, a HCDR2, and a HCDR3 having an amino acid sequence of SEQ ID NOs:36, 37, and 38, respectively. In another embodiment, provided herein is a binding region that binds CD3s, comprising a VL comprising a LCDR1, a LCDR2, and a LCDR3 having an amino acid sequence of SEQ ID NOs:39, 40, and 41, respectively. In another embodiment, provided herein is a binding region that binds CD3s, comprising: (i) a VH comprising a HCDR1, a HCDR2, and a HCDR3 having an amino acid sequence of SEQ ID NOs:36, 37, and 38, respectively, and (ii) a VL comprising a LCDR1 , a LCDR2, and a LCDR3 having an amino acid sequence of SEQ ID NOs:39, 40, and 41, respectively.

[0118] In one embodiment, provided herein is a binding region that binds CD3s, comprising a VH comprising a HCDR1, a HCDR2, and a HCDR3 having an amino acid sequence of SEQ ID NOs:42, 43, and 44, respectively. In another embodiment, provided herein is a binding region that binds CD3s, comprising a VL comprising a LCDR1, a LCDR2, and a LCDR3 having anamino acid sequence of SEQ ID NOs:45, 46, and 31, respectively. In another embodiment, provided herein is a binding region that binds CD3s, comprising: (i) a VH comprising a HCDR1, a HCDR2, and a HCDR3 having an amino acid sequence of SEQ ID NOs:42, 43, and 44, respectively, and (ii) a VL comprising a LCDR1 , a LCDR2, and a LCDR3 having an amino acid sequence of SEQ ID NOs:45, 46, and 31, respectively.

[0119] In some embodiments, the CD3s-binding domain further comprises one or more framework regions. Framework regions described herein are determined based upon the boundaries of the CDR numbering system. In other words, if the CDRs are determined by, e.g., Kabat, IMGT, or Chothia, then the framework regions are the amino acid residues surrounding the CDRs in the variable region in the format, from the N-terminus to C-terminus: FR1-CDR1- FR2-CDR2-FR3-CDR3-FR4. For example, FR1 is defined as the amino acid residues N- terminal to the CDR1 amino acid residues as defined by, e.g., the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system, FR2 is defined as the amino acid residues between CDR1 and CDR2 amino acid residues as defined by, e.g., the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system, FR3 is defined as the amino acid residues between CDR2 and CDR3 amino acid residues as defined by, e.g., the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system, and FR4 is defined as the amino acid residues C-terminal to the CDR3 amino acid residues as defined by, e.g., the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system. In some embodiments, the CD3s-binding domain further comprises one or more framework regions of SEQ ID NO:47 or 48.

[0120] In some embodiments, the CD3s-binding domain provided herein comprises a VH comprising the amino acid sequence of SEQ ID NO:47, and a VL comprising the amino acid sequence of SEQ ID NO:48.7.5.1 Polyclonal Antibodies[000121] In some embodiments, the ENPP3 x CD3 antibodies of the present disclosure may comprise polyclonal antibodies. Methods of preparing polyclonal antibodies are known to the skilled artisan. Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and / or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent may include a ENPP3 polypeptide, a CD3 polypeptide, or a fusion protein thereof. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized or to immunize the mammal with the protein and one or more adjuvants. Examples of such immunogenic proteins include, but are not limited to, keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants which may be employed include Ribi, CpG, Poly 1C, Freund’s complete adjuvant, and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation. The mammal can then be bled, and the serum assayed for ENPP3antibody titer. If desired, the mammal can be boosted until the antibody titer increases or plateaus. Additionally or alternatively, lymphocytes may be obtained from the immunized animal for fusion and preparation of monoclonal antibodies from hybridoma as described below.7.5.2 Monoclonal Antibodies[000122] The antibodies of the present disclosure may alternatively be monoclonal antibodies. Monoclonal antibodies may be made using the hybridoma method first described by Kohler et aL, 1975, Nature 256:495-97, or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).[000123] In the hybridoma method, a mouse or other appropriate host animal, such as a hamster, is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. In some embodiments, the immunizing antigen is a human protein or a fragment thereof. In some embodiments, the immunizing antigen is a mouse protein or a fragment thereof. In some embodiments, the immunizing antigen is a cyno protein or a fragment thereof. After immunization, lymphocytes are isolated and then fused with a myeloma cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice 59-103 (1986)).[000124] The hybridoma cells thus prepared are seeded and grown in a suitable culture medium which, in certain embodiments, contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells (also referred to as fusion partner). For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the selective culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which prevent the growth of HGPRT-deficient cells.[000125] Exemplary fusion partner myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a selective medium that selects against the unfused parental cells. Exemplary myeloma cell lines are murine myeloma lines, such as SP-2 and derivatives, for example, X63-Ag8-653 cells available from the American Type Culture Collection (Manassas, VA), and those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center (San Diego, CA). Human myeloma and mouse-human heteromyeloma cell lines also have beendescribed for the production of human monoclonal antibodies (Kozbor, 1984, Immunol. 133:3001-05; and Brodeur et aL, Monoclonal Antibody Production Techniques and Applications 51-63 (1987)).[000126] Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. The binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as RIA or ELISA. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis described in Munson et al., 1980, Anal. Biochem. 107:220-39.[000127] Once hybridoma cells that produce antibodies of the desired specificity, affinity, and / or activity are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, supra . Suitable culture media for this purpose include, for example, DMEM or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal, for example, by i.p. injection of the cells into mice.[000128] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures such as, for example, affinity chromatography (e.g., using protein A or protein G-Sepharose) or ionexchange chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis, etc. [000129] DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells can serve as a source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells, such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et aL, 1993, Curr. Opinion in Immunol. 5:256-62 and Pliickthun, 1992, Immunol. Revs. 130:151-88.[000130] Table 2In a further embodiment, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in, for example, Antibody Phage Display: Methods and Protocols (O’Brien and Aitken eds., 2002). In principle,synthetic antibody clones are selected by screening phage libraries containing phages that display various fragments of antibody variable region (Fv) fused to phage coat protein. Such phage libraries are screened against the desired antigen. Clones expressing Fv fragments capable of binding to the desired antigen are adsorbed to the antigen and thus separated from the nonbinding clones in the library. The binding clones are then eluted from the antigen and can be further enriched by additional cycles of antigen adsorption / elution.[000131] Variable domains can be displayed functionally on phage, either as single-chain Fv (scFv) fragments, in which VH and VL are covalently linked through a short, flexible peptide, or as Fab fragments, in which they are each fused to a constant domain and interact non-covalently, as described, for example, in Winter et al. , 1994, Ann. Rev. Immunol. 12:433-55.[000132] Repertoires of VH and VL genes can be separately cloned by PCR and recombined randomly in phage libraries, which can then be searched for antigen-binding clones as described in Winter et al. , supra. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned to provide a single source of human antibodies to a wide range of non-self and also self antigens without any immunization as described by Griffiths et al., 1993, EMBO J 12:725-34. Finanaivenaive libraries can also be made synthetically by cloning the unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro as described, for example, by Hoogenboom and Winter, 1992, J. Mol. Biol. 227:381-88.[000133] Screening of the libraries can be accomplished by various techniques known in the art. For example, ENPP3 (e.g., a ENPP3 polypeptide, fragment, or epitope) can be used to coat the wells of adsorption plates, expressed on host cells affixed to adsorption plates or used in cell sorting, conjugated to biotin for capture with streptavidin-coated beads, or used in any other method for panning display libraries. The selection of antibodies with slow dissociation kinetics (e.g., good binding affinities) can be promoted by use of long washes and monovalent phage display as described in Bass et al., 1990, Proteins 8:309-14 and WO 92 / 09690, and by use of a low coating density of antigen as described in Marks etal., 1992, Biotechnol. 10:779-83.[000134] Anti-ENPP3 and anti-CD3 antibodies can be obtained by designing a suitable antigen screening procedure to select for the phage clone of interest followed by construction of a fulllength anti-ENPP3 or anti-CD3 antibody clone using VH and / or VL sequences (e.g., the Fv sequences), or various CDR sequences from VH and VL sequences, from the phage clone of interest and suitable constant region (e.g., Fc) sequences described in Kabat et al., supra. [000135] In another embodiment, anti-ENPP3 or anti-CD3 antibody is generated by using methods as described in Bowers etal., 2011, Proc Natl Acad Sci USA. 108:20455-60, e.g., the SHM-XHL™ platform (AnaptysBio, San Diego, CA). Briefly, in this approach, a fully human library of IgGs is constructed in a mammalian cell line (e.g., HEK293) as a starting library. Mammalian cells displaying immunoglobulin that binds to a target peptide or epitope are selected (e.g., by FACS sorting), then activation-induced cytidine deaminase (AID)-triggered somatic hypermutation is reproduced in vitro to expand diversity of the initially selected pool of antibodies. After several rounds of affinity maturation by coupling mammalian cell surface display with in vitro somatic hypermutation, high affinity, high specificity anti-ENPP3 antibodies are generated. Further methods that can be used to generate antibody libraries and / or antibody affinity maturation are disclosed, e.g., in U.S. Patent Nos. 8,685,897 and 8,603,930, and U.S. Publ. Nos. 2014 / 0170705, 2014 / 0094392, 2012 / 0028301, 2011 / 0183855, and 2009 / 0075378, each of which are incorporated herein by reference.7.5.3 Humanized Antibodies[000136] In some embodiments, bispecific antibodies or antigen binding fragments provided herein can be humanized bispecific antibodies or antigen binding fragments that bind ENPP3 and CD3, including human and / or cynomolgus ENPP3 and CD3. For example, humanized antibodies of the present disclosure may comprise one or more CDRs as shown in Table 1 and Table 2. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody can have one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization may be performed, for example, following the method of Jones et al., 1986, Nature 321:522-25; Riechmann et al. , 1988, Nature 332:323-27; and Verhoeyen et al., 1988, Science 239:1534-36), by substituting hypervariable region sequences for the corresponding sequences of a human antibody.[000137] In some cases, the humanized antibodies are constructed by CDR grafting, in which the amino acid sequences of the six CDRs of the parent non-human antibody (e.g., rodent) aregrafted onto a human antibody framework. For example, Padlan et al. determined that only about one third of the residues in the CDRs actually contact the antigen, and termed these the “specificity determining residues,” or SDRs (Padlan etal., 1995, FASEB J. 9: 133-39). In the technique of SDR grafting, only the SDR residues are grafted onto the human antibody framework (see, e.g., Kashmiri et al., 2005, Methods 36:25-34).[000138] The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies can be important to reduce antigenicity. For example, according to the so-called “best- fit” method, the sequence of the variable domain of a non-human (e.g., rodent) antibody is screened against the entire library of known human variable-domain sequences. The human sequence that is closest to that of the rodent may be selected as the human framework for the humanized antibody (Sims et al., 1993, J. Immunol. 151 :2296-308; and Chothia et al., 1987, J. Mol. Biol. 196:901-17). Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et al. , 1992, Proc. Natl. Acad. Sci. USA 89:4285-89; and Presta et al., 1993, J. Immunol. 151:2623-32). In some cases, the framework is derived from the consensus sequences of the most abundant human subclasses, VL6 subgroup I (VL6I) and VH subgroup III (VHIII). In another method, human germline genes are used as the source of the framework regions.[000139] In an alternative paradigm based on comparison of CDRs, called superhumanization, FR homology is irrelevant. The method consists of comparison of the non-human sequence with the functional human germline gene repertoire. Those genes encoding the same or closely related canonical structures to the murine sequences are then selected. Next, within the genes sharing the canonical structures with the non-human antibody, those with highest homology within the CDRs are chosen as FR donors. Finally, the non-human CDRs are grafted onto these FRs (see, e.g., Tan etal., 2002, J. Immunol. 169:1119-25).[000140] It is further generally desirable that antibodies be humanized with retention of their affinity for the antigen and other favorable biological properties. To achieve this goal, according to one method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available whichillustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. These include, for example, WAM (Whitelegg and Rees, 2000, Protein Eng. 13:819-24), Modeller (Sail and Blundell, 1993, J. Mol. Biol. 234:779-815), and Swiss PDB Viewer (Guex and Peitsch, 1997, Electrophoresis 18:2714-23). Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, e.g., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.[000141] Another method for antibody humanization is based on a metric of antibody humanness termed Human String Content (HSC). This method compares the mouse sequence with the repertoire of human germline genes, and the differences are scored as HSC. The target sequence is then humanized by maximizing its HSC rather than using a global identity measure to generate multiple diverse humanized variants (Lazar et al. , 2007, Mol. Immunol. 44:1986-98). [000142] In addition to the methods described above, empirical methods may be used to generate and select humanized antibodies. These methods include those that are based upon the generation of large libraries of humanized variants and selection of the best clones using enrichment technologies or high throughput screening techniques. Antibody variants may be isolated from phage, ribosome, and yeast display libraries as well as by bacterial colony screening (see, e.g., Hoogenboom, 2005, Nat. Biotechnol. 23:1105-16; Dufner et al., 2006, Trends Biotechnol. 24:523-29; Feldhaus et al., 2003, Nat. Biotechnol. 21: 163-70; and Schlapschy et al., 2004, Protein Eng. Des. Sei. 17:847-60).[000143] In the FR library approach, a collection of residue variants are introduced at specific positions in the FR followed by screening of the library to select the FR that best supports the grafted CDR. The residues to be substituted may include some or all of the “Vernier” residues identified as potentially contributing to CDR structure (see, e.g., Foote and Winter, 1992, J. Mol. Biol. 224:487-99), or from the more limited set of target residues identified by Baca et al. (1997, J. Biol. Chem. 272: 10678-84).[000144] In FR shuffling, whole FRs are combined with the non-human CDRs instead of creating combinatorial libraries of selected residue variants (see, e.g., Dall’Acqua et al., 2005,Methods 36:43-60). The libraries may be screened for binding in a two-step process, first humanizing VL, followed by VH. Alternatively, a one-step FR shuffling process may be used. Such a process has been shown to be more efficient than the two-step screening, as the resulting antibodies exhibited improved biochemical and physicochemical properties including enhanced expression, increased affinity, and thermal stability (see, e.g., Damschroder et al., 2007, Mol. Immunol. 44:3049-60).[000145] The “humaneering” method is based on experimental identification of essential minimum specificity determinants (MSDs) and is based on sequential replacement of non-human fragments into libraries of human FRs and assessment of binding. It begins with regions of the CDR3 of non-human VH and VL chains and progressively replaces other regions of the non-human antibody into the human FRs, including the CDR1 and CDR2 of both VH and VL. This methodology typically results in epitope retention and identification of antibodies from multiple subclasses with distinct human V-segment CDRs. Humaneering allows for isolation of antibodies that are 91-96% homologous to human germline gene antibodies (see, e.g., Alfenito, Cambridge Healthtech Institute’s Third Annual PEGS, The Protein Engineering Summit, 2007). [000146] The “human engineering” method involves altering a non-human antibody or antibody fragment, such as a mouse or chimeric antibody or antibody fragment, by making specific changes to the amino acid sequence of the antibody so as to produce a modified antibody with reduced immunogenicity in a human that nonetheless retains the desirable binding properties of the original non-human antibodies. Generally, the technique involves classifying amino acid residues of a non-human (e.g., mouse) antibody as “low risk,” “moderate risk,” or “high risk” residues. The classification is performed using a global risk / reward calculation that evaluates the predicted benefits of making particular substitution (e.g., for immunogenicity in humans) against the risk that the substitution will affect the resulting antibody’s folding. The particular human amino acid residue to be substituted at a given position (e.g., low or moderate risk) of a non-human (e.g., mouse) antibody sequence can be selected by aligning an amino acid sequence from the non-human antibody’s variable regions with the corresponding region of a specific or consensus human antibody sequence. The amino acid residues at low or moderate risk positions in the non-human sequence can be substituted for the corresponding residues in the human antibody sequence according to the alignment. Techniques for making human engineered proteins are described in greater detail in Studnicka et al., 1994, Protein Engineering 7:805-14;U.S. Pat. Nos. 5,766,886; 5,770,196; 5,821,123; and 5,869,619; and PCT Publication WO 93 / 11794.7.5.4 Human Antibodies[000147] Human ENPP3 -binding and CD3 -binding domains can be constructed by combining Fv clone variable domain sequence(s) selected from human-derived phage display libraries with known human constant domain sequences(s). Alternatively, human monoclonal ENPP3-binding and CD3-binding domains of the present disclosure can be made by the hybridoma method. Human myeloma and mouse- human heteromyeloma cell lines for the production of human monoclonal antibodies have been described, for example, by Kozbor, 1984, J. Immunol.133:3001-05; Brodeur etal., Monoclonal Antibody Production Techniques and Applications 51- 63 (1987); and Boerner et al., 1991, J. Immunol. 147:86-95.[000148] It is also possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. Transgenic mice that express human antibody repertoires have been used to generate high-affinity human sequence monoclonal antibodies against a wide variety of potential drug targets (see, e.g., Jakobovits, A., 1995, Curr. Opin. Biotechnol. 6(5):561-66; Bruggemann and Taussing, 1997, Curr. Opin. Biotechnol. 8(4):455-58; U.S. Pat. Nos. 6,075,181 and 6,150,584; and Lonberg et al., 2005, Nature Biotechnol. 23: 1117-25).[000149] Alternatively, the human antibody may be prepared via immortalization of human B lymphocytes producing an antibody directed against a target antigen (e.g., such B lymphocytes may be recovered from an individual or may have been immunized in vitro) (see, e.g, Cole et al., Monoclonal Antibodies and Cancer Therapy (1985); Boerner et al., 1991, J. Immunol. 147(l):86-95; and U.S. Pat. No. 5,750,373).[000150] Gene shuffling can also be used to derive human antibodies from non-human, for example, rodent, antibodies, where the human antibody has similar affinities and specificities to the starting non-human antibody. According to this method, which is also called “epitope imprinting” or “guided selection,” either the heavy or light chain variable region of a non-human antibody fragment obtained by phage display techniques as described herein is replaced with a repertoire of human V domain genes, creating a population of non-human chain / human chain scFv or Fab chimeras. Selection with antigen results in isolation of a non-human chain / human chain chimeric scFv or Fab wherein the human chain restores the antigen binding site destroyedupon removal of the corresponding non-human chain in the primary phage display clone (e.g., the epitope guides (imprints) the choice of the human chain partner). When the process is repeated in order to replace the remaining non-human chain, a human antibody is obtained (see, e.g., PCT WO 93 / 06213; and Osbourn etal., 2005, Methods 36:61-68). Unlike traditional humanization of non-human antibodies by CDR grafting, this technique provides completely human antibodies, which have no FR or CDR residues of non-human origin. Examples of guided selection to humanize mouse antibodies towards cell surface antigens include the folate-binding protein present on ovarian cancer cells (see, e.g., Figini et al., 1998, Cancer Res. 58:991-96) and CD147, which is highly expressed on hepatocellular carcinoma (see, e.g, Bao et al., 2005, Cancer Biol. Ther. 4: 1374-80).[000151] A potential disadvantage of the guided selection approach is that shuffling of one antibody chain while keeping the other constant could result in epitope drift. In order to maintain the epitope recognized by the non-human antibody, CDR retention can be applied (see, e.g., Klimka et al., 2000, Br. J. Cancer. 83:252-60; and Beiboer et al., 2000, J. Mol. Biol. 296:833-49). In this method, the non-human HCDR3 is commonly retained, as this CDR may be at the center of the antigen-binding site and may be the most important region of the antibody for antigen recognition. In some instances, however, HCDR3 and LCDR3, as well as HCDR2, LCDR2, and LCDR1 of the non-human antibody may be retained.7.5.5 Antibody Fragments[000152] The present disclosure provides antibodies and antibody fragments that bind to ENPP3. In certain circumstances there are advantages of using antibody fragments, rather than whole antibodies. The smaller size of the fragments allows for rapid clearance, and may lead to improved access to cells, tissues, or organs. For a review of certain antibody fragments, see Hudson etal., 2003, Nature Med. 9: 129-34.[000153] Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., 1992, J. Biochem. Biophys. Methods 24:107-17; and Brennan et al., 1985, Science 229:81-83). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv, scFv and spFv antibody fragments can all be expressed in and secreted from E. colt or yeast cells, thus allowing the facile production of large amounts of these fragments. Antibody fragments can be isolated from the antibody phage libraries discussed above.Alternatively, Fab’-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab’)2 fragments (Carter et al., 1992, Bio / Technology 10: 163-67). According to another approach, F(ab’)2 fragments can be isolated directly from recombinant host cell culture. Fab and F(ab’)2 fragment with increased in vivo half-life comprising salvage receptor binding epitope residues are described in, for example, U.S. Pat. No. 5,869,046. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In certain embodiments, an antibody is a single chain Fv fragment (scFv) (see, e.g., WO 93 / 16185; U.S. Pat. Nos. 5,571,894 and 5,587,458). In certain embodiments, an antibody is a stapled single chain Fv fragment (spFv) (see, e.g., WO2021030657). Fv and scFv have intact combining sites that are devoid of constant regions; thus, they may be suitable for reduced nonspecific binding during in vivo use. scFv fusion proteins may be constructed to yield fusion of an effector protein at either the amino or the carboxy terminus of an scFv (See, e.g., Borrebaeck ed., supra). The antibody fragment may also be a “linear antibody,” for example, as described in the references cited above. Such linear antibodies may be multi- specific, such as bispecific.[000154] Antibodies provided herein include, but are not limited to, immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, for example, molecules that contain an antigen binding site that bind to an ENPP3 or CD3 epitope. The immunoglobulin molecules provided herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2) of immunoglobulin molecule.[000155] Variants and derivatives of antibodies include antibody functional fragments that retain the ability to bind to a ENPP3 epitope. In some embodiments, a CD3 -binding domain is an antigen binding fragment of an antibody. In some embodiments, the CD3 -binding domain is a portion of an intact antibody. In some embodiments, the CD3 -binding domain is stapled single chain antibody molecules(e.g., spFv).[000156] In some embodiments, the CD3s binding spFv comprises a VH comprising the sequence of SEQ ID NO:47 and a VL comprising the sequence of SEQ ID NO:48. In some embodiments, the spFv comprises an amino acid sequence of SEQ ID NO:49.Table 3: CD3s-Binding spFv7.6 ENPP3 x CD3 Bispecific Antibodies[000157] In some embodiments, the ENPP3 -binding domain comprises an Fab. In some embodiments, the CD3 -binding domain comprise an spFv. In some embodiments, the CD3- binding domain comprises an spFv-Fc. In some embodiments, the ENPP3 -binding domain comprises an Fab, and the CD3-binding domain comprises an spFv. In some embodiments, the ENPP3-binding domain comprises an Fab and the CD3-binding domain comprises an spFv-Fc. [000158] In some embodiments, the ENPP3 -binding domain is fused or conjugated to a first immunoglobulin (Ig) constant region or a fragment of the first Ig constant region and / or the CD3 -binding domain is fused or conjugated to a second immunoglobulin (Ig) constant region or a fragment of the second Ig constant region.[000159] In some embodiments, the fragment of the first Ig constant region and / or the fragment of the second Ig constant region comprises a Fc region. In some embodiments, the fragment of the first Ig constant region and / or the fragment of the second Ig constant region comprises a CH2 domain. In some embodiments, the fragment of the first Ig constant region and / or the fragment of the second Ig constant region comprises a CH3 domain. In some embodiments, the fragment of the first Ig constant region and / or the fragment of the second Ig constant region comprises the CH2 domain and the CH3 domain.[000160] In some embodiments, the fragment of the first Ig constant region and / or the fragment of the second Ig constant region comprises at least portion of a hinge, the CH2 domain and theCH3 domain. In some embodiments, the fragment of the Ig constant region comprises the hinge, the CH2 domain and the CH3 domain.[000161] In some embodiments, the first Ig constant region or the fragment of the first Ig constant region and the second Ig constant region or the fragment of the second Ig constant region is an IgGl isotype.[000162] The first Ig constant region or the fragment of the first Ig constant region and the second Ig constant region or the fragment of the second Ig constant region can further be engineered as described herein. In some embodiments, the first Ig constant region or the fragment of the first Ig constant region and the second Ig constant region or the fragment of the second Ig constant region comprises at least one mutation that results in reduced binding of the bispecific antibody to a FcyR.[000163] In some embodiments, the at least one mutation that results in reduced binding of the bispecific antibody to the FcyR is L234A / L235A / D265S, wherein residue numbering is according to the EU index.[000164] In some embodiments, the bispecific protein comprises at least one mutation in a CH3 domain of the first Ig constant region or in a CH3 domain of the fragment of the first Ig constant region and / or at least one mutation in a CH3 domain of the second Ig constant region or in a CH3 domain of the fragment of the second Ig constant region. In some embodiments, the at least one mutation in a CH3 domain of the first Ig constant region or in a CH3 domain of the fragment of the first Ig constant region and / or at least one mutation in a CH3 domain of the second Ig constant region or in a CH3 domain of the fragment of the second Ig constant region is selected from the group consisting of T366W and T366S / L368A / Y407V, wherein residue numbering is according to the EU index.[000165] In some embodiments, the bispecific binding proteins provided herein are antibodies having a full-length antibody structure. “Full length antibody” refers to an antibody having two full length antibody heavy chains and two full length antibody light chains. A full-length antibody heavy chain (HC) consists of well-known heavy chain variable and constant domains VH, CHI, hinge, CH2, and CH3. A full-length antibody light chain (LC) consists of well-known light chain variable and constant domains VL and CL. The full-length antibody can be lacking the C-terminal lysine (K) in either one or both heavy chains. “Fab-arm” or “half molecule” refers to one heavy chain-light chain pair that specifically binds an antigen.[000166] Full length bispecific antibodies can be generated for example using Fab arm exchange (or half molecule exchange) between two monospecific bivalent antibodies by introducing substitutions at the heavy chain CH3 interface in each half molecule to favor heterodimer formation of two antibody half molecules having distinct specificity either in vitro in cell-free environment or using co-expression. The Fab arm exchange reaction is the result of a disulfide-bond isomerization reaction and dissociation-association of CH3 domains. The heavy chain disulfide bonds in the hinge regions of the parental monospecific antibodies are reduced. The resulting free cysteines of one of the parental monospecific antibodies form an inter heavychain disulfide bond with cysteine residues of a second parental monospecific antibody molecule and simultaneously CH3 domains of the parental antibodies release and reform by dissociationassociation. The CH3 domains of the Fab arms can be engineered to favor heterodimerization over homodimerization. The resulting product is a bispecific antibody having two Fab arms or half molecules which each bind a distinct epitope, i.e. an epitope on ENPP3 and an epitope on CD3.[000167] ‘ ‘Homodimerization” refers to an interaction of two heavy chains having identical CH3 amino acid sequences. “Homodimer” refers to an antibody having two heavy chains with identical CH3 amino acid sequences. “Heterodimerization” refers to an interaction of two heavy chains having non-identical CH3 amino acid sequences. “Heterodimer” refers to an antibody having two heavy chains with non-identical CH3 amino acid sequences.[000168] The “knob-in-hole” strategy (see, e.g., International Publication No. WO 2006 / 028936) can be used to generate full length bispecific antibodies. Briefly, selected amino acids forming the interface of the CH3 domains in human IgG can be mutated at positions affecting CH3 domain interactions to promote heterodimer formation. An amino acid with a small side chain (hole) is introduced into a heavy chain of an antibody specifically binding a first antigen and an amino acid with a large side chain (knob) is introduced into a heavy chain of an antibody specifically binding a second antigen. After co-expression of the two antibodies, a heterodimer is formed as a result of the preferential interaction of the heavy chain with a “hole” with the heavy chain with a “knob.” Exemplary CH3 substitution pairs forming a knob and a hole are (expressed as modified position in the first CH3 domain of the first heavy chain / modified position in the second CH3 domain of the second heavy chain): T366W / T366S L368 A_Y407V.[000169] The CrossMAb technology can be used to generate full length bispecific antibodies provided herein. CrossMAbs, in addition to utilizing the “knob-in-hole” strategy to promoter Fab arm exchange, have in one of the half arms the CHI and the CL domains exchanged to ensure correct light chain pairing of the resulting bispecific antibody (see e.g. U.S. Patent No. 8,242,247).[000170] Other cross-over strategies can be used to generate full length bispecific antibodies provided herein by exchanging variable or constant, or both domains between the heavy chain and the light chain or within the heavy chain in the bispecific antibodies, either in one or both arms. These exchanges include for example VH-CH1 with VL-CL, VH with VL, CH3 with CL and CH3 with CHI as described in International Publication Nos. WO 2009 / 080254, WO 2009 / 080251, WO 2009 / 018386 and WO 2009 / 080252.[000171] LUZ-Y technology can be utilized to generate bispecific antibodies provided herein. In this technology, a leucine zipper is added into the C terminus of the CH3 domains to drive the heterodimer assembly from parental mAbs that is removed post-purification as described in Wramk et al., (2012) J Biol Chem 287(52): 42221-9.[000172] SEEDbody technology can be utilized to generate bispecific antibodies provided herein. SEEDbodies have, in their constant domains, select IgG residues substituted with IgA residues to promote heterodimerization as described in U.S. Patent No. US20070287170.[000173] In addition to methods described above, binding agents provided herein can be generated in vitro in a cell-free environment by introducing asymmetrical mutations in the CH3 regions of two monospecific homodimeric antibodies and forming the bispecific heterodimeric antibody from two parent monospecific homodimeric antibodies in reducing conditions to allow disulfide bond isomerization according to methods described in PCT Pat. Publ. No. WO 2011 / 131746.[000174] In some embodiments described herein, the bispecific ENPP3 x CD3 bispecific antibody comprises at least one substitution in an antibody CH3 constant domain. Substitutions are typically made at the DNA level to a molecule such as the constant domain of the antibody using standard methods.[000175] The antibodies provided herein can be engineered into various well-known antibody forms.[000176] In some embodiments, the ENPP3 x CD3 binding protein provided herein has the structure as illustrated in Figure 1. In specific embodiments, the ENPP3 x CD3 binding protein provided herein comprises a spFv that binds CD3s and a Fab that binds ENPP3, and the binding protein further comprises a Fc region. In specific embodiments, the ENPP3 x CD3 binding protein provided herein comprises: (i) a first polypeptide comprising a spFv that binds CD3s that is linked to one arm of an antibody Fc region, (ii) a second polypeptide comprising the VH domain that binds ENPP3 that is linked to the other arm of an antibody Fc region, and (iii) a third polypeptide comprising the VL domain that binds ENPP3, wherein the VH domain and the VL domain forms a Fab that binds ENPP3, and the first polypeptide and the second polypeptide forms a Fc region.[000177] In some embodiments, the ENPP3 x CD3 binding protein provided herein comprises one or more constant region mutation.[000178] In some embodiments, the Fc regions comprise one or more modifications that facilitate heterodimerization of the first and second polypeptides. In some embodiments, one arm of the Fc region comprises T366W substitution, and the other arm of the Fc region comprises the T366S, L368A, Y407V substitutions. In some embodiments, the Fc region comprises one or more mutations that reduces binding affinity of the Fc domain to Fc receptors. In particular embodiments, one arm of the Fc region comprises L234A, L235A, and D265S (AAS) mutations. In particular embodiments, the second arm of the Fc region comprises L234A, L235A, and D265S (AAS) mutations. In particular embodiments, each the first and second arms of the Fc region comprises L234A, L235A, and D265S (AAS) mutations.[000179] In some embodiments, the ENPP3 x CD3 binding protein provided herein comprises: (i) a first polypeptide comprising a spFv that binds CD3s that is linked to one arm of an antibody Fc region, (ii) a second polypeptide comprising the VH domain that binds ENPP3 that is linked to the other arm of an antibody Fc region, and (iii) a third polypeptide comprising the VL domain that binds ENPP3, wherein the VH domain and the VL domain forms a Fab that binds ENPP3, and the first polypeptide and the second polypeptide forms a Fc region; further wherein the Fc region of the first polypeptide comprises the T366S, L368A, Y407V substitutions, and the Fc region of the second polypeptide comprises the T366W substitution. In specific embodiments described in this paragraph, the Fc region of the first polypeptide further comprises the L234A, L235A, and D265S (AAS) mutations. In specific embodiments described in this paragraph, theFc region of the second polypeptide further comprises the L234A, L235A, and D265S (AAS) mutations. In specific embodiments described in this paragraph, both Fc regions of the first and second polypeptides further comprise the L234A, L235A, and D265S (AAS) mutations. [000180] In alternative embodiments, the ENPP3 x CD3 binding protein provided herein comprises: (i) a first polypeptide comprising a spFv that binds CD3s that is linked to one arm of an antibody Fc region, (ii) a second polypeptide comprising the VH domain that binds ENPP3 that is linked to the other arm of an antibody Fc region, and (iii) a third polypeptide comprising the VL domain that binds ENPP3, wherein the VH domain and the VL domain forms a Fab that binds ENPP3, and the first polypeptide and the second polypeptide forms a Fc region; further wherein the Fc region of the first polypeptide comprises the T366W substitution and the Fc region of the second polypeptide comprises the T366S, L368A, Y407V substitutions. In specific embodiments described in this paragraph, the Fc region of the first polypeptide further comprises the L234A, L235A, and D265S (AAS) mutations. In specific embodiments described in this paragraph, the Fc region of the second polypeptide further comprises the L234A, L235A, and D265S (AAS) mutations. In specific embodiments described in this paragraph, both Fc regions of the first and second polypeptides further comprise the L234A, L235A, and D265S (AAS) mutations.[000181] In specific embodiments, the ENPP3 x CD3 binding protein provided herein comprises: (i) a first polypeptide comprising a spFv that binds CD3s that is linked to one arm of an antibody Fc region, (ii) a second polypeptide comprising the VH domain that binds ENPP3 that is linked to the other arm of an antibody Fc region, and (iii) a third polypeptide comprising the VL domain that binds ENPP3, wherein the VH domain and the VL domain forms a Fab that binds ENPP3, and the first polypeptide and the second polypeptide forms a Fc region. In some embodiments, the first polypeptide comprises an amino acid sequence of SEQ ID NO: 50. In some embodiments, the second polypeptide comprising an amino acid sequence of SEQ ID NO:24. In some embodiments, the third polypeptide comprising an amino acid sequence of SEQ ID NO:25.[000182] In a specific embodiment, the ENPP3 x CD3 binding protein provided herein comprises: (i) a first polypeptide comprising a spFv that binds CD3s that is linked to one arm of an antibody Fc region, (ii) a second polypeptide comprising the VH domain that binds ENPP3 that is linked to the other arm of an antibody Fc region, and (iii) a third polypeptide comprisingthe VL domain that binds ENPP3, wherein the VH domain and the VL domain forms a Fab that binds ENPP3, and the first polypeptide and the second polypeptide forms a Fc region, wherein the first polypeptide comprises an amino acid sequence of SEQ ID NO: 50, the second polypeptide comprises an amino acid sequence of SEQ ID NO:24, and the third polypeptide comprises an amino acid sequence of SEQ ID NO:25 (see Table 4A). In some embodiments, the ENPP3 x CD3 binding protein provided herein is encoded by nucleic acid sequences shown in Table 4B.Table 4A: Sequences of NPP3B815Table 4B: DNA Sequences Encoding NPP3B815[000183] In some embodiments, the ENPP3 x CD3 binding protein provided is in the spFv format as described in International Patent Application Publication No. WO2021030657, the content of which is herein incorporated by reference in its entirety.5.7 Polynucleotides, Vectors, and Host Cells[000184] Also disclosed are isolated polynucleotides that encode the ENPP3 x CD3 binding protein provided herein. The isolated polynucleotides capable of encoding the ENPP3 x CD3 binding protein provided herein may be included on the same, or different, vectors to produce antibodies or antigen-binding fragments of the invention.[000185] In some embodiments, the polynucleotides of the invention include a polynucleotide encoding a leader sequence. Any leader sequence known in the art may be employed. The polynucleotide encoding the leader sequence may include a restriction endonuclease cleavage site or a translation initiation site.[000186] Also provided are vectors comprising the polynucleotides of the invention. The vectors can be expression vectors. The expression vector may contain one or more additional sequences such as but not limited to regulatory sequences (e.g., promoter, enhancer), a selection marker, and a polyadenylation signal.[000187] Recombinant expression vectors within the scope of the description include synthetic, or cDNA-derived nucleic acid fragments that encode at least one recombinant protein which may be operably linked to suitable regulatory elements.[000188] The transcriptional and translational control sequences in expression vectors to be used in transforming vertebrate cells may be provided by viral sources. Exemplary vectors may be constructed as described by Okayama and Berg, 3 Mol. Cell. Biol. 280 (1983).[000189] In some embodiments, the antibody- or antigen-binding fragment-coding sequence is placed under control of a powerful constitutive promoter. In addition, many viral promoters function constitutively in eukaryotic cells and are suitable for use with the described embodiments. In one embodiment, the coding sequence of the bispecific antibody of the invention is placed under control of an inducible promoter.[000190] Vectors described herein may contain one or more Internal Ribosome Entry Site(s) (IRES). Inclusion of an IRES sequence into fusion vectors may be beneficial for enhancing expression of some proteins. In some embodiments the vector system will include one or more polyadenylation sites, which may be upstream or downstream of any of the aforementioned nucleic acid sequences.[000191] The vectors may comprise selection markers. A nucleic acid sequence encoding a selection marker or the cloning site may be upstream or downstream of a nucleic acid sequence encoding a polypeptide of interest or cloning site.[000192] The vectors described herein may be used to transform various cells with the genes encoding the described antibodies or antigen-binding fragments. For example, the vectors may be used to generate bispecific ENPP3 x CD3 antibodies or antigen-binding fragment-producing cells. Thus, the invention also provides a host cell comprising the vectors of the invention. [000193] Numerous techniques are known in the art for the introduction of foreign genes into cells and may be used to construct the recombinant cells for purposes of carrying out the described methods, in accordance with the various embodiments described and exemplified herein. The technique used should provide for the stable transfer of the heterologous gene sequence to the host cell, such that the heterologous gene sequence is heritable and expressible by the cell progeny, and so that the necessary development and physiological functions of the recipient cells are not disrupted. Techniques which may be used include but are not limited to chromosome transfer (e.g., cell fusion, chromosome mediated gene transfer, micro cell mediated gene transfer), physical methods (e.g., transfection, spheroplast fusion, microinjection, electroporation, liposome carrier), viral vector transfer (e.g., recombinant DNA viruses, recombinant RNA viruses) and the like (described in Cline, 29 Pharmac. Ther. 69-92 (1985)). Calcium phosphate precipitation and polyethylene glycol (PEG)-induced fusion of bacterial protoplasts with mammalian cells may also be used to transform cells.[000194] Cells suitable for use in the expression of the antibodies or antigen-binding fragments described herein are preferably eukaryotic cells, more preferably cells of plant, rodent, or human origin. In addition, expression of antibodies may be accomplished using hybridoma cells. Methods for producing hybridomas are well established in the art.[000195] Cells transformed with expression vectors of the invention may be selected or screened for recombinant expression of the antibodies or antigen-binding fragments of theinvention. Recombinant-positive cells are expanded and screened for subclones exhibiting a desired phenotype, such as high-level expression, enhanced growth properties, or the ability to yield proteins with desired biochemical characteristics, for example, due to protein modification or altered post-translational modifications. These phenotypes may be due to inherent properties of a given subclone or to mutation. Mutations may be effected through the use of chemicals, UV-wavelength light, radiation, viruses, insertional mutagens, inhibition of DNA mismatch repair, or a combination of such methods.[000196] Table 1 shows polynucleotide sequences encoding the VH / VL amino acid sequences for an exemplary ENPP3 binding arm. Table 2 shows polynucleotide sequences encoding the VH / VL amino acid sequences for an exemplary CD3s binding arm. Table 4B shows DNA sequences encoding the NPP3B815 antibody.[000197] In some embodiments, the ENPP3 binding arm comprises a VH encoded by a polynucleotide sequence as set forth in SEQ ID NO: 52. In some embodiments, the ENPP3 binding arm comprises a VL encoded by a polynucleotide sequence as set forth in SEQ ID NO 53.[000198] In some embodiments, the CD3s binding arm comprises a VH encoded by a polynucleotide sequence as set forth in SEQ ID NO: 56. In some embodiments, the CD3s binding arm comprises a VL encoded by a polynucleotide sequence as set forth in SEQ ID NO: 57. [000199] In some embodiments, the bispecific ENPP3 CD3s binding molecule comprises an ENPP3 binding arm comprising a VH encoded by a polynucleotide sequence as set forth in SEQ ID NO: 52 and a VL encoded by a polynucleotide sequence as set forth in SEQ ID NO: 53 and a CD3s binding arm comprising a VH encoded by a polynucleotide sequence as set forth in SEQ ID NO: 56 and a VL encoded by a polynucleotide sequence as set forth in SEQ ID NO: 57.[000200] In some embodiments, the binding agent is encoded by a first polynucleotide encoding a spFv that binds CD3s, a CH2 domain, and a CH3 domain; a second polynucleotide encoding a VH domain that binds ENPP3, a CH2, domain and a CH3 domain; and a third polynucleotide encoding a VL domain that binds ENPP3. In some embodiments, the first polynucleotide encoding a spFv that binds CD3s comprises SEQ ID NO: 58. In some embodiments, the first polynucleotide encoding a spFv that binds CD3s, a CH2 domain, and a CH3 domain comprises SEQ ID NO: 59. In some embodiments, the second polynucleotide encoding a VH domain that binds ENPP3, a CH2 domain, and a CH3 domain comprises SEQ IDNO: 54. In some embodiments, the third polynucleotide encoding a VL domain that binds ENPP3 comprises SEQ ID NO: 55.5.8 Pharmaceutical Compositions[000201] In another general aspect, provided is a pharmaceutical composition comprising the ENPP3 x CD3 bispecific antibody provided herein and a pharmaceutically acceptable excipient. In another general aspect, provided is a pharmaceutical composition comprising a nucleic acid encoding the ENPP3 x CD3 bispecific antibody provided herein or a fragment or a portion thereof and a pharmaceutically acceptable excipient. In another general aspect, provided is a pharmaceutical composition comprising the ENPP3 x CD3 bispecific antibody provided herein and a pharmaceutically acceptable excipient.[000202] In another general aspect, provided herein is a method of producing a pharmaceutical composition comprising a binding agent or an antigen binding region thereof provided herein, comprising combining a binding agent or an antigen binding region thereof with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.5.9 Methods of Use[000203] The functional activity of ENPP3 x CD3 bispecific antibody provided herein can be characterized by methods known in the art and as described herein. Methods for characterizing binding agents include, but are not limited to, affinity and specificity assays including Biacore, ELISA, and OctetRed analysis; binding assays to detect the binding of antibodies to target cells by FACS; binding assays to detect the binding of antibodies to the target antigen on cells. According to particular embodiments, the methods for characterizing binding agents include those described below.[000204] An ENPP3 x CD3 bispecific antibody of the disclosure is useful in a variety of applications including, but not limited to, therapeutic treatment methods, such as treatment of cancer. In some embodiments, the cancer is a renal cell cancer (RCC), a clear cell RCC (CCRCC), a papillary renal cancer, endometrioid ovarian cancer, endometrioid uterine carcinoma, colorectal adenocarcinoma (CRC), and lung adenocarcinoma.[000205] In some embodiments, the therapeutic treatment methods comprise immunotherapy for cancer. In some embodiments, the ENPP3 binding agent is useful for activating, promoting,increasing, and / or enhancing an immune response to a cancer or cancer cell. In some embodiments, the ENPP3 binding agent is useful for activating, promoting, increasing, and / or enhancing an immune response to a tumor or tumor cell. In some embodiments, the ENPP3 binding agent is useful for activating, promoting, increasing, and / or enhancing a T cell response to a cancer or cancer cells. In some embodiments, the ENPP3 binding agent is useful for activating, promoting, increasing, and / or enhancing a T cell response to a tumor or tumor cell. The methods of use may be in vitro, ex vivo, or in vivo methods.[000206] In one aspect, provided herein is a method of directing a T cell to a target cell expressing ENPP3 by contacting the T cell with an effective amount of an ENPP3 x CD3 binding agent provided herein. In some embodiments, wherein the CD3 binding region binds the T cell. In some embodiments, the ENPP3-binding domain binds the target cell. In some embodiments, the target cell is a tumor or cancer cell. In some embodiments, the cancer is a renal cell cancer (RCC), a clear cell RCC (CCRCC), a papillary renal cancer, endometrioid ovarian cancer, endometrioid uterine carcinoma, colorectal adenocarcinoma (CRC), and lung adenocarcinoma.[000207] In another aspect, provided herein is a method of directing a T cell to a cancer or tumor cell, comprising contacting the T cell with an effective amount of a pharmaceutical composition comprising an ENPP3 x CD3 binding agent provided herein, wherein the CD3 binding region binds the T cell. In some embodiments, the directed T cell incudes cytokine release. In some embodiments, cytokine release is increased compared to a reference. In some embodiments, said cytokine release is determined by measuring IL-ip, IL-2, IL-4, IL-6, IL-8, IL- 10, IL-12, IL-13, TNE-a, IFN-y, or any combination thereof. In some embodiments, the directed T cell induces cytokine release, and release of one or more of IL- 1 [3, IL-2, IL-4, IL-6, IL-8, IL- 10, IL-12, IL-13, TNE-a, and ILN-y is increased as compared to a reference. In some embodiments, a cytokine reference is: (a) a cytokine measured in a corresponding normal cell or issue; (b) a cytokine measured in a neighboring non-cancerous cell or tissue in the same subject; or (c) a cytokine measured in a corresponding cell or tissue measured in a cohort of healthy subjects. In some embodiments, the directed T cell induces apoptosis in the cancer or tumor cell. In some embodiments, when the T cell is directed to the cancer or tumor cell, the T cell induces differential cytotoxicity and cytokine release. That is, a method of directing a T cell to a cancer or tumor cell results in T-cell dependent cytotoxicity (TDCC) that is inversely related to T cellcytokine release. For example, in some embodiments, TDCC is increased compared to a reference and cytokine release is decreased compared to a reference. In some embodiments, said TDCC reference is: (a) TDCC measured in a corresponding normal cell or tissue; (b) TDCC measured in a neighboring non-cancerous cell or tissue in the same subject; or (c) TDCC measured in a corresponding cell or tissue measured in a cohort of healthy subjects. In some embodiments, said TDCC is determined by measuring apoptosis. In some embodiments, caspase mediated apoptosis is increased. In some embodiments, a cytokine reference is: (a) a cytokine measured in a corresponding normal cell or issue; (b) a cytokine measured in a neighboring non- cancerous cell or tissue in the same subject; or (c) a cytokine measured in a corresponding cell or tissue measured in a cohort of healthy subjects. In some embodiments, said cytokine release is determined by measuring IL- 10, IL-2, IL-4, IL-6, IL-8, IL- 10, IL- 12, IL-13, TNF-a, IFN-y, or any combination thereof.[000208] In some embodiments, the cancer cell expresses ENPP3. In some embodiments, the cancer cell expresses high levels of ENPP3 compared to a reference expression level. In some embodiments, the cancer cell expresses low levels of ENPP3 compared to a reference expression level. In some embodiments, said reference expression level of ENPP3 is: (a) a predetermined expression level of ENPP3; (b) an ENPP3 expression level in a corresponding normal cell or issue; (c) an ENPP3 expression level measured in a neighboring non-cancerous cell or tissue in the same subject; or (d) an ENPP3 expression level in a corresponding cell or tissue measured in a cohort of healthy subjects. In some embodiments, said expression level of ENPP3 is determined by measuring the protein expression level of ENPP3.[000209] In one aspect, provided herein is a method of activating a T cell, comprising contacting the T cell with an effective amount of the ENPP3 x CD3 binding protein provided herein, wherein the CD3 binding region binds the T cell. In another aspect, provided herein is a method of activating a T cell, comprising contacting the T cell with a pharmaceutical composition comprising an ENPP3 x CD3 binding protein provided herein.[000210] In some embodiments, T cell activation is measured by expression of certain markers by the T cells. In some embodiments, T cell activation is measured by the percentage of T cells positive for a particular marker in a population of T cells. In some embodiments, the T cell activation marker is CD25. In some embodiments, the T cell activation marker is CD69. In some embodiments, the method of activating the T cell increases expression of CD25, CD69 or bothCD25 and CD69 in the T cells. In some embodiments, the method of activating the T cell increases the percentage of CD25+ T cells, CD69+ T cells, and / or CD25+ / CD69+ T cell in the population of T cells.[000211] In one aspect, provided herein is a method of targeting an antigen on the surface of a target cell, the method comprising contacting the target cell with an effective amount of an ENPP3 x CD3 binding protein provided herein, wherein the ENPP3 -binding domain binds to the target cell. In another aspect, provided herein is a method of targeting an antigen on the surface of a target cell, the method comprising contacting the target cell with an effective amount of a pharmaceutical composition comprising a ENPP3 x CD3 binding protein provided herein, wherein the ENPP3-binding domain binds to the target cell. In some embodiments, provided herein is a method of targeting an antigen on the surface of a target cell, the method comprising contacting the target cell with an effective amount of a pharmaceutical composition comprising a ENPP3 x CD3 binding protein provided herein. In some embodiments, the target cell is a cancer or tumor cell that expresses ENPP3. In some embodiments, the target cell expresses high levels of ENPP3 compared to a reference expression level. In some embodiments, the target cell expresses low levels of ENPP3 compared to a reference expression level. In some embodiments, said reference expression level of ENPP3 is: (a) a predetermined expression level of ENPP3; (b) an ENPP3 expression level in a corresponding normal cell or issue; (c) an ENPP3 expression level measured in a neighboring non-cancerous cell or tissue in the same subject; or (d) an ENPP3 expression level in a corresponding cell or tissue measured in a cohort of healthy subjects. In some embodiments, said expression level of ENPP3 is determined by measuring the protein expression level of ENPP3. In some embodiments, the cancer is a renal cell cancer (RCC), a clear cell RCC (CCRCC), a papillary renal cancer, endometrioid ovarian cancer, endometrioid uterine carcinoma, colorectal adenocarcinoma (CRC), and lung adenocarcinoma. [000212] In one aspect, provided herein is a method of killing or inhibiting the proliferation of a cancer or tumor cell, comprising contacting the cancer or tumor cell with a ENPP3 x CD3 binding protein provided herein. In another aspect, provided herein is a method of killing or inhibiting the proliferation of a cancer or tumor cell, comprising contacting the cancer or tumor cell with a pharmaceutical composition comprising a ENPP3 x CD3 binding protein provided herein. In some embodiments, the ENPP3 x CD3 binding protein activates a T cell. In some embodiments, the CD3 binding region activates the T cell. In some embodiments, the activatedT cell induces apoptosis in the cancer or tumor cell. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a renal cell cancer (RCC), a clear cell RCC (CCRCC), a papillary renal cancer, endometrioid ovarian cancer, endometrioid uterine carcinoma, colorectal adenocarcinoma (CRC), and lung adenocarcinoma.[000213] In some embodiments, the cancer or tumor cell expresses high levels of ENPP3 compared to a reference expression level. In some embodiments, the cancer or tumor cell expresses low levels of ENPP3 compared to a reference expression level. In some embodiments, said reference expression level of ENPP3 is: (a) a predetermined expression level of ENPP3; (b) an ENPP3 expression level in a corresponding normal cell or issue; (c) an ENPP3 expression level measured in a neighboring non-cancerous cell or tissue in the same subject; or (d) an ENPP3 expression level in a corresponding cell or tissue measured in a cohort of healthy subjects. In some embodiments, said expression level of ENPP3 is determined by measuring the protein expression level of ENPP3.[000214] In one aspect, provided herein is a method of treating a cancer or tumor in a subject, comprising administering an effective amount of a ENPP3 x CD3 binding protein provided herein. In another aspect, provided herein is a method of treating a cancer or tumor in a subject, comprising administering an effective amount of a pharmaceutical composition comprising a ENPP3 x CD3 binding protein provided herein or the pharmaceutical composition provided herein. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a renal cell cancer (RCC), a clear cell RCC (CCRCC), a papillary renal cancer, endometrioid ovarian cancer, endometrioid uterine carcinoma, colorectal adenocarcinoma (CRC), and lung adenocarcinoma.[000215] In some embodiments, the cancer or tumor cell expresses ENPP3. In some embodiments, the cancer or tumor cell expresses high levels of ENPP3 compared to a reference expression level. In some embodiments, the cancer or tumor cell expresses low levels of ENPP3 compared to a reference expression level. In some embodiments, said reference expression level of ENPP3 is: (a) a predetermined expression level of ENPP3; (b) an ENPP3 expression level in a corresponding normal cell or issue; (c) an ENPP3 expression level measured in a neighboring non-cancerous cell or tissue in the same subject; or (d) an ENPP3 expression level in a corresponding cell or tissue measured in a cohort of healthy subjects. In some embodiments,said expression level of ENPP3 is determined by measuring the protein expression level of ENPP3.[000216] In another aspect, provided herein is a use of the ENPP3 x CD3 binding protein provided herein in the manufacture of a medicament for treatment of a cancer or tumor in a subject thereof. In yet a further aspect, provided herein is a binding agent for use in the treatment of a cancer or tumor. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a renal cell cancer (RCC), a clear cell RCC (CCRCC), a papillary renal cancer, endometrioid ovarian cancer, endometrioid uterine carcinoma, colorectal adenocarcinoma (CRC), and lung adenocarcinoma.[000217] In some embodiments, the cancer or tumor cell expresses ENPP3. In some embodiments, the cancer or tumor cell expresses high levels of ENPP3 compared to a reference expression level. In some embodiments, the cancer or tumor cell expresses low levels of ENPP3 compared to a reference expression level. In some embodiments, said reference expression level of ENPP3 is: (a) a predetermined expression level of ENPP3; (b) an ENPP3 expression level in a corresponding normal cell or issue; (c) an ENPP3 expression level measured in a neighboring non-cancerous cell or tissue in the same subject; or (d) an ENPP3 expression level in a corresponding cell or tissue measured in a cohort of healthy subjects. In some embodiments, said expression level of ENPP3 is determined by measuring the protein expression level of ENPP3.[000218] In one aspect, provided herein is a method of treating a subject having a ENPP3- expressing cancer or tumor, comprising administering or providing for administration of an effective amount of the ENPP3 x CD3 binding protein to the subject wherein the subject has a ENPP3 -expressing cancer or tumor. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a renal cell cancer (RCC), a clear cell RCC (CCRCC), a papillary renal cancer, endometrioid ovarian cancer, endometrioid uterine carcinoma, colorectal adenocarcinoma (CRC), and lung adenocarcinoma. In some embodiments, said expression level of ENPP3 is determined by measuring the protein expression level of ENPP3. In some embodiments, the subject is a subject in need thereof. In some embodiments, the subject is a human. In specific embodiments, the subject is administered an effective amount.[000219] In some embodiments, before the first administration, the method comprises administering the ENPP3 x CD3 bispecific antibody to a subject with measurable or evaluablecancer. In some embodiments, before the first administration, the subject has a measurable lesion. In some embodiments, before the first administration, the subject has a measurable lesion per Response Evaluation Criteria in Solid Tumors (RECIST) vl.l. In some embodiments, before the first administration, the subject has endometrioid ovarian cancer and has a disease evaluable per RECIST vl.l. In some embodiments, before the first administration, the subject has endometrioid ovarian cancer and has a cancer antigen (CA at least 125 greater than twice the upper limit of normal (ULN).[000220] According to particular embodiments, the pharmaceutical compositions described herein are formulated to be suitable for the intended route of administration to a subject. For example, the pharmaceutical compositions described herein can be formulated to be suitable for intravenous, subcutaneous, or intramuscular administration.[000221] In some embodiments, the method comprises administering an ENPP3 x CD3 bispecific antibody to a subject. In some embodiments, the subject has an ENPP3 -expressing cancer or tumor. In some embodiments, the expression level of ENPP3 is determined by measuring the protein expression level of ENPP3. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a renal cell cancer (RCC), a clear cell RCC (CCRCC), a papillary renal cancer, endometrioid ovarian cancer, endometrioid uterine carcinoma, colorectal adenocarcinoma (CRC), and lung adenocarcinoma. In some embodiments, the subject is a subject in need of treatment thereof. In some embodiments, the subject is a human. In some embodiments, the ENPP3 x CD3 bispecific antibody or antigen binding fragment is administered in a dose selected from the group consisting of 0.017 mg, 0.05 mg, 0.15 mg, 0.5 mg, 0.65 mg, 0.8 mg, 0.175 mg, 1.75 mg. 2 mg, 4 mg, 6 mg, 21 mg, 70 mg, and 100 mg. [000222] In some embodiments, the method comprises administering the ENPP3 x CD3 bispecific antibody to the subject once a week (Q1W). In some embodiments, the ENPP3 x CD3 bispecific antibody or antigen binding fragment is administered in a dose selected from the group consisting of 0.017 mg Q1W, 0.05 mg Q1W, 0.15 mg Q1W, 0.5 mg Q1W, 0.65 mg Q1W, 0.8 mg Q1W, 0.175 mg Q1W, 1.75 mg Q I W. 2 mg Q1W, 4 mg Q1W, 6 mg Q1W, 21 mg Q1W, 70 mg Q1W, and 100 mg Q1W. In some embodiments, the method comprises administering the ENPP3 x CD3 bispecific antibody to the subject once every two weeks (Q2W). In some embodiments, the ENPP3 x CD3 bispecific antibody or antigen binding fragment is administered in a dose selected from the group consisting of 0.017 mg Q2W, 0.05 mg Q2W, 0.15 mg Q2W,0.5 mg Q2W, 0.65 mg Q2W, 0.8 mg Q2W, 0.175 mg Q2W, 1.75 mg Q2W, 2 mg Q2W, 4 mg Q2W, 6 mg Q2W, 21 mg Q2W, 70 mg Q2W, and 100 mg Q2W. In some embodiments, the method comprises administering the ENPP3 x CD3 bispecific antibody to the subject once every three weeks (Q3W). In some embodiments, the ENPP3 x CD3 bispecific antibody or antigen binding fragment is administered in a dose selected from the group consisting of 0.017 mg Q3W, 0.05 mg Q3W, 0.15 mg Q3W, 0.5 mg Q3W, 0.65 mg Q3W, 0.8 mg Q3W, 0.175 mg Q3W, 1.75 mg Q3W, 2 mg Q3W, 4 mg Q3W, 6 mg Q3W, 21 mg Q3W, 70 mg Q3W, and 100 mg Q3W. [000223] In some embodiments, the ENPP3 x CD3 bispecific antibody or antigen binding fragment is administered at a dose which comprises a step-up dose and a target dose. In some embodiments, the step-up dose comprises one or more step-up doses. In some embodiments, the one or more step-up doses is a fraction of the target dose. In some embodiments, the one or more step-up doses is about 0.15 mg each. In some embodiments, the step-up dose is one step-up dose of about 0.15 mg. In some embodiments, the one or more step-up doses is two step-up doses: a first step-up dose of about 0.15 mg and a second step-up dose of about 0.65 mg.[000224] In some embodiments, the ENPP3 x CD3 bispecific antibody or antigen binding fragment is administered at a step-up dose of about 0.15 mg, followed by a target dose of about 0.5 mg. In some embodiments, the ENPP3 x CD3 bispecific antibody or antigen binding fragment is administered at a step-up dose of about 0.15 mg, followed by a target dose of about 0.65 mg. In some embodiments, the ENPP3 x CD3 bispecific antibody or antigen binding fragment is administered at a step-up dose of about 0.15 mg, followed by a second step-up dose of about 0.65 mg, followed by a target dose of about 2 mg.[000225] In some embodiments, the method comprises administering the ENPP3 x CD3 bispecific antibody to the subject subcutaneously. In some embodiments, the method comprises administering the ENPP3 x CD3 bispecific antibody to the subject intravenously.[000226] In case of conflict, the specification, including definitions, will control. As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a peptide sequence” or “a treatment,” includes a plurality of such sequences, treatments, and so forth. It is further noted that the claims can be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology such as“solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.[000227] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges can independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.[000228] As used herein, numerical values are often presented in a range format throughout this document. The use of a range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention unless the context clearly indicates otherwise. Accordingly, the use of a range expressly includes all possible subranges, all individual numerical values within that range, and all numerical values or numerical ranges including integers within such ranges and fractions of the values or the integers within ranges, unless the context clearly indicates otherwise. This construction applies regardless of the breadth of the range and in all contexts throughout this patent document. Thus, for example, reference to a range of 90-100% includes 91-99%, 92-98%, 93-95%, 91-98%, 91-97%, 91-96%, 91-95%, 91- 94%, 91-93%, and so forth. Reference to a range of 90-100% also includes 91%, 92%, 93%, 94%, 95%, 96%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so forth. In addition, reference to a range of 1-3, 3-5, 5-10, 10- 20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120, 120-130, 130- 140, 140-150, 150-160, 160-170, 170-180, 180-190, 190-200, 200-225, 225-250 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. In a further example, reference to a range of 25-250, 250-500, 500-1000, 1000-2500, 2500-5000, 5000-25,000, or 5000-50,000 includes any numerical value or range within or encompassing such values, e.g., 25, 26, 27, 28, 29...250, 251, 252, 253, 254....500, 501, 502, 503, 504... , etc. The use of a series of ranges includes combinations of the upper and lower ranges to provide another range. This construction applies regardless of the breadth of the range and in all contexts throughout this patent document. Thus, for example, reference to a series of ranges such as 5-10, 10-20, 20-30, 30-40, 40-50, 50-75, 75-100, 100-150, includes ranges such as 5-20, 5-30, 5-40, 5-50, 5-75, 5-100, 5-150, and 10-30, 10-40, 10-50, 10-75, 10-100, 10-150, and 20-40, 20-50, 20-75, 20-100, 20-150, and so forth.[000229] For the sake of conciseness, certain abbreviations are used herein. One example is the single letter abbreviation to represent amino acid residues. The amino acids and their corresponding three letter and single letter abbreviations are as follows: alanine Ala (A) arginine Arg (R) asparagine Asn (N) aspartic acid Asp (D) cysteine Cys (C) glutamic acid Glu (E) glutamine Gin (Q) glycine Gly (G) histidine His (H) isoleucine He (I) leucine Leu (L) lysine Lys (K) methionine Met (M) phenylalanine Phe (F) proline Pro (P) serine Ser (S) threonine Thr (T) tryptophan Trp (W) tyrosine Tyr (Y) valine Vai (V)[000230] The invention is generally disclosed herein using affirmative language to describe the numerous embodiments. The invention also specifically includes embodiments in which particular subject matter is excluded, in full or in part, such as substances or materials, method steps and conditions, protocols, procedures, assays or analysis. Thus, even though the invention is generally not expressed herein in terms of what the invention does not include, aspects that are not expressly included in the invention are nevertheless disclosed herein.[000231] Particular embodiments of this invention are described herein. Upon reading the foregoing description, variations of the disclosed embodiments may become apparent toindividuals working in the art, and it is expected that those skilled artisans may employ such variations as appropriate. Accordingly, it is intended that the invention be practiced otherwise than as specifically described herein, and that the 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.[000232] All publications, patent applications, accession numbers, and other references cited in this specification are herein incorporated by reference in its entirety as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided can be different from the actual publication dates which can need to be independently confirmed.[000233] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the descriptions in the Experimental section are intended to illustrate but not limit the scope of invention described in the claims.6. EMBODIMENTS1. A bispecific antibody comprising a first antigen binding region that binds to ENPP3 (ENPP3 -binding domain) and a second antigen binding region that binds to CD3s (CD3 -binding domain) , wherein the ENPP3 -binding domain comprises a VH and VL selected from the group consisting of a VH domain comprising a HCDR1, HCDR2, and HCDR3 of a VH as set forth in SEQ ID NO:22, and a VL domain comprising a LCDR1, LCDR2, and LCDR3 of a VL as set forth in SEQ ID NO: 23.2. The bispecific antibody of embodiment 1, wherein in the CD3-binding domain comprises a VH domain comprising a HCDR1, HCDR2, and HCDR3 of a VH as set forth inSEQ ID NO:47, and a VL domain comprising a LCDR1, LCDR2, and LCDR3 of a VL as set forth in SEQ ID NO: 48.3. The bispecific antibody of embodiment 1, wherein the ENPP3 -binding domain comprises CDR sequences selected from the group consisting of:(a) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence of SEQ ID NO:2, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 3, the LCDR1 comprises the amino acid sequence of SEQ ID NO:4, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 5, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 6;(b) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 7, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 8, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 3, the LCDR1 comprises the amino acid sequence of SEQ ID NO:4, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 5, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 6;(c) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 9, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 10, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 3, the LCDR1 comprises the amino acid sequence of SEQ ID NO:4, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 5, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 6;(d) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 11, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 12, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 13, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 14, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 15, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 16;(e)the HCDR1 comprises the amino acid sequence of SEQ ID NO: 17, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 18, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 19, the LCDR1 comprises the amino acid sequence of SEQ ID NO:20, the LCDR2 comprises the amino acid sequence of GAS , and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 6.4. The bispecific antibody of embodiment 2, wherein the second antigen binding region that binds to CD3s comprises CDR sequences selected from the group consisting of:(a) the HCDR1 comprises the amino acid sequence of SEQ ID NO:26, the HCDR2 comprises the amino acid sequence of SEQ ID NO:27, the HCDR3 comprises the amino acid sequence of SEQ ID NO:28, the LCDR1 comprises the amino acid sequence of SEQ ID NO:29, the LCDR2 comprises the amino acid sequence of SEQ ID NO:30, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:31;(b) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 32, the HCDR2 comprises the amino acid sequence of SEQ ID NO:33, the HCDR3 comprises the amino acid sequence of SEQ ID NO:28, the LCDR1 comprises the amino acid sequence of SEQ ID NO:29 the LCDR2 comprises the amino acid sequence of SEQ ID NO:30, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:31;(c) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 34, the HCDR2 comprises the amino acid sequence of SEQ ID NO:35, the HCDR3 comprises the amino acid sequence of SEQ ID NO:28, the LCDR1 comprises the amino acid sequence of SEQ ID NO:29, the LCDR2 comprises the amino acid sequence of SEQ ID NO:30, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:31;(d) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 36, the HCDR2 comprises the amino acid sequence of SEQ ID NO:37, the HCDR3 comprises the amino acid sequence of SEQ ID NO:38, the LCDR1 comprises the amino acid sequence of SEQ ID NO:39, the LCDR2 comprises the amino acid sequence of SEQ ID NO:40, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:41; and(e) the HCDR1 comprises the amino acid sequence of SEQ ID NO:42, the HCDR2 comprises the amino acid sequence of SEQ ID NO:43, the HCDR3 comprises the amino acid sequence of SEQ ID NO:44, the LCDR1 comprises the amino acid sequence of SEQ ID NO:45, the LCDR2 comprises the amino acid sequence of DSS and the LCDR3 comprises the amino acid sequence of SEQ ID NO:31.5. The bispecific antibody of any one of embodiments 1 to 4, wherein(i)the first binding region comprises a VH domain comprising the amino acid sequence of SEQ ID NO:22, and a VL domain comprising the amino acid sequence of SEQ ID NO:23; and(ii)the second binding region comprises a VH domain comprising the amino acid sequence of SEQ ID NO:47, and a VL domain comprising the amino acid sequence of SEQ ID NO:48.6. The bispecific antibody of any one of embodiments 1-5, wherein the ENPP3 -binding domain comprises a Fab, and the CD3 -binding domain comprises a stapled scFv fragment (spFv).7. The bispecific antibody of embodiment 19, wherein the spFv comprises at least one disulfide bond between the VH or VL and the linker.8. The bispecific antibody of any one of embodiments 1-7, wherein the binding agent further comprises an immunoglobulin (Ig) constant region, a fragment of the Ig constant region, wherein optionally the fragment of the Ig constant region is an Fc region or an CH3 domain.9. A bispecific antibody comprising(i) a first polypeptide comprising a spFv that binds CD3s, a CH2 domain and a CH3 domain;(ii) a second polypeptide comprising a VH domain that binds ENPP3, a CH2 domain and a CH3 domain; and(iii) a third polypeptide comprising a VL domain that binds ENPP3; wherein the spFv that binds CD3s comprises a VH domain comprising a HCDR1, HCDR2, and HCDR3 of a VH as set forth in SEQ ID NO: 47, and a VL domain comprising a LCDR1, LCDR2, and LCDR3 of a VL as set forth in SEQ ID NO:48; and wherein the second and third polypeptide comprising a VH and VL that bind to ENPP3, wherein the VH and VL that bind to ENPP3 are selected from the group consisting of a VH domain comprising a HCDR1 , HCDR2, and HCDR3 of a VH as set forth in SEQ IDNO:22, and a VL domain comprising a LCDR1, LCDR2, and LCDR3 of a VL as set forth in SEQ ID NO: 23.10. The bispecific antibody of embodiment 22, wherein:(i) the spFv that binds CD3s comprises CDRs selected from the group consisting of:(a) the HCDR1 comprises the amino acid sequence of SEQ ID NO:26, the HCDR2 comprises the amino acid sequence of SEQ ID NO:27, the HCDR3 comprises the amino acid sequence of SEQ ID NO:28, the LCDR1 comprises the amino acid sequence of SEQ ID NO:29, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 30, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:31;(b) the HCDR1 comprises the amino acid sequence of SEQ ID NO:32, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 33, the HCDR3 comprises the amino acid sequence of SEQ ID NO:28, the LCDR1 comprises the amino acid sequence of SEQ ID NO:29 the LCDR2 comprises the amino acid sequence of SEQ ID NO: 30, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:31;(c) the HCDR1 comprises the amino acid sequence of SEQ ID NO:34, the HCDR2 comprises the amino acid sequence of SEQ ID NO:35, the HCDR3 comprises the amino acid sequence of SEQ ID NO:28, the LCDR1 comprises the amino acid sequence of SEQ ID NO:29, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 30, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:31;(d) the HCDR1 comprises the amino acid sequence of SEQ ID NO:36, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 37, the HCDR3 comprises the amino acid sequence of SEQ ID NO:38, the LCDR1 comprises the amino acid sequence of SEQ ID NO:39, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 40, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:41; and(e) the HCDR1 comprises the amino acid sequence of SEQ ID NO:42, the HCDR2 comprises the amino acid sequence of SEQ ID NO:43, the HCDR3 comprises the amino acid sequence of SEQ ID NO:44, the LCDR1 comprises the amino acid sequence of SEQ ID NO:45, the LCDR2 comprises the amino acid sequence of DSS and the LCDR3 comprises the amino acid sequence of SEQ ID NO:31; and(ii) the Fab that binds ENPP3 comprises:(a) in the VH domain that binds ENPP3, HCDR1 comprises the amino acid sequence of SEQ ID NO: 1 ; HCDR2 comprises the amino acid sequence of SEQ ID NO:2; HCDR3 comprises the amino acid sequence of SEQ ID NO:3; and in the VL domain that binds ENPP3, LCDR1 comprises the amino acid sequence of SEQ ID NO:4; LCDR2 comprises the amino acid sequence of SEQ ID NO:5; and LCDR3 comprises the amino acid sequence of SEQ ID NO: 6;(b) in the VH domain that binds ENPP3, HCDR1 comprises the amino acid sequence of SEQ ID NO:7; HCDR2 comprises the amino acid sequence of SEQ ID NO:8; HCDR3 comprises the amino acid sequence of SEQ ID NO:3; and in the VL domain that binds ENPP3, LCDR1 comprises the amino acid sequence of SEQ ID NO:4; LCDR2 comprises the amino acid sequence of SEQ ID NO:5; and LCDR3 comprises the amino acid sequence of SEQ ID NO: 6;(c) in the VH domain that binds ENPP3, HCDR1 comprises the amino acid sequence of SEQ ID NOV; HCDR2 comprises the amino acid sequence of SEQ ID NO:10; HCDR3 comprises the amino acid sequence of SEQ ID NO:3; and in the VL domain that binds ENPP3, LCDR1 comprises the amino acid sequence of SEQ ID NO:4; LCDR2 comprises the amino acid sequence of SEQ ID NO:5; and LCDR3 comprises the amino acid sequence of SEQ ID NO:6;(d) in the VH domain that binds ENPP3, HCDR1 comprises the amino acid sequence of SEQ ID NO:11; HCDR2 comprises the amino acid sequence of SEQ ID NO: 12; HCDR3 comprises the amino acid sequence of SEQ ID NO: 13; and in the VL domain that binds ENPP3, LCDR1 comprises the amino acid sequence of SEQ ID NO: 14; LCDR2 comprises the amino acid sequence of SEQID NO: 15; and LCDR3 comprises the amino acid sequence of SEQ ID NO: 16; and(e) in the VH domain that binds ENPP3, HCDR1 comprises the amino acid sequence of SEQ ID NO: 17; HCDR2 comprises the amino acid sequence of SEQ ID NO: 18; HCDR3 comprises the amino acid sequence of SEQ ID NO: 19; and in the VL domain that binds ENPP3, LCDR1 comprises the amino acid sequence of SEQ ID NO:20; LCDR2 comprises the amino acid sequence of GAS ; and LCDR3 comprises the amino acid sequence of SEQ ID NO: 6.11. The bispecific antibody of embodiment 9 or 10, wherein the spFv that binds CD3s comprises a VH comprising the amino acid sequence of SEQ ID NO:47, and a VL comprising the amino acid sequence of SEQ ID NO:48; the VH that binds ENPP3 comprises the amino acid sequence of SEQ ID NO:22, and the VL that binds ENPP3 comprises the amino acid sequence of SEQ ID NO: 23.12. The bispecific antibody of any one of embodiments 9 to 11, wherein the spFv that binds CD3s comprises the amino acid sequence of SEQ ID NO:49; the VH that binds ENPP3 comprises the amino acid sequence of SEQ ID NO:22, and the VL that binds ENPP3 comprises the amino acid sequence of SEQ ID NO:23.13. The bispecific antibody of any one of embodiments 9 to 12, wherein:(i) the first polypeptide comprising a spFv that binds CD3s, a CH2 domain and a CH3 domain comprises the amino acid sequence of SEQ ID NO: 50;(ii) the second polypeptide comprising a VH domain that binds ENPP3, a CH2 domain and a CH3 domain comprises the amino acid sequence of SEQ ID NO:24; and(iii) the third polypeptide comprising a VL domain that binds ENPP3 comprises the amino acid sequence of SEQ ID NO:25.14. The bispecific antibody of any one of embodiments 9 to 13, wherein the spFv comprises at least one disulfide bond between the VH or VL and the linker.15. A composition comprising the bispecific antibody of any one of embodiments 1 to 14, and a pharmaceutically acceptable carrier.16. A polynucleotide comprising nucleotide sequences encoding a VH, a VL, or both a VH and a VL of the bispecific antibody of any one of embodiments 1 to 15.17. A vector comprising the polynucleotide of embodiment 16.18. A cell comprising the polynucleotide of embodiment 16.19. A method of treating cancer in a subject in need thereof, comprising administering to the subject a bispecific antibody comprising an ENPP3 -binding domain and a CD3 -binding domain, wherein:A) the ENPP3 -binding domain comprises the heavy chain complementarity determining region 1 (HCDR1), first HCDR2, and first HCDR3 of the variable chain heavy region (VH) SEQ ID NO:22 and the light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3 of the variable chain light region (VL) of SEQ ID NO; andB) the CD3 -binding domain comprises the HCDR1, HCDR2, and HCDR3 of the VH of SEQ ID NO: 47 and the LCDR1, LCDR2, and LCDR3, of the VH of SEQ ID NO:48; and wherein the cancer is a solid tumor.20. The method of embodiment 19, wherein:A) the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the ENPP3- binding domain comprise the amino acid sequences of: i) SEQ ID NO:1, 2, 3, 4, 5, and 6, respectively; ii) SEQ ID NO: 7, 8, 3, 4, 5, and 6, respectively;iii) SEQ ID N0:9, 10, 3, 4, 5, and 6, respectively; iv) SEQ ID NO:11, 12, 13, 14, 15, and 16, respectively; or v) SEQ ID NO: 17, 18, 19, 20, 21, and 6, respectively; andB) the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the CD3- binding domain comprise the amino acid sequences of: i) SEQ ID NO:26, 27, 28, 29, 30, and 31, respectively; n) SEQ ID NO:32, 33, 28, 29, 30, and 31, respectively; iii) SEQ ID NO:34, 35, 28, 29, 30, and 31, respectively; iv) SEQ ID NO:36, 37, 38, 39, 40, and 41, respectively; or v) SEQ ID NO:42, 43, 44, 45, 46, and 31, respectively; and wherein the cancer is a solid tumor.21. The method of embodiment 19 or 20, wherein the cancer is selected from the group consisting of renal cell cancer (RCC), lung adenocarcinoma, endometrioid ovarian cancer, endometrioid uterine carcinoma, and colorectal adenocarcinoma (CRC).22. The method of any one of embodiments 19-21, wherein the ENPP3 -binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 22 and a VL comprising the amino acid sequence of SEQ ID NO:23.23. The method of any one of embodiments 19-22, wherein the CD3 -binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 47 and a VL comprising the amino acid sequence of SEQ ID NO:48.24. The method of any one of embodiments 19-23, wherein the CD3 -binding domain comprises an spFv comprising the amino acid sequence of SEQ ID NO:49.25. The method of any one of embodiments 19-24, wherein the ENPP3-binding domain comprises a heavy chain (HC) peptide comprising the amino acid sequence of SEQ ID NO:24 and a light chain (LC) peptide comprising the amino acid sequence of SEQ ID NO:25.26. The method of any one of embodiments 19-25, wherein the CD3-binding domain comprises an spFv-Fc fusion peptide comprising an amino acid sequence of SEQ ID NO: 50.27. The method of any one of embodiments 19-26, wherein the subject has measurable or evaluable cancer.28. The method of any one of embodiments 19-27, wherein the subject has a measurable lesion per Response Evaluation Criteria in Solid Tumors (RECIST) vl.l.29. The method of any one of embodiments 19-28, wherein the cancer is ovarian cancer and the subject has disease evaluable per RECIST vl .1 or a cancer antigen (CA) at least 125 greater than twice the upper limit of normal (ULN).30. The method of any one of embodiments 19-29, wherein the bispecific antibody is administered at a dose selected from the group consisting of: 0.017 mg, 0.05 mg, 0.15 mg, 0.5 mg, 0.65 mg, 0.8 mg, 1.75 mg, 2 mg, 4 mg, 6 mg, 21 mg, 70 mg, and 100 mg.31. The method of embodiments 30, wherein the bispecific antibody is administered at a dose of 0.017 mg.32. The method of embodiments 30, wherein the bispecific antibody is administered at a dose of 0.05 mg.33. The method of embodiments 30, wherein the bispecific antibody is administered at a dose of 0.15 mg.34. The method of embodiments 30, wherein the bispecific antibody is administered at a dose of 0.5 mg.35. The method of embodiments 30, wherein the bispecific antibody is administered at a dose of 0.65 mg.36. The method of embodiments 30, wherein the bispecific antibody is administered at a dose of 0.8 mg.37. The method of embodiments 30, wherein the bispecific antibody is administered at a dose of 1.75 mg.38. The method of embodiments 30, wherein the bispecific antibody is administered at a dose of 2 mg.39. The method of embodiments 30, wherein the bispecific antibody is administered at a dose of 4 mg.40. The method of embodiments 30, wherein the bispecific antibody is administered at a dose of 6 mg.41. The method of embodiments 30, wherein the bispecific antibody is administered at a dose of 21 mg.42. The method of embodiments 30, wherein the bispecific antibody is administered at a dose of 70 mg.43. The method of embodiments 30, wherein the bispecific antibody is administered at a dose of 100 mg.44. The method of any one of embodiments 30-43, wherein the dose comprises a step-up dose and a target dose.45. The method of any one of embodiments 30-43, wherein the step-up dose comprises one or more step-up doses.46. The method of any one of embodiments 30-43, wherein the one or more step-up doses is a fraction of the target dose.47. The method of any one of embodiments 30-43, wherein the one or more step-up doses is about 0.15 mg each.48. The method of any one of embodiments 45, wherein the step-up dose is one step-up dose of about 0.15 mg.49. The method of any one of embodiments 30-43, wherein the one or more step-up doses is two step-up doses, and wherein the first step-up dose is about 0.15 mg and the second step-up dose is about 0.65 mg.50. The method of any one of embodiments 19-49, wherein the bispecific antibody is administered once every week.51. The method of any one of embodiments 19-49, wherein the bispecific antibody is administered once every two weeks.52. The method of any one of embodiments 19-49, wherein the bispecific antibody is administered once every three weeks.53. The method of any one of embodiments 19-52, wherein the bispecific antibody is administered subcutaneously.54. The method of embodiment 21, wherein the cancer is renal cell cancer (RCC).55. The method of embodiment 21, wherein the cancer is lung adenocarcinoma.56. The method of embodiment 21, wherein the cancer is endometrioid ovarian cancer.57. The method of embodiment 21, wherein the cancer is endometrioid uterine carcinoma.58. The method of embodiment 21, wherein the cancer is colorectal adenocarcinoma.7. EXAMPLES[000234] The following is a description of various methods and materials used in the studies, and are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below were performed and are all of the experiments that may be performed. It is to be understood that exemplary descriptions written in the present tense were not necessarily performed, but rather that the descriptions can be performed to generate the data and the like associated with the teachings of the present invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental errors and deviations should be accounted for.[000235] Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius (°C), and pressure is at or near atmospheric. Standard abbreviations are used, including the following: bp = base pair(s); kb = kilobase(s); s or sec = second(s); min = minute(s); h or hr = hour(s); aa = amino acid(s); kb = kilobase(s); nt = nucleotide(s); pg = picogram; ng = nanogram; pg = microgram; mg = milligram; g = gram; kg = kilogram; pl or pL = picoliter(s); dl or dL = deciliter; pl or pL = microliter; ml or mL = milliliter; 1 or L = liter; pM = micromolar; mM = millimolar; M = molar; kDa = kilodalton; i.m. = intramuscular(ly); i.p. = intraperitoneal(ly); SC or SQ = subcutaneous(ly); QD = daily; BID = twice daily; QW = weekly; TIW = three times a week; QM = monthly; HPLC = high performance liquid chromatography; BW = body weight; U = unit; ns = not statistically significant; PBS = phosphate-buffered saline; PCR = polymerase chain reaction; NHS = N-Hydroxysuccinimide; HSA = human serum albumin; BSA = bovine serum albumin; DMEM = Dulbeco’s Modification of Eagle’s Medium; GC = genome copy; EDTA = ethylenediaminetetraacetic acid.[000236] The following experimental methods were employed throughout the Examples described herein.Example 1: Generation of antibodies binding to ENPP3[000237] Anti-ENPP3 antibodies were generated using Ablexis transgenic mouse platform, using techniques known in the art. Briefly, Ablexis Kappa mice were immunized using a plasmid comprising full length human ENPP3 DNA sequence. B cells were recovered following standard protocols, and the supernatants from the sorted B cells were screened by protein MSD for binding to human ENPP3 (Uniprot ID: 014638). Fifteen binders were selected based on binding to human ENPP3. Eleven binders were selected for further characterization (Table 1). The binding affinities for select binders to human and cynomolgus monkey ENPP3 are shown in Table 5.Table 5: Binding affinities of select binders to human and cyno ENPP3.[000238] Next, the binders were assessed for, among other things, CDR sequence similarity and biophysics characteristics like binding affinity to human and cyno ENPP3, hydrophobicity, non-specific binding, CIC, conformational stability and SPR based binding to other ENPP family members.Example 2. Generation and characterization of bi-specific ENPP3 x CD3 antibodies. Engineering of the ENPP3 x CD3 antibodies[000239] The ENPP3 binding arm of ENPP3 x CD3 bispecific antibodies was engineered to comprise an anti-ENPP3 Fab arm derived from ENPP3 mAb described in Example 1. To prepare the bispecific antibody, the VH1 and VL1 of the ENPP3 mAb were engineered in VH1- CHl-hinge-CH2-CH3 (Heavy Chain 1, HC1) and VL1-CL (Light Chain 1, LC1) formats respectively and expressed as IgGl. The Fc silencing mutation L234A / L235A / D265S were introduced in the Fc region of HC1. Mutations designed to promote selective heterodimerization (“hole” mutation T366S, L368A and Y407V) were also engineered in the Fc domain of HC1. The parental anti-ENPP3 variable regions were formatted as a Fab in the bispecific antibody.[000240] The CD3 binding arms of ENPP3 x CD3 antibodies were engineered using VH and VL domains of CD3B2030-N106A antibody, disclosed in W02022 / 201053, which isincorporated herein by reference in its entirety. The sequences of CD3B2030-N106A antibody are shown in Table 5 A. The CD3B2030-N106A is a low-medium affinity CD3 binding arm. The VH and VL of the CD3B2030-N106A antibody were engineered as either a scFv domain or a stabilized (or stapled) scFv domain herein described as spFv, in VL-linker-VH-Fc orientation.[000241] The generation of the scFv fragment derived from the CD3B2030-N106A antibody is described in W02022 / 201053, which is incorporated herein by reference in its entirety.[000242] The stabilized scFv (spFv) was generated using the VH and the VL of CD3B2030- N106A binder by engineering disulfide bonds between the VH and the linker and between the and the linker. Two structurally conserved surface exposed framework positions (anchor points) that are not involved in antigen binding, were identified, one on the VH at position Hl 05 and one on the VL at position L42 (Chothia numbering), and mutated into cysteine (Cys) residues to generate the VH of SEQ ID NO:47 and VL of SEQ ID NO:48. A flexible linker of sequence GGGSGGSGGCPPCGGSGG (SEQ ID NO: 62) comprising two Cys residues was used to conjugate the VL and the VH in the VL-linker-VH (LH) format yielding the spFv of SEQ ID NO:49. The distance and location of the Cys residues of the linker and the Cys residues of the VH and the VL is critical for the formation of the disulfide bonds between the Cys residues of the Linker and each anchor point of the VH and the VL.[000243] The stapled scFv in the VL-Linker-VH was further engineered into a Heavy Chain 2 (HC2) and expressed as IgGl . Additionally, the Fc silencing mutation L234A / L235A / D265S and the heterodimerization (“knob” mutation T366W) were engineered in the Fc domain of HC2.Example 3: Characterization of bi-specific ENPP3 x CD3 antibodies[000244] Bispecific antibody (BsAb) NPP3B815 was further characterized. The NPP3B815 antibody comprises an anti-CD3 single-chain fragment variable featuring a “stapled” linker (spFv) on heavy chain 1 (HC1), derived from CD3B2030 N106A CD3 binder. The spFv comprises a linker having a central “C1PPC2” motif (SEQ ID NO: 63) wherein Ci forms a disulfide bond with (in the case of the spFv in the “light-heavy” orientation) an engineered cysteine at position L42 (Chothia numbering) in the variable light domain and C2 forms a disulfide bond with an engineered cysteine at position Hl 05 in the variable heavy domain. Heavy chain 1 comprises the “knob” mutation: T366W. Heavy chain 2 (HC2) comprises the anti-ENPP3 Fab-region, derived from the NPP3B56 antibody and the “hole” mutations: T366S,L368A, Y407V. Table 1 shows CDR and VH / VL amino acid sequences for the ENPP3 binding arm. Table 3A shows CDR and VH / VL amino acid sequences for the CD3 binding arm. Table 5 A shows full length sequences of the NPP3B815 antibody. Table 5B shows DNA sequences encoding the NPP3B815 antibody.[000245] NPP3B815 was assessed for, among other things, purity, binding affinity, thermal stability, solubility, serum stability, nonspecific binding, viscosity, and aggregation potential. NPP3B815 maintained high purity, showed no conformational changes after 2 weeks at 37°C stress test, exhibited high solubility with acceptable viscosity, stability, and high percent monomer (at 150 mg / mL) at 40°C, maintained target binding in human serum, and showed no evidence for nonspecific binding. In summary, NPP3B815 met the ideal or acceptable criteria as demonstrated by the results from different biophysical assays, and the overall intrinsic properties were in favor of manufacturability.[000246] CD3 specific CD3B2030-N106A-LH scFv was generated by a genetic fusion of variable region light chain (VL) and variable region heavy chain (VH) with a flexible linker. The scFv recapitulates the antigen binding specificity and largely the affinity of a parental Fv. However, scFv-containing molecules are prone to aggregation due to low thermal stability and transient separation and intermolecular VL / VH reassociation (‘breathing’).[000247] Both of these liabilities were addressed by scFv ‘stapling’, abbreviated as spFv. To this end, 2 disulfide bonds were engineered between the flexible linker and anchor positions of the VL and VH domains (one on each). This novel strategy is compatible in both VL-VH and VH-VL orientations for almost all Fv domains. Extensive characterization of several molecules demonstrates that spFv molecules not only retain the same binding and function with improved biophysical properties but stapling significantly improved protein quality and aggregation of therapeutics observed in scFv.[000248] The spFv consists of a linker having a central ‘C1PPC2’ motif (SEQ ID NO: 63) wherein Cl forms a disulfide bond with (in the case of the spFv in the Tight- heavy’ orientation) an engineered cysteine at Position 42 (Chothia numbering) in the VL domain and C2 forms a disulfide bond with an engineered cysteine at Position 105 in the VH domain. HC1 features the ‘knob’ mutation T366W. HC2 features the anti-ENPP3 Fab region, and the ‘hole’ mutations T366S, L368A, and Y407V.[000249] The BsAb was developed to evaluate the therapeutic potential of targeting ENPP3 for T-cell redirection. NPP3B815 was developed for the treatment of advanced solid tumors where ENPP3 is known to be highly expressed on the cell surface.[000250] NPP3B815 was generated by co-expression of the anti-CD3e spFv ‘knob’ HC with the anti-ENPP3 Fab HC containing the ‘hole’ and paired with the light chain (LC). The anti- CD3e variable region was derived from Cris7, identified in wild-type mice and humanized in scFv format to identify humanized variants with higher thermal stability than parental Cris7 in scFv format with a range of CD3e affinities. Briefly, the murine complementarity determining region (CDR) regions were grafted into the IGHV1-69*O2-IGHJ1-O1 and IGKV3-l l*02-IGKJ4- 01 human germlines followed by human framework adaption as described previously. The parental HC contained an NG sequence in CDR3 at amino acid Positions 106-107 (Positions 100B-100C in Kabat numbering), representing a potential risk. This risk was eliminated by mutation N106A. Although several mutations could eliminate this risk, N106A was selected based on weaker affinity of the N106A variant compared to the parental v- region, since weaker affinity towards CD3e may be associated with lower toxicity in patients. The anti- CD3 v-region was then formatted into a spFv in the final molecule NPP3B815. The anti-ENPP3 variable region was discovered by immunizing transgenic mice (Ablexis) with a plasmid expressing full-length ENPP3 (Genedata DNA batch ID VB000066101). The parental anti- ENPP3 variable region, featured in the NPP3B56 mAb was not modified and was formatted as a Fab in the final molecule NPP3B815.Intrinsic Design Properties of select ENPP3 x CD3 bispecific antibodies[000251] NPP3B815 exhibited, among other characteristics, a favorable biophysical profile with high affinity (i.e., 920 pM) to human ENPP3 and low affinity (i.e., 110 nM) to human CD3, high purity, good conformational stability, low hydrophobicity, and no nonspecific binding. Furthermore, NPP3B815 exhibited good stability profiles in serum, under ex vivo physiological conditions, under PTM-inducing forced degradation studies, and under thermal stress at high concentrations. In summary, NPP3B815 exhibited low risk for development with favorable intrinsic properties of manufacturability.Selection of ENPP3-expressing Cancer Cell Lines for NPP3B815 in Vitro and in Vivo Assessment[000252] ENPP3 surface expression and receptor density were initially evaluated on a panel of 13 in vitro established RCC and HCC cell lines by flow cytometry using a commercial ENPP3 antibody (clone NP4D6) binding to a similar epitope as the lead ENPP3 -binding domain arm within NPP3B815. As represented in Figure 2, ENPP3 endogenous expression ranged from negative (determined as < lower limit of detection [LLOD], 2,969 receptors / cell) in Cakil, HEK293T and RXF393 (PDX) cell lines, low (<15,000 receptors / cell) in 786-0, ACHN, Huh7 and Hep3B, medium (15,000-40,000 receptors / cell) in KMRC20, HepG2 and VMRCRCW, medium-high (50,000-100,000 receptors / cell) in TUHR4TKB and TUHR10TKB and high (>100,000 receptors / cell) in A704 cell lines. Endogenous ENPP3 was knocked out (using clustered regularly interspaced short palindromic repeats [CRISPR]) in HepG2 cell line, thus generating the negative cell line HepG2 ENPP3-KO. Conversely, ENPP3 was overexpressed in the CHO-K1 cell line to generate the overexpression cell line CHO-K1 ENPP3OE.[000253] To confirm if similar ENPP3 expression was detectable by NPP3B815 on these cell lines, direct- labeled ENPP3 x Null BsAb (NPP3B812), which has the same ENPP3 binder NPP3B56 as NPP3B815, was used for flow cytometry-based receptor density studies. The level of ENPP3 expression with NPP3B812 was observed to be comparable to that seen with the commercial antibody in the cell lines tested. While these cell lines represent a range of ENPP3 expression, most of them having lower expression than the ccRCC tumors. Among these cell lines, A704, VMRCRCW, and HepG2 ENPP3-KO cell lines were used for most of the key in vitro functional studies as they represent high, medium, and negative ENPP3 expression levels, respectively.[000254] Two of these cell lines (i.e., VMRCRCW and HepG2) have also been established as in vivo CDX models to use for NPP3B815 efficacy studies. Ex vivo ENPP3 expression was measured by IHC and flow cytometry (dissociated tumors) at a tumor volume equivalent to the tumor at randomization in these 2 CDX tumor models. IHC showed ENPP3 positivity in both CDX tumors and receptor density was found to be 10,600 (VMRCRCW) and 22,000 (HepG2) ENPP3 receptors per cell (Figure 3). To explore a higher ENPP3 expressing tumor model for in vivo studies, several positive ccRCC PDX models were identified by IHC; ENPP3 expressionwas evaluated ex vivo in the ccRCC PDX model RXF488, which showed high ENPP3 expression by IHC and receptor density measurements of 130,000 receptors / cell.In Vitro Binding of NPP3B815 to Tumor Cells and T Cells[000255] In vitro cell binding of NPP3B815 (NPP3B56 x CD3B2030-N106A) was assessed on high (A704), medium (VMRCRCW), and negative endogenous ENPP3 -expressing cell lines (Table 6). NPP3B815 exhibited a dose-dependent binding to both positive cell lines, A704 and VMRCRCW, with EC50 values of 1.01 nM and 0.5 nM respectively (Figure 4 and Table 7). NPP3B815 did not exhibit binding to the ENPP3 -negative cell line HepG2 ENPP3-KO. No binding was seen to any of the cell lines with the Null x CD3 (79C3B615) or isotype control (79C3B613) antibodies.Table 6: ENPP3 expression (receptor density) in different cancer cell lines[000256] Further, binding of NPP3B815 to human T cells isolated from 6 different healthy donors was assessed. A dose-dependent binding was observed to T cells from all 6 donors, with an average EC50 (± standard error of the mean) of 177±18.5 nM (Figure 4 and Table 7). As expected, no binding was seen with isotype control antibody 79C3B613 to any of the T-cell donors.[000257] To evaluate the specificity of NPP3B815 to ENPP3 compared to other ENPP family members, cell binding was assessed on CH0K1 cells overexpressing ENPP1, ENPP2, or ENPP3 (Figure 5). NPP3B815 exhibitied a dose-dependent binding to only the CH0K1 cells overexpressing ENPP3, with a binding affinity (EC50) of 0.5 nM. NPP3B815 did not exhibit any binding to the ENPP3 -negative cell line CH0K1 Parental or CHOK1 cellls overexpressing ENPP1 or ENPP2 (Figure 5 and Table 7). No binding was seen to any of the cell lines with the isotype control (79C3B613) antibody.Table 7: Cell Binding ECso values for NPP3B815 across target cells and T-cells.ECx, x% effective concentration; ND, not determined.ECso values were not determined if the dose-response curve did not satisfy one or both of the following criteria: R2has to be >0.9 and the log (95% confidence interval) difference needs to be <1.2.NPP3B815-induced Cytotoxicity and T-cell Activation on a Panel of Cancer Cell Lines with Different Levels of Endogenous ENPP3 Expression[000258] The ability of NPP3B815 (NPP3B56 x CD3B2030-N106A) to induce T-cell- mediated tumor cell killing was assessed using an Incucyte (live cell time-lapse) instrument against a panel of cancer cell lines (Table 8) with different ENPP3 expression levels: high (A704), medium-high (TUHR10TKB, TUHR4TKB), medium (VMRCRCW, HepG2), and negative (HepG2 ENPP3-KO). NPP3B815-induced dose-dependent T-cell-mediated cytotoxicity at an Effector to Target cell (E:T) ratio of 3: 1 on all the ENPP3 -positive cell lines (A704, VMRCRCW, HepG2, TUHR4TKB, and TUHR1 OTKB), while no killing was seen against the negative cell line (HepG2 ENPP3-KO) (Figure 6). As expected, no T-cell-mediated tumor cell killing was seen with either Null x CD3 (79C3B615) or ENPP3 x Null (NPP3B812) control antibodies in any of the cell lines tested. The EC50 and maximum activity values for tumor cell killing are shown in Table 8, as the cell lines with higher ENPP3 expression had lower EC50 values with high max tumor cell killing.[000259] To assess the level of T cell activation induced by the ENPP3 x CD3 bispecific antibody, CD25 expression was measured on T cells by flow cytometry at 48 hours post antibody treatment (Figure 7). A dose-dependent increase in T cell activation (E:T ratio = 3: 1) was seen with NPP3B815 on all the ENPP3 -positive cell lines tested (A704, VMRCRCW, and HepG2, while no T cell activation was seen with NPP3B815 in the negative cell line (HepG2ENPP3-KO) or with the control antibodies Null x CD3 and ENPP3 x Null in any of the cell lines tested. The EC50 and maximum activity values for T-cell activation are shown in Table 8.Table 8:ECso and maximum activity values for tumor cell killing and T-cell activation in aECx, x% effective concentration; Max, maximal; ND, not determined.EC50, EC90, and Max killing values were not determined if the dose-response curve did not satisfy one or both of the following criteria: R2 has to be >0.9 and the log (95% confidence interval) difference needs to be <1.2.[ELN E066020, E069414, E063183, E063179]NPP3B815-induced Cytotoxicity and T cell Activation Across Multiple T cell Donors and E:T Ratios[000260] NPP3B815-induced T-cell-mediated tumor cell killing of ENPP3 -positive cell lines A704 (high) and VMRCRCW (medium) and of ENPP3 -negative cell line HepG2 ENPP3-KO was evaluated in the presence of T cells isolated from 6 different healthy human donors (tested at E:T ratio = 3:1) (Figure 8A). Dose-dependent T-cell-mediated cytotoxicity was seen with NPP3B815 across all 6 donors, with some variability seen in EC50 values and maximum cell killing between the different donors (Table 9). As expected, no T-cell-mediated tumor cell killing was seen with Null x CD3 control antibody 79C3B615 against all cell lines and T-cell donors tested. Like A704, dose-dependent T-cell-mediated cytotoxicity was seen with NPP3B815 across all donors in the ENPP3-medium cell line, VMRCRCW, while no killing was observed against the negative cell line (HepG2 ENPP3-KO). EC50 and maximum killing values are shown in Table 9.[000261] In addition to the cytotoxicity assessment described above, NPP3B815-induced T-cell activation (CD25 expression by flow cytometry at 48 hours) was evaluated with the same 6 T-cell donors at an E:T ratio of 3: 1 (Figure 8B). A dose-dependent increase in T-cell activation was seen with NPP3B815 across all 6 T-cell donors in both ENPP3 -positive cell lines (A704 and VMRCRCW). No T-cell activation was seen with NPP3B815 in the negative cell line (HepG2ENPP3-K0) or with the Null x CD3 control antibody 79C3B615 against all cell lines tested. The EC50 and maximum values for T-cell activation are shown in Table 10.[000262] NPP3B815-induced cytotoxicity was also evaluated at lower, more physiologically relevant E:T ratios of 1 : 1 and 1:3, and a dose-dependent killing was observed in the ENPP3-high cell line, A704 at these E:T ratios (Figure 9). A modest decrease in the maximum killing activity of NPP3B815 was seen at the lower E:T ratios of 1: 1 (41%) and 1 :3 (17%), compared to that observed in the ENPP3-high cell line A704 at an E:T ratio of 3:1 (71%), when measured at 72 hours (Table 9). NPP3B815Interestingly, with longer incubation time (5 days vs 3 days), the maximum killing in A704 at the lower E:T ratios 1 :3 increased from 41% to 91% for E:T = 1: 1 and from 17% to 65% 35% for E:T = 1 :3, leading to similar max killing as with higher E:T ratios. Similar cytotoxicity assays at lower E:T ratios were also preformed in the ENPP3 -medium cell line VMRCRCW and the negative cell line, HepG2 ENPP3-KO. A dose-dependent killing was observed in VMRCRCW at the lower E:T ratios, while no killing was observed against the negative cell line HepG2 ENPP3-KO (Figure 9). As expected, no T-cell-mediated tumor cell killing was seen with the Null x CD3 control antibody 79C3B615 at any of the E:T ratios or cell lines tested. The EC50 and maximum activity values for tumor cell killing are shown in Table 9.Table 9: EC50 values for tumor cell cytotoxicity across T-cell donors and E:T ratios.ECx, x% effective concentration; Max, maximal; ND, not determined.ECso and Max killing values were not determined if the dose-response curve did not satisfy one or both of the following criteria: R2has to be >0.9 and the log (95% confidence interval) difference needs to be <1.2.Table 10: ECso values for T-cell activation across donors (E;T ratio = 3:1).ECso and Max activity values were not determined if the dose-response curve did not satisfy one or both of the following criteria: R2has to be >0.9 and the log (95% confidence interval) difference needs to be <1.2.NPP3B815-induced Cytokine Release in Presence of T Cells and Tumor Cells [000263] To further characterize T cell activation by NPP3B815, cytokine release profiles of 10 established inflammatory cytokines were measured using the Human Vplex Proinflammatory Panel (Meso Scale Discovery [MSD]) in the ENPP3-high cell line A704 at 48 hours post incubation with T cells from 6 different healthy human donors at an E: T ratio of 3 : 1. A dosedependent increase in all pro-inflammatory cytokines tested was seen with NPP3B815 across all 6 T cell donors with some variability in response seen between donors (Figure 10). Some cytokines such as interferon (IFN)-y, interleukin (IL)-2, IL- 10, IL-6, and tumor necrosis factor (TNF)-a exhibited a strong dose-dependent induction with NPP3B815 treatment (Figure 10 and Table 11), while others (IL-10, IL-12p70, IL-4, IL-13, IL-8) had a modest to low dose-dependent induction. Concentrations of induced cytokines widely vary upon NPP3B815 treatment from the lowest induced cytokine (IL-10) in the single-digit pg / mL to the highest (IFN-y) in the 3-digit ng / mL range. The Null x CD3 control antibody 79C3B615 induced low / no cytokine response as expected, except in the case of IL-8, which showed high background signal.Table 11: EC50 values for cytokine release with T-cell donors.ECX, x% effective concentration; Max, maximal; ND, not determined; PBMC, peripheral blood mononuclear cell. EC50, EC90, and Max killing values were not determined if the dose-response curve did not satisfy one or both of the following criteria: R2has to be >0.9 and the log (95% confidence interval) difference needs to be <1.2.NPP3B815-induced Cytotoxicity from PBMC Donors[000264] NPP3B815-induced tumor cell killing was evaluated in the presence of PBMCs isolated from corresponding 6 donors from which human T-cell were previously isolated. Dosedependent cytotoxicity was seen with NPP3B815 treatment in the presence of all 6 PBMC donors in the ENPP3-high cell line A704 at an E:T ratio of 5:1 (Figure 11). Dose-dependent cytotoxicity was also seen with NPP3B815 treatment in the presence of all 6 PBMC donors in the ENPP3-medim cell line VMRCRCW, while no killing was observed against the negative cell line HepG2 ENPP3-KO.[000265] Further, NPP3B815-induced cytotoxicity in the presence of PBMCs was also evaluated at lower, more physiologically relevant E: T ratios of 3 : 1 and 1 : 1. A dose-dependent killing of ENPP3 -positive cell line A704 was observed at the lower E:T ratios in the presence of all 6 PBMC donors (Figure 11). A moderate decrease in the maximum killing activity of NPP3B815 was seen at the lowest E:T ratio of 1:1 (44%), when compared to that observed at an E:T ratio of 5: 1 (82%) and 3: 1 (78%). As expected, no tumor cell killing was seen with the Null x CD3 control antibody 79C3B615 in the presence of any of the PBMC donors and cell lines tested (Figure 11). EC50 and maximum killing values are shown in Table 12.Table 12: EC50 values for tumor ce 1 cytotoxicity with PBMC donors.ECx, x% effective concentration; Max, maximal; ND, not determined; PBMC, peripheral blood mononuclear cell. ECso, EC90, and Max killing values were not determined if the dose-response curve did not satisfy one or both of the following criteria: R2has to be >0.9 and the log (95% confidence interval) difference needs to be <1.2.Cell Binding and Cytotoxicity with cyno ENPP3 expressing cells.[000266] NPP3B815’s ENPP3-binding arm is cynomolgus monkey cross-reactive, but its CD3- binding arm is human specific. Therefore, a tool molecule was generated (i.e., NPP3B847) containing the same ENPP3 -binding arm as NPP3B815 but including a cynomolgus cross- reactive CD3 -binding arm, CD3B219 (unstapled, scFv). In vitro cell binding of the tool molecue NPP3B847 was assessed on human and cyno T-cells as well as a cyno ENPP3 cell line, HepG2- KO cyENPP3 OE. NPP3B847 exhibited a dose-dependent binding to the cyno ENPP3 overexpressing cell line HepG2-KO cyENPP3 OE with an EC50 value of 1.6 nM, with no binding seen with the isotype control (79C3B613) antibody (Table 13). Further, comparable dose-dependent binding of NPP3B847 was observed with both human and cynomolgus monkey T cells, with binding affinity (EC50) values being 4.5 nM and 5.2 nM, respectively (Table 13).Table 13: Summary of molecular characteristics of NPP3B847[000267] An assessment of the in vitro functional activity of the tool molecule NPP3B847 was conducted by assessing T-cell activation and T-cell-mediated tumor cell cytotoxicity in the presence of human or cynomolgus monkey T cells. These readouts were measured by flowcytometry in HepG2 ENPP3-K0 cells overexpressing cyno ENPP3 (Figure 12 and Table 14) and compared to the activity of the clinical candidate NPP3B815 in the presence of human T-cells. [000268] NPP3B847-induced T-cell-mediated cell killing of HepG3-KO cyno ENPP3-OE cell line was evaluated in the presence of cyno T cells isolated from 4 different donors (tested at E:T ratio = 3:1) (Figure 12). Dose-dependent T-cell-mediated cytotoxicity was seen with NPP3B847 across all 4 donors, with some variability seen in EC50 values (0.05 nM - 0.14 nM) between the different donors (Table 14). As expected, no T-cell-mediated tumor cell killing was seen with Null x CD3 control antibody NPP3B41 with cyno T-cell donors tested. Further, NPP3B847 induced T-cell-mediated cell killing of HepG2-KO cyno ENPP3-OE cell line was compared to that of NPP3B815 using human T-cells as effector cells at an E:T ratio = 3: 1 (Figure 31). Dosedependent T-cell-mediated cytotoxicity was seen with both NPP3B847 and NPP3B815 at EC50 values of 0.016 nM and 0.079 nM respectively, with no killing seen with Null x CD3 control antibody NPP3B41 with human T-cells (Table 14).[000269] In addition to assessing cytotoxicity, NPP3B847-induced T-cell activation (CD25 expression by flow cytometry at 48 hours) was evaluated with the same 4 cyno T-cell donors at an E:T ratio of 3:1 (Figure 12). A dose-dependent increase in T-cell activation (EC50 = 0.005 nM - 0.033 nM) was seen with NPP3B847 across all 4 T-cell donors tested in the HepG3-KO cyno ENPP3-OE cell line and no activity was seen with Null x CD3 control antibody NPP3B41. Further, human T-cell activation was evaluated with both NPP3B847 and NPP3B815 using HepG2-KO cyno ENPP3-OE cell line at an E:T ratio = 3: 1 (Figure 12). Dose-dependent human T-cell-activation was seen with both NPP3B847 and NPP3B815 at an EC50 value of 0.004 nM and 0.023 nM, respectively, with no activation seen with Null x CD3 control antibody NPP3B41. The EC50 values for cyno and human T-cell activation with NPP3B815 and NPP3B41 are shown in Table 14.Table 14: Summary of EC50 values from cytotoxicity and T-cell activation assays using NPP3B815 and NPP3B847.cyno, cynomolgus monkey; cytotox., cytotoxicity; ECso, 50% effective concentration; N / A, not applicable.In Vivo Efficacy[000270] The antitumor activity of the ENPP3 x CD3 stapled (spFv) BsAb NPP3B815 compared with Null x CD3 control antibody was evaluated in the established ENPP3-low (-10,600 receptors / cell) human RCC CDX model VMRCRCW or the ENPP3 -medium (-22,000 receptors / cell) human HCC HepG2 model, respectively. Efficacy studies were performed in female immune-compromised NSG mice humanized with donor CD3+ pan T cells. Twice-weekly treatment with NPP3B815 administered intraperitoneally (IP) was initiated after SC tumors were established and 1 day post IP Tcell engraftment. Engraftment of human T cells can lead to body weight loss due to eventual graft-versus-host disease (GvHD); however, treatment with NPP3B815 did not result in significant body weight loss as compared to the Null x CD3 -treated control group. In the HepG2 tumor model, body weight loss due to tumor-induced cachexia was observed across all groups.[000271] In Study ONC2022-035, mice bearing established SC VMRCRCW xenografts were IP dosed with NPP3B815 twice weekly at 10, 1, 0.1, and 0.01 mg / kg or Null x CD3 control antibody (10 mg / kg) for a total of 8 doses (n=10 / group). Significant antitumor efficacy was observed with NPP3B815 treatment at 10 and 1 mg / kg over time (p<0.05) with 70% and 75% A tumor growth inhibition (TGI), respectively, as compared to Null x CD3-antibody-treated control mice on Day 39 post tumor implantation (Figure 13A). Treatment with NPP3B815 at 0.1 and 0.01 mg / kg resulted in 58% and 42% ATGI, respectively, as compared to the Null x CD3- antibody -treated animals on Day 39. While statistically significant (p<0.05) the data at these lower NPP3B815 doses are not biologically significant (Johnson et al., 2001, Br J Cancer, 84(10): 1424-1431). The lack of biologically significant efficacy (i.e., >60% TGI) observed from treatment with NPP3B815 at 0.1 and 0.01 mg / kg demonstrated that 1 mg / kg is the minimally efficacious dose against low ENPP3 target level model. All efficacy results are summarized in Table 15.Table 15: Summary of efficacy results for NPP3B815 or NPP3B194.CR, complete response; F, female; IP, intraperitoneal; M, male; NSG, non-obese diabetic (NOD) severe combined immunodeficiency (scid) gamma or NOD.Cg- / , / 7lt / c'"' / Il2rmIW]l / SzJ: q3d-q4d, twice a week; SC, subcutaneous; TGI, tumor growth inhibition. p<0.05 versus control except where noted as not significant (ns).Example 4: Treatment of Solid Tumors with NPP3B815[000272] NPP3B815 is a T cell-redirecting bispecific antibody that binds CD3 and ENPP3 for the treatment of advanced-stage solid tumors with approximately 50% or greater prevalence of ENPP3 expression. Exemplary solid tumors include kidney cancer (clear cell and papillary histologies), lung adenocarcinoma, endometrioid uterine and ovarian cancers, and CRC.[000273] Safety, pharmacokinetics (PK), pharmacodynamics (PD), and antitumor activity of NPP3B815 administered to participants with solid tumors that have an overall high prevalence of ENPP3 are examined. Retrospective evaluation of ENPP3 expression in these tumors is performed to understand the relationship between ENPP3 expression, PD effects, and antitumor activity.Arms[000274] In Part 1 (dose escalation) participants receive NPP3B815. The dose is escalated sequentially until the recommended phase 2 dose (RP2D) regimen(s) have been identified. In Part 2 (dose expansion) participants receive NPP3B815 at the RP2D regimen(s) determined in Part 1.OBJECTIVES AND ENDPOINTSOVERALL DESIGN[000275] This is an open-label, multi-center study to determine the safety, PK, PD, and preliminary antitumor activity of NPP3B815 in participants with advanced-stage solid tumors from selected tumor types with approximately 50% or greater prevalence of ENPP3 expression. The study is conducted in 2 parts: Dose Escalation (Part 1) and Dose Expansion (Part 2).[000276] A target group of 140 participants are treated. The actual number of participants treated in Part 1 depends on the number of cohorts explored and the number of participants enrolled in each cohort. The actual number of participants treated in Part 2 depends on the number of cohorts tested and whether more than 1 RP2D is selected.[000277] The study treatment is administered subcutaneously (SC) at a dose assigned by the sponsor according to the dose escalation or cohort expansion strategy outlined in the sections below.[000278] During the study, safety is monitored by the SET at each dose and at regular intervals during cohort expansion. Cumulative data from subsequent treatment cycles are monitored for late-onset toxicities. SET decisions are based on the review of all available data including, but not limited to, PK, PD, safety, and preliminary antitumor activity.Observation Period[000279] The observation period begins after each study treatment administration. All participants in Part 1 and Part 2 require observation for at least 2 hours post administration of Treatment Doses 2 to 3, inclusive. Observation may be extended to administrations beyond Dose 3 by the investigator or treating physician if needed.Provisional Dosing Table[000280] A dosing table (Table 16) is provided below depicting certain dose levels.Table 16: Provisional Dosing TablePart 1 (Dose Escalation)[000281] Part 1 is intended to identify the RP2D regimen(s) of NPP3B815. The FIH starting dose is Dose Level 1 corresponding to the MABEL. Subsequent doses are selected based on the review of all available data including, but not limited to, PK, PD, safety, and preliminary clinical activity. The initial dosing schedule is Q3W.[000282] After the starting dose, each dose escalation cohort is approved by the SET based on DLT assessment and review of available data using an adaptive dose escalation strategy.Determination of the RP2D regimen(s)[000283] The RP2D regimen(s) are determined after review of all available PK, PD, safety, and efficacy data and are a dose level at which the isotonic estimate of the DLT rate is below or equal to the target rate of 28%. At least 6 participants should have been assessed for DLTs at the provisional RP2D(s) dose level. One or more RP2D regimen may be selected.Part 2 (Dose Expansion)[000284] In Part 2, NPP3B815 are administered, at the RP2D regimen(s) as determined in Part 1, in disease specific cohorts to participants with the following diseases: kidney cancer, lung adenocarcinoma, endometrioid uterine and ovarian cancers, and CRC. Participants are enrolled in each cohort to confirm the safety, PK / PD, and preliminary clinical activity of NPP3B815.STUDY POPULATIONInclusion Criteria:Ages Eligible for Study: 18 Years and older Sexes Eligible for Study: All Gender Based: No.Gender Eligibility Criteria: N / A Accepts Healthy Volunteers: No• Have histologically or cytologically confirmed, metastatic, or unresectable solid tumor of one of the following types: a) Renal cell cancer (RCC)-clear cell or papillary carcinoma; b) Endometrioid ovarian cancer c) Endometrioid uterine carcinoma; d) Colorectal adenocarcinoma (CRC); e) Lung adenocarcinoma.• Have measurable or evaluable disease: Part 1- Either measurable or evaluable disease; Part 2- At least 1 measurable lesion per RECIST vl.1. Participants with ovarian cancer without a measurable lesion must have disease evaluable per Response Evaluation Criteria in Solid Tumors (RECIST vl.l) or have cancer antigen (CA) 125 greater than (>) 2*upper limit of normal (ULN) during screening.• All participants in Part 1 and Part 2 must consent to provide an archived tumor tissue sample at screening.• Have an Eastern Cooperative Oncology Group (ECOG) performance status of 0 to 1 at screening.• Be willing and able to adhere to the lifestyle restrictions specified in this protocol.Exclusion Criteria:• Active Central Nervous System (CNS) involvement with the exception of locally treated brain metastases that are clinically stable and asymptomatic for > 2 weeks and who are off or receiving low-dose corticosteroid treatment (less than or equal to [<=] 10 milligrams [mg] prednisone or equivalent) for at least 2 weeks prior to start of study treatment.• Toxicity from prior anticancer therapy that has not resolved to Grade <=1 (except alopecia, vitiligo, Grade <=2 peripheral neuropathy, or endocrinopathies that are stable on hormone replacement).• History of Grade greater than or equal to (>=) 2 immune-related AE(s) with prior immunotherapy that led to discontinuation of previous immunotherapy with the exception of Grade 2 and Grade 3 immune-related AEs that responded to treatment and that did not recur following rechallenge. Endocrinopathies that are stable on hormone replacement therapy or that have resolved are allowed.• History of solid organ or hematologic stem cell transplantation.• Any episode of partial or complete bowel obstruction requiring hospitalization within 30 days prior to first dose of study treatment.Table 17: NPP3B815 AdministrationExample 5: Preliminary Safety Results of an Ectonucleotide Pyrophosphatase / Phosphodiesterase Family Member 3 (ENPP3) x CD3 Bispecific Antibody in Patients (pts) with Advanced Solid Tumors.Introduction / Objective:[000285] ENPP3, a Type II transmembrane protein, is a target with primarily apical expression in normal epithelial tissues, with non-apical expression in basophils, mast cells, adrenal gland, and a subset of small blood vessels. While the role of ENPP3 in cancer remains unclear, it is postulated that the restricted apical expression in normal tissue prevents potential off-tumor effects, while allowing selective targeting of depolarized tumor ENPP3 expression. ENPP3 is highly prevalent in several solid tumors including renal cell carcinoma (RCC), lung adenocarcinoma, epithelioid endometrial and ovarian cancers, and colorectal cancer (CRC). NPP3B815 is a bispecific antibody targeting ENPP3 and the CD3 T cell receptor that demonstrated ENPP3 -expression-dependent potent in vitro T-cell activation, cytotoxicity, and anti-tumor activity (with complete tumor regressions) in in vitro and in vivo models. Preclinical safety assessment with a tool molecule using the therapeutic ENPP3 binding arm but with a cynomolgus CD3 cross-reactive arm identified modality consistent toxicities.Materials / Methods:[000286] NPP3B815 is being evaluated in this Phase 1 study to establish safety, pharmacokinetics (PK) / pharmacodynamics (PD), and anti-tumor activity in pts with advanced solid tumors with approximately 50% or greater prevalence of ENPP3 expression. Archival tissue is being collected and analyzed retrospectively for ENPP3 expression. The study is being conducted in 2 parts: Dose Escalation (Part 1) and Dose Expansion (Part 2). In Part 1, study treatment is administered SC with dose escalation guided by a BOIN statistical design.Results:[000287] Twenty-one patients have been treated in Part 1 of study (clear cell RCC n=9, papillary RCC n=2, lung n=l, CRC n=6, endometrioid uterine cancer n=l and endometrioid ovarian cancer n=2) at various doses ranging from 0.017mg - 0.5mg SC Q 3W without step-up dosing and up to 0.65mg with step-up dosing. Two dose limiting toxicities (DLTs) were reported at 0.5mg dose without step-up dosing (Grade 3 cytokine release syndrome (CRS) and Grade 3 maculopapular rash). The most common adverse reactions are rash reported in 7 (33.3%) pts, followed by injection site reactions (ISR) and fatigue both reported in 5 (23.8%) pts and CRS reported in 4 (19%) pts. CRS events were reported for pts above the 0.15mg dose and were Grade 1, 2 and 3.Discussion / Conclusion:[000288] Preliminary clinical data from dose escalation demonstrates a safety profile expected for T-cell redirectors. DLT’s observed at the 0.5mg dose led to implementation of step-up dosing to mitigate CRS. From cohort 6 onwards a minimum of 3 lung adenocarcinoma patients will be enrolled in each cohort with the aim of identifying a new targeted agent for lung adenocarcinoma and other solid tumors.[000289][000290]

Claims

WHAT IS CLAIMED IS:

1. A method of treating cancer in a subject in need thereof, comprising administering to the subject a bispecific antibody comprising an ENPP3 -binding domain and a CD3 -binding domain, wherein:A) the ENPP3 -binding domain comprises the heavy chain complementarity determining region 1 (HCDR1 ), the HCDR2, and the HCDR3 of a heavy chain variable region (VH) of SEQ ID NO:22, and the light chain complementarity determining region 1 (LCDR1 ), the LCDR2, and the LCDR3 of a light chain variable region (VL) of SEQ ID NO:23; andB) the CD3 -binding domain comprises the HCDR1, the HCDR2, and the HCDR3 of a VH or SEQ ID NO: 47, and the LCDR1 , the LCDR2, and the LCDR3 of a VL of SEQ ID NO:48; and wherein the cancer is a solid tumor.

2. The method of claim 1 , wherein:A) the ENPP3 -binding domain comprises a HCDR1, a HCDR2, a HCDR3, a LCDR1, a LCDR2, and a LCDR3 comprising the amino acid sequences of: i) SEQ ID NO:1, 2, 3, 4, 5, and 6, respectively; ii) SEQ ID NO: 7, 8, 3, 4, 5, and 6, respectively; iii) SEQ ID NOV, 10, 3, 4, 5, and 6, respectively; iv) SEQ ID NO:11, 12, 13, 14, 15, and 16, respectively; or v) SEQ ID NO: 17, 18, 19, 20, 21, and 6, respectively; andB) the CD3 -binding domain comprises a HCDR1, a HCDR2, a HCDR3, a LCDR1, a LCDR2, and a LCDR3 comprising the amino acid sequences of: i) SEQ ID NO:26, 27, 28, 29, 30, and 31, respectively; n) SEQ ID NO:32, 33, 28, 29, 30, and 31, respectively; iii) SEQ ID NO:34, 35, 28, 29, 30, and 31, respectively; iv) SEQ ID NO:36, 37, 38, 39, 40, and 41, respectively; or v) SEQ ID NO:42, 43, 44, 45, 46, and 31, respectively.

3. The method of claim 1 or 2, wherein the cancer is selected from the group consisting of renal cell cancer (RCC), lung adenocarcinoma, endometrioid ovarian cancer, endometrioid uterine carcinoma, and colorectal adenocarcinoma (CRC).

4. The method of any one of claims 1-3, wherein the ENPP3 -binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 22 and a VL comprising the amino acid sequence of SEQ ID NO:23.

5. The method of any one of claims 1-4, wherein the CD3-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 47 and a VL comprising the amino acid sequence of SEQ ID NO:48.

6. The method of any one of claims 1-5, wherein the CD3 -binding domain comprises an spFv comprising the amino acid sequence of SEQ ID NO:49.

7. The method of any one of claims 1-6, wherein the ENPP3 -binding domain comprises a first heavy chain (HC) peptide comprising the amino acid sequence of SEQ ID NO: 24 and a first light chain (LC) peptide comprising the amino acid sequence of SEQ ID NO:25.

8. The method of any one of claims 1-7, wherein the CD3 -binding domain comprises an spFv-Fc fusion peptide comprising an amino acid sequence of SEQ ID NO: 50.

9. The method of any one of claims 1-8, wherein the subject has measurable or evaluable cancer.

10. The method of any one of claims 1-9, wherein the subject has a measurable lesion per Response Evaluation Criteria in Solid Tumors (RECIST) vl.l.

11. The method of any one of claims 1-10, wherein the cancer is ovarian cancer and the subject has disease evaluable per RECIST vl.l or a cancer antigen (CA) at least 125 greater than twice the upper limit of normal (ULN).

12. The method of any one of claims 1-11, wherein the bispecific antibody is administered at a dose selected from the group consisting of: 0.017 mg, 0.05 mg, 0.15 mg, 0.5 mg, 0.65 mg, 0.8 mg, 1.75 mg, 2 mg, 4 mg, 6 mg, 21 mg, 70 mg, and 100 mg.

13. The method of claim 12, wherein the bispecific antibody is administered at a dose of 0.017 mg.

14. The method of claim 12, wherein the bispecific antibody is administered at a dose of 0.05 mg.

15. The method of claim 12, wherein the bispecific antibody is administered at a dose of 0.15 mg.

16. The method of claim 12, wherein the bispecific antibody is administered at a dose of 0.5 mg.

17. The method of claim 12, wherein the bispecific antibody is administered at a dose of 0.65 mg.

18. The method of claim 12, wherein the bispecific antibody is administered at a dose of 0.8 mg.

19. The method of claim 12, wherein the bispecific antibody is administered at a dose of 1.75 mg.

20. The method of claim 12, wherein the bispecific antibody is administered at a dose of 2 mg.

21. The method of claim 12, wherein the bispecific antibody is administered at a dose of4 mg.

22. The method of claim 12, wherein the bispecific antibody is administered at a dose of6 mg.

23. The method of claim 12, wherein the bispecific antibody is administered at a dose of 21 mg.

24. The method of claim 12, wherein the bispecific antibody is administered at a dose of 70 mg.

25. The method of claim 12, wherein the bispecific antibody is administered at a dose of 100 mg.

26. The method of any one of claims 12-25, wherein the dose comprises a step-up dose and a target dose.

27. The method of any one of claims 12-26, wherein the step-up dose comprises one or more step-up doses.

28. The method of any one of claims 1-27, wherein the bispecific antibody is administered once every week.

29. The method of any one of claims 1-27, wherein the bispecific antibody is administered once every two weeks.

30. The method of any one of claims 1-27, wherein the bispecific antibody is administered once every three weeks.

31. The method of any one of claims 1-30, wherein the bispecific antibody is administered subcutaneously.

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