Bispecific antibody constructs that bind ALPP / alppl2 and CD3

Abstract

Bispecific antibody constructs comprising a first binding domain, which binds to human alkaline phosphatase, placental type (ALPP) and / or alkaline phosphatase, germ cell type (ALPPL2), on the surface of a tumor cell and a second binding domain, which binds to human CD3 on the surface of a T cell.

Description

BISPECIFIC ANTIBODY CONSTRUCTS THAT BIND ALPP / ALPPL2 AND CD3CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U. S. Provisional Application No. 63 / 898,891. filed October 14. 2025, and U. S. Provisional Application No. 63 / 745.932, filed January 16, 2025, which are incorporated herein by reference in its entirety.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The contents of the electronic sequence listing (26093-WO-PCT_SL.xml; Size: 607,449 bytes; and Date of Creation: March 31, 2025) are herein incorporated by reference in their entirety.FIELD

[0003] The present invention relates to bispecific antibody constructs comprising a first binding domain, which binds to human alkaline phosphatase, placental type (ALPP) and / or alkaline phosphatase, germ cell type (ALPPL2), herein after “ALPP / L2”, on the surface of a tumor cell and a second binding domain, which binds to human CD3 on the surface of a T cell. The bispecific antibody constructs are herein referred to as “ALPP / L2 T-cell engagers” or "‘ALPP / L2 TCE”.BACKGROUND

[0004] Alkaline phosphatases are a group of dimeric metalloenzymes that catalyze the hydrolysis of phosphomonoesters (Le Du et al. J. Bio Chem. 2001, 276, 9158-9165). In humans there are four isozymes, placental (herein referred to as ALPP but also known as PLAP), germ cell type (herein referred to as ALPPL2, but also known as ALPG or GCAP), intestinal (ALPI) and tissue non-specific (herein referred to as ALPL, but also known as TNAP).

[0005] These four isozymes are all glycoproteins that are anchored to the cell surface via a C-terminal glycophosphatidylinositol post translational modification. ALPP, ALPPL2 and ALPI are all located on chromosome 2, whereas ALPL is located on chromosome 1. ALPP and ALPPL2 share high sequence homology at 98 percent and have a similar expression profile, being expressed in the placenta but largely absent in normal adult tissue. ALPI has relatively high homology to ALPP / ALPPL2 at around 87 percent. This isoform is widely expressed throughout the intestinal tract and has an important role in gut mucosal defense. The fourth isozyme, ALPLis widely expressed in many tissues including bone, liver, and kidney. ALPL has a lower homology to ALPP / ALPPL2 at around 57%.

[0006] Upregulation of ALPP and ALPPL2 has been observed at the mRNA level and at the protein level in a broad range of cancers including ovarian cancer, endometrial cancer, cervical cancer, gastric cancer, esophageal cancer, bladder cancer, lung cancer, pancreatic cancer, and testicular cancer (Reiswich et al. J of Pathol. Clin. Res., 2021, 7, 577-589). High levels of ALPP / ALPPL2 have also been linked to poor prognosis in gastric and ovarian cancers (Orsaria et al. Cancer Biomarkers 2016, 17, 479-486; Liu et al. Human Pathol. 2019, 86, 49-56). Given its expression levels on cancer cells compared to its relative absence on normal tissue, ALPP / ALPPL2 provides an opportunity to provide cancer therapies that selectively target cancer cells over non-cancer cells.

[0007] Antibodies targeting ALPP and / or ALPPL2 have previously been described (See Ravenni et al. MAbs, 2014, 6 (1), 86-94; WO2017095823 (University of California);WO2021158178 (Agency for Science. Technology, and Research); WO2022197890 (Seagen Inc ); W02023049150 (University of California; WO2023215746 (Javelin Oncology, Inc ); and WO2024133763 (Almac Discovery Ltd.).

[0008] CD3 (cluster of differentiation 3) is a protein complex and co-receptor found on T cells and plays a critical role in activation of T cells. The CD3 complex is composed of four distinct chains: CD3y, CD35, and two CD3e chains. These chains combine with the T-cell receptor (TCR) and CD3-zeta (q-chain) to create a signal that initiates activation in T lymphocytes.Together, the TCR, CD3-zeta, and other CD3 molecules form the TCR complex.

[0009] The use of antibodies targeting CD3 has proven to be effective in clustering CD3 molecules on T cells, resulting in T cell activation similar to the activation that occurs when the TCR binds to MHC molecules loaded with peptides. Consequently, CD3-specific antibodies have been proposed for therapies aiming to activate T cells.

[0010] Bispecific antibodies have been developed to simultaneously engage CD3 and a tumor antigen. These antibodies redirect T cells towards targeted tumor cells, leading to their destruction. Examples of such bispecific antibodies include the BiTE and DART formats, which bind monovalently to CD3 and the tumor antigen. Additionally, there are IgG-like formats, like BEAT antibodies, where one binding arm attaches to the tumor antigen while the second arm binds to CD3 on T cells. The latter formats have the benefit of extended in vivo half-life because of the Fc domain, which confers recycling properties thereby offering the ability for less frequent dosing. However, designing Fc based bispecific molecules requires use of unique technologies to promote heterodimer formation, such as DuoBody, Azy metric, Knob-into-Hole, and the like. Forsome of these technologies, designing the bispecific with one arm as a single chain Fv (scFv) provides less complex design and allows faster construct screenings. However, scFvs are prone for aggregate formation during production resulting in lower yield and thereby higher cost of goods.SUMMARY

[0011] The present invention provides bispecific antibody constructs comprising a first binding domain, which binds to human alkaline phosphatase, placental type (ALPP) and / or alkaline phosphatase, germ cell t pe (ALPPL2), herein after “ALPP / L2”, on the surface of a tumor cell and a second binding domain, which binds to human CD3 on the surface of a T cell. These anti-ALPP / L2 x anti-CD3 bispecific antibodies are T-cell engagers (TCE) referred to herein as ALPP / L2 TCE, bispecific antibody, or ALPP / L2 TCE BsAb. The ALPP / L2 TCE bispecific antibodies may be used to treat proliferative diseases or tumors that display ALPP and / or ALPPL2 on the cell surface. Examples of proliferative diseases include but are not limited to mesothelioma, testicular cancer, endometrial cancer, pancreatic cancer, ovarian cancer, non-small cell lung cancer (NSCLC), gastric cancer, bladder cancer, head and neck cancer, cervical cancer, breast cancer, esophageal cancer, cholangiocarcinoma, prostate cancer, or colon cancer.

[0012] The present invention provides bispecific antibodies comprising a first binding domain, which binds to human ALPP and / or ALPPL2 on the surface of a target cell, and a second binding domain, which binds to human CD3 on the surface of a T cell, wherein the first binding domain comprises: (a) a first heavy chain variable domain (VH) comprising complementarity determining regions (CDRs) VH-CDR1, VH-CDR2, and VH-CDR3 and a first light chain variable domain (VL) comprising CDRs VL-CDR1, VL-CDR2, and VL-CDR3, wherein (1) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 3, SEQ IDNO: 4, and SEQ ID NO: 5, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 6, SEQ IDNO: 7, and SEQ ID NO: 8, respectively;(2) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 11, SEQ IDNO: 12, and SEQ ID NO: 13, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 14, SEQ IDNO: 15, and SEQ ID NO: 16. respectively;(3) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 19, SEQ IDNO: 20, and SEQ ID NO: 21, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 22, SEQ IDNO: 23, and SEQ ID NO: 24, respectively;(4) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 27, SEQ IDNO: 28, and SEQ ID NO: 29. respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 30, SEQ IDNO: 31. and SEQ ID NO: 32, respectively;(5) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 35, SEQ IDNO: 36, and SEQ ID NO: 37. respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 38, SEQ IDNO: 39. and SEQ ID NO: 40, respectively;(6) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 43, SEQ IDNO: 44, and SEQ ID NO: 45, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 46, SEQ IDNO: 47, and SEQ ID NO: 48, respectively;(7) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 51, SEQ IDNO: 52, and SEQ ID NO: 53, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 54, SEQ IDNO: 55, and SEQ ID NO: 56. respectively;(8) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 59, SEQ IDNO: 60, and SEQ ID NO: 61, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 62, SEQ IDNO: 63, and SEQ ID NO: 64. respectively;(9) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 67, SEQ IDNO: 68, and SEQ ID NO: 69, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 70, SEQ IDNO: 71, and SEQ ID NO: 72, respectively;(10) VH-CDR1, VH-CDR2. and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 75, SEQ IDNO: 76, and SEQ ID NO: 77, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 78, SEQ IDNO: 79, and SEQ ID NO: 80, respectively;(11) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 83, SEQ IDNO: 84, and SEQ ID NO: 85, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 86, SEQ IDNO: 87, and SEQ ID NO: 88, respectively;(12) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 91, SEQ IDNO: 92, and SEQ ID NO: 93, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 94, SEQ IDNO: 95, and SEQ ID NO: 96, respectively;(13) VH-CDR1, VH-CDR2. and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 99, SEQ IDNO: 100, and SEQ ID NO: 101, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 102, SEQ IDNO: 103, and SEQ ID NO: 104, respectively;(14) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 107, SEQ IDNO: 108, and SEQ ID NO: 109, respectively, and VL-CDRL VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 110, SEQ IDNO: 111, and SEQ ID NO: 112, respectively;(15) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 115, SEQ IDNO: 116. and SEQ ID NO: 117, respectively, and VL-CDR1. VL-CDR2. and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 118, SEQ IDNO: 119, and SEQ ID NO: 120, respectively;(16) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 123, SEQ IDNO: 124. and SEQ ID NO: 125, respectively, and VL-CDR1, VL-CDR2. and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 126, SEQ IDNO: 127, and SEQ ID NO: 128, respectively;(17) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 131, SEQ IDNO: 132, and SEQ ID NO: 133, respectively, and VL-CDR1, VL-CDR2. and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 134, SEQ IDNO: 135, and SEQ ID NO: 136, respectively;(18) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 139, SEQ IDNO: 140, and SEQ ID NO: 141, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 142, SEQ IDNO: 143, and SEQ ID NO: 144, respectively;(19) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 147, SEQ IDNO: 148, and SEQ ID NO: 149, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 150, SEQ IDNO: 151, and SEQ ID NO: 152, respectively;(20) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 155, SEQ IDNO: 156, and SEQ ID NO: 157, respectively, and VL-CDR1, VL-CDR2,and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO 158, SEQ IDNO: 159, and SEQ ID NO: 160, respectively;(21) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 163, SEQ IDNO: 164, and SEQ ID NO: 165, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 166, SEQ IDNO: 167, and SEQ ID NO: 168, respectively;(22) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 171, SEQ IDNO: 172, and SEQ ID NO: 173, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 174, SEQ IDNO: 175, and SEQ ID NO: 176, respectively;(23) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 179, SEQ IDNO: 180, and SEQ ID NO: 181, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 182, SEQ IDNO: 183, and SEQ ID NO: 184, respectively;(24) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 187, SEQ IDNO: 188, and SEQ ID NO: 189, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 190, SEQ IDNO: 191, and SEQ ID NO: 192, respectively;(25) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 195, SEQ IDNO: 196, and SEQ ID NO: 197, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 198, SEQ IDNO: 199, and SEQ ID NO: 200, respectively;(26 VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 203, SEQ IDNO: 204, and SEQ ID NO: 205, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 206, SEQ IDNO: 207, and SEQ ID NO: 208, respectively;(27) VH-CDR1, VH-CDR2. and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 211, SEQ IDNO: 212, and SEQ ID NO: 213, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 214, SEQ IDNO: 215, and SEQ ID NO: 216, respectively;(28) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 219, SEQ IDNO: 220, and SEQ ID NO: 221, respectively, and VL-CDRL VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 222, SEQ IDNO: 223, and SEQ ID NO: 224, respectively;(29) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 227, SEQ IDNO: 228, and SEQ ID NO: 229, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 230, SEQ IDNO: 231, and SEQ ID NO: 232, respectively;(30) VH-CDR1, VH-CDR2. and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 235, SEQ IDNO: 236, and SEQ ID NO: 237, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 238, SEQ IDNO: 239, and SEQ ID NO: 240, respectively;(31) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 243, SEQ IDNO: 244, and SEQ ID NO: 245, respectively, and VL-CDRL VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 246, SEQ IDNO: 247, and SEQ ID NO: 248, respectively;(32) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 251, SEQ IDNO: 252. and SEQ ID NO: 253. respectively, and VL-CDR1. VL-CDR2. and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 254, SEQ IDNO: 255, and SEQ ID NO: 256, respectively;(33) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 259, SEQ IDNO: 260. and SEQ ID NO: 261, respectively, and VL-CDR1, VL-CDR2. and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 262, SEQ IDNO: 263, and SEQ ID NO: 264, respectively;(34) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 267, SEQ IDNO: 268, and SEQ ID NO: 269, respectively, and VL-CDR1, VL-CDR2. and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 270, SEQ IDNO: 271, and SEQ ID NO: 272, respectively;(35) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 275, SEQ IDNO: 276, and SEQ ID NO: 277, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 278, SEQ IDNO: 279, and SEQ ID NO: 280, respectively;(36) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 283, SEQ IDNO: 284, and SEQ ID NO: 285, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 286, SEQ IDNO: 287, and SEQ ID NO: 288, respectively;(37) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 291, SEQ IDNO: 292, and SEQ ID NO: 293, respectively, and VL-CDR1, VL-CDR2,and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 294, SEQ IDNO: 295, and SEQ ID NO: 296, respectively;(38) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 299 SEQ IDNO: 300, and SEQ ID NO: 301, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 302, SEQ IDNO: 303, and SEQ ID NO: 304, respectively;(39) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 307, SEQ IDNO: 308, and SEQ ID NO: 309, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 310, SEQ IDNO: 311, and SEQ ID NO: 312, respectively;(40) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 315, SEQ IDNO: 316, and SEQ ID NO: 317, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 318, SEQ IDNO: 319, and SEQ ID NO: 320, respectively;(41) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 323, SEQ IDNO: 324, and SEQ ID NO: 325, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 326, SEQ IDNO: 327, and SEQ ID NO: 328, respectively;(42) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 341, SEQ IDNO: 342, and SEQ ID NO: 343, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 344, SEQ IDNO: 345, and SEQ ID NO: 346, respectively;(43) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 349, SEQ IDNO: 350, and SEQ ID NO: 351, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 352, SEQ IDNO: 353, and SEQ ID NO: 354, respectively;(44) VH-CDR1, VH-CDR2. and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 357, SEQ IDNO: 358, and SEQ ID NO: 359, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 360, SEQ IDNO: 361, and SEQ ID NO: 362, respectively;(45) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 365, SEQ IDNO: 366, and SEQ ID NO: 367, respectively, and VL-CDRL VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 368, SEQ IDNO: 369, and SEQ ID NO: 370, respectively; or(46) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 373, SEQ IDNO: 374, and SEQ ID NO: 375, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 376, SEQ IDNO: 377, and SEQ ID NO: 378, respectively; and (b) wherein the second binding domain comprises a second VH comprising CDRs VH-CDR1. VH-CDR2. and VH-CDR3 and a second VL comprising CDRs VL-CDR1, VL-CDR2, and VL-CDR3, wherein VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 424, SEQ IDNO: 425, and SEQ ID NO: 426, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 428, SEQ IDNO: 429, and SEQ ID NO: 430, respectively; and wherein the first binding domain is linked to the N-terminus of a first heavy chain constant region (CH) comprising at least a CH2-CH3 domain and the second binding domain is linked to the N-terminus of a second CH comprising at least CH2-CH3 domains; wherein the CH3 domains of the first CH and the second CH each comprise one or more mutations promoting heterodimerization of the first and second CH3 domains to form the bispecific antibody.

[0013] In certain embodiments of the bispecific antibody, the bispecific antibody comprises (a) the VH-CDR1, VH-CDR2, and VH-CDR3 of the first binding domain comprise the amino acid sequence set forth in SEQ ID NO: 3, SEQ IDNO: 4, and SEQ ID NO: 5, respectively, and the VL-CDR1, VL-CDR2, and VL-CDR3 of the first binding domain comprise the amino acid sequence set forth in SEQ ID NO: 6, SEQ IDNO: 7, and SEQ ID NO: 8, respectively; (b) VH-CDR1, VH-CDR2, and VH-CDR3 of the first binding domain comprise the amino acid sequence set forth in SEQ ID NO: 11, SEQ IDNO: 12, and SEQ ID NO: 13, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 of the first binding domain comprise the amino acid sequence set forth in SEQ ID NO: 14, SEQ IDNO: 15, and SEQ ID NO: 16, respectively; or (c) VH-CDR1, VH-CDR2, and VH-CDR3 of the first binding domain comprise the amino acid sequence set forth in SEQ ID NO:19, SEQ IDNO: 20, and SEQ ID NO: 21, respectively, and VL-CDR1, VL-CDR2. and VL-CDR3 of the first binding domain comprise the amino acid sequence set forth in SEQ ID NO: 22, SEQ IDNO: 23, and SEQ ID NO: 24, respectively.

[0014] In certain embodiments of the bispecific antibody, the first binding domain comprises: (a) a VH comprising the amino acid sequence set forth in SEQ ID NO: 1 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 2:(b) a VH comprising the amino acid sequence set forth in SEQ ID NO: 9 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 10;(c) a VH comprising the amino acid sequence set forth in SEQ ID NO: 17 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 18;(d) a VH comprising the amino acid sequence set forth in SEQ ID NO: 25 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 26;(e) a VH comprising the amino acid sequence set forth in SEQ ID NO: 33 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 34;(f) a VH comprising the amino acid sequence set forth in SEQ ID NO: 41 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 42;(g) a VH comprising the amino acid sequence set forth in SEQ ID NO: 49 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 50;(h) a VH comprising the amino acid sequence set forth in SEQ ID NO: 57 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 58;(i) a VH comprising the amino acid sequence set forth in SEQ ID NO: 65 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 66;(j) a VH comprising the amino acid sequence set forth in SEQ ID NO: 73 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 74;(k) a VH comprising the amino acid sequence set forth in SEQ ID NO: 81 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 82;(l) a VH comprising the amino acid sequence set forth in SEQ ID NO: 89 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 90;(m) a VH comprising the amino acid sequence set forth in SEQ ID NO: 97 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 98;(n) a VH comprising the amino acid sequence set forth in SEQ ID NO: 105 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 106;(o) a VH comprising the amino acid sequence set forth in SEQ ID NO: 113 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 114;(p) a VH comprising the amino acid sequence set forth in SEQ ID NO: 121 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 122;(q) a VH comprising the amino acid sequence set forth in SEQ ID NO: 129 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 130;(r) a VH comprising the amino acid sequence set forth in SEQ ID NO: 137 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 138;(s) a VH comprising the amino acid sequence set forth in SEQ ID NO: 145 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 146;(t) a VH comprising the amino acid sequence set forth in SEQ ID NO: 153 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 154;(u) a VH comprising the amino acid sequence set forth in SEQ ID NO: 161 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 162;(v) a VH comprising the amino acid sequence set forth in SEQ ID NO: 169 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 170;(w) a VH comprising the amino acid sequence set forth in SEQ ID NO: 177 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 178;(x) a VH comprising the amino acid sequence set forth in SEQ ID NO: 185 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 186;(y) a VH comprising the amino acid sequence set forth in SEQ ID NO: 193 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 194;(z) a VH comprising the amino acid sequence set forth in SEQ ID NO: 201 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 202;(aa) a VH comprising the amino acid sequence set forth in SEQ ID NO: 209 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 210;(bb) a VH comprising the amino acid sequence set forth in SEQ ID NO: 217 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 218;(cc) a VH comprising the amino acid sequence set forth in SEQ ID NO: 225 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 226;(dd) a VH comprising the amino acid sequence set forth in SEQ ID NO: 233 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 234;(ee) a VH comprising the amino acid sequence set forth in SEQ ID NO: 241 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 242;(ff) a VH comprising the amino acid sequence set forth in SEQ ID NO: 249 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 250;(gg) a VH comprising the amino acid sequence set forth in SEQ ID NO: 257 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 258;(hh) a VH comprising the amino acid sequence set forth in SEQ ID NO: 265 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 266;(ii) a VH comprising the amino acid sequence set forth in SEQ ID NO: 273 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 272;(jj) a VH comprising the amino acid sequence set forth in SEQ ID NO: 281 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 282;26093(kk) a VH comprising the amino acid sequence set forth in SEQ ID NO: 289 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 290;(11) a VH comprising the amino acid sequence set forth in SEQ ID NO: 297 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 298;(mm) a VH comprising the amino acid sequence set forth in SEQ ID NO: 305 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 306;(nn) a VH comprising the amino acid sequence set forth in SEQ ID NO: 313 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 314;(oo) a VH comprising the amino acid sequence set forth in SEQ ID NO: 321 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 322;(pp) a VH comprising the amino acid sequence set forth in SEQ ID NO: 329 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 340;(qq) a VH comprising the amino acid sequence set forth in SEQ ID NO: 347 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 348;(rr) a VH comprising the amino acid sequence set forth in SEQ ID NO: 355 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 356;(ss) a VH comprising the amino acid sequence set forth in SEQ ID NO: 363 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 364; or(tt) a VH comprising the amino acid sequence set forth in SEQ ID NO: 371 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 372;(uu) a VH comprising the amino acid sequence set forth in SEQ ID NO: 1 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 419;(vv) a VH comprising the amino acid sequence set forth in SEQ ID NO: 420 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 421; or(ww) a VH comprising the amino acid sequence set forth in SEQ ID NO: 422 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 18; andwherein the second binding domain comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 423 or 604 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 427 or 605.

[0015] In certain embodiments of the bispecific antibody, the first binding domain comprises: (a) a VH comprising the amino acid sequence set forth in SEQ ID NO: 1 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 2;(b) a VH comprising the amino acid sequence set forth in SEQ ID NO: 9 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 10;26093(c) a VH comprising the amino acid sequence set forth in SEQ ID NO: 17 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 18;(d) a VH comprising the amino acid sequence set forth in SEQ ID NO: 1 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 419;(e) a VH comprising the amino acid sequence set forth in SEQ ID NO: 420 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 421; or(f) a VH comprising the amino acid sequence set forth in SEQ ID NO: 422 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 18; andwherein the second binding domain comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 423 or 604 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 427 or 605.

[0016] In certain embodiments of the bispecific antibody, the first binding domain is a Fab comprising a first VH-CH and VL-CL pair and the second binding domain is an scFv comprising from the N-terminus to the C-terminus VL-VH-CH’ or VH-VL-CH’, wherein the first CH comprises a first CHI domain, first Hinge, first CH2 domain, and first CH3 domain and the CH’ is a truncated CH comprising a deletion of CHI and comprising at least a second CH2 domain and second CH3 domain, wherein the CH and CH’ domains each comprise one or more amino acid substitutions in their CH3 domains that promote heterodimerization of the CH and CH’ domains to form the bispecific antibody.

[0017] In certain embodiments of the bispecific antibody, (a) the first CH3 domain comprises amino acid substitutions S354C and T366W and the second CH3 domain comprises amino acid substitutions Y349C, T366S, L368A, and Y407V; (b) the first CH3 domain comprises amino acid substitutions Y349C, T366S, L368A, and Y407V to form the knob and the second CH3 domain comprises amino acid substitutions S354C and T366W; (c) the first CH3 domain comprises amino acid substitution 409W amino acid substitution and the second CH3 domain comprises amino acid substitutions D399 and F405T; (d) the first CH3 domain comprises amino acid substitutions D399 and F405T amino acid substitutions and the second CH3 domain comprises amino acid substitution 409W; (e) the first CH3 domain comprises amino acid substitution 357W and the second CH3 domain comprises amino acid substitution Y349S; (f) the first CH3 domain comprises amino acid substitutions Y349S and the second CH3 domain comprises amino acid substitution 357W; (g) the first CH3 domain comprises amino acid substitutions T350V, L351Y, F405A, and Y407V and the second CH3 domain comprises amino acid substitutions T350, T366, K392 and T394W; (h) the first CH3 domain comprises amino acid substitutions T350, T366, K392 and T394W and the second CH3 domain comprises amino acid26093substitutions T350V, L351Y, F405A, and Y407V; (i) the first CH3 domain comprises amino acid substitutions T350V, L351Y, F405A, and Y407V and the second CH3 domain comprises amino acid substitutions T350V and T366W; or (j) the first CH3 domain comprises amino acid substitutions T350V and T366W to and the second CH3 domain comprises amino acid substitutions T350V. L351Y, F405A, and Y407V; wherein the first and second CH3 domains form a heterodimeric Fc region; and wherein the amino acid numbering is according to the EU numbering scheme.

[0018] In certain embodiments of the bispecific antibody, the first binding domain is a first Fab comprising a first VH-CH and VL-CL pair and the second binding domain is a second Fab comprising a second VH-CH and VL-CL pair, wherein the CH of the first VH-CH and a VL-CL pair and the second VH-CH and VL-CL pair each comprise one or more amino acid substitutions that promote heterodimerization to form the bispecific antibody.

[0019] In certain embodiments of the bispecific antibody, (a) the first CH3 domain comprises amino acid substitutions S354C and T366W and the second CH3 domain comprises amino acid substitutions Y349C, T366S, L368A, and Y407V; (b) the first CH3 domain comprises amino acid substitutions Y349C, T366S, L368A, and Y407V to form the knob and the second CH3 domain comprises amino acid substitutions S354C and T366W; (c) the first CH3 domain comprises amino acid substitution 409W amino acid substitution and the second CH3 domain comprises amino acid substitutions D399 and F405T; (d) the first CH3 domain comprises amino acid substitutions D399 and F405T amino acid substitutions and the second CH3 domain comprises amino acid substitution 409W; (e) the first CH3 domain comprises amino acid substitution 357W and the second CH3 domain comprises amino acid substitution Y349S; (I) the first CH3 domain comprises amino acid substitutions Y349S and the second CH3 domain comprises amino acid substitution 357W; (g) the first CH3 domain comprises amino acid substitutions T350V, L351Y, F405A, and Y407V and the second CH3 domain comprises amino acid substitutions T350, T366, K392 and T394W; (h) the first CH3 domain comprises amino acid substitutions T350. T366, K392 and T394W and the second CH3 domain comprises amino acid substitutions T350V, L351Y, F405A, and Y407V; (i) the first CH3 domain comprises amino acid substitutions T350V, L351Y, F405A, and Y407V and the second CH3 domain comprises amino acid substitutions T350V and T366W; or (j) the first CH3 domain comprises amino acid substitutions T350V and T366W to and the second CH3 domain comprises amino acid substitutions T350V, L351Y, F405A, and Y407V; wherein the first and second CH3 domains form a heterodimeric Fc region; and wherein the amino acid numbering is according to the EU numbering scheme.26093

[0020] In certain embodiments of the bispecific antibody, the CH is of the human IgGl or IgG4 isotype comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, insertions, deletions, or combinations thereof and the first and second CL is of the human kappa or lambda isotype comprising 1, 2, 3, 4, 5, 6, 7. 8, 9, or 10 amino acid substitutions, insertions, deletions, or combinations thereof.

[0021] In certain embodiments of the bispecific antibody, (a) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 381 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 392; (b) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 382 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 393; (c) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 383 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 394; (d) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 384 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 395; (e) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 385 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 396; (f) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 386 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 397; (g) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 387 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 398; (h) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 388 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 399; (i) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 389 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 400; (j) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 390 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 401; (k) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 392 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 381; (1) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 393 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 382; (m) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 394 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 383; (n) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 395 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 384; (o) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 396 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 385; (p) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 397 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 386; (q) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 398 and second CH26093comprises the amino acid sequence set forth in SEQ ID NO: 387; (r) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 399 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 388; (s) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 400 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 389; or (t) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 401 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 390.

[0022] In certain embodiments of the bispecific antibody, the CH’ comprises amino acids 8-15 of the Hinge region, CH2 domain, and CH3 domain.

[0023] In certain embodiments of the bispecific antibody, (a) the CH comprises the amino acid sequence set forth in SEQ ID NO: 381 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 431; (b) the CH comprises the amino acid sequence set forth in SEQ ID NO: 382 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 432; (c) the CH comprises the amino acid sequence set forth in SEQ ID NO: 383 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 433; (d) the CH comprises the amino acid sequence set forth in SEQ ID NO: 384 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 434; (e) the CH comprises the amino acid sequence set forth in SEQ ID NO: 385 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 435; (f) the CH comprises the amino acid sequence set forth in SEQ ID NO: 386 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 436; (g) the CH comprises the amino acid sequence set forth in SEQ ID NO: 387 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 437; (h) the CH comprises the amino acid sequence set forth in SEQ ID NO: 388 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 438; (i) the CH comprises the amino acid sequence set forth in SEQ ID NO: 389 and CH comprises the amino acid sequence set forth in SEQ ID NO: 439; (j) the CH comprises the amino acid sequence set forth in SEQ ID NO: 390 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 440; (k) the CH comprises the amino acid sequence set forth in SEQ ID NO: 392 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 441; (1) the CH comprises the amino acid sequence set forth in SEQ ID NO: 393 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 442; (m) the CH comprises the amino acid sequence set forth in SEQ ID NO: 394 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 443; (n) the CH comprises the amino acid sequence set forth in SEQ ID NO: 395 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 444; (o) the CH comprises the amino acid sequence set forth in SEQ ID NO: 396 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 445; (p) the CH comprises the amino acid sequence set forth in SEQ ID NO: 397 and CH’ comprises the amino acid sequence set forth in26093SEQ ID NO: 446; (q) the CH comprises the amino acid sequence set forth in SEQ ID NO: 398 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 447; (r) the CH comprises the amino acid sequence set forth in SEQ ID NO: 399 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 448; (s) the CH comprises the amino acid sequence set forth in SEQ ID NO: 400 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 449; or (t) the CH comprises the amino acid sequence set forth in SEQ ID NO: 401 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 450.

[0024] In certain embodiments of the bispecific antibody, the CL comprises the kappa isotype.

[0025] In certain embodiments, the CL comprises 1. 2, 3, 4. 5, 6, 7, 8, 9, or 10 amino acid substitutions, insertions, deletions, or combinations thereof compared to the amino acid sequence of the native CL.

[0026] In certain embodiments of the bispecific antibody, the CH and CH’ comprises: (a) a substitution of the amino acids at positions 252, 254, and 256 of the CH and the CH' with amino acids Tyr (Y), Thr (T), and Glu (E), respectively, wherein the numbering is according to the EU numbering scheme; (b) a substitution of the amino acids at positions 233 and 235 of the CH and the CH’ with the amino acid Ala (A), wherein the numbering is according to the EU numbering scheme; (c) a substitution of the amino acids at positions 234 and 235 of the CH and the CH’ with the amino acid A and at position 265 with Ser (S), wherein the numbering is according to the EU numbering scheme; (d) a substitution of the amino acids at positions 234 and 235 of the CH and the CH’ with the amino acid A and at position 329 with Gly (G), wherein the numbering is according to the EU numbering scheme; (e) a substitution of the amino acid at position 235 of the CH and the CH’ with the amino acid E, wherein the numbering is according to the EU numbering scheme; (f) a substitution of the amino acid at position 265 of the CH and the CH’ with the amino acid A, wherein the numbering is according to the EU numbering scheme; (g) a substitution of the amino acid at position 265 of the CH and the CH’ with the amino acid A and at position 297 with the amino acid G, wherein the numbering is according to the EU numbering scheme; (h) a substitution of the amino acid at position 297 of the CH and the CH’ with any amino acid except for Asn (N), wherein the numbering is according to the EU numbering scheme; or (i) a substitution of the amino acid at position 297 of the CH and the CH’ with the amino acid A, at position 356 with Glu (E), at position 358 with Met (M), wherein the numbering is according to the EU numbering scheme.

[0027] In certain embodiments of the bispecific antibody, the CH and CH’ comprise a C-terminal lysine or lack a C-terminal lysine or a C-terminal gly cine-ly sine dipeptide. In certain embodiments, the N-terminal amino acid of the VH is pyroglutamate.26093

[0028] Further provided is a bispecific antibody comprising: a first arm, which binds to human ALPP and / or ALPPL2 on the surface of a target cell, and a second arm, which binds to human CD3 on the surface of a T cell, comprising (a) a first arm comprising a heavy chain (HC) comprising the amino acid sequence set forth in SEQ ID NO: 458 associated with a light chain (LC) comprising the amino acid sequence set forth in SEQ ID NO: 459 and a second arm comprising an scFv-CH’ comprising the amino acid sequence set forth in SEQ ID NO: 461; (b) a first arm comprising an HC comprising the amino acid sequence set forth in SEQ ID NO: 510 associated with an LC comprising the amino acid sequence set forth in SEQ ID NO: 453 and a second arm comprising an scFv-CH’ comprising the amino acid sequence set forth in SEQ ID NO: 509; (c) a first arm comprising an HC comprising the amino acid sequence set forth in SEQ ID NO: 511 associated with an LC comprising the amino acid sequence set forth in SEQ ID NO: 453 and a second arm comprising an scFv-CH’ comprising the amino acid sequence set forth in SEQ ID NO: 508; (d) a first arm comprising an HC comprising the amino acid sequence set forth in SEQ ID NO: 512 associated with an LC comprising the amino acid sequence set forth in SEQ ID NO: 456 and a second arm comprising an scFv-CH’ comprising the amino acid sequence set forth in SEQ ID NO: 508; (e) a first arm comprising an HC comprising the amino acid sequence set forth in SEQ ID NO: 513 associated with an LC comprising the amino acid sequence set forth in SEQ ID NO: 456 and a second arm comprising an scFv-CH’ comprising the amino acid sequence set forth in SEQ ID NO: 509; (f) a first arm comprising an HC comprising the amino acid sequence set forth in SEQ ID NO: 514 associated with an LC comprising the amino acid sequence set forth in SEQ ID NO: 459 and a second arm comprising an scFv-CH’ comprising the amino acid sequence set forth in SEQ ID NO: 509; or (g) a first arm comprising an HC comprising the amino acid sequence set forth in SEQ ID NO: 515 associated with an LC comprising the amino acid sequence set forth in SEQ ID NO: 459 and a second arm comprising an scFv-CH’ comprising the amino acid sequence set forth in SEQ ID NO: 508.

[0029] In a further embodiment of the bispecific antibody, a bispecific antibody is provided comprising a heavy chain (HC), a light chain (LC), and an scFv linked to a truncated HC constant domain (scFv-CH’), wherein the HC comprises the amino acid sequence set forth in SEQ ID NO: 451 or 460, the LC comprising the amino acid sequence set forth in SEQ ID NO: 453, and the scFv-CH' comprising the amino acid sequence set forth in SEQ ID NO: 462.

[0030] In a further embodiment of the bispecific antibody, a bispecific antibody is provided comprising a heavy chain (HC), a light chain (LC), and an scFv linked to a truncated HC constant domain (scFv-CH’), wherein the HC comprises the amino acid sequence set forth in26093SEQ ID NO: 457, the LC comprising the amino acid sequence set forth in SEQ ID NO: 459, and the scFv-CH’ comprising the amino acid sequence set forth in SEQ ID NO: 462.

[0031] In a further embodiment of the bispecific antibody, a bispecific antibody is provided comprising a heavy chain (HC), a light chain (LC), and an scFv linked to a truncated HC constant domain (scFv-CH’), wherein the HC comprises the amino acid sequence set forth in SEQ ID NO: 454, the LC comprising the amino acid sequence set forth in SEQ ID NO: 456, and the scFv-CH’ comprising the amino acid sequence set forth in SEQ ID NO: 461.

[0032] In a further embodiment of the bispecific antibody, a bispecific antibody is provided comprising a heavy chain (HC), a light chain (LC), and an scFv linked to a truncated HC constant domain (scFv-CH’), wherein the HC comprises the amino acid sequence set forth in SEQ ID NO: 458, the LC comprising the amino acid sequence set forth in SEQ ID NO: 459, and the scFv-CH’ comprising the amino acid sequence set forth in SEQ ID NO: 461.

[0033] In a further embodiment of the bispecific antibody, a bispecific antibody is provided comprising a heavy chain (HC), a light chain (LC), and an scFv linked to a truncated HC constant domain (scFv-CH’), wherein the HC comprises the amino acid sequence set forth in SEQ ID NO: 510, the LC comprising the amino acid sequence set forth in SEQ ID NO: 453, and an scFv-CH’ comprising the amino acid sequence set forth in SEQ ID NO: 509.

[0034] In a further embodiment of the bispecific antibody, a bispecific antibody is provided comprising a heavy chain (HC), a light chain (LC), and an scFv linked to a truncated HC constant domain (scFv-CH’), wherein the HC comprises the amino acid sequence set forth in SEQ ID NO: 511, the LC comprising the amino acid sequence set forth in SEQ ID NO: 453, and an scFv-CH' comprising the amino acid sequence set forth in SEQ ID NO: 508.

[0035] In a further embodiment of the bispecific antibody, a bispecific antibody is provided comprising a heavy chain (HC), a light chain (LC), and an scFv linked to a truncated HC constant domain (scFv-CH’), wherein the HC comprises the amino acid sequence set forth in SEQ ID NO: 512, the LC comprising the amino acid sequence set forth in SEQ ID NO: 456, and the scFv-CH' comprising the amino acid sequence set forth in SEQ ID NO: 508.

[0036] In a further embodiment, a bispecific antibody is provided comprising a heavy chain (HC), a light chain (LC), and an scFv linked to a truncated HC constant domain (scFv-CH'), wherein the HC comprises the amino acid sequence set forth in SEQ ID NO: 514, the LC comprising the amino acid sequence set forth in SEQ ID NO: 459, and the scFv-CH’ comprising the amino acid sequence set forth in SEQ ID NO: 509.

[0037] In a further embodiment, a bispecific antibody is provided comprising a heavy chain (HC), a light chain (LC), and an scFv linked to a truncated HC constant domain (scFv-CH'), wherein the HC comprises the amino acid sequence set forth in SEQ ID NO: 515, the LC comprising the amino acid sequence set forth in SEQ ID NO: 459, and the scFv-CH' comprising the amino acid sequence set forth in SEQ ID NO: 508.

[0038] Further provided is a pharmaceutical composition comprising the bispecific antibody of herein and a pharmaceutically acceptable carrier or diluent.

[0039] Further provided is a method for treating a proliferative disease in an individual in need of the treatment comprising administering to the individual a therapeutically effective amount of the bispecific antibody herein or a pharmaceutical composition comprising the bispecific antibody to treat the proliferative disease.

[0040] In certain embodiments of the method, the proliferative disease comprises cells that display ALPP and / or ALPPL2 on the cell surface. In certain embodiments, the proliferative disease comprises a tumor that displays ALPP and / or ALPPL2 on the surface of the cells thereof.

[0041] In certain embodiments of the method, the proliferative disease or tumor is a cancer or malignancy selected from the group consisting of mesothelioma, testicular cancer, endometrial cancer, pancreatic cancer, ovarian cancer, non-small cell lung cancer (NSCLC), gastric cancer, bladder cancer, head and neck cancer, cervical cancer, breast cancer, esophageal cancer, cholangiocarcinoma, prostate cancer, and colon cancer.

[0042] Further provided is use of the bispecific antibody herein or a pharmaceutical composition comprising the bispecific antibody for the manufacture of a medicament for treatment of a proliferative disease.

[0043] In certain embodiments of the use, the proliferative disease comprises cells that display ALPP and / or ALPPL2 on the cell surface. In certain embodiments, the proliferative disease comprises a tumor that displays ALPP and / or ALPPL2 on the surface of the cells thereof.

[0044] In certain embodiments of the use, the proliferative disease or tumor is a cancer or malignancy selected from the group consisting of mesothelioma, testicular cancer, endometrial cancer, pancreatic cancer, ovarian cancer, non-small cell lung cancer (NSCLC), gastric cancer, bladder cancer, head and neck cancer, cervical cancer, breast cancer, esophageal cancer, cholangiocarcinoma, prostate cancer, and colon cancer.

[0045] Further provided is a bispecific antibody disclosed herein or a pharmaceutical composition comprising the bispecific antibody for use in the treatment a proliferative disease.

[0046] In certain embodiments, the proliferative disease comprises cells that display ALPP and / or ALPPL2 on the cell surface. In certain embodiments, the proliferative disease comprises a tumor that displays ALPP and / or ALPPL2 on the surface of the cells thereof.26093

[0047] In certain embodiments, the proliferative disease or tumor is a cancer or malignancy selected from the group consisting of mesothelioma, testicular cancer, endometrial cancer, pancreatic cancer, ovarian cancer, non-small cell lung cancer (NSCLC), gastric cancer, bladder cancer, head and neck cancer, cervical cancer, breast cancer, esophageal cancer, cholangiocarcinoma. prostate cancer, and colon cancer.

[0048] Further provided is a combination therapy for treating a proliferative disease comprising the bispecific antibody disclosed herein or a pharmaceutical composition comprising the bispecific antibody and a second therapeutic agent.

[0049] In certain embodiments of combination therapy, the proliferative disease comprises cells that display ALPP and / or ALPPL2 on the cell surface. In certain embodiments, the proliferative disease comprises a tumor that displays ALPP and / or ALPPL2 on the surface of the cells thereof.

[0050] In certain embodiments of combination therapy, the proliferative disease or tumor is a cancer or malignancy selected from the group consisting of mesothelioma, testicular cancer, endometrial cancer, pancreatic cancer, ovarian cancer, non-small cell lung cancer (NSCLC), gastric cancer, bladder cancer, head and neck cancer, cervical cancer, breast cancer, esophageal cancer, cholangiocarcinoma, prostate cancer, and colon cancer.

[0051] The present invention further provides a combination therapy for treating a proliferative disease in an individual in need of the treatment comprising administering to the individual a therapeutically effective amount of a bispecific antibody disclosed herein or pharmaceutical composition thereof and a second therapeutic agent to treat the proliferative disease. In certain embodiments of the combination therapy, the second therapeutic agent is a chemotherapy agent or a therapeutic antibody other than an antibody that binds ALPP and / or ALPPL2.

[0052] In certain embodiments of the combination therapy, the proliferative disease comprises a tumor that displays ALPP and / or ALPPL2 on the surface of the cells thereof. In certain embodiments of the combination therapy, the proliferative disease or tumor is a cancer or malignancy selected from the group consisting of mesothelioma, testicular cancer, endometrial cancer, pancreatic cancer, ovarian cancer, non-small cell lung cancer (NSCLC), gastric cancer, bladder cancer, head and neck cancer, cervical cancer, breast cancer, esophageal cancer, cholangiocarcinoma, prostate cancer, and colon cancer.

[0053] Further provided is a nucleic acid molecule encoding the heavy chain (HC), light chain (LC). or scFv-Fc of a bispecific antibody disclosed herein. Further provided is an expression vector comprising one or more of the nucleic acid molecules. Further provided is a host cell comprising the one or more nucleic acid molecules.26093

[0054] Further provided is a host cell comprising a first nucleic acid molecule encoding the heavy chain (HC) of a bispecific antibody herein, a second nucleic acid molecule encoding the light chain (LC) of a bispecific antibody herein; and a third nucleic acid molecule encoding the scFv-CH’ of a bispecific antibody herein, wherein the CH3 region of the HC and the scFv comprise one or more mutations promoting heterodimerization.

[0055] Further provided is a method for producing a bispecific antibody herein comprising (a) providing the host cell herein; (b) cultivating the host cell in a medium under conditions suitable for expressing the bispecific antibody; and (c) isolating the bispecific antibody from the host cells and / or medium.

[0056] Further provided is a bispecific antibody herein conjugated to a detectable moiety. In certain embodiments, the detectable moiety is detectable by magnetic resonance imaging (MRI) or by X-ray imaging.

[0057] Further provided is a method for detecting ALPP and / or ALPPL2 on the surface of a cell in an individual comprising administering to the individual the bispecific antibody herein conjugated to a detectable moiety and detecting the cells in the individual that bind the bispecific antibody.

[0058] The present invention further provides a composition comprising a bispecific antibody disclosed herein or pharmaceutical composition thereof and a hyaluronan degrading enzyme. In certain embodiments of the composition, the hyaluronan degrading enzyme is a soluble hyaluronidase. In certain embodiments of the composition, the soluble hyaluronidase is soluble pH20. In certain embodiments, of the composition the soluble hyaluronidase is hyaluronidase (recombinant human) or berahyaluronidase alfa.

[0059] The present invention further provides a kit comprising a bispecific antibody disclosed herein or pharmaceutical composition thereof and a hyaluronan degrading enzyme. In certain embodiments of the kit, the bispecific antibody and the hyaluronan degrading enzyme are provided in separate containers or the bispecific antibody and the hyaluronan degrading enzyme are provided as a mixture in a single container. In certain embodiments of the kit, the bispecific antibody and the hyaluronan degrading enzyme are provided in separate chambers of a dualchamber injection device or the bispecific antibody and the hyaluronan degrading enzyme are provided as a mixture in a single chamber of a single-chamber injection device. In certain embodiments of the kit, the hyaluronan degrading enzyme is a soluble hyaluronidase. In certain embodiments of the kit, the soluble hyaluronidase is soluble pH20. In certain embodiments of the kit, the soluble hyaluronidase is hyaluronidase (recombinant human) or berahyaluronidase alfa.26093

[0060] The present invention further provides a method for treating a proliferative disease in an individual in need of the treatment comprising administering to the individual a therapeutically effective amount of the bispecific antibody of any one of claims 1-15 or the pharmaceutical composition of claim 16 and a hyaluronan degrading enzyme to treat the proliferative disease. In certain embodiments of the method, the proliferative disease comprises cells that display ALPP and / or ALPPL2 on the cell surface. In certain embodiments of the method, the proliferative disease comprises a tumor that displays ALPP and / or ALPPL2 on the surface of the cells thereof. In certain embodiments of the method, the proliferative disease or tumor is a cancer or malignancy selected from the group consisting of mesothelioma, testicular cancer, endometrial cancer, pancreatic cancer, ovarian cancer, non-small cell lung cancer (NSCLC), gastric cancer, bladder cancer, head and neck cancer, cervical cancer, breast cancer, esophageal cancer, cholangiocarcinoma, prostate cancer, and colon cancer. In certain embodiments of the method, the hyaluronan degrading enzyme is a soluble hyaluronidase. In certain embodiments of the method, the soluble hyaluronidase is soluble pH20. In certain embodiments of the method, the soluble hyaluronidase is hyaluronidase (recombinant human) or berahyaluronidase alfa. In certain embodiments of the method, the bispecific antibody and the hyaluronan degrading enzyme are administered to the individual sequentially or simultaneously. In certain embodiments of the method, the bispecific antibody and the hyaluronan degrading enzyme are mixed to form a mixture and the mixture is administered to the individual. In certain embodiments of the method, the bispecific antibody and the hyaluronan degrading enzyme are administered to the individually systemically. In certain embodiments of the method, the bispecific antibody and the hyaluronan degrading enzyme are administered to the individually subcutaneously or intramuscularly.

[0061] The present invention further provides a combination therapy for treating a proliferative disease in an individual in need of the treatment comprising administering to the individual (a) a therapeutically effective amount of a bispecific antibody disclosed herein or pharmaceutical composition thereof, (b) a second therapeutic agent, and (c) a hyaluronan degrading enzyme, to treat the proliferative disease. In certain embodiments of the combination therapy, the second therapeutic agent is a chemotherapy agent or a therapeutic antibody other than an antibody that binds ALPP and / or ALPPL2. In certain embodiments of the combination therapy, the proliferative disease comprises a tumor that displays ALPP and / or ALPPL2 on the surface of the cells thereof. In certain embodiments of the combination therapy, the proliferative disease or tumor is a cancer or malignancy selected from the group consisting of mesothelioma, testicular cancer, endometrial cancer, pancreatic cancer, ovarian cancer, non-small cell lung cancer26093(NSCLC), gastric cancer, bladder cancer, head and neck cancer, cervical cancer, breast cancer, esophageal cancer, cholangiocarcinoma, prostate cancer, and colon cancer. In certain embodiments of the combination therapy, the hyaluronan degrading enzyme is a soluble hyaluronidase. In certain embodiments of the combination therapy, the soluble hyaluronidase is soluble pH20. In certain embodiments of the combination therapy, the soluble hyaluronidase is hyaluronidase (recombinant human) or berahyaluronidase alfa. In certain embodiments of the combination therapy, the bispecific antibody and the hyaluronan degrading enzyme are administered to the individual sequentially or simultaneously. In certain embodiments of the combination therapy, the bispecific antibody and the hyaluronan degrading enzyme are mixed to form a mixture and the mixture is administered to the individual. In certain embodiments of the combination therapy, ALPP / L2 binder and the hyaluronan degrading enzyme are administered to the individually systemically. In certain embodiments of the combination therapy, the bispecific antibody and the hyaluronan degrading enzyme are administered to the individually subcutaneously or intramuscularly.

[0062] The summary of the technology described above is non-limiting and other features and advantages of the technology will be apparent from the following detailed description, and from the claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Fig. 1A shows a dendrogram of the ALPP / L2 binder binning results shown Table 14 of Example 5. The ALPP / L2 binder clones associated with the sample ID numbers shown in the figure are shown in Table 14.

[0064] Fig. IB shows a community plot summarizing the ALPP / L2 binder binning results shown Table 9 of Example 5. The ALPP / L2 binder clones associated with the sample ID numbers shown in the figure are shown in Table 14.

[0065] Fig. 2A shows a ribbon diagram of human ALPPL2 showing the location of the epitopes bound by ALPP / L2 binders 16B5, 17A7. 21H7, and 9G10 as inferred from hydrogen / deuterium exchange (HDX) protein mapping. ALPP / L2 binder 12F3 and its epitope with respect to binding human ALPP are disclosed in WO2022197890.

[0066] Fig. 2B shows a ribbon diagram of human ALPPL2 showing the location of the epitopes bound by ALPP / L2 binders 24F4, 10B3, and 9G10 as inferred from hydrogen / deuterium exchange (HDX) protection mapping.

[0067] Fig. 3 show s dose-dependent potency of ALPP / L2 TCE in a T cell dependent cytotoxicity' (TDCC) assay.26093

[0068] Fig. 4A shows that 24F4 ALPP / L2 TCE. 10B3 ALPP / L2 TCE, and 9G10 ALPP / L2 TCE mediated human T cell activation as shown by the dose-dependent induction of IL-6 in aT-cell potency assay. Error bar + / - SEM.

[0069] Fig. 4B shows that 24F4 ALPP / L2 TCE, 10B3 ALPP / L2 TCE, and 9G10 ALPP / L2 TCE mediated human T cell activation as shown by the dose-dependent induction of TNFa in a T-cell potency assay. Error bar + / - SEM.

[0070] Fig. 4C shows that 24F4 ALPP / L2 TCE, 10B3 ALPP / L2 TCE, and 9G10 ALPP / L2 TCE mediated human T cell activation as shown by the dose-dependent induction of IFNy in a T-cell potency assay. Error bar + / - SEM.

[0071] Fig. 5A shows that 24F4 ALPP / L2 TCE, 10B3 ALPP / L2 TCE, and 9G10 ALPP / L2 TCE induce target-dependent T cell-mediated killing of Capan2 tumor cells. Error bar + / - SEM.

[0072] Fig. 5B shows that 24F4 ALPP / L2 TCE, 10B3 ALPP / L2 TCE, and 9G10 ALPP / L2 TCE induce target-dependent T cell-mediated killing of MDA-MB-468 tumor cells. Error bar + / -SEM.

[0073] Fig. 5C shows that 24F4 ALPP / L2 TCE, 10B3 ALPP / L2 TCE, and 9G10 ALPP / L2 TCE induce target-dependent T cell-mediated killing of HPPAFII tumor cells. Error bar + / -SEM

[0074] Fig. 6 shows the in vivo efficacy of ALPP / L2 TCE in HPAFII-bearing PBMC-engrafted mice.

[0075] Fig. 7 shows that deletion of ALPP / L2 in cancer cell lines does not impact tumor growth.DETAILED DESCRIPTION

[0076] The present invention provides bispecific antibody constructs comprising a first binding domain, which binds to human alkaline phosphatase, placental type (ALPP) and / or alkaline phosphatase, germ cell type (ALPPL2), herein after “ALPP / L2”, on the surface of a tumor cell and a second binding domain, which binds to human CD3 on the surface of a T cell. These anti-ALPP / L2 x anti-CD3 bispecific antibodies are T-cell engagers (TCE) referred to herein as ALPP / L2 TCE, bispecific antibody, or ALPP / L2 TCE BsAb.

[0077] T cell engagers (TCEs) are a class of therapeutic molecules designed to redirect killing of T cells to tumor associated antigen (TAA)-expressing cells. TCEs are very potent molecules exhibiting picomolar in vitro potency and robust efficacy in preclinical models. As of August 2024, nine TCEs have been approved by the FDA, including two for treatment of solid tumors. However, clinical development of TCEs has been hindered by on-target / off-tumor toxicityincluding cytokine release syndrome (CRS) and normal tissue damage. ALPP / L2 are novel TAAs expressed in normal placenta and overexpressed in multiple cancer indications such as ovarian, endometrial, colorectal, non-small cell lung cancer (NSCLC), mesothelioma, testicular, gastric, pancreatic, bladder cancer. The tumor specificity of ALPP / L2 expression targets the ALPP / L2 TCE to malignant cells at clinically active doses, while sparing normal tissues.

[0078] As an example, over 25,000 women in the United States died from ovarian or endometrial cancer in 2024. Surgical resection and platinum-based chemotherapy have been the standard of care therapies for decades. While anti-angiogenic therapies for ovarian cancer, PARP inhibition in BRCA-mutated cancers, and immune checkpoint blockade in endometrial cancer have been recent advances, these gynecological malignancies still represent an area of high unmet medical need. Novel treatment modalities such as T cell engagers, which generally have non-overlapping toxicities with other standard of care therapies, may represent a critical addition to the therapeutic arsenal against gynecological malignancies.

[0079] Currently, the TCE landscape is concentrated in Hematologic malignancies. To date there has been limited success with TCEs in solid tumors. Of the 11 TCE bispecific antibodies that have gained regulator}' approval, the solid tumor setting accounts for two of the approvals, including the latest FDA approval of tarlatamab in small cell lung cancer (SCLC). Recent trends show higher interest in developing TCEs in solid tumors, with -66% of Phase II trials evaluating TCEs focused on solid tumor malignancies.

[0080] However, the unmet medical need in ALPP / L2 expressing tumors remains high. Newly diagnosed estimates in the United States for 2023 indicate the largest populations with ALPP / L2 expression are lung, endometrial, and gastric cancers. In terms of ALPP / L2 expression levels, ovarian, endometrial, and bladder cancers rank highest.

[0081] The ALPP / L2 TCE bispecific antibodies disclosed herein provide a potential for increased depth of response (more potent modality); improved durability (no acquired resistance to payload), and increase efficacy in combination with chemotherapy, antibody-drug conjugates (ADCs), immune-oncology (IO), or other targeted therapies.

[0082] The present disclosure further provides an engineered anti-CD3 scFv derived from SP34.185 optimized for lower aggregation and also better thermal stability in the scFv format.Definitions

[0083] Listed below are definitions of various terms used herein. These definitions apply to the terms as they are used throughout this specification and claims, unless otherwise limited in specific instances, either individually or as part of a larger group.

[0084] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and peptide chemistry are those well-known and commonly employed in the art.

[0085] As used herein, the articles “a” and '“an” refer to one or to more than one (i. e.. to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Furthermore, use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting.

[0086] As used herein, the term “about” in quantitative terms refers to plus or minus 10% of the value it modifies (rounded up to the nearest whole number if the value is not sub-dividable, such as a number of molecules or nucleotides).

[0087] All ranges disclosed herein are inclusive of the recited endpoint and independently combinable (for example, the range of “from 50 mg to 500 mg” is inclusive of the endpoints, 50 mg and 500 mg, and all the intermediate values). The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value; they are sufficiently imprecise to include values approximating these ranges and / or values.

[0088] As used herein, the term “comprising” may include the embodiments “consisting of’ and “consisting essentially of.” The terms “comprise(s),” “include(s).” “having,” “has,” “may.” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that require the presence of the named ingredients / steps and permit the presence of other ingredients / steps. However, such description should be construed as also describing compositions or processes as “consisting of and “consisting essentially of the enumerated components, which allows the presence of only the named components or compounds, along with any acceptable carriers or fluids, and excludes other components or compounds.

[0089] As used herein, the terms “ALPP,” “alkaline phosphatase,” “alkaline phosphatase, placental,” “ALPase.” or “PLAP” are used interchangeably, and, unless otherwise specified, include any naturally occurring variants (e.g., splice variants, allelic variants), isoforms, and vertebrate species homologs of human ALPP. The term encompasses “full length,” unprocessed ALPP as well as any form of ALPP that results from processing within a cell, e.g., “proprotein” comprising amino acids 23-535 and “mature” protein form comprising amino acids 23-506. The amino acid sequence of an exemplary human ALPP is provided in Uniprot ID: P05187 or RefSeq ID: NM_001632. The amino acid sequence of one specific example of human ALPP protein is set forth in SEQ ID NO: 379.

[0090] As used herein, the terms “ALPPL2,” “alkaline phosphatase, placental-like 2,” or “alkaline phosphatase, germ cell” are used interchangeably, and, unless otherwise specified, include any naturally occurring variants (e.g., splice variants, allelic variants), isoforms, and vertebrate species homologs of human ALPPL2. The term encompasses “full length,” unprocessed ALPPL2 as well as any form of ALPPL2 that results from processing within a cell, e.g., “proprotein” comprising amino acids 20-532 and “mature” protein form comprising amino acids 20-503. The amino acid sequence of an exemplary human ALPPL2 is provided in Uniprot ID: P10696 or RefSeq ID: NM_031313. The amino acid sequence of one specific example of a human ALPPL2 protein is set forth in SEQ ID NO: 380.

[0091] As used herein, the terms “ALPI” or “alkaline phosphatase, intestinal” are used interchangeably, and, unless otherwise specified, include any naturally occurring variants (e.g., splice variants, allelic variants), isoforms, and vertebrate species homologs of human ALPI. The term encompasses “full length,” unprocessed ALPI as well as any form of ALPI that results from processing within a cell, e.g., “proprotein” and “mature” protein forms. The amino acid sequence of an exemplary human ALPI is provided in Uniprot ID: P09923 or RefSeq ID: NM 001631.

[0092] As used herein, the terms “ALPL”, alkaline phosphatase, liver / bone / kidney”, or “alkaline phosphatase, tissue non-specific” are used interchangeably, and, unless otherwise specified, include any naturally occurring variants (e.g.. splice variants, allelic variants), isoforms, and vertebrate species homologs of human ALPL. The term encompasses “full length,” unprocessed ALPL as well as any form of ALPL that results from processing within a cell, e.g., “proprotein” and “mature” protein forms. The amino acid sequence of an exemplary human ALPL is provided in Uniprot ID: P05186 or RefSeq ID: NM_000478.

[0093] As used herein, the term “ALPP / L2 binder” refers to a molecule disclosed herein that binds ALPP and / or ALPPL2. An ALPP / L2 binder may be an antibody or antigen-binding fragment thereof that specifically binds ALPP and / or ALPPL2 w ith no detectable binding to ALPI or ALPL and are referred to herein as anti-ALPP / L2 antibodies or anti-ALPP / L2-binding fragments thereof. Because ALPP and ALPPL2 share high sequence homology at 98 percent and have a similar expression profile, being expressed in the placenta but largely absent in normal adult tissue, the ALPP / L2 binders disclosed herein specifically bind both ALPP and ALPPL2 with no detectable binding to ALPI or ALPL.

[0094] As used herein, the term "affinity" refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, "binding affinity" refers to intrinsic binding affinity which reflects a 1: 1 interaction betw een members of a binding pair (e.g.,26093antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including KinExA and surface plasmon resonance (SPR; Biacore™). Specific illustrative and exemplary embodiments for measuring binding affinity are described in the following.

[0095] As used herein, the term "administration" and "treatment," as it applies to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to contact of an exogenous pharmaceutical, therapeutic, diagnostic agent, or composition comprising a human MSLN binder as disclosed herein to the animal, human, subject, cell, tissue, organ, or biological fluid. Treatment of a cell encompasses contact of a reagent to the cell, as well as contact of a reagent to a fluid, where the fluid is in contact with the cell. " Administration" and "treatment" also means in vitro and ex vivo treatments, e.g., of a cell, by a reagent, diagnostic, binding compound, or by another cell. The term "subject" includes any organism, preferably an animal, more preferably a mammal (e.g., human, rat, mouse, dog, cat. rabbit). In a preferred embodiment, the term “subject” refers to a human.

[0096] As used herein, the term ‘'amino acid” refers to a simple organic compound containing both a carboxyl ( — COOH) and an amino ( —NH2) group. Amino acids are the building blocks for proteins, polypeptides, and peptides. Amino acids occur in L-form and D-form, with the L-form in naturally occurring proteins, polypeptides, and peptides. Amino acids and their code names are set forth in the following chart.Amino acid Three letter One lettercode codeAlanine Ala AArginine Arg RAsparagine Asn NAspartic acid Asp DCysteine Cys CGlutamine Gin QGlutamic acid Glu EGlvcine Glv GHistidine His HIsoleucine He ILeucine Leu LLysine Lvs KMethionine Met MPhenylalanine Phe FProline Pro PSerine Ser SThreonine Thr T26093Amino acid Three letter One lettercode codeTryptophan Trp WTyrosine Tyr YValine Val V

[0097] As used herein, the term "antibody" or “immunoglobulin” as used herein refers to a glycoprotein comprising at least two heavy chains (HCs) and two light chains (LCs) interconnected by disulfide bonds. A typical IgGl or IgG4 antibody is divalent and contains two identical LCs, two identical HCs, and a total of 16 disulfide bonds, including four inter-chain disulfide bonds in the hinge region of the HC and 12 intra-chain disulfide bonds associated with 12 individual domains of the antibody. There are two inter-chain disulfide bonds that connect the cysteine residue of one hinge region to the other hinge region and there is one disulfide bond that connects a cysteine residue in the constant domain of one light chain to the cysteine residue in one hinge region and one disulfide bond that connects a cysteine residue in the constant domain of the other light chain to the cysteine residue in the other hinge region. Each HC is comprised of a heavy chain variable region or domain (VH) and a heavy chain constant region or domain (CH). Each light chain is comprised of an LC variable region or domain (VL) and a LC constant domain (CL). In certain naturally occurring IgG, IgD, and IgA antibodies, the heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. In general, the basic antibody structural unit for antibodies is a Y-shaped tetramer comprising two HC / LC pairs (2H). Each tetramer includes two identical pairs of polypeptide chains, each pair having one LC (about 25 kDa) and HC chain (about 50-70 kDa) (H+L). Each HC: LC pair comprises one VH: VL pair. A single VH: VL pair may be referred to by the term “Fab”. The term “Fab” as used herein refers to “fragment antigen binding region.” Thus, each antibody tetramer comprises two Fabs, one per each arm of the Y-shaped antibody.

[0098] The human VH includes seven family members: VH1, VH2, VH3, VH4, VH5, VH6, and VH7; and the human VL includes 16 family members: Vκ1, Vκ2, Vκ3, Vκ4, Vκ5, Vκ6, Vλ1, Vλ2, Vλ3, Vλ4, Vλ5, Vλ6, Vλ7, Vλ8, Vλ9, and Vλ10. Each of these family members can be further divided into particular subtypes. The VH and VL can be further subdivided into regions of hypervariability, termed complementarity determining region (CDR) areas, interspersed with regions that are more conserved, termed framework regions (FR). Each VH is composed of three CDR regions (VH-CDR1, VH-CDR2. VH-CDR3) and four FR regions, arranged from aminoterminus to carboxy-terminus in the following order: FR-H1, VH-CDR1, VH-FR2, VH-CDR2, VH-FR3, VH-CDR3, VH-FR4. Each VL is composed of three CDR regions (VL-CDR1, VL-26093CDR2. VL-CDR3) and four FR regions, arranged from amino-terminus to carboxy -terminus in the following order: VL-FR1, VL-CDR1, VL-FR2, VL-CDR2, VL-FR3, VL-CDR3, VL-FR4. Numbering of the amino acids in a VH and VL may be determined using the Kabat numbering scheme. See Béranger et al., Ed. Ginetoux, Correspondence between the IMGT unique numbering for C-DOMAIN. the IMGT exon numbering, the Eu and Kabat numberings: Human IGHG, created: 17 / 05 / 2001, Version: 08 / 06 / 2016, which is accessible atwww.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html).

[0099] The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. Typically, the numbering of the amino acids in the heavy chain constant domain begins with number 118, which is in accordance with the Eu numbering scheme. The Eu numbering scheme is based upon the amino acid sequence of human IgGl (Eu). which has a constant domain that begins at amino acid position 118 of the amino acid sequence of the IgGl described in Edelman et al., Proc. Natl. Acad. Sci. USA. 63: 78-85 (1969), and is shown for the IgGl, IgG2, IgG3, and IgG4 constant domains in Béranger et al., op. cit.

[0100] The CDRs of the variable regions of the heavy and light chains form a binding domain that interacts with an antigen. A number of methods are available in the art for defining CDR sequences of the antibody variable domains (See Dondelinger et al.. Frontiers in Immunol. 9: Article 2278 (2018)).

[0101] Kabat, Chothia, AbM, Contact, and IMGT are all numbering schemes for antibodies and T cell receptors that differ in the way they are developed and the information they convey.

[0102] The Kabat numbering scheme is based on sequence variability and is the most commonly used scheme (See Kabat et al. Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) (defining the CDR regions of an antibody by sequence). It is based on alignments of known antibody sequences and defines variability as the number of different amino acids at a given position. However, the positions where insertions occur in VL-CDR1 and VH-CDR1 don't match the structural insertion positions.

[0103] The Chothia numbering scheme is based on the location of the structural loop region (See Chothia & Lesk, J. Mol. Biol. 196: 901-917 (1987); Al-Lazikani et al., J. Mol. Biol. 273: 927-948 (1997)). It is similar to Kabat but places VL-CDR1 and VH-CDR1 insertions at the structurally correct positions.26093

[0104] AbM numbering scheme is a compromise between the Kabat and Chothia used by Oxford Molecular's AbM antibody modelling software (see Karu et al., ILAR Journal 37: 132-141 (1995).

[0105] Contact numbering scheme is based on an analysis of the available complex crystal structures (See www.bioinf.org.uk: Prof. Andrew C. R. Martin's Group; Abhinandan & Martin, Mol. Immunol. 45:3832-3839 (2008)).

[0106] IMGT (ImMunoGeneTics) numbering scheme is a standardized numbering system for all the protein sequences of the immunoglobulin superfamily, including variable domains from antibody light and heavy chains as well as T cell receptor chains from different species and counts residues continuously from 1 to 128 based on the germ-line V sequence alignment (see Giudicelli et al., Nucleic Acids Res. 25:206-11 (1997); Lefranc, Immunol Today 18:509(1997); Lefranc et al., Dev Comp Immunol. 27:55-77 (2003)).

[0107] The following general rules disclosed in www.bioinf.org.uk: Prof. Andrew C. R.Martin's Group and reproduced in Tables 1 and 2 below provide guidance for defining the CDRs in an antibody sequence that includes those amino acids that specifically interact with the amino acids comprising the epitope in the antigen to which the antibody binds. There are rare examples where these generally constant features do not occur; however, the Cys residues are the most conserved feature.Table 1VL-CDR1Start About amino acid residue 24Residue before Usually a CvsResidue after Usually a Trp. Typically, Trp-Tyr-Gln, but also, Trp- Leu-Gln, Trp-Phe-Gln, or Trp-Tvr-LeuLength 10 to 17 amino acid residuesVL-CDR2Start Usually 16 amino acid residues after the end of VL- CDR1Residues before Generally, Ile-Tyr, but also, Val-Tyr, Ile-Lys, or Ile-PheLength Usually seven amino acid residuesVL-CDR3Start Usually 33 amino acid residues after end of VL-CDR2Residue before Usually CvsResidues after Usually Phe-Gly-Xaa-GlyLength Seven to 11 amino acid residuesVH-CDR1Start About amino acid residue 26 (usually four amino acidresidues after a Cys) [Chothia / AbM definition]; Kabatdefinition starts five amino acid residues later26093Residues before Usually Cys-Xaa-Xaa-XaaResidues after Usually aTrp. Typically Trp-Val, but also, Trp-Ile or Trp-AlaLength 10 to 12 amino acid residues [AbM definition]; Chothia definition excludes the last four amino acid residues VH-CDR2Start Usually 15 amino acid residues after the end of Kabat / AbM definition) of VH- CDR1Residues before Typically Leu-Glu-Trp-Ile-Gly (SEQ ID NO: 608), but a number of variationsResidues after Lys / Arg-Leu / Ile / Val / Phe / Thr / Ala-Thr / Ser / Ile / Ala Length Kabat definition 16 to 19 amino acid residues; AbM (and recent Chothia) definition ends seven amino acid residues earlierVLH-CDR3Start Usually 33 amino acid residues after end of VH- CDR2(usually two amino acid residues after a Cys) Residues before Usually Cvs-Xaa-Xaa (typically Cys-Ala-Arg) Residues after Usually Trp-Gly-Xaa-GlyLength Three to 25 amino acid residuesTable 2VL-CDR Kabat AbM Chothial Contact^ IMGT LoopLI L24-L34 L24-L34 L24-L34 L30-L36 L27—L32 L2 L50-L56 L50-L56 L50-L56 L46-L55 L50- L52 L3 L89-L97 L89-L97 L89-L97 L89-L96 L89- L97 VH-CDR Kabat AbM Chothia^ Contact^ IMGT LoopHl H31-H35B H26- H26- H30— H26- (Kabat H35B H32.34 H35B H35B Numbering)^Hl H31-H35 H26-H35 H26- H30— H26- (Chothia H32 H35 H33 Numbering)H2 H50-H65 H50-H58 H52- H47— H51—H56 H58 H5626093H3 H95-H102 H95— H95- H93— H93- H102 H102 H101 H102ISome of these numbering schemes (particularly for Chothia loops) varydepending on the individual publication examined.2 Any of the numbering schemes can be used for these CDR definitions, exceptthe Contact numbering scheme uses the Chothia or Martin (Enhanced Chothia)definition.^The end of the Chothia VH-CDR1 loop when numbered using the Kabatnumbering convention varies between H32 and H34 depending on the lengthof the loop. (This is because the Kabat numbering scheme places the insertionsat H35A and H35B.)If neither H35A nor H35B is present, the loop ends at H32If only H35A is present, the loop ends at H33If both H35A and H35B are present, the loop ends at H34

[0108] In general, the state of the art recognizes that in many cases, the VH-CDR3 region of the heavy chain is the primary determinant of antibody specificity, and examples of specific antibody generation based on HV-CDR3 alone are known in the art (e.g., Beiboer et al., J. Mol. Biol. 296: 833-849 (2000); Klimka et al., British J. Cancer 83: 252-260 (2000); Rader et al., Proc. Natl. Acad. Sci. USA 95: 8910-8915 (1998); Xu et al., Immunity 13: 37-45 (2000).

[0109] The entire amino acid sequence of the VH and VL are commonly numbered according to Kabat while the three CDRs within the variable region may be defined according to any one of the aforementioned numbering schemes. In particular embodiments, the numbering of the amino acid positions in the VH and VL may be sequential beginning with amino acid position 1 and continuing sequentially to the end of the sequence or according to Kabat. Unless specified otherwise, the amino acid positions in the VH and VL herein are defined according to sequential numbering.

[0110] The numbering of the amino acid positions in the heavy chain constant domain may be sequential beginning with amino acid position 1 and continuing sequentially to the end of the sequence or according to IMGT. Kabat, or Eu numbering. The IgGl heavy chain constant domain amino acid sequence has 330 amino acids, which may be sequentially numbered 1 to 330. The corresponding sequence numbered according to the EU numbering scheme begins with position number 118 and ends with position number 447. Unless specified otherwise, the amino acid positions in the heavy and light chains herein are defined according to sequential numbering.

[0111] The numbering of the amino acid positions in the light chain constant domain may be sequential beginning with amino acid position 1 and continuing sequentially to the end of the sequence or according to IMGT, Kabat, or Eu numbering. The IgGl light chain constant domain26093amino acid sequence has 107 amino acids, which may be sequentially numbered 1 to 107. The corresponding sequence numbered according to the EU numbering scheme begins with position number 108 and ends with position number 214. Unless specified otherwise, the amino acid positions in the heavy and light chains herein are defined according to sequential numbering.

[0112] As used herein, the term " Fc region”, or “Fc” as used herein is the crystallizable fragment region obtained from an antibody that comprises the CH2 and CH3 domains of the antibody. Since an antibody is a dimer comprising two VH-CH1-CH2-CH3 protein chains, the Fc region is a dimer comprising the CH2-CH3 domains of both VH-CH1-CH2-CH3 protein chains. In an antibody, the two Fc regions are held together by two or more disulfide bonds and by¬ hydrophobic interactions of the CH3 domains. The Fc region may be obtained by digesting an antibody with the protease papain. Typically, amino acids of the CH2-CH3 domains of the Fc region are numbered according to the Eu numbering convention (See Edelmann et al., Biochem.63: 78-85 (1969)).

[0113] As used herein, the term "antigen" as used herein refers to any foreign substance which induces an immune response in the body. In the present invention, an antigen refers to ALPP and / or ALPPL2 or a fragment of ALPP and / or ALPPL2.

[0114] As used herein, the term “antigen binding fragment” refers to a polypeptide or polypeptides comprising a fragment of a full-length antibody, which retains the ability to specifically bind to the antigen bound by the full-length antibody, and / or to compete with the full-length antibody for specifically binding to the antigen. Examples of antigen binding fragments include but are not limited to Fab fragment, Fab’ fragment, F(ab’)2 fragment, Fv region, and scFv.

[0115] As used herein, the terms “Fab”, " Fab fragment", or " Fab molecule” may be used interchangeably and refer to an antigen binder comprising one antibody LC (VL and CL) and the CHI and VH of one antibody HC. The HC component of a Fab molecule cannot form a disulfide bond with another HC molecule. A " Fab fragment" can also be the product of papain cleavage of an antibody.

[0116] As used herein, the term " Fab1fragment" refers to an antigen binder comprising one antibody LC and a portion or fragment of one antibody HC that contains the VH and the CHI domain up to a region between the CHI and CH2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form a F(ab')2 molecule.

[0117] As used herein, the term " F(ab')2 fragment" refers to an antigen binder comprising two antibody LCs and two HCs containing the VH and the CHI domain up to a region between the CHI and CH2 domains, such that an interchain disulfide bond is formed between the two HCs.26093An F(ab')2 fragment thus is composed of two Fab' fragments that are held together by a disulfide bond between the two HCs. An " F(ab')2 fragment" can be the product of pepsin cleavage of an antibody.

[0118] As used herein, the term " Fv region" refers to an antigen binder comprising the VH and VL of an antibody but lacks the constant regions.

[0119] As used herein, the term “ScFv” or “single-chain variable fragment” refers to a fusion protein comprising a VH and VL fused or linked together by a short linker peptide of ten to about 25 amino acids. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH to the C-terminus of the VL, or N-terminus of the VL to the C-terminus of the VH. The scFv retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker.

[0120] These and other potential constructs are described at Chan & Carter (2010) Nat. Rev. Immunol. 10: 301. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. Antigen-binding fragments can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.

[0121] As used herein, the term "isolated” antibodies or antigen-binding fragments thereof are at least partially free of other biological molecules from the cells or cell cultures in which they are produced. Such biological molecules include nucleic acids, proteins, lipids, carbohydrates, or other material such as cellular debris and growth medium. An isolated antibody or antigenbinding fragment may further be at least partially free of expression system components such as biological molecules from a host cell or of the growth medium thereof. Generally, the term "isolated" is not intended to refer to a complete absence of such biological molecules or to an absence of water, buffers, or salts or to components of a pharmaceutical formulation that includes the antibodies or fragments.

[0122] As used herein, the term "monoclonal antibody" refers to a population of substantially homogeneous antibodies, i.e., the antibody molecules comprising the population are identical in amino acid sequence except for possible naturally occurring mutations that may be present in minor amounts. In contrast, conventional (polyclonal) antibody preparations typically include a multitude of different antibodies having different amino acid sequences in their variable domains that are often specific for different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be26093made by the hybridoma method first described by Kohler et al., Nature 256: 495 (1975) or may be made by recombinant DNA methods (see, e g., U. S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 352: 624-628 (1991), and Marks et al., J. Mol. Biol. 222: 581-597 (1991), for example. See also Presta, J. Allergy Clin. Immunol. 116: 731 (2005).

[0123] As used herein, the term “bispecific antibody” or “BsAb) refers to an antibody that has two distinct binding domains that can bind to two antigens or two epitopes (an antigen part) of the same antigen simultaneously. In particular embodiments, the binding domains may comprise Fabs. scFvs, or a combination of Fab and scFv.

[0124] As used herein, the term ‘'T-cell engagers” or “TCE” refers to bispecific antibodies (“BsAbs”) comprising a first arm comprising a first binding domain that binds a tumor-associated antigen (“TAA”) displayed on the surface of a tumor cell and a second arm comprising a second binding domain that binds CD3 displayed on the surface of a T-cell, which directly activates T-cells and their anti -tumor features, ultimately resulting in the lysis of the targeted tumor cells.

[0125] As used herein, the term “ALPP / L2 TCE” refers to an anti-ALPP / L2 x anti-CD3 bispecific antibody comprising a first arm comprising a first binding domain that binds to ALPP and / or ALPPL2 displayed on the surface of certain tumor cells and a second arm comprising a second binding domain that binds CD3 displayed on the surface of a T-cell, which directly activate T-cells and their anti-tumor features, ultimately resulting in the lysis of tumor cells that display ALPP and / or ALPPL2 on the surface of the cell.

[0126] As used herein, the term "gene" is used broadly to refer to any segment of nucleic acid associated with a biological function. Thus, genes include coding sequences and / or the regulatory sequences required for their expression. For example, "gene" refers to a nucleic acid fragment that expresses mRNA, functional RNA, or specific protein, including regulatory sequences. " Genes" also include nonexpressed DNA segments that, for example, form recognition sequences for other proteins. " Genes" can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters. Genes include both naturally occurring nucleotide sequences encoding a molecule of interest and synthetically derived nucleotide sequences encoding a molecule of interest, for example, complementary DNA (cDNA) obtained from a messenger RNA (mRNA) nucleotide sequence.

[0127] As used herein, the term “germline” or "germline sequence" refers to a sequence of unrearranged immunoglobulin DNA sequences. Any suitable source of unrearranged26093immunoglobulin sequences may be used. Human germline sequences may be obtained, for example, from JOINSOLVER® germline databases on the website for the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the United States National Institutes of Health. Mouse germline sequences may be obtained, for example, as described in Giudicelli et al.. Nucleic Acids Res. 33: D256-D261 (2005).

[0128] As used herein, the term ‘'library” as used herein is, typically, a collection of related but diverse polynucleotides that are, in general, in a common vector backbone. For example, a light chain or heavy chain immunoglobulin library may contain polynucleotides, in a common vector backbone, that encode light and / or heavy chain immunoglobulins, which are diverse but related in their nucleotide sequence; for example, which immunoglobulins are functionally diverse in their abilities to form complexes with other immunoglobulins, e.g., in an antibody display system of the present invention, and bind a particular antigen.

[0129] As used herein, the term “polynucleotides” discussed herein form part of the present invention. A "polynucleotide", “polynucleic acid”, "nucleic acid " or "nucleic acid molecule" include DNA and RNA, single- or double-stranded. Polynucleotides e g., encoding an immunoglobulin chain or component of the antibody display system of the present invention, may, in an embodiment of the invention, be flanked by natural regulatory (expression control) sequences, or may be associated with heterologous sequences, including promoters, internal ribosome entry sites (IRES) and other ribosome binding site sequences, enhancers, response elements, suppressors, signal sequences, polyadenylation sequences, introns, 5’- and 3'-non-coding regions, and the like.

[0130] Polynucleotides e.g., encoding an immunoglobulin chain or component of the antibody display system of the present invention, may be operably associated with a promoter. A “promoter” or “promoter sequence” is, in an embodiment of the invention, a DNA regulatory region capable of binding an RNA polymerase in a cell (e.g., directly or through other promoterbound proteins or substances) and initiating transcription of a coding sequence. A promoter sequence is, in general, bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at any level. Within the promoter sequence may be found a transcription initiation site (conveniently defined, for example, by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. The promoter may be operably associated with other expression control sequences, including enhancer and repressor sequences or with a nucleic acid of the invention. Promoters which may be used to control gene expression include, but are not limited to, cytomegalovirus (CMV) promoter (U. S.26093Patent Nos. 5,385,839 and 5,168,062), the SV40 early promoter region (Benoist, et al.. Nature 290: 304-310 (1981)), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell 22: 787-797 (1980)), the herpes thymidine kinase promoter (Wagner et al.. Proc. Natl. Acad. Sci. USA 78: 1441-1445 (1981)), the regulatory sequences of the metallothionein gene (Brinster et al., Nature 296: 39-42 (1982)); prokaryotic expression vectors such as the β-lactamase promoter (Villa-Komaroff et al.. Proc. Natl. Acad. Sci. USA 75: 3727-3731 (1978)), or the tac promoter (DeBoer et al., Proc. Natl. Acad. Sci. USA 80: 21-25 (1983)); see also " Useful proteins from recombinant bacteria" in Scientific American 242: 74-94 (1980); and promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter or the alkaline phosphatase promoter.

[0131] As used herein, the terms "vector", "cloning vector" and "expression vector" include a vehicle (e.g., a plasmid) by which a DNA or RNA sequence can be introduced into a host cell so as to transform the host and, optionally, promote expression and / or replication of the introduced sequence. Polynucleotides encoding an immunoglobulin chain or component of the antibody display system of the present invention may, in an embodiment of the invention, be in a vector.

[0132] As used herein, the terms "cell," "cell line," and "cell culture" are used interchangeably and all such designations include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that not all progeny will have precisely identical DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.

[0133] As used herein, the term "control sequences" or “regulatory sequences” refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for expression in eukaryotes, for example, include a promoter, operator or enhancer sequences, transcription termination sequences, and polyadenylation sequences for expression of a messenger RNA encoding a protein and a ribosome binding site for facilitating translation of the messenger RNA.

[0134] As used herein, a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence, e g., a regulatory sequence. For example, DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or26093a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0135] As used herein, the term "encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.

[0136] As used herein, the term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence.

[0137] As used herein, the term "treat" or "treating" means to administer a therapeutic agent, such as a composition containing any of the ALPP / L2 binders of the present invention, topically, subcutaneously, intramuscular, intradermally, or systemically to an individual in need. The amount of a therapeutic agent that is effective to treat cancer or proliferative disease in the individual may vary according to factors such as the injury or disease state, age, and / or weight of the individual, and the ability of the therapeutic agent to elicit a desired response in the individual. Whether the therapeutic objective has been achieved can be assessed by the individual and / or any clinical measurement typically used by physicians or other skilled healthcare providers to assess the severity or progression status of the treatment. Thus, the terms denote that a beneficial result has been or will be conferred on a human or animal individual in need.

[0138] As used herein, the term "treatment," as it applies to a human or veterinary individual, refers to both therapeutic treatment and prophylactic or preventative measures in the management and care of a patient or condition to prevent, cure, ameliorate, or slow the progression of a26093medical condition. This can include the use of drugs, biologies, diagnostic assays to assist in formulation of treatment regimes, or other medical procedures. A physician or veterinarian typically orders treatment as part of a care plan after a diagnosis. " Treatment" as it applies to a human or veterinary individual, encompasses contact of the ALPP / L2 TCEs of the present invention to a human or animal subject.

[0139] As used herein, the term ‘'therapeutically effective amount” refers to a quantity of a specific substance sufficient to achieve a desired effect in an individual being treated. For instance, this may be the amount necessary to inhibit or reduce the severity of a disease or disorder in an individual.

[0140] As used herein, the term ‘'combination therapy” refers to treatment of a human or animal individual comprising administering a first therapeutic agent and a second therapeutic agent consecutively or concurrently to the individual wherein at least one of the therapeutic agents is a bispecific antibody of the present invention. In general, the first and second therapeutic agents are administered to the individual separately and not as a mixture; however, there may be embodiments where the first and second therapeutic agents are mixed prior to administration.

[0141] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “engineered cells,” '‘transformants,” and '‘transformed cells,” which include the primary engineered (e.g., transformed) cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein. As appropriate, the host cells can be stably or transiently transfected with a polynucleotide encoding a fusion protein, as described herein.Bispecific Antibodies (ALPP / L2 TCEs)

[0142] The bispecific antibodies of the present invention (ALPP / L2 TCEs) comprise a first binding domain, which binds to human ALPP and / or ALPPL2 on the surface of a target cell, and a second binding domain, which binds to human CD3 on the surface of a T cell. The bispecific antibodies herein are ALPP / L2 T-cell engagers (ALPP / L2 TCEs) since they can simultaneously bind ALPP and / or ALPPL2 and CD3. The first binding domain comprises a first heavy chain variable domain (VH) comprising the complementarity determining regions (VH-CDRs) 1, 2, and 3; and a first light chain variable domain (VL) comprising CDRs (VL-CDRs) 1, 2, and 3,26093both of an ALPP / L2 binder disclosed herein. The second binding domain comprises a second VH comprising the VH-CDRs 1, 2, and 3; and a second VL comprising the VL-CDRs 1, 2, and 3, both of a CD3 binder disclosed herein.

[0143] In a further embodiment, the first binding domain comprises the first VH and first VL of an ALPP / L2 binder disclosed herein and the second binding domain comprises the second VH and second VL of a CD3 binder disclosed herein. In particular embodiments, the first binding domain comprises the first VH and first VL in a Fab format and the second binding domain comprises the second VH and second VL in a Fab format. In particular embodiments, the first binding domain comprises the first VH and first VL in a Fab format and the second binding domain comprises the second VH and second VL in an scFv format. In particular embodiments, the first binding domain comprises the first VH and first VL in an scFv format and the second binding domain comprises the second VH and second VL in a Fab format. In particular embodiments, the first binding domain comprises the first VH and first VL in an scFv format and the second binding domain comprises the second VH and second VL in an scFv format.

[0144] The scFv format may comprise a structure from the N-terminus to the C-terminus selected from VL-linker-VH or VH-linker-VL, wherein the linker is a short flexible linker.

[0145] The first binding domain of the bispecific antibody is linked to the N-terminus of a first heavy chain constant region (CH) comprising at least first CH2 and CH3 domains and the second binding domain is linked to the N-terminus of a second CH comprising at least second CH2 and CH3 domains; wherein CH3 domains of the first CH and the second CH each comprise one or more mutations promoting heterodimerization of the first and second CH3 domains to form a heterodimeric Fc region at the base of the Y structure of the bispecific antibody with a first arm comprising the first binding domain and a second arm comprising the second binding domain. As used herein, the term “Fc region’’ refers to the heterodimer formed by the association of a first CH3 domain with a second CH3 domain.

[0146] In certain embodiments of the bispecific antibody, the first binding domain is a Fab comprising a first VH-CH and VL-CL pair and the second binding domain is an scFv comprising from the N-terminus to the C-terminus VL-VH-CH’ or VH-VL-CH’, wherein the CH comprises CHI, Hinge, CH2, and CH3 domains and the CH’ is a truncated CH comprising at least CH2 and CH3 domains. The CH and CH’ each comprise one or more amino acid substitutions in their CH3 domains that promote heterodimerization of the CH and CH' to form a bispecific antibody with a first arm comprising the first binding domain and a second arm comprising the second binding domain. In further embodiments, the CH’ comprises at least amino acids 8 to 15 of the Hinge and the CH2 and CH3 domains.26093

[0147] In certain embodiments of the bispecific antibody, the first binding domain is a first Fab comprising a first VH-CH and VL-CL pair and the second binding domain is a second Fab comprising a second VH-CH and VL-CL pair, wherein the CH3 domain of the first VH-CH and VL-CL pair and the CH3 domain of the second VH-CH and VL-CL pair each comprise one or more amino acid substitutions that promote heterodimerization to form a heterodimeric Fc region of the bispecific antibody with a first arm comprising the first binding domain and a second arm comprising the second binding domain.

[0148] In certain embodiments of the bispecific antibody, the first binding domain is an scFv comprising from the N-terminus to the C-terminus VL-VH-CH' or VH-VL-CH’ and the second binding domain is a Fab comprising a first VH-CH and VL-CL pair, wherein the CH comprises CHI, Hinge, CH2, and CH3 domains and the CH’ is a truncated CH comprising at least CH2 and CH3 domains. The CH and CH’ each comprise one or more amino acid substitutions in their CH3 domains that promote heterodimerization of the CH and CH’ to form an Fc region of a bispecific antibody with a first arm comprising the first binding domain and a second arm comprising the second binding domain. In further embodiments, the CH’ comprises at least amino acids 8 to 15 of the Hinge and the CH2 and CH3 domains.

[0149] In certain embodiments of the bispecific antibody, the first binding domain is an scFv comprising from the N-terminus to the C-terminus a first VL-VH-CH' or VH-VL-CH’ and the second binding domain is an scFv comprising from the N-terminus to the C-terminus a second VL-VH-CH’ or VH-VL-CH’, wherein the first and second CH’ are each a truncated CH comprising at least CH2 and CH3 domains. The first CH’ and second CH’ each comprise one or more amino acid substitutions in their CH3 domains that promote heterodimerization of the first CH’ and second CH’ to form an Fc region of a bispecific antibody with a first arm comprising the first binding domain and a second arm comprising the second binding domain. In further embodiments, the first and second CH’ comprise at least amino acids 8 to 15 of the Hinge and the CH2 and CH3 domains.

[0150] In certain embodiments of the bispecific antibodies, the CH. CH’, and CL further independently comprise one or more amino acid substitutions, deletions, insertions, or combinations thereof. In certain embodiments, the CH, CH’, and CL further independently comprise 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, insertions, or combinations thereof.

[0151] In certain embodiments of the bispecific antibodies, the CH and CH’ are of an IgGl, IgG2, IgG3, or IgG4 isotype and CL is of the human kappa or human lambda isotype. In further embodiments, the VH is linked to the CH of the IgGl or IgG4 isoty pe and the VL is linked to the26093CL of the human kappa (e.g., the amino acid sequence shown in SEQ ID NO: 391) or human lambda isotype. In further embodiments, the IgGl or IgG4 isotype may include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, insertions, deletions, or combinations thereof compared to the native human IgGl or IgG4 isotype. In certain embodiments, the CH and CH’ are of the IgGl isotype and may in certain embodiments, comprise 1, 2. 3, 4, 5. 6, 7, 8. 9, or 10 amino acid substitutions, insertions, deletions, or combinations thereof compared to the amino acid sequence of the native IgGl isotype. In certain embodiments, the CL is of the kappa isotype and may in particular embodiments, comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, insertions, deletions, or combinations thereof compared to the amino acid sequence of the native kappa isotype.

[0152] As used herein, the first CH or CH’ comprises a first CH3 domain and the second CH or CH’ comprises a second CH3 domain. The first CH3 domain and second CH3 domain comprise one or more amino acid substitutions that promote formation of an Fc region heterodimeric CH: CH or CH: CH’ pair, which provides the bispecific antibody when the first CH or CH’ is linked to a first binding domain and the second CH or CH’ is linked to a second binding domain. Amino acid substitutions that facilitate formation of heterodimeric Fc regions are well known in the art and may be used to construct the bispecific antibodies of the present invention.

[0153] Amino acid modifications that promote formation of a heterodimeric Fc region of a bispecific antibody have been disclosed in WO9850431 (incorporated herein by reference), which discloses a knob-in-hole (KIH) method for creating Fc region heterodimers. In the KIH method, one CH3 domain of the heterodimer pair comprises amino acid substitutions that create a protuberance that extends outward from surface of the CH3 domain (knob) that fits into a hole created by appropriate amino acid substitutions in the other CH3 domain of the heterodimeric Fc region, which promotes heterodimer formation over homodimer formation. An example of amino acid substitutions that may be used include S354C and T366W amino acid substitutions to a first CH3 domain to form the knob and Y349C, T366S, L368A, and Y407V amino acid substitutions in a second CH3 domain to form the hole (amino acid numbering according to the EU numbering scheme) wherein the first and second CH3 domains form a heterodimer Fc region. Thus, in certain embodiments of the bispecific antibody, the first CH3 domain comprises the amino acid substitutions S354C and T366W and the second CH3 domain comprises the amino acid substitutions Y349C. T366S, L368A, and Y407V or the first CH3 domain comprises the amino acid substitutions Y349C, T366S, L368A, and Y407V and the second CH3 domain comprises the amino acid substitutions S354C and T366W.26093

[0154] WO2014084607 (incorporated herein by reference) discloses another KJH in which the heterodimeric Fc region comprises a first CH3 domain comprising a K409W amino acid substitution to form the knob and a second CH3 domain comprising D399V and F405T amino acid substitutions to form the hole (amino acid numbering according to the EU numbering scheme) wherein the first and second CH3 domains form a heterodimeric Fc region. Thus, in certain embodiments of the bispecific antibody, the first CH3 domain comprises the amino acid substitutions K409W and the second CH3 domain comprises the amino acid substitutions D399V and F405T or the first CH3 domain comprises the amino acid substitutions D399V and F405T and the second CH3 domain comprises the amino acid substitutions K409W.

[0155] WO2013063702 (incorporated herein by reference) discloses KIH in which the heterodimeric Fc region comprises a first CH3 domain comprising amino acid T350V, L351Y, F405A, and Y407V amino acid substitutions, and the second CH3 domain comprises T350V, T366W, K392M, and T394W amino acid substitutions (amino acid numbering according to the EU numbering scheme) wherein the first and second CFI3 domains form a heterodimeric Fc region. Thus, in certain embodiments of the bispecific antibody, the first CH3 domain comprises the amino acid substitutions T350V, L351Y, F405A, and Y407V and the second CH3 domain comprises the amino acid substitutions T350V, T366W, K392M, and T394W or the first CH3 domain comprises the amino acid substitutions T350V, T366W, K392M. and T394W and the second CH3 domain comprises the amino acid substitutions T350V, L351Y, F405A, and Y407V.

[0156] WO2013063702 (incorporated herein by reference) discloses KIH in which the heterodimeric Fc region comprises a first CH3 domain comprising amino acid T350V, L351Y, F405A, and Y407V amino acid substitutions, and the second CH3 domain comprises T350V and T366W amino acid substitutions (amino acid numbering according to the EU numbenng scheme) wherein the first and second CH3 domains form a heterodimeric Fc region. Thus, in certain embodiments of the bispecific antibody, the first CH3 domain comprises the amino acid substitutions T350V, L351Y, F405A, and Y407V and the second CH3 domain comprises the amino acid substitutions T350V. and T366W or the first CH3 domain comprises the amino acid substitutions T350V and T366W, and the second CH3 domain comprises the amino acid substitutions T350V, L351Y, F405A, and Y407V.

[0157] Other methods for promoting Fc region heterodimeric pairs useful for making the bispecific antibodies include, but are not limited to, electrostatic steering as disclosed in W02009089004 or protein isoelectric point modifications as disclosed in WO2013055809 and methods disclosed in WO20122058786, WO2013096291, and WO2011131746, all ofwhich are incorporated herein by reference.26093

[0158] In certain embodiments of the bispecific antibody, the Fc region is of IgGl origin and comprises a first CH3 domain comprising S354C and T366W amino acid substitutions to form the knob and a second CH3 domain comprising Y349C, T366S, L368A, and Y407V amino acid substitutions to form the hole wherein the first and second CH3 domains form a heterodimeric Fc region and wherein the amino acid numbering is according to the EU numbering scheme.

[0159] In certain embodiments of the bispecific antibody, the Fc region is of IgGl origin and comprises a first CH3 domain comprising Y349C, T366S, L368A, and Y407V amino acid substitutions to form the knob and a second CH3 domain comprising S354C and T366W amino acid substitutions to form the hole wherein the first and second CH3 domains form a heterodimeric Fc region and wherein the amino acid numbering is according to the EU numbering scheme.

[0160] In certain embodiments of the bispecific antibody, the Fc region is of IgGl origin and comprises a first CH3 domain comprising 409W amino acid substitution to form the knob and a second CH3 domain comprising D399 and F405T amino acid substitutions to form the hole wherein the first and second CH3 domains form a heterodimeric Fc region and wherein the amino acid numbering is according to the EU numbering scheme.

[0161] In certain embodiments of the bispecific antibody, the Fc region is of IgGl origin and comprises a first CH3 domain comprising D399 and F405T amino acid substitutions to form the knob and a second CH3 domain comprising 409W amino acid substitution to form the hole wherein the first and second CH3 domains form a heterodimeric Fc region and wherein the amino acid numbering is according to the EU numbering scheme.

[0162] In certain embodiments of the bispecific antibody, the Fc region is of IgGl origin and comprises a first CH3 domain comprising 357W amino acid substitution to form the knob and a second CH3 domain comprising Y349S amino acid substitution to form the hole wherein the first and second CH3 domains form a heterodimeric Fc region and wherein the amino acid numbering is according to the EU numbering scheme.

[0163] In certain embodiments of the bispecific antibody, the Fc region is of IgGl origin and comprises a first CH3 domain comprising Y349S amino acid substitution to form the knob and a second CH3 domain comprising 357W amino acid substitution to form the hole wherein the first and second CH3 domains form a heterodimeric Fc region and wherein the amino acid numbering is according to the EU numbering scheme.

[0164] In certain embodiments of the bispecific antibody, the Fc region is of IgGl origin and comprises a first CH3 domain comprising T350V, L351Y, F405A, and Y407V amino acid substitutions to form the knob and a second CH3 domain comprising T350, T366, K392 and26093T394W amino acid substitutions to form the hole wherein the first and second CH3 domains form a heterodimeric Fc region and wherein the amino acid numbering is according to the EU numbering scheme.

[0165] In certain embodiments of the bispecific antibody, the Fc region is of IgGl origin and comprises a first CH3 domain comprising T350, T366, K.392 and T394W amino acid substitutions to form the knob and a second CH3 domain comprising T350V, L351Y, F405A, and Y407V amino acid substitutions to form the hole wherein the first and second CH3 domains form a heterodimeric Fc region and wherein the amino acid numbering is according to the EU numbering scheme.

[0166] In certain embodiments of the bispecific antibody, the Fc region is of IgGl origin and comprises a first CH3 domain comprising T350V, L351Y, F405A, and Y407V amino acid substitutions to form the knob and a second CH3 domain comprising T350V and T366W amino acid substitutions to form the hole wherein the first and second CH3 domains form a heterodimeric Fc region and wherein the amino acid numbering is according to the EU numbering scheme.

[0167] In certain embodiments of the bispecific antibody, the Fc region is of IgGl origin and comprises a first CH3 domain comprising amino acid substitutions T350V and T366W to form the knob and a second CH3 domain comprising T350V. L351 Y, F405A, and Y407V amino acid substitutions to form the hole wherein the first and second CH3 domains form a heterodimeric Fc region and wherein the amino acid numbering is according to the EU numbering scheme.

[0168] In certain embodiments, the first and second CH (CH2 and / or CH3 thereof) domains further comprise a substitution of the amino acids that confer a longer circulation half-life by virtue of more favorable binding to hFcRn. Fc regions containing the amino acid substitutions M428L / N434S (LS mutant), L309D / Q311H / N434S (DHS mutant), H433K / N434F (KF mutant), or M252Y / S254T / T256E (YTE mutant) confer 10- to 12-fold higher affinity for FcRn at pH 5.8, result in the greatest reported increase in antibody half-life (2- to 4-fold in circulation) in mice and in non-human primates (See Lee et al., Nat. Communications 10. 5031 (2019); Fc positions according to the EU numbering scheme numbering scheme). Thus, the present invention includes embodiments that comprises any one of these half-life extension amino acid substitutions.

[0169] In certain embodiments, the first and second CH (CH2 and / or CH3 thereof) domains of the bispecific antibodies further comprise one or more amino acid substitutions that reduce or ablate effector function of the Fc region. In certain embodiments, the first and second CH domains are of IgGl origin and further comprise:26093(a) E233A and L235A amino acid substitutions wherein the numbering is according to the EU numbering scheme;(b) L234A L235A D265S substitutions wherein the numbering is according to the EU numbering scheme;(c) L234A L235A P329G substitutions wherein the numbering is according to the EU numbering scheme;(d) L235E substitution wherein the numbering is according to the EU numbering scheme; (e) D265A substitution wherein the numbering is according to the EU numbering scheme; (f) D265A N297G substitutions wherein the numbering is according to the EU numbering scheme;(g) N297X, wherein X is any amino acid other than N substitution wherein the numbering is according to the EU numbering scheme; or(h) N297A / D356E / E358M substitutions wherein the numbering is according to the EU numbering scheme.

[0170] In certain embodiments, the first and second CH domains comprise (i) amino acid substitutions that confer a longer circulation half-life of bispecific antibodies and (ii) amino acid substitution or substitutions that reduce or ablate effector function of the Fc region of the bispecific antibody.

[0171] In certain embodiments of the invention, the CH3 domains of the Fc region of the IgGl or IgG4 heavy chain constant domains as disclosed herein comprise a C-terminal lysine or lack either a C-terminal lysine or a C-terminal glycine-lysine dipeptide. In some embodiments, the N-terminal amino acid of the antibody variable domains may undergo cyclization to pyroglutamate. Thus, in a composition comprising a particular antibody disclosed herein, the composition may comprise a population of antibody species wherein each species may independently comprise a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine and / or comprise an N-terminal glutamine or glutamic acid or cyclization of the N-terminal amino acid to pyroglutamate.ALPP / L2 Binders

[0172] The ALPP / L2 binders providing the first binding domain for the bispecific antibodies disclosed herein comprise (i) a heavy chain variable domain (VH) comprising VH complementarity determining regions (VH-CDRs) 1, 2, and 3; and (ii) a light chain variable domain (VL) comprising VL complementarity determining regions (VL-CDRs) 1, 2, and 3,26093wherein the ALPP / L2 binders comprise any one of the 46 possible combinations of VH-CDR / VL-CDR amino acid sequence sets shown in Table 3.Table 3VH CDR Amino Acid SEQ ID VL CDR Amino Acid SEQ ID Sequence NO: Sequence NO:CDR1 SYAMS 3 RASQSVSSSYLS 6 CDR2 AISISGTYTYYADSVKG 4 GTSTRAT 7 CDR3 AHSTYPYYYYYYMDV 5 HQDYNFPLT 8 CDR1 IYTMK 11 RASQSVSNSYLA 14 CDR2 AISASGGSTSYSDSLKG 12 GASSRAA 15 CDR3 DPLPYNWNFFYYYMDV 13 QQYGRSPLT 16 CDR1 NYAMS 19 RASQSVSSYLA 22 CDR2 AISGSGGSTYYADSVKG 20 RGASNRAT 23 CDR3 DPLPANWNYYYYMDV 21 QQRSNWPLT 24 CDR1 SYWMS 27 RASQGIRNDLG 30 CDR2 NINQDGSEKNYVDSVQG 28 AASSLQS 31 CDR3 DYSNYDDYYYYFYMDV 29 LQDYNYPWT 32 CDR1 SYWMT 35 RASQGIRDDLA 38 CDR2 NIKQDGSEENYVDSVKG 36 AASNLQS 39 CDR3 DTVTTGFYHYFYMDV 37 LQDYIYPYT 40 CDR1 SYGMH 43 RASQGISNALA 46 CDR2 VIWYDGSNKYYADSVKG 44 DASSLES 47 CDR3 ENNWHLNYFFYYMDV 45 QQFNNYIYT 48 CDR1 SYGMH 51 RASQDINNVLA 54 CDR2 VIWYDGSNKYYADSVKG 52 DASSLES 55 CDR3 ENNWHLNYYFYYMDV 53 QHFNDFIYT 56 CDR1 SYGMH 59 RASQGISSALA 62 CDR2 AVIWYDGSNKYYADSVKG 60 DASSLEG 63 CDR3 SENNWNGFYNFFYMDV 61 QQFNNDLYT 64 CDR1 RYWMS 67 RASQGIRNDLG 70 CDR2 NIKQDGSEKNYVDTVKG 68 AASSLQS 71 CDR3 DYDFWNGYYLYYSMDV 69 LQDYNYPYT 72 CDR1 SYGMH 75 GASQSVSSSYLA 78 CDR2 VIWYDGSNKYYADSVKG 76 DASSRAS 79 CDR3 ENNWNGYYHYYYMDV 77 QQYGSSLYT 80 CDR1 SYGMH 83 RASQGIRSALA 86 CDR2 FIWYDGSKKYYADSVKG 84 DASSLES 87 CDR3 ENNWNGYYNFYYMDV 85 RQQFNNYYT 88 CDR1 SYGMH 91 GASQSVSNNYLA 94 CDR2 VIWYDGSNKYYADSVKG 92 DASSRAT 95 CDR3 ENNWNGYYYYYYMDV 93 QQYGSSLYT 96 CDR1 PYAMH 99 GASQSISGSYLA 10226093CDR2 VIWYDTINKYYADSVKG 100 DASSRAT 103 CDR3 ENNWNGYYSYYYMDV 101 QHYENSLYT 104 CDR1 SYGMH 107 RASQDISRVLA 110 CDR2 VIWYDGSNKYYVDSVKG 108 DASSLES 111 CDR3 ENNWNNYYHFYYMDV 109 QQFNNYMYT 112 CDR1 SYYWT 115 RASQSISNNYLS 118 CDR2 YIYYSGSANYNPSLKG 116 GASTRAT 119 CDR3 GRRGYSGNGDYYYYMDV 117 QQDFNLPLT 120 CDR1 NFAMG 123 RASQDIRYDLG 126 CDR2 RISSSGRDTFYTDSVKG 124 GASSLHS 127 CDR3 LGPV 125 LQLNNFPYT 128 CDR1 GYWMT 131 RASQNIRNDLG 134 CDR2 NINQDGNEKNYVDSVKG 132 AASSLQS 135 CDR3 DTSNYDFYYYYFYMDV 133 LQDYNYPYT 136 CDR1 SYAMN 139 RASQSVSTYLV 142 CDR2 AISGSGGSTYYADSVKG 140 DASNRAT 143 CDR3 DPLPANWNYFYYMDV 141 QQRSNWPLT 144 CDR1 SYYWS 147 RASQSISSSYLS 150 CDR2 RIYSSGSTNYNPSLKSR 148 GVSSRAT 151 CDR3 FSMVRGVMGYMDV 149 QQDYNLPYT 152 CDR1 VYGMH 155 RASQGISSALA 158 CDR2 VIWYDGTNKYYADSVKG 156 DASRLES 159 CDR3 ENNWNAYYHFHYMDV 157 QQFNNNLYT 160 CDR1 SYWMS 163 RTSQGIRNDLG 166 CDR2 NINQDGSEKYYVDSVKG 164 AASNLQS 167 CDR3 DASNYDGYYYYFYMDV 165 LQDSNYPYT 168 CDR1 SYYWT 171 RASQSISDSYLS 174 CDR2 YIFYSGSTNYNPSLKG 172 GASTRAT 175 CDR3 GRRGYRGNGDYYYYMDV 173 QQDFNLPLT 176 CDR1 SYGMH 179 RASQGISSALA 182 CDR2 IIWYDGTNKYYADSVKG 180 DASSLES 183 CDR3 ENNWNGYYHFYYMDV 181 QQFNNYLYT 184 CDR1 DLSMY 187 RASQGISNYLA 190 CDR2 GFDPEAGETIYAQKFQG 188 DASSLQS 191 CDR3 GRYCFSTSCSFNYNYYMDV 189 QQYNSFPPT 192 CDR1 SYTMK 195 RASQSVSSSYLA 198 CDR2 AISGSGGSTYYADSVKG 196 GASS RAT 199 CDR3 DPLPYNWSFYYYYMDV 197 QQYGSSPLT 200 CDR1 TYGFN 203 RASQSVGSFLA 206 CDR2 WISAYNGDTRYAQKFQG 204 GASTRAT 207 CDR3 GTTVTTPYYYYSYMDV 205 QQYINWPLT 20826093CDR1 SYAMS 211 RASQSISSYLN 214 CDR2 AISGSGGSSKNGNSVKG 212 DASSLQS 215 CDR3 GTWPWPGDHMDV 213 QQSYSTPPT 216 CDR1 NYAMS 219 KSSQSLLHSDGKTYLY 222 CDR2 GVRNSGSGTYYADSVKG 220 EVSNRFS 223 CDR3 GGTPVTAPYYYYYYMDV 221 MQSIQLPYT 224 CDR1 TISPYW 227 RASQGIRDDLG 230 CDR2 NINQDGSEKHYVDSVKG 228 AASSLQS 231 CDR3 DDSVYDSYYYYFYMDV 229 LQDYTYPYT 232 CDR1 ELSMH 235 RASQSVSSTSFS 238 CDR2 GFDPEDGGTIFAQKFQG 236 GASTRAT 239 CDR3 WGSYYRWFDP 237 HQDYNLPFT 240 CDR1 SYWMS 243 RASQGIRNDLA 246 CDR2 NINQDGNEKNYVDSVKG 244 AASSLQS 247 CDR3 DTSNYDLYSYYFYMDV 245 LQDYIYPYT 248 CDR1 SYGMH 251 RASQGISNTLA 254 CDR2 LIWYDGSNEYYADSVKG 252 DASRLEG 255 CDR3 ENNWNGRYYFYYMDV 253 QQFNNYPT 256 CDR1 SYTMK 259 RASQSVSSSYLA 262 CDR2 AISGSGGSTYYADSVKG 260 GASS RAT 263 CDR3 DPLPYNWNFYYYYMDV 261 QQYGSSPLT 264 CDR1 SYALS 267 RASQGISSALA 270 CDR2 VISGNGI ITYYADSVKG 268 DASSLES 271 CDR3 DDSSYYGLGSFPN 269 QQFNNYPYS 272 CDR1 NYAMS 275 RASQSVNNNLA 278 CDR2 AISGSGGSTYYADSVKG 276 GASTRAT 279 CDR3 DGGVPVVPYLYYYYMDV 1T1 QQYNNWPFT 280 CDR1 IYTMK 283 RASQSVSSSYLS 286 CDR2 AISASGGSTSYSDSVKG 284 GASTRAT 287 CDR3 DPLPYNWSFFYYYMDV 285 QQDYNLIT 288 CDR1 TYVMK 291 RASQSVSSSYLA 294 CDR2 AISGSGGSTSYTDSVKG 292 GASSRAT 295 CDR3 DPLPYNWSFYFYYMDV 293 QQYGSSPLT 296 CDR1 SYWMT 299 RASQGIRNDLG 302 CDR2 NIKQDGSEKHYVDSVKG 300 AASTLQS 303 CDR3 DTSNYDLYYYYFYMDV 301 LQDNSYPYT 304 CDR1 SYWMS 307 RASQGIRNDLG 310 CDR2 NVNQDGSEQNFVDSVKG 308 AASSLQS 311 CDR3 DASNYDGYYYYYYTDV 309 DYNYPYT 312 CDR1 SYVMS 315 TLSSGHSSYAIA 318 CDR2 AISGSGDRTYYADSVKG 316 LNSDGSH 319 CDR3 AAGYCTNGVCLYYYYMDV 317 QTWGTGIRV 32026093CDR1 GSDYWV 323 RASQSVRSSYLS 326 CDR2 SIYYSGSTYYNPALKS 324 GASTRAT 327 CDR3 RGNDDYYYFYMDV 325 QQDYNLPMYT 328 CDR1 TYAMS 341 RASQSVSSNLA 344 CDR2 AISGSGGSRYYADSVKG 342 GASTRAT 345 CDR3 DGGVPVVPYLYYYYMDV 343 QHYNNWPFT 346 CDR1 SYGMH 349 RASQGINSALA 352 CDR2 FISYDGKNKYYIDSVRG 350 DASRLES 353 CDR3 ENNWNDFYNYYYMDVW 351 QQFNNYMYT 354 CDR1 SYYWS 357 RASQGIRNDLG 360 CDR2 GRIYTSGSTNYNPSLKS 358 AASSLQ 361 CDR3 EWYYYYMDV 359 LQDYNYPYT 362 CDR1 SYWMS 365 RASQGIRDDLG 368 CDR2 NIKQDGSEKNYVDAVKG 366 AASTLQS 369 CDR3 DNDNWNGFYYYYSMDV 367 LQDNNYPYT 370 CDR1 PYWMT 373 RASQGIRDDLG 376 CDR2 NINQDGNEKNYVDSVKG 374 AASSLQS 377 CDR3 DDSVYDSYYYYFYMGV 375 LQDYNYPYT 378 CDR sequences defined by Kabat numbering scheme.

[0173] The first arm of the bispecific antibody comprises any one of the VH: VL pairs comprising the CDR amino acid sequences set forth in Table 3 wherein the VH of the pair is linked to a CH comprising an amino acid sequenced show n in Table 4 and the VL of the pair is linked to a human LC Kappa constant domain comprising the amino acid sequence set forth in SEQ ID NO: 391.| Table 4 | Description CH Amino Acid SEQ Description CH Amino Acid SEQ Sequence ID Sequence ID (KIH: knob) NO: (KIH: hole) NO:Human IgGl ASTKGPSVFPLAPSS 381 Human IgGl ASTKGPSVFPLAPSS 392 HC constant KSTSGGTAALGCLVK HC constant KSTSGGTAALGCLVK domain DYFPEPVTVSWNSGA domain DYFPEPVTVSWNSGA (KIH: knob LTSGVHTFPAVLQSS (KIH: Hole LTSGVHTFPAVLQSS S354C GLYSLSSWTVPSSS Y349C T366S GLYSLSSWTVPSSS T366W) LGTQTYICNVNHKPS L368A LGTQTYICNVNHKPS NTKVDKKVEPKSCDK Y407V) NTKVDKKVEPKSCDK THTCPPCPAPELLGG THTCPPCPAPELLGG PSVFLFPPKPKDTLM PSVFLFPPKPKDTLM I SRTPEVTCVWDVS I SRTPEVTCVWDVS HEDPEVKFNWYVDGV HEDPEVKFNWYVDGV EVHNAKTKPREEQYN EVHNAKTKPREEQYN STYRWSVLTVLHQD STYRWSVLTVLHQD WLNGKEYKCKVSNKA WLNGKEYKCKVSNKA LPAPIEKTISKAKGQ LPAPIEKTISKAKGQ PREPQVYTLPPCRDE PREPQVCTLPPSRDE26093LTKNQVSLWCLVKGF LTKNQVSLSCAVKGF YPSDIAVEWESNGQP YPSDIAVEWESNGQP ENNYKTTPPVLDSDG ENNYKTTPPVLDSDG SFFLYSKLTVDKSRW SFFLVSKLTVDKSRW QQGNVFSCSVMHEAL QQGNVFSCSVMHEAL HNHYTQKSLSLSPGK HNHYTQKSLSLSPGKHuman IgGl ASTKGPSVFPLAPSS 382 Human IgGl ASTKGPSVFPLAPSS 393 HC Constant KSTSGGTAALGCLVK HC Constant KSTSGGTAALGCLVK domain DYFPEPVTVSWNSGA domain (YTE) DYFPEPVTVSWNSGA (YTE) (KIH: LTSGVHTFPAVLQSS (KIH: Hole LTSGVHTFPAVLQSS knob S354C GLYSLSSWTVPSSS Y349C T366S GLYSLSSWTVPSSS T366W) LGTQTYICNVNHKPS L368A LGTQTYICNVNHKPS NTKVDKKVEPKSCDK Y407V) NTKVDKKVEPKSCDK THTCPPCPAPELLGG THTCPPCPAPELLGG PSVFLFPPKPKDTLY PSVFLFPPKPKDTLY I TRE P E VT CVWDVS ITREPEVT CVWDVS HEDPEVKFNWYVDGV HEDPEVKFNWYVDGV EVHNAKTKPREEQYN EVHNAKTKPREEQYN STYRWSVLTVLHQD STYRWSVLTVLHQD WLNGKEYKCKVSNKA WLNGKEYKCKVSNKA LPAPIEK ISKAKGQ LPAPIEKTISKAKGQ PREPQVYTLPPCRDE PREPQVCTLPPSRDE LTKNQVSLWCLVKGF LTKNQVSLSCAVKGF YPSDIAVEWESNGQP YPSDIAVEWESNGQP ENNYKTTPPVLDSDG ENNYKTTPPVLDSDG SFFLYSKLTVDKSRW SFFLVSKLTVDKSRW QQGNVFSCSVMHEAL QQGNVFSCSVMHEAL HNHYTQKSLSLSPGK HNHYTQKSLSLSPGKHuman IgGl ASTKGPSVFPLAPSS 383 Human IgGl ASTKGPSVFPLAPSS 394 HC Constant KSTSGGTAALGCLVK HC Constant KSTSGGTAALGCLVK domain DYFPEPVTVSWNSGA domain DYFPEPVTVSWNSGA (E233A / L23 LTSGVHTFPAVLQSS (E233A / L235 LTSGVHTFPAVLQSS 5A) (KIH: GLYSLSSWTVPSSS A) (KIH: Hole GLYSLSSWTVPSSS knob S354C LGTQTYICNVNHKPS Y349C T366S LGTQTYICNVNHKPS T366W) NTKVDKKVEPKSCDK L368A NTKVDKKVEPKSCDK THTCPPCPAPALAGG Y407V) THTCPPCPAPALAGG PSVFLFPPKPKDTLM PSVFLFPPKPKDTLM I S RT P E VT CVWDVS I SRTPEVT CVWDVS HEDPEVKFNWYVDGV HEDPEVKFNWYVDGV EVHNAKTKPREEQYN EVHNAKTKPREEQYN STYRWSVLTVLHQD STYRWSVLTVLHQD WLNGKEYKCKVSNKA WLNGKEYKCKVSNKA LPAPIEKTISKAKGQ LPAPIEKTISKAKGQ PREPQVYTLPPCRDE PREPQVCTLPPSRDE LTKNQVSLWCLVKGF LTKNQVSLSCAVKGF YPSDIAVEWESNGQP YPSDIAVEWESNGQP ENNYKTTPPVLDSDG ENNYKTTPPVLDSDG SFFLYSKLTVDKSRW SFFLVSKLTVDKSRW QQGNVFSCSVMHEAL QQGNVFSCSVMHEAL HNHYTQKSLSLSPGK HNHYTQKSLSLSPGKHuman IgGl ASTKGPSVFPLAPSS 384 Human IgGl ASTKGPSVFPLAPSS 395 HC Constant KSTSGGTAALGCLVK HC Constant KSTSGGTAALGCLVK domain DYFPEPVTVSWNSGA domain DYFPEPVTVSWNSGA26093(L234A LTSGVHTFPAVLQSS (L234A LTSGVHTFPAVLQSS L235A GLYSLSSWTVPSSS L235A GLYSLSSWTVPSSS D265S) LGTQTYICNVNHKPS D265S) (KIH: LGTQTYICNVNHKPS (KIH: knob NTKVDKKVEPKSCDK Hole Y349C NTKVDKKVEPKSCDK S354C THTCPPCPAPEAAGG T366S L368A THTCPPCPAPEAAGG T366W) PSVFLFPPKPKDTLM Y407V) PSVFLFPPKPKDTLM I S RT P E VT CVWS VS I SRTPEVTCVWSVS HEDPEVKFNWYVDGV HEDPEVKFNWYVDGV EVHNAKTKPREEQYN EVHNAKTKPREEQYN STYRWSVLTVLHQD STYRWSVLTVLHQD WLNGKEYKCKVSNKA WLNGKEYKCKVSNKA LPAPIEKTISKAKGQ LPAPIEKTISKAKGQ PREPQVYTLPPCRDE PREPQVCTLPPSRDE LTKNQVSLWCLVKGF LTKNQVSLSCAVKGF YPSDIAVEWESNGQP YPSDIAVEWESNGQP ENNYKTTPPVLDSDG ENNYKTTPPVLDSDG SFFLYSKLTVDKSRW SFFLYSKLTVDKSRW QQGNVFSCSVMHEAL QQGNVFSCSVMHEAL HNHYTQKSLSLSPGK HNHYTQKSLSLSPGKHuman IgGl ASTKGPSVFPLAPSS 385 Human IgGl ASTKGPSVFPLAPSS 396 HC Constant KSTSGGTAALGCLVK HC Constant KSTSGGTAALGCLVK domain DYFPEPVTVSWNSGA domain DYFPEPVTVSWNSGA (L234A LTSGVHTFPAVLQSS (L234A LTSGVHTFPAVLQSS L235A GLYSLSSWTVPSSS L235A GLYSLSSWTVPSSS P329G) LGTQTYICNVNHKPS P329G) (KIH: LGTQTYICNVNHKPS (KIH: knob NTKVDKKVEPKSCDK Hole Y349C NTKVDKKVEPKSCDK S354C THTCPPCPAPEAAGG T366S L368A THTCPPCPAPEAAGG T366W) PSVFLFPPKPKDTLM Y407V) PSVFLFPPKPKDTLM I SRTPEVTCVWSVS I SRTPEVTCVWSVS HEDPEVKFNWYVDGV HEDPEVKFNWYVDGV EVHNAKTKPREEQYN EVHNAKTKPREEQYN STYRWSVLTVLHQD STYRWSVLTVLHQD WLNGKEYKCKVSNKA WLNGKEYKCKVSNKA LGAPIEKTISKAKGQ LGAPIEKTISKAKGQ PREPQVYTLPPCRDE PREPQVCTLPPSRDE LTKNQVSLWCLVKGF LTKNQVSLSCAVKGF YPSDIAVEWESNGQP YPSDIAVEWESNGQP ENNYKTTPPVLDSDG ENNYKTTPPVLDSDG SFFLYSKLTVDKSRW SFFLYSKLTVDKSRW QQGNVFSCSVMHEAL QQGNVFSCSVMHEAL HNHYTQKSLSLSPGK HNHYTQKSLSLSPGKHuman IgGl ASTKGPSVFPLAPSS 386 Human IgGl ASTKGPSVFPLAPSS 397 HC Constant KSTSGGTAALGCLVK HC Constant KSTSGGTAALGCLVK domain DYFPEPVTVSWNSGA domain DYFPEPVTVSWNSGA (L235E) LTSGVHTFPAVLQSS (L235E) LTSGVHTFPAVLQSS (KIH: knob GLYSLSSWTVPSSS (KIH: Hole GLYSLSSWTVPSSS S354C LGTQTYICNVNHKPS Y349C T366S LGTQTYICNVNHKPS T366W) NTKVDKKVEPKSCDK L368A NTKVDKKVEPKSCDK THTCPPCPAPELEGG Y407V) THTCPPCPAPELEGG PSVFLFPPKPKDTLM PSVFLFPPKPKDTLM I S RT P E VT CVWDVS I SRTPEVT CVWDVS HEDPEVKFNWYVDGV HEDPEVKFNWYVDGV EVHNAKTKPREEQYN EVHNAKTKPREEQYN STYRWSVLTVLHQD STYRWSVLTVLHQD26093WLNGKEYKCKVSNKA WLNGKEYKCKVSNKA LPAPIEKTISKAKGQ LPAPIEKTISKAKGQ PREPQVYTLPPCRDE PREPQVCTLPPSRDE LTKNQVSLWCLVKGF LTKNQVSLSCAVKGF YPSDIAVEWESNGQP YPSDIAVEWESNGQP ENNYKTTPPVLDSDG ENNYKTTPPVLDSDG SFFLYSKLTVDKSRW SFFLVSKLTVDKSRW QQGNVFSCSVMHEAL QQGNVFSCSVMHEAL HNHYTQKSLSLSPGK HNHYTQKSLSLSPGKHuman IgGl ASTKGPSVFPLAPSS 387 Human IgGl ASTKGPSVFPLAPSS 398 HC Constant KSTSGGTAALGCLVK HC Constant KSTSGGTAALGCLVK domain DYFPEPVTVSWNSGA domain DYFPEPVTVSWNSGA (D265A) LTSGVHTFPAVLQSS (D265A) LTSGVHTFPAVLQSS (KIH: knob GLYSLSSWTVPSSS (KIH: Hole GLYSLSSWTVPSSS S354C LGTQTYICNVNHKPS Y349C T366S LGTQTYICNVNHKPS T366W) NTKVDKKVEPKSCDK L368A NTKVDKKVEPKSCDK THTCPPCPAPELLGG Y407V) THTCPPCPAPELLGG PSVFLFPPKPKDTLM PSVFLFPPKPKDTLM ISR PEVTCVWAVS ISRTPEVTCVWAVS HEDPEVKFNWYVDGV HEDPEVKFNWYVDGV EVHNAKTKPREEQYN EVHNAKTKPREEQYN STYRWSVLTVLHQD STYRWSVLTVLHQD WLNGKEYKCKVSNKA WLNGKEYKCKVSNKA LPAPIEKTISKAKGQ LPAPIEKTISKAKGQ PREPQVYTLPPCRDE PREPQVCTLPPSRDE LTKNQVSLWCLVKGF LTKNQVSLSCAVKGF YPSDIAVEWESNGQP YPSDIAVEWESNGQP ENNYKTTPPVLDSDG ENNYKTTPPVLDSDG SFFLYSKLTVDKSRW SFFLVSKLTVDKSRW QQGNVFSCSVMHEAL QQGNVFSCSVMHEAL HNHYTQKSLSLSPGK HNHYTQKSLSLSPGKHuman IgGl ASTKGPSVFPLAPSS 388 Human IgGl ASTKGPSVFPLAPSS 399 HC Constant KSTSGGTAALGCLVK HC Constant KSTSGGTAALGCLVK domain DYFPEPVTVSWNSGA domain DYFPEPVTVSWNSGA (D265A LTSGVHTFPAVLQSS (D265A LTSGVHTFPAVLQSS N297G) GLYSLSSWTVPSSS N297G) (KIH: GLYSLSSWTVPSSS (KIH: knob LGTQTYICNVNHKPS Hole Y349C LGTQTYICNVNHKPS S354C NTKVDKKVEPKSCDK T366S L368A NTKVDKKVEPKSCDK T366W) THTCPPCPAPELLGG Y407V) THTCPPCPAPELLGG PSVFLFPPKPKDTLM PSVFLFPPKPKDTLM ISRTPEVTCVWAVS ISRTPEVTCVWAVS HEDPEVKFNWYVDGV HEDPEVKFNWYVDGV EVHNAKTKPREEQYG EVHNAKTKPREEQYG STYRWSVLTVLHQD STYRWSVLTVLHQD WLNGKEYKCKVSNKA WLNGKEYKCKVSNKA LPAPIEKTISKAKGQ LPAPIEKTISKAKGQ PREPQVYTLPPCRDE PREPQVCTLPPSRDE LTKNQVSLWCLVKGF LTKNQVSLSCAVKGF YPSDIAVEWESNGQP YPSDIAVEWESNGQP ENNYKTTPPVLDSDG ENNYKTTPPVLDSDG SFFLYSKLTVDKSRW SFFLVSKLTVDKSRW QQGNVFSCSVMHEAL QQGNVFSCSVMHEAL HNHYTQKSLSLSPGK HNHYTQKSLSLSPGK26093Human IgGl ASTKGPSVFPLAPSS 389 Human IgGl ASTKGPSVFPLAPSS 400 HC Constant KSTSGGTAALGCLVK HC Constant KSTSGGTAALGCLVK domain DYFPEPVTVSWNSGA domain DYFPEPVTVSWNSGA (N297X, LTSGVHTFPAVLQSS (N297X, LTSGVHTFPAVLQSS wherein X is GLYSLSSWTVPSSS wherein X is GLYSLSSWTVPSSS any amino LGTQTYICNVNHKPS any amino LGTQTYICNVNHKPS acid other NTKVDKKVEPKSCDK acid other than NTKVDKKVEPKSCDK than N) THTCPPCPAPELLGG N) (KIH: Hole THTCPPCPAPELLGG (KIH: knob PSVFLFPPKPKDTLM Y349C T366S PSVFLFPPKPKDTLM S354C I S RT P E VT CVWDVS L368A I SRTPEVT CVWDVS T366W) HEDPEVKFNWYVDGV Y407V) HEDPEVKFNWYVDGV EVHNAKTKPREEQYX EVHNAKTKPREEQYX STYRWSVLTVLHQD STYRWSVLTVLHQD WLNGKEYKCKVSNKA WLNGKEYKCKVSNKA LPAPIEKTISKAKGQ LPAPIEKTISKAKGQ PREPQVYTLPPCRDE PREPQVCTLPPSRDE LTKNQVSLWCLVKGF LTKNQVSLSCAVKGF YPSDIAVEWESNGQP YPSDIAVEWESNGQP ENNYKTTPPVLDSDG ENNYKTTPPVLDSDG SFFLYSKLTVDKSRW SFFLYSKLTVDKSRW QQGNVFSCSVMHEAL QQGNVFSCSVMHEAL HNHYTQKSLSLSPGK HNHYTQKSLSLSPGKHuman IgGl ASTKGPSVFPLAPSS 390 Human IgGl ASTKGPSVFPLAPSS 401 HC Constant KSTSGGTAALGCLVK HC Constant KSTSGGTAALGCLVK domain DYFPEPVTVSWNSGA domain DYFPEPVTVSWNSGA (N297A / D35 LTSGVHTFPAVLQSS (N297A / D356 LTSGVHTFPAVLQSS 6E / L358M) GLYSLSSWTVPSSS E / L358M) GLYSLSSWTVPSSS (KIH: knob LGTQTYICNVNHKPS (KIH: Hole LGTQTYICNVNHKPS S354C NTKVDKKVEPKSCDK Y349C T366S NTKVDKKVEPKSCDK T366W) THTCPPCPAPELLGG L368A THTCPPCPAPELLGG PSVFLFPPKPKDTLM Y407V) PSVFLFPPKPKDTLMI S RT P E VT CVWDVS I SRTPEVT CVWDVS HEDPEVKFNWYVDGV HEDPEVKFNWYVDGV EVHNAKTKPREEQYA EVHNAKTKPREEQYA STYRWSVLTVLHQD STYRWSVLTVLHQD WLNGKEYKCKVSNKA WLNGKEYKCKVSNKA LPAPIEKTISKAKGQ LPAPIEKTISKAKGQ PREPQVYTLPPCREE PREPQVYTLPPSREE MTKNQVSLWCLVKGF MTKNQVSLSCAVKGF YPSDIAVEWESNGQP YPSDIAVEWESNGQP ENNYKTTPPVLDSDG ENNYKTTPPVLDSDG SFFLYSKLTVDKSRW SFFLYSKLTVDKSRW QQGNVFSCSVMHEAL QQGNVFSCSVMHEAL HNHYTQKSLSLSPGK HNHYTQKSLSLSPGKThe CH amino acid substitution positions are numbered according to the Eu numbering scheme. The CH amino acid substitutions are shown in bold.

[0174] The amino acid sequences for the CHs shown in Table 4 may lack a C-terminal lysine residue or the C-terminal di-peptide glycine-lysine. Amino acid sequences 468-487 are variant CH regions that correspond to the CH regions shown in Table 4 but which lack a C-terminal lysine residue.26093

[0175] In a further embodiment the ALPPL2 binder comprises any one of the 46 combinations of VH / VL amino acid sequences shown in Table 5. In particular embodiments, the VH-FR1, VH-FR2, VH-FR3, and VH-FR4 of the VH may further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, insertions, or combinations thereof. In particular embodiments, the VL-FR1, VL-FR2, VL-FR3, and VL-FR4 of the VL may further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, insertions, or combinations thereof. In particular embodiments, the VH-FR1, VH-FR2, VH-FR3, and VH-FR4 of the VH and the VL-FR1, VL-FR2, VL-FR3, and VL-FR4 of the VL may further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, insertions, or combinations thereof.Table 5Clone V.. H.. A. mi.no A. ci.d. S „equence SE. Q v. ID VL „ Amino Acid SE. Q \ IDMNO: Sequence NO:24F4 EVQLLESGGGLVQPGGSL 1 KIVMTQSPATLSLSPGE 2RLSCAASGFTFSSYAMSW RATLSCRASQSVSSSYL VRQAPGKGLEWVSAISIS SWYQQKPGQAPRLLMYG GTYTYYADSVKGRFTISR TSTRATDIPARFSGSGS DNSENTLFLQMNSLRAED GTDFTLTISSLQPEDFA TAVYYCAKAHSTYPYYYY VYYCHQDYNFPLTFGGG YYMDVWGKGT TVTVS S TKVEIK10B3 EVQLLESGGGLVQPGGSL 9 EIVLTQSPGTLSLSPGE 10RLSCAASGFTFSIYTMKW RATLSCRASQSVSNSYL VRQAPGKGLDWVSAISAS AWYQQKPGQAPRLI IYG GGSTSYSDSLKGRFTISR ASSRAAGIPDRFSGSGS DNSKNTVNLQMNSLRTED GTDFTLTISRLEPEDFA SAVYYCAKDPLPYNWNFF IYHCQQYGRSPLTFGGG YYYMDVWGKGT TVTVS S TKVEIK9G10 EVQLLESGGGLVQPGGSL 17 EIVLTQSPATLSLSPGE 18RLSCAASVSTFNNYAMSW RATLSCRASQSVSSYLA VRQAPGKGLEWVSAISGS WYQQKPGQAPRLLIYGA GGSTYYADSVKGRFTISR SNRATGI PARFSGSGSG DNSKNTLYLQMNSLRAED TDFTLTISSLEPEDFAV TAVYYCAKDPLPANWNYY YYCQQRSNWPLTFGGGT YYMDVWGKGT TVTVS S KVEIK8A7 EVQLMDSGGDLVQPGGSL 25 AIQMTQSPSSLSASVGD 26RLSCAASGFTFSSYWMSW RVTITCRASQGIRNDLG VRQAPGKGLEWVANINQD WYQQKPGKAPKLLIYAA GSEKNYVDSVQGRFTISR SSLQSGVPSRFSGSGSG DNAKKSLYLQMNSLRAED TDFTLTISSLQPEDFAT TAVYYCARDYSNYDDYYY YYCLQDYNYPWTFGQGT YFYMDVWGKGT TVTVS S KVEIK16H2 EVQLVESGGGLVQPGGSL 33 AIQMTQSPSSLSASVGD 34RLSCAASGFTFSSYWMTW RVTIACRASQGIRDDLA VRQSPGKGLEWVANIKQD WFQQKPGKAPKVLIYAA GSEENYVDSVKGRFTISR SNLQSGVPSRFSGSGSG26093DN AKN S L Y L QMN S L RAD D TDFTLTIRSLQPEDSAT TAVYYCARDTVTTGFYHY YYCLQDYIYPYTFGQGT FYMDVWGKGT TVTVS S RLEIK11C7 QVQLVESGGGWQPGRSL 41 AIQLTQSPSSLSASVGD 42RLSCAASGLTFSSYGMHW RVT I T CRASQGI SNALA VRQAPGKGLEWVAVIWYD WYQQKPGKAPKLLIYDA GSNKYYAD SVKGR F T I S R SSLESGVPSRFSGSGSG DNSKNTLYLQMNSLRAED TDFTLTISSLQPEDFAT TAVYYCARENNWHLNYFF YYCQQFNNYIYTFGQGT YYMDVWGKGT TVTVS S KLEIK5A1 QVQLVESGGGWQPGRSL 49 AIQLTQSPSSLSASVGD 50RLSCAASGFTFSSYGMHW RVT I T CRASQD INNVLA VRQAPGKGLEWVAVIWYD WYQQKPGKAPKLLIYDA GSNKYYAD SVKGR F T I S R SSLESGVPLRFRGSGSG DNSKNTLFLQMNSLRAED TDFTLTINSLQPEDFAN TAVYYCARENNWHLNYYF YYCQHFNDFIYTFGQGT YYMDVWGKGT TVTVS S KLEIK8G6 QVQLVESGGGWQPGRSL 57 AIQLTQSPSSLSASVGD 58RL S CAAS GL T F S S YGMHW RVT I T CRASQGI S SALA VRQAPGKGLEWVAVIWYD WYQQKPGKAPKLLIYDA GSNKYYAD SVKGR F T I S R SSLEGGVPSRFSGSGSG DNSKNTLYLQMNSLRAED TDFTLTIRSLQPEDFAT TAVYYCARENNWNGFYNF YYCQQFNNDLYTFGQGT FYMDV GKGT TVTVS S KLEIK50F5 EVQLVESGGGLVQPGGSL 65 AIQMTQSPSSLSASVGD 66RLSCAASGFTFSRYWMSW RVT I T CRASQGIRNDLG VRQAPGKGLEWVANIKQD WYQQKPGKAPNLLIYAA GSEKNYVDTVKGRFTISR SSLQSGVPSRFSGSGSG DNAKNSLYLLMNSLRAED TDFTLTISSLQPEDFAT TAVYYCARDYDFWNGYYL YYCLQDYNYPYTFGQGT YYSMDVWGT GT TVTVS S KLEIK5E8 QVQLVESGGGWQPGRSL 73 EIVLTQSPATLSLSPGE 74RLSCSASGFTFSSYGMHW RATL S CGASQSVS S SYL VRQAPGKGLEWVAVIWYD AWYQQKPGLAPRLLIYD GSNKYYAD SVKGR F T I S R ASSRASGIPDRFRGSGS DNSKNTLYLQMNSLSADD GTDFTLTISRLEPEDFA TALYYCARENNWNGYYHY VY Y CQQYGS SLYT F GQ G YYMDVWGKGT TVTVS S TKLQIK14A11 QVQLVESGGGWQPGRSL 81 AIQLTQSPSSLSASVGD 82RLSCAASGFTFSSYGMHW RVT I T CRASQGIRSALA VRQAPGKGLEWVAFIWYD WYQQKPGKAPTLLIYDA GSKKYYAD SVKGR F T I S R SSLESGVPRRFSGSGSG DNSKNTLYLQMNSLRVED TDFTLTISSLQPEDFAT TAVYYCARENNWNGYYNF YYCQQFNNYYTFGQGTK YYMDVWGKGTTVIVSS LEIK19A3 QVQLVESGGGWQPGRSL 89 EIVLTQSPATLSLSPGE 90RLSCAASGFTFSSYGMHW RAT L S CGASQSVSNNYL VRQAPGKGLEWVAVIWYD AWYQQKPGLAPRLLIYD GSNKYYAD SVKGR F T I S R ASSRATGIPVRFSGSGS26093DNSKNTLDLQMNSLRAED GTDFTLSISRLEPEDFA TAVYYCARENNWNGYYYY VY Y CQQYGS SLYT F GQ G YYMDVWGKGT TVTVS S TKLEIK15B6 QVQLVESGGGWQPGRSL 97 EIVLTQSPATLSLSPGE 98RLSCAASGFTFSPYAMHW RAT L S CGASQS I SGSYL VRQAPGKGLEWVAVIWYD AWYQQKPGLAPRLLIYD T INKYYAD SVKGR F T I S R ASSRATGTPVRFSGSGS DNSKNTLFLQMNSLRAED GTDFTLTISRLEPEDFA TAMYYCARENNWNGYYSY VY Y CQHYENSLYT F GQ G YYMDVWGKGT KVT VS S TKLEIR6E6 QVQLVESGGGWQPGRSL 105 AIQLTQSPSSLSASVGD 106RLSCAASGFTFSSYGMHW RVT I T CRASQD I SRVLA VRQAPGKGLEWVAVIWYD WYQQKPGKAPKLLIYDA GSNKYYVD SVKGR F T I S R SSLESGVPRRFSGRGSG DNSKNTLYLQMNSLRAED TEFTLTISNLQPEDFAT TAVYYCARENNWNNYYHF YFCQQFNNYMYTFGQGT YYMDVWGKGTAVTVS S KLEIK17H6 QVQLQESGPGLVKPSETL 113 EIVMTQSPATLSLSPGE 114SLTCTVSGGS I ISYYWTW RAT L S CRASQS I SNNYL IRQPPGKGLEWIGYIYYS SWYQQIYGQAPRLLIYG GSANYNPSLKGRVT T S VD ASTRATGIPARFSGSGS TSKSQFSLKLSSVTAADT GTDFTLTISSLQPEDFA AVY Y CARGRRGYS GNGD Y VYYCQQDFNLPLTFGGG YYYMDVWGKGT TVTVS S TKVEIK10F6 EVQLLESGGGWQPGGSL 121 DIQMTQSPSSLSASIGD 122RL S C WS G F T F SNFAMGW TVT I T CRASQD IRYDLG VRQAPGRGLEWVARISSS WYQQKPGKAPKRLIYGA GRDTFYTDSVKGRFSISR SSLHSGVPSRFSGSRSG DNSNNTLYLQLSSLRAED TEFTLTISSLQPEDFAT TAVYYCASLGPVWGNGTT YYCLQLNNFPYTFGQGT VTVSS KLEIR4F4 EVQLVESGGGLVQPGGSL 129 AIQMTQSPSSLSASVGD 130RL S CVAS GL T I SGYWMTW RVT I TCRASQNIRNDLG VRQAPGKGLEWVANINQD WYQQKPGKAPKLLIYAA GNEKNYVD SVKGR F T I S R SSLQSGVPSRFSGSGSG DN AKN S L Y L QMN S L RAE D TDFTLTISSLQPEDFAT TAIYYCVKDTSNYDFYYY YYCLQDYNYPYTFGQGT YFYMDVWGKGT TVTVS S KLEIK4B10 EVQLLESGGGLVQPGGSL 137 EIVLTQSPATLSLSPGE 138RLSCAASGITISSYAMNW RAT L S CRASQSVS T YLV VRQAPGKGLEWVSAISGS WYQQKPGQAPRLLIYDA GGS T YYAD SVKGR F T I S R SNRATGIPARFSGSGSG DNSKNTLYLQMNSLRAED TDFTLTISSLDPEDFAG TAVYYCAKDPLPANWNYF YYCQQRSNWPLTFGGGT YYMDV GKGT TVTVS S KVEIK49G5 QVQLQESGPGLVKPSETL 145 EIVMTQSPATLSLSPGE 146SLTCTVSGGS I SSYYWSW RAT L S CRASQS I S S SYL IRQPAGKGLEWIGRIYSS SWYQQTPGQAPRLLIYG GSTNYNPSLKSRVTMSED VSSRATGIPARFSGSGSTSKNQISLNLSSVTAADT GTDFTLTISSLQPEDFA AVYYCARFSMVRGVMGYM VY Y CQQD YNLPYT F GQ G DVWGKGTTVTVSS TKLEIE7C8 QVQLVESGGGWQPGRSL 153 AIQLTQSPSSLSASVGD 154RL S CEAS GL T F SVYGMHW RVT 11 CRASQGI S SALA VRQAPGKGLEWVAVIWYD WYQQAPGKAPKLLIYDA GTNKY YAD S VKGR F T I S R SRLESGVPLRFSGSGSG DNSKNTLYLQMNSLRAED TDFTLTISSLQPEDFAT TAVYHCARENNWNAYYHF YYCQQFNNNLYTFGQGT HYMDVWGKGTSVTVSS KLEIK9B12 EVQLVESGGGLVQPGGSL 161 AIQMTQSPSSLSASVGD 162RLACAASGFTISSYWMSW RVT I TCRTSQGIRNDLG VRQAPGKGLEWVANINQD WYQQKPGKAPKLLIYAA GSEKYYVD SVKGR F T I S R SNLQSGVPSRFSGSGSG DNAKNSLSLQMNSLRAED TDFALTISSLQPEDFAT TAVYYCARDASNYDGYYY YYCLQDSNYPYTFGQGT YFYMDV GKGT TVTVS S KLEIK16B5 QVQLQESGPGLVKPSETL 169 EILMTQSPATLSLSPGE 170SLTCTVSGGS I ISYYWTW RATLSCRASQSISDSYL IRQPPGKGLEWIGYIFYS SWYQQIPGQAPRLLIYG GSTNYNPSLKGRVTLSVD ASTRATGVPARFSGSGS TSKNQFSLKLSSVTAADT GTDFTLTISSLQPEDFA AVYYCARGRRGYRGNGDY VYYCQQDFNLPLTFGGG YYYMDVWGKGT TVTVS S TKVEIK3G8 QVQLVESGGGWQPGRSL 177 AIQLTQSPSSLSASVGD 178RISCAASGLTFSSYGMHW RVT I T CRASQGI S SALA VRQAPGKGLEWVAIIWYD WYQQKPGKVPKLLIYDA GTNKY YAD SVKGR F T I S R SSLESRVPSRFSGSGSG DNSKNTLYLQMNSLRAED TDFTLTISSLQPEDFAT TAVYYCARENNWNGYYHF YYCQQFNNYLYTFGQGT YYMDVWGKGT TVTVS S KLEIK17E4 QVQLVQSGAEVKKPGASV 185 DIQMTQSPSSLSASVGD 186KVSCKVSEYTLTDLSMYW RVT I T CRASQGI SNYLA VRQAPGKGLEWMGGFDPE WFQQKPGKAPKSLIYDA AGET I YAQKFQGRVTMT E SSLQSGVPSKFTGSGSG DTSTDTAYMELSSLRSED TDFTFTISSLQPEDFAT TAVYYCAIGRYCFSTSCS YYCQQYNSFPPTFGQGT FNYNYYMDVWGKGTTVTV KVEIK SS25G8 EVQLLESGGGLVQPGGSL 193 EIVLTQSPGTLSLSPGE 194RLSCAASGFIFSSYTMKW RAT L S CRASQSVS S SYL VRQAPGKGLEWVSAISGS AWYHQKPGQAPRLLIYG GGS T YYAD SVKGR F T I S R ASSRATGIPDRFSGSGS DNSKNTLYLQMNSLRAED GTDFTLTISRLEPEDFA TAVYYCAKDPLPYNWSFY VYYCQQYGSSPLTFGGG YYYMDVWGKGT TVTVS S TKVEIK16C2 QVQLVQSGAEVKKPGASV 201 EIVMTQSPATLSVSPGE 202KVSCKASGFTLTTYGFNW RAT L S CRASQSVGS FLA VRQAPGQGLEWMGWISAY WYQQKPGQAPRLLIYGANGDTRYAQKFQGRVTMT T STRATGIPARFSGSGSG DTSTSTAYMELWSLRSDD TEFTLTISSLQSEDFAV TAVYYCARGTTVTTPYYY YYCQQYINWPLTFGGGT YSYMDVLGKGTTVTVSS KVEIK21H7 EVQLLESGGGLVQPGGSL 209 DIQMTQSPSSLSASVGD 210RLSCAASGFTFSSYAMSW RVT I T CRASQS I S S YLN VRQAPGKGLEWVAAISGS WYQQKPGKAPRLLIYDA GGS SKNGNSVKGR F T I S R SSLQSGVPSRFSGRGSG DNSKNTLYLLMSILRAED TDFTLTISSLQPEDFAT TAI Y YCAKGTWPWPGD YYCQQSYSTPPTFGQGT HMD VWGKGT TVTVS S KVEIK32H6 EVQLLESGGGLVQPGGSL 217 DIVMTQTPLSLSVTPGQ 218RLSCAASGFTFGNYAMSW PAS I S CKSSQSLLHSDG VRQAPGRGLEWVSGVRNS KTYLYWYLQKPGQPPQL GS GT YYAD SVKGR F T I S R L I YEVSNRFS GVP DRF S DNSKNTLYLQMNSLRAED GSGSGTDFTLKISRVEA TAVYYCAKGGTPVTAPYY E D VGVY Y CMQS I QLPYT YYYYMDVWGKGTSVAVS S FGQGTKLEIK2A2 EVQLVESGGGLVQPGGSL 225 AIQMTQSPSSLSASVGD 226RL S CAAS GFT I SPYWMT W RVT I S CRASQGIRDDLG VRQAPGKGLEWVANINQD WYQQKPGKAPELLIYAA GSEKHYVD SVKGR F T I S R SSLQSGVPSRFSGSGSG DNVHNSLFLQMNSLRAED TDFTLTISSLQPEDFAT TAVYYCVRDDSVYDSYYY YYCLQDYTYPYTFGQGT YFYMDV GKGT TVTVS S KLEIK12C6 QVQLVQSGPEVKKPGASV 233 EIVMTQSPATLSLSPGE 234KVSCKVSGYTLTELSMHW RATLSCRASQSVSSTSF VRQAPGKGLEWMGGFDPE SWYQQKPGQAPRLLIFG DGGT I FAQKFQGRVTMT E ASTRATGIPARFSGSGS DTSTDTAYMELSSLRSED GTDFTLTISSLQPEDFA TAVYYCAGWGSYYRWFDP VYYCHQDYNLPFTFGPG WGQGTLVTVSS TKVDIK12F1 EVQLVESGGGLVQPGGSL 241 AIQMTQSPSSLSASVGD 242RLSCAASGFTFSSYWMSW RVT I T CRASQGIRNDLA VRQAPGKGLEWVANINQD WYQQKPGKAPNLLIYAA GNEKNYVD SVKGR F T I S R SSLQSGVPSRFSGSGSG DN VKN S LH L QMN S L RAE D TDFTLTISSLQPEDFAT SAVYFCARDTSNYDLYSY YYCLQDYIYPYTFGQGT YFYMDVWGKGT TVTVS S KLEIK16F2 QVQLVESGGGWQPGRSL 249 AIQLTQSPSSLSASVGD 250RLSCAASGLTFSSYGMHW RVT I T CRASQGI SNTLA VRQAPGMGLEWVALIWYD WYQQKPGKPPKLLIYDA GSNE YYAD SVKGR FT I S R SRLEGGVPLRFSGSGSG DNFKNTLYLQMNSLRAED TDFTLTISSLQPEDFAT TAVYYCARENNWNGRYYF YYCQQFNNYPTFGGGTK YYMD VWGKGT TVTVS S VEIK14B12 EVQLLESGGGLVQPGGSL 257 EIVLTQSPGTLSLSPGE 258RL S CAAS GF I F S S YTMKW RAT L S CRASQSVS S SYL VRQAPGKGLEWVSAISGS AWYHQKPGQAPRLLIYGGGS T YYAD SVKGR F T I S R ASSRATGIPDRFSGSGS DNSKNTLYLHVNSLRAED GTDFTLTISRLEPEDFA TAVYYCAKDPLPYNWNFY VYYCQQYGSSPLTFGGG YYYMDVWGKGT TVTVS S TKVEIK10G2 EVQLLESGGGLVQPGGSL 265 AIQLTQSPSSLSASVGD 266RLSCAASGFTFSSYALSW RVT I T CRASQGI S SALA VRQTPGKGLEWVSVISGN WYQQKPGKTPKLLIYDA GIITYYADSVKGRFTISR SSLESRVPSRFSGSGSG DNSKNTLYLQMNSLRAED TDFTLTISSLQPEDFAT TAVYYCAKDDSSYYGLGS YYCQQFNNYPYSFGQGT FPNWFDPWGQGTLVTVSS KLEIK6E10 EGQLLESGGGLVQPGGSL 273 EIVMTQSPATLSVSPGE 274RLSCAASGFTFNNYAMSW RAT L S CRASQSVNNNLA VRQAPGKGLEWVSAISGS WYQQKPGQAPRLLIFGA GGS T YYAD SVKGR F T I S R STRATGLPARFSGSGSG DNSKNTLYLQMNSLRAED TEFTLTISSLQSEDFAV TAVYYCAKDGGVPWPYL YYCQQYNNWPFTFGQGT YYYYMDVWGKGTTVTVS S KLEIK10A9 EVQLLESGGGLVQPGGSL 281 EIVMTQSPATLSLSPGE 282RLSCAASGFTFSIYTMKW RAT L S CRASQSVS S SYL VRQAPGKGL E WVS Al SAS SWYQQKPGQAPRLLIYG GGSTSYSDSVKGRFTISR ASTRATGIPARFSGSGS DNSKNTVNLQMNSLRTED GTDFTLTISSLQPEDFA SAVYYCAKDPLPYNWSFF VYYCQQDYNLITFGQGT YYYMDVWGKGT TVTVS S RLEIK35D5 EVQLLESGGGLVQSGGSL 289 EIVLTQSPGTLSLSPGE 290RL S CVAS GF I FNTYVMKW RAT L S CRASQSVS S SYL VRQAPGRGLEWVSAISGS AWYQQKPGQAPRLLIYG GGS T S YTD SVKGR F T VS R ASSRATGIPDRFSGSGS DNSKNTLYLQMTSLRAED GTDFTLTISRLEPEDFA TAVYYCAKDPLPYNWSFY VYYCQQYGSSPLTFGGG FYYMDVWGKGT TVTVS S TKVELK17A7 EVQLVESGGGLVQPGGSL 297 AIQMTQSPSSLSASVGD 298RLSCAASGFTFSSYWMTW RVT I T CRASQGIRNDLG VRQAPGKGLEWVANIKQD WYQQKPGKAPKLLIYAA GSEKHYVD SVKGR F T I S R STLQSGVPSRFSGSGSG DN AKN S L Y L QMN S L RAE D TDFTLTISSLQPEDFAT TAVYYCARDTSNYDLYYY YYCLQDNSYPYTFGQGT YFYMDVWGKGT TVTVS S KLEIK18A11 EVQLVESGGGLVQPGGSL 305 AIQMTQSPSSLSASVGD 306RLSCAASGFTFSSYWMSW R I T I T CRASQGIRNDLG VRQAPGKGLEWVANVNQD WYQQKPGKAPKLLIYAA GSEQNFVDSVKGRFTISR SSLQSGVPSRFSGSGSG DN AKN S VH L QMN S L RAE D TDFTLTISSLQPEDFAT TAVYYCARDASNYDGYYY YYCLQDYNYPYTFGQGT YYYTDVWGKGT TVTVS S KLEIK49G11 EVQLLESGGGLVQPGGSL 313 QLVLTQSPSASASLGAS 314RLSCAASGFTFRSYVMSW VKLTCTLSSGHSSYAIA VRQAPGKGLEWVSAISGS WHQQQPEKGPRYLMKLN26093GDRT YYAD SVKGR F T I S R SDGSHSKGDGIPDRFSG DNSKNTVYLQVKSLRAED SSSGAERYLTISSLQSE TAGYYCAKAAGYCTNGVC DEADYYCQTWGTGIRVF LYYYYMDVWGKGT TVTVS GGGTKLTVLS3A9 QLQLQESGPGLVKPSETL 321 EIVMTQSPATLSLSPGE 322SLTCTVSGGS I SSGSDYW RAT L S CRASQSVRS SYL VWIRQPPGKGLEWIGSIY SWYQQKPGQAPRLLIYG YSGSTYYNPALKSRVT I S ASTRATGIPARFSGSGS VDTSKNQFSLKLSSVTAA GTDFTLTISSLQPEDFA D T AVY Y CARRGNDD YYYF VYYCQQDYNLPMYTFGQ YMDVWGKGTTVTVSA GTKLEIK13H8 EVHLLESGGGLVQPGGSL 329 KIVMTQSPATLSVSPGE 340RLSCAASGFTFSTYAMSW RAT L S CRASQSVS SNLA VRQAPGKGLEWVSAISGS WYQQKPGQAPRLLIYGA GGSRYYAD SVKGR F I S R STRATGIPARFSGSGSG DNSKNTLYLQMNSLRAED TEFTLTISSLQSEDFAV TAVYYCAKDGGVPWPYL YYCQHYNNWPFTFGQGT YYYYMDVWGKGTTVTVS S KLEIK14B8.1 QVQLVESGGGWQPGRSL 347 AIQLTQSPSSLSASVRD 348RLSCAASGFTFSSYGMHW RVT I T CRASQGINSALA VRQVPGKGLEWVAFISYD WYQQKPGKAPKLLIYDA GKNKYY ID SVRGR F I S R SRLESRVPSRFSGSGSG DNSKNTLFLQMNSLRAED TDFTLTISSLQPEDFAT TAVYYCARENNWNDFYNY YYCQQFNNYMYTFGQGT YYMDVWGKGT TVTVS S KLEIK7F11.1 QVQLQESGPGLVKPSETL 355 AIQMTQSPSSLSASVGD 356SLTCTVSGGS I SSYYWSW RVT I T CRASQGIRNDLG IRQPAGKGLEWIGRIYTS WYQQKPGKAPKLLIYAA GSTNYNPSLKSRVTMSGD SSLQSGVPSRFSGSGSG TSKNQFSLKLTSVTAADT TDFTLTISSLQPEDFAT AVYYCAREWYYYYMDVW YYCLQDYNYPYTFGQGT GKGTTVTVSS KLEIK35D7.1 EVQLVESGGGLVQPGGSL 363 AIQMTQSPSSLSASVGD 364RL S CAAS GF S L S S YWMS W RVT I T CRASQGIRDDLG VRQAPGKGLEWVANIKQD WYQQTPGKAPKLLIYAA GSEKNYVDAVKGR F T I S R STLQSGVPSRFSGSGSG DN AKN S L Y L HMN S L RVE D TDFTLTISSLQPEDFAT TAVYYCARDNDNWNGFYY YYCLQDNNYPYTFGQGT YYSMDVWGKGT TVTVS S KLEIK13B3 EVQLVESGGGLVQPGGSL 371 AIQMTQSPSSLSASVGD 372RLSCVASGFTFGPYWMTW RVT I T CRASQGIRDDLG VRQAPGKGLEWVANINQD WYQQKPGKAPELLIYAA GNEKNYVD SVKGR F T I S R SSLQSGVPSRFSGSGSG DNVKNSLFLQMNSLRAED TDFTLTISSLQPEDFAT TAVYYCVRDDSVYDSYYY YYCLQDYNYPYTFGQGT YFYMGVWGGGTAVTVS S KLEIK2609324F4 VH + VL EVQLLESGGGLVQPGGSL 1 EIVLTQSPATLSLSPGE 419 (K1E Q79E M4L RLSCAASGFTFSSYAMSW RATLSCRASQSVSSSYL M48I D57G) VRQAPGKGLEWVSAISIS SWYQQKPGQAPRLLIYG GTYTYYADSVKGRFTISR TSTRATGIPARFSGSGS DNSENTLFLQMNSLRAED GTDFTLTISSLEPEDFA TAVYYCAKAHSTYPYYYY VYYCHQDYNFPLTFGGG YYMDVWGKGT TVTVS S TKVEIK10B3 VH (D46E EVQLLESGGGLVQPGGSL 420 EIVLTQSPGTLSLSPGE 421 T84AN79Y RLSCAASGFTFSIYTMKW RATLSCRASQSVSNSYLS87T) + VL VRQAPGKGLEWVSAISAS AWYQQKPGQAPRLI IYG (I85V H87Y) GGSTSYSDSLKGRFTISR ASSRAAGIPDRFSGSGS DNSKNTVYLQMNSLRAED GTDFTLTISRLEPEDFA T AVY YCAKDPLPYNWNFF VYYCQQYGRSPLTFGGG YYYMDVWGKGT TVTVS S TKVEIK9G10 VH EVQLLESGGGLVQPGGSL 422 EIVLTQSPATLSLSPGE 18 (V26G) + VL RLSCAASGSTFNNYAMSW RATLSCRASQSVSSYLA VRQAPGKGLEWVSAISGS WYQQKPGQAPRLLIYGA GGSTYYADSVKGRFTISR SNRATGI PARFSGSGSG DNSKNTLYLQMNSLRAED TDFTLTISSLEPEDFAV TAVYYCAKDPLPANWNYY YYCQQRSNWPLTFGGGT YYMDVWGKGT TVTVS S KVEIKThe VH and VL CDR sequences are shown in bold. CDR sequences defined by Kabat numbering scheme.

[0176] The first arm of the bispecific antibody comprising any one of the VH: VL pairs set forth in Table 5 wherein the VH of the pair is linked to a CH comprising an amino acid sequence selected from the amino acid sequences show n in Table 4 (or CH variant that lacks a C-terminal lysine residue) and the VL of the pair is linked to a human LC Kappa constant domain comprising the amino acid sequence set forth in SEQ ID NO: 391.CD3 Binders

[0177] A widely used antibody specific for human CD3 with cross reactivity to non-human primate CD3 is the mouse monoclonal antibody SP34 (Y oshino et al., Exp. Anim 49: 97-110 (2000); Conrad et al.. Cytometry 71 A: 925-33 (2007)). Because this antibody is derived from mouse, many humanized versions have been generated and incorporated in various forms into bispecific molecules, including some that are in the clinic. International patent publications W02008119565, W02008119566, W02008119567, W02008119568. W02010037836, and W02010037837 disclose anti-CD3 humanized antibody IC2 and WO2022125576 discloses an humanized anti-CD3 scFv SP34.185 in which the variable domains are arranged in the VH-VL orientation, each of IC2 and SP34.185 comprising a VH / VL pair in which the VH comprises VH-CDR 1, 2, and 3 shown in Table 6 and the VL comprises VL-CDR 1, 2, and 3 shown in Table 6.The VH and VL of I2C and SP34.185, each comprise amino acid sequences set forth in SEQ ID26093NO: 604 and SEQ ID NO: 605, respectively, as shown in Table 7. The VH / VL pairs have been used to construct various bispecific antibodies comprising an arm that binds a tumor associated antigen (TAA) and an arm that binds human and non-human primate CD3.

[0178] The CD3 binder herein may comprise the VH and VL of any anti-CD3 antibody and may be used to construct the ALPP / L2 TCEs disclosed herein. In particular embodiments, the second binding domain that binds CD3 comprises a Fab or an scFv. In further embodiments, the VH and VL comprising the scFv format are optimized for lower aggregation and thermal stability while maintaining affinity for CD3.

[0179] The CD3 binders providing the second binding domain for the bispecific antibodies disclosed herein comprise (i) a VH comprising VH-CDRs 1, 2, and 3; and (ii) a VL comprising VL-CDRs 1, 2, and 3, wherein the VH-CDRs and VL-CDRs comprise the amino acid sequences set shown in Table 6.Table 6VH CDR Amino Acid SEQ ID VL CDR Amino Acid SEQ ID Sequence NO: Sequence NO:CDR1 KYAMN 424 GSSTGAVTSGNYPN 428CDR2 RIRSKYNNYATYYADSVKD 425 GTKFLAP 429CDR3 HGNFGNSYI SYWAY 426 VLWYSNRWV 430CDR sequences defined by Kabat numbering scheme.

[0180] In a further embodiment, the CD3 binder comprises the VH / VL amino acid sequences of scFv SP34.185 or engineered scFv SP34.185 as shown in Table 7. The VH of the engineered SP34.185 has an N87S amino acid substitution as shown in Table 7 and the VL of the engineered SP34.185 has a G57W amino acid substitution as shown in Table 7. In particular embodiments, the VH-FR1, VH-FR2, VH-FR3, and VH-FR4 of the VH may further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, insertions, or combinations thereof. In particular embodiments, the VL-FR1, VL-FR2, VL-FR3, and VL-FR4 of the VL may further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, insertions, or combinations thereof. In particular embodiments, the VH-FR1, VH-FR2, VH-FR3, and VH-FR4 of the VH and the VL-FR1, VL-FR2, VL-FR3, and VL-FR4 of the VL may further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9. or 10 amino acid substitutions, deletions, insertions, or combinations thereof.Table 7C,,onst.ruct. V.. H.. A. mm. o A. ci.d.cSeqMuence SE. Q A ID VL „ Amino Acid SE. Q ID NO: Sequence NO:SP34.185 EVQLVESGGGLVQPGGSL 604 QTWTQEPSLTVSPGGT 605KLSCAASGFTFNKYAMNW VTLTCGSSTGAVTSGNY VRQAPGKGLEWVARIRSK PNWVQQKPGQAPRGLIG YNNYATYYADSVKDRFTI GTKFLAPGTPARFSGSL26093SRDDSKNTAYLQMNNLKT LGGKAALTLSGVQPEDE EDTAVYYCVRHGNFGNSY AEYYCVLWYSNRWVFGG ISYWAYWGQGTLVTVSS GTKLTVLEngineered EVQLVESGGGLVQPGGSL 423 QTWTQEPSLTVSPGGT 427 SP34.185 KLSCAASGFTFNKYAMNW VTLTCGSSTGAVTSGNY VRQAPGKGLEWVARIRSK PNWVQQKPGQAPRGLIG YNNYATYYADSVKDRFTI GTKFLAPWTPARFSGSL SRDDSKNTAYLQMNSLKT LGGKAALTLSGVQPEDE EDTAVYYCVRHGNFGNSY AEYYCVLWYSNRWVFGG ISYWAYWGQGTLVTVSS GTKLTVLThe VH and VL CDRs are shown in bold. CDR sequences defined by Kabat numbering scheme.The N87S and G57W substitutions in the engineered VH and VL. respectively, are underlined.

[0181] The CD3 binders herein may comprise a VH of Table 7 linked to a CH comprising the amino acid sequence set forth in SEQ ID NO: 467 and a VL of Table 7 linked to a human CL comprising the amino acid sequence set forth in SEQ ID NO: 391. In certain embodiments, the VH and the VL may each independently comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, insertions, deletions, or combinations thereof.

[0182] In further embodiments, the CH comprises a substitution of the amino acids at positions 252, 254. and 256 of the constant domain of the heavy chain with amino acids Tyr (Y), Thr (T), and Glu (E), respectively (M252Y, S254T, T256E substitution) wherein the numbering is according to the EU numbering scheme (The positions according to sequential number are 256, 258, and 260, respectively).

[0183] In further embodiments, the CH is of IgGl isotype and comprises E233A and L235A amino acid substitutions wherein the numbering is according to the EU numbering scheme.

[0184] In further embodiments, the CH is of IgGl isotype and comprises L234A L235A D265S substitutions wherein the numbering is according to the EU numbering scheme.

[0185] In further embodiments, the CH is of IgGl isotype and comprises L234A L235A P329G substitutions wherein the numbering is according to the EU numbering scheme.

[0186] In further embodiments, the CH is of IgGl isotype and comprises an L235E substitution wherein the numbering is according to the EU numbering scheme.

[0187] In further embodiments, the CH is of IgGl isotype and comprises D265A substitution wherein the numbering is according to the EU numbering scheme.

[0188] In further embodiments, the CH is of IgGl isotype and comprises D265A N297G substitutions wherein the numbering is according to the EU numbering scheme.

[0189] In further embodiments, the CH is of IgGl isotype and comprises N297X, wherein X is any amino acid other than N substitution wherein the numbering is according to the EU numbering scheme.26093

[0190] In further embodiments, the CH is of IgGl isotype and comprises N297A / D356E / L358M substitutions wherein the numbering is according to the EU numbering scheme.

[0191] In certain embodiments of the invention, the CH3 domains of the IgGl or IgG4 heavy chain constant domains as disclosed herein may comprise a C-terminal lysine or lack either a C-terminal lysine or a C-terminal glycine-lysine dipeptide. In some embodiments, the N-terminal amino acid of the antibody variable domains may undergo cyclization to pyroglutamate. Thus, in a composition comprising a particular antibody disclosed herein, the composition may comprise a population of antibody species wherein each species may independently comprise a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine and / or comprise an N-terminal glutamine or glutamic acid or cyclization of the N-terminal amino acid to pyroglutamate.

[0192] For certain bispecific antibodies as disclosed herein, the second arm of the bispecific antibody comprising a VH: VL pair having the CDR amino acid sequences as set forth in Table 6 or the VH and VL sequences as set forth in Table 7. Table 7 comprises embodiments in which the VH is linked to a CH comprising an amino acid sequenced shown in Table 4 (or CH variant that lacks a C-terminal lysine residue) and the VL is linked to a human LC Kappa constant domain comprising the amino acid sequence set forth in SEQ ID NO: 391 with the proviso that when the CH of the first arm that binds ALPP and / or ALPPL2 comprises a knob mutation as set forth in Table 4, the CH of second arm that binds CD3 comprises a hole mutation at set forth in Table 4 that pairs with the CH of the first arm, and when the CH of the first arm that binds ALPP and / or ALPPL2 comprises a hole mutation as set forth in Table 4, the CH of second arm that binds CD3 comprises a knob mutation at set forth in Table 4 that pairs with the CH of the first arm, with the further proviso that for any first arm CH: second arm CH pair, both the CH of the first arm and the CH of the second arm comprise the same amino acid substitutions that reduce or ablate effector function.

[0193] In certain embodiments, the CD3 binding portion of the second arm is in an scFv format wherein the scFv comprises form the N-terminus to the C-terminus the structure VL-linker-VH (LH orientation) or VH-linker-VL (HL orientation), wherein the linker is a flexible peptide comprising 15 to 40 amino acids. In certain embodiments, the linker comprises primarily glycine and serine residues. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID No: 465. An exemplary scFv in the LH orientation comprises the amino acid sequence set forth in SEQ ID NO: 463 and an exemplary scFv in the HL orientation comprises the amino acid sequence set forth in SEQ ID NO: 464, as shown Table 8 below.26093_ Table 8 _scFv comprising engineered SP34.185 VH / VL Amino Acid SequencesSEQ SEQ LH orientation ID HL orientation IDNO: NO:QTWTQEPSLTVSPGGTVT 463 QEVQLVESGGGLVQPGGSL 464 LTCGSSTGAVTSGNYPNWV KLSCAASGFTFNKYAMNWV QQKP GQAP RGL I GGTKFLA RQAP GKGL EWVARIRSKYN PWTPARFSGSLLGGKAALT NYATYYADSVKDRFTISRD LSGVQPEDEAEYYCVLWYS DSKNTAYLQMNSLKTEDTA NRWVFGGGTKLTVLGGGGS VYYCVRHGNFGNSYISYWA GGGGSGGGGSGGGGSEVQL YWGQGTLVTVSSGGGGSGG VE S GGGL VQP GGS L KL S CA GGSGGGGSGGGGST W QE ASGFTFNKYAMNWVRQAPG PSLTVSPGGTVTLTCGSST KGL E VARIRSKYNNYAT Y GAVT S GNYPNWVQQ K P GQ A YADSVKDRFTISRDDSKNT PRGLIGGTKFLAPWTPARF AYLQMNSLKTEDTAVYYCV SGSLLGGKAALTLSGVQPE RHGNFGNSYISYWAYWGQG DEAEYYCVLWYSNRWVFGG TLVTVSS GTKLTVL_ scFv comprising SP34.185 VH / VL Amino Acid Sequences _ QTWTQEPSLTVSPGGTVT 606 QEVQLVESGGGLVQPGGSL 607 LTCGSSTGAVTSGNYPNWV KLSCAASGFTFNKYAMNWV QQKP GQAP RGL I GGTKFLA RQAP GKGL EWVARIRSKYN PGTPARFSGSLLGGKAALT NYATYYADSVKDRFTISRD LSGVQPEDEAEYYCVLWYS DSKNTAYLQMNNLKTEDTA NRWVFGGGTKLTVLGGGGS VYYCVRHGNFGNSYISYWA GGGGSGGGGSGGGGSEVQL YWGQGTLVTVSSGGGGSGG VE S GGGL VQP GGS L KL S CA GGSGGGGSGGGGST WTQE ASGFTFNKYAMNWVRQAPG PSLTVSPGGTVTLTCGSST KGL E VARIRSKYNNYAT Y GAVT S GNYPNWVQQ K P GQ A YADSVKDRFTISRDDSKNT PRGLIGGTKFLAPGTPARF AYLQMNNLKTEDTAVYYCV SGSLLGGKAALTLSGVQPE RHGNFGNSYISYWAYWGQG DEAEYYCVLWYSNRWVFGG TLVTVSS _ GTKLTVL _The scFv CDRs are shown in bold. CDR sequences defined by Kabat numberingscheme.

[0194] In certain embodiments, the scFv comprising the second arm is provided as an scFv-CH’ fusion protein in which the scFv is linked at the C-terminus to the N-terminus of a truncated CH, which comprises at least the CH2 and CH3 domains (CH’).

[0195] In certain embodiments the CH’ further includes the Hinge region or a portion thereof, for example amino acids 8 to 15 of the Hinge region. In certain embodiments the scFv is linked to the CH’ by a peptide linker.

[0196] In certain embodiments the scFv is linked to the CH’ by a peptide linker comprising the amino acid sequence set forth in SEQ ID NO: 466.

[0197] In certain embodiments, the CH’ is selected from an amino acid sequence set forth in Table 9 with the proviso that when the CH of the first arm that binds ALPP and / or ALPPL2 comprises a knob mutation as set forth in Table 4, the CH’ of second arm that binds CD326093comprises a hole mutation at set forth in Table 9 that pairs with the CH of the first arm, and when the CH of the first arm that binds ALPP and / or ALPPL2 comprises a hole mutation as set forth in Table 4, the CH’ of second arm that binds CD3 comprises a knob mutation at set forth in Table 9 that pairs with the CH of the first arm, with the further proviso that for any CH: CH’ pair (Fc region), both the CH and the CH’ comprise the same amino acid substitutions that reduce or ablate effector function.Tabic 9Description CH’ Amino acid SEQ Description CH’ Amino Acid SEQ Sequence ID Sequence ID (KIH: Hole) NO: (KIH: knob) NO:Human IgGl THTCPPCPAPELLGGP 431 Human IgGl THTCPPCPAPELLGG 441 HC 8-15 Hinge SVFLFPPKPKDTLMIS HC 8-15 PSVFLFPPKPKDTLM+ CH2-CH3 RTPEVTCVWDVSHED Hinge + CH2- ISRTPEVTCVWDVS (KIH: Hole PEVKFNWYVDGVEVHN CH3 HEDPEVKFNWYVDGV Y349C T366S AKTKPREEQYNSTYRV (KIH: knob EVHNAKTKPREEQYN L368AY407V) VSVLTVLHQDWLNGKE S354C STYRWSVLTVLHQD YKCKVSNKALPAPIEK T366W) WLNGKEYKCKVSNKA TISKAKGQPREPQVCT LPAPIEKTISKAKGQ LPPSRDELTKNQVSLS PREPQVYTLPPCRDE CAVKGFYPSDIAVEWE LTKNQVSLWCLVKGF SNGQPENNYKTTPPVL YPSDIAVEWESNGQP DSDGSFFLVSKLTVDK ENNYKTTPPVLDSDG SRWQQGNVFSCSVMHE SFFLYSKLTVDKSRW ALHNHYTQKSLSLSPG QQGNVFSCSVMHEAL K HNHYTQKSLSLSPGKHuman IgGl THTCPPCPAPELLGGP 432 Human IgGl THTCPPCPAPELLGG 442 HC 8-15 Hinge SVFLFPPKPKDTLYIT HC 8-15 PSVFLFPPKPKDTLY+ CH2-CH3 REPEVTCVWDVSHED Hinge + CH2- ITREPEVTCVWDVS (YTE) (KIH: PEVKFNWYVDGVEVHN CH3 (YTE) HEDPEVKFNWYVDGV HoleY349C AKTKPREEQYNSTYRV (KIH: knob EVHNAKTKPREEQYN T366S L368A VSVLTVLHQDWLNGKE S354C STYRWSVLTVLHQD Y407V) YKCKVSNKALPAPIEK T366W) WLNGKEYKCKVSNKA TISKAKGQPREPQVCT LPAPIEKTISKAKGQ LPPSRDELTKNQVSLS PREPQVYTLPPCRDE CAVKGFYPSDIAVEWE LTKNQVSLWCLVKGF SNGQPENNYKTTPPVL YPSDIAVEWESNGQP DSDGSFFLVSKLTVDK ENNYKTTPPVLDSDG SRWQQGNVFSCSVMHE SFFLYSKLTVDKSRW ALHNHYTQKSLSLSPG QQGNVFSCSVMHEAL K HNHYTQKSLSLSPGKHuman IgGl THTCPPCPAPALAGGP 433 Human IgGl THTCPPCPAPALAGG 443 HC 8-15 Hinge SVFLFPPKPKDTLMIS HC 8-15 PSVFLFPPKPKDTLM+ CH2-CH3 RTPEVTCVWDVSHED Hinge + CH2- ISRTPEVTCVWDVS (E233A / L235A PEVKFNWYVDGVEVHN CH3 HEDPEVKFNWYVDGV) (KIH: Hole AKTKPREEQYNSTYRV (E233A / L235 EVHNAKTKPREEQYN Y349C T366S VSVLTVLHQDWLNGKE A) (KIH: knob STYRWSVLTVLHQD L368AY407V) YKCKVSNKALPAPIEK S354C WLNGKEYKCKVSNKA TISKAKGQPREPQVCT T366W) LPAPIEKTISKAKGQ26093LPPSRDELTKNQVSLS PREPQVYTLPPCRDE CAVKGFYPSDIAVEWE LTKNQVSLWCLVKGF SNGQPENNYKTTPPVL YPSDIAVEWESNGQP DSDGSFFLVSKLTVDK ENNYKTTPPVLDSDG SRWQQGNVFSCSVMHE SFFLYSKLTVDKSRW ALHNHYTQKSLSLSPG QQGNVFSCSVMHEAL K HNHYTQKSLSLSPGKHuman IgGl THTCPPCPAPEAAGGP 434 Human IgGl THTCPPCPAPEAAGG 444 HC 8-15 Hinge SVFLFPPKPKDTLMIS HC 8-15 PSVFLFPPKPKDTLM + CH2-CH3 RTPEVTCVWSVSHED Hinge + CH2- I SRTPEVTCVWSVS (L234A L235A P E VK FNW Y VD GVE VHN CH3 (L234A HEDPEVKFNWYVDGV D265S) (KIH: AKTKPREEQYNSTYRV L235A EVHNAKTKPREEQYN Hole Y349C VSVLTVLHQDWLNGKE D265S) (KIH: STYRWSVLTVLHQD T366S L368A YKCKVSNKALPAPIEK knob S354C WLNGKEYKCKVSNKA Y407V) TISKAKGQPREPQVCT T366W) LPAPIEKTISKAKGQ LPPSRDELTKNQVSLS PREPQVYTLPPCRDE CAVKGFYPSDIAVEWE LTKNQVSLWCLVKGF SNGQPENNYKTTPPVL YPSDIAVEWESNGQP DSDGSFFLVSKLTVDK ENNYKTTPPVLDSDG SRWQQGNVFSCSVMHE SFFLYSKLTVDKSRW ALHNHYTQKSLSLSPG QQGNVFSCSVMHEAL K HNHYTQKSLSLSPGKHuman IgGl THTCPPCPAPEAAGGP 435 Human IgGl THTCPPCPAPEAAGG 445 HC 8-15 Hinge SVFLFPPKPKDTLMIS HC 8-15 PSVFLFPPKPKDTLM + CH2-CH3 RTPEVTCVWSVSHED Hinge + CH2- I SRTPEVTCVWSVS (L234A L235A P E VK FN Y VD GVE VHN CH3 (L234A HEDPEVKFNWYVDGV P329G) (KIH: AKTKPREEQYNSTYRV L235A EVHNAKTKPREEQYN Hole Y349C VSVLTVLHQDWLNGKE P329G) (KIH: STYRWSVLTVLHQD T366S L368A YKCKVSNKALGAPIEK knob S354C WLNGKEYKCKVSNKA Y407V) TISKAKGQPREPQVCT T366W) LGAPIEKTISKAKGQ LPPSRDELTKNQVSLS PREPQVYTLPPCRDE CAVKGFYPSDIAVEWE LTKNQVSLWCLVKGF SNGQPENNYKTTPPVL YPSDIAVEWESNGQP DSDGSFFLVSKLTVDK ENNYKTTPPVLDSDG SRWQQGNVFSCSVMHE SFFLYSKLTVDKSRW ALHNHYTQKSLSLSPG QQGNVFSCSVMHEAL K HNHYTQKSLSLSPGKHuman IgGl THTCPPCPAPELEGGP 436 Human IgGl THTCPPCPAPELEGG 446 HC 8-15 Hinge SVFLFPPKPKDTLMIS HC 8-15 PSVFLFPPKPKDTLM + CH2-CH3 RTPEVTCVWDVSHED Hinge + CH2- I SRTPEVTCVWDVS (L235E) (KIH: P E VK FNW Y VD GVE VHN CH3 (L235E) HEDPEVKFNWYVDGV Hole Y349C AKTKPREEQYNSTYRV (KIH: knob EVHNAKTKPREEQYN T366S L368A VSVLTVLHQDWLNGKE S354C STYRWSVLTVLHQD Y407V) YKCKVSNKALPAPIEK T366W) WLNGKEYKCKVSNKA TISKAKGQPREPQVCT LPAPIEKTISKAKGQ LPPSRDELTKNQVSLS PREPQVYTLPPCRDE CAVKGFYPSDIAVEWE LTKNQVSLWCLVKGF SNGQPENNYKTTPPVL YPSDIAVEWESNGQP DSDGSFFLVSKLTVDK ENNYKTTPPVLDSDG SRWQQGNVFSCSVMHE SFFLYSKLTVDKSRW ALHNHYTQKSLSLSPG QQGNVFSCSVMHEAL K HNHYTQKSLSLSPGK26093Human IgGl THTCPPCPAPELLGGP 437 Human IgGl THTCPPCPAPELLGG 447 HC 8-15 Hinge SVFLFPPKPKDTLMIS HC 8-15 PSVFLFPPKPKDTLM + CH2-CH3 RTPEVTCVVVAVSHED Hinge + CH2- I SRTPEVTCVWAVS (D265A) (KIH: P E VK FNW Y VD GVE VHN CH3 (D265A) HEDPEVKFNWYVDGV Hole Y349C AKTKPREEQYNSTYRV (KIH: knob EVHNAKTKPREEQYN T366S L368A VSVLTVLHQDWLNGKE S354C STYRWSVLTVLHQD Y407V) YKCKVSNKALPAPIEK T366W) WLNGKEYKCKVSNKA TISKAKGQPREPQVCT L PAP IE KT IS KAKGQ LPPSRDELTKNQVSLS PREPQVYTLPPCRDE CAVKGFYPSDIAVEWE LTKNQVSLWCLVKGF SNGQPENNYKTTPPVL YPSDIAVEWESNGQP DSDGSFFLVSKLTVDK ENNYKTTPPVLDSDG SRWQQGNVFSCSVMHE SFFLYSKLTVDKSRW ALHNHYTQKSLSLSPG QQGNVFSCSVMHEAL K HNHYTQKSLSLSPGKHuman IgGl THTCPPCPAPELLGGP 438 Human IgGl THTCPPCPAPELLGG 448 HC 8-15 Hinge SVFLFPPKPKDTLMIS HC 8-15 PSVFLFPPKPKDTLM + CH2-CH3 RTPEVTCVVVAVSHED Hinge + CH2- I SRTPEVTCVWAVS (D265A P E VK FNW Y VD GVE VHN CH3 (D265A HEDPEVKFNWYVDGV N297G) (KIH: AKTKPREEQYGSTYRV N297G) (KIH: EVHNAKTKPREEQYG Hole Y349C VSVLTVLHQDWLNGKE knob S354C STYRWSVLTVLHQD T366S L368A YKCKVSNKALPAPIEK T366W) WLNGKEYKCKVSNKA Y407V) TISKAKGQPREPQVCT L PAP IE KT IS KAKGQ LPPSRDELTKNQVSLS PREPQVYTLPPCRDE CAVKGFYPSDIAVEWE LTKNQVSLWCLVKGF SNGQPENNYKTTPPVL YPSDIAVEWESNGQP DSDGSFFLVSKLTVDK ENNYKTTPPVLDSDG SRWQQGNVFSCSVMHE SFFLYSKLTVDKSRW ALHNHYTQKSLSLSPG QQGNVFSCSVMHEAL K HNHYTQKSLSLSPGKHuman IgGl THTCPPCPAPELLGGP 439 Human IgGl THTCPPCPAPELLGG 449 HC 8-15 Hinge SVFLFPPKPKDTLMIS HC 8-15 PSVFLFPPKPKDTLM + CH2-CH3 RTPEVTCVWDVSHED Hinge + CH2- I SRTPEVTCVWDVS (N297X, P E VK FNW Y VD GVE VHN CH3 (N297X, HEDPEVKFNWYVDGV wherein X is AKTKPREEQYXSTYRV wherein X is EVHNAKTKPREEQYX any amino acid VSVLTVLHQDWLNGKE any amino STYRWSVLTVLHQD other than N) YKCKVSNKALPAPIEK acid other than WLNGKEYKCKVSNKA (KIH: Hole TISKAKGQPREPQVCT N) (KIH: knob L PAP IE KT IS KAKGQ Y349C T366S LPPSRDELTKNQVSLS S354C PREPQVYTLPPCRDE L368AY407V) CAVKGFYPSDIAVEWE T366W) LTKNQVSLWCLVKGF SNGQPENNYKTTPPVL YPSDIAVEWESNGQP DSDGSFFLVSKLTVDK ENNYKTTPPVLDSDG SRWQQGNVFSCSVMHE SFFLYSKLTVDKSRW ALHNHYTQKSLSLSPG QQGNVFSCSVMHEAL K HNHYTQKSLSLSPGKHuman IgGl THTCPPCPAPELLGGP 440 Human IgGl THTCPPCPAPELLGG 450 HC 8-15 Hinge SVFLFPPKPKDTLMIS HC 8-15 PSVFLFPPKPKDTLM + CH2-CH3 RTPEVTCVWDVSHED Hinge + CH2- I SRTPEVTCVWDVS (N297A / D356 PEVKFNWYVDGVEVHN CH3 HEDPEVKFNWYVDGV E / L358M) AKTKPREEQYASTYRV (N297A / D356 EVHNAKTKPREEQYA (KIH: Hole VSVLTVLHQDWLNGKE E / L358M) STYRWSVLTVLHQD Y349C T366S YKCKVSNKALPAPIEK (KIH: knob WLNGKEYKCKVSNKA L368AY407V) T I S KAKGQ PRE PQVCT L PAP IE KT IS KAKGQ26093LPPSREEMTKNQVSLS S354C PREPQVYTLPPCREE CAVKGFYPSDIAVEWE T366W) MTKNQVSLWCLVKGF SNGQPENNYKTTPPVL YPSDIAVEWESNGQP DSDGSFFLVSKLTVDK ENNYKTTPPVLDSDG SRWQQGNVFSCSVMHE SFFLYSKLTVDKSRW ALHNHYTQKSLSLSPG QQGNVFSCSVMHEAL_ K _ HNHYTQKSLSLSPGK The CH’ amino acid substitution positions are numbered according to the Eu numbering scheme. The CH' amino acid substitutions are shown in bold.

[0198] The amino acid sequences for the CH’s shown in Table 4 may lack a C-terminal lysine residue or the C-terminal di-peptide glycine-lysine. Amino acid sequences 488-507 are variant CH' domains that correspond to the CH' domains shown in Table 4 but which lack a C-terminal lysine residue.

[0199] Exemplary scFv-CH’ constructs are shown in Table 10.Table 10Description ScFv-CH’ Amino Acid SEQ ScFv-CH’ Amino Acid SEQ Sequence ID Sequence ID NO: (K-minus) NO:Anti-CD3 scFv- QTVVTQEPSLTVSPGGT 461 QTVVTQEPSLTVSPGGT 508CH’ (Engineered VTLTCGSS TGAVT S GNY VTLTCGSSTGAVTSGNYSP34.185, LH PNWVQQKPGQAPRGL I G PNWVQQKPGQAPRGL I Gorientation) + GTKFLAP W TPARFSGSL GTKFLAPW TPARFSGSLHuman IgGl HC 8- LGGKAALTLSGVQPEDE LGGKAALTLSGVQPEDE15 Hinge + CH2- AEYYCVLWYSNRWVFGG AEYYCVLWYSNRWVFGGCH3 (L234A GTKL T VL GGGGSGGGGS GTKL T VL GGGGSGGGGSL235A GGGGSGGGGS^VQN^ S GGGGSGGGGSEVQLVES D265S)(KIH: knob GGGLVQPGGSLKLSCAA GGGLVQPGGSLKLSCAAS354C T366W) S GET FNKYAMNWVRQAP S GFT FNKYAMNWVRQAPGKGL EWVARIRSKYNNY GKGL EWVARIRSKYNNY ATYYADSVKDRFT I SRD ATYYADSVKDRFT I S RD DSKNTAYLQMNSLKTED DSKNTAYLQMNSLKTED TAVYYCVRHGNFGNSYI TAVYYCVRHGNFGNSYI SYWAYWGQGTLVTVSSS SYWAYWGQGTLVTVS S S GGSGGGGSGGTHT C P P C GGSGGGGSGGTHTCPPC PAPEAAGGPSVFLFPPK PAPEAAGGPSVFLFPPK PKDTLMISRTPEVTCW PKDTLMISRTPEVTCW VSVS HE D P E VKFN YVD VSVSHEDPEVKFNWYVD GVE VHNAKT KP RE EQ YN GVE VHNAKT KP RE EQ YN STYRWSVLTVLHQDWL STYRWSVLTVLHQDWL NGKEYKCKVSNKALPAP NGKEYKCKVSNKALPAP IEKTISKAKGQPREPQV IEKTISKAKGQPREPQV YTLPPCRDELTKNQVSL YTLPPCRDELTKNQVSL WCLVKGFYPSDIAVEWE WCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLD SNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSR SDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALH WQQGNVFSCSVMHEALH NHYTQKSLSLSPGK NHYTQKSLSLSPG26093Anti-CD3 scFv- QTWTQEPSLTVSPGGT 462 QTWTQEPSLTVSPGGT 509CH’ (Engineered VTLTCGSS TGAVT S GNY VTLTCGSSTGAVTSGNYSP34.185, LH PNWVQQKPGQAPRGL I G PNWVQQKPGQAPRGL I Gorientation) + GTKFLAP W TPARFSGSL GTKFLAPW TPARFSGSLHuman IgGl HC 8- LGGKAALTLSGVQPEDE LGGKAALTLSGVQPEDE15 Hinge + CH2- AEYYCVLWYSNRWVFGG AEYYCVLWYSNRWVFGGCH3 (L234A GTKL T VL GGGGSGGGGS GTKL T VL GGGGSGGGGSL235A GGGGSGGGGSEVQLVE S GGGGSGGGGSEVQLVES D265S)(KIH: Hole GGGLVQPGGSLKLSCAA GGGLVQPGGSLKLSCAAY349C T366S S GET FNKYAMNWVRQAP S GFT FNKYAMNWVRQAP L368AY407V) GKGL EWVARIRSKYNNY GKGL EWVARIRSKYNNY ATYYADSVKDRFT I SRD ATYYADSVKDRFT I S RD DSKNTAYLQMNSLKTED DSKNTAYLQMNSLKTED TAVYYCVRHGNFGNSYI TAVYYCVRHGNFGNSYI SYWAYWGQGTLVTVSSS SYWAYWGQGTLVTVS S S GGSGGGGSGGTHT C PPC GGSGGGGSGGTHTCPPC PAPEAAGGPSVFLFPPK PAPEAAGGPSVFLFPPK PKDTLMISRTPEVTCW PKDTLMISRTPEVTCW VSVS HE D P E VKFN YVD VSVSHEDPEVKFNWYVD GVE VHNAKT KP RE EQ YN GVE VHNAKT KP RE EQ YN STYRWSVLTVLHQDWL STYRWSVLTVLHQDWL NGKEYKCKVSNKALPAP NGKEYKCKVSNKALPAP IEKTISKAKGQPREPQV IEKTISKAKGQPREPQV CTLPPSRDELTKNQVSL CTLPPSRDELTKNQVSL SCAVKGFYPSDIAVEWE SCAVKGFYPSDIAVEWE SNGQPENNYKTTPPVLD SNGQPENNYKTTPPVLD SDGSFFLVSKLTVDKSR SDGSFFLVSKLTVDKSR WQQGNVFSCSVMHEALH WQQGNVFSCSVMHEALH NHYTQKSLSLSPGK NHYTQKSLSLSPGThe CH’ amino acid substitution positions are numbered according to the Eu numberingscheme. The scFv CDRs and CH’ amino acid substitutions are shown in bold.Exemplary bispecific antibodies

[0200] In certain embodiments, the exemplary bispecific antibody comprises a first arm comprising a first binding domain that binds ALPP / L2 and a second binding domain comprising a second binding domain that binds CD3. The first binding domain comprises a Fab comprising a VH: VL pair and the second binding domain comprises an scFv comprising from the N-terminus to the C-terminus the structure VL-linker-VH; wherein the C-terminus of the VH of the Fab is linked to the N-terminus of a CH comprising from the N-terminus to the C-terminus, the structure CH1-Hinge-CH2-CH3, to provide the structure of VH-Cl-Hinge-CH2-CH3 and the C-terminus of the VL of the Fab is linked to the N-terminus of a CL to provide the structure VL-CL; wherein the VH-Cl-Hinge-CH2-CH3 and the VL-CL together provide the first arm; and wherein the C-terminus of the scFv is linked to the N-terminus of truncated CH (CH’) comprising from the N-terminus to the C-terminus the structure AHinge-CH2-CH3 to provide the structure scFv-AHinge-CH2-CH3 (wherein AHinge consists of amino acids 8 to 15 of the Hinge).

[0201] Exemplary embodiments of bispecific antibodies include the following embodiments.26093

[0202] A bispecific antibody comprising (a) a first arm comprising a VH-CH: VL-CL pair wherein the VH and VL are a pair selected from the group consisting of the VH-VL pairs comprising an amino acid sequence set forth in Table 3 or 5; a CH comprising a knob structure wherein the CH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 381-390; and a CL comprising the amino acid sequence set forth in SEQ ID NO: 391; and (b) a second arm comprising an scFv comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 463 and 464 or SEQ ID NO: 606 and 607 and a CH’ comprising a hole structure wherein the CH’ comprises an amino acid sequence selected from the group consisting of SEQ ID Nos: 431-440; with the proviso that the CH and CH’ comprise the same CH2 amino acid substitutions.

[0203] A bispecific antibody comprising (a) a first arm comprising a VH-CH: VL-CL pair wherein the VH and VL are a pair selected from the group consisting of the VH-VL pairs comprising an amino acid sequence set forth in Table 3 or 5; a CH comprising a hole structure wherein the CH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 392-401; and a CL comprising the amino acid sequence set forth in SEQ ID NO: 391; and (b) a second arm comprising an scFv comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 463 and 464 or SEQ ID NO: 606 and 607 and a CH’ comprising a knob structure wherein the CH’ comprises an amino acid sequence selected from the group consisting of SEQ ID Nos: 441-450; with the proviso that the CH and CH’ comprise the same CH2 amino acid substitutions.

[0204] A bispecific antibody comprising (a) a first arm comprising a VH-CH: VL-CL pair wherein the VH and VL are a pair selected from the group consisting of the VH-VL pairs comprising an amino acid sequence set forth in Table 3 or 5; a CH comprising a knob structure wherein the CH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 468-477; and a CL comprising the amino acid sequence set forth in SEQ ID NO: 391; and (b) a second arm comprising an scFv comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 463 and 464 or SEQ ID NO: 606 and 607 and a CH’ comprising a hole structure wherein the CH’ comprises an amino acid sequence selected from the group consisting of SEQ ID Nos: 488-497; with the proviso that the CH and CH’ comprise the same CH2 amino acid substitutions.

[0205] A bispecific antibody comprising (a) a first arm comprising a VH-CH: VL-CL pair wherein the VH and VL are a pair selected from the group consisting of the VH-VL pairs comprising an amino acid sequence set forth in Table 3 or 5; a CH comprising a hole structure wherein the CH comprises an amino acid sequence selected from the group consisting of SEQ ID26093NOs: 478-487; and a CL comprising the amino acid sequence set forth in SEQ ID NO: 391; and (b) a second arm comprising an scFv comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 463 and 464 or SEQ ID NO: 606 and 607 and a CH’ comprising a knob structure wherein the CH’ comprises an amino acid sequence selected from the group consisting of SEQ ID Nos: 498-507; with the proviso that the CH and CH’ comprise the same CH2 amino acid substitutions.

[0206] A bispecific antibody comprising (a) a first arm comprising a VH-CH: VL-CL pair wherein the VH and VL are a pair selected from the group consisting of the VH-VL pairs comprising an amino acid sequence set forth in Table 3 or 5; a CH comprising a ZW8 6A structure wherein the CH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 524-532; and a CL comprising the amino acid sequence set forth in SEQ ID NO: 391; and (b) a second arm comprising an scFv comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 463 and 464 or SEQ ID NO: 606 and 607 and a CH’ comprising aZW857B structure wherein the CH’ comprises an amino acid sequence selected from the group consisting of SEQ ID Nos: 533-542; with the proviso that the CH and CH’ comprise the same CH2 amino acid substitutions.

[0207] A bispecific antibody comprising (a) a first arm comprising a VH-CH: VL-CL pair wherein the VH and VL are a pair selected from the group consisting of the VH-VL pairs comprising an amino acid sequence set forth in Table 3 or 5; a CH comprising a ZW857B structure wherein the CH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 543-552; and a CL comprising the amino acid sequence set forth in SEQ ID NO: 391; and (b) a second arm comprising an scFv comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 463 and 464 or SEQ ID NO: 606 and 607 and a CH’ comprising aZW856A structure wherein the CH’ comprises an amino acid sequence selected from the group consisting of SEQ ID Nos: 553-562; with the proviso that the CH and CH’ comprise the same CH2 amino acid substitutions.

[0208] A bispecific antibody comprising (a) a first arm comprising a VH-CH: VL-CL pair wherein the VH and VL are a pair selected from the group consisting of the VH-VL pairs comprising an amino acid sequence set forth in Table 3 or 5; a CH comprising a ZW856A structure wherein the CH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 563-572; and a CL comprising the amino acid sequence set forth in SEQ ID NO: 391; and (b) a second arm comprising an scFv comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 463 and 464 or SEQ ID NO: 606 and 607 and a CH’ comprising aZW857B structure wherein the CH’ comprises an amino acid sequence selected26093from the group consisting of SEQ ID Nos: 573-582; with the proviso that the CH and CH’ comprise the same CH2 amino acid substitutions.

[0209] A bispecific antibody comprising (a) a first arm comprising a VH-CH: VL-CL pair wherein the VH and VL are a pair selected from the group consisting of the VH-VL pairs comprising an amino acid sequence set forth in Table 3 or 5; a CH comprising a ZW857B structure wherein the CH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 583-592; and a CL comprising the amino acid sequence set forth in SEQ ID NO: 391; and (b) a second arm comprising an scFv comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 463 and 464 a or SEQ ID NO: 606 and 607 nd a CH' comprising aZW856A structure wherein the CH’ comprises an amino acid sequence selected from the group consisting of SEQ ID Nos: 593-602; with the proviso that the CH and CH’ comprise the same CH2 amino acid substitutions.

[0210] A bispecific antibody comprising (a) a first arm comprising a VH-CH: VL-CL pair wherein the VH-CH comprises the amino acid sequence set forth in SEQ ID NO: 333 and a VL-CL comprising the amino acid sequence set forth in SEQ ID NO: 453; and (b) a second arm comprising an scFv-CH’ comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 332 and 333.

[0211] A bispecific antibody comprising (a) a first arm comprising a VH-CH: VL-CL pair wherein the VH-CH comprises the amino acid sequence set forth in SEQ ID NO: 335 and a VL-CL comprising the amino acid sequence set forth in SEQ ID NO: 453; and (b) a second arm comprising an scFv-CH’ comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 330 and 331.

[0212] A bispecific antibody comprising (a) a first arm comprising a VH-CH: VL-CL pair wherein the VH-CH comprises the amino acid sequence set forth in SEQ ID NO: 337 and a VL-CL comprising the amino acid sequence set forth in SEQ ID NO: 456; and (b) a second arm comprising an scFv-CH' comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 330 and 331.

[0213] A bispecific antibody comprising (a) a first arm comprising a VH-CH: VL-CL pair wherein the VH-CH comprises the amino acid sequence set forth in SEQ ID NO: 336 and a VL-CL comprising the amino acid sequence set forth in SEQ ID NO: 456; and (b) a second arm comprising an scFv-CH’ comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 332 and 333.

[0214] A bispecific antibody comprising (a) a first arm comprising a VH-CH: VL-CL pair wherein the VH-CH comprises the amino acid sequence set forth in SEQ ID NO: 338 and a VL-26093CL comprising the amino acid sequence set forth in SEQ ID NO: 459; and (b) a second arm comprising an scFv-CH’ comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 332 and 333.

[0215] A bispecific antibody comprising (a) a first arm comprising a VH-CH: VL-CL pair wherein the VH-CH comprises the amino acid sequence set forth in SEQ ID NO: 339 and a VL-CL comprising the amino acid sequence set forth in SEQ ID NO: 459; and (b) a second arm comprising an scFv-CH’ comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 330 and 331.

[0216] A bispecific antibody 24F4 ALPP / L2 (HC knob / LC: K1E Q79E M4L M48I D57G / LH_scFv_CD3 hole)-IgGl L234A L235A D265S K+ / Kappa) comprising a first arm comprising a VH-CH comprising the amino acid sequence set forth in SEQ ID NO: 451 associated with a VL-CL comprising the amino acid sequence set forth in SEQ ID NO: 453 and a second arm comprising an scFv-CH' comprising the amino acid sequence set forth in SEQ ID NO: 462.

[0217] A bispecific antibody 24F4 ALPP / L2 (HC hole / LC: K1E Q79E M4L M48I D57G / LH_scFv_CD3 knob)-IgGl L234A L235A D265S K+ / Kappa) comprising a first arm comprising a VH-CH comprising the amino acid sequence set forth in SEQ ID NO: 452 associated with a VL-CL comprising the amino acid sequence set forth in SEQ ID NO: 453 and a second arm comprising an scFv-CH' comprising the amino acid sequence set forth in SEQ ID NO: 461.

[0218] A bispecific antibody 10B3 ALPP / L2 (HC: D46E T84A N79Y S87T hole / LC: I85V H87Y / LH_CD3 scFv_knob)-IgGl L234A L235A D265S K+ / Kappa) comprising a first arm comprising a VH-CH comprising the amino acid sequence set forth in SEQ ID NO: 454 associated with a VL-CL comprising the amino acid sequence set forth in SEQ ID NO: 456 and a second arm comprising an scFv-CH’ comprising the amino acid sequence set forth in SEQ ID NO: 461.

[0219] A bispecific antibody 10B3 ALPP / L2 (HC: D46E T84A N79Y S87T knob / LC: I85V H87Y / LH scFv CD3 hole)-IgGl L234A L235A D265S K+ / Kappa) comprising a first arm comprising a VH-CH comprising the amino acid sequence set forth in SEQ ID NO: 455 associated with a VL-CL comprising the amino acid sequence set forth in SEQ ID NO: 456 and a second arm comprising an scFv-CH’ comprising the amino acid sequence set forth in SEQ ID NO: 462.

[0220] A bispecific antibody 9G10 ALPP / L2 TCE (HC: V26G knob / LC / LH_scFv_CD3 hole)-IgGl L234A L235A D265S K+ / Kappa) comprising a first arm comprising a VH-CH comprising the amino acid sequence set forth in SEQ ID NO: 457 associated with a VL-CL comprising the26093amino acid sequence set forth in SEQ ID NO: 459 and a second arm comprising an scFv-CH’ comprising the amino acid sequence set forth in SEQ ID NO: 462.

[0221] A bispecific antibody 9G10 ALPP / L2 TCE (HC: V26G hole / LC / LH_scFv_CD3 knob)-IgGl L234A L235 A D265S K- / Kappa) comprising a first arm comprising a VH-CH comprising the amino acid sequence set forth in SEQ ID NO: 458 associated with a VL-CL comprising the amino acid sequence set forth in SEQ ID NO: 459 and a second arm comprising an scFv-CH’ comprising the amino acid sequence set forth in SEQ ID NO: 461.

[0222] A bispecific antibody 24F4 ALPP / L2 (HC knob / LC: K1E Q79E M4L M48I D57G I LH_scFv_CD3 hole)-IgGl L234A L235A D265S K- / Kappa) comprising a first arm comprising a VH-CH comprising the amino acid sequence set forth in SEQ ID NO: 510 associated with a VL-CL comprising the amino acid sequence set forth in SEQ ID NO: 453 and a second arm comprising an scFv-CH’ comprising the amino acid sequence set forth in SEQ ID NO: 509.

[0223] A bispecific antibody 24F4 ALPP / L2 (HC hole / LC: K1E Q79E M4L M48I D57G / LH_scFv_CD3 knob)-IgGl L234A L235A D265S K- / Kappa) comprising a first arm comprising a VH-CH comprising the amino acid sequence set forth in SEQ ID NO: 511 associated with a VL-CL comprising the amino acid sequence set forth in SEQ ID NO: 453 and a second arm comprising an scFv-CH' comprising the amino acid sequence set forth in SEQ ID NO: 508.

[0224] A bispecific antibody 10B3 ALPP / L2 (HC: D46E T84A N79Y S87T hole / LC: I85V H87Y / LH_scFv_CD3 knob)-IgGl L234A L235A D265S K- / Kappa) comprising a first arm comprising a VH-CH comprising the amino acid sequence set forth in SEQ ID NO: 512 associated with a VL-CL comprising the amino acid sequence set forth in SEQ ID NO: 456 and a second arm comprising an scFv-CH’ comprising the amino acid sequence set forth in SEQ ID NO: 508.

[0225] A bispecific antibody 10B3 ALPP / L2 (HC: D46E T84A N79Y S87T knob / LC: I85V H87Y / LH_scFv_CD3 hole)-IgGl L234A L235A D265S K- / Kappa) comprising a first arm comprising a VH-CH comprising the amino acid sequence set forth in SEQ ID NO: 513 associated with a VL-CL comprising the amino acid sequence set forth in SEQ ID NO: 456 and a second arm comprising an scFv-CH’ comprising the amino acid sequence set forth in SEQ ID NO: 509.

[0226] A bispecific antibody 9G10 ALPP / L2 TCE (HC: V26G knob / LC / LH_scFv_CD3 hole)-IgGl L234A L235 A D265S K- / Kappa) comprising a first arm comprising a VH-CH comprising the amino acid sequence set forth in SEQ ID NO: 514 associated with a VL-CL comprising the amino acid sequence set forth in SEQ ID NO: 459 and a second arm comprising an scFv-CH’ comprising the amino acid sequence set forth in SEQ ID NO: 509.26093

[0227] A bispecific antibody 9G10 ALPP / L2 TCE (HC: V26G hole / LC / LH_scFv_CD3 knob)-IgGl L234A L235 A D265S K- / Kappa) comprising a first arm comprising a VH-CH comprising the amino acid sequence set forth in SEQ ID NO: 515 associated with a VL-CL comprising the amino acid sequence set forth in SEQ ID NO: 459 and a second arm comprising an scFv-CH’ comprising the amino acid sequence set forth in SEQ ID NO: 508.Nucleic acid molecules encoding the components of the bispecific antibodies disclosed herein

[0228] Further provided are nucleic acid molecules that encode the components comprising the bispecific antibodies disclosed herein. The HC of the first arm of the bispecific antibody is encoded by a first nucleic acid molecule and the light chain of the first arm of the bispecific antibody is encoded by a second nucleic acid molecule. The scFv-CH’ of the second arm of the first arm of the bispecific antibody is encoded by a third nucleic acid molecule.

[0229] In certain embodiments, the HC, LC, and scFv or scFv-CH’ are expressed as a fusion protein in which the N-terminus of the HC, LC, and scFv or scFv-CH’ are each fused at the N-tenninus to a leader peptide to facilitate the transport of their respective encoded protein through the secretory pathway. In general, the leader peptide is cleaved from the HC, LC, and scFv or scFv-CH' as they migrate through the secretory pathway. Examples of leader / signal peptides that may be used include those comprising the amino acid sequence set forth in SEQ ID NO: 402 or SEQ ID NO: 403. Thus, in certain embodiments, the aforementioned nucleic acid molecules may comprise a polynucleotide encoding a leader peptide in which the 3’ end is linked to the 5‘ end of the nucleic acid molecule encoding their respective protein.

[0230] The nucleic acid molecules disclosed herein may include one or more substitutions that optimize one or more of the codons for enhancing the expression of the nucleic acid molecule in a particular host cell, e.g., yeast or fungal host cell, non-human mammalian host cell, human host cell, insect host cell, or prokary ote host cell.

[0231] Further provided are expression cassettes comprising one or more of the nucleic acid molecules encoding the components of the bispecific antibodies disclosed herein. In certain embodiments, a nucleic acid molecule encoding the HC or HC fused to a leader peptide is inserted between a nucleic acid molecule comprising a promoter element and a nucleic acid molecule comprising a transcription termination element to provide an expression cassette for expressing the HC. In certain embodiments, a nucleic acid molecule encoding the LC or LC fused to a leader peptide is inserted between a nucleic acid molecule comprising a promoter element and a nucleic acid molecule comprising a transcription termination element to provide an26093expression cassete for expressing the LC. In certain embodiments, a nucleic acid molecule encoding the scFv or scFv-CH’ fused to a leader peptide is inserted between a nucleic acid molecule comprising a promoter element and a nucleic acid molecule comprising a transcription termination element to provide an expression cassete for expressing the scFv or scFv-CH'.

[0232] Further provided are expression vectors comprising one or more of the nucleic acid molecules encoding the components of the bispecific antibodies disclosed herein. The expression vector may be a plasmid or a virus.

[0233] In certain embodiments, a nucleic acid molecule encoding the HC or HC fused to a leader peptide is inserted between a nucleic acid molecule comprising a promoter element and a nucleic acid molecule comprising a transcription termination element of an expression vector to provide an expression vector comprising an expression cassete for expressing the HC. In certain embodiments, a nucleic acid molecule encoding the LC or LC fused to a leader peptide is inserted between a nucleic acid molecule comprising a promoter element and a nucleic acid molecule comprising a transcription termination element of an expression vector to provide an expression vector comprising an expression cassete for expressing the LC. In certain embodiments, a nucleic acid molecule encoding the scFv-CH’ or scFv-CH’ fused to a leader peptide is inserted between a nucleic acid molecule comprising a promoter element and a nucleic acid molecule comprising a transcription termination element of an expression vector to provide an expression vector comprising an expression cassete for expressing the scFv or scFv-CH’.

[0234] In certain embodiments, a single nucleic acid molecule comprising an expression cassette encoding the HC or HC fused to a leader peptide and an expression cassette encoding theLC or LC fused to a leader peptide is provided.

[0235] In certain embodiments, a single nucleic acid molecule comprising an expression cassete encoding the HC or HC fused to a leader peptide, an expression cassete encoding the LC or LC fused to a leader peptide, and an expression cassete encoding the scFv or scFv-CH’ fused to a leader peptide is provided.

[0236] In certain embodiments, a single nucleic acid molecule comprising an expression cassete encoding the HC or HC fused to a leader peptide and an expression cassete encoding the scFv or scFv-CH’ fused to a leader peptide is provided.

[0237] In certain embodiments, a single nucleic acid molecule comprising an expression cassete encoding the LC or LC fused to a leader peptide and an expression cassette encoding the scFv or scFv-CH’ fused to a leader peptide is provided.Methods for making a bispecific antibody disclosed herein26093

[0238] Recombinant methods for making a bispecific antibody disclosed herein comprises introducing into a host cell expression cassettes encoding the HC, the LC, and the scFv-CH’. The expression cassettes may be provided by of the aforementioned expression vectors. The host cell is then cultured under conditions and a time period suitable for expression of the HC, LC, and scFv-CH’ followed by isolating the bispecific antibody from the host cell and / or medium in which the host cell is grown. See e.g., W02004041862, WO2006122786, W02008020079, WO2008142164 or W02009068627. During transport through the secretory system and secretion for the host cell, the HC, LC, and scFv-CH’ assemble into the bispecific antibody. Further provided are the host cells that comprise one or more of the HC, LC, and scFv-CH' expression cassettes.

[0239] Eukaryotic and prokaryotic host cells, including mammalian cells as hosts for expression of the bispecific antibodies are w ell known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, but are not limited to, Chinese hamster ovary (CHO) cells, NSO cells, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells and a number of other cell lines. Thus, mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse, and hamster cells. Appropriate ell lines for expressing the bispecific antibodies are selected by determining which cell lines have high expression levels. Other cell lines that may be used are insect cell lines (e.g., Spodoptera frugiperda or Trichoplusia ni), amphibian cells, bacterial cells, plant cells and fungal cells. Fungal cells include yeast and filamentous fungus cells including, for example, Pichia pastoris, Saccharomyces cerevisiae, and Trichoderma reesei. Further provided herewith is any host cell comprising one or more nucleic acid molecules or expression cassettes encoding the bispecific antibodies disclosed herein.

[0240] Further, expression of a bispecific antibody from cell lines can be enhanced using a number of known techniques. For example, the glutamine synthetase gene expression system (the GS system) is a common approach for enhancing expression under certain conditions. The GS system is discussed in whole or part in connection with European Patent Nos. 0216846B1, 0256055B1, 0323997B1, and 0338841B1. Thus, in an embodiment, the mammalian host cells lack a glutamine synthetase gene and are grow n in the absence of glutamine in the medium wherein, however, the nucleic acid molecule encoding the immunoglobulin chain comprises a glutamine synthetase gene w hich complements the lack of the gene in the host cell. Such host cells may comprise the nucleic acid molecule(s) or expression cassettes, or expression vector(s)26093as discussed herein as well as expression methods, as discussed herein, for making a bispecific antibody disclosed herein using such host cells.

[0241] Methods for purifying bispecific antibody disclosed herein comprise introducing a sample (e.g., culture medium, cell lysate or cell lysate fraction, e.g., a soluble fraction of the lysate) comprising the bispecific antibody to a purification medium (e.g.. cation-exchange medium, anion-exchange medium and / or hydrophobic exchange medium) and either collecting purified bispecific antibody from the flow-through fraction of said sample that does not bind to the medium; or, discarding the flow-through fraction and eluting bispecific antibody bound from the medium and collecting the eluate. In certain embodiments, the medium is in a column to which the sample is applied. In an embodiment, the purification method is conducted following recombinant expression of the bispecific antibody in a host cell, e.g., wherein the host cell is first lysed and, optionally, the lysate is purified of insoluble materials prior to purification on a medium; or wherein the bispecific antibody is secreted into the culture medium by the host cell and the medium or a fraction thereof is applied to the purification medium.

[0242] In general, glycoproteins produced in a particular cell line or transgenic animal will have a glycosylation pattern that is characteristic for glycoproteins produced in the cell line or transgenic animal. Therefore, the particular glycosylation pattern of bispecific antibody will depend on the particular cell line or transgenic animal used to produce the bispecific antibody.

[0243] The bispecific antibodies disclosed herein further include embodiments comprising N-linked glycans that are typically added to immunoglobulins produced in Chinese hamster ovary cells (CHO N-linked glycans) or to engineered yeast cells (engineered yeast N-linked glycans), such as, for example, Pichia pastoris. For example, in an embodiment, the bispecific antibody comprises one or more of the ‘‘engineered yeast N-linked glycans7’ or “CHO N-linked glycans” (e g., GO and / or GO-F and / or G1 and / or Gl-F and / or G2-F and / or Man5). In an embodiment, the bispecific antibody comprises the engineered yeast N-linked glycans, i.e., GO and / or G1 and / or G2, optionally, further including Man5. In an embodiment, the bispecific antibodies comprise the CHO N-linked glycans, i.e., GO-F. Gl-F and G2-F, optionally, further including GO and / or G1 and / or G2 and / or Man5. In an embodiment, about 80% to about 95% (e.g., about 80-90%, about 85%, about 90% or about 95%) of all N-linked glycans on the bispecific antibodies are engineered yeast N-linked glycans or CHO N-linked glycans. See Nett et al. Yeast. 28: 237-252 (2011); Hamilton et al. Science. 313: 1441-1443 (2006); Hamilton et al. Curr Opin Biotechnol.18(5): 387-392 (2007). For example, in an embodiment, an engineered yeast cell is GFI5.0 or YGLY8316 or strains set forth in U. S. Patent No. 7,795,002 or Zha et al. Methods Mol Biol. 988: 31-43 (2013). See also International Patent Application Publication No. WO2013066765.26093Pharmaceutical compositions comprising bispecific antibodies that bind ALPP / L2 and CD3

[0244] Pharmaceutical compositions comprising one or more bispecific antibodies and a pharmaceutically acceptable carrier wherein the carrier may be a diluent, adjuvant, excipient, or vehicle with which the bispecific antibody is administered. Such vehicles may be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. For example, 0.4% saline and 0.3% glycine may be used. These solutions are sterile and generally free of particulate matter. They may be sterilized by conventional, well-known sterilization techniques (e.g., filtration). The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, stabilizing, thickening, lubricating and coloring agents, etc. The concentration of the bispecific antibody(s) in such pharmaceutical formulation may vary widely, i.e.. from less than about 0.5%, usually to at least about 1% to as much as 15 or 20% by weight and will be selected primarily based on required dose, fluid volumes, viscosities, etc., according to the particular mode of administration selected. Suitable vehicles and formulations, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in Remington: The Science and Practice of Pharmacy, 21.sup.st Edition, Troy, D. B. ed., Lipincott Williams and Wilkins, Philadelphia, Pa. 2006. Part 5, Pharmaceutical Manufacturing pp 691-1092, see especially pp. 958-989.

[0245] The mode of administration of the bispecific antibody of the present invention or pharmaceutical composition comprising the bispecific antibody may be any suitable route such as parenteral administration, e.g.. intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, pulmonary, transmucosal (oral, intranasal, intravaginal, rectal) or other means appreciated by the skilled artisan, as well known in the art.

[0246] The bispecific antibody may be administered to an individual (e.g., patient) by any suitable route, for example parentally by intravenous (i.v.) infusion or bolus injection, intramuscularly or subcutaneously, or intraperitoneally. An i.v. infusion may be given over for, example, 15, 30, 60, 90, 120, 180, or 240 minutes, or from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours.

[0247] The administration of the bispecific antibody or a pharmaceutical composition comprising the bispecific antibody may be repeated after one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, five weeks, six weeks, seven weeks, two months, three months, four months, five months, six months or longer.26093Repeated courses of treatment are also possible, as is chronic administration. The repeated administration may be at the same dose or at a different dose.

[0248] The bispecific antibody or a pharmaceutical composition comprising the bispecific antibody may be administered by maintenance therapy, such as, e.g., once a week for a period of 6 months or more.

[0249] The bispecific antibody or a pharmaceutical composition comprising the bispecific antibody may also be administered prophylactically in order to reduce the risk of developing cancer, delay the onset of the occurrence of an event in cancer progression, and / or reduce the risk of recurrence when a cancer is in remission. This may be especially useful in patients wherein it is difficult to locate a tumor that is known to be present due to other biological factors.

[0250] The bispecific antibody or pharmaceutical composition comprising the bispecific antibody may be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional protein preparations and well known lyophilization and reconstitution techniques can be employed.Combination therapy treatments

[0251] Combination therapies comprising a bispecific antibody disclosed herein or pharmaceutical composition comprising the bispecific antibody disclosed herein and another or second therapeutic agent (e.g., small molecule or antibody that is not a bispecific antibody) may be used for the treatment any proliferative disease, in particular, the treatment of cancer or malignancy. In particular embodiments, the combination therapy further in combination with a chemotherapy step may be used for the treatment of any proliferative disease in which ALPP and / or ALPPL2 is expressed on the cell surface, in particular, the treatment of cancer or malignancy. Exemplary cancers or malignancies that display ALPP and / or ALPPL2 on the cell surface include but are not limited to, mesothelioma, testicular cancer, endometrial cancer, pancreatic cancer, ovarian cancer, non-small cell lung cancer (NSCLC), gastric cancer, and a colon cancer.Combination therapy comprising a bispecific antibody and chemotherapy

[0252] The combination therapy comprising bispecific antibody or a pharmaceutical composition comprising the bispecific antibody may be administered to an individual having a proliferative disease (e.g., cancer or malignancy) in combination with chemotherapy. The individual may undergo the chemotherapy at the same time the individual is undergoing a combination therapy disclosed herein. The individual may undergo a combination therapy26093disclosed herein after the individual has completed chemotherapy. The individual may be administered the chemotherapy after completion of the combination therapy. The combination therapy may also be administered to an individual having recurrent or metastatic cancer with disease progression or relapse cancer and who is undergoing chemotherapy or who has completed chemotherapy.

[0253] The chemotherapy may include a chemotherapy agent selected from the group consisting of:(i) alkylating agents, including but not limited to, bifunctional alky lators. cyclophosphamide, mechlorethamine, chlorambucil, and melphalan;(ii) monofunctional alkylators, including but not limited to, dacarbazine, nitrosoureas, and temozolomide (oral dacarbazine);(iii) anthracycline or alkylcycline;(iv) cytoskeletal disruptors (taxanes), including but not limited to, paclitaxel, docetaxel, abraxane, and taxotere;(v) epothilones, including but not limited to, ixabepilone, and utidelone;(vi) histone deacetylase inhibitors, including but not limited to, vorinostat, and romidepsin;(vii) inhibitors of topoisomerase i, including but not limited to, irinotecan, and topotecan;(viii) inhibitors of topoisomerase ii, including but not limited to, etoposide, teniposide, and tafluposide;(ix) kinase inhibitors, including but not limited to, bortezomib, erlotinib, gefitinib, imatinib, vemurafenib, and vismodegib;(x) nucleotide analogs and precursor analogs, including but not limited to, azacitidine, azathioprine, fluoropyrimidines (e.g., such as capecitabine, carmofur, doxifl uridine, fluorouracil, and tegafur) cytarabine, gemcitabine, hydroxyurea, mercaptopurine, methotrexate, and tioguanine (formerly thioguanine);(xi) peptide antibiotics, including but not limited to, bleomycin and actinomycin; a platinum-based agent, including but not limited to, carboplatin, cisplatin, and oxaliplatin;(xii) retinoids, including but not limited to, tretinoin, alitretinoin, and bexarotene; and(xiii) vinca alkaloids and derivatives, including but not limited to, vinblastine, vincristine, vindesine, and vinorelbine.26093

[0254] Selecting a dose of the chemotherapy agent for chemotherapy depends on several factors, including the serum or tissue turnover rate of the agent, the level of symptoms, the immunogenicity of the agent, and the accessibility of the target cells, tissue or organ in the individual being treated.

[0255] The dose of the additional therapeutic agent should be an amount that provides an acceptable level of side effects. Accordingly, the dose amount and dosing frequency of each additional therapeutic agent will depend in part on the particular therapeutic agent, the severity’ of the cancer being treated, and patient characteristics. Guidance in selecting appropriate doses of antibodies, cytokines, and small molecules are available. See. e.g., Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY; Baert et al. (2003) New Engl. J. Med. 348:601-608; Milgrom et al. (1999) New Engl. J. Med. 341:1966-1973; Slamon et al. (2001) New Engl. J. Med. 344:783-792; Beniaminovitz et al. (2000) New Engl. J. Med. 342:613-619; Ghosh et al. (2003) New Engl. J. Med. 348:24-32; Lipsky et al. (2000) New Engl. J. Med. 343:1594-1602; Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57th Ed); Medical Economics Company; ISBN: 1563634457; 57th edition (November 2002). Determination of the appropriate dose regimen may be made by the clinician, e.g., using parameters or factors known or suspected in the art to affect treatment or predicted to affect treatment, and will depend, for example, on the individual's clinical history (e.g., previous therapy), the type and stage of the cancer to be treated and biomarkers of response to one or more of the therapeutic agents in the combination therapy.

[0256] Thus, embodiments of the combination therapy disclosed herein further includes a chemotherapy step comprising platinum-containing chemotherapy, e.g., pemetrexed and platinum chemotherapy or carboplatin and either paclitaxel or nab-paclitaxel. In particular embodiments, the combination therapy with a chemotherapy step may be used for treating at least NSCLC and HNSCC.

[0257] The combination therapy further in combination w ith a chemotherapy step may be used for the treatment of any proliferative disease in which ALPP and / or ALPPL2 is expressed on the cell surface, in particular, the treatment of cancer or malignancy. Exemplary cancers or malignancies that display ALPP and / or ALPPL2 on the cell surface include but are not limited to, mesothelioma, testicular cancer, endometrial cancer, pancreatic cancer, ovarian cancer, non-small cell lung cancer (NSCLC), gastric cancer, and a colon cancer.26093Combination therapy comprising bispecific antibody and a therapeutic antibody

[0258] The bispecific antibody or a pharmaceutical composition comprising the bispecific antibody may be administered in combination with one or more therapeutic antibodies that binds a target other than ALPP and / or ALPPL2 for the treatment of cancer or proliferative disease. The individual may undergo treatment with the therapeutic antibody at the same time the individual is undergoing a combination therapy disclosed herein. The individual may undergo the combination therapy after the individual has completed treatment with the therapeutic antibody. The individual may be administered the treatment with the therapeutic antibody after completion of the combination therapy. The combination therapy may also be administered to an individual having recunent or metastatic cancer with disease progression or relapse cancer and who is undergoing chemotherapy or who has completed chemotherapy. In certain embodiments, the therapeutic agent targets the programmed death 1 receptor or ligand, PD-1 and PD-L1, respectively. In certain embodiments, the therapeutic agent targets Cytotoxic T-lymphocyte associated protein 4 (CTLA-4), Lymphocyte activation gene 3 (LAG-3), T-cell immunoglobulin and mucin domain-containing molecule 3 (TIM-3), B and T lymphocyte attenuator (BTLA), and T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory' motif (ITIAM) domains (TIGIT).

[0259] Exemplary anti-PD-1 antibodies that may be used in a combination therapy with the bispecific antibodys disclosed herein include any antibody that binds PD-1 and inhibits PD-1 from binding PD-L1 and / or PD-L2 or binds PD-L1 or PD-L2 and inhibits it from binding PD-1. In a particular embodiment, the exemplary' anti-PD-1 antibody is pembrolizumab (KEYTRUDA). In a particular embodiment, the exemplary’ anti-PD-1 antibody is nivolumab (OPDIVO). In a particular embodiment, the exemplary anti-PD-1 antibody is cemiplimab (L1BTAYO). In a particular embodiment, the exemplary’ anti-PD-Ll antibody’ is durvalumab (IMFINZI). In a particular embodiment, the exemplary' anti-PD-Ll antibody is atezolizumab (TECENTRIQ). In a particular embodiment, the exemplary anti-PD-Ll antibody is avelumab (BAVENCIO).

[0260] In a particular embodiment, the exemplary anti-CTLA4 antibody is tremelimumab (IMJUDO). In a particular embodiment, the exemplary anti-CTLA4 antibody is ipilimumab (YERVOY). In a particular embodiment, the exemplary' anti-LAG-3 antibody is relatlimab (OPDUALAG).

[0261] The combination therapy may be used for the treatment of any proliferative disease in which ALPP and / or ALPPL2 is expressed on the cell surface, in particular, the treatment of cancer or malignancy. Exemplary cancers or malignancies that display ALPP and / or ALPPL2 on the cell surface include but are not limited to, mesothelioma, testicular cancer, endometrial26093cancer, pancreatic cancer, ovarian cancer, non-small cell lung cancer (NSCLC), gastric cancer, and colon cancer.Injection device for administering a bispecific antibody

[0262] An injection device is provided that comprises any one of the bispecific antibodies or a pharmaceutical composition comprising any one of the bispecific antibodies. An injection device is a device that introduces a substance into the body of a patient via a parenteral route, e.g., intramuscular, subcutaneous or intravenous. For example, an injection device may be a syringe (e.g., pre-filled with the pharmaceutical composition, such as an auto-injector) which, for example, includes a cylinder or barrel for holding fluid to be injected (e.g., comprising any one of the bispecific antibodies or pharmaceutical compositions comprising any one of the bispecific antibodies), a needle for piecing skin and / or blood vessels for injection of the fluid; and a plunger for pushing the fluid out of the cylinder and through the needle bore. In an embodiment, an injection device comprising any one of the bispecific antibodies or a pharmaceutical composition comprising any one of the bispecific antibodies is an intravenous (IV) injection device. Such a device includes a composition comprising the bispecific antibody or a pharmaceutical composition in a cannula or trocar / needle which may be attached to a tube which may be attached to a bag or reservoir for holding fluid (e.g.. saline; or lactated ringer solution comprising NaCl, sodium lactate, KC1, CaC12 and optionally including glucose) introduced into the body of the subject through the cannula or trocar / needle.

[0263] The bispecific antibodies or pharmaceutical compositions comprising the bispecific antibodies may, in an embodiment, be introduced into the device once the trocar and cannula are inserted into the vein of a subject and the trocar is removed from the inserted cannula. The IV device may, for example, be inserted into a peripheral vein (e g., in the hand or arm); the superior vena cava or inferior vena cava, or within the right atrium of the heart (e.g., a central IV); or into a subclavian, internal jugular, or a femoral vein and, for example, advanced toward the heart until it reaches the superior vena cava or right atrium (e.g.. a central venous line). In an embodiment of the invention, an injection device is an autoinjector; a jet injector or an external infusion pump. A jet injector uses a high-pressure narrow jet of liquid, which penetrates the epidermis to introduce the bispecific antibody or a pharmaceutical composition comprising the bispecific antibody to a patient's body. External infusion pumps are medical devices that deliver the bispecific antibody or a pharmaceutical composition comprising the bispecific antibody into a patient’s body in controlled amounts. External infusion pumps may be powered electrically or mechanically. Different pumps operate in different ways, for example, a syringe pump holds fluid in the26093reservoir of a syringe, and a moveable piston controls fluid delivery, an elastomeric pump holds fluid in a stretchable balloon reservoir, and pressure from the elastic walls of the balloon drives fluid delivery'. In a peristaltic pump, a set of rollers pinches down on a length of flexible tubing, pushing fluid forward. In a multi-channel pump, fluids can be delivered from multiple reservoirs at multiple rates.Kits comprising the bispecific antibody or pharmaceutical composition thereof

[0264] Further provided are kits comprising one or more components that include, but are not limited to, a bispecific antibody or a pharmaceutical composition comprising the bispecific antibody in association with one or more additional components including, but not limited to, a further therapeutic agent, as discussed herein. The bispecific antibody or a pharmaceutical composition comprising the bispecific antibody and / or the therapeutic agent can be formulated as a pure composition or in combination with a pharmaceutically acceptable carrier, in a pharmaceutical composition.

[0265] In one embodiment, the kit includes the bispecific antibody or a pharmaceutical composition comprising the bispecific antibody in one container (e.g., in a sterile glass or plastic vial) and a further therapeutic agent in another container (e.g., in a sterile glass or plastic vial).

[0266] In another embodiment, the kit comprises a combination, including bispecific antibody or a pharmaceutical composition comprising the bispecific antibody in combination with one or more therapeutic agents formulated together, optionally, in a pharmaceutical composition, in a single, common container.

[0267] If the kit includes a pharmaceutical composition for parenteral administration to a subject, the kit can include a device for performing such administration. For example, the kit can include one or more hypodermic needles or other injection devices as discussed above. Thus, further included is a kit comprising an injection device and the bispecific antibody or a pharmaceutical composition comprising the bispecific antibody, e.g., wherein the injection device includes the bispecific antibody or a pharmaceutical composition comprising the bispecific antibody, or wherein the bispecific antibody or pharmaceutical composition comprising the bispecific antibody is in a separate vessel.

[0268] The kit can include a package insert including information concerning the pharmaceutical composition and dosage form in the kit. Generally, such information aids patients and physicians in using the enclosed pharmaceutical compositions and dosage forms effectively and safely. For example, the following information regarding a combination of the invention may be supplied in the insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy26093parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdosage, proper dosage and administration, how supplied, proper storage conditions, references, manufacturer / distributor information and patent information.Combination therapy comprising co-administration with hyaluronan degrading enzymes Administration of a bispecific antibody disclosed herein may be by any suitable route and may be facilitated by agents such as hyaluronan degrading enzymes, including hyaluronidases, including soluble PH20 polypeptides, and variants thereof. For systemic administration, the facilitating agents may be modified to increase pharmacological properties, such as serum half-life, by modifying the agents, such as with polymers, for example, polyethylene glycol (PEG). See, e.g., U. S. Pat. Nos.7, 767, 429; 8,431,380; 7,871,607, International Patent Application Publication No. W02020022791, U. S. Patent Application Publication No. US20060104968 and European Patent No.l858926Bl, and in numerous other patents and publications. In certain embodiments, administration is subcutaneous or intramuscular.Accordingly, specific embodiments relate to (a) pharmaceutical compositions or fixed-dose pharmaceutical compositions comprising a bispecific antibody disclosed herein and any one of a hyaluronan degrading enzyme, hyaluronidase, soluble hyaluronidase, soluble PH20 polypeptide, or a variant of any of the foregoing; (b) pharmaceutical compositions or fixed-dose pharmaceutical compositions comprising a bispecific antibody disclosed herein, a therapeutic agent other than a bispecific antibody (e.g., any one or more of the therapeutic agents mentioned supra.), and any one of a hyaluronan degrading enzyme, hyaluronidase, soluble hyaluronidase, soluble PH20 polypeptide, or a variant of any of the foregoing; (c) kits comprising a pharmaceutical composition comprising a bispecific antibody disclosed herein and any one of a hyaluronan degrading enzyme, hyaluronidase, soluble hyaluronidase, soluble PH20 polypeptide, or a variant of any of the foregoing, and instructions for use; (d) kits comprising a first vial comprising a bispecific antibody disclosed herein and a second vial comprising any one of a hyaluronan degrading enzyme, hyaluronidase, soluble hyaluronidase, soluble PH20 polypeptide, or a variant of any of the foregoing, and instructions for use.In certain embodiments of the aforementioned compositions and kits, the soluble hyaluronidase is a soluble PH20 hyaluronidase assigned the nonproprietary' name “hyaluronidase (human recombinant)”by the United States Adopted Name (USAN) Council, which is also known by its chemical name "36-482-hyaluronoglucosaminidase" or “hyaluronidase 1 (human sperm surface protein PH20)-(l-447) peptide” and which is currently marketed by Halozyme under the tradename Hylenex™. In certain embodiments, the hyaluronidase is 3-305-Hyaluronidase PH-2026093(human) fusion protein with 302-322 -hyaluronidase HYAL-1 (human) fusion protein with 327-433-hyaluronidase PH-20 (human) (ACI), which is assigned the nonproprietary name “berahyaluronidase alfa” by the USAN Council and which is currently marketed by Alteogen under the tradename Berahyaluronidase™. In particular embodiments, the pharmaceutical composition comprises a bispecific antibody disclosed herein and a soluble PH20 polypeptide or a variant thereof.Exemplary embodiments

[0269] Embodiment 1. A bispecific antibody comprising a first binding domain, which binds to human ALPP and / or ALPPL2 on the surface of a target cell, and a second binding domain, which binds to human CD3 on the surface of a T cell, wherein the first binding domain comprises (a) a first heavy chain variable domain (VH) comprising complementarity determining regions (CDRs) VH-CDR1, VH-CDR2, and VH-CDR3 and a first light chain variable domain (VL) compnsing CDRs VL-CDR1, VL-CDR2, and VL-CDR3. wherein(1) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 3, SEQ IDNO: 4, and SEQ ID NO: 5, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 6, SEQ IDNO: 7, and SEQ ID NO: 8, respectively:(2) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 11, SEQ IDNO: 12, and SEQ ID NO: 13, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 14, SEQ IDNO: 15, and SEQ ID NO: 16. respectively;(3) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 19, SEQ IDNO: 20, and SEQ ID NO: 21, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 22, SEQ IDNO: 23, and SEQ ID NO: 24, respectively;(4) VH-CDR1. VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 27, SEQ IDNO: 28, and SEQ ID NO: 29, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 30, SEQ IDNO: 31, and SEQ ID NO: 32, respectively;(5) VH-CDR1. VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 35, SEQ IDNO: 36, and SEQ ID NO: 37, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 38, SEQ IDNO: 39, and SEQ ID NO: 40, respectively;26093(6) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 43, SEQ IDNO: 44, and SEQ ID NO: 45, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 46, SEQ IDNO: 47, and SEQ ID NO: 48, respectively;(7) VH-CDR1. VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 51, SEQ IDNO: 52, and SEQ ID NO: 53, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 54, SEQ IDNO: 55, and SEQ ID NO: 56, respectively;(8) VH-CDR1. VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 59, SEQ IDNO: 60, and SEQ ID NO: 61, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 62, SEQ IDNO: 63, and SEQ ID NO: 64, respectively;(9) VH-CDR1. VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 67. SEQ IDNO: 68, and SEQ ID NO: 69, respectively, and VL-CDR1, VL-CDR2. and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 70, SEQ IDNO: 71, and SEQ ID NO: 72, respectively;(10) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 75, SEQ IDNO: 76, and SEQ ID NO: 77, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 78, SEQ IDNO: 79, and SEQ ID NO: 80, respectively;(11) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 83, SEQ IDNO: 84, and SEQ ID NO: 85, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 86, SEQ IDNO: 87, and SEQ ID NO: 88, respectively;(12) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 91, SEQ IDNO: 92, and SEQ ID NO: 93, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 94, SEQ IDNO: 95, and SEQ ID NO: 96, respectively;(13) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 99, SEQ IDNO: 100, and SEQ ID NO: 101, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 102, SEQ IDNO: 103, and SEQ ID NO: 104, respectively;(14) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 107, SEQ IDNO: 108, and SEQ ID NO: 109, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 110, SEQ IDNO: 111, and SEQ ID NO: 112, respectively;(15) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 115, SEQ IDNO: 116, and SEQ ID NO: 117, respectively, and VL-CDR1, VL-CDR2. and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 118, SEQ IDNO: 119, and SEQ ID NO: 120, respectively;(16) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 123, SEQ IDNO: 124, and SEQ ID NO: 125, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 126, SEQ IDNO: 127, and SEQ ID NO: 128, respectively;(17) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 131, SEQ IDNO: 132, and SEQ ID NO: 133, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 134, SEQ IDNO: 135, and SEQ ID NO: 136, respectively;(18) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 139, SEQ IDNO: 140, and SEQ ID NO: 141, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 142, SEQ IDNO: 143, and SEQ ID NO: 144, respectively;(19) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 147, SEQ IDNO: 148, and SEQ ID NO: 149, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 150, SEQ IDNO: 151, and SEQ ID NO: 152, respectively;(20) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 155, SEQ IDNO: 156, and SEQ ID NO: 157, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO 158, SEQ IDNO: 159, and SEQ ID NO: 160, respectively;(21) VH-CDR1. VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 163, SEQ IDNO: 164, and SEQ ID NO: 165, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 166, SEQ IDNO: 167, and SEQ ID NO: 168, respectively;(22) VH-CDR1. VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 171, SEQ IDNO: 172, and SEQ ID NO: 173, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 174, SEQ IDNO: 175, and SEQ ID NO: 176, respectively;(23) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 179, SEQ IDNO: 180, and SEQ ID NO: 181, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 182, SEQ IDNO: 183, and SEQ ID NO: 184, respectively;(24) VH-CDR1. VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 187, SEQ IDNO: 188, and SEQ ID NO: 189, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 190, SEQ IDNO: 191, and SEQ ID NO: 192, respectively;(25) VH-CDR1. VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 195, SEQ IDNO: 196, and SEQ ID NO: 197, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 198, SEQ IDNO: 199, and SEQ ID NO: 200, respectively;(26 VH-CDR1. VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 203, SEQ IDNO: 204, and SEQ ID NO: 205, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 206, SEQ IDNO: 207, and SEQ ID NO: 208, respectively;(27) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 211, SEQ IDNO: 212, and SEQ ID NO: 213, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 214, SEQ IDNO: 215, and SEQ ID NO: 216, respectively;(28) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 219, SEQ IDNO: 220, and SEQ ID NO: 221, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 222, SEQ IDNO: 223, and SEQ ID NO: 224, respectively;(29) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 227, SEQ IDNO: 228, and SEQ ID NO: 229, respectively, and VL-CDR1, VL-CDR2. and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 230, SEQ IDNO: 231, and SEQ ID NO: 232, respectively;(30) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 235, SEQ IDNO: 236, and SEQ ID NO: 237, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 238, SEQ IDNO: 239, and SEQ ID NO: 240, respectively;(31) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 243, SEQ IDNO: 244, and SEQ ID NO: 245, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 246, SEQ IDNO: 247, and SEQ ID NO: 248, respectively;(32) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 251, SEQ IDNO: 252, and SEQ ID NO: 253, respectively, and VL-CDR1, VL-CDR2. and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 254, SEQ IDNO: 255, and SEQ ID NO: 256, respectively;(33) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 259, SEQ IDNO: 260, and SEQ ID NO: 261, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 262, SEQ IDNO: 263, and SEQ ID NO: 264, respectively;(34) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 267, SEQ IDNO: 268, and SEQ ID NO: 269, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 270, SEQ IDNO: 271, and SEQ ID NO: 272, respectively;(35) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 275, SEQ IDNO: 276, and SEQ ID NO: 277, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 278, SEQ IDNO: 279, and SEQ ID NO: 280, respectively;(36) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 283, SEQ IDNO: 284, and SEQ ID NO: 285, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 286, SEQ IDNO: 287, and SEQ ID NO: 288, respectively;(37) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 291, SEQ IDNO: 292, and SEQ ID NO: 293, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 294, SEQ IDNO: 295, and SEQ ID NO: 296, respectively;(38) VH-CDR1. VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 299 SEQ IDNO: 300, and SEQ ID NO: 301, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 302, SEQ IDNO: 303, and SEQ ID NO: 304, respectively;(39) VH-CDR1. VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 307, SEQ IDNO: 308, and SEQ ID NO: 309, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 310, SEQ IDNO: 311, and SEQ ID NO: 312, respectively;26093(40) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 315, SEQ IDNO: 316, and SEQ ID NO: 317, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 318, SEQ IDNO: 319, and SEQ ID NO: 320, respectively;(41) VH-CDR1. VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 323, SEQ IDNO: 324, and SEQ ID NO: 325, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 326, SEQ IDNO: 327, and SEQ ID NO: 328, respectively;(42) VH-CDR1. VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 341, SEQ IDNO: 342, and SEQ ID NO: 343, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 344, SEQ IDNO: 345, and SEQ ID NO: 346, respectively;(43) VH-CDR1. VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 349, SEQ IDNO: 350, and SEQ ID NO: 351, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 352, SEQ IDNO: 353, and SEQ ID NO: 354, respectively;(44) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 357, SEQ IDNO: 358, and SEQ ID NO: 359, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 360, SEQ IDNO: 361, and SEQ ID NO: 362, respectively;(45) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 365, SEQ IDNO: 366, and SEQ ID NO: 367, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 368, SEQ IDNO: 369, and SEQ ID NO: 370, respectively; or(46) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 373, SEQ IDNO: 374, and SEQ ID NO: 375, respectively, and VL-CDR1, VL-CDR2. and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 376, SEQ IDNO: 377, and SEQ ID NO: 378, respectively; and(b) wherein the second binding domain comprises a second VH comprising CDRs VH-CDR1, VH-CDR2, and VH-CDR3 and a second VL comprising CDRs VL-CDR1, VL-CDR2, and VL-CDR3, wherein VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 424, SEQ IDNO: 425, and SEQ ID NO: 426, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 428, SEQ IDNO: 429, and SEQ IDNO: 430, respectively; and26093wherein the first binding domain is linked to the N-terminus of a first heavy chain constant region (CH) comprising at least a CH2-CH3 domain and the second binding domain is linked to the N-terminus of a second CH comprising at least CH2-CH3 domains; wherein the CH3 domains of the first CH and the second CH each comprise one or more mutations promoting heterodimerization of the first and second CH3 domains to form the bispecific antibody.

[0270] Embodiment 2. The bispecific antibody of embodiment 1, wherein(a) the VH-CDR1, VH-CDR2, and VH-CDR3 of the first binding domain comprise the amino acid sequence set forth in SEQ ID NO: 3, SEQ IDNO: 4, and SEQ ID NO: 5, respectively, and the VL-CDR1. VL-CDR2, and VL-CDR3 of the first binding domain comprise the amino acid sequence set forth in SEQ ID NO: 6, SEQ IDNO: 7, and SEQ ID NO: 8, respectively;(b) VH-CDR1, VH-CDR2, and VH-CDR3 of the first binding domain comprise the amino acid sequence set forth in SEQ ID NO: 11, SEQ IDNO: 12, and SEQ ID NO: 13, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 of the first binding domain comprise the amino acid sequence set forth in SEQ ID NO: 14, SEQ IDNO: 15, and SEQ ID NO: 16, respectively;(c) VH-CDR1, VH-CDR2, and VH-CDR3 of the first binding domain comprise the amino acid sequence set forth in SEQ ID NO: 19, SEQ IDNO: 20, and SEQ ID NO: 21, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 of the first binding domain comprise the amino acid sequence set forth in SEQ ID NO: 22, SEQ IDNO: 23, and SEQ ID NO: 24, respectively.

[0271] Embodiment 3. The bispecific antibody of embodiment 1, wherein the first binding domain comprises(a) a VH comprising the amino acid sequence set forth in SEQ ID NO: 1 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 2;(b) a VH comprising the amino acid sequence set forth in SEQ ID NO: 9 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 10;(c) a VH comprising the amino acid sequence set forth in SEQ ID NO: 17 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 18;(d) a VH comprising the amino acid sequence set forth in SEQ ID NO: 25 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 26;(e) a VH comprising the amino acid sequence set forth in SEQ ID NO: 33 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 34;(I) a VH comprising the amino acid sequence set forth in SEQ ID NO: 41 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 42;(g) a VH comprising the amino acid sequence set forth in SEQ ID NO: 49 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 50;26093(h) a VH comprising the amino acid sequence set forth in SEQ ID NO: 57 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 58;(i) a VH comprising the amino acid sequence set forth in SEQ ID NO: 65 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 66;(j) a VH comprising the amino acid sequence set forth in SEQ ID NO: 73 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 74;(k) a VH comprising the amino acid sequence set forth in SEQ ID NO: 81 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 82;(l) a VH comprising the amino acid sequence set forth in SEQ ID NO: 89 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 90;(m) a VH comprising the amino acid sequence set forth in SEQ ID NO: 97 and a VL comprising the ammo acid sequence set forth in SEQ ID NO: 98;(n) a VH comprising the amino acid sequence set forth in SEQ ID NO: 105 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 106;(o) a VH comprising the amino acid sequence set forth in SEQ ID NO: 113 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 114;(p) a VH comprising the amino acid sequence set forth in SEQ ID NO: 121 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 122;(q) a VH comprising the amino acid sequence set forth in SEQ ID NO: 129 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 130;(r) a VH comprising the amino acid sequence set forth in SEQ ID NO: 137 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 138;(s) a VH comprising the amino acid sequence set forth in SEQ ID NO: 145 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 146;(t) a VH comprising the amino acid sequence set forth in SEQ ID NO: 153 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 154;(u) a VH comprising the amino acid sequence set forth in SEQ ID NO: 161 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 162;(v) a VH comprising the amino acid sequence set forth in SEQ ID NO: 169 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 170;(w) a VH comprising the amino acid sequence set forth in SEQ ID NO: 177 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 178;(x) a VH comprising the amino acid sequence set forth in SEQ ID NO: 185 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 186;26093(y) a VH comprising the amino acid sequence set forth in SEQ ID NO: 193 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 194;(z) a VH comprising the amino acid sequence set forth in SEQ ID NO: 201 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 202;(aa) a VH comprising the amino acid sequence set forth in SEQ ID NO: 209 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 210;(bb) a VH comprising the amino acid sequence set forth in SEQ ID NO: 217 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 218;(cc) a VH comprising the amino acid sequence set forth in SEQ ID NO: 225 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 226;(dd) a VH comprising the amino acid sequence set forth in SEQ ID NO: 233 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 234;(ee) a VH comprising the amino acid sequence set forth in SEQ ID NO: 241 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 242;(ff) a VH comprising the amino acid sequence set forth in SEQ ID NO: 249 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 250;(gg) a VH comprising the amino acid sequence set forth in SEQ ID NO: 257 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 258;(hh) a VH comprising the amino acid sequence set forth in SEQ ID NO: 265 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 266;(ii) a VH comprising the amino acid sequence set forth in SEQ ID NO: 273 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 272;(jj) a VH comprising the amino acid sequence set forth in SEQ ID NO: 281 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 282;(kk) a VH comprising the amino acid sequence set forth in SEQ ID NO: 289 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 290;(11) a VH comprising the amino acid sequence set forth in SEQ ID NO: 297 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 298;(mm) a VH comprising the amino acid sequence set forth in SEQ ID NO: 305 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 306;(nn) a VH comprising the amino acid sequence set forth in SEQ ID NO: 313 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 314;(oo) a VH comprising the amino acid sequence set forth in SEQ ID NO: 321 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 322;26093(pp) a VH comprising the amino acid sequence set forth in SEQ ID NO: 329 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 340;(qq) a VH comprising the amino acid sequence set forth in SEQ ID NO: 347 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 348;(rr) a VH comprising the amino acid sequence set forth in SEQ ID NO: 355 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 356;(ss) a VH comprising the amino acid sequence set forth in SEQ ID NO: 363 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 364; or(tt) a VH comprising the amino acid sequence set forth in SEQ ID NO: 371 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 372;(uu) a VH comprising the amino acid sequence set forth in SEQ ID NO: 1 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 419;(vv) a VH comprising the amino acid sequence set forth in SEQ ID NO: 420 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 421; or(ww) a VH comprising the amino acid sequence set forth in SEQ ID NO: 422 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 18; andwherein the second binding domain comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 423 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 427.

[0272] Embodiment 4. The bispecific antibody of embodiment 1, wherein the first binding domain comprises(a) a VH comprising the amino acid sequence set forth in SEQ ID NO: 1 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 2;(b) a VH comprising the amino acid sequence set forth in SEQ ID NO: 9 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 10;(c) a VH comprising the amino acid sequence set forth in SEQ ID NO: 17 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 18;(d) a VH comprising the amino acid sequence set forth in SEQ ID NO: 1 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 419;(e) a VH comprising the amino acid sequence set forth in SEQ ID NO: 420 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 421; or(f) a VH comprising the amino acid sequence set forth in SEQ ID NO: 422 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 18; and26093wherein the second binding domain comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 423 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 427.

[0273] Embodiment 5. The bispecific antibody of embodiment 1, wherein the first binding domain is a Fab comprising a first VH-CH and VL-CL pair and the second binding domain is an scFv comprising from the N-terminus to the C-terminus VL-VH-CH’ or VH-VL-CH’, wherein the first CH comprises a first CHI domain, first Hinge, first CH2 domain, and first CH3 domain and the CH’ is a truncated CH comprising a deletion of CHI and comprising at least a second CH2 domain and second CH3 domain, wherein the CH and CH' domains each comprise one or more amino acid substitutions in their CH3 domains that promote heterodimerization of the CH and CH’ domains to form the bispecific antibody.

[0274] Embodiment 6. The bispecific antibody of embodiment 5, wherein(a) the first CH3 domain comprises amino acid substitutions S354C and T366W and the second CH3 domain comprises amino acid substitutions Y349C, T366S, L368A, and Y407V; (b) the first CH3 domain comprises amino acid substitutions Y349C, T366S, L368A, and Y407V to form the knob and the second CH3 domain comprises amino acid substitutions S354C and T366W;(c) the first CH3 domain comprises amino acid substitution 409W amino acid substitution and the second CH3 domain comprises amino acid substitutions D399 and F405T;(d) the first CH3 domain comprises amino acid substitutions D399 and F405T amino acid substitutions and the second CH3 domain comprises amino acid substitution 409W;(e) the first CH3 domain comprises amino acid substitution 357W and the second CH3 domain comprises amino acid substitution Y349S;(f) the first CH3 domain comprises amino acid substitutions Y349S and the second CH3 domain comprises amino acid substitution 357W;(g) the first CH3 domain comprises amino acid substitutions T350V, L351Y, F405A, and Y407V and the second CH3 domain comprises amino acid substitutions T350, T366, K392 and T394W;(h) the first CH3 domain comprises amino acid substitutions T350, T366, K392 and T394W and the second CH3 domain comprises amino acid substitutions T350V, L351Y, F405A, and Y407V;(i) the first CH3 domain comprises amino acid substitutions T350V, L351Y, F405A, and Y407V and the second CH3 domain comprises amino acid substitutions T350V and T366W; or26093(j) the first CH3 domain comprises amino acid substitutions T350V and T366W to and the second CH3 domain comprises amino acid substitutions T350V, L351Y, F405A, and Y407V; wherein the first and second CH3 domains form a heterodimeric Fc region; and wherein the amino acid numbering is according to the EU numbering scheme.

[0275] Embodiment 7. The bispecific antibody of embodiment 1, wherein the first binding domain is a first Fab comprising a first VH-CH and VL-CL pair and the second binding domain is a second Fab comprising a second VH-CH and VL-CL pair, wherein the CH of the first VH-CH and a VL-CL pair and the second VH-CH and VL-CL pair each comprise one or more amino acid substitutions that promote heterodimerization to form the bispecific antibody.

[0276] Embodiment 8. The bispecific antibody of embodiment 7, wherein(a) the first CH3 domain comprises amino acid substitutions S354C and T366W and the second CH3 domain comprises amino acid substitutions Y349C, T366S, L368A, and Y407V; (b) the first CH3 domain comprises amino acid substitutions Y349C, T366S, L368A, and Y407V to form the knob and the second CH3 domain comprises amino acid substitutions S354C and T366W;(c) the first CH3 domain comprises amino acid substitution 409W amino acid substitution and the second CH3 domain comprises amino acid substitutions D399 and F405T;(d) the first CH3 domain comprises amino acid substitutions D399 and F405T amino acid substitutions and the second CH3 domain comprises amino acid substitution 409 W;(e) the first CH3 domain comprises amino acid substitution 357W and the second CH3 domain comprises amino acid substitution Y349S;(f) the first CH3 domain comprises amino acid substitutions Y349S and the second CH3 domain comprises amino acid substitution 357W;(g) the first CH3 domain comprises amino acid substitutions T350V, L351Y, F405A, and Y407V and the second CH3 domain comprises amino acid substitutions T350, T366, K392 and T394W;(h) the first CH3 domain comprises amino acid substitutions T350, T366, K392 and T394W and the second CH3 domain comprises amino acid substitutions T350V, L351Y, F405A, and Y407V;(i) the first CH3 domain comprises amino acid substitutions T350V, L351Y, F405A, and Y407V and the second CH3 domain comprises amino acid substitutions T350V and T366W; or (j) the first CH3 domain comprises amino acid substitutions T350V and T366W to and the second CH3 domain comprises amino acid substitutions T350V, L351Y, F405A, and Y407V;26093wherein the first and second CH3 domains form a heterodimeric Fc region; and wherein the amino acid numbering is EU numbering scheme.

[0277] Embodiment 9. The bispecific antibody of embodiment 1, wherein the CH is of the human IgGl or IgG4 isotype comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, insertions, deletions, or combinations thereof and the first and second CL is of the human kappa or lambda isotype comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, insertions, deletions, or combinations thereof.

[0278] Embodiment 10. The bispecific antibody of embodiment 1, wherein(a) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 381 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 392;(b) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 382 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 393;(c) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 383 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 394;(d) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 384 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 395;(e) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 385 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 396;(f) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 386 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 397;(g) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 387 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 398;(h) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 388 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 399;(i) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 389 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 400;(j) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 390 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 401;(k) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 392 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 381;(l) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 393 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 382;(m) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 394 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 383;26093(n) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 395 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 384;(o) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 396 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 385;(p) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 397 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 386;(q) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 398 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 387;(r) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 399 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 388;(s) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 400 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 389; or(t) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 401 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 390.

[0279] Embodiment 11. The bispecific antibody of embodiment 5, wherein CH’ comprises amino acids 8-15 of the Hinge region, CH2 domain, and CH3 domain.

[0280] Embodiment 12. The bispecific antibody of embodiment 5, wherein(a) the CH comprises the amino acid sequence set forth in SEQ ID NO: 381 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 431;(b) the CH comprises the amino acid sequence set forth in SEQ ID NO: 382 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 432;(c) the CH comprises the amino acid sequence set forth in SEQ ID NO: 383 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 433;(d) the CH comprises the amino acid sequence set forth in SEQ ID NO: 384 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 434;(e) the CH comprises the amino acid sequence set forth in SEQ ID NO: 385 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 435;(f) the CH comprises the amino acid sequence set forth in SEQ ID NO: 386 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 436;(g) the CH comprises the amino acid sequence set forth in SEQ ID NO: 387 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 437;(h) the CH comprises the amino acid sequence set forth in SEQ ID NO: 388 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 438;26093(i) the CH comprises the amino acid sequence set forth in SEQ ID NO: 389 and CH' comprises the amino acid sequence set forth in SEQ ID NO: 439;(j) the CH comprises the amino acid sequence set forth in SEQ ID NO: 390 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 440;(k) the CH comprises the amino acid sequence set forth in SEQ ID NO: 392 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 441;(l) the CH comprises the amino acid sequence set forth in SEQ ID NO: 393 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 442;(m) the CH comprises the amino acid sequence set forth in SEQ ID NO: 394 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 443;(n) the CH comprises the amino acid sequence set forth in SEQ ID NO: 395 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 444;(o) the CH comprises the amino acid sequence set forth in SEQ ID NO: 396 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 445;(p) the CH comprises the amino acid sequence set forth in SEQ ID NO: 397 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 446;(q) the CH comprises the amino acid sequence set forth in SEQ ID NO: 398 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 447;(r) the CH comprises the amino acid sequence set forth in SEQ ID NO: 399 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 448;(s) the CH comprises the amino acid sequence set forth in SEQ ID NO: 400 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 449; or(t) the CH compnses the amino acid sequence set forth in SEQ ID NO: 401 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 450.

[0281] Embodiment 13. The bispecific antibody of embodiment 5 or 7, wherein the CL comprises the kappa isotype.

[0282] Embodiment 14. The bispecific antibody of embodiment 13. wherein the CL comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, insertions, deletions, or combinations thereof compared to the amino acid sequence of the native CL.

[0283] Embodiment 15. The bispecific antibody of embodiment 5, wherein the CH and CH’ comprises:(a) a substitution of the amino acids at positions 252, 254, and 256 of the CH and the CH’ with amino acids Tyr (Y), Thr (T), and Glu (E), respectively, wherein the numbering is EU numbering scheme;26093(b) a substitution of the amino acids at positions 233 and 235 of the CH and the CH’ with the amino acid Ala (A), wherein the numbering is according to the EU numbering scheme;(c) a substitution of the amino acids at positions 234 and 235 of the CH and the CH’ with the amino acid A and at position 265 with Ser (S), wherein the numbering is according to the EU numbering scheme;(d) a substitution of the amino acids at positions 234 and 235 of the CH and the CH’ with the amino acid A and at position 329 with Gly (G), wherein the numbering is according to the EU numbering scheme;(e) a substitution of the amino acid at position 235 of the CH and the CH' with the amino acid E, wherein the numbering is according to the EU numbering scheme;(f) a substitution of the amino acid at position 265 of the CH and the CH’ with the amino acid A, w herein the numbering is according to the EU numbering scheme;(g) a substitution of the amino acid at position 265 of the CH and the CH’ with the amino acid A and at position 297 with the amino acid G. w herein the numbering is according to the EU numbering scheme;(h) a substitution of the amino acid at position 297 of the CH and the CH’ with any amino acid except for Asn (N), wherein the numbering is according to the EU numbering scheme; or (i) a substitution of the amino acid at position 297 of the CH and the CH' with the amino acid A, at position 356 with Glu (E), at position 358 with Met (M), wherein the numbering is according to the EU numbering scheme.

[0284] Embodiment 16. The bispecific antibody of embodiment 5, wherein the CH and CH’ comprise a C-terminal lysine or lack a C-terminal lysine or a C-terminal gly cine-ly sine dipeptide.

[0285] Embodiment 17. The bispecific antibody of any one of the foregoing embodiments, w herein the N-terminal amino acid of the VH is pyroglutamate.

[0286] Embodiment 18. A bispecific antibody comprising: a first arm, which binds to human ALPP and / or ALPPL2 on the surface of a target cell, and a second arm, which binds to human CD3 on the surface of a T cell, comprising(a) a first arm comprising a heavy chain (HC) comprising the amino acid sequence set forth in SEQ ID NO: 458 associated with a light chain (LC) comprising the amino acid sequence set forth in SEQ ID NO: 459 and a second arm comprising an scFv-CH’ comprising the amino acid sequence set forth in SEQ ID NO: 461;(b) a first arm comprising an HC comprising the amino acid sequence set forth in SEQ ID NO: 510 associated with an LC comprising the amino acid sequence set forth in SEQ ID NO:26093453 and a second arm comprising an scFv-CH’ comprising the amino acid sequence set forth in SEQ ID NO: 509;(c) a first arm comprising an HC comprising the amino acid sequence set forth in SEQ ID NO: 511 associated with an LC comprising the amino acid sequence set forth in SEQ ID NO: 453 and a second arm comprising an scFv-CH’ comprising the amino acid sequence set forth in SEQ ID NO: 508;(d) a first arm comprising an HC comprising the amino acid sequence set forth in SEQ ID NO: 512 associated with an LC comprising the amino acid sequence set forth in SEQ ID NO: 456 and a second arm comprising an scFv-CH' comprising the amino acid sequence set forth in SEQ ID NO: 508;(e) a first arm comprising an HC comprising the amino acid sequence set forth in SEQ ID NO: 513 associated with an LC comprising the amino acid sequence set forth in SEQ ID NO: 456 and a second arm comprising an scFv-CH’ comprising the amino acid sequence set forth in SEQ ID NO: 509;(f) a first arm comprising an HC comprising the amino acid sequence set forth in SEQ ID NO: 514 associated with an LC comprising the amino acid sequence set forth in SEQ ID NO: 459 and a second arm comprising an scFv-CH’ comprising the amino acid sequence set forth in SEQ ID NO: 509; or(g) a first arm comprising an HC comprising the amino acid sequence set forth in SEQ ID NO: 515 associated with an LC comprising the amino acid sequence set forth in SEQ ID NO: 459 and a second arm comprising an scFv-CH’ comprising the amino acid sequence set forth in SEQ ID NO: 508.

[0287] Embodiment 19. A bispecific antibody is provided comprising a heavy chain (HC), a light chain (LC), and an scFv linked to a truncated HC constant domain (scFv-CH’), wherein the HC comprises the amino acid sequence set forth in SEQ ID NO: 451, the LC comprising the amino acid sequence set forth in SEQ ID NO: 453, and the scFv-CH' comprising the amino acid sequence set forth in SEQ ID NO: 462; or wherein the HC comprises the amino acid sequence set forth in SEQ ID NO: 457, the LC comprising the amino acid sequence set forth in SEQ ID NO: 459, and the scFv-CH’ comprising the amino acid sequence set forth in SEQ ID NO: 462; or wherein the HC comprises the amino acid sequence set forth in SEQ ID NO: 454, the LC comprising the amino acid sequence set forth in SEQ ID NO: 456, and the scFv-CH’ comprising the amino acid sequence set forth in SEQ ID NO: 461.

[0288] Embodiment 20. A bispecific antibody is provided comprising a heavy chain (HC), a light chain (LC), and an scFv linked to a truncated HC constant domain (scFv-CH’), wherein the26093HC comprises the amino acid sequence set forth in SEQ ID NO: 510, the LC comprising the amino acid sequence set forth in SEQ ID NO: 453, and the scFv-CH' comprising the amino acid sequence set forth in SEQ ID NO: 509; or wherein the HC comprises the amino acid sequence set forth in SEQ ID NO: 514, the LC comprising the amino acid sequence set forth in SEQ ID NO: 459, and the scFv-CH’ comprising the amino acid sequence set forth in SEQ ID NO: 509; or wherein the HC comprises the amino acid sequence set forth in SEQ ID NO: 512, the LC comprising the amino acid sequence set forth in SEQ ID NO: 456, and the scFv-CH’ comprising the amino acid sequence set forth in SEQ ID NO: 508.

[0289] Embodiment 21. A pharmaceutical composition comprising the bispecific antibody of any one of embodiments 1-20 and a pharmaceutically acceptable carrier or diluent.

[0290] Embodiment 22. A method for treating a proliferative disease in an individual in need of the treatment comprising administering to the individual a therapeutically effective amount of the bispecific antibody of embodiment 1 or a pharmaceutical composition comprising the bispecific antibody to treat the proliferative disease.

[0291] Embodiment 23. The method of embodiment 22, wherein the proliferative disease comprises cells that display ALPP and / or ALPPL2 on the cell surface.

[0292] Embodiment 24. The method of embodiment 22, wherein the proliferative disease comprises a tumor that displays ALPP and / or ALPPL2 on the surface of the cells thereof.

[0293] Embodiment 25. The method of embodiment 22 or 23, wherein the proliferative disease or tumor is a cancer or malignancy selected from the group consisting of mesothelioma, testicular cancer, endometrial cancer, pancreatic cancer, ovarian cancer, non-small cell lung cancer (NSCLC), gastric cancer, bladder cancer, head and neck cancer, cervical cancer, breast cancer, esophageal cancer, cholangiocarcinoma. prostate cancer, and colon cancer.

[0294] Embodiment 26. Use of the bispecific antibody of any one of embodiments 1-20 or a pharmaceutical composition comprising the bispecific antibody for the manufacture of a medicament for treatment of a proliferative disease.

[0295] Embodiment 27. The use of embodiment 26. wherein the proliferative disease comprises cells that display ALPP and / or ALPPL2 on the cell surface.

[0296] Embodiment 28. The use of embodiment 26, wherein the proliferative disease comprises a tumor that displays ALPP and / or ALPPL2 on the surface of the cells thereof.

[0297] Embodiment 29. The use of embodiment 26 or 27. wherein the proliferative disease or tumor is a cancer or malignancy selected from the group consisting of mesothelioma, testicular cancer, endometrial cancer, pancreatic cancer, ovarian cancer, non-small cell lung cancer26093(NSCLC), gastric cancer, bladder cancer, head and neck cancer, cervical cancer, breast cancer, esophageal cancer, cholangiocarcinoma, prostate cancer, and colon cancer.

[0298] Embodiment 30. The bispecific antibody of any one of embodiments 1-20 or a pharmaceutical composition comprising the bispecific antibody for treatment a proliferative disease.

[0299] Embodiment 31. The bispecific antibody of embodiment 30, wherein the proliferative disease comprises cells that display ALPP and / or ALPPL2 on the cell surface.

[0300] Embodiment 32. The bispecific antibody of embodiment 30, wherein the proliferative disease comprises a tumor that displays ALPP and / or ALPPL2 on the surface of the cells thereof.

[0301] Embodiment 33. The bispecific antibody of embodiment 30 or 31, wherein the proliferative disease or tumor is a cancer or malignancy selected from the group consisting of mesothelioma, testicular cancer, endometrial cancer, pancreatic cancer, ovarian cancer, non-small cell lung cancer (NSCLC). gastric cancer, bladder cancer, head and neck cancer, cervical cancer, breast cancer, esophageal cancer, cholangiocarcinoma, prostate cancer, and colon cancer.

[0302] Embodiment 34. A combination therapy for treating a proliferative disease comprising the bispecific antibody of any one of embodiments 1-20 or a pharmaceutical composition comprising the bispecific antibody and a second therapeutic agent.

[0303] Embodiment 35. The combination therapy of embodiment 34, wherein the second therapeutic agent is a chemotherapy agent or a therapeutic antibody other than an antibody that binds ALPP and / or ALPPL2.

[0304] Embodiment 36. The combination therapy of embodiment 34 or 35, wherein the proliferative disease comprises cells that display ALPP and / or ALPPL2 on the cell surface.

[0305] Embodiment 37. The combination therapy of embodiment 34, wherein the proliferative disease comprises a tumor that displays ALPP and / or ALPPL2 on the surface of the cells thereof.

[0306] Embodiment 38. The combination therapy of embodiment 34, 35, or 36, wherein the proliferative disease or tumor is a cancer or malignancy selected from the group consisting of mesothelioma, testicular cancer, endometrial cancer, pancreatic cancer, ovarian cancer, non-small cell lung cancer (NSCLC), gastric cancer, bladder cancer, head and neck cancer, cervical cancer, breast cancer, esophageal cancer, cholangiocarcinoma, prostate cancer, and colon cancer.

[0307] Embodiment 39. A nucleic acid molecule encoding the heavy chain (HC), light chain (LC). or scFv-Fc of a bispecific antibody of any one of embodiments 1-20.

[0308] Embodiment 40. An expression vector comprising one or more of the nucleic acid molecules of embodiment 39.26093

[0309] Embodiment 41. A host cell comprising the one or more nucleic acid molecules of embodiment 39.

[0310] Embodiment 42. A host cell comprising a first nucleic acid molecule encoding the heavy chain (HC) of a bispecific antibody of any one of embodiments 1-20, a second nucleic acid molecule encoding the light chain (LC) of a bispecific antibody of any one of embodiments 1-20; and a third nucleic acid molecule encoding the scFv-CH’ of a bispecific antibody of any one of embodiments 1-20, wherein the CH3 region of the HC and the scFv comprise one or more mutations promoting heterodimerization.

[0311] Embodiment 43. A method for producing a bispecific antibody of any one of embodiments 1-20 comprising (a) providing the host cell of embodiment 42; (b) cultivating the host cell in a medium under conditions suitable for expressing the bispecific antibody; and (c) isolating the bispecific antibody from the host cells and / or medium.

[0312] Embodiment 44. The bispecific antibody of any one of embodiments 1-20 conjugated to a detectable moiety.

[0313] Embodiment 45. The bispecific antibody of embodiment 44, wherein the detectable moiety is detectable by magnetic resonance imaging (MRI) or by X-ray imaging.

[0314] Embodiment 46. A method for detecting ALPP and / or ALPPL2 on the surface of a cell in an individual comprising administering to the individual the bispecific antibody of embodiment 44 or 45 and detecting the cells in the individual that bind the bispecific antibody.EXAMPLES

[0315] The following examples are meant to be illustrative and should not be construed as further limiting. The contents of the figures and all references, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference.EXAMPLE 1ALPP / L2 binder discovery and optimization

[0316] The ALPP / L2 binders disclosed herein were discovered through the utilization of the Trianni Mouse™ (Trianni), a transgenic mouse in which the relevant human immunoglobulin sequences had been introduced into the genome by genetic engineering. Through use of such technology, chimeric monoclonal antibodies containing the full repertoire of human heavy- and light-chain variable domains and the retention of the mouse CH and CL domains (chimeric anti-ALPP / L2 antibodies) were produced.26093

[0317] Essentially, a cohort of Trianni Mouse™ (AbCellera, Vancouver, BC) was immunized with mRNA encoding human ALPPL2. To select Trianni mice producing chimeric ALPPL2-specific antibodies, sera from immunized mice was screened by ELISA for binding to recombinant human ALPP2 or ALPP. Briefly, an ELISA plate coated with recombinant human ALPPL2 or ALPP was incubated with dilutions of serum from immunized mice for one hour at room temperature, the assay plate was washed, and specific antibody binding was detected with HRP-labeled anti-mouse IgG antibody. Plates was read using an ELISA reader (Biotek).

[0318] Generation of Hybridomas: To generate hybridomas producing chimeric anti-human ALPP / ALPPL2 antibodies, splenocytes and draining lymph node cells harvested from immunized mice were fused to an appropriate immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas were screened for production of chimeric ALPP / ALPPL2-specific antibodies.

[0319] For example, single cell suspensions of splenocytes and lymph node cells from immunized mice were fused to equal number of Sp2 / 0 non-mouse IgG secreting myeloma cells (ATCC, CRL 1581) by electrofusion. Cells were plated in flat bottom 96-well tissue culture plates, followed by about 2 weeks of incubation in selection medium (HAT medium), then switched to hybridoma culture medium. Approximately 10-14 days after cell plating, supernatants from individual wells were screened by ELISA as described above. The chimeric antibody-secreting hybridomas were transferred to 24-well plates, screened again, and if still positive for anti-ALPP / ALPPL2 activity, the hybridomas were subcloned by limiting dilution or sorting using a single cell sorter. The stable subclones were then cultured in vitro to generate small amounts of chimeric antibodies to be used for purification and characterization. Hybridoma cell lines that were reactive for the antigen of interest were expanded. Sequencing of the human VH and VL of the chimeric antibodies was accomplished by RNA isolation, followed by cDNA sequencing of the human VH and human VL using Sanger sequencing methods. Fully human anti-ALPP / L2 monoclonal antibodies (the human VH and VL fused to human CH / CL) were generated for recombinant expression. Sixty-six unique VH / VL pair sequences were identified of which approximately 47 VH / VL pairs were confirmed to be binders of human ALPP / ALPPL2. Of the confirmed binders, 16 were determined to be non-internalizing and 31 were determined to be internalizing. The fully human anti-ALPP / L2 monoclonal antibodies so identified are designated herein as ALPP / L2 binders.EXAMPLE 2Cell Binding / Specificity of the ALPP / L2 Binders- Ill -26093

[0320] In this example purified ALPP / L2 binders were evaluated in an EC50FACS Binding Assay to human ALPPL2, human ALPP, human ALPI, human ALPL, rhesus ALPP, mouse ALPPL2.Cell binding'.

[0321] Human ALPPL2-CHOK1 Clone 4F11 was grown in 175 cm2flask using media receipt DMEM / F12 + 10% heat inactivated FBS, 50 pg / mL Gentamicin, and 1.0 mg / mL G418. Human ALPP-CHOK1 Clone 1C5, Human ALPI-CHOK1 Clone 2F6, Human ALPL-CHOK1 Clone 4E7, Rhesus ALPP-CHOK1 Clone 4D9, and Mouse ALPP-CHOK1 Clone 5E9 were grown in 175 cm2 flask using media receipt F12K + 10% heat inactivated FBS, 50 pg / mL Gentamicin, and 8.0 pg / mL Puromycin.

[0322] All cells were harvested using 0.25% Trypsin / EDTA (Mouse ALPP-CHOK1 Clone 5E9) or TrypLE™ Express and washed twice with phosphate buffered saline (PBS) buffer. Cell trace dye (CellTrace™-Violet Thermo Scientific, Catalog # C34557 and CellTrace™-Far Red Thermo Scientific Catalog # 34564) was diluted at optimized concentrations in PBS (1 mL staining volume / lOM cells) and used to resuspend cell pellets prepared from the different recombinant cell lines. Cells were incubated with the dyes at 37° C for 20 minutes in the dark, shaking. These staining reactions were stopped by adding warm DMEM / F12 (Dulbecco’s Modified Eagles Medium with Ham’s F12) complete medium with 10% fetal bovine serum (FBS), using 5X the original staining volume, and incubated at 37° C for 10 minutes, shaking. Stained cells were spun down and washed once with 10 mL PBS and resuspended in Fluorescence- Activated Cell Sorter (FACS) buffer (2.0% fetal bovine serum in Hanks Balanced Salt Solution (HBSS)). Cells were then checked to confirm both positive staining with the dyes and that each recombinant cell line demonstrated a separate fluorescent intensity using Intellicyt® iQue Screener PLUS.

[0323] The different recombinant cell lines were then mixed by resuspending in FACS buffer and aliquoted into 96-V-well plates (50 pL / well, 30x104cells / well). In separate 96-V-well plates, purified ALPP / L2 binders and control antibodies were diluted in FACS buffer with starting concentration of 10 pg / mL and tittered 1:3, 8 total points. Diluted ALPP / L2 binders and control antibodies were added to cells for 30 minutes and then the cells were pelleted in a centrifuge, the supernatant fraction decanted, and the cells washed IX with FACS buffer. Finally, the cells were stained with a fluorescently labeled secondary' antibody (specific to the antibody Fc domain) for 15 minutes and then the cells w ere pelleted in a centrifuge, the supernatant fraction decanted, and26093the cells washed 2X with FACS buffer. Cells resuspended in 150 pL FACS buffer were then analyzed using a Intellicyt® iQue Screener PLUS.

[0324] The results are shown in Table 11.Table 11Cell binding / specificity of ALPP / L2 BindersCell binding EC50 (pg / mL, xl0‘3)Human Human Rhesus Mouse Human HumanClone Name ALPPL2 ALPP ALPP ALPPL2 ALPI 293 ALPL CHOK1 CHOK1 CHOK1 CHOK1 CHOK1 25G8 2.4 1.3 3.6 NB NB NB 29D2 3.5 4.3 10 NB NB NB 16C2 15 11 37 NB 4.64 NB 8A7 1 3.8 120 NB NB NB 16H2 0.72 0.83 8.3 NB NB NB 11C7 36 52 68 NB NB NB 5A1 22 54 49 NB NB NB 8G6 16 46 31 NB NB NB 50F5 4.7 3.3 31 NB NB NB 12G9 0.0004317 1.5 170 NB NB NB 5E8 7.3 19 23 NB NB NB 14A11 42 100 260 NB NB NB 19 A3 110 34 94 NB NB NB 14H5 17 20 110 NB NB NB 15B6 80 25 96 NB NB NB Very17E4 1590 1730 2350 NB Weak NB Very6E6 11 11 9.2 NB Weak NB 15B4 0.2 7.1 17 NB NB NB 17H6 0.00001481 180 140 NB NB NB 10F6 0.00003769 0.59 150 NB NB NB 4F4 0.45 0.93 13 NB NB NB 4B10 4.3 70 73 NB NB NB 49G5 100 150 220 NB NB NB 7C8 66 150 650 NB Weak NB 9B12 0.54 48 370 NB NB NB 6F5 0.66 2.2 28 NB NB NB 16B5 0.64 8.7 59 NB NB NB Weak Binder19E5 (no fit) 4570 NB NB NB NB 3G8 20 45 29 NB NB NB 17E4 0.23 5.3 7.3 NB NB NB 24F4 4.1 16 8 NB NB NB 9G10 1.3 2.5 16 NB NB NB 21H7 2.8 3.3 2 Very' Weak NB NB2.5F8 1.141E-14 0.92 2.2 NB NB NB26093StrongBinder (no32H6 fit) 0.63 0.11 NB NB NB 2A2 0.8 6.7 8.3 NB NB NB StrongBinder (no12C6 fit) 3.7 NB NB NB NB 12F1 0.48 2.4 4.3 NB NB NB 14C6 NB NB NB NB NB NB 16F2 23 67 50 NB NB NB 14B12 1.5 5.7 3.5 NB NB NB 16C2 1.5 2.9 9.4 NB NB NB 10G2 14 34 19 NB NB NB 6E10 8.4 23 25 NB NB NB 10B3 0.72 3.8 4.7 NB NB NB 10A9 20 8.9 64 NB NB NB 35D5 3.5 5.6 5.3 NB NB NB 17A7 1.5 2.3 2.6 NB NB NB 18A11 0.13 1.2 8.4 NB NB NB 49G11 NB 1660 NB NB NB NB 3A4 NB NB NB NB NB NB 46F5 NB 550 NB NB NB NB 21G1 NB 400 NB NB NB NB 3A9 0.35 4 8.7 Very Weak NB NB 13H8 1.8 5.9 11 NB NB NB 14E4 NB NB NB NB NB NB 14B8.1 12 25 26 NB NB NB 7F11.1 NB NB NB NB NB NB 7D11.1 NB NB NB NB NB NB 35D7.1 0.8 5 8.4 NB NB NB 10H9 NB NB NB NB NB NB 13B3 0.0011 1.3 3.1 NB NB NB 14B8.2 NB NB NB NB NB NB 7F11.2 NB NB NB NB NB NB 7D11.2 NB NB NB NB NB NB 35D7.2 NB NB NB NB NB NBNote: NB - no bindingEXAMPLE 3Binding Affinity of the ALPP / L2 binders by Surface Plasmon Resonance

[0325] Binding of the ALPP / L2 binders to human, rhesus, and mouse ALPP / L2 proteins was performed using SPR (surface plasmon resonance) on a Biacore™ 4000 instrument. Briefly, an anti -mouse Fc antibody (Cytiva. # 29215281) was immobilized on a CM5 sensor chip (Cytiva, 29149603). ALPP / L2 binders were diluted to 10 nM and captured for 60 seconds. Human, rhesus, or mouse ALPP / L2 protein was then injected for 180 seconds at concentrations of 22 to 200 nM and allowed to dissociate for 240 seconds. Data were fit using Biacore™ Evaluation26093software, version 1.1 and a 1: 1 fitting model (Langmuir Binding, Rmax = global. R1 = constant, offset = 0). The results are presented in Table 12.Table 12Binding affinity of ALPP / L2 binders to human ALPPL2 and Rhesus ALPP Human Human Human Rhesus RhesusRhesus ALPP Clone Name ALPPL2 ALPPL2 ALPPL2 ALPP ALPPKD (M) ka (1 / Ms) kd (1 / s) KD (M) ka (1 / Ms) kd (1 / s)25 G8 1.90E+05 2.67E-04 1.41E-09 3.63E+05 1.99E-04 5.48E-10 29D2 NB NB NB NB NB NB 16C2 1.90E+05 2.32E-04 1.22E-09 2.10E+05 1.64E-04 7.78E-10 8A7 3.05E+05 2.75E-04 9.02E-10 1.87E+06 3.53E-02 1.89E-08 16H2 5.02E+05 8.94E-05 1.78E-10 7.86E+05 1.24E-04 1.58E-10 11C7 6.89E+06 2.23E-04 3.24E-11 1.43E+06 6.47E-03 4.52E-09 5A1 7.35E+05 4.63E-04 6.30E-10 1.14E+10 1.10E-01 9.64E-12 8G6 1.61E+07 2.03E-04 1.26E-11 3.11E+06 1.56E-03 5.00E-10 50F5 9.78E+04 3.70E-04 3.78E-09 2.20E+05 3.28E-04 1.49E-09 12G9 NB NB NB NB NB NB 5E8 2.19E+05 3.98E-04 1.82E-09 1.34E+05 3.66E-03 2.73E-08 14A11 2.22E+05 3.41E-04 1.54E-09 4.98E+04 6.07E-03 1.22E-07 19A3 1.29E+07 2.03E-03 1.57E-10 1.20E+05 5.11E-03 4.28E-08 14H5 NB NB NB NB NB NB 15B6 4.72E+05 5.51E-04 1.17E-09 2.39E+05 6.21E-03 2.60E-08 17E4 NB NB NB NB NB NB 6E6 2.67E+05 2.52E-04 9.44E-10 2.40E+05 2.82E-04 1.17E-09 15B4 NB NB NB NB NB NB 17H6 1.95E+05 3.74E-04 1.92E-09 3.00E+05 4.54E-04 1.52E-09 10F6 2.07E+05 3.75E-04 1.82E-09 1.91E+05 4.02E-04 2.11E-09 4F4 4.30E+05 2.83E-04 6.59E-10 5.72E+05 2.50E-04 4.36E-10 4B10 7.00E+06 1.62E-04 2.32E-11 5.75E+05 6.39E-04 1.11E-09 49G5 6.36E+04 4.74E-04 7.45E-09 4.00E+04 1.30E-03 3.26E-08 7C8 7.61E+05 9.94E-05 1.31E-10 3.58E+05 3.89E-04 1.09E-09 9B12 2.66E+05 1.90E-04 7.13E-10 7.41E+05 8.88E-04 1.20E-09 6F5 NB NB NB NB NB NB 16B5 2.22E+05 2.86E-04 1.29E-09 2.67E+05 2.87E-04 1.08E-09 19E5 NB NB NB NB NB NB 3G8 3.99E+05 2.52E-04 6.31E-10 5.60E+05 5.62E-04 1.00E-09 17E4 1.26E+09 4.14E-04 3.30E-13 7.87E+03 4.87E-06 6.19E-10 24F4 3.80E+05 3.80E-05 1.00E-10 5.48E+05 2.31E-04 4.22E-10 9G10 3.25E+07 4.64E-04 1.43E-11 3.21E+10 3.75E+00 1.17E-10 21H7 2.16E+05 5.55E-04 2.57E-09 2.38E+05 3.16E-04 1.33E-09 2.5F8 NB NB NB NB NB NB 32H6 1.83E+05 3.24E-04 1.78E-09 2.46E+05 2.92E-04 1.19E-092A2 2.36E+05 3.73E-04 1.58E-09 2.93E+05 2.06E-04 7.04E-102609312C6 4.08E+07 4.91E-04 1.20E-11 NB NB NB 12F1 2.67E+07 8.72E-06 3.27E-13 1.41E+09 1.82E-01 1.29E-10 14C6 NB NB NB NB NB NB 16F2 4.70E+06 1.41E-04 3.00E-11 4.55E+08 6.08E-02 1.34E-10 14B12 1.68E+05 2.58E-04 1.53E-09 3.30E+05 1.95E-04 5.91E-10 16C2 NB NB NB NB NB NB 10G2 2.23E+05 3.06E-04 1.37E-09 2.00E+05 2.89E-04 1.44E-09 6E10 3.14E+05 3.90E-04 1.24E-09 4.85E+04 1.68E-02 3.47E-07 10B3 3.32E+05 2.29E-04 6.90E-10 3.03E+06 6.57E-05 2.17E-11 10A9 1.15E+05 9.96E-03 8.64E-08 NB NB NB 35D5 3.73E+05 2.05E-05 5.49E-11 1.20E+09 1.50E-01 1.25E-10 17A7 3.48E+05 5.34E-05 1.54E-10 1.35E+06 4.68E-03 3.45E-09 18A11 2.51E+05 2.43E-04 9.66E-10 3.31E+05 3.39E-04 1.03E-09 Low49G11 0.00E+00 0.00E+00 Capture NB NB Low Capture 3A4 NB NB NB NB NB NB 46F5 NB NB NB NB NB NB 21G1 NB NB NB NB NB NB 3A9 8.06E+08 3.65E-02 4.52E-11 4.56E+08 3.04E-02 6.68E-11 13H8 3.31E+05 1.24E-04 3.75E-10 4.00E+04 8.63E-02 2.15E-06 14E4 NB NB NB NB NB NB 14B8.1 1.07E+05 3.01E-04 2.81E-09 2.09E+05 1.55E-04 7.41E-10 Low7F11.1 0.00E+00 0.00E+00 Capture NB NB Low Capture 7D11.1 NB NB NB NB NB NB 35D7.1 1.91E+05 2.63E-04 1.38E-09 2.28E+05 2.26E-04 9.92E-10 10H9 NB NB NB NB NB NB 13B3 2.70E+09 4.12E-02 1.53E-11 4.59E+05 6.48E-05 1.41E-10 14B8.2 NB NB NB NB NB NB 7F11.2 NB NB NB NB NB NB 7D11.2 NB NB NB NB NB NB 35D7.2 NB NB NB NB NB NBNote: NB - no bindingEXAMPLE 4Physicochemical Properties of the ALPP / L2 Binders

[0326] The hydrophobicity and thermal stability of the ALPP / L2 binders were determined using Hydrophobic interaction chromatography (HIC) and Nano differential scanning fluorimetry (nanoDSF).HIC procedure

[0327] To determine the hydrophobicity of the ALPP / L2 binders, 50 pg of an ALPP / L2 binder at 1 mg / mL was mixed 1: 1 (v / v) with a 2 M ammonium sulfate, 100 mM sodium phosphate, pH260937.0 solution and held at ambient temperature for 20 to 30 minutes. The prepared ALPP / L2 binder was subsequently filtered through a 0.22 pm PVDF spin column at 8000 g for 1 minute prior to loading on a Dionex™ UltiMate™ 3000 HPLC (ThermoFisher, Waltham, Massachusetts, USA) with a ProPac™ HIC-10 column equilibrated in 1 M ammonium sulfate, 100 mM sodium phosphate, pH 7.0 (mobile phase A). The ALPP / L2 binder was eluted using an inverted gradient from mobile phase A to 100 mM sodium phosphate, pH 7.0 (mobile phase B). The column temperature was maintained at 30 °C during the total run time of 45 minutes. The elution was followed by recording the absorbance at OD 280 nm as a function of time; the data were then exported and analyzed using the Empower software. The retention time of each sample was compared to a reference and is characteristic of the sample’s hydrophobicity with longer elution times correlating with higher degree of hydrophobicity.NanoDSF procedure

[0328] ALPP / L2 binders were analyzed by nanoDSF to determine thermal stability. Nano-DSF is a method for measuring ultra-high-resolution protein s tabi 1 i ty using intrinsic tryptophan or tyrosine fluorescence. All nano-DSF studies were performed using the Nanotemper Prometheus NT.48 or Panta instrument. ALPP / L2 binders (~10 pL at 0.5-1 mg / mL) were loaded by capillarity into standard grade nano-DSF capillaries, placed on the Prometheus capillary holder and subjected to a temperature ramping of 1 °C / minute from 20 °C to 95 °C.

[0329] The melting point (Tm) onset (°C) and Tm (°C) values were automatically calculated by the nanoDSF software and indicate the structural stability of the ALPP / L2 binders and were obtained by monitoring the intrinsic tryptophan and tyrosine fluorescence at the emission wavelengths of 330 nm and 350 nm. To generate an unfolding curve, the ratio of the fluorescence intensities (F350 nm / F330 nm) was plotted vs. temperature or time. The thermal stability of an ALPP / L2 binder was described by the thermal unfolding transition midpoint Tm (°C), at which half of the ALPP / L2 binder population is unfolded. The Tm corresponds to the inflection point of the unfolding curve and was determined via the derivative of the curve.

[0330] The aggregation point Tagg (°C) is representative of the colloidal stability’ of the ALPP / L2 binders and was obtained by monitoring the back-reflection of near ultraviolet (UV) light using back reflection optics. The back-reflection optics use of near UV light scattering by protein aggregates, and thus only non-scattered light reaches the detector. The reduction of back reflected light is therefore a direct measure for aggregation of an ALPP / L2 binder.

[0331] The hydrophobicity’ and thermal stability of the ALPP / L2 binders are presented in Table 1326093Table 13Physicochemical properties of ALPP / L2 bindersThermal stability (Nano Hydrophobicity (H1C) DSF) Control ~26 min RT Clone Tm onset Tml RT(min) Main peak% Note (°C) (°C)25 G8 60.7 66.8 27.02 72.12 Multiple peaks 29D2 58.3 64.0 27.96 91.31 Multiple peaks 8A7 60.8 69.0 22.24 10016H2 53.8 65.3 18.82 10050F5 61.1 68.5 27.69 10012G9 60.9 68.1 22.43 98.1314H5 52.6 57.5 18.58 62.52 Multiple peaks 15B4 58.2 65.6 25.49 10017H6 58.4 66.7 27.10 98.2510F6 57.7 66.4 13.80 1004F4 54.3 66.0 >40 0 No elution 4B10 56.4 63.9 21.06 92.21 Multiple peaks 9B12 60.3 66.4 24.21 1006F5 59.6 67.0 >40 0 No elution 16B5 57.1 65.2 24.42 72.2917E4 57.6 65.5 22.29 1009G10 58.9 67.7 16.65 10021H7 58.7 65.9 16.09 88.692.5F8 60.3 65.1 19.27 1002A2 52.3 65.9 28.38 10012F1 59.6 65.9 30.72 10014B12 57.0 64.3 27.27 10016C2 58.7 64.3 31.81 10010G2 59.0 64.9 26.24 1006E10 63.0 68.5 23.56 10035D5 53.4 61.1 29.52 71.9 Multiple peaks 17A7 58.8 65.1 27.22 10018A11 59.0 65.5 27.05 1003A9 58.9 66.5 28.85 99.0135D7.1 58.6 66.0 23.42 10013B3 59.0 64.1 35.30 100 Broad peak 5E8 60.2 65.6 32.65 10015B6 60.3 66.7 >40 0 No elution 2. B3 51.8 60.5 24.59 10011C7 59.6 69.6 30.90 1005A1 56.9 71.0 38.30 1008G6 61.3 68.0 34.80 1005E8 58.9 65.4 33.20 1002609314A11 61.0 68.5 27.50 10019 A3 58.3 64.1 >40 100 No peaks15B6 59.7 66.5 >40 10049G5 56.7 74.1 18.10 1003G8 60.8 65.8 >40 100 No peaks24F4 53.7 69.2 >40 10032H6 56.7 67.3 18.50 10016F2 58.6 63.8 34.30 10010B3 51.6 60.8 24.60 10010A9 55.6 65.7 >40 100 No peaks13H8 61.9 67.7 22.10 10014B8.1 60.5 69.0 25.90 100NOTE: ALPP / L2 binders with poor / no expression or weak binders not analyzed. RT -Retention timeEXAMPLE 5Identification of Internalizing and Non-internalizing ALP P / L2 binders

[0332] ALPP / L2 binders that are internalized when bound ALPP or ALPPL2 on the cell surface from those ALPP / L2 binders that are not internalized were identified using an ALPPL2 Fab-ZAP Internalization Assav.ALPPL2 Fab-ZAP Internalization Assay:

[0333] ALPPL2-CHOK1 Clone 4F 11 was grown in 175 cm2flask using media receipt previously described.

[0334] Day 1 - Cells were harvested as described in Example 2. Cells were counted and adjusted to a cell density of IxlO6cells / 20 mL in media. Cells were then diluted in a 1:4 ratio in media (e.g., 10 mL cells + 40 mL media). Cells were plated in a flat clear bottom white tissue culture treated 96 well plates (Coming Catalog 3903). To plate out cells at 100 pL, using the 1:4 diluted tube, added 90 pL / well for a final cell density of 900 cells / well plus 10 pL of media. Cells were only added to center 60 wells due to evaporation (4-5 day assay set-up). Plates were incubated overnight at 37 °C.

[0335] Day 2 - Using Fab-ZAP human kit (Advanced Targeting System Catalog IT-51) Saporin reagent (Catalog PR-01, Lot 131-140, Concentration 1.0 mg / rnL, Molecular Weight 30 kDa), control IgG-SAP (Catalog IT-67, Lot 110-67. 1.9 mg / mL, Molecular Weight 107 kDa), and Human Fab-ZAP (Catalog IT-51, Lot 130-57, Concentration 2.2 mg / mL, Molecular Weight 106 kDa) was prepared per recommendation by the manufacturer’s guidelines. Human Fab-ZAP was spiked in media at a final concentration of 60 nM and added to a separate tissue culture26093plate. The ALPP / L2 binders were titered 1:20, 8-points, at a starting concentration of 666.7 nM in media with spiked human Fab-ZAP. Diluted ALPP / L2 binders plus prepared Human Fab-ZAP were added to Human ALPPL2-CHOK1 Clone 4F11 white tissue culture treated 96 well plates. Plates incubated at 37°C for 72 hours.

[0336] Day 5 - CellTiter-Glo® Luminescent Cell Viability Assay Kit (Promega PR-G7572) was added 1:1 volume and read on Envision to determine which ALPP / L2 binders w ere internalized and which w ere not internalized. The results are shown in Table 14.Table 14Non-Internalizing ALPP / L2 InternalizingBinders ALPP / L2 BindersClone Name Clone Name11C7 25 G85A1 29D28G6 16C25E8 8A714A11 16H219A3 50F515B6 6E649G5 15B43G8 17H624F4 10F632H6 4F416F2 4B1010B3 7C810A9 9B1213H8 16B517E49G1021H72A212C612F114B1210G26E1035D517A718A1149G113A914B8.17F11.135D7.113B326093EXAMPLE 6Epitope Binning of the ALPP / L2 Binders

[0337] Binning was conducted at Aviva Biosciences by high throughput surface plasmon resonance (SPR) using the Carterra* LSA (Carterra Inc. Salt Lake City, UT) system using the manufacturer’s instructions and sensor chip HC30M, briefly as follows.

[0338] A biosensor surface coated with goat anti-human IgG Fc was prepared on an HC30M sensor chip (Carterra*, Salt Lake City, UT) via amine-coupling using the Carterra* LSA’s surface array preparation protocol. The running buffer for chip preparation was 25 mM MES (2-(N-morpholino)ethanesulfonic acid) pH 5.5. 150 mM NaCl, and 0.05% (v / v) Tween® -20. The entire sensor surface was first activated with a mixture of 40 mM EDC (1 -Ethyl-3-[3-dimethyl aminopropyl] carbodiimide hydrochloride) and 10 mM sulfo-NHS (1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride / N-hydroxy sulfosuccinimide) in 100 mM MES buffer at pH 5.5 for 5 min. ALPP / L2 binders were diluted to approximately 10 pg / rnL in 10 mM sodium acetate pH 4.5. 0.05% Tween®-20 in duplicate were coupled to unique spots on the EDC-sulfo-NHS-modified surface for 10 minutes, followed by a 5 minute injection of 1 M ethanolamine-HCl (pH 8.5) to quench any remaining reactive esters. ALPP / L2 binders were then captured at 50 pg / mL using the 96-channel printhead before being crosslinked to the surface with BS3 ((bis(sulfosuccinimidyl)suberate)) for 8 minutes followed by blocking with 1 M ethanolamine-HCl (pH 8.5). The resulting array of covalently coupled ALPP / L2 binders and other anti-ALPP / L2 antibodies on the sensor surface was used for subsequent epitope binning measurements. Binning in this study was performed in a “classical sandwich format” at 25 °C in lx HBS-TE (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.01% Tween® 20;)+ 0.5 mg / mL BSA using the single flow cell mode of the Carterra® LSA instrument. Each binning experiment cycle involved first a 10-minute injection of human IgG Kappa to block any available Fc sites followed by 5 minute injection of the ALPP or ALPPL2 at 30 nM, then follow ed by a 5 minute injection of the secondary antibody at 50 pg / mL. The array surface was regenerated with two 30 second pulses of regeneration solution after each binning cycle. The raw high-throughput epitope binning results were processed using the Carterra® LSA epitope software to generate a heat map as well as other visualizations such as a combined dendrogram and net...

Claims

WHAT IS CLAIMED IS:

1. A bispecific antibody comprising a first binding domain, which binds to human ALPP and / or ALPPL2 on the surface of a target cell, and a second binding domain, which binds to human CD3 on the surface of a T cell, wherein the first binding domain comprises(a) a first heavy chain variable domain (VH) comprising complementarity determining regions (CDRs) VH-CDR1, VH-CDR2, and VH-CDR3 and a first light chain variable domain (VL) comprising CDRs VL-CDR1, VL-CDR2, and VL-CDR3, wherein(1) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 3, SEQ IDNO: 4, and SEQ ID NO: 5, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 6, SEQ IDNO: 7, and SEQ ID NO: 8, respectively;(2) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 11, SEQ IDNO: 12, and SEQ ID NO: 13, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 14, SEQ IDNO:

15. and SEQ ID NO: 16, respectively;(3) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 19, SEQ IDNO: 20, and SEQ ID NO: 21, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 22, SEQ IDNO: 23, and SEQ ID NO: 24, respectively;(4) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 27, SEQ IDNO: 28, and SEQ ID NO:

29. respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 30, SEQ IDNO: 31, and SEQ ID NO: 32, respectively;(5) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 35, SEQ IDNO:

36. and SEQ ID NO:

37. respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 38, SEQ IDNO: 39, and SEQ ID NO: 40, respectively;(6) VH-CDRL VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 43, SEQ IDNO: 44, and SEQ ID NO: 45, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 46, SEQ IDNO:

47. and SEQ ID NO: 48, respectively;(7) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 51, SEQ IDNO: 52, and SEQ ID NO: 53, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 54, SEQ IDNO:

55. and SEQ ID NO: 56, respectively;(8) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 59, SEQ IDNO: 60, and SEQ ID NO: 61, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 62, SEQ IDNO: 63, and SEQ ID NO: 64, respectively;(9) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 67, SEQ IDNO: 68, and SEQ ID NO: 69, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 70, SEQ IDNO: 71, and SEQ ID NO: 72, respectively;(10) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 75, SEQ IDNO: 76, and SEQ ID NO:

77. respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 78, SEQ IDNO: 79, and SEQ ID NO: 80, respectively;(11) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 83, SEQ IDNO:

84. and SEQ ID NO:

85. respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 86, SEQ IDNO: 87, and SEQ ID NO: 88, respectively;(12) VH-CDR1, VH-CDR2. and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 91, SEQ IDNO: 92, and SEQ ID NO: 93, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise theamino acid sequence set forth in SEQ ID NO: 94, SEQ 1DNO: 95, and SEQ ID NO: 96, respectively;(13) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 99, SEQ IDNO: 100, and SEQ ID NO: 101, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 102, SEQ IDNO: 103, and SEQ ID NO: 104, respectively;(14) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO:

107. SEQ IDNO: 108, and SEQ ID NO:

109. respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 110, SEQ IDNO: 111, and SEQ ID NO: 112, respectively;(15) VH-CDR1, VH-CDR2. and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO:

115. SEQ IDNO: 116, and SEQ ID NO:

117. respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 118, SEQ IDNO: 119, and SEQ ID NO:

120. respectively;(16) VH-CDR1, VH-CDR2. and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 123, SEQ IDNO: 124, and SEQ ID NO: 125, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 126, SEQ IDNO: 127, and SEQ ID NO:

128. respectively;(17) VH-CDRL VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 131, SEQ IDNO: 132, and SEQ ID NO: 133, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 134, SEQ IDNO: 135, and SEQ ID NO:

136. respectively;(18) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 139, SEQ IDNO: 140, and SEQ ID NO: 141, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO:

142. SEQ IDNO: 143, and SEQ ID NO: 144, respectively;(19) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 147, SEQ IDNO: 148, and SEQ ID NO: 149,respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 150, SEQ IDNO: 151, and SEQ ID NO: 152, respectively;(20) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO:

155. SEQ IDNO: 156, and SEQ ID NO:

157. respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO 158, SEQ IDNO: 159, and SEQ ID NO: 160, respectively;(21) VH-CDR1, VH-CDR2. and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 163, SEQ IDNO: 164, and SEQ ID NO: 165, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 166, SEQ IDNO: 167, and SEQ ID NO:

168. respectively;(22) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 171, SEQ IDNO: 172, and SEQ ID NO: 173, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 174, SEQ IDNO: 175, and SEQ ID NO:

176. respectively;(23) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 179, SEQ IDNO: 180, and SEQ ID NO: 181, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO:

182. SEQ IDNO: 183, and SEQ ID NO:

184. respectively;(24) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 187, SEQ IDNO: 188, and SEQ ID NO: 189, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO:

190. SEQ IDNO: 191, and SEQ ID NO: 192, respectively;(25) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 195, SEQ IDNO: 196, and SEQ ID NO: 197, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 198, SEQ IDNO: 199, and SEQ ID NO: 200, respectively;26093(26 VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 203, SEQ IDNO: 204, and SEQ ID NO: 205, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO:

206. SEQ IDNO: 207, and SEQ ID NO:

208. respectively;(27) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 211, SEQ IDNO: 212, and SEQ ID NO: 213, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO:

214. SEQ IDNO: 215, and SEQ ID NO: 216, respectively;(28) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 219, SEQ IDNO: 220, and SEQ ID NO: 221, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 222, SEQ IDNO: 223, and SEQ ID NO: 224, respectively;(29) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 227, SEQ IDNO: 228, and SEQ ID NO: 229, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 230, SEQ IDNO: 231, and SEQ ID NO: 232, respectively;(30) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO:

235. SEQ IDNO: 236, and SEQ ID NO:

237. respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 238, SEQ IDNO: 239, and SEQ ID NO: 240, respectively;(31) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO:

243. SEQ IDNO: 244, and SEQ ID NO:

245. respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 246, SEQ IDNO: 247, and SEQ ID NO: 248, respectively;(32) VH-CDR1, VH-CDR2. and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 251, SEQ IDNO: 252, and SEQ ID NO: 253, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise theamino acid sequence set forth in SEQ ID NO: 254, SEQ IDNO: 255, and SEQ ID NO: 256, respectively;(33) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 259, SEQ IDNO: 260, and SEQ ID NO: 261, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 262, SEQ IDNO: 263, and SEQ ID NO: 264, respectively;(34) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO:

267. SEQ IDNO: 268, and SEQ ID NO:

269. respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 270, SEQ IDNO: 271, and SEQ ID NO: 272, respectively;(35) VH-CDR1, VH-CDR2. and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO:

275. SEQ IDNO: 276, and SEQ ID NO:

277. respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 278, SEQ IDNO: 279, and SEQ ID NO:

280. respectively;(36) VH-CDR1, VH-CDR2. and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 283, SEQ IDNO: 284, and SEQ ID NO: 285, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 286, SEQ IDNO: 287, and SEQ ID NO:

288. respectively;(37) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 291, SEQ IDNO: 292, and SEQ ID NO: 293, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 294, SEQ IDNO: 295, and SEQ ID NO:

296. respectively;(38) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 299 SEQ IDNO: 300, and SEQ ID NO: 301, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO:

302. SEQ IDNO: 303, and SEQ ID NO: 304, respectively;(39) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 307, SEQ IDNO: 308, and SEQ ID NO: 309,respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 310, SEQ IDNO: 311, and SEQ ID NO: 312, respectively;(40) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO:

315. SEQ IDNO: 316, and SEQ ID NO:

317. respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 318, SEQ IDNO: 319, and SEQ ID NO: 320, respectively;(41) VH-CDR1, VH-CDR2. and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 323, SEQ IDNO: 324, and SEQ ID NO: 325, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 326, SEQ IDNO: 327, and SEQ ID NO:

328. respectively;(42) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 341, SEQ IDNO: 342, and SEQ ID NO: 343, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 344, SEQ IDNO: 345, and SEQ ID NO:

346. respectively;(43) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 349, SEQ IDNO: 350, and SEQ ID NO: 351, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO:

352. SEQ IDNO: 353, and SEQ ID NO:

354. respectively;(44) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 357, SEQ IDNO: 358, and SEQ ID NO: 359, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO:

360. SEQ IDNO: 361, and SEQ ID NO: 362, respectively;(45) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 365, SEQ IDNO: 366, and SEQ ID NO: 367, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 368, SEQ IDNO: 369, and SEQ ID NO: 370, respectively; or26093(46) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 373, SEQ IDNO: 374, and SEQ ID NO: 375, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO:

376. SEQ IDNO: 377, and SEQ ID NO: 378, respectively; and(b) wherein the second binding domain comprises a second VH comprising CDRs VH-CDR1, VH-CDR2, and VH-CDR3 and a second VL comprising CDRs VL- CDR1, VL-CDR2, and VL-CDR3, wherein VH-CDR1, VH-CDR2, and VH- CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 424, SEQ IDNO: 425, and SEQ ID NO: 426, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 428, SEQ IDNO: 429, and SEQ ID NO: 430, respectively; andwherein the first binding domain is linked to the N-terminus of a first heavy chain constant region (CH) comprising at least a CH2-CH3 domain and the second binding domain is linked to the N-terminus of a second CH comprising at least CH2-CH3 domains; wherein the CH3 domains of the first CH and the second CH each comprise one or more mutations promoting heterodimerization of the first and second CH3 domains to form the bispecific antibody.

2. The bispecific antibody of claim 1, wherein(a) the VH-CDR1, VH-CDR2, and VH-CDR3 of the first binding domain comprise the amino acid sequence set forth in SEQ ID NO: 3, SEQ IDNO: 4, and SEQ ID NO: 5, respectively, and the VL-CDR1, VL-CDR2, and VL-CDR3 of the first binding domain comprise the amino acid sequence set forth in SEQ ID NO: 6, SEQ IDNO: 7, and SEQ ID NO: 8, respectively;(b) VH-CDR1, VH-CDR2, and VH-CDR3 of the first binding domain comprise the amino acid sequence set forth in SEQ ID NO: 11, SEQ IDNO: 12, and SEQ ID NO: 13, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 of the first binding domain comprise the amino acid sequence set forth in SEQ ID NO: 14, SEQ IDNO: 15, and SEQ ID NO: 16, respectively;(c) VH-CDR1, VH-CDR2, and VH-CDR3 of the first binding domain comprise the amino acid sequence set forth in SEQ ID NO: 19, SEQ IDNO: 20, and SEQ ID NO: 21, respectively, and VL-CDR1, VL-CDR2, and VL-CDR3 of the firstbinding domain comprise the amino acid sequence set forth in SEQ ID NO: 22, SEQ IDNO: 23, and SEQ ID NO: 24, respectively.

3. The bispecific antibody of claim 1, wherein the first binding domain comprises(a) a VH comprising the amino acid sequence set forth in SEQ ID NO: 1 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 419:(b) a VH comprising the amino acid sequence set forth in SEQ ID NO: 9 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 10;(c) a VH comprising the amino acid sequence set forth in SEQ ID NO: 17 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 18;(d) a VH comprising the amino acid sequence set forth in SEQ ID NO: 25 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 26;(e) a VH comprising the amino acid sequence set forth in SEQ ID NO: 33 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 34;(f) a VH comprising the amino acid sequence set forth in SEQ ID NO: 41 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 42;(g) a VH comprising the amino acid sequence set forth in SEQ ID NO: 49 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 50;(h) a VH comprising the amino acid sequence set forth in SEQ ID NO: 57 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 58;(i) a VH comprising the amino acid sequence set forth in SEQ ID NO: 65 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 66;(j) a VH comprising the amino acid sequence set forth in SEQ ID NO: 73 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 74;(k) a VH comprising the amino acid sequence set forth in SEQ ID NO: 81 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 82;(l) a VH comprising the amino acid sequence set forth in SEQ ID NO: 89 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 90;(m) a VH comprising the amino acid sequence set forth in SEQ ID NO: 97 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 98;(n) a VH comprising the amino acid sequence set forth in SEQ ID NO: 105 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 106;(o) a VH comprising the amino acid sequence set forth in SEQ ID NO: 113 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 114;(p) a VH comprising the amino acid sequence set forth in SEQ ID NO: 121 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 122:(q) a VH comprising the amino acid sequence set forth in SEQ ID NO: 129 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 130;(r) a VH comprising the amino acid sequence set forth in SEQ ID NO: 137 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 138:(s) a VH comprising the amino acid sequence set forth in SEQ ID NO: 145 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 146;(t) a VH comprising the amino acid sequence set forth in SEQ ID NO: 153 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 154;(u) a VH comprising the amino acid sequence set forth in SEQ ID NO: 161 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 162;(v) a VH comprising the amino acid sequence set forth in SEQ ID NO: 169 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 170;(w) a VH comprising the amino acid sequence set forth in SEQ ID NO: 177 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 178;(x) a VH comprising the amino acid sequence set forth in SEQ ID NO: 185 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 186;(y) a VH comprising the amino acid sequence set forth in SEQ ID NO: 193 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 194;(z) a VH comprising the amino acid sequence set forth in SEQ ID NO: 201 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 202;(aa) a VH comprising the amino acid sequence set forth in SEQ ID NO: 209 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 210:(bb) a VH comprising the amino acid sequence set forth in SEQ ID NO: 217 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 218;(cc) a VH comprising the amino acid sequence set forth in SEQ ID NO: 225 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 226:(dd) a VH comprising the amino acid sequence set forth in SEQ ID NO: 233 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 234;(ee) a VH comprising the amino acid sequence set forth in SEQ ID NO: 241 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 242;(ff) a VH comprising the amino acid sequence set forth in SEQ ID NO: 249 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 250;26093(gg) a VH comprising the amino acid sequence set forth in SEQ ID NO: 257 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 258:(hh) a VH comprising the amino acid sequence set forth in SEQ ID NO: 265 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 266;(ii) a VH comprising the amino acid sequence set forth in SEQ ID NO: 273 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 272:(jj) a VH comprising the amino acid sequence set forth in SEQ ID NO: 281 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 282;(kk) a VH comprising the amino acid sequence set forth in SEQ ID NO: 289 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 290;(11) a VH comprising the amino acid sequence set forth in SEQ ID NO: 297 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 298;(mm) a VH comprising the amino acid sequence set forth in SEQ ID NO: 305 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 306;(nn) a VH comprising the amino acid sequence set forth in SEQ ID NO: 313 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 314;(oo) a VH comprising the amino acid sequence set forth in SEQ ID NO: 321 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 322;(pp) a VH comprising the amino acid sequence set forth in SEQ ID NO: 329 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 340;(qq) a VH comprising the amino acid sequence set forth in SEQ ID NO: 347 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 348;(rr) a VH comprising the amino acid sequence set forth in SEQ ID NO: 355 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 356:(ss) a VH comprising the amino acid sequence set forth in SEQ ID NO: 363 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 364; or(tt) a VH comprising the amino acid sequence set forth in SEQ ID NO: 371 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 372:(uu) a VH comprising the amino acid sequence set forth in SEQ ID NO: 1 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 2;(vv) a VH comprising the amino acid sequence set forth in SEQ ID NO: 420 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 421; or(ww) a VH comprising the amino acid sequence set forth in SEQ ID NO: 422 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 18; and26093wherein the second binding domain comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 423 or 604 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 427 or 605.

4. The bispecific antibody of claim 1, wherein the first binding domain comprises(a) a VH comprising the amino acid sequence set forth in SEQ ID NO: 1 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 419;(b) a VH comprising the amino acid sequence set forth in SEQ ID NO: 9 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 10;(c) a VH comprising the amino acid sequence set forth in SEQ ID NO: 17 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 18;(d) a VH comprising the amino acid sequence set forth in SEQ ID NO: 1 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 2;(e) a VH comprising the amino acid sequence set forth in SEQ ID NO: 420 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 421; or (f) a VH comprising the amino acid sequence set forth in SEQ ID NO: 422 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 18; and wherein the second binding domain comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 423 or 604 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 427 or 605.

5. The bispecific antibody of claim 1, wherein the first binding domain is a Fab comprising a first VH-CH and VL-CL pair and the second binding domain is an scFv comprising from the N-terminus to the C-terminus VL-VH-CH’ or VH-VL-CH’, wherein the first CH comprises a first CHI domain, first Hinge, first CH2 domain, and first CH3 domain and the CH’ is a truncated CH comprising a deletion of CHI and comprising at least a second CH2 domain and second CH3 domain, wherein the CH and CH’ domains each comprise one or more amino acid substitutions in their CH3 domains that promote heterodimerization of the CH and CH’ domains to form the bispecific antibody.

6. The bispecific antibody of claim 5, wherein(a) the first CH3 domain comprises amino acid substitutions S354C and T366W and the second CH3 domain comprises amino acid substitutions Y349C, T366S, L368A, and Y407V;26093(b) the first CH3 domain comprises amino acid substitutions Y349C, T366S, L368A.and Y407V to form the knob and the second CH3 domain comprises amino acid substitutions S354C and T366W;(c) the first CH3 domain comprises amino acid substitution 409W amino acid substitution and the second CH3 domain comprises amino acid substitutions D399 and F405T;(d) the first CH3 domain comprises amino acid substitutions D399 and F405T amino acid substitutions and the second CH3 domain comprises amino acid substitution 409W;(e) the first CH3 domain comprises amino acid substitution 357W and the second CH3 domain comprises amino acid substitution Y349S;(f) the first CH3 domain comprises amino acid substitutions Y349S and the second CH3 domain comprises amino acid substitution 357W;(g) the first CH3 domain comprises amino acid substitutions T350V, L351Y. F405A, and Y407V and the second CH3 domain comprises amino acid substitutions T350, T366, K392 and T394W;(h) the first CH3 domain comprises amino acid substitutions T350, T366. K392 and T394W and the second CH3 domain comprises amino acid substitutions T350V, L351Y, F405A, and Y407V;(i) the first CH3 domain comprises amino acid substitutions T350V, L351Y, F405A, and Y407V and the second CH3 domain comprises amino acid substitutions T350V and T366W; or(j) the first CH3 domain comprises amino acid substitutions T350V and T366W to and the second CH3 domain comprises amino acid substitutions T350V, L351Y, F405A, and Y407V;wherein the first and second CH3 domains form a heterodimeric Fc region; and wherein the amino acid numbering is according to the EU numbering scheme.

7. The bispecific antibody of claim 1, wherein the first binding domain is a first Fab comprising a first VH-CH and VL-CL pair and the second binding domain is a second Fab comprising a second VH-CH and VL-CL pair, wherein the CH of the first VH-CH and a VL-CL pair and the second VH-CH and VL-CL pair each comprise one or more amino acid substitutions that promote heterodimerization to form the bispecific antibody.260938. The bispecific antibody of claim 7, wherein(a) the first CH3 domain comprises amino acid substitutions S354C and T366W and the second CH3 domain comprises amino acid substitutions Y349C, T366S, L368A, and Y407V;(b) the first CH3 domain comprises amino acid substitutions Y349C, T366S, L368A.and Y407V to form the knob and the second CH3 domain comprises amino acid substitutions S354C and T366W;(c) the first CH3 domain comprises amino acid substitution 409W amino acid substitution and the second CH3 domain comprises amino acid substitutions D399 and F405T;(d) the first CH3 domain comprises amino acid substitutions D399 and F405T amino acid substitutions and the second CH3 domain comprises amino acid substitution 409W;(e) the first CH3 domain comprises amino acid substitution 357W and the second CH3 domain comprises amino acid substitution Y349S;(f) the first CH3 domain comprises amino acid substitutions Y349S and the second CH3 domain comprises amino acid substitution 357W;(g) the first CH3 domain comprises amino acid substitutions T350V, L351Y. F405A, and Y407V and the second CH3 domain comprises amino acid substitutions T350, T366, K392 and T394W;(h) the first CH3 domain comprises amino acid substitutions T350, T366, K392 and T394W and the second CH3 domain comprises amino acid substitutions T350V, L351Y. F405A, and Y407V;(i) the first CH3 domain comprises amino acid substitutions T350V, L351Y, F405A, and Y407V and the second CH3 domain comprises amino acid substitutions T350V and T366W; or(j) the first CH3 domain comprises amino acid substitutions T350V and T366W to and the second CH3 domain comprises amino acid substitutions T350V, L351Y, F405A, and Y407V;wherein the first and second CH3 domains form a heterodimeric Fc region; and wherein the amino acid numbering is according to the EU numbering scheme.

9. The bispecific antibody of claim 1, wherein the CH is of the human IgGl or IgG4 isotype comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, insertions, deletions, or26093combinations thereof and the first and second CL is of the human kappa or lambda isotype comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, insertions, deletions, or combinations thereof.

10. The bispecific antibody of claim 1, wherein(a) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 381 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 392; (b) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 382 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 393; (c) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 383 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 394; (d) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 384 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 395; (e) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 385 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 396; (f) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 386 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 397; (g) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 387 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 398; (h) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 388 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 399; (i) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 389 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 400; (j) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 390 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 401; (k) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 392 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 381; (l) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 393 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 382; (m) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 394 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 383; (n) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 395 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 384;26093(o) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 396 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 385; (p) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 397 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 386; (q) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 398 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 387; (r) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 399 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 388; (s) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 400 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 389; or (t) the first CH comprises the amino acid sequence set forth in SEQ ID NO: 401 and second CH comprises the amino acid sequence set forth in SEQ ID NO: 390.

11. The bispecific antibody of claim 5, wherein CH’ comprises amino acids 8-15 of the Hinge region, CH2 domain, and CH3 domain.

12. The bispecific antibody of claim 5, wherein(a) the CH comprises the amino acid sequence set forth in SEQ ID NO: 381 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 431;(b) the CH comprises the amino acid sequence set forth in SEQ ID NO: 382 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 432;(c) the CH comprises the amino acid sequence set forth in SEQ ID NO: 383 and CH' comprises the amino acid sequence set forth in SEQ ID NO: 433;(d) the CH comprises the amino acid sequence set forth in SEQ ID NO: 384 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 434;(e) the CH comprises the amino acid sequence set forth in SEQ ID NO: 385 and CH' comprises the amino acid sequence set forth in SEQ ID NO: 435;(f) the CH comprises the amino acid sequence set forth in SEQ ID NO: 386 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 436;(g) the CH comprises the amino acid sequence set forth in SEQ ID NO: 387 and CH' comprises the amino acid sequence set forth in SEQ ID NO: 437;(h) the CH comprises the amino acid sequence set forth in SEQ ID NO: 388 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 438;26093(i) the CH comprises the amino acid sequence set forth in SEQ ID NO: 389 and CH' comprises the amino acid sequence set forth in SEQ ID NO: 439;(j) the CH comprises the amino acid sequence set forth in SEQ ID NO: 390 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 440;(k) the CH comprises the amino acid sequence set forth in SEQ ID NO: 392 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 441;(l) the CH comprises the amino acid sequence set forth in SEQ ID NO: 393 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 442;(m) the CH comprises the amino acid sequence set forth in SEQ ID NO: 394 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 443;(n) the CH comprises the amino acid sequence set forth in SEQ ID NO: 395 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 444;(o) the CH comprises the amino acid sequence set forth in SEQ ID NO: 396 and CH' comprises the amino acid sequence set forth in SEQ ID NO: 445;(p) the CH comprises the amino acid sequence set forth in SEQ ID NO: 397 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 446;(q) the CH comprises the amino acid sequence set forth in SEQ ID NO: 398 and CH' comprises the amino acid sequence set forth in SEQ ID NO: 447;(r) the CH comprises the amino acid sequence set forth in SEQ ID NO: 399 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 448;(s) the CH comprises the amino acid sequence set forth in SEQ ID NO: 400 and CH' comprises the amino acid sequence set forth in SEQ ID NO: 449; or (t) the CH compnses the amino acid sequence set forth in SEQ ID NO: 401 and CH’ comprises the amino acid sequence set forth in SEQ ID NO: 450.

13. The bispecific antibody of claim 5 or 7, wherein the CL comprises the kappa isotype.

14. The bispecific antibody of claim 13, wherein the CL comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, insertions, deletions, or combinations thereof compared to the amino acid sequence of the native CL.

15. The bispecific antibody of claim 5, wherein the CH and CH’ comprises:26093(a) a substitution of the amino acids at positions 252, 254. and 256 of the CH and the CH’ with amino acids Tyr (Y), Thr (T), and Glu (E), respectively, wherein the numbering is according to the EU numbering scheme;(b) a substitution of the amino acids at positions 233 and 235 of the CH and the CH’ with the amino acid Ala (A), wherein the numbering is according to the EU numbering scheme;(c) a substitution of the amino acids at positions 234 and 235 of the CH and the CH’ with the amino acid A and at position 265 with Ser (S), wherein the numbering is according to the EU numbering scheme;(d) a substitution of the amino acids at positions 234 and 235 of the CH and the CH’ with the amino acid A and at position 329 with Gly (G), wherein the numbering is according to the EU numbering scheme;(e) a substitution of the amino acid at position 235 of the CH and the CH’ with the amino acid E. wherein the numbering is according to the EU numbering scheme; (f) a substitution of the amino acid at position 265 of the CH and the CH’ with the amino acid A, wherein the numbering is according to the EU numbering scheme; (g) a substitution of the amino acid at position 265 of the CH and the CH’ with the amino acid A and at position 297 with the amino acid G, wherein the numbering is according to the EU numbering scheme;(h) a substitution of the amino acid at position 297 of the CH and the CH’ with any amino acid except for Asn (N), wherein the numbering is according to the EU numbering scheme: or(i) a substitution of the amino acid at position 297 of the CH and the CH’ with the amino acid A, at position 356 with Glu (E), at position 358 with Met (M), wherein the numbering is according to the EU numbering scheme.

16. The bispecific antibody of claim 5, wherein the CH and CH’ comprise a C-terminal lysine or lack a C-terminal lysine or a C-terminal glycine-lysine dipeptide.

17. The bispecific antibody of any one of the foregoing claims, wherein the N-terminal amino acid of the VH is pyroglutamate.

18. A bispecific antibody comprising: a heavy chain (HC), a light chain (LC), and an scFv linked to a truncated HC constant domain (scFv-CH’), wherein the HC comprises the amino acid26093sequence set forth in SEQ ID NO: 451, the LC comprises the amino acid sequence set forth in SEQ ID NO: 453, and the scFv-CH’ comprises the amino acid sequence set forth in SEQ ID NO: 462; or wherein the HC comprises the amino acid sequence set forth in SEQ ID NO: 457, the LC comprises the amino acid sequence set forth in SEQ ID NO: 459, and the scFv-CH’ comprises the amino acid sequence set forth in SEQ ID NO: 462; or wherein the HC comprises the amino acid sequence set forth in SEQ ID NO: 454, the LC comprises the amino acid sequence set forth in SEQ ID NO: 456, and the scFv-CH’ comprises the amino acid sequence set forth in SEQ ID NO: 461.

19. A bispecific antibody comprising: a first arm, which binds to human ALPP and / or ALPPL2 on the surface of a target cell, and a second arm, which binds to human CD3 on the surface of a T cell, comprising(a) a first arm comprising a heavy chain (HC) comprising the amino acid sequence set forth in SEQ ID NO: 510 associated with a light chain (LC) comprising the amino acid sequence set forth in SEQ ID NO: 453 and a second arm comprising an an scFv linked to a truncated HC constant domain (scFv-CH’) comprising the amino acid sequence set forth in SEQ ID NO: 509;(b) a first arm comprising an HC comprising the amino acid sequence set forth in SEQ ID NO: 511 associated with an LC comprising the amino acid sequence set forth in SEQ ID NO: 453 and a second arm comprising an scFv-CH’ comprising the amino acid sequence set forth in SEQ ID NO: 508;(c) a first arm comprising an HC comprising the amino acid sequence set forth in SEQ ID NO: 512 associated with an LC comprising the amino acid sequence set forth in SEQ ID NO: 456 and a second arm comprising an scFv-CH’ comprising the amino acid sequence set forth in SEQ ID NO: 508;(d) a first arm comprising an HC comprising the amino acid sequence set forth in SEQ ID NO: 513 associated with an LC comprising the amino acid sequence set forth in SEQ ID NO: 456 and a second arm comprising an scFv-CH’ comprising the amino acid sequence set forth in SEQ ID NO: 509;(e) a first arm comprising an HC comprising the amino acid sequence set forth in SEQ ID NO: 514 associated with an LC comprising the amino acid sequence set forth in SEQ ID NO: 459 and a second arm comprising an scFv-CH’ comprising the amino acid sequence set forth in SEQ ID NO: 509; or26093(f) a first arm comprising an HC comprising the amino acid sequence set forth in SEQ ID NO: 515 associated with an LC comprising the amino acid sequence set forth in SEQ ID NO: 459 and a second arm comprising an scFv-CH’ comprising the amino acid sequence set forth in SEQ ID NO: 508.

20. A bispecific antibody comprising: a heavy chain (HC), a light chain (LC), and an scFv linked to a truncated HC constant domain (scFv-CH’), wherein the HC comprises the amino acid sequence set forth in SEQ ID NO: 510, the LC comprising the amino acid sequence set forth in SEQ ID NO: 453, and the scFv-CH’ comprising the amino acid sequence set forth in SEQ ID NO: 509; or wherein the HC comprises the amino acid sequence set forth in SEQ ID NO: 514, the LC comprising the amino acid sequence set forth in SEQ ID NO: 459, and the scFv-CH’ comprising the amino acid sequence set forth in SEQ ID NO: 509; or wherein the HC comprises the amino acid sequence set forth in SEQ ID NO: 512, the LC comprising the amino acid sequence set forth in SEQ ID NO: 456, and the scFv-CH’ comprising the amino acid sequence set forth in SEQ ID NO: 508.

21. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1-20 and a pharmaceutically acceptable carrier or diluent.

22. A method for treating a proliferative disease or tumor in an individual in need of the treatment comprising administering to the individual a therapeutically effective amount of the bispecific antibody of claim 1 or a pharmaceutical composition comprising the bispecific antibody to treat the proliferative disease.

23. The method of claim 22, wherein the proliferative disease comprises cells that display ALPP and / or ALPPL2 on the cell surface.

24. The method of claim 22, wherein the proliferative disease comprises a tumor that displays ALPP and / or ALPPL2 on the surface of the cells thereof.

25. The method of claim 22 or 23, wherein the proliferative disease or tumor is a cancer or malignancy selected from the group consisting of mesothelioma, testicular cancer, endometrial cancer, pancreatic cancer, ovarian cancer, non-small cell lung cancer (NSCLC), gastric cancer,26093bladder cancer, head and neck cancer, cervical cancer, breast cancer, esophageal cancer, cholangiocarcinoma, prostate cancer, and colon cancer.

26. Use of the bispecific antibody of any one of claims 1-20 or a pharmaceutical composition comprising the bispecific antibody for the manufacture of a medicament for treatment of a proliferative disease or tumor.

27. The use of claim 26, wherein the proliferative disease comprises cells that display ALPP and / or ALPPL2 on the cell surface.

28. The use of claim 26, wherein the proliferative disease comprises a tumor that displays ALPP and / or ALPPL2 on the surface of the cells thereof.

29. The use of claim 26 or 27, wherein the proliferative disease or tumor is a cancer or malignancy selected from the group consisting of mesothelioma, testicular cancer, endometrial cancer, pancreatic cancer, ovarian cancer, non-small cell lung cancer (NSCLC), gastric cancer, bladder cancer, head and neck cancer, cervical cancer, breast cancer, esophageal cancer, cholangiocarcinoma. prostate cancer, and colon cancer.

30. The bispecific antibody of any one of claims 1-20 or a pharmaceutical composition comprising the bispecific antibody for treatment a proliferative disease or tumor.

31. The bispecific antibody of claim 30, wherein the proliferative disease comprises cells that display ALPP and / or ALPPL2 on the cell surface.

32. The bispecific antibody of claim 30, wherein the proliferative disease comprises a tumor that displays ALPP and / or ALPPL2 on the surface of the cells thereof.

33. The bispecific antibody of claim 30 or 31, wherein the proliferative disease or tumor is a cancer or malignancy selected from the group consisting of mesothelioma, testicular cancer, endometrial cancer, pancreatic cancer, ovarian cancer, non-small cell lung cancer (NSCLC), gastric cancer, bladder cancer, head and neck cancer, cervical cancer, breast cancer, esophageal cancer, cholangiocarcinoma, prostate cancer, and colon cancer.2609334. A combination therapy for treating a proliferative disease or tumor comprising the bispecific antibody of any one of claims 1-20 or a pharmaceutical composition comprising the bispecific antibody and a second therapeutic agent.

35. The combination therapy of claim 34, wherein the second therapeutic agent is a chemotherapy agent or a therapeutic antibody other than an antibody that binds ALPP and / or ALPPL2.

36. The combination therapy of claim 34 or 35, wherein the proliferative disease comprises cells that display ALPP and / or ALPPL2 on the cell surface.

37. The combination therapy of claim 34, wherein the proliferative disease comprises a tumor that displays ALPP and / or ALPPL2 on the surface of the cells thereof.

38. The combination therapy of claim 34, 35, or 36, wherein the proliferative disease or tumor is a cancer or malignancy selected from the group consisting of mesothelioma, testicular cancer, endometrial cancer, pancreatic cancer, ovarian cancer, non-small cell lung cancer (NSCLC), gastric cancer, bladder cancer, head and neck cancer, cervical cancer, breast cancer, esophageal cancer, cholangiocarcinoma, prostate cancer, and colon cancer.

39. A nucleic acid molecule encoding the heavy chain (HC), light chain (LC), or scFv-Fc of a bispecific antibody of any one of claims 1-20.

40. An expression vector comprising one or more of the nucleic acid molecules of claim 39.

41. A host cell comprising one or more nucleic acid molecules of claim 39.

42. A host cell comprising a first nucleic acid molecule encoding the heavy chain (HC) of a bispecific antibody of any one of claims 1 -20, a second nucleic acid molecule encoding the light chain (LC) of a bispecific antibody of any one of claims 1-20; and a third nucleic acid molecule encoding the scFv-CH' of a bispecific antibody of any one of claims 1-20, wherein the CH3 region of the HC and the scFv comprise one or more mutations promoting heterodimerization.2609343. A method for producing a bispecific antibody of any one of claims 1-20 comprising (a) providing the host cell of claim 42; (b) cultivating the host cell in a medium under conditions suitable for expressing the bispecific antibody; and (c) isolating the bispecific antibody from the host cells and / or medium.

44. The bispecific antibody of any one of claims 1-20 conjugated to a detectable moiety.

45. The bispecific antibody of claim 44, wherein the detectable moiety is detectable by magnetic resonance imaging (MRI) or by X-ray imaging.

46. A method for detecting ALPP and / or ALPPL2 on the surface of a cell in an individual comprising administering to the individual the bispecific antibody of claim 44 or 45 and detecting the cells in the individual that bind the bispecific antibody.

47. A composition comprising a bispecific antibody of any one of claims 1-20 or a pharmaceutical composition comprising the bispecific antibody, and a hyaluronan degrading enzyme.

48. The composition of claim 47, wherein the hyaluronan degrading enzyme is a soluble hyaluronidase.

49. The composition of claim 48, wherein the soluble hyaluronidase is soluble pH20.

50. The composition of claim 48, wherein the soluble hyaluronidase is hyaluronidase (recombinant human) or berahyaluronidase alfa.

51. A kit comprising a bispecific antibody of any one of claims 1-20 or a pharmaceutical composition comprising the bispecific antibody and a hyaluronan degrading enzyme.

52. The kit of claim 51, wherein the bispecific antibody and the hyaluronan degrading enzyme are provided in separate containers or the bispecific antibody and the hyaluronan degrading enzyme are provided as a mixture in a single container.2609353. The kit of claim 51, wherein the bispecific antibody and the hyaluronan degrading enzyme are provided in separate chambers of a dual-chamber injection device or the bispecific antibody and the hyaluronan degrading enzyme are provided as a mixture in a single chamber of a single-chamber injection device.

54. The kit of claim 51, wherein the hyaluronan degrading enzyme is a soluble hyaluronidase.

55. The kit of claim 54, wherein the soluble hyaluronidase is soluble pH20.

56. The kit of claim 54, wherein the soluble hyaluronidase is hyaluronidase (recombinant human) or berahyaluronidase alfa.

57. A method for treating a proliferative disease in an individual in need of the treatment comprising administering to the individual a therapeutically effective amount of the bispecific antibody of any one of claims 1-20 or a pharmaceutical composition comprising the bispecific antibody and a hyaluronan degrading enzyme to treat the proliferative disease.

58. The method of claim 57, wherein the proliferative disease comprises cells that display ALPP and / or ALPPL2 on the cell surface.

59. The method of claim 57, wherein the proliferative disease comprises a tumor that displays ALPP and / or ALPPL2 on the surface of the cells thereof.

60. The method of claim 58 or 59, wherein the proliferative disease or tumor is a cancer or malignancy selected from the group consisting of mesothelioma, testicular cancer, endometrial cancer, pancreatic cancer, ovarian cancer, non-small cell lung cancer (NSCLC), gastric cancer, bladder cancer, head and neck cancer, cervical cancer, breast cancer, esophageal cancer, cholangiocarcinoma, prostate cancer, and colon cancer.

61. The method of claim 57, wherein the hyaluronan degrading enzyme is a soluble hyaluronidase.

62. The method of claim 61, wherein the soluble hyaluronidase is soluble pH20.2609363 The method of claim 61. wherein the soluble hyaluronidase is hyaluronidase (recombinant human) or berahyaluronidase alfa.

64. The method of claim 57, wherein the bispecific antibody and the hyaluronan degrading enzyme are administered to the individual sequentially or simultaneously.

65. The method of claim 57, wherein the bispecific antibody and the hyaluronan degrading enzyme are mixed to form a mixture and the mixture is administered to the individual.

66. The method of claim 57, wherein the bispecific antibody and the hyaluronan degrading enzyme are administered to the individually systemically.

67. The method of claim 57, wherein the bispecific antibody and the hyaluronan degrading enzyme are administered to the individually subcutaneously or intramuscularly.

68. A CD3 binder comprising a heavy chain variable domain (VH) comprising the amino acid sequences set forth in SEQ ID NO: 423 and a light chain variable domain (VL) comprising the amino acid sequence set forth in SEQ ID NO: 427.

69. The CD3 binder of claim 68, wherein the CD3 binder is an antibody comprising the VH and VL or a CD3-binding fragment thereof comprising the VH and VL.

70. The CD3 binder of claim 69, wherein the CD3-binding fragment thereof is a Fab or an scFv.

71. The CD3 binder of claim 68. wherein the scFv comprises the amino acid sequence set forth in SEQ ID NO: 463 or SEQ ID NO: 464.

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