ALPP and / or alppl2 binders and their use

Abstract

ALPP / L2 binders that bind alkaline phosphatase, placental type (ALPP) and / or alkaline phosphatase, germ cell type (ALPPL2) are disclosed. In specific embodiments, the ALPP / L2 binders may be anti-ALPP / L2 antibodies or antigen-binding fragments thereof. The ALPP / L2 binders are useful for treating proliferative diseases that comprise cells that display ALPP / L2 on the cell surface.

Description

ALPP AND / OR ALPPL2 BINDERS AND THEIR USECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 898880, filed October 14, 2025 and U.S. Provisional Application No. 63 / 745,914, 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 (26092-WO-PCT_SL.xmL Size: 384,574 bytes; and Date of Creation: March 27, 2025) are herein incorporated by reference in their entirety.FIELD

[0003] The present invention relates to molecules that bind alkaline phosphatase, placental type (ALPP) and / or alkaline phosphatase, germ cell type (ALPPL2), herein after “ALPP / L2 binders”, and methods of using them. In specific embodiments, the ALPP / L2 binders may be anti-ALPP / L2 antibodies or antigen-binding fragments thereof.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 (ALP1) 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 and 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, ALPL is widely expressed in many tissues including bone, liver, and kidney. ALPL has a lower homology to ALPP and 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; International Patent Application Publication Nos. WO2017095823 (University of California); WO2021154534 (Promab / Forevertek);WO2021158178 (Agency for Science. Technology, and Research); WO2022197890 (Seagen Inc.); WO2023049150 (University of California; WO2023215746 (Javelin Oncology, Inc.); and WO2024133763 (Almac Discovery Ltd.).SUMMARY

[0008] The present invention provides improved ALPP / L2 binders, which may be derived from antibodies or antigen-binding fragments thereof that bind ALPP and / or ALPPL2. The ALPP / L2 binders may comprise a variety of embodiments, e.g., a bivalent whole antibody comprising two identical anti-ALPP / L2 VH / VL pairs or Fabs, a monovalent antibody comprising a single anti-ALPP / L2 VH / VL pair or Fab or single-chain variable fragment (scFv), a bispecific antibody comprising an anti-ALPP / L2 Fab or scFv and a Fab or scFv directed to another target, an antibody-drug conjugate comprising a bivalent or monovalent anti-ALPP / L2 antibody conjugated to a therapeutic agent, or a chimeric antigen receptor (CAR) for CAR-based T-cell therapy comprising an anti-ALPP / L2 scFv.

[0009] The ALPP / L2 binder of any one of the embodiments disclosed herein may be used to target tumor cells in a variety of ways. The ALPP / L2 binder of any one of the embodiments disclosed herein are also useful for detection of tumor cells and may be used for example in a companion diagnostic for detecting ALPP and / or ALPPL2. The ALPP / L2 binder of any one of the embodiments disclosed herein bind to cells that express or overexpress ALPP and / or ALPPL2 while having no detectable binding to cells that express or overexpress ALPP or ALPI. Exemplary non-limiting cells expressing ALPP and / or ALPPL2 on the cell surface include but are not limited to, mesothelioma cells, testicular cancer cells, endometrial cancer cells, pancreaticcancer cells, ovarian cancer cells, non-small cell lung cancer (NSLC) cells, gastric cancer cells, and a colon cancer cells.

[0010] Provided is an ALPP / L2 binder comprising a heavy chain variable domain (VH) comprising complementarity determining regions (CDRs) VH-CDR1, VH-CDR2, and VH-CDR3 and a light chain variable domain (VL) comprising CDRs VL-CDR1. VL-CDR2, and VL-CDR3, wherein(a) 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;(b) 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;(c) 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;(d) 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;(e) 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;(I) 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;(g) 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;(h) 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;(i) 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;(j) 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;(k) 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;(l) 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;(m) 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;(n) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 107, SEQ IDNO: 108, and SEQ IDNO: 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;(o) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 115, SEQ IDNO: 116, and SEQ IDNO: 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;(p) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 123, SEQ IDNO: 124, and SEQ IDNO: 125, respectively, and VL-CDR1, VL-CDR2, and26092VL-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 126, SEQ IDNO: 127, and SEQ ID NO: 128, respectively;(q) 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;(r) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 139, SEQ IDNO: 140, and SEQ IDNO: 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;(s) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 147, SEQ IDNO: 148, and SEQ IDNO: 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;(t) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 155, SEQ IDNO: 156, and SEQ IDNO: 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;(u) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 163, SEQ IDNO: 164, and SEQ IDNO: 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;(v) VH-CDR1, VH-CDR2, and VH-CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 171, SEQ IDNO: 172, and SEQ IDNO: 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;(w) 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;(x) 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;(y) 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;(z) 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;(aa) 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;(bb) 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;(cc) 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;(dd) 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;(ee) 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;(ff) 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;(gg) 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;(hh) 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;(ii) 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;(jj) 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;(kk) 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;(11) 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;(mm) 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;(nn) 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;(oo) 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;(pp) 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;(qq) 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;(rr) 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;(ss) 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(tt) 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.

[0011] In a further embodiment of the ALPP / L2 binder, the ALPP / L2 binder 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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.

[0012] In a further embodiment of the ALPP / L2 binder, the ALPP / L2 binder comprises an antibody in which the VH is covalently linked to a heavy chain constant domain (CH) comprising a CHI domain, CH2 domain, and CH3 domain and the VL is covalently linked to a light chain constant domain (CL).

[0013] In a further embodiment of the ALPP / L2 binder, the CH comprises an IgGl, IgG2, IgG3, or IgG4 isotype and the CL comprises a human kappa or human lambda isotype.

[0014] In a further embodiment of the ALPP / L2 binder, the CH comprises the IgGl or IgG4 isotype and the CL comprises a human kappa or human lambda isotype.

[0015] In a further embodiment of the ALPP / L2 binder, the IgGl or IgG4 isoty pe comprises 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.

[0016] In a further embodiment of the ALPP / L2 binder, the IgGl isotype comprises 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 isotype.

[0017] In a further embodiment of the ALPP / L2 binder, the CL comprises the kappa isotype

[0018] In a further embodiment of the ALPP / L2 binder, the kappa isotype 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 kappa isotype.

[0019] In a further embodiment of the ALPP / L2 binder, the constant domain of the human IgGl comprises:(a) a substitution of the amino acids at positions 252, 254, and 256 of the CH with amino acids Tyr (Y), Thr (T), and Glu (E), respectively, wherein the numbering is according to Eu;26092(b) a substitution of the amino acids at positions 233 and 235 with the amino acid Ala (A), wherein the numbering is according to Eu;(c) a substitution of the amino acids at positions 234 and 235 with the amino acid A and at position 265 with Ser (S), wherein the numbering is according to Eu;(d) a substitution of the amino acids at positions 234 and 235 with the amino acid A and at position 329 with Gly (G), wherein the numbering is according to Eu;(e) a substitution of the amino acid at position 235 with the amino acid E, wherein the numbering is according to Eu;(f) a substitution of the amino acid at position 265 with the amino acid A, wherein the numbering is according to Eu;(g) a substitution of the amino acid at position 265 with the amino acid A and at position 297 with the amino acid G, wherein the numbering is according to Eu;(h) a substitution of the amino acid at position 297 with any amino acid except for Asn (N), wherein the numbering is according to Eu; or(i) a substitution of the amino acid at position 297 with the amino acid A, at position 356 with Glu (E), at position 358 with Met (M), wherein the numbering is according to Eu;

[0020] In a further embodiment of the ALPP / L2 binder, the CH comprise a C -terminal lysine or lacks a C-terminal lysine or a C-terminal gly cine-ly sine dipeptide.

[0021] In a further embodiment of the ALPP / L2 binder, the N-terminal amino acid of the VH is pyroglutamate.

[0022] In a further embodiment of the ALPP / L2 binder, the ALPP / L2 binder is an antigenbinding fragment of an antibody selected from the group consisting of a Fab fragment, a Fab’ fragment, a F(ab’)2 fragment, a scFab, an Fv region, and an ScFv.

[0023] In a further embodiment of the ALPP / L2 binder, the ALPP / L2 binder comprises an ScFv.

[0024] In a further embodiment of the ALPP / L2 binder, the ALPP / L2 binder comprises a chimeric antigen receptor T (CAR-T) cell or chimeric antigen receptor natural killer (CAR-NK) cell comprising the ScFv.

[0025] Further provided is a pharmaceutical composition comprising the ALPP / L2 binder disclosed herein and a pharmaceutically acceptable carrier or diluent.

[0026] 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 an ALPP / L2 binder or pharmaceutical composition thereof to treat the proliferative disease. In a particular embodiment, the proliferative disease comprises cells that display ALPP and / or26092ALPPL2 on the cell surface, i.e.. the cells are ALPP / L2 positive. In particular embodiments of the method, the proliferative disease is an ALPP / L2 positive tumor. In further embodiments, the proliferative disease is an ALPP / L2 positive 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.

[0027] Further provided is use of an ALPP / L2 binder or pharmaceutical composition thereof for the manufacture of a medicament for treatment of a proliferative disease. In a particular embodiment, the proliferative disease comprises cells that display ALPP and / or ALPPL2 on the cell surface, i.e., cells that are ALPP / L2 positive. In particular embodiments of the use, the proliferative disease is an ALPP / L2 positive tumor. In further embodiments, the proliferative disease is an ALPP / L2 positive 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.

[0028] Further provided is an ALPP / L2 binder disclosed herein or pharmaceutical composition thereof for use in the treatment of a proliferative disease. In a particular embodiment, the proliferative disease comprises cells that display ALPP and / or ALPPL2 on the cell surface, i.e., the cells are ALPP / L2 positive. In particular embodiments of the ALPP / L2 binder disclosed herein or pharmaceutical composition thereof, the proliferative disease is an ALPP / L2 positive tumor. In further embodiments, the proliferative disease is an ALPP / L2 positive 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.

[0029] Further provided is a combination therapy for treating a proliferative disease comprising an ALPP / L2 binder disclosed herein or a pharmaceutical composition thereof and a second therapeutic agent. In particular embodiments of the combination thereapy, the proliferative disease comprises cells that display ALPP and / or ALPPL2 on the cell surface, i.e., the cells are ALPP / L2 positive. In a further embodiment, the proliferative disease is an ALPP / L2 positive tumor. In further embodiments, the proliferative disease is an ALPP / L2 positive 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,26092bladder cancer, head and neck cancer, cervical cancer, breast cancer, esophageal cancer, cholangiocarcinoma, prostate cancer, and a colon cancer.

[0030] 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 an ALPP / L2 binder 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.

[0031] 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.

[0032] Further provided is a nucleic acid molecule encoding an ALPP / L2 binder disclosed herein.

[0033] Further provided is an expression vector comprising one or more of nucleic acid molecules encoding an ALPP / L2 binder disclosed herein.

[0034] Further provided is a host cell comprising a nucleic acid molecule encoding an ALPP / L2 binder disclosed herein or an expression vector encoding an ALPP / L2 binder disclosed herein.

[0035] Further provided is a method for producing an ALPP / L2 binder comprising (a) providing the host cell comprising a nucleic acid molecule encoding an ALPP / L2 binder disclosed herein or an expression vector encoding an ALPP / L2 binder disclosed herein; (b) cultivating the host cell in a medium under conditions suitable for expressing the ALPP / L2 binder; and (c) isolating the ALPP / L2 binder from the medium.

[0036] Further provided is an ALPP / L2 binder disclosed herein conjugated to a detectable moiety. In a further embodiment, the detectable moiety is detectable by magnetic resonance imaging (MRI) or by X-ray imaging. 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 ALPP / L2 binder disclosed herein conjugated to a detectable moiety and detecting the cells in the individual that bind the ALPP / L2 binder.26092

[0037] The present invention further provides a composition comprising an ALPP / L2 binder 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.

[0038] The present invention further provides a kit comprising an ALPP / L2 binder disclosed herein or the pharmaceutical composition thereof and a hyaluronan degrading enzyme. In certain embodiments of the kit, the ALPP / L2 binder and the hyaluronan degrading enzyme are provided in separate containers or the ALPP / L2 binder and the hyaluronan degrading enzyme are provided as a mixture in a single container. In certain embodiments of the kit, the ALPP / L2 binder and the hyaluronan degrading enzyme are provided in separate chambers of a dual-chamber injection device or the ALPP / L2 binder and the hyaluronan degrading enzy me 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.

[0039] 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 ALPP / L2 binder disclosed herein or the pharmaceutical composition thereof and a hyaluronan degrading enz me 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 pH200. In certain embodiments of the method, the soluble hyaluronidase is hyaluronidase (recombinant human) or berahyaluronidase alfa. In certain embodiments of the method, the ALPP / L2 binder and the hyaluronan degrading enzy me are administered to the individual sequentially or simultaneously. In certain embodiments of the26092method, the ALPP / L2 binder 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 ALPP / L2 binder and the hyaluronan degrading enzy me are administered to the individually systemically. In certain embodiments of the method, the ALPP / L2 binder and the hyaluronan degrading enzyme are administered to the individually subcutaneously or intramuscularly.

[0040] 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 an ALPP / L2 binder disclosed herein or pharmaceutical composition thereof, (b) a second therapeutic agent, and (c) a hyaluronan degrading enz me, 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 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 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 ALPP / L2 binder and the hyaluronan degrading enzyme are administered to the individual sequentially or simultaneously. In certain embodiments of the combination therapy, the ALPP / L2 binder 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 ALPP / L2 binder and the hyaluronan degrading enzy me are administered to the individually subcutaneously or intramuscularly.

[0041] 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.26092BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Fig. 1A shows a dendrogram of the ALPP / L2 binder binning results shown in Table 9 of Example 5. The ALPP / L2 binder clones associated with the sample ID numbers shown in the figure are shown in Table 9.

[0043] Fig. 1B shows a community plot summarizing the ALPP / L2 binder binning results shown in Table 9 of Example 5. The ALPP / L2 binder clones associated with the sample ID numbers shown in the figure are shown in Table 9.

[0044] 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 International Patent Application Publication No. WO2022197890.

[0045] 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.DETAILED DESCRIPTION

[0046] The present invention provides improved ALPP / L2 binders, which may be derived from antibodies or antigen-binding fragments thereof that bind ALPP and / or ALPPL2. The ALPP / L2 binders may comprise a variety of embodiments, e.g., a bivalent whole antibody comprising two identical anti-ALPP / L2 VH / VL pairs or Fabs, a monovalent antibody comprising a single anti-ALPP / L2 VH / VL pair or Fab or single-chain variable fragment (scFv), a bispecific antibody comprising an anti-ALPP / L2 Fab or scFv and a Fab or scFv directed to another target, an antibody-drug conjugate comprising an anti-ALPP / L2 bivalent or monovalent antibody conjugated to a therapeutic agent, or a chimeric antigen receptor (CAR) for CAR-based T-cell therapy comprising an anti-ALPP / L2 scFv. The ALPP / L2 binder of any one of the embodiments disclosed herein may be used to target tumor cells in a variety of ways. The ALPP / L2 binder of any one of the embodiments disclosed herein are also useful for detection of tumor cells and may be used for example in a companion diagnostic for detecting ALPP and / or ALPPL2. The ALPP / L2 binder of any one of the embodiments disclosed herein bind to cells that express or overexpress ALPP and / or ALPPL2 while having no detectable binding to cells that express or overexpress ALPP or ALPI. Exemplary non-limiting cells expressing ALPP and / or ALPPL2 on the cell surface include but are not limited to, mesothelioma cells, testicular cancer cells,26092endometrial cancer cells, pancreatic cancer cells, ovarian cancer cells, non-small cell lung cancer (NSLC) cells, gastric cancer cells, and colon cancer cells.

[0047] The ALPP / L2 binders may be used in a monotherapy for the treatment of any proliferative disease in which ALPP and / or ALPPL2 is expressed on the cell surface, in particular, the treatment of a cancer or malignancy that displays ALPP and / or ALPPL2 on the surface of the cells comprising the 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 colon cancer.

[0048] The ALPP / L2 binders may be used in a combination therapy with a chemotherapy agent 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 that display ALPP and / or ALPPL2 on the surface of the cells comprising the 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 colon cancer.

[0049] The ALPP / L2 binders may be used in a combination therapy with another or second therapeutic antibody that binds a target other than ALPP and / or ALPPL2 for the treatment of any proliferative disease in which ALPP and / or ALPPL2 is expressed on the cell surface, in particular, the treatment of a cancer or malignancy that express ALPP and / or ALPPL2 on the surface of the cells comprising the 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 colon cancer.Definitions

[0050] 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.

[0051] 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.26092

[0052] 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.

[0053] 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).

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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,”26092unprocessed 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.

[0058] 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.

[0059] 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 vanants, 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.

[0060] As used herein, the terms "overexpress" and “highly express” may be used interchangeably and means to transcribe and translate more genetic product than normal (such as in normal cells), the process of which is often a characteristic of cancer or malignant cells.

[0061] 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 with 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.

[0062] 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 between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented26092by 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.

[0063] 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 ALPP / L2 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.

[0064] 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 Gln QGlutamic acid Glu EGlycine Gly GHistidine His HIsoleucine Ile ILeucine Leu LLysine Lys KMethionine Met MPhenylalanine Phe FProline Pro PSerine Ser SThreonine Thr TTryptophan Trp WTyrosine Tyr Y26092Amino acid Three letter One lettercode codeValine Val V

[0065] 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.

[0066] 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-CDR2, 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-FR4.26092Numbering of the amino acids in a VH and VL may be determined using the Kabat numbering scheme. See Beranger 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 at www.imgt.org / IMGTScientificChart / Numbering / Hu IGHGnber.html).

[0067] 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 Beranger et al., op. cit.

[0068] 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)).

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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).26092

[0073] 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)).

[0074] 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)).

[0075] 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 CysResidue after Usually a Trp. Typically, Trp-Tyr-Gln. but also, Trp- Leu-Gln, Trp-Phe-Gln, or Trp-Tyr-LeuLength 10 to 17 amino acid residuesVL-CDR2Start Usually 16 amino acid residues after the end of VL- CDR1Residues before Generally, lle-Tyr, but also, Val-Tvr. lle-Lys, or Ile-Phe Length Usually seven amino acid residuesVL-CDR3Start Usually 33 amino acid residues after end of VL-CDR2 Residue before Usually CysResidues after Usually Phe-Glv-Xaa-GlyLength Seven to 11 amino acid residuesVH-CDR1Start About amino acid residue 26 (usually four amino acid residues after a Cys) [Chothia / AbM definition]; Kabat definition starts five amino acid residues laterResidues before Usually Cys-Xaa-Xaa-XaaResidues after Usually a Trp. Typically Trp-Val, but also, Trp-Ile orTrp- AlaLength 10 to 12 amino acid residues [AbM definition]; Chothiadefinition excludes the last four amino acid residues26092VH-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: 427), 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 Cys-Xaa-Xaa (typically Cys-Ala-Arg) Residues after Usually Trp-Glv-Xaa-GlyLength Three to 25 amino acid residuesTable 2VL-CDR Kabat AbM Chothia1Contact2IMGT 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 Chothia1Contact2IMGT LoopHl H31-H35B H26- H26- H30- H26- (Kabat H35B H32.34 H35B H35B Numbering)3Hl H31-H35 H26-H35 H26- H30- H26- (Chothia H32 H35 H33 Numbering)H2 H50-H65 H50-H58 H52- H47- H51 —H56 H58 H56 H3 H95-H102 H95— H95— H93- H93—H102 H102 H101 H1021Some of these numbering schemes (particularly for Chothia loops) vary depending on the individual publication examined.2 Any of the numbering schemes can be used for these CDR definitions, except the Contact numbering scheme uses the Chothia or Martin (Enhanced Chothia) definition.^The end of the Chothia VH-CDR1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop. (This is because the Kabat numbering scheme places the insertions at H35A and H35B.)If neither 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

[0076] 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 speci ficity. 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).

[0077] 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.

[0078] 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 Eu 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.

[0079] 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 domain amino acid sequence has 107 amino acids, which may be sequentially numbered 1 to 107. The corresponding sequence numbered according to Eu begins with position number 108 and endswith position number 214. Unless specified otherwise, the amino acid positions in the heavy and light chains herein are defined according to sequential numbering.

[0080] 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)).

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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 Fab1fragments to form a F(ab’)2 molecule.

[0085] 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. An F(ab')2 fragment thus is composed of two Fab' fragments that are held together by a disulfidebond between the two HCs. An " F(ab')2 fragment" can be the product of pepsin cleavage of an antibody.

[0086] 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. The VH and VL chains of the Fv fragments are held together by non-covalent interactions.

[0087] 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. This protein retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker.

[0088] As used herein, the term “scFab” refers to a single-chain Fab in which the N-terminus of the VH is covalently linked to the C-terminus of the VL by a short linker peptide of ten to about 25 amino acids or the N-terminus of the VL is covalently linked to the C-terminus of the VH by a short linker peptide of ten to about 25 amino acids.

[0089] As used herein, the term "diabody" refers to an antigen binder comprising a small antibody fragment with two antigen-binding regions, which fragments comprise a VH connected to a VL in the same polypeptide chain (VH-VL or VL-VH). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementarity domains of another chain and create two antigen-binding regions. Diabodies are described more fully in, e.g., EP 404,097; WO 93 / 11161; and Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448. For a review of engineered antibody variants generally see Holliger and Hudson (2005) Nat. Biotechnol. 23:1126-1136.

[0090] 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 uti 1 ity 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.

[0091] 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 as26092biological 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.

[0092] 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 be made 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. Patent 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).

[0093] As used herein, the term "chimeric antigen receptor" (CAR) refers to a recombinant polypeptide comprising at least an extracellular domain that binds specifically to an antigen or a target, a transmembrane domain and an intracellular T cell receptor-activating signaling domain. Engagement of the extracellular domain of the CAR with the target antigen on the surface of a target cell results in clustering of the CAR and delivers an activation stimulus to the CAR-containing cell. CARs redirect the specificity of immune effector cells and trigger proliferation, cytokine production, phagocytosis and / or production of molecules that can mediate cell death of the target antigen-expressing cell in a maj or histocompatibility (MHC)-independent manner.

[0094] As used herein, the term "extracellular antigen binding domain," "extracellular domain." or "extracellular ligand binding domain" when used in reference to a CAR refers to the part of a CAR that is located outside of the cell membrane and is capable of binding to an antigen, target or ligand.

[0095] As used herein, the term "hinge region" when used in reference to a CAR refers to the part of a CAR that connects two adjacent domains of the CAR protein, e.g., the extracellular domain and the transmembrane domain.26092

[0096] As used herein, the term "transmembrane domain" refers to the portion of a CAR that extends across the cell membrane and anchors the CAR to cell membrane.

[0097] As used herein, the term "intracellular T cell receptor-activating signaling domain", "cytoplasmic signaling domain," or "intracellular signaling domain" refers to the part of a CAR that is located inside of the cell membrane and is capable of transducing an effector signal.

[0098] As used herein, the term "immune cell" or "immune effector cell" refers to a cell that is involved in an immune response, e.g., in the promotion of an immune effector response.Examples of immune cells include T cells, B cells, natural killer (NK) cells, mast cells, and myeloid-derived phagocytes. According to particular embodiments, the engineered immune cells are T cells, and are referred to as CAR-T cells because they are engineered to express CARs of the invention. According to particular embodiments, the engineered immune cells are NK cells, and are referred to as CAR-NK cells because they are engineered to express CARs of the invention.

[0099] As used herein, the term "engineered immune cell" refers to an immune cell, also referred to as an immune effector cell, that has been genetically modified by the addition of extra genetic material in the form of DNA or RNA to the total genetic material of the cell. According to embodiments herein, the engineered immune cells have been genetically modified to express a human ALPP / ALPPL2-targeting CAR according to the invention.

[0100] As used herein, the term "stimulator}’ molecule" refers to a molecule expressed by a T cell that provides the primary cytoplasmic signaling sequence(s) that regulate primary activation of the T cell receptor (TCR) complex in a stimulatory way for at least some aspect of the T cell signaling pathway. Stimulatory molecules comprise two distinct classes of cytoplasmic signaling sequence, those that initiate antigen-dependent primary activation (referred to as "primary signaling domains"), and those that act in an antigen-independent manner to provide a secondary of co-stimulatory signal (referred to as "co-stimulatory signaling domains").

[0101] As used herein, the term "immune cell" or "immune effector cell" refers to a cell that is involved in an immune response, e.g., in the promotion of an immune effector response.Examples of immune cells include T cells, B cells, natural killer (NK) cells, mast cells, and myeloid-derived phagocytes. According to particular embodiments, the engineered immune cells are T cells, and are referred to as CAR-T cells because they are engineered to express CARs comprising an ScFv disclosed herein that targets human ALPP and / or ALPPL2; or the engineered immune cells are NK cells and are referred to as CAR-NK cells because they are engineered to express CARs comprising an ScFv disclosed herein that targets human ALPP and / or ALPPL2.26092

[0102] 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 know n 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.

[0103] As used herein, the term “germline” or "germline sequence" refers to a sequence of unrearranged immunoglobulin DNA sequences. Any suitable source of unrearranged immunoglobulin 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).

[0104] 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.

[0105] 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.26092

[0106] 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. Patent 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 0-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.

[0107] 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.

[0108] As used herein, the terms "cell," "cell line," and "cell culture" are used interchangeably and all such designations include progeny. Thus, the yvords "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 function26092or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.

[0109] 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.

[0110] 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; or a 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.

[0111] 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.

[0112] As used herein, the term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence.26092

[0113] 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 ty pically 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.

[0114] As used herein, the term "treatment," as it applies to a human or veterinary individual, refers to therapeutic treatment, as well as diagnostic applications. " Treatment" as it applies to a human or veterinary individual, encompasses contact of the ALPP / L2 binders of the present invention to a human or animal subject.

[0115] 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.

[0116] 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 an ALPP / L2 binder 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.

[0117] 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.26092ALPP / L2 Binders

[0118] The ALPP / L2 binders disclosed herein comprise (i) a heavy chain variable domain (VH) comprising VH complementarity determining regions (VH-CDRs) 1, 2, and 3; and (ii) alight chain variable domain (VL) comprising VL complementarity determining regions (VL-CDRs) 1, 2, and 3, wherein 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.Tabic 3SEQID SEQIDVHNO:VLNO: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 1 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 AVI YDGSNKYYADSVKG 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 8626092CDR2 FIWYDGSKKYYADSVKG 84 DASSLES 87 CDR3 ENNWNGYYNFYYMDV 85 RQQFNNYYT 88 CDR1 SYGMH 91 GASQSVSNNYLA 94 CDR2 VIWYDGSNKYYADSVKG 92 DASS RAT 95 CDR3 ENNWNGYYYYYYMDV 93 QQYGSSLYT 96 CDR1 PYAMH 99 GASQSISGSYLA 102 CDR2 VIWYDTINKYYADSVKG 100 DASS RAT 103 CDR3 ENNWNGYYSYYYMDV 101 QHYENSLYT 104 CDR1 SYGMH 107 RASQDISRVLA HO CDR2 VIWYDGSNKYYVDSVKG 108 DASSLES 111 CDR3 ENNWNNYYHFYYMDV 109 QQFNNYMYT 112 CDR1 SYYWT 115 RASQSISNNYLS 118 CDR2 YIYYSGSANYNPSLKG 116 GAS RAT 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 I IWYDGTNKYYADSVKG 180 DASSLES 183 CDR3 ENNWNGYYHFYYMDV 181 QQFNNYLYT 184 CDR1 DLSMY 187 RASQGISNYLA 190 CDR2 GFDPEAGETIYAQKFQG 188 DASSLQS 191 CDR3 GRYCFSTSCSFNYNYYMDV 189 QQYNSFPPT 19226092CDR1 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 208 CDR1 SYAMS 211 RASQSISSYLN 214 CDR2 AISGSGGSSKNGNSVKG 212 DASSLQS 215 CDR3 GTVVPVVPGDHMDV 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 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 GASS RAT 295 CDR3 DPLPYNWSFYFYYMDV 293 QQYGSSPLT 296 CDR1 SYWMT 299 RASQGIRNDLG 302 CDR2 NIKQDGSEKHYVDSVKG 300 AASTLQS 303 CDR3 DTSNYDLYYYYFYMDV 301 LQDNSYPYT 30426092CDR1 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 320 CDR1 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 DGGVPWPYLYYYYMDV 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 EVVYYYYMDV 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

[0119] In a further embodiment, the ALPP / L2 binder may comprise any one of the 46 combinations of VH / VL amino acid sequence pairs shown in Table 4. 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 4SEQID SEQIDC|°neVHNg. VLNQ. 24F4 EVQLLESGGGLVQPGGSL 1 KIVMTQSPATLSLSPGERA 2RLSCAASGFTFSSYAMSW TLSCRASQSVSSSYLSWYQ26092VRQAPGKGLEWVSAISIS QKPGQAPRLLMYGTSTRAT GT YT YYAD SVKGR F T I S R DIPARFSGSGSGTDFTLTI DNSENTLFLQMNSLRAED S S L Q P E D FAVY Y CHQD YNF TAVYYCAKAHSTYPYYYY PLTFGGGTKVEIK YYMDVWGKGT TVTVS S10B3 EVQLLESGGGLVQPGGSL 9 EIVLTQSPGTLSLSPGERA 10RLSCAASGFTFSIYTMKW TL S CRASQSVSNS YLAWYQ VRQAPGKGL DWVS Al SAS QKPGQAPRLI IYGASSRAA GGSTSYSDSLKGRFTISR GIPDRFSGSGSGTDFTLTI DNSKNTVNLQMNSLRTED SRLEPEDFAIYHCQQYGRS SAVYYCAKDPLPYNWNFF PLTFGGGTKVEIK YYYMDVWGKGT TVTVS S9G10 EVQLLESGGGLVQPGGSL 17 EIVLTQSPATLSLSPGERA 18RL S CAAS VS T FNNYAMS W TL S CRASQSVS S YLAW YQQ VRQAPGKGLEWVSAISGS KPGQAPRLLIYGASNRATG GGS T YYAD SVKGR F T I S R IPARFSGSGSGTDFTLTIS DNSKNTLYLQMNSLRAED S L E P E D FAVY Y CQQRSNWP TAVYYCAKDPLPANWNYY LTFGGGTKVEIK YYMDV GKGT TVTVS S8A7 EVQLMDSGGDLVQPGGSL 25 AIQMTQSPSSLSASVGDRV 26RLSCAASGFTFSSYWMSW TIT CRASQGIRNDLGW YQQ VRQAPGKGLEWVANINQD KPGKAPKLLIYAASSLQSG GSEKNYVD SVQGR F T I S R VPSRFSGSGSGTDFTLTIS DNAKKSLYLQMNSLRAED S LQ P E D FAT Y YCLQDYNYP TAVYYCARDYSNYDDYYY WTFGQGTKVEIK YFYMDVWGKGT TVTVS S16H2 EVQLVESGGGLVQPGGSL 33 AIQMTQSPSSLSASVGDRV 34RLSCAASGFTFSSYWMTW TIACRASQGIRDDLAWFQQ VRQSPGKGLEWVANIKQD KPGKAPKVLIYAASNLQSG GSEENYVD SVKGR F T I S R VPSRFSGSGSGTDFTLTIR DN AKN S L Y L QMN S L RAD D SLQPEDSATYYCLQDYIYP TAVYYCARDTVTTGFYHY YTFGQGTRLEIK FYMDV GKGT TVTVS S11C7 QVQLVESGGGWQPGRSL 41 AIQLTQSPSSLSASVGDRV 42RLSCAASGLTFSSYGMHW TIT CRASQGI SNALAW YQQ VRQAPGKGLEWVAVIWYD KPGKAPKLLIYDASSLESG GSNKY YAD SVKGR F T I S R VPSRFSGSGSGTDFTLTIS DNSKNTLYLQMNSLRAED SLQPEDFATYYCQQFNNYI TAVYYCARENNWHLNYFF YTFGQGTKLEIK YYMDV GKGT TVTVS S5A1 QVQLVESGGGWQPGRSL 49 AIQLTQSPSSLSASVGDRV 50RLSCAASGFTFSSYGMHW TIT CRASQDINNVLAW YQQ VRQAPGKGLEWVAVIWYD KPGKAPKLLIYDASSLESG GSNKY YAD SVKGR F T I S R VPLRFRGSGSGTDFTLTIN DNSKNTLFLQMNSLRAED SLQPEDFANYYCQHFNDFI TAVYYCARENNWHLNYYF YTFGQGTKLEIK YYMDVWGKGT TVTVS S8G6 QVQLVESGGGWQPGRSL 57 AIQLTQSPSSLSASVGDRV 58RLSCAASGLTFSSYGMHW TIT CRASQGI S SALAW YQQ26092VRQAPGKGLEWVAVIWYD KPGKAPKLLIYDASSLEGG GSNKY YAD S VKGR F T I S R VPSRFSGSGSGTDFTLTIR DNSKNTLYLQMNSLRAED SLQPEDFATYYCQQFNNDL TAVYYCARENNWNGFYNF YTFGQGTKLEIK FYMDVWGKGT TVTVS S50F5 EVQLVESGGGLVQPGGSL 65 AIQMTQSPSSLSASVGDRV 66RLSCAASGFTFSRYWMSW TIT CRASQGIRNDLGW YQQ VRQAPGKGLEWVANIKQD KPGKAPNLLIYAASSLQSG GSEKNYVDTVKGRFTISR VPSRFSGSGSGTDFTLTIS DN AKN SLYLLMNSLRAED S LQ P E D FAT Y YCLQDYNYP TAVYYCARDYDFWNGYYL YTFGQGTKLEIK YYSMDVWGT GT TVTVS S5E8 QVQLVESGGGWQPGRSL 73 EIVLTQSPATLSLSPGERA 74RLSCSASGFTFSSYGMHW TLSCGASQSVSSSYLAWYQ VRQAPGKGLEWVAVIWYD QKPGLAPRLLIYDASSRAS GSNKY YAD S VKGR F T I S R GIPDRFRGSGSGTDFTLTI DNSKNTLYLQMNSLSADD S RL E P E D FAVY Y CQQYGS S TALYYCARENNWNGYYHY LYTFGQGTKLQIK YYMDVWGKGT TVTVS S14A11 QVQLVESGGGWQPGRSL 81 AIQLTQSPSSLSASVGDRV 82RLSCAASGFTFSSYGMHW TIT CRASQGIRSALAW YQQ VRQAPGKGLEWVAFIWYD KPGKAPTLLIYDASSLESG GSKKYYAD SVKGR F T I S R VPRRFSGSGSGTDFTLTIS DNSKNTLYLQMNSLRVED SLQPEDFAIYYCQQFNNYY TAVYYCARENNWNGYYNF TFGQGTKLEIK YYMDVWGKGTTVIVSS19A3 QVQLVESGGGWQPGRSL 89 EIVLTQSPATLSLSPGERA 90RLSCAASGFTFSSYGMHW TL S CGASQSVSNNYLAWYQ VRQAPGKGLEWVAVIWYD QKPGLAPRLLIYDASSRAT GSNKY YAD SVKGR F T I S R GIPVRFSGSGSGTDFTLSI DNSKNTLDLQMNSLRAED S RL E P E D FAVY Y CQQYGS S TAVYYCARENNWNGYYYY LYTFGQGTKLEIK YYMDV GKGT TVTVS S15B6 QVQLVESGGGWQPGRSL 97 EIVLTQSPATLSLSPGERA 98RL S C AA S G F T F S P YAMH W TLSCGASQSISGSYLAWYQ VRQAPGKGLEWVAVIWYD QKPGLAPRLLIYDASSRAT T INKY YAD SVKGR F T I S R GTPVRFSGSGSGTDFTLTI DNSKNTLFLQMNSLRAED S RL E P E D FAVY Y CQHYENS TAMYYCARENNWNGYYSY LYTFGQGTKLEIR YYMDV GKGT KVT VS S6E6 QVQLVESGGGWQPGRSL 105 AIQLTQSPSSLSASVGDRV 106RLSCAASGFTFSSYGMHW TIT CRASQDI SRVLAW YQQ VRQAPGKGLEWVAVIWYD KPGKAPKLLIYDASSLESG GSNKY YVD SVKGR F T I S R VPRRFSGRGSGTEFTLTIS DNSKNTLYLQMNSLRAED NLQ P E D FAT Y FCQQFNNYM TAVYYCARENNWNNYYHF YTFGQGTKLEIK YYMDVWGKGTAVTVS S17H6 QVQLQESGPGLVKPSETL 113 EIVMTQSPATLSLSPGERA 114SLTCTVSGGS I ISYYWTW TL S CRASQS I SNNYLS WYQ26092IRQPPGKGLEWIGYIYYS QIYGQAPRLLIYGASTRAT GSANYNPSLKGRVT T S VD GIPARFSGSGSGTDFTLTI TSKSQFSLKLSSVTAADT SSLQPEDFAVYYCQQDFNL AVY Y CARGRRGYS GNGD Y PLTFGGGTKVEIK YYYMDVWGKGT TVTVS S10F6 EVQLLESGGGVVQPGGSL 121 DIQMTQSPSSLSASIGDTV 122RLSCVVSGFTFSNFAMGW TIT CRASQDIRYDLG YQQ VRQAPGRGLEWVARISSS KPGKAPKRLIYGASSLHSG GRDTFYTDSVKGRFSISR VPSRFSGSRSGTEFTLTIS DNSNNTLYLQLSSLRAED S LQ P E D FAT Y YCLQLNNFP TAVYYCASLGPVWGNGTT YTFGQGTKLEIR VTVSS4F4 EVQLVESGGGLVQPGGSL 129 AIQMTQSPSSLSASVGDRV 130RLSCVASGLTISGYWMTW TIT CRASQNIRNDLG YQQ VRQAPGKGLEWVANINQD KPGKAPKLLIYAASSLQSG GNEKNYVDSVKGRFTISR VPSRFSGSGSGTDFTLTIS DN AKN S L Y L QMN S L RAE D S LQ P E D FAT Y YCLQDYNYP TAIYYCVKDTSNYDFYYY YTFGQGTKLEIK YFYMDVWGKGT TVTVS S4B10 EVQLLESGGGLVQPGGSL 137 EIVLTQSPATLSLSPGERA 138RLSCAASGITISSYAMNW TLSCRASQSVSTYLVWYQQ VRQAPGKGLEWVSAISGS KPGQAPRLLIYDASNRATG GGS T YYAD SVKGR F T I S R IPARFSGSGSGTDFTLTIS DNSKNTLYLQMNSLRAED SLOPED F AGY Y CQQRSNWP TAVYYCAKDPLPANWNYF LTFGGGTKVEIK YYMDVWGKGT TVTVS S49G5 QVQLQESGPGLVKPSETL 145 EIVMTQSPATLSLSPGERA 146SLTCTVSGGS I SSYYWSW TLSCRASQSISSSYLSWYQ IRQPAGKGLEWIGRIYSS QTPGQAPRLLIYGVSSRAT GSTNYNPSLKSRVTMSED GIPARFSGSGSGTDFTLTI TSKNQISLNLSSVTAADT S S L Q P E D FAVY Y CQQD YNL AVYYCARFSMVRGVMGYM PYTFGQGTKLEIE DVWGKGTTVTVSS7C8 QVQLVESGGGWQPGRSL 153 AIQLTQSPSSLSASVGDRV 154RL S CEAS GL T F SVYGMHW TIIC RASQGI S SALAW YQQ VRQAPGKGLEWVAVIWYD APGKAPKLLIYDASRLESG GTNKY YAD SVKGR F T I S R VPLRFSGSGSGTDFTLTIS DNSKNTLYLQMNSLRAED SLQPEDFATYYCQQFNNNL TAVYHCARENNWNAYYHF YTFGQGTKLEIK HYMDV GKGTS VTVSS9B12 EVQLVESGGGLVQPGGSL 161 AIQMTQSPSSLSASVGDRV 162RLACAASGFTISSYWMSW TIT CRTSQGIRNDLG YQQ VRQAPGKGLEWVANINQD KPGKAPKLLIYAASNLQSG GSEKYYVD SVKGR F T I S R VPSRFSGSGSGTDFALTIS DNAKNSLSLQMNSLRAED SLQPEDFATYYCLQDSNYP TAVYYCARDASNYDGYYY YTFGQGTKLEIK YFYMDVWGKGT TVTVS S16B5 QVQLQESGPGLVKPSETL 169 EILMTQSPATLSLSPGERA 170SLTCTVSGGS I ISYYWTW TLSCRASQSISDSYLSWYQ26092IRQPPGKGLEWIGYIFYS Q I P GQAP RLL I YGASTRAT GSTNYNPSLKGRVTLSVD GVPARFSGSGSGTDFTLTI TSKNQFSLKLSSVTAADT SSLQPEDFAVYYCQQDFNL AVYYCARGRRGYRGNGDY PLTFGGGTKVEIK YYYMDVWGKGT TVTVS S3G8 QVQLVESGGGWQPGRSL 177 AIQLTQSPSSLSASVGDRV 178R I S CAAS GL T F S S YGMHW TIT CRASQGI S SALA YQQ VRQAPGKGLEWVAIIWYD KPGKVPKLLIYDASSLESR GTNKY YAD S VKGR F T I S R VPSRFSGSGSGTDFTLTIS DNSKNTLYLQMNSLRAED SLQPEDFATYYCQQFNNYL TAVYYCARENNWNGYYHF YTFGQGTKLEIK YYMDVWGKGT TVTVS S17E4 QVQLVQSGAEVKKPGASV 185 DIQMTQSPSSLSASVGDRV 186KVSCKVSEYTLTDLSMYW TIT CRASQGI SNYLAW FQQ VRQAPGKGLEWMGGFDPE KPGKAPKSLIYDASSLQSG AGET I YAQKFQGRVTMT E VPSKFTGSGSGTDFTFTIS DTSTDTAYMELSSLRSED SLQPEDFATYYCQQYNSFP TAVYYCAIGRYCFSTSCS PTFGQGTKVEIK FNYNYYMDVWGKGTTVTV SS25 G8 EVQLLESGGGLVQPGGSL 193 EIVLTQSPGTLSLSPGERA 194RLSCAASGFIFSSYTMKW TL S CRASQSVS S S YLAWYH VRQAPGKGLEWVSAISGS QKPGQAPRLLIYGASSRAT GGS T YYAD SVKGR F T I S R GIPDRFSGSGSGTDFTLTI DNSKNTLYLQMNSLRAED S RL E P E D FAVY Y CQQYGS S TAVYYCAKDPLPYNWSFY PLTFGGGTKVEIK YYYMDVWGKGT TVTVS S16C2 QVQLVQSGAEVKKPGASV 201 EIVMTQSPATLSVSPGERA 202KVSCKASGFTLTTYGFNW TLSCRASQSVGSFLAWYQQ VRQAPGQGLEWMGWISAY KPGQAPRLLIYGASTRATG NGDTRYAQKFQGRVTMT T IPARFSGSGSGTEFTLTIS DTSTSTAYMELWSLRSDD S LQ S E D FAVY YCQQYINWP TAVYYCARGTTVTTPYYY LTFGGGTKVEIK YSYMDVLGKGTTVTVSS21H7 EVQLLESGGGLVQPGGSL 209 DIQMTQSPSSLSASVGDRV 210RLSCAASGFTFSSYAMSW TITCRASQSISSYLNWYQQ VRQAPGKGLEWVAAISGS KPGKAPRLLIYDASSLQSG GGS SKNGN SVKGR F T I S R VPSRFSGRGSGTDFTLTIS DNSKNTLYLLMSILRAED SLQPEDFATYYCQQSYSTP TAI Y YCAKGTVVPVVPGD PTFGQGTKVEIK HMDVWGKGT TVTVS S32H6 EVQLLESGGGLVQPGGSL 217 DIVMTQTPLSLSVTPGQPA 218RLSCAASGFTFGNYAMSW SIS CKSSQSLLHSDGKTYL VRQAPGRGLEWVSGVRNS YWYLQKPGQPPQLLIYEVS GS GT YYAD SVKGR F T I S R NRFSGVPDRFSGSGSGTDF DNSKNTLYLQMNSLRAED T L K I S RVE AE D VGVY Y CMQ TAVYYCAKGGTPVTAPYY SIQLPYTFGQGTKLEIK YYYYMDVWGKGTSVAVSS260922A2 EVQLVESGGGLVQPGGSL 225 AIQMTQSPSSLSASVGDRV 226RLSCAASGFTISPYWMTW T I S CRASQGIRDDLG YQQ VRQAPGKGLEWVANINQD KPGKAPELLIYAASSLQSG GSEKHYVD SVKGR F T I S R VPSRFSGSGSGTDFTLTIS DNVHNSLFLQMNSLRAED S LQ P E D FAT Y YCLQDYTYP TAVYYCVRDDSVYDSYYY YTFGQGTKLEIK YFYMDVWGKGT TVTVS S12C6 QVQLVQSGPEVKKPGASV 233 EIVMTQSPATLSLSPGERA 234KVSCKVSGYTLTELSMHW TLSCRASQSVSSTSFSWYQ VRQAPGKGLEWMGGFDPE QKPGQAPRLLIFGASTRAT DGGT I FAQKFQGRVTMT E GIPARFSGSGSGTDFTLTI DTSTDTAYMELSSLRSED S S L Q P E D FAVY Y CHQD YNL TAVYYCAGWGSYYRWFDP PFTFGPGTKVDIK WGQGTLVTVSS12F1 EVQLVESGGGLVQPGGSL 241 AIQMTQSPSSLSASVGDRV 242RLSCAASGFTFSSYWMSW T I T CRASQGIRNDLA YQQ VRQAPGKGLEWVANINQD KPGKAPNLLIYAASSLQSG GNEKNYVD SVKGR F T I S R VPSRFSGSGSGTDFTLTIS DN VKN S LH L QMN S L RAE D SLQPEDFATYYCLQDYIYP SAVYFCARDTSNYDLYSY YTFGQGTKLEIK YFYMDVWGKGT TVTVS S16F2 QVQLVESGGGWQPGRSL 249 AIQLTQSPSSLSASVGDRV 250RLSCAASGLTFSSYGMHW TIT CRASQGI SNTLA YQQ VRQAPGMGLEWVALIWYD KPGKPPKLLIYDASRLEGG GSNEYYADSVKGRFTISR VPLRFSGSGSGTDFTLTIS DNFKNTLYLQMNSLRAED SLQPEDFATYYCQQFNNYP TAVYYCARENNWNGRYYF TFGGGTKVEIK YYMDV GKGT TVTVS S14B12 EVQLLESGGGLVQPGGSL 257 EIVLTQSPGTLSLSPGERA 258RLSCAASGFIFSSYTMKW TL S CRASQSVS S S YLAWYH VRQAPGKGLEWVSAISGS QKPGQAPRLLIYGASSRAT GGS T YYAD SVKGR F T I S R GIPDRFSGSGSGTDFTLTI DNSKNTLYLHVNSLRAED S RL E P E D FAVY Y CQQYGS S TAVYYCAKDPLPYNWNFY PLTFGGGTKVEIK YYYMDVWGKGT TVTVS S10G2 EVQLLESGGGLVQPGGSL 265 AIQLTQSPSSLSASVGDRV 266RLSCAASGFTFSSYALSW TIT CRASQGI S SALA YQQ VRQTPGKGLEWVSVISGN KPGKTPKLLIYDASSLESR GIITYYADSVKGRFTISR VPSRFSGSGSGTDFTLTIS DNSKNTLYLQMNSLRAED SLQPEDFATYYCQQFNNYP TAVYYCAKDDSSYYGLGS YSFGQGTKLEIK FPNWFDPWGQGTLVTVSS6E10 EGQLLESGGGLVQPGGSL 273 EIVMTQSPATLSVSPGERA 274RLSCAASGFTFNNYAMSW TL S CRASQSVNNNLA YQQ VRQAPGKGLEWVSAISGS KPGQAPRLLIFGASTRATG GGS T YYAD SVKGR F T I S R LPARFSGSGSGTEFTLTIS DNSKNTLYLQMNSLRAED S LQ S E D FAVY YCQQYNNWP TAVYYCAKDGGVPVVPYL FTFGQGTKLEIK YYYYMDVWGKGTTVTVS S2609210A9 EVQLLESGGGLVQPGGSL 281 EIVMTQSPATLSLSPGERA 282RLSCAASGFTFSIYTMKW TL S CRASQSVS S S YLS WYQ VRQAPGKGL E WVS Al SAS QKPGQAPRLLIYGASTRAT GGSTSYSDSVKGRFTISR GIPARFSGSGSGTDFTLTI DNSKNTVNLQMNSLRTED S S L Q P E D FAVY Y CQQD YNL SAVYYCAKDPLPYNWSFF ITFGQGTRLEIK YYYMDVWGKGT TVTVS S35D5 EVQLLESGGGLVQSGGSL 289 EIVLTQSPGTLSLSPGERA 290RL S CVAS GF I FNT YVMKW TL S CRASQSVS S SYLAWYQ VRQAPGRGLEWVSAISGS QKPGQAPRLLIYGASSRAT GGS T S YTD SVKGR F T VS R GIPDRFSGSGSGTDFTLTI DNSKNTLYLQMTSLRAED S RL E P E D FAVY Y CQQYGS S TAVYYCAKDPLPYNWSFY PLTFGGGTKVELK FYYMDVWGKGT TVTVS S17A7 EVQLVESGGGLVQPGGSL 297 AIQMTQSPSSLSASVGDRV 298RLSCAASGFTFSSYWMTW TIT CRASQGIRNDLG YQQ VRQAPGKGLEWVANIKQD KPGKAPKLLIYAASTLQSG GSEKHYVD SVKGR F T I S R VPSRFSGSGSGTDFTLTIS DN AKN S L Y L QMN S L RAE D S LQ P E D FAT Y YCLQDNSYP TAVYYCARDTSNYDLYYY YTFGQGTKLEIK YFYMDVWGKGT TVTVS S18A11 EVQLVESGGGLVQPGGSL 305 AIQMTQSPSSLSASVGDRI 306RLSCAASGFTFSSYWMSW TIT CRASQGIRNDLG YQQ VRQAPGKGLEWVANVNQD KPGKAPKLLIYAASSLQSG GSEQNFVDSVKGRFT I S R VPSRFSGSGSGTDFTLTIS DN AKN S VH L QMN S L RAE D SLQPEDFATYYCLQDYNYP TAVYYCARDASNYDGYYY YTFGQGTKLEIK YYYTDVWGKGT TVTVS S49G11 EVQLLESGGGLVQPGGSL 313 QLVLTQSPSASASLGASVK 314RLSCAASGFTFRSYVMSW LTCTLSSGHSSYAIAWHQQ VRQAPGRGLEWVSAISGS QPEKGPRYLMKLNSDGSHS GDRT YYAD SVKGR F T I S R KGDGIPDRFSGSSSGAERY DNSKNTVYLQVKSLRAED LTISSLQSEDEADYYCQTW TAGYYCAKAAGYCTNGVC GTGIRVFGGGTKLTVL LYYYYMDVWGKGT TVTVSS3A9 QLQLQESGPGLVKPSETL 321 EIVMTQSPATLSLSPGERA 322SLTCTVSGGS I SSGSDYW TL S CRASQSVRS S YLS W YQ VWIRQPPGKGLEWIGSIY QKPGQAPRLLIYGASTRAT YSGSTYYNPALKSRVT I S GIPARFSGSGSGTDFTLTI VDTSKNQFSLKLSSVTAA S S L Q P E D FAVY Y CQQD YNL DTAVYYCARRGNDDYYYF PMYTFGQGTKLEIK YMDVWGKGTTVTVSA13H8 EVHLLESGGGLVQPGGSL 329 KIVMTQSPATLSVSPGERA 340RLSCAASGFTFSTYAMSW TL S CRASQSVS SNLA YQQ VRQAPGRGLEWVSAISGS KPGQAPRLLIYGASTRATG GGSRYYAD SVKGR F T I S R IPARFSGSGSGTEFTLTIS DNSKNTLYLQMNSLRAED S LQ S E D FAVY YCQHYNNWP TAVYYCAKDGGVPVVPYL FTFGQGTKLEIK YYYYMDVWGKGTTVTVS S2609214B8.1 QVQLVESGGGWQPGRSL 347 AIQLTQSPSSLSASVRDRV 348RLSCAASGFTFSSYGMHW TIT CRASQGINSALA YQQ VRQVPGKGLEWVAFISYD KPGKAPKLLIYDASRLESR GKNKYY ID SVRGR F T I S R VPSRFSGSGSGTDFTLTIS DNSKNTLFLQMNSLRAED S LQ P E D FAT Y YCQQFNNYM TAVYYCARENNWNDFYNY YTFGQGTKLEIK YYMDVWGKGT TVTVS S7F11.1 QVQLQESGPGLVKPSETL 355 AIQMTQSPSSLSASVGDRV 356SLTCTVSGGS I SSYYWSW TIT CRASQGIRNDLG YQQ IRQPAGKGLEWIGRIYTS KPGKAPKLLIYAASSLQSG GSTNYNPSLKSRVTMSGD VPSRFSGSGSGTDFTLTIS TSKNQFSLKLTSVTAADT S LQ P E D FAT Y YCLQDYNYP AVYYCAREVVYYYYMDVW YTFGQGTKLEIK GKGTTVTVSS35D7.1 EVQLVESGGGLVQPGGSL 363 AIQMTQSPSSLSASVGDRV 364 RLSCAASGFSLSSYWMSW TIT CRASQGIRDDLG YQQ VRQAPGKGLEWVANIKQD TPGKAPKLLIYAASTLQSG GSEKNYVDAVKGRFTISR VPSRFSGSGSGTDFTLTIS DN AKN S L Y L HMN S L RVE D SLQPEDFATYYCLQDNNYP TAVYYCARDNDNWNGFYY YTFGQGTKLEIK YYSMDVWGKGT TVTVS S13B3 EVQLVESGGGLVQPGGSL 371 AIQMTQSPSSLSASVGDRV 372RLSCVASGFTFGPYWMTW TIT CRASQGIRDDLGW YQQ VRQAPGKGLEWVANINQD KPGKAPELLIYAASSLQSG GNEKNYVD SVKGR F T I S R VPSRFSGSGSGTDFTLTIS DNVKNSLFLQMNSLRAED S LQ P E D FAT Y YCLQDYNYP TAVYYCVRDDSVYDSYYY YTFGQGTKLEIKYFYMGVWGGGTAVTVSSVH-CDR and VL-CDR sequences are shown in

[0120] In particular embodiments, the ALPP / L2 binder is an antibody comprising a VH covalently linked to a CH comprising CHI, CH2, and CH3 and a VL covalently linked to a CL. In particular embodiments, the VH is linked to the CH of an IgGl, IgG2, IgG3, or IgG4 isotype and the VL is linked to the CL 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 the CL 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 lgG4 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 particular embodiments, the CH is of the IgGl isoty pe 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 IgGl isotype. In particular 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,26092insertions, deletions, or combinations thereof compared to the amino acid sequence of the native kappa isotype. An antibody comprises the association of two VH-CH1-CH2-CH3 proteins to form a Y structure wherein the CH3 domains form a dimeric Fc region at the base of the Y structure. As used herein, the term “Fc region'’ refers to the dimer formed by the association of two CH3 domains.

[0121] In particular embodiments, the VH is linked to the constant domain of a human IgGl comprising the amino acid sequence set forth in SEQ ID NO: 381 or variant thereof comprising an S375C substitution and having the amino acid sequence shown in SEQ ID NO: 392.

[0122] In further embodiments, the constant domain of the human IgGl 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 Eu (The positions according to sequential number are 256, 258, and 260, respectively), to provide a heavy chain constant domain comprising a “YTE” substitution and having the amino acid sequence set forth in SEQ ID NO: 382 or variant thereof comprising an S375C substitution and having the amino acid sequence shown in SEQ ID NO: 393.

[0123] In further embodiments, the human IgGl heavy chain constant domain comprises E233A and L235A amino acid substitutions wherein the numbering is according to Eu, to provide a heavy chain constant domain having the amino acid sequence set forth in SEQ ID NO: 383 or variant thereof comprising an S375C substitution and having the amino acid sequence shown in SEQ ID NO: 394.

[0124] In further embodiments, the human IgGl heavy chain constant domain comprises L234A L235A D265S substitutions wherein the numbering is according to Eu, to provide a heavy chain constant domain having the amino acid sequence set forth in SEQ ID NO: 384 or variant thereof comprising an S375C substitution and having the amino acid sequence shown in SEQ ID NO: 395.

[0125] In further embodiments, the human IgGl heavy chain constant domain comprises L234A L235A P329G substitutions wherein the numbering is according to Eu, to provide a heavy chain constant domain having the amino acid sequence set forth in SEQ ID NO: 385 or variant thereof comprising an S375C substitution and having the amino acid sequence show n in SEQ ID NO: 396.

[0126] In further embodiments, the human IgGl heavy chain constant domain comprises an L235E substitution wherein the numbering is according to Eu, to provide a heavy chain constant domain having the amino acid sequence set forth in SEQ ID NO: 386 or variant thereof26092comprising an S375C substitution and having the amino acid sequence shown in SEQ ID NO: 397.

[0127] In further embodiments, the human IgGl heavy chain constant domain comprises D265A substitution wherein the numbering is according to Eu, to provide a heavy chain constant domain having the amino acid sequence set forth in SEQ ID NO: 387 or variant thereof comprising an S375C substitution and having the amino acid sequence shown in SEQ ID NO: 398.

[0128] In further embodiments, the human IgGl heavy chain constant domain comprises D265A N297G substitutions wherein the numbering is according to Eu, to provide a heavy chain constant domain having the amino acid sequence set forth in SEQ ID NO: 388 or variant thereof comprising an S375C substitution and having the amino acid sequence shown in SEQ ID NO: 399.

[0129] In further embodiments, the human IgGl heavy chain constant domain comprises N297X, wherein X is any amino acid other than N substitution wherein the numbering is according to Eu, to provide a heavy chain constant domain having the amino acid sequence set forth in SEQ ID NO: 389 or variant thereof comprising an S375C substitution and having the amino acid sequence shown in SEQ ID NO: 400.

[0130] In further embodiments, the human IgGl heavy chain constant domain comprises N297A / D356E / L358M substitutions wherein the numbering is according to Eu, to provide a heavy chain constant domain having the amino acid sequence set forth in SEQ ID NO: 390 or variant thereof comprising an S375C substitution and having the amino acid sequence shown in SEQ ID NO: 401.

[0131] In particular embodiments of the invention, 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.ScFv fusion proteins that bind ALPP and / or ALPPL2

[0132] In particular embodiments, the VH and VL disclosed herein are expressed as an ScFv fusion protein in which the VL and VH domains are linked together by a peptide linker. The26092peptide linker joins the carboxyl terminus of one variable region domain to the amino terminus of the other variable domain without compromising the fidelity of the VH–VL paring and antigenbinding sites. Thus, the ScFv may comprise a fusion protein in which the C-terminus of a VL is linked by a peptide linker to the N-terminus of a VH or a fusion protein in which the C-terminus of a VH is linked by a peptide linker to the N-terminus of a VL. Peptide linkers for linking the variable domains can vary from 10 to 25 amino acids in length and are typically, but not always, composed of hydrophilic amino acids such as glycine (G) and serine (S) having the structure G4S (SEQ ID NO: 419), for example, (G4S)n(SEQ ID NO: 420), wherein n is 1, 2, 3, 4, or 5. Peptide linkers of shorter lengths (0-4 amino acids) have also been used; however, ScFv bearing shorter linkers may form multimers. Generally, the (G4S)3 peptide comprising three repeating G4S units (G4S)3?’ disclosed as SEQ ID NO: 421) is used as an ScFv peptide linker (See for example, Leath et al., Int. J. Oncol. 24:765-771 (2004); Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993); Iliades et al., FEBS Lett. 409:437-441 (1997)).

[0133] Exemplary ScFv fusion proteins comprise the structure VL-(G4S)n-VH or VH-(G4S)n-VL, wherein n is 1, 2, 3, 4, or 5 (“(G4S)n” is disclosed as SEQ ID NO: 420), and wherein the VL / VH pair comprise a VL-CDR / VH-CDR pair selected from the combinations shown in Table 3. In a further embodiment, the exemplary ScFv fusion proteins comprise the structure VL-(G4S)n-VH or VH-(G4S)n-VL, wherein n is 1, 2, 3, 4, or 5 (“(G4S)n’’ is disclosed as SEQ ID NO: 420), and wherein the VL / VH pair is selected from the combinations shown in Table 4.

[0134] The ScFvs disclosed herein may be provided in a Bispecific T-cell engager (BiTE®) format comprising a CD3 binder (ScFv) linked by a peptide linker to an ScFv that binds ALPP and / or ALPPL2 as disclosed herein. When both arms of the BiTE® molecule are bound to their specific targets, the T cells are induced to release perforin that forms pores in the wall of the target cells, and toxic molecules called granzymes enter into the target cells through the pore, leading to the death of the target cell.

[0135] ScFvs disclosed herein may also be fused to cellular toxins, radioisotopes, cytokines, and enzymes for cancer, autoimmune, and / or inflammatory therapeutic applications. In particular embodiments, the peptide linker may comprise 1 to 10 G4S peptide units (SEQ ID NO: 422).

[0136] In further embodiments, the ScFvs disclosed herein may be linked to or inserted in different locations of an intact IgG molecule to confer dual epitope binding. For example, a bispecific antibody may be provided comprising two heterodimeric heavy chain constant domains wherein the N-terminus of one heavy chain constant domain is fused to the C-terminus of an ScFv disclosed herein and the N-terminus of the other heavy chain constant domain is fused26092to the C-terminus of an ScFv that targets an antigen other than ALPP and / or ALPPL2 or a Fab’ that targets an antigen other than ALPP and / or ALPPL2.Chimeric Antigen Receptors (CARs)

[0137] In other general aspects, cells are provided that expresses an ALPP / L2 binder on the cell surface. The ALPP / L2 binder expressed on the cell surface may be a whole antibody, a bispecific antibody, a Fab’, or an ScFv. The cell expressing the ALPP and / or ALPPL2 on the cell surface may be an immune cell, for example, a T cell or an NK cell. In a particular embodiment, the cell expresses on the cell surface a chimeric antigen receptor (CAR), wherein the CAR comprises an ALPP / L2 binder, which in particular embodiments may be an ScFv.

[0138] In one aspect, the CAR comprises: (a) an extracellular domain comprising an ScFv that specifically binds to ALPP and / or ALPPL2; (b) a transmembrane domain; and (c) an intracellular signaling domain.

[0139] In some embodiments, in a nascent CAR, the extracellular domain is preceded by a signal or leader peptide at the N-terminus. Any suitable signal or leader peptide can be used in the invention. The signal peptide can be derived from a natural, synthetic, semi-synthetic or recombinant source. Examples of signal or leader peptides include those show n in SEQ ID NO: 402 and 403.

[0140] According to particular embodiments, the CAR may further comprise a hinge region connecting the extracellular domain and the transmembrane domain. The hinge region functions to move the extracellular domain away from the surface of the engineered immune cell to enable proper cell / cell contact, binding to the target or antigen, and activation (Patel et al.. Gene Therapy 6: 412-419 (1999)). Any suitable hinge region can be used in a CAR. It can be derived from a natural, synthetic, semi-synthetic, or recombinant source. Examples of a suitable hinge region for a CAR is a peptide comprising the amino acid sequence GS repeated up to six times, or a fragment thereof, or a hinge region of an IgGl or IgG4 antibody, or a hinge region from a CD8 protein, or a derivative thereof. A CD8 hinge comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 423, or the amino acid sequence set forth in SEQ ID NO: 423.

[0141] Any suitable transmembrane domain can be used in a CAR. The transmembrane domain can be derived from a natural, synthetic, semi-synthetic or recombinant source. According to some embodiments, the transmembrane domain is a transmembrane domain from molecules such as CD8, CD28, CD4, CD2, GMCSFR and the like. For example, a CD8 transmembrane domain26092comprising an amino acid sequence at least 90% identical to SEQ ID NO: 424. preferably the amino acid sequence of SEQ ID NO: 424.

[0142] Any suitable intracellular signaling domain can be used in a CAR. In particular embodiments, the entire intracellular signaling domain is used. In other particular embodiments, a truncated portion of the signaling domain that transduces the effector signal is used. According to particular embodiments, the intracellular signaling domain generates a signal that promotes an immune effector function of the CAR-expressing cell, e.g., a CAR-T cell, including, but not limited to, proliferation, activation and / or differentiation, or killing of target cells in the case of CAR-NK cells. In particular embodiments, the signal promotes, e.g., cytolytic activity, helper activity, and / or cytokine secretion of the CAR-T cell. The intracellular signaling domain may comprise afunctional signaling domain derived from CD3ζ, TCRζ, FcRγ, FcRβ, CD3β, CD3δ, CD3ε, CD16, CD22, CD27, CD28, CD30, CD79a, CD79b, CD134 (also known as TNFRSF4 or OX-40), 4-1BB (CD137), CD278 (also known as ICOS), FceRI, DAP10, DAP12, ITAM domains or CD66d, and the like. According to particular embodiments, the intracellular signaling domain comprises a primary signaling domain, e.g., CD3ζ, and one or more co-stimulatory signaling domains, e.g., 4-BB.

[0143] An exemplary' CAR may comprise (a) an extracellular domain comprising an ScFv that specifically binds ALPP and / or ALPPL2; (b) a CD8 transmembrane domain; and (c) an intracellular signaling domain comprising a co-stimulatory domain comprising the functional signaling domain of 4-1BB (e.g., the amino acid sequence set forth in SEQ ID NO: 425), and a primary' signaling domain comprising the functional signaling domain of CD3ζ (e.g., the amino acid sequence set forth in SEQ ID NO: 426), wherein the CAR optionally further comprises a hinge region connecting the extracellular domain and the transmembrane domain.Nucleic acid molecules encoding ALPP / L2 binders

[0144] Further provided are nucleic acid molecules that encode the ALPP / L2 binders of the present invention. In particular embodiments, the ALPP / L2 binder comprises a VH encoded by a first nucleic acid molecule and a VL encoded by a second nucleic acid molecule. In particular embodiments, the ALPP / L2 binder is an antibody in which the heavy chain is encoded by a first nucleic acid molecule and the light chain is encoded by a second nucleic acid molecule.

[0145] In particular embodiments, the heavy chain and light chain (or VH and VL) are expressed as a fusion protein in which the N-terminus of the heavy chain and light chain (or VH and VL) are fused at the N-terminus to a leader peptide to facilitate the transport of the ALPP / L2 binder through the secretory pathway. In particular embodiments, the N-terminus of the ScFv26092fusion protein is fused at the N-terminus to a leader or signal peptide to facilitate the transport of the ScFv 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 particular embodiments, the aforementioned nucleic acid molecules may comprise a polynucleotide encoding a leader peptide linked to the 5’ end of the nucleic acid molecule encoding the ALPP / L2 binder.

[0146] 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 prokaryote host cell.Methods for making an ALPP / L2 binder

[0147] Recombinant methods for making an ALPP / L2 binder of the present invention comprise introducing into a host cell (i) an expression vector comprising nucleic acid molecule(s) that encode the VH and VL of an ALPP / L2 binder or the heavy chain and light chain of an ALPP / L2 binder, or (ii) two expression vectors comprising nucleic acid molecules, one vector comprising a nucleic acid molecule encoding the VH of an ALPP / L2 binder or the heavy chain of an ALPP / L2 binder, the other vector comprising a nucleic acid molecule encoding the VL of an ALPP / L2 binder or the light chain of ALPP / L2 binder. The nucleic acid molecules or polynucleotides encoding the VH, VL, heavy chain, or light chain are operably linked to a promoter and other transcription and translation regulatory sequences. The host cell is cultured under conditions and a time period suitable for expression of the nucleic acid molecules followed by isolating the ALPP / L2 binder from the host cell and / or medium in which the host cell is grown. See e.g., International Patent Application Publication Nos. W02004041862, WO2006122786, W02008020079, WO2008142164 or W02009068627. The expression vector may be a plasmid or viral vector. The invention also relates to host cells that comprise such nucleic acid molecules encoding the ALPP / L2 binders (host cells comprising nucleic acid molecules encoding the VH and VL or nucleic acid molecules encoding the heavy chain and light chain) or components thereof (host cells comprising nucleic acid molecule encoding solely the VH or heavy chain or solely the VL or light chain).

[0148] Eukaryotic and prokaryotic host cells, including mammalian cells as hosts for expression of the ALPP / L2 binder are well 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 hamster26092kidney (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. In particular cell lines are selected through 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 an ALPP / L2 binder disclosed herein or comprising one or more nucleic acid molecules encoding such ALPP / L2 binder or comprising an expression vector that comprises one or more nucleic acid molecules encoding such ALPP / L2 binder.

[0149] Further, expression of an ALPP / L2 binder from production 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 grown in the absence of glutamine in the medium wherein, however, the nucleic acid molecule encoding the immunoglobulin chain comprises a glutamine synthetase gene which complements the lack of the gene in the host cell. Such host cells may comprise the ALPP / L2 binder or nucleic acid molecule(s) or expression vector(s) as discussed herein as well as expression methods, as discussed herein, for making the ALPP / L2 binder using such a host cell.

[0150] Methods for purifying an ALPP / L2 binder comprise introducing a sample (e.g., culture medium, cell lysate or cell lysate fraction, e.g., a soluble fraction of the lysate) comprising the ALPP / L2 binder to a purification medium (e.g., cation-exchange medium, anion-exchange medium and / or hydrophobic exchange medium) and either collecting purified ALPP / L2 binder from the flow-through fraction of said sample that does not bind to the medium; or, discarding the flow-through fraction and eluting bound ALPP / L2 binder from the medium and collecting the eluate. In an embodiment, 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 ALPP / L2 binder 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 ALPP / L2 binder is secreted into the culture medium by the host cell and the medium or a fraction thereof is applied to the purification medium.26092

[0151] 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 ALPP / L2 binder will depend on the particular cell line or transgenic animal used to produce the ALPP / L2 binder. ALPP / L2 binders comprising only non-fucosylated N-glycans may be advantageous, because non-fucosylated antibodies have been shown to typically exhibit more potent efficacy than their fucosylated counterparts both in vitro and in vivo (See for example, Shinkawa et al., J. Biol. Chem. 278: 3466-3473 (2003); U. S. Patent Nos. 6,946,292 and 7,214,775). These ALPP / L2 binders with non-fucosylated N-glycans are not likely to be immunogenic because their carbohydrate structures are a normal component of the population that exists in human serum IgG.

[0152] ALPP / L2 binders 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 ALPP / L2 binder comprises one or more of the “engineered yeast N-linked glycans” 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 ALPP / L2 binder comprises the engineered yeast N-linked glycans, i.e.. GO and / or G1 and / or G2, optionally, further including Man5. In an embodiment, the ALPP / L2 binders 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 ALPP / L2 binders 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 Opm 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.Pharmaceutical compositions comprising ALPP / L2 binders

[0153] Pharmaceutical compositions comprising one or more ALPP / L2 binders and a pharmaceutically acceptable carrier wherein the carrier may be a diluent, adjuvant, excipient, or vehicle with which the ALPP / L2 binder 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% glycine26092may 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 ALPP / L2 binder(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.

[0154] The mode of administration of the ALPP / L2 binder of the present invention or pharmaceutical composition comprising the ALPP / L2 binder 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.

[0155] The ALPP / L2 binder 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, 1, 30, 60, 90, 120, 180, or 240 minutes, or from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours.

[0156] The administration of the ALPP / L2 binder or a pharmaceutical composition comprising the ALPP / L2 binder 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. Repeated courses of treatment are also possible, as is chronic administration. The repeated administration may be at the same dose or at a different dose.

[0157] The ALPP / L2 binder or a pharmaceutical composition comprising the ALPP / L2 binder may be administered by maintenance therapy, such as, e.g., once a week for a period of 6 months or more.

[0158] The ALPP / L2 binder or a pharmaceutical composition comprising the ALPP / L2 binder 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 recurrence26092when 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.

[0159] The ALPP / L2 binder or pharmaceutical composition comprising the ALPP / L2 binder 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

[0160] Combination therapies comprising an ALPP / L2 binder disclosed herein or pharmaceutical composition comprising the ALPP / L2 binder disclosed herein and another or second therapeutic agent (e.g., small molecule or antibody that is not an ALPP / L2 binder) may be used for the treatment of 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 an ALPP / L2 binder and chemotherapy

[0161] The combination therapy comprising ALPP / L2 binder or a pharmaceutical composition comprising the ALPP / L2 binder 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 therapy disclosed 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.

[0162] The chemotherapy may include a chemotherapy agent selected from the group consisting of:(i) alkylating agents, including but not limited to, bifunctional alkylators, cyclophosphamide, mechlorethamine, chlorambucil, and melphalan;26092(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, doxifluridine, 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.

[0163] 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.

[0164] 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) Monoclonal26092Antibodies, 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.

[0165] 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.

[0166] 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 ALPP / L2 binder and a therapeutic antibody

[0167] The ALPP / L2 binder or a pharmaceutical composition comprising the ALPP / L2 binder 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 recurrent or metastatic cancer with disease progression or relapse cancer and who is26092undergoing chemotherapy or who has completed chemotherapy. In particular embodiments, the therapeutic agent targets the programmed death 1 receptor or ligand, PD-1 and PD-L1, respectively. In particular 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).

[0168] Exemplary anti-PD-1 antibodies that may be used in a combination therapy with the ALPP / L2 binders 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 (LIBTAYO). 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-L1 antibody is avelumab (BAVENCIO).

[0169] 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).

[0170] 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, endometrial cancer, pancreatic cancer, ovarian cancer, non-small cell lung cancer (NSCLC), gastric cancer, and colon cancer.Injection device for administering an ALPP / L2 binder

[0171] An injection device is provided that comprises any one of the ALPP / L2 binders or a pharmaceutical composition comprising any one of the ALPP / L2 binders. 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 of26092the ALPP / L2 binders or pharmaceutical compositions comprising any one of the ALPP / L2 binders), 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 ALPP / L2 binders or a pharmaceutical composition comprising any one of the ALPP / L2 binders is an intravenous (IV) injection device. Such a device includes a composition comprising said ALPP / L2 binder 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.

[0172] The ALPP / L2 binders or a pharmaceutical compositions comprising the ALPP / L2 binders 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 ALPP / L2 binder or a pharmaceutical composition comprising the ALPP / L2 binder to a patient's body. External infusion pumps are medical devices that deliver the ALPP / L2 binder or a pharmaceutical composition comprising the ALPP / L2 binder 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 the reservoir 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 ALPP / L2 binder or pharmaceutical composition thereof

[0173] Further provided are kits comprising one or more components that include, but are not limited to, an ALPP / L2 binder or a pharmaceutical composition comprising the ALPP / L2 binder in association with one or more additional components including, but not limited to, a furthertherapeutic agent, as discussed herein. The ALPP / L2 binder or a pharmaceutical composition comprising the ALPP / L2 binder and / or the therapeutic agent can be formulated as a pure composition or in combination with a pharmaceutically acceptable carrier, in a pharmaceutical composition.

[0174] In one embodiment, the kit includes the ALPP / L2 binder or a pharmaceutical composition comprising the ALPP / L2 binder 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).

[0175] In another embodiment, the kit comprises a combination, including ALPP / L2 binder or a pharmaceutical composition comprising the ALPP / L2 binder in combination with one or more therapeutic agents formulated together, optionally, in a pharmaceutical composition, in a single, common container.

[0176] 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 inj ection device and the ALPP / L2 binder or a pharmaceutical composition comprising the ALPP / L2 binder, e.g., wherein the injection device includes the ALPP / L2 binder or a pharmaceutical composition comprising the ALPP / L2 binder, or wherein the ALPP / L2 binder or pharmaceutical composition comprising the ALPP / L2 binder is in a separate vessel.

[0177] 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, efficacy parameters, 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

[0178] Administration of an ALPP / L2 binder 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 halflife, 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.1858926B1, and in numerous other patents and publications. In certain embodiments, administration is subcutaneous or intramuscular.

[0179] Accordingly, specific embodiments relate to (a) pharmaceutical compositions or fixed-dose pharmaceutical compositions comprising an AEPP / L2 binder 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 an AEPP / L2 binder disclosed herein, a therapeutic agent other than an ALPP / E2 binder (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 an ALPP / E2 binder 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 an AEPP / L2 binder 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.

[0180] 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)-( 1-447) peptide’7and which is currently marketed by Halozyme under the tradename Hylenex™. In certain embodiments, the hyaluronidase is 3-305-Hyaluronidase PH-20 (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 an AEPP / L2 binder disclosed herein and a soluble PH20 polypeptide or a variant thereof.EXAMPLES

[0181] The following examples describe the discovery and characterization of the new class of ALPP / L2 binders are meant to be illustrative and should not be construed as further limiting. Thecontents 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

[0182] 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.

[0183] 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 w as incubated with dilutions of serum from immunized mice for one hour at room temperature, the assay plate was w ashed, and specific antibody binding w as detected with HRP-labeled anti-mouse IgG antibody. Plates was read using an ELISA reader (Biotek).

[0184] 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.

[0185] 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 w ere reactive for the antigen of interest were expanded. Sequencing of the human26092VH 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

[0186] In this example purified ALPP / L2 binders were evaluated in an EC50 FACS Binding Assay to human ALPPL2, human ALPP, human ALPL human ALPL, rhesus ALPP, mouse ALPPL2.Cell binding'.

[0187] 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 cm2flask using media receipt F12K + 10% heat inactivated FBS, 50 pg / mL Gentamicin, and 8.0 pg / mL Puromycin.

[0188] All cells were harvested using 0.25% Trypsin / EDTA (Mouse ALPP-CHOK1 Clone 5E9) or TrypLETMExpress 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 Balanced26092Salt 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.

[0189] The different recombinant cell lines were then mixed by resuspending in FACS buffer and aliquoted into 96-V-well plates (50 pL / well, 30x104 cells / 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 region) for 15 minutes and then the cells w ere pelleted in a centrifuge, the supernatant fraction decanted, and the cells washed 2X with FACS buffer. Cells resuspended in 150 pL FACS buffer were then analyzed using a Intellicyt® iQue Screener PLUS.

[0190] The results are shown in Table 5.Table 5: Cell binding / specificity of ALPP / L2 BindersCell binding EC50(μg / mL, x10-3)Human Human Rhesus Mouse Hinn an Clone Name ALPPL2 ALPP ALPP ALPPL2 Human ALPL ALPI 293CHOK1 CHOK1 CHOK1 CHOK1 CHOK1 25 G8 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 Very- 6E6 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 NB4F4 0.45 0.93 13 NB NB NB260924B10 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 NB 5F8 1.141E-14 0.92 2.2 NB NB NB StrongBinder (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 NB7D11.2 NB NB NB NB NB NB2609235D7.2 | NB | NB | NB | NB | NB | NBNote: NB - no bindingEXAMPLE 3Binding Affinity of the ALPP / L2 binders by Surface Plasmon Resonance

[0191] 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 region 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™ Evaluation software, version 1.1 and a 1:1 fitting model (Langmuir Binding, Rmax = global, RI = constant, offset = 0). The results are presented in Table 6.Table 6: Binding 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) 25G8 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 19 A3 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-0949G5 6.36E+04 4.74E-04 7.45E-09 4.00E+04 1.30E-03 3.26E-08260927C8 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 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-09 2A2 2.36E+05 3.73E-04 1.58E-09 2.93E+05 2.06E-04 7.04E-10 12C6 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-09Low49G11 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-10Low7F11.1 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 NB35D7.2 NB NB NB NB NB NB26092Note: NB - no bindingEXAMPLE 4Physicochemical Properties of the ALPP / L2 Binders

[0192] 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

[0193] 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, pH 7.0 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

[0194] ALPP / L2 binders were analyzed by nanoDSF to determine thermal stability. Nano-DSF is a method for measuring ultra-high-resolution protein stability' using intrinsic tryptophan or ty rosine 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.

[0195] 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 fluorescence26092intensities (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.

[0196] 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.

[0197] The hydrophobicity and thermal stability of the ALPP / L2 binders are presented in TableTable 7: Physicochemical properties of ALPP / L2 binders Thermal stability (Nano Hydrophobicity (HIC)DSF) Control ~26 min RTClone 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 88.695F8 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 100260926E10 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 10B3 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 10014A11 61.0 68.5 27.50 10019 A3 58.3 64.1 >40 100 No peaks 15B6 59.7 66.5 >40 10049G5 56.7 74.1 18.10 1003G8 60.8 65.8 >40 100 No peaks 24F4 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 peaks 13H8 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 ALPP / L2 binders

[0198] 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 Assay.ALPPL2 Fab-ZAP Internalization Assay:

[0199] ALPPL2-CHOK1 Clone 4F11 was grown in 175 cm2flask using media receipt previously described.

[0200] Day 1 - Cells were harvested as described in Example 2. Cells were counted and adjusted to a cell density of 1x106cells / 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 tissue26092culture 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.

[0201] 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 / mL, 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 culture plate. The ALPP / L2 binders were tittered 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.

[0202] 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 were internalized and which were not internalized. The results are shown in Table 8.Table 8Non-Internalizing ALPP / L2 InternalizingBinders ALPP / L2 BindersClone Name Clone Name11C7 25 G85A1 29D28G6 16C25E8 2.8A714A11 16H219A3 50F515B6 6E649G5 15B43G8 17H624F4 10F632H6 4F416F2 4B1010B3 7C810A9 9B1213H8 16B517E49G1021H72A22609212C612F114B1210G26E1035D517A718A1149G113A914B8.17F11.135D7.113B3EXAMPLE 6Epitope Binning of the ALPP / L2 Binders

[0203] 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.

[0204] A biosensor surface coated with goat anti -human IgG Fc region 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-dimethylaminopropyl]carbodiimide hydrochloride) and 10 mM sulfo-NHS (l-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 / mL 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 ug / 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 experiment26092cycle involved first a 10 minutes 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 followed 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 network / community plots.

[0205] The binning was able to sort the ALPP / L2 binders into 12 bins. Binning data is shown in Table 9. A dendrogram and community plot summarizing the binning results is shown in Fig.1A and Fig. IB.Table 9: Epitope binningSample ID Clone ka (1 / Ms) kd (1 / s) KD (M) bin# DAB014614 25G8 1.90E+05 2.67E-04 1.41E-09 1 DAB014635 4B10 7.00E+06 1.62E-04 2.32E-11 1 DAB014645 9G10 3.25E+07 4.64E-04 1.43E-11 1 DAB014615 29D2 low capture 2 DAB014616 16C2 1.90E+05 2.32E-04 1.22E-09 3 DAB014617 8A7 3.05E+05 2.75E-04 9.02E-10 4 DAB014618 16H2 5.02E+05 8.94E-05 1.78E-10 4 DAB014622 50F5 9.78E+04 3.70E-04 3.78E-09 4 DAB014623 12G9 low capture 4 DAB014634 4F4 4.30E+05 2.83E-04 6.59E-10 4 DAB014638 9B12 2.66E+05 1.90E-04 7.13E-10 4 DAB014639 6F5 low capture 4 DAB014646 21H7 2.16E+05 5.55E-04 2.57E-09 4 DAB014647 5F8 low capture 4 DAB014649 2A2 2.36E+05 3.73E-04 1.58E-09 4 DAB014651 12F1 2.67E+07 8.72E-06 3.27E-13 4 DAB014655 16C2 low capture 4 DAB014661 17A7 3.48E+05 5.34E-05 1.54E-10 4 DAB014662 18A11 2.51E+05 2.43E-04 9.66E-10 4 DAB014673 35D7.1 1.91E+05 2.63E-04 1.38E-09 4 DAB014675 13B3 2.70E+09 4.12E-02 1.53E-11 4 DAB014619 11C7 6.89E+06 2.23E-04 3.24E-11 5 DAB014620 5A1 7.35E+05 4.63E-04 6.30E-10 5 DAB014624 5E8 2.19E+05 3.98E-04 1.82E-09 5 DAB014625 14A11 2.22E+05 3.41E-04 1.54E-09 5 DAB014626 19A3 1.29E+07 2.03E-03 1.57E-10 5 DAB014628 15B6 4.72E+05 5.51E-04 1.17E-09 5 DAB014630 6E6 2.67E+05 2.52E-04 9.44E-10 5 DAB014636 49G5 6.36E+04 4.74E-04 7.45E-09 5DAB014637 7C8 7.61E+05 9.94E-05 1.31E-10 526092DAB014642 3G8 3.99E+05 2.52E-04 6.31E-10 5 DAB014670 14B8.1 1.07E+05 3.01E-04 2.81E-09 5 DAB014621 8G6 1.61E+07 2.03E-04 1.26E-11 6 DAB014631 15B4 low capture 7 DAB014632 17H6 1.95E+05 3.74E-04 1.92E-09 7 DAB014640 16B5 2.22E+05 2.86E-04 1.29E-09 7 DAB014633 10F6 2.07E+05 3.75E-04 1.82E-09 8 DAB014643 17E4 1.26E+09 4.14E-04 3.30E-13 9 DAB014650 12C6 4.08E+07 4.91E-04 1.20E-11 9 DAB014644 24F4 3.80E+05 3.80E-05 1.00E-10 10 DAB014648 32H6 1.83E+05 3.24E-04 1.78E-09 10 DAB014653 16F2 4.70E+06 1.41E-04 3.00E-11 10 DAB014656 10G2 2.23E+05 3.06E-04 1.37E-09 10 DAB014657 6E10 3.14E+05 3.90E-04 1.24E-09 10 DAB014668 13H8 3.31E+05 1.24E-04 3.75E-10 10 DAB014654 14B12 1.68E+05 2.58E-04 1.53E-09 11 DAB014658 10B3 3.32E+05 2.29E-04 6.90E-10 11 DAB014660 35D5 3.73E+05 2.05E-05 5.49E-11 11DAB014667 3A9 8.06E+08 3.65E-02 4.52E-11 12 NOTE: ALPP / L2 binders with poor / no expression or weak binders not analyzed.EXAMPLE 7Epitope Mapping of Several AI. PP 1.2 Binders

[0206] Hydrogen / Deuterium (H / D) exchange (HDX) was used to identify the epitopes on ALPPL2 recognized by ALPP / L2 binders derived from clones 24F4, 2.10B3, 9G10, 16B5. 17A7, 9G10, 21H7. 12F1, 13H8, and 14B8.1, 24F4, 10B3 were constructed in a mAb format comprising a HC in which a VH was fused to an IgGl CH comprising L234A, L235A, and D265S amino acid substitutions and a LC in which a VL was fused to CL kappa. 9G10 was constructed in a Fab format comprising a HC in which a VH was fused to a wild-type IgG1 CH and a LC in which a VL was fused to CL kappa. PLAP h2-IgGl L234A L235A D265S / LC Kappa PLAPh2 comprises a HC comprising the amino acid sequence of SEQ ID NO: 418 and a LC comprising the amino acid sequence set forth in SEQ ID NO: 417. The amino acid sequences for the VH and VL of PLAPh2 have been disclosed in U. S. Patent No. 12030957, U. S. Patent No.12037407, and U. S. Patent Application Publication No. US20220348688.

[0207] The contact areas between ALPP / L2 binders and human ALPPL2 were determined by hydrogen deuterium exchange mass spectrometry (HDX-MS) analysis. HDX-MS measures the exchange of deuterium with hydrogen into the amide backbone of the protein. One factor influencing the exchange rate is the hydrogen's exposure to solvent. Comparison of the exchange26092levels in the antigen when the antibody is bound can identify regions of the protein where the antibody is binding.Pepsin / XIII digestion and Liquid Chromatography-Mass Spectrometry

[0208] Approximately 2.1 micrograms (40 pmole) of the recombinant ALPPL2 protein in 100 pL of control buffer (50 mM phosphate, 100 mM sodium chloride at pH 7.4) was denatured by adding 100 pL of 8 M urea, 1.25 M TCEP (tris(2-carboxyethyl)phosphine) buffer (final pH was 2.5), which was incubated for 3 minutes at 20 °C. Then, the mixture was subjected to on-column pepsin / XIII digestion using a pepsin / XIII column (2.1 x 30 mm, NovaBioAssays, Woburn, MA, NBA2014002). The resultant peptides were trapped and desalted on an ACQUITY UPLC BEH C18 VanGuard pre-column (130A, 1.7 pm, 2.1 mmx 5 mm, 186003975, Waters Corporation, Milford, MA) for 3.5 min at 160 pL / minutes. Peptides were then eluted from the trap using a 2% - 32% gradient of acetonitrile (with 0.3% formic acid) over 12.5 minutes at a flow rate of 150 pL / minute and are separated on a 50 x 1 mm C8 column (3 pm, NBA2014015, NovaBioAssays).

[0209] An ultra-performance liquid chromatography-mass spectroscopy (UPLC-MS) system comprised of a Waters ACQUITY UPLC coupled to an Orbitrap Exploris™ 480 Mass Spectrometer (Thermofisher) was used. Solvent A was 0.3% formic acid in water. The injection valve, enzyme column and their related connecting tubing were stored inside a cooling box maintained at 20 °C. The second switching valve, C8 column and their related connecting tubing were inside another chilled circulating box maintained at -6 °C. Peptide identification was done through searching MS / MS data against the ALPPL2 amino acid sequence with Byonic search algorithm from Protein Metrics. The mass tolerance for the precursor and product ions were 10 ppm and 0.02 Da, respectively.HDX-MS for the recombinant ALP PL2 with and without the presence of the ALPP / L2 binder

[0210] All the ALPP / L2 binders were buffer exchanged with PBS PH 7.4 buffer in 30 kDa Amicon® ultra centrifugal filter by centrifugation at 14.000 g for 8 minutes for three times. The concentrations of ALPP / L2 binder before and after buffer exchange were measured by nanodrop.

[0211] The protein complex samples were prepared by mixing ALPPL2: ALPP / L2 binder at a final 1:2 molar ratio, with ALPP or ALPPL2 was kept at 0.21 mg / mL (4 pM), and the final concentration of ligand was kept at 1.17 mg / mL (8 pM).

[0212] Ten microliters of ALPPL2 protein alone, or 10 pL of ALPPL2 and ALPP / L2 binder mixture was incubated with 90 pL deuterium oxide (deuterated) labeling buffer (50 mM sodium phosphate, 100 mM sodium chloride at pH 7.0) for 0 seconds (s), 15 s, 60 s, 600 s, or 3600 s at2609220 °C. Hydrogen / deuterium exchange was quenched by adding 100 µL of 8 M urea, 1.25 M TCEP buffer (final pH was 2.5). Subsequently, the quenched samples were subjected to on-column pepsin and protease type XIII digestion and liquid chromatography-mass spectroscopy (LC-MS) analysis as described above. The mass spectra were recorded in mass spectroscopy (MS) only mode.

[0213] Raw MS data was processed using HDX WorkBench, software for the analysis of H / D exchange MS data (Pascal et al., J. Am. Soc. Mass Spectrom. 2012, 23 (9), 1512-1521). The deuterium levels were calculated using the average mass difference between the deuterated peptide and its undeuterated form (to).

[0214] By comparing the deuterium uptake profile for ALPPL2 and ALPP / L2 binder complex vs. ALPPL2 alone, the potential residues responsible for ligand binding were identified on APPL2. The amino acids protected from hydrogen / deuterium exchange (H / D protection) by the various antibodies is shown in Table 10 and in Fig. 2A and Fig. 2B.Table 10: Epitopes identified HDXDescription H / D Protection Epitope InternalBin izing APPL2 epitope:Human-x-[ALPPL2_H]-mAb-(24F4)-IgGl RQAAEALGAAKKLQ 10 No L234A L235A D265S / LC Kappa (AA 33-46; SEQ ID NO: 404)ALPPL2 epitopes:RILKGQKKDKLGPETFLHuman-x-[ALPPL2_H]-mAb-(10B3)-IgGl (AA 72-88; SEQ ID NO: 405) 11 No L234A L235A D265S / LC Kappa GGYPLRGSSIFGL(AA 384-396; SEQ ID NO: 406)ALPPL2 epitope:QKKDKLGPETFHuman-x-[ALPPL2_H]-Fab-(9G10)-IgGl / (AA 77-87; SEQ ID NO: 408) 1 Yes LC Kappa GGYPLRGSSIF(AA 384-394; SEQ ID NO: 409)ALPPL2 epitope:IHHuman-x- [ALPPL2 H] -Fab-( 16B5)-IgGl / (AA 297-298) 7 Yes Kappa TLDPSL(AA 302-307; SEQ ID NO: 412)ALPPL2 epitope:Human-x-[ALPPL2_H]-Fab-(17A7)-IgGl / APGKAR 4 Yes Kappa(AA 397-402; SEQ ID NO: 413)ALPPL2 epitope:PVEHuman-x-[ALPPL2_H]-mAb-(21H7)-IgGl (AA 22-24) 4 Yes / Kappa QKKDKLGPETF(AA 77-87; SEQ ID NO: 414)Human-x-[ALPPL2_H]-mAb-(12Fl)-IgGl / ND 4 YesKappa26092(equivalent to 9G10)ALPPL2 epitope:KLQPAQTAAKNHuman-x-[ALPPL2_H]-mAb-(13H8) -IgGl (AA 44-54; SEQ ID NO: 415) 10 No / Kappa NPRGFFL(AA 321-327; SEQ ID NO: 416)ALPPL2 epitope:KLQPAQTAAKNHuman-x-[ALPPL2_H]-mAb-(14B8.1) - (AA 44-54; SEQ ID NO: 415) 5 No IgGl / Kappa NPRGFFL(AA 321-327; SEQ ID NO: 416)ALPPL2 epitope:Humanized-x-[ALPPL2_H]-mAb-(PLAPAPGKARDRKAYTVLL ND NDh2)-IgGl L234A L235A D265S / LC Kappa(AA 397-411; SEQ ID NO: 407)ALPP epitope:LDPSVTHLMGLFEPGDMKYEIHRHumanized mAb 12F3* 7 Yes DSTLDPSLMEMTEAALRLLS(AA 280-321; SEQ ID NO: 410)Amino acid (AA) positions according to the amino acid sequence shown for the proprotein + signal sequence.*Humanized mAb 12F3 has been disclosed in International Patent Application Publication No. WO2022197890 and the amino acid sequence of the epitope it binds is shown in Fig. 25 thereof. Note that amino acid R in SEQ ID NO: 410 of ALPP is the amino acid L in the corresponding amino acid sequence of ALPPL2 (See SEQ ID NO: 411).ND: not determinedSEQUENCES SEQ Description SequenceID (CH and CH’ amino acid numbering is according to Eu NO: numbering scheme; CDRs are defined according to Kabat) 1 24F4 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPG (VH) KGLEWVSAISISGTYTYYADSVKGRFTISRDNSENTLFLQMN SLRAEDTAVYYCAKAHSTYPYYYYYYMDVWGKGTTVTVSS2 24F4 KIVMTQS PATLSLSPGERATLSCRASQSVSSSYLSWYQQKPG (VL) QAPRLLMYGTSTRATDIPARFSGSGSGTDFTLTISSLQPEDF AVYYCHQDYNFPLTFGGGTKVEIK3 24F4 SYAMS(VH-CDR1)4 24F4 AISISGTYTYYADSVKG(VH-CDR2)5 24F4 AHSTYPYYYYYYMDV(VH-CDR2)6 24F4 RASQSVSSSYLS(VL-CDR1)7 24F4 GTSTRAT(VL-CDR2)8 24F4 HQDYNFPLT(VL-CDR3)9 10B3 (VH) EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYTMKWVRQAPGKGLDWVSAISASGGSTSYSDSLKGRFTISRDNSKNTVNLQMNSLRTEDSAVYYCAKDPLPYNWNFFYYYMDVWGKGTTVTVSS2609210 10B3 (VL) EIVLTQS PGTLSLSPGERATLSCRASQSVSNSYLAWYQQKPG QAPRLIIYGASSRAAGIPDRFSGSGSGTDFTLTISRLEPEDF AIYHCQQYGRSPLTFGGGTKVEIK11 10B3 (VH- IYTMKCDR1)12 10B3 (VH- AISASGGSTSYSDSLKGCDR2)13 10B3 (VH- DPLPYNWNFFYYYMDVCDR3)14 10B3 (VL-CDR1) RASQSVSNSYLA15 10B3 (VL-CDR2) GASSRAA16 10B3 (VL-CDR3) QQYGRSPLT17 9G10 (VH) EVQLLESGGGLVQPGGSLRLSCAASVSTFNNYAMSWVRQAPG KGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMN SLRAEDTAVYYCAKDPLPANWNYYYYMDVWGKGTTVTVSS18 9G10 EIVLTQS PATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQ (VL) APRLLIYGASNRATGIPARFSGSGSGTDFTLTISSLEPEDFA VYYCQQRSNWPLTFGGGTKVEIK19 9G10 (VH- NYAMSCDR1)20 9G10 (VH- AISGSGGSTYYADSVKGCDR2)21 9G10 (VH- DPLPANWNYYYYMDVCDR3)22 9G10 (VL- RASQSVSSYLACDR1)23 9G10 (VL- RGASNRATCDR2)24 9G10 (VL- QQRSNWPLTCDR3)25 8A7 (VH) EVQLMDSGGDLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPG KGLEWVANINQDGSEKNYVDSVQGRFTISRDNAKKSLYLQMN SLRAEDTAVYYCARDYSNYDDYYYYFYMDVWGKGTTVTVSS26 8A7 (VL) AIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGK APKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCLQDYNYPWTFGQGTKVEIK27 8A7 (VH-CDR1) SYWMS28 8A7 (VH-CDR2) NINQDGSEKNYVDSVQG29 8A7 (VH-CDR3) DYSNYDD YYYYFYMDV30 8A7 (VL-CDR1) RASQGIRNDLG31 8A7 (VL-CDR2) AASSLQS32 8A7 (VL-CDR3) LQDYNYPWT33 16H2 (VH) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMTWVRQSPG KGLEWVANIKQDGSEENYVDSVKGRFTISRDNAKNSLYLQMN S L RADDT AVY Y CARDTVTTGFYHYFYMDVWGKGT TVTVS S34 16H2 (VL) AIQMTQSPSSLSASVGDRVTIACRASQGIRDDLAWFQQKPGK APKVLIYAASNLQSGVPSRFSGSGSGTDFTLTIRSLQPEDSA TYYCLQDYIYPYTFGQGTRLEIK35 16H2 (VH- SYWMTCDR1)2609236 16H2 (VH- NIKQDGSEENYVDSVKGCDR2)37 16H2 (VH- DTVTTGFYHYFYMDVCDR3)38 16H2 (VL- RASQGIRDDLACDR1)39 16H2 (VL- AASNLQSCDR2)40 16H2 (VL- LQDYIYPYTCDR3)41 11C7 (VH) QVQLVESGGGWQPGRSLRLSCAASGLTFSSYGMHWVRQAPG KGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMN SLRAEDTAVYYCAKENNWHLNYFFYYMDVWGKGTTVTVSS42 11C7 (VL) AIQLTQSPSSLSASVGDRVTITCRASQGISNALAWYQQKPGK APKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQFNNYIYTFGQGTKLEIK43 11C7 (VH- SYGMHCDR1)44 11C7 (VH- VIWYDGSNKYYADSVKGCDR2)45 11C7 (VH- ENNWHLNYFFYYMDVCDR3)46 11C7 (VL-CDR1) RASQGISNALA47 11C7 (VL-CDR2) DASSLES48 11C7 (VL-CDR3) QQFNNYIYT49 5A1 (VH) QVQL VE S G G GWQ P G R S L RL S CAAS G FT F S SYGMH WVRQA P G KGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLFLQMN SLRAEDTAVYYCAKENNWHLNYYFYYMDVWGKGTTVTVSS50 5A1 (VL) AIQLTQSPSSLSASVGDRVTITCRASQDINNVLAWYQQKPGK APKLLIYDASSLESGVPLRFRGSGSGTDFTLTINSLQPEDFA NYYCQHFNDFIYTFGQGTKLEIK51 51 5A1 (VH-CDR1) SYGMH52 5A1 (VH-CDR2) VIWYDGSNKYYADSVKG53 5A1 (VH-CDR3) ENNWHLNYYFYYMDV54 5A1 (VL-CDR1) RASQDINNVLA55 5A1 (VL-CDR2) DASSLES56 5A1 (VL-CDR3) QHFNDFIYT57 8G6 (VH) QVQLVESGGGWQPGRSLRLSCAASGLTFSSYGMHWVRQAPG KGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMN SLRAEDTAVYYCARENNWNGFYNFFYMDVWGKGTTVTVSS58 8G6 (VL) AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGK APKLLIYDASSLEGGVPSRFSGSGSGTDFTLTIRSLQPEDFA TYYCQQFNNDLYTFGQGTKLEIK59 8G6 (VH-CDR1) SYGMH60 8G6 (VH-CDR2) AVIWYDGSNKYYADSVKG61 8G6 (VH-CDR3) SENNWNGFYNFFYMDV62 8G6 (VL-CDR1) RASQGISSALA63 8G6 (VL-CDR2) DASSLEG64 8G6 (VL-CDR3) QQFNNDLYT2609265 50F5 EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPG (VH) KGLEWVANIKQDGSEKNYVDTVKGRFTISRDNAKNSLYLLMN SLRAEDTAVYYCARDYDFWNGYYLYYSMDVWGTGTTVTVSS66 50F5 AIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGK (VL) APNLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCLQDYNYPYTFGQGTKLEIK67 50F5 RYWMS(VH-CDR1)68 50F5 NIKQDGSEKNYVDTVKG(VH-CDR2)69 50F5 DYDFWNGYYLYYSMDV(VH-CDR3)70 50F5 RASQGIRNDLG(VL-CDR1)71 50F5 AASSLQS(VL-CDR2)72 50F5 LQDYNYPYT(VL-CDR3)73 5E8 (VH) QVQLVESGGGWQPGRSLRLSCSASGFTFSSYGMHWVRQAPG KGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMN SLSADDTALYYCARENNWNGYYHYYYMDVWGKGTTVTVSS74 5E8 (VL) EIVLTQS PATLSLSPGERATLSCGASQSVSSSYLAWYQQKPG LAPRLLIYDASSRASGIPDRFRGSGSGTDFTLTISRLEPEDF AVYYCQQYGSSLYTFGQGTKLQIK75 5E8 (VH-CDR1) SYGMH76 5E8 (VH-CDR2) VIWYDGSNKYYADSVKG77 5E8 (VH-CDR3) ENNWNGYYHYYYMDV78 5E8 (VL-CDR1) GASQSVSSSYLA79 5E8 (VL-CDR2) DASSRAS80 5E8 (VL-CDR3) QQYGSSLYT81 14A11 (VH) QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMHWVRQAPG KGLEWVAFIWYDGSKKYYADSVKGRFTISRDNSKNTLYLQMN SLRVEDTAVYYCARENNWNGYYNFYYMDVWGKGTTVIVSS82 14A11 (VL) AIQLTQSPSSLSASVGDRVTITCRASQGIRSALAWYQQKPGK APTLLIYDASSLESGVPRRFSGSGSGTDFTLTISSLQPEDFA IYYCQQFNNYYTFGQGTKLEIK83 14A11 (VH- SYGMHCDR1)84 14A11 (VH- FIWYDGSKKYYADSVKGCDR2)85 14A11 (VH- ENNWNGYYNFYYMDVCDR3)86 14 All (VL- RASQGIRSALACDR1)87 14A11 (VL- DASSLESCDR2)88 14A11 (VL- RQQFNNYYTCDR3)2609289 19A3 (VH) QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTTSRDNSKNTLDLQMNSLRAEDTAVYYCARENNWNGYYYYYYMDVWGKGTTVTVSS90 19 A3 (VL) E I VL T Q S P AT L S L S P G E RAT L S C GAS Q S VSNN YL AW Y Q Q K P G LAPRLLIYDASSRATGIPVRFSGSGSGTDFTLSISRLEPEDF AVYYCQQYGSSLYTFGQGTKLEIK91 19A3 (VH- SYGMHCDR1)92 19A3 (VH- VIWYDGSNKYYADSVKGCDR2)93 19A3 (VH- ENNWNGYYYYYYMDVCDR3)94 19 A3 (VL- GASQSVSNNYLACDR1)95 19 A3 (VL- DASSRATCDR2)96 19 A3 (VL- QQYGSSLYTCDR3)97 15B6 (VH) QVQLVESGGGVVQPGRSLRLSCAASGFTFSPYAMHWVRQAPGKGLEWVAVIWYDTINKYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAMYYCARENNWNGYYSYYYMDVWGKGTKVTVSS98 15B6 (VL) ETVLTQS PATLSLSPGERATLSCGASQSISGSYLAWYQQKPG LAPRLLLYDASSRATGTPVRFSGSGSGTDFTLTLSRLEPEDF AVYYCQHYENSLYTFGQGTKLETR99 15B6 (VH- PYAMHCDR1)100 15B6 (VH- VIWYDTINKYYADSVKGCDR2)101 15B6 (VH- ENNWNGYYSYYYMDVCDR3)102 15B6 (VL-CDR1) GASQSISGSYLA103 15B6 (VL-CDR2) DASSRAT104 15B6 (VL-CDR3) QHYENSLYT105 6E6 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARENNWNNYYHFYYMDVWGKGTAVTVSS106 6E6 AIQLTQSPSSLSASVGDRVTITCRASQDISRVLAWYQQKPGK (VL) APKLLTYDASSLESGVPRRFSGRGSGTEFTLTTSNLQPEDFA TYFCQQFNNYMYTFGQGTKLEIK107 6E6 SYGMH(VH-CDR1)108 6E6 VIWYDGSNKYYVDSVKG(VH-CDR2)109 6E6 ENNWNNYYHFYYMDV(VH-CDR3)110 6E6 RASQDISRVLA(VL-CDR1)111 6E6 DASSLES(VL-CDR2)112 6E6 QQFNNYMYT26092(VL-CDR3)113 17H6 (VH) QVQLQESGPGLVKPSETLSLTCTVSGGSI ISYYWTWIRQPPG KGLEWIGYIYYSGSANYNPSLKGRVTTSVDTSKSQFSLKLSS VTAADTAVYYCARGRRGYSGNGDYYYYMDVWGKGTTVTVSS114 17H6 (VL) EIVMTQS PATLSLSPGERATLSCRASQSISNNYLSWYQQIYG QAPRLLIYGASTRATGIPARFSGSGSGTDFTLTISSLQPEDF AVYYCQQDFNLPLTFGGGTKVEIK115 17H6 (VH- SYYWTCDR1)116 17H6 (VH- YIYYSGSANYNPSLKGCDR2)117 17H6 (VH- GRRGYSGNGDYYYYMDVCDR3)118 17H6 (VL- RASQSISNNYLSCDR1)119 17H6 (VL- GASTRATCDR2)120 17H6 (VL- QQDFNLPLTCDR3)121 10F6 (VH) EVQLLESGGGWQPGGSLRLSCVVSGFTFSNFAMGWVRQAPG RGLEWVARISSSGRDTFYTDSVKGRFSISRDNSNNTLYLQLS SLRAEDTAVYYCASLGPVWGNGTTVTVSS122 10F6 (VL) DIQMTQSPSSLSASIGDTVTITCRASQDIRYDLGWYQQKPGK APKRLIYGASSLHSGVPSRFSGSRSGTEFTLTISSLQPEDFA TYYCLQLNNFPYTFGQGTKLEIR123 10F6 (VH-CDR1) NFAMG124 10F6 (VH-CDR2) RISSSGRDTFYTDSVKG125 10F6 (VH-CDR3) LGPV126 10F6 (VL-CDR1) RASQDIRYDLG127 10F6 (VL-CDR2) GASSLHS128 10F6 (VL-CDR3) LQLNNFPYT129 4F4 (VH) EVQLVESGGGLVQPGGSLRLSCVASGLTISGYWMTWVRQAPG KGL E WVANINQDGNEKNYVD SVKGR FT I S RDNAKN S L Y L QMN S L RAE DT A I Y Y C VKDT SN YD F Y Y Y YFYMD VWG KG T T VT VS S130 4F4 (VL) AIQMTQSPSSLSASVGDRVTITCRASQNIRNDLGWYQQKPGK APKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCLQDYNYPYTFGQGTKLEIK131 4F4 (VH-CDR1) GYWMT132 4F4 NINQDGNEKNYVDSVKG(VH-CDR2)133 4F4 (VH-CDR3) DT SNYDF YYYYFYMDV134 4F4 (VL-CDR1) RASQNIRNDLG135 4F4 (VL-CDR2) AASSLQS136 4F4 (VL-CDR3) LQDYNYPYT137 4B10 EVQLLESGGGLVQPGGSLRLSCAASGITISSYAMNWVRQAPG (VH) KGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMN S LRAE DT AVY Y CAKDPLPANWNYFYYMDVWGKGT TVTVS S138 4B10 EIVLTQS PATLSLSPGERATLSCRASQSVSTYLVWYQQKPGQ (VL) APRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLDPEDFAGYYCQQRSNWPLTFGGGTKVEIK26092139 4B10 SYAMN(VH-CDR1)140 4B10 AISGSGGSTYYADSVKG(VH-CDR2)141 4B10 DPLPANWNYFYYMDV(VH-CDR3)142 4B10 RASQSVSTYLV(VL-CDR1)143 4B10 DASNRAT(VL-CDR2)144 4B10 QQRSNWPLT(VL-CDR3)145 49G5 (VH) QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPAG KGLEWIGRIYSSGSTNYNPSLKSRVTMSEDTSKNQISLNLSS VTAADTAVY YCARFSMVRGVMGYMDVWGKGT TVTVS S146 49G5 (VL) EIVMTQS PATLSLSPGERATLSCRASQSISSSYLSWYQQTPG QAPRLLIYGVSSRATGIPARFSGSGSGTDFTLTISSLQPEDF AVYYCQQDYNLPYTFGQGTKLEIE147 49G5 (VH- SYYWSCDR1)148 49G5 (VH- RIYSSGSTNYNPSLKSRCDR2)149 49G5 (VH- FSMVRGVMGYMDVCDR3)150 49G5 (VL- RASQSISSSYLSCDR1)151 49G5 (VL- GVSSRATCDR2)152 49G5 (VL- QQDYNLPYTCDR3)153 7C8 (VH) QVQLVESGGGVVQPGRSLRLSCEASGLTFSVYGMHWVRQAPGKGLEWVAVIWYDGTNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYHCARENNWNAYYHFHYMDVWGKGTSVTVSS154 7C8 (VL) AIQLTQSPSSLSASVGDRVTIICRASQGISSALAWYQQAPGK APKLLIYDASRLESGVPLRFSGSGSGTDFTLTISSLQPEDFA T Y Y CQQFNNNL YT FGQGT KL E I K155 7C8 (VH-CDR1) VYGMH156 2.7C8 VIWYDGTNKYYADSVKG(VH-CDR2)157 7C8 (VH-CDR3) ENNWNAYYHFHYMDV158 7C8 (VL-CDR1) RASQGISSALA159 7C8 (VL-CDR2) DASRLES160 7C8 (VL-CDR3) QQFNNNLYT161 9B12 (VH) EVQLVESGGGLVQPGGSLRLACAASGFTISSYWMSWVRQAPG KGLEWVANINQDGSEKYYVDSVKGRFT I S RDNAKNS LS LQMN S L RAE DT AVY Y C ARDASN YD G Y Y Y YF YMD VWG KGTTVTVSS162 9B12 (VL) AIQMTQSPSSLSASVGDRVTITCRTSQGIRNDLGWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTDFALTISSLQPEDFATYYCLQDSNYPYTFGQGTKLEIK26092163 9B12 (VH- SYWMSCDR1)164 9B12 (VH- NINQDGSEKYYVDSVKGCDR2)165 9B12 (VH- DASNYDGYYYYFYMDVCDR3)166 9B12 (VL-CDR1) RTSQGIRNDLG167 9B12 (VL-CDR2) AASNLQS168 9B12 (VL-CDR3) LQDSNYPYT169 16B5 (VH) QVQLQESGPGLVKPSETLSLTCTVSGGSI ISYYWTWIRQPPG KGLEWIGYIFYSGSTNYNPSLKGRVTLSVDTSKNQFSLKLSS VTAADTAVYYCARGRRGYRGNGDYYYYMDVWGKGTTVTVSS170 16B5 (VL) EILMTQSPATLSLSPGERATLSCRASQSISDSYLSWYQQIPG QAPRLLIYGASTRATGVPARFSGSGSGTDFTLTISSLQPEDF AVYYCQQDFNLPLTFGGGTKVEIK171 16B5 (VH- SYYWTCDR1)172 16B5 (VH- YIFYSGSTNYNPSLKGCDR2)173 16B5 (VH- GRRGYRGNGDYYYYMDVCDR3)174 16B5 (VL-CDR1) RASQSISDSYLS175 16B5 (VL-CDR2) GASTRAT176 16B5 (VL-CDR3) QQDFNLPLT177 3G8 (VH) QVQLVESGGGWQPGRSLRISCAASGLTFSSYGMHWVRQAPG KGLEWVAIIWYDGTNKYYADSVKGRFTISRDNSKNTLYLQMN S LRAE DT AVY Y CARENNWNGYYHFYYMDVWGKGT TVTVS S178 3G8 (VL) AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGK VPKLLIYDASSLESRVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQFNNYLYTFGQGTKLEIK179 3G8 (VH-CDR1) SYGMH180 3G8 (VH-CDR2) I IWYDGTNKYYADSVKG181 3G8 (VH-CDR3) ENNWNGYYHFYYMDV182 3G8 (VL-CDR1) RASQGISSALA183 3G8 (VL-CDR2) DASSLES184 3G8 (VL-CDR3) QQFNNYLYT185 17E4 QVQLVQS GAEVKKPGASVKVS CKVS E YTLTDLSMYWVRQAPG (VH) KGLEWMGGFDPEAGETIYAQKFQGRVTMTEDTSTDTAYMELS SLRSEDTAVYYCAIGRYCFSTSCSFNYNYYMDVWGKGTTVTV SS186 17E4 DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWFQQKPGK (VL) APKSLIYDASSLQSGVPSKFTGSGSGTDFTFTISSLQPEDFA TYYCQQYNSFPPTFGQGTKVEIK187 17E4 DLSMY(VH-CDR1)188 17E4 GFDPEAGETIYAQKFQG(VH-CDR2)189 17E4 GRYCFSTSCSFNYNYYMDV(VH-CDR3)190 17E4 RASQGISNYLA26092(VL-CDR1)191 17E4 DASSLQS(VL-CDR2)192 17E4 QQYNSFPPT(VL-CDR3)193 25G8 (VH) EVQLLESGGGLVQPGGSLRLSCAASGFIFSSYTMKWVRQAPG KGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMN S L RAE DT AVY Y CAKDPLPYNWSFYYYYMDVWGKGTTVTVSS194 25 G8 (VL) EIVLTQS PGTLSLSPGERATLSCRASQSVSSSYLAWYHQKPG QAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDF AVYYCQQYGSSPLTFGGGTKVEIK195 25G8 (VH- SYTMKCDR1)196 25G8 (VH- AISGSGGSTYYADSVKGCDR2)197 25G8 (VH- DPLPYNWSFYYYYMDVCDR3)198 25G8 (VL- RASQSVSSSYLACDR1)199 25 G8 (VL- GASSRATCDR2)200 25 G8 (VL- QQYGSSPLTCDR3)201 16C2 QVQLVQS GAEVKKPGASVKVS CKAS GFTLTTYGFNWVRQAPG (VH) QGL E WMGWI SAYNGDTRYAQKFQGRVTMT TDTSTSTAYMELW S LRS DDT AVYYCARGTTVTTPYYYYSYMDVLGKGTTVTVS S202 16C2 EIVMTQS PATLSVSPGERATLSCRASQSVGSFLAWYQQKPGQ (VL) APRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFA VYYCQQYINWPLTFGGGTKVEIK203 16C2 TYGFN(VH-CDR1)204 16C2 WI SAYNGDTRYAQKFQG(VH-CDR2)205 16C2 GTTVTTPYYYYSYMDV(VH-CDR3)206 16C2 RASQSVGSFLA(VL-CDR1)207 16C2 GASTRAT(VL-CDR2)208 16C2 QQYINWPLT(VL-CDR3)209 21H7 (VH) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPG KGLEWVAAISGSGGSSKNGNSVKGRFTISRDNSKNTLYLLMSILRAEDTAIYYCAKGTVVPVVPGDHMDVWGKGTTVTVSS 210 21H7 (VL) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGK APRLLIYDASSLQSGVPSRFSGRGSGTDFTLTISSLQPEDFA TYYCQQSYSTPPTFGQGTKVEIK211 21H7 (VH- SYAMSCDR1)26092212 21H7 (VH- AISGSGGSSKNGNSVKGCDR2)213 21H7 (VH- GTVVPVVPGDHMDVCDR3)214 21H7 (VL- RASQSISSYLNCDR1)215 21H7 (VL- DASSLQSCDR2)216 21 H7 (VL- QQSYSTPPTCDR3)217 32H6 (VH) EVQLLESGGGLVQPGGSLRLSCAASGFTFGNYAMSWVRQAPG RGLEWVSGVRNSGSGTYYADSVKGRFTISRDNSKNTLYLQMN S LRAE DT AVY Y CAKGGTPVTAPYYYYYYMDVWGKGT S VAVS S218 32H6 DIVMTQTPLSLSVTPGQPASISCKSSQSLLHSDGKTYLYWYL (VL) QKPGQPPQLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVE AEDVGVYYCMQSIQLPYTFGQGTKLEIK219 32H6 NYAMS(VH-CDR1)220 32H6 GVRNSGSGTYYADSVKG(VH-CDR2)221 32H6 GGTPVTAPYYYYYYMDV(VH-CDR3)222 32H6 KSSQSLLHSDGKTYLY(VL-CDR1)223 32H6 EVSNRFS(VL-CDR2)224 32H6 MQSIQLPYT(VL-CDR3)225 2A2 (VH) EVQLVESGGGLVQPGGSLRLSCAASGFTISPYWMTWVRQAPG KGLEWVANINQDGSEKHYVDSVKGRFTISRDNVHNSLFLQMN S L RAE DT AVY Y CVRDDSVYDS YYYYFYMDVWGKGTTVT VS S226 2A2 (VL) AIQMTQSPSSLSASVGDRVTISCRASQGIRDDLGWYQQKPGK APELLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCLQDYTYPYTFGQGTKLEIK227 2A2 (VH-CDR1) TISPYW228 2A2 (VH-CDR2) NINQDGSEKHYVDSVKG229 2A2 (VH-CDR3) DDSVYDSYYYYFYMDV230 2A2 (VL-CDR1) RASQGIRDDLG231 2A2 (VL-CDR2) AASSLQS232 2A2 (VL-CDR3) LQDYTYPYT233 12C6 (VH) QVQLVQS GPEVKKPGASVKVS CKVS GYTLTELSMHWVRQAPG KGLEWMGGFDPEDGGTIFAQKFQGRVTMTEDTSTDTAYMELS S LRS E DT AVY Y CAGWGS YYRWFDPWGQGT LVTVS S234 12C6 (VL) EIVMTQS PATLSLSPGERATLSCRASQSVSSTSFSWYQQKPG QAPRLLIFGASTRATGIPARFSGSGSGTDFTLTISSLQPEDF AVYYCHQDYNLPFTFGPGTKVDIK235 12C6 (VH- ELSMHCDR1)236 12C6 (VH- GFDPEDGGTIFAQKFQGCDR2)26092237 12C6 (VH- WGSYYRWFDPCDR3)238 12C6 (VL-CDR1) RASQSVSSTSFS239 12C6 (VL-CDR2) GASTRAT240 12C6 (VL-CDR3) HQDYNLPFT241 12F1 (VH) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPG KGL E WVANINQDGNEKNYVD SVKGR FT I S RDNVKN S LH L QMN SLRAEDSAVYFCARDTSNYDLYSYYFYMDVWGKGTTVTVSS242 12F1 (VL) AIQMTQSPSSLSASVGDRVTITCRASQGIRNDLAWYQQKPGK APNLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCLQDYIYPYTFGQGTKLEIK243 12F1 (VH-CDR1) SYWMS244 12F1 (VH-CDR2) NINQDGNEKNYVDSVKG245 12F1 (VH-CDR3) DTSNYDLYSYYFYMDV246 12F1 (VL-CDR1) RASQGIRNDLA247 12F1 (VL-CDR2) AASSLQS248 12F1 (VL-CDR3) LQDYIYPYT249 16F2 (VH) QVQLVESGGGWQPGRSLRLSCAASGLTFSSYGMHWVRQAPG MGLEWVALIWYDGSNEYYADSVKGRFTISRDNFKNTLYLQMN S LRAE DT AVY Y CARENNWNGRYYFYYMDVWGKGT TVTVS S250 16F2 (VL) AIQLTQSPSSLSASVGDRVTITCRASQGISNTLAWYQQKPGK PPKLLIYDASRLEGGVPLRFSGSGSGTDFTLTISSLQPEDFA TYYCQQFNNYPTFGGGTKVEIK251 16F2 (VH-CDR1) SYGMH252 16F2 (VH-CDR2) LIWYDGSNEYYADSVKG253 16F2 (VH-CDR3) ENNWNGRYYFYYMDV254 16F2 (VL-CDR1) RASQGISNTLA255 16F2 (VL-CDR2) DASRLEG256 16F2 (VL-CDR3) QQFNNYPT257 14B12 (VH) EVQLLESGGGLVQPGGSLRLSCAASGFIFSSYTMKWVRQAPG KGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLHVN SLRAEDTAVYYCAKDPLPYNWNFYYYYMDVWGKGTTVTVSS258 14B12 (VL) EIVLTQS PGTLSLSPGERATLSCRASQSVSSSYLAWYHQKPG QAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDF AVYYCQQYGSSPLTFGGGTKVEIK259 14B12 (VH- SYTMKCDR1)260 14B12 (VH- AISGSGGSTYYADSVKGCDR2)261 14B12 (VH- DPLPYNWNFYYYYMDVCDR3)262 14B12 (VL- RASQSVSSSYLACDR1)263 14B12 (VL- GASSRATCDR2)264 14B12 (VL- QQYGSSPLTCDR3)265 10G2 (VH) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYALSWVRQTPGKGLEWVSVISGNGIITYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDDSSYYGLGSFPNWFDPWGQGTLVTVSS26092266 10G2 (VL) AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGK TPKLLIYDASSLESRVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQFNNYPYSFGQGTKLEIK267 10G2 (VH- SYALSCDR1)268 10G2 (VH- VI SGNGI ITYYADSVKGCDR2)269 10G2 (VH- DDSSYYGLGSFPNCDR3)270 10G2 (VL- RASQGISSALACDR1)271 10G2 (VL- DASSLESCDR2)272 10G2 (VL- QQFNNYPYSCDR3)273 6E10 (VH) EGQLLESGGGLVQPGGSLRLSCAASGFTFNNYAMSWVRQAPG KGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMN S LRAE DT AVY Y CAKDGGVPVVPYLYYYYMDVWGKGTTVTVSS274 6E10 (VL) E I VMT QS P AT L S VS P G E RAT L S CRASQSVNNNLAWY QQKP GQ APRLLIFGASTRATGLPARFSGSGSGTEFTLTISSLQSEDFA VYYCQQYNNWPFTFGQGTKLEIK275 6E10 (VH- NYAMSCDR1)276 6E10 (VH- AISGSGGSTYYADSVKGCDR2)277 6E10 (VH- DGGVPVVPYLYYYYMDVCDR3)278 6E10 (VL-CDR1) RASQSVNNNLA279 6E10 (VL-CDR2) GASTRAT280 6E10 (VL-CDR3) QQYNNWPFT281 10A9 (VH) EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYTMKWVRQAPG KGLEWVSAISASGGSTSYSDSVKGRFTISRDNSKNTVNLQMN SLRTEDSAVYYCAKDPLPYNWSFFYYYMDVWGKGTTVTVSS282 10A9 EIVMTQS PATLSLSPGERATLSCRASQSVSSSYLSWYQQKPG (VL) QAPRLLIYGASTRATGIPARFSGSGSGTDFTLTISSLQPEDF AVYYCQQDYNLITFGQGTRLEIK283 10A9 IYTMK(VH-CDR1)284 10A9 Al SASGGSTSYSDSVKG(VH-CDR2)285 10A9 DPLPYNWSFFYYYMDV(VH-CDR3)286 10A9 RASQSVSSSYLS(VL-CDR1)287 10A9 GASTRAT(VL-CDR2)288 10A9 QQDYNLIT(VL-CDR3)26092289 35D5 (VH) EVQLLESGGGLVQSGGSLRLSCVASGFIFNTYVMKWVRQAPG RGLEWVSAISGSGGSTSYTDSVKGRFTVSRDNSKNTLYLQMT S L RAE DT AVY Y CARD PL P YNWS F YF Y YMD VWG KGTTVTVSS290 35D5 (VL) EIVLTQS PGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPG QAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDF AVYYCQQYGSSPLTFGGGTKVELK291 35D5 (VH- TYVMKCDR1)292 35D5 (VH- Al SGSGGSTSYTDSVKGCDR2)293 35D5 (VH- DPLPYNWSFYFYYMDVCDR3)294 35D5 (VL- RASQSVSSSYLACDR1)295 35D5 (VL- GASSRATCDR2)296 35D5 (VL- QQYGSSPLTCDR3)297 17A7 (VH) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMTWVRQAPG KGLEWVANIKQDGSEKHYVDSVKGRFTISRDNAKNSLYLQMN SLRAEDTAVYYCARDTSNYDLYYYYFYMDVWGKGTTVTVSS298 17A7 (VL) AIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGK APKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCLQDNSYPYTFGQGTKLEIK299 17A7 (VH- SYWMTCDR1)300 17A7 (VH- NIKQDGSEKHYVDSVKGCDR2)301 17A7 (VH- DT SNYDL YYYYFYMDVCDR3)302 17A7 (VL- RASQGIRNDLGCDR1)303 17A7 (VL- AASTLQSCDR2)304 17A7 (VL- LQDNSYPYTCDR3)305 18A11 (VH) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPG KGLEWVANVNQDGSEQNFVDSVKGRFT I S RDNAKNSVHLQMN SLRAEDTAVYYCARDASNYDGYYYYYYTDVWGKGTTVTVSS306 18 All (VL) AIQMTQSPSSLSASVGDRITITCRASQGIRNDLGWYQQKPGK APKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCLQDYNYPYTFGQGTKLEIK307 18A11 (VH- SYWMSCDR1)308 18A11 (VH- NVNQDGSEQNFVDSVKGCDR2)309 18A11 (VH- DASNYDGYYYYYYTDVCDR3)310 18A11 (VL- RASQGIRNDLGCDR1)26092311 18A11 (VL- AASSLQSCDR2)312 18A11 (VL- DYNYPYTCDR3)313 49G11 (VH) EVQLLESGGGLVQPGGSLRLSCAASGFTFRSYVMSWVRQAPG KGLEWVSAISGSGDRTYYADSVKGRFTISRDNSKNTVYLQVK SLRAEDTAGYYCAKAAGYCTNGVCLYYYYMDVWGKGTTVTVSS314 49G11 (VL) QLVLTQSPSASASLGASVKLTCTLSSGHSSYAIAWHQQQPEK GPRYLMKLNSDGSHSKGDGIPDRFSGSSSGAERYLTISSLQS EDEADYYCQTWGTGIRVFGGGTKLTVL315 49G11 (VH- SYVMSCDR1)316 49G11 (VH- Al SGSGDRTYYADSVKGCDR2)317 49G11 (VH- AAGYCTNGVCLYYYYMDVCDR3)318 49G11 (VL- TLSSGHSSYAIACDR1)319 49G11 (VL- LNSDGSHCDR2)320 49G11 (VL- QTWGTGIRVCDR3)321 3A9 (VH) QLQLQESGPGLVKPSETLSLTCTVSGGSISSGSDYWVWIRQP PGKGLEWIGSIYYSGSTYYNPALKSRVTISVDTSKNQFSLKL SSVTAADTAVYYCARRGNDDYYYFYMDVWGKGTTVTVSA322 3A9 (VL) EIVMTQS PATLSLSPGERATLSCRASQSVRSSYLSWYQQKPG QAPRLLIYGASTRATGIPARFSGSGSGTDFTLTISSLQPEDF AVY Y CQQD YNL PMYT F G QGT KL E I K323 3A9 (VH-CDR1) GSDYWV324 3A9 (VH-CDR2) SIYYSGSTYYNPALKS325 3A9 (VH-CDR3) RGNDDYYYFYMDV326 3A9 (VL-CDR1) RASQSVRSSYLS327 3A9 (VL-CDR2) GASTRAT328 3A9 (VL-CDR3) QQDYNLPMYT329 13H8 (VH) EVHLLESGGGLVQPGGSLRLSCAASGFTFSTYAMSWVRQAPG KGLEWVSAISGSGGSRYYADSVKGRFTISRDNSKNTLYLQMN S LRAE DT AVY Y CAKDGGVPVVPYLYYYYMDVWGKGTTVTVSS340 13H8 (VL) KIVMTQS PATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQ APRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFA VYYCQHYNNWPFTFGQGTKLEIK341 13H8 (VH- TYAMSCDR1)342 13H8 (VH- Al SGSGGSRYYADSVKGCDR2)343 13H8 (VH- DGGVPVVPYLYYYYMDVCDR3)344 13H8 (VL- RASQSVSSNLACDR1)26092345 13H8 (VL- GASTRATCDR2)346 13H8 (VL- QHYNNWPFTCDR3)347 14B8.1 (VH) QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMHWVRQVPG KGLEWVAFISYDGKNKYYIDSVRGRFTISRDNSKNTLFLQMN S LRAE DT AVY Y CARENNWNDFYNYYYMDVWGKGT TVTVS S348 14B8.1 (VL) AIQLTQSPSSLSASVRDRVTITCRASQGINSALAWYQQKPGK APKLLIYDASRLESRVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQFNNYMYTFGQGTKLEIK349 14B8.1 (VH- SYGMHCDR1)350 14B8.1 (VH- FI SYDGKNKYYIDSVRGCDR2)351 14B8.1 (VH- ENNWNDFYNYYYMDVWCDR3)352 14B8.1 (VL- RASQGINSALACDR1)353 14B8.1 (VL- DASRLESCDR2)354 14B8.1 (VL- QQFNNYMYTCDR3)355 7F11.1 (VH) QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPAG KGLEWIGRIYTSGSTNYNPSLKSRVTMSGDTSKNQFSLKLTS VT AADTAVY YCAREVVYYYYMDVWGKGTTVTVS S356 7F11.1 (VL) AIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGK APKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCLQDYNYPYTFGQGTKLEIK357 7F11.1 (VH- SYYWSCDR1)358 7F11.1 (VH- GRIYTSGSTNYNPSLKSCDR2)359 7F11.1 (VH- EVVYYYYMDVCDR3)360 7F11.1 (VL- RASQGIRNDLGCDR1)361 7F11.1 (VL- AASSLQCDR2)362 7F11.1 (VL- LQDYNYPYTCDR3)363 35D7.1 (VH) EVQLVESGGGLVQPGGSLRLSCAASGFSLSSYWMSWVRQAPG KGLEWVANIKQDGSEKNYVDAVKGRFTISRDNAKNSLYLHMN SLRVEDTAVYYCARDNDNWNGFYYYYSMDVWGKGTTVTVSS364 35D7.1 (VL) AIQMTQSPSSLSASVGDRVTITCRASQGIRDDLGWYQQTPGK APKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCLQDNNYPYTFGQGTKLEIK365 35D7.1 (VH- SYWMSCDR1)366 35D7.1 (VH- NIKQDGSEKNYVDAVKGCDR2)367 35D7.1 (VH- DNDNWNGFYYYYSMDVCDR3)368 35D7.1 (VL- RASQGIRDDLGCDR1)369 35D7.1 (VL- AASTLQSCDR2)370 35D7.1 (VL- LQDNNYPYTCDR3)371 13B3 (VH) EVQLVESGGGLVQPGGSLRLSCVASGFTFGPYWMTWVRQAPG KGL E WVANINQDGNEKNYVD SVKGR FT I S RDNVKN S L FL QMN SLRAEDTAVYYCVRDDSVYDSYYYYFYMGVWGGGTAVTVSS372 13B3 (VL) AIQMTQSPSSLSASVGDRVTITCRASQGIRDDLGWYQQKPGK APELLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCLQDYNYPYTFGQGTKLEIK373 13B3 (VH- PYWMTCDR1)374 13B3 (VH- NINQDGNEKNYVDSVKGCDR2)375 13B3 (VH- DDSVYDSYYYYFYMGVCDR3)376 13B3 (VL-CDR1) RASQGIRDDLG377 13B3 (VL-CDR2) AASSLQS378 13B3 (VL-CDR3) LQDYNYPYT379 Human ALPP MLGPCMLLLLLLLGLRLQLSLGIIPVEEENPDFWNREAAEAL (signal sequence: GAAKKLQ PAQT AAKNL 11 FL GDGMGVS TVT AARI LKGQKKDK 1 -22)(proprotein: LGPEI PLAMDRFPYVALSKT YNVDKHVPDSGATATAYLCGVK 23-535) GN FQT I GL S AAARFNQCNTT RGNEVI S VMNRAKKAGKS VGW (mature protein: TTTRVQHASPAGTYAHTVNRNWYSDADVPASARQEGCQDIAT 23-506) QLISNMDIDVILGGGRKYMFRMGTPDPEYPDDYSQGGTRLDG KN LVQE WL AKRQGARY VWNRT ELMQASLDPSVTHLMGLFEPG DMKYEIHRDSTLDPSLMEMTEAALRLLSRNPRGFFLFVEGGR IDHGHHESRAYRALTETIMFDDAIERAGQLTSEEDTLSLVTA DHSHVFS FGGY PLRGS S I FGLAPGKARDRKAYTVLLYGNGPG YVLKDGARPDVTESESGSPEYRQQSAVPLDEETHAGEDVAVF ARGPQAHLVHGVQEQTFIAHVMAFAACLEPYTACDLAPPAGT TDAAHPGRSWPALLPLLAGTLLLLETATAP380 Human ALPPL2 MQGPWVLLLLGLRLQLSLGI I PVEEENPDFWNRQAAEALGAA (signal sequence: KKLQPAQTAAKNLIIFLGDGMGVSTVTAARILKGQKKDKLGP l-19)(proprotein: ETFLAMDRFPYVALSKTYSVDKHVPDSGATATAYLCGVKGNF 20-532) QTIGLSAAARFNQCNTTRGNEVISVMNRAKKAGKSVGWTTT (mature protein: RVQHASPAGAYAHTVNRNWYSDADVPASARQEGCQDIATQLI 20-503) SNMDIDVILGGGRKYMFPMGTPDPEYPDDYSQGGTRLDGKNL VQEWLAKHQGARYVWNRTELLQASLDPSVTHLMGLFEPGDMK YEIHRDSTLDPSLMEMTEAALLLLSRNPRGFFLFVEGGRIDH GHHESRAYRALTETIMFDDAIERAGQLTSEEDTLSLVTADHS HVFS FGGY PLRGS S I FGLAPGKARDRKAYTVLLYGNGPGYVL KDGARPDVTESESGSPEYRQQSAVPLDGETHAGEDVAVFARG P Q AH L VH GVQE QT F I AH VMA FAAC L E P YT AC D L A P RAG T T DAAHPGPSVVPALLPLLAGTLLLLLGTATAP381 Human IgGl HC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN constant domain SGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTP EVT CVWDVS H E D PE VKFNW YVDGVEVH NAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK382 Human IgGl HC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN Constant domain SGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICN (YTE) VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF P P KPKDT L YI TRE PEVT CVWDVS H E D PEVKFNWYVDGVEVH NAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK383 Human IgGl HC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN Constant domain SGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICN (E233A / L235A) VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPALAGGPSVFLF PPKPKDTLMISRTP EVT CVWDVS H E D PE VKFNW YVDGVEVH NAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK384 Human IgGl HC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN Constant domain SGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICN (L234A L235A VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLF D265S) PPKPKDTLMISRTP EVT CVWSVS H E D PE VKFNW YVDGVEVH NAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK385 Human IgGl HC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN Constant domain SGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICN (L234AL235A VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLF P329G) PPKPKDTLMISRTP EVT CVWSVS H E D PE VKFNW YVDGVEVH NAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKA LGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK386 Human IgGl HC ASTKGPSVFPLAPSSKSTSGGTLELGCLVKDYFPEPVTVSWN Constant domain SGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICN (L235E) VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELEGGPSVFLF PPKPKDTLMISRTP EVT CVWDVS H E D PE VKFNW YVDGVEVH NAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK387 Human IgGl HC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN Constant domain SGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICN(D265A) VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP P KP KDT LM I S RT P EVT CVWAVS H E D PE VKFNW YVDGVEVH NAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK388 Human IgGl HC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN Constant domain SGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICN (D265AN297G) VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTP EVT CVWAVS H E D PE VKFNW YVDGVEVH NAKTKPREEQYGSTYRWSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK389 Human IgGl HC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN Constant domain SGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICN (N297X, wherein VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF X is any amino PPKPKDTLMISRTP EVT C WVDVS H E D PE VKFNW YVDGVEVH acid other than N) NAKTKPREEQYXSTYRWSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK390 Human IgGl HC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN Constant domain SGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICN (N297A / D356E / L VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF 358M) PPKPKDTLMISRTP EVT C WVDVS H E D PE VKFNW YVDGVEVH NAKTKPREEQYASTYRWSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK391 Human LC Kappa RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWK constant domain VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLS S PVTKS FNRGEC392 Human IgGl HC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN constant domain SGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICN (S375C) VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTP EVT C WVDVS H E D PE VKFNW YVDGVEVH NAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPCDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK393 Human IgGl HC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN Constant domain SGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICN (YTE S375C) VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF P P KPKDT L YI TRE PEVT CVWDVS H E D PEVKFNWYVDGVEVH NAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPCDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK394 Human IgGl HC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN Constant domain SGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPALAGGPSVFLF26092(E233A / L235A P P KP KDT LM I S RT P EVT CVWDVS H E D PE VKFNW YVDGVEVH S375C) NAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPCDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK395 Human IgGl HC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN Constant domain SGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICN (L234AL235A VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLF D265S S375C) PPKPKDTLMISRTP EVT CVWSVS H E D PE VKFNW YVDGVEVH NAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPCDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK396 Human IgGl HC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN Constant domain SGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICN (L234A L235A VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLF P329G S375C) PPKPKDTLMISRTP EVT CVWSVS H E D PE VKFNW YVDGVEVH NAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKA LGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPCDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK397 Human IgGl HC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN Constant domain SGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICN (L235E S375C) VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELEGGPSVFLF PPKPKDTLMISRTP EVT CVWDVS H E D PE VKFNW YVDGVEVH NAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPCDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK398 Human IgGl HC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN Constant domain SGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICN (D265A S375C) VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTP EVT CVWAVS H E D PE VKFNW YVDGVEVH NAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPCDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK399 Human IgGl HC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN Constant domain SGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICN (D265AN297G VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF S375C) PPKPKDTLMISRTP EVT CVWAVS H E D PE VKFNW YVDGVEVH NAKTKPREEQYGSTYRWSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPCDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK400 Human IgGl HC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN Constant domain SGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICN (N297X, wherein VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF X is any amino PPKPKDTLMISRTP EVT CVWDVS H E D PE VKFNW YVDGVEVH acid other than N NAKTKPREEQYXSTYRWSVLTVLHQDWLNGKEYKCKVSNKAS375C) LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV26092KGFYPCDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK401 Human IgGl HC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN Constant domain SGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICN (N297A / D356E / L VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF 358M S375C) PPKPKDTLMISRTP EVT CVWDVS H E D PE VKFNW YVDGVEVH NAKTKPREEQYASTYRWSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV KGFYPCDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK402 Leader peptide A MSVPTQVLGLLLLWLTDARC403 Leader peptide B MEWSWVFLFFLSVTTGVHS404 APPL2 epitope RQAAE AL GAAKKL Q(AA 33-46 ofALPPL2 in SEQIDNO: 380)405 ALPPL2 epitope RILKGQKKDKLGPETFL(AA 72-88 ofALPPL2 in SEQIDNO: 380)406 ALPPL2 epitope GGYPLRGSSIFGL(AA 384-396 ofALPPL2 in SEQIDNO: 380)407 ALPPL2 epitope APGKARDRKAYTVLL(AA 397-411 ofALPPL2 in SEQIDNO: 380)408 ALPPL2 epitope QKKDKLGPETF(AA 77-87 ofALPPL2 in SEQIDNO: 380)409 ALPPL2 epitope GGYPLRGSSIF(AA 384-394 ofALPPL2 in SEQIDNO: 380)410 ALPP epitope LDPSVTHLMGLFEPGDMKYEIHRDSTLDPSLMEMTEAALRLL (AA 280-321 of SALPPL2 in SEQIDNO: 379)411 ALPPL2 epitope LDPSVTHLMGLFEPGDMKYEIHRDSTLDPSLMEMTEAALLLL (AA 277-319 of SALPPL2 in SEQIDNO: 380)412 ALPPL2 epitope TLDPSL(AA 302-307 ofALPPL2 in SEQIDNO: 380)26092413 ALPPL2 epitope APGKAR(AA 397-402 ofALPPL2 in SEQIDNO: 380)414 ALPPL2 epitope QKKDKLGPETF(AA 77-87 ofALPPL2 in SEQIDNO: 380)415 ALPPL2 epitope KLQPAQTAAKN(AA 44-54 ofALPPL2 in SEQIDNO: 380)416 ALPPL2 epitope NPRGFFL(AA 321-327 ofALPPL2 in SEQIDNO: 380)417 PLAPh2 LC DIQMTQS PSSLSASVGDRVTITCRASENIYSYVAWYQQKPGK (CL=Kappa) APKLLIYNAKSLASGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQHHYVSPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLK SGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC418 PLAPh2 HC QVQLQESGPGLVKPSETLSLTCTVSGFSLTSYGVSWIRQPAG (CH=IgGl KGLEWIGVIWEDGSTNYHSALISRVTMSVDTSKNQFSLKLSS L234AL235A VTAADTAVYYCARPH YGS S YVGAME YWGAGTTVTVS S AS T KG D265S) PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK DTLMISRTP E VT C VWS VS HEDPEVKFNWYVD GVE VH N AKT K PREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK419 G4S GGGGS420 (G4S)n, n=l-5 GGGGSGGGGSGGGGSGGGGSGGGGS421 (G4S)3GGGGSGGGGSGGGGS422 (G4S)1.10GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGG GGSGGGGS423 CD 8 hinge TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDF ACDIY424 CD8 signaling IWAPLAGTCGVLLLSLVITLYCKdomain425 4-1BB RGRKKLLYI FKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL intracellulardomain426 CD3-zeta RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRD intracellular PEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKdomain GHDGLYQGLSTATKDTYDALHMQALPPR26092427 Residuestypically beforeVH-CDR2 LEIWG26092

[0215] While the present invention is described herein with reference to illustrated embodiments, it should be understood that the invention is not limited hereto. Those having ordinary skill in the art and access to the teachings herein will recognize additional modifications and embodiments within the scope thereof. Various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the claims. Therefore, the present invention is limited only by the language of the specification and the claims attached herein.

[0216] Patents, patent applications, publications, product descriptions, and protocols are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties for all purposes.

[0217] Other embodiments of the present invention are within the following claims.

Claims

WHAT IS CLAIMED IS:

1. An ALPP / L2 binder comprising a heavy’ chain variable domain (VH) comprising complementarity determining regions (CDRs) VH-CDR1, VH-CDR2, and VH-CDR3 and a light chain variable domain (VL) comprising CDRs VL-CDR1, VL-CDR2, and VL-CDR3, wherein (a) 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;(b) 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;(c) 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;(d) 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;(e) 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;(I) 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;(g) 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;(h) 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;(i) 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;(j) 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;(k) 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;(l) 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;(m) 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; (n) 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; (o) 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; (p) 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; (q) 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; (r) 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; (s) 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; (t) 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; (u) 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; (v) 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; (w) 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; (x) 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;26092(y) 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; (z) 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; (aa) 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; (bb) 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; (cc) 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; (dd) 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; (ee) 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; (ff) 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; (gg) 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,26092and 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; (hh) 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; (ii) 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; (jj) 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; (kk) 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; (11) 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; (mm) 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; (nn) 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; (oo) 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;(pp) 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; (qq) 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; (rr) 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; (ss) 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 (tt) 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.

2. The ALPP / L2 binder of claim 1, wherein the ALPP / L2 binder 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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;26092(x) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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: 274: (jj) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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.

3. The ALPP / L2 binder of claim 1. wherein the ALPP / L2 binder comprises an antibody in which the VH is covalently linked to a heavy chain constant domain (CH) comprising a CHI domain, CH2 domain, and CH3 domain and the VL is covalently linked to a light chain constant domain (CL).

4. The ALPP / L2 binder of claim 3, wherein the CH comprises an IgGl, IgG2, IgG3, or IgG4 isotype and the CL comprises a human kappa or human lambda isotype.

5. The ALPP / L2 binder of claim 4, wherein the CH comprises the IgGl or IgG4 isotype and the CL comprises a human kappa or human lambda isotype.

6. The ALPP / L2 binder of claim 5, wherein the IgGl or IgG4 isotype comprises 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.

7. The ALPP / L2 binder of claim 5, wherein the IgGl isotype comprises 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 isotype.

8. The ALPP / L2 binder of claim 4, wherein the CL comprises the kappa isotype.

9. The ALPP / L2 binder of claim 4, wherein the kappa isotype 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 kappa isotype.

10. The ALPP / L2 binder of claim 7. the constant domain of the human IgGl comprises:(a) a substitution of the amino acids at positions 252, 254, and 256 of the CH with amino acids Tyr (Y), Thr (T), and Glu (E), respectively, wherein the numbering is according to Eu;(b) a substitution of the amino acids at positions 233 and 235 with the amino acid Ala (A), wherein the numbering is according to Eu;(c) a substitution of the amino acids at positions 234 and 235 with the amino acid A and at position 265 with Ser (S), wherein the numbering is according to Eu;(d) a substitution of the amino acids at positions 234 and 235 with the amino acid A and at position 329 with Gly (G), wherein the numbering is according to Eu; (e) a substitution of the amino acid at position 235 with the amino acid E, wherein the numbering is according to Eu;(f) a substitution of the amino acid at position 265 with the amino acid A, wherein the numbering is according to Eu;(g) a substitution of the amino acid at position 265 with the amino acid A and at position 297 with the amino acid G, wherein the numbering is according to Eu; (h) a substitution of the amino acid at position 297 with any amino acid except for Asn (N), wherein the numbering is according to Eu; or(i) a substitution of the amino acid at position 297 with the amino acid A, at position 356 with Glu (E), at position 358 with Met (M), wherein the numbering is according to Eu.

11. The ALPP / L2 binder of claim 1-10, wherein the CH comprise a C-terminal lysine or lacks a C-terminal lysine or a C-terminal glycine-lysine dipeptide.

12. The ALPP / L2 binder of claim 1-11, wherein the N-terminal amino acid of the VH is pyroglutamate.2609213. The ALPP / L2 binder of claim 1. wherein the ALPP / L2 binder is an antigen-binding fragment of an antibody selected from the group consisting of a Fab fragment, a Fab’ fragment, a F(ab’)2 fragment, a scFab, an Fv region, and an ScFv.

14. The ALPP / L2 binder of claim 13, wherein the ALPP / L2 binder comprises an ScFv.

15. The ALPP / L2 binder of claim 14, wherein the ALPP / L2 binder comprises a chimeric antigen receptor T (CAR-T) cell or chimeric antigen receptor natural killer (CAR-NK) cell comprising the ScFv.

16. A pharmaceutical composition comprising the ALPP / L2 binder of any one of claims 1-15 and a pharmaceutically acceptable carrier or diluent.

17. 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 ALPP / L2 binder of any one of claims 1-15 or the pharmaceutical composition of claim 16 to treat the proliferative disease.

18. The method of claim 17, wherein the proliferative disease comprises cells that display ALPP and / or ALPPL2 on the cell surface.

19. The method of claim 17, wherein the proliferative disease comprises a tumor that displays ALPP and / or ALPPL2 on the surface of the cells thereof.

20. The method of claim 17 or 18, 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.

21. Use of the ALPP / L2 binder of any one of claims 1-15 or the pharmaceutical composition of claim 16 for the manufacture of a medicament for treatment of a proliferative disease.2609222. The use of claim 21, wherein the proliferative disease comprises cells that display ALPP and / or ALPPL2 on the cell surface.

23. The use of claim 21, wherein the proliferative disease comprises a tumor that displays ALPP and / or ALPPL2 on the surface of the cells thereof.

24. The use of claim 21 or 22, 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.

25. The ALPP / L2 binder of any one of claims 1-15 or the pharmaceutical composition of claim 16 for treatment a proliferative disease.

26. The ALPP / L2 binder of claim 25, wherein the proliferative disease comprises cells that display ALPP and / or ALPPL2 on the cell surface.

27. The ALPP / L2 binder of claim 25, wherein the proliferative disease comprises a tumor that displays ALPP and / or ALPPL2 on the surface of the cells thereof.

28. The ALPP / L2 binder of claim 25 or 26, 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.

29. A combination therapy for treating a proliferative disease comprising the ALPP / L2 binder of any one of claims 1-15 or the pharmaceutical composition of claim 16 and a second therapeutic agent.

30. The combination therapy of claim 29, wherein the second therapeutic agent is a chemotherapy agent or a therapeutic antibody other than an antibody that binds ALPP and / or ALPPL2.- Ill -2609231. The combination therapy of claim 29 or 30, wherein the proliferative disease comprises cells that display ALPP and / or ALPPL2 on the cell surface.

32. The combination therapy of claim 29, wherein the proliferative disease comprises a tumor that displays ALPP and / or ALPPL2 on the surface of the cells thereof.

33. The combination therapy of claim 29, 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.

34. A nucleic acid molecule encoding the ALPP / L2 binder of any one of claims 1-14.

35. An expression vector comprising one or more of the nucleic acid molecules of claim 34.

36. A host cell comprising the nucleic acid molecule of claim 34 or expression vector of claim 35.

37. A method for producing an ALPP / L2 binder comprising (a) providing the host cell of claim 36; (b) cultivating the host cell in a medium under conditions suitable for expressing the ALPP / L2 binder; and (c) isolating the ALPP / L2 binder from the medium.

38. The ALPP / L2 binder of any one of claims 1-14 conjugated to a detectable moiety.

39. The ALPP / L2 binder of claim 38, wherein the detectable moiety is detectable by magnetic resonance imaging (MRI) or by X-ray imaging.

40. A method for detecting ALPP and / or ALPPL2 on the surface of a cell in an individual comprising administering to the individual the ALPP / L2 binder of claim 38 or 39 and detecting the cells in the individual that bind the ALPP / L2 binder.2609241. A composition comprising an ALPP / L2 binder of any one of claims 1-15 or the pharmaceutical composition of claim 16, and a hyaluronan degrading enzyme.

42. The composition of claim 41, wherein the hyaluronan degrading enzyme is a soluble hyaluronidase.

43. The composition of claim 42, wherein the soluble hyaluronidase is soluble pH20.

44. The composition of claim 42, wherein the soluble hyaluronidase is hyaluronidase (recombinant human) or berahyaluronidase alfa.

45. A kit comprising an ALPP / L2 binder of any one of claims 1-15 or the pharmaceutical composition of claim 16 and a hyaluronan degrading enzyme.

46. The kit of claim 45, wherein the ALPP / L2 binder and the hyaluronan degrading enzyme are provided in separate containers or the ALPP / L2 binder and the hyaluronan degrading enzyme are provided as a mixture in a single container.

47. The kit of claim 45, wherein the ALPP / L2 binder and the hyaluronan degrading enzyme are provided in separate chambers of a dual-chamber injection device or the ALPP / L2 binder and the hyaluronan degrading enzy me are provided as a mixture in a single chamber of a singlechamber injection device.

48. The kit of claim 45, wherein the hyaluronan degrading enzyme is a soluble hyaluronidase.

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

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

51. 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 ALPP / L2 binder of any one of claims 1-15 or the pharmaceutical composition of claim 16 and a hyaluronan degrading enzyme to treat the proliferative disease.2609252. The method of claim 51, wherein the proliferative disease comprises cells that display ALPP and / or ALPPL2 on the cell surface.

53. The method of claim 51. wherein the proliferative disease comprises a tumor that displays ALPP and / or ALPPL2 on the surface of the cells thereof.

54. The method of claim 51 or 52, 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.

55. The method of claim 51, wherein the hyaluronan degrading enzyme is a soluble hyaluronidase.

56. The method of claim 55, wherein the soluble hyaluronidase is soluble pH20.57 The method of claim 55, wherein the soluble hyaluronidase is hyaluronidase (recombinant human) or berahyaluronidase alfa.

58. The method of claim 51, wherein the ALPP / L2 binder and the hyaluronan degrading enzyme are administered to the individual sequentially or simultaneously.

59. The method of claim 51, wherein the ALPP / L2 binder and the hyaluronan degrading enzyme are mixed to form a mixture and the mixture is administered to the individual.

60. The method of claim 51, wherein the ALPP / L2 binder and the hyaluronan degrading enzyme are administered to the individually systemically.

61. The method of claim 51, wherein the ALPP / L2 binder and the hyaluronan degrading enzyme are administered to the individually subcutaneously or intramuscularly.

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