(B) Identification of the target antigen bound by the tumor specific antibody M25. One of the antibodies, M25, showed high specificity against an antigen we identify here as ALPPL2. Immunohistochemistry on normal human tissues found that ALPPL2 is expressed only on placental trophoblasts but not any other normal tissues. This significant tissue specificity and broad tumor type coverage suggests that ALPPL2 could be an excellent cell surface target for therapeutic development against mesothelioma. To evaluate therapeutic potential of ALPPL2 targeting, an ALPPL2-targeted antibody-drug conjugate was developed Magnolol and demonstrated potent and specific tumor killing in vitro and in vivo against both epithelioid and sarcomatoid mesothelioma. Thus, ALPPL2 belongs to a rare Vegfc class of cell surface antigens classified as truly tumor-specific and is well suited for therapy development against ALPPL2-expressing tumors. Keywords: ALPPL2, ALPP, ALPI, ALPL, alkaline phosphatase family, alkaline phosphatase placental-like 2, tumor-specific cell surface antigen, mesothelioma, ALPPL2-expressing tumors, antibody-drug conjugate, phage antibody display, yeast antibody display, affinity maturation Introduction Mesothelioma is a rare, asbestos-related tumor (1) that arises predominantly from serosal cells of the pleura and, to a lesser extent, the peritoneum, pericardium, and tunica vaginalis (2). About 3,000 new cases are diagnosed annually in the United States (3,4). Current approved treatments for malignant mesothelioma include chemotherapy, radiation therapy, or combination treatments (4,5). However, malignant mesothelioma is incurable, and the overall five-year survival rate is about 8% (6,7). Malignant mesothelioma can be classified into three histological types, epithelioid (~50-60%), sarcomatoid Magnolol (fibrous, ~10-20%), and mixed (biphasic, ~30-40%) (2,8). Epithelioid mesothelioma tends to have a better prognosis than the other two types. Sarcomatoid mesothelioma is particularly recalcitrant to treatment, progresses most aggressively and has the shortest median overall survival (2,9). Currently, the best studied cell surface antigen for mesothelioma is mesothelin, a GPI-anchored molecule, which is the only biomarker approved by the FDA for this disease (10,11). Mesothelin, however, is rarely expressed by sarcomatoid mesothelioma (12). In addition, mesothelin is also expressed by normal mesothelium, raising concerns for on-target toxicity on normal tissues. Therefore new targets are needed for developing novel therapies against this incurable malignancy. An ideal cell surface Magnolol target for mesothelioma should carry the following desired properties: (1) it is expressed in all subtypes of mesothelioma but not normal tissues. (2) it is a true tumor antigen that is indicated by mesothelioma Magnolol cells residing in their cells microenvironment, as opposed to cell collection artifacts. We have previously developed methods for selecting phage human being antibody display libraries directly on tumor issues derived from individual specimens (13C15) following counter-selection on normal cells and cells. In the case of mesothelioma, we recognized a panel of novel human being single-chain variable fragments (scFvs) that bind to both epithelial and sarcomatous types but not normal mesothelium (13), a definite differentiation from antibodies binding to mesothelin that is indicated by epithelial mesothelioma but hardly ever by sarcomatous mesothelioma (12), and also by normal mesothelium (16). One of the antibodies, M25, showed the highest specificity, binding to a novel cell surface target indicated by all subtypes of mesothelioma (13). We hereby statement the recognition of the prospective antigen bound by M25 as human being alkaline phosphatase, placental-like 2 (ALPPL2), a member of the human being Magnolol alkaline phosphatase family. Among the four users of this family, ALPPL2 and placental alkaline phosphate (ALPP) are virtually identical in amino acid sequence (98% homology) and have a highly restricted cells expression pattern, expressing in placental trophoblasts only. Both share high homology with the intestinal alkaline phosphatase (ALPI) (87% homology), and some homology with the tissue-nonspecific liver/bone/kidney phosphatase ALPL (57% homology) (Henthorn et al., 1988). We display that M25 binds specifically to ALPPL2 and ALPP but not ALPI or ALPL. We performed considerable immunohistochemistry (IHC) studies and showed that ALPPL2 is definitely indicated in mesothelioma but.
