A Monoclonal Antibody Against Neem Leaf Glycoprotein Recognizes Carcinoembryonic Antigen (CEA) and Restricts CEA Expressing Tumor Growth Arnab Das,*w Subhasis Barik,w Saptak Banerjee,w Anamika Bose,z Koustav Sarkar,y Jaydip Biswas,8 Rathindranath Baral,w and Smarajit Pal* Summary: Carcinoembryonic antigen (CEA) is one of the promis- ing tumor antigens mainly associated with carcinoma of the colon, lung, breast, etc. and received wide attention for cancer immuno- therapy. Neem leaf glycoprotein (NLGP), an effective immuno- modulator, is able to generate humoral and cellular immune responses in murine tumor models. We have generated a mono- clonal antibody (mAb) against NLGP by fusing NLGP-immunized mice splenocytes with nonsecretory myeloma cells. A highly anti- NLGP mAb secreting clone (1C8; IgG2a in nature) has been identified and propagated in culture. 1C8 recognizes human CEA as good as NLGP by enzyme linked immunosorbent assay, West- ern blotting, and immunoprecipitation. 1C8 detects CEA on colon cancer tissues by immunochistochemistry. By flow cytometry, 1C8 specifically reacts with CEA + human (Colo-205, HCT-116, and HT-29) and mouse (CT-26) colon cancer cells, but it showed minimum reactivity with CEA  human (MCF7, SiHa, and SCC084) and mouse (B16MelF10) cancer cells. This anti-NLGP 1C8 mAb revealed significant antitumor activity and better sur- vivability in vivo in animals bearing mouse (CT-26 in BALB/c) and human (Colo-205 in athymic nude) CEA + cancer cells. 1C8 has no direct influence on proliferation and migration of CEA + cells, however, NK cell–dependent strong antibody-dependent cellular cytotoxicity reaction toward CEA + cells and normalization of angiogenesis are chiefly associated with tumor growth restriction. Obtained results provided a new immunotherapeutic approach for the effective management of CEA + tumors. Key Words: colon cancer, carcinoembryonic antigen, monoclonal antibody, neem leaf glycoprotein, antibody-dependent cellular cytotoxicity (J Immunother 2014;37:394–406) C arcinoembryonic antigen (CEA) was one of the first tumor antigens, isolated from colon cancer patients. 1 It is a 180 kDa glycoprotein consisting of B60% carbohy- drate and variation on it exhibits considerable hetero- geneity. 2 It also acts as an adhesion molecule and may play a role in metastasis. 3,4 The generation of monoclonal anti- bodies (mAbs) against CEA allowed the detection of the overexpressed CEA protein in a variety of adenocarcinomas, including gastric, colonic, rectal, ovarian, breast, etc. 5–7 As a self oncofetal protein, CEA is weakly immunogenic in cancer patients, although, it generates both antibody 8 and T-cell 9 responses. Exploiting such knowledge several vaccine strategies have been formulated and tested, including generation of anti-CEA antibodies, 10,11 anti-idiotypic antibodies mim- icking CEA, 12–14 use of CEA peptides and recombinant protein, 15–18,19 recombinant microorganisms expressing CEA, 20 CEA-pulsed dendritic cells, 21–23 etc. 24–26 to increase vaccine effectiveness. These strategies use CEA as a target molecule in various forms of active specific and passive immunotherapy. In case of CEA expressing tumors, CEA appeared as a self-antigen by the immune system, thus, patients with CEA + tumors are typically immunologically “tolerant” to CEA. 12 Passive approach that involves infu- sion of huge amount of anti-CEA antibody is often unsuccessful as it generates neutralizing antibodies. 27 Thus, alternate approach to target CEA is still in search. Despite the existing lacunae in the understanding, relationship of the CEA and plant materials is explored. Stoger et al 28 successfully expressed a single-chain Fv antibody (ScFvT84.66) against CEA in the staple cereal crops—rice and wheat. Transient expression of a tumor- specific single-chain fragment and a chimeric antibody against human CEA was reported in tobacco leaves. 29 Production of antibodies in plants against human CEA was also reviewed. 30 In our effort to elucidate the adjuvant function of a preparation from neem (Azadirachta indica) leaves [neem leaf preparation (NLP)] in enhancement of antibody response against breast tumor–associated anti- gen, 31 melanoma surface antigen, 32 and CEA, 33,34 we sur- prisingly found that the serum polyclonal antibody against this NLP reacts specifically with human CEA with mini- mum reactivity with other antigens. 35 As, clinical sig- nificance of the CEA recognition by anti-NLP antibody was not explored, here, we attempted to generate and characterize a mAb against neem leaf glycoprotein (NLGP). Successful generation of mAb eventually showed its recognition to human CEA that have an enormous significance in the induction of anti-CEA antitumor immunity. In association with the in vivo testing of anti- NLGP mAb for restriction of CEA + tumor growth, mechanism of antitumor action is also partially explored. MATERIALS AND METHODS Reagents and Media Human CEA was procured from Aspen Bio Inc., CO. Anti-human CEA antibody, anti-mouse peroxidise-labeled IgG, CEA peptide (101-115), fluorescent stain DAPI, MTT, Received for publication March 28, 2014; accepted May 29, 2014. From the *Clinical Biochemistry Unit; Departments of wImmuno- regulation and Immunodiagnostics; 8Surgical Oncology and Med- ical Oncology, Chittaranjan National Cancer Institute; zDepartment of Molecular Medicine, Bose Institute, Kolkata, India; and yPediatric Hematology Oncology, University of Iowa Children’s Hospital, IA. Reprints: Smarajit Pal, Clinical Biochemistry Unit, Chittaranjan National Cancer Institute (CNCI), 37, S. P. Mukherjee Road, Kolkata 700026, India (e-mail: psmarajit@hotmail.com). Supplemental Digital Content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Website, www.immunotherapy-journal.com). Copyright r 2014 by Lippincott Williams & Wilkins BASIC STUDY 394 | www.immunotherapy-journal.com J Immunother  Volume 37, Number 8, October 2014