Preparation of (177Lu-DOTA)n-PAMAM-[Nimotuzumab-F(ab’)2] as a Therapeutic Radioimmunoconjugate for EGFR Overexpressed Cancers Treatment

Titis Sekar Humani, Sutari Sutari, Triningsih Triningsih, Martalena Ramli, Rien Ritawidya, Ratna Dini Haryuni

Abstract


Intact monoclonal antibodies with a high molecular weight tend to have a poor pharmacokinetic profile and tumor penetration, and potential for eliciting host antibody responses. F(ab’)2 fragments smaller than intact monoclonal antibodies that still maintain antigen binding could solve this problem. The objective of this study was to optimize the digestion process of nimotuzumab, an anti-EGFR monoclonal antibody, into its F(ab’)2 fragment and investigate its potential as a therapeutic radioimmunoconjugate. Optimal conditions for digestion of nimotuzumab to its F(ab’)2 fragment were found to be 6 hours of digestion time with a pH of 3.5 and 1:100 mol ratio of pepsin to nimotuzumab. The purity of the F(ab’)2-nimotuzumab was confirmed by SDS-PAGE and HPLC analysis. Prior to its labeling with lutetium-177 radionuclide, the nimotuzumab-F(ab’)2 was conjugated to DOTA-PAMAM dendrimer [DOTA denotes 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, PAMAM denotes poly(amidoamine)] to form conjugate of (DOTA)n-PAMAM-[nimotuzumab-F(ab’)2]. Radiolabeling of DOTA-PAMAM-[nimotuzumab-F(ab’)2] conjugate with 177Lu resulted in (177Lu-DOTA)n-PAMAM-[nimotuzumab-F(ab’)2] with radiochemical purity > 99% after purification with a PD-10 column. Further studies still need to be performed in order to confirm the potential of this radioimmunoconjugate as a radioimmunotherapeutic agent for EGFR overexpressed cancers.


Keywords


dendrimer PAMAM; EGFR; nimotuzumab-F(ab’)2; pepsin digestion; 177Lu-DOTA-PAMAM-[nimotuzumab-F(ab’)2]

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References


Mendelsohn, J. Targeting the Epidermal Growth Factor Receptor for Cancer Therapy, Journal of Clinical Oncology, 20(18), pp. 1s-13s, 2002.

Mendelsohn, J. & Baselga, J., Epidermal Growth Factor Receptor Targeting in Cancer, Seminars in Oncology, 33(4), pp. 369-385, 2006.

Allan, D.G.P., Nimotuzumab: Evidence of Clinical Benefit without Rash, The Oncologist, 10(9), pp. 760-761, 2005.

Talavera, A., Friemann, R., Gómez-Puerta, S., Martinez-Fleites, C., Garrido, G., Rabasa, A., López-Requena, A., Pupo, A., Johansen, R.F., Sánchez, O., Krengel, U. & Moreno, E., Nimotuzumab, An Antitumor Antibody that Targets The Epidermal Growth Factor Receptor, Blocks Ligand Binding while Permitting The Active Receptor Conformation, Cancer Research, 69(14), pp. 5851-5859, 2009.

Diaz, A., Preclinical Efficacy of Nimotuzumab, An Anti-EGFR Monoclonal Antibody as A Single Agent Therapy in Human GBM U87MG Xenografts, Journal of Cancer Therapy, 03(04), pp. 245-255, 2012.

Crombet, T., Osorio, M., Cruz, T., Roca, C., del Castillo, R., Mon, R., Iznaga-Escobar, N., Figueredo, R., Koropatnick, J., Renginfo, E., Fernández, E., Alvárez, D., Torres, O., Ramos, M., Leonard, I., Pérez, R. & Lage, A., Use of The Humanized Anti-Epidermal Growth Factor Receptor Monoclonal Antibody h-R3 in Combination with Radiotherapy in The Treatment of Locally Advanced Head and Neck Cancer Patients, Journal of Clinical Oncology, 22(9), pp. 1646-1654, 2004.

Takeda, M., Okamoto, I., Nishimura, Y. & Nakagawa, K., Nimotuzumab, A Novel Monoclonal Antibody to The Epidermal Growth Factor Receptor, in The Treatment of Non-Small Cell Lung Cancer, Lung Cancer : Targets and Therapy, 2, pp. 59-67, 2011.

Westphal, M., Heese, O., Steinbach, J.P., Schnell, O., Schackert, G., Mehdorn, M., Schulz, D., Simon, M., Schlegel, U., Senft, C., Geletneky, K., Braun, C., Hartung, J.G., Reuter, D., Metz, M.W., Bach, F. & Pietsch, T., A Randomised, Open Label Phase III Trial with Nimotuzumab, An Anti-Epidermal Growth Factor Receptor Monoclonal Antibody in The Treatment of Newly Diagnosed Adult Glioblastoma, European Journal of Cancers, 51(4), pp. 522-532, 2015.

Reddy, B.K.M., Lokesh, V., Vidyasagar, M.S., Shenoy, K., Babu, K.G., Shenoy, A., Naveen, T., Joseph, B., Bonanthaya, R., Nanjundappa, P. Bapsy, P., Loknatha, Shetty, J., Prasad, K. & Pasha, C.R., Tanvir, Nimotuzumab Provides Survival Benefit to Patients with Inoperable Advanced Squamous Cell Carcinoma of The Head and Neck: A Randomized, Open-Label, Phase IIb, 5-Year Study in Indian Patients, Oral Oncology, 50(5), pp. 498-505, 2014.

Ahmad, Z.A., Yeap, S.K., Ali, A.M., Ho, W.Y., Alitheen, N.B.M. & Hamid, M., ScFv Antibody: Principles and Clinical Application, Clinical and Developmental Immunology, 2012, pp. 1-15, 2012.

Hong, H., Zhang, Y., Orbay, H., Valdovinos, H.F., Nayak, T.R., Bean, J., Theuer, C.P., Barnhart, T.E. & Cai, W., Positron Emission Tomography Imaging of Tumor Angiogenesis with A (61/64)Cu-Labeled F(ab’)2 antibody Fragment, Mol Pharm, 10(2), pp. 709-716, 2013.

El-sayed, M., Kiani, M.F., Naimark, M.D., Hikal, A.H. & Ghandehari, H., Extravasation of Poly(amidoamine) (PAMAM) Dendrimers Across Microvascular Network Endothelium, 18(1), pp. 18-23, 2001.

Pourianazar, N. Taghavi. Mutlu, P. & Gunduz, U., Bioapplications of Poly(amidoamine) (PAMAM) Dendrimers in Nanomedicine, Journal of Nanoparticle Research, 16(4), pp. 1-38, 2014.

Haryuni, R. D., Bahtiar, A., Soenarjo, S., Harahap, Y., Mutalib, A. & Ramli, M., Fragmentation of Nimotuzumab for Preparation of I- F (ab’)2 -Nimotuzumab as A Precursor for Radiopharmaceutical for Cancer Therapy, Atom Indonesia Journal, 40(1), pp. 13-21, 2014.

Ritawidya, R., Setyowati, S., Rustendi, C.T., Maskur, Ramli, M., Hidayat, B., Subur, M., Mutalib, A. & Mashyur, J., Preparation Method of (DOTA)n-[Dendrimer PAMAM]-(Trastuzumab)m Conjugate as Radiopharmaceutical Compound for Radioimmunotherapy of (177Lu-DOTA)n-[Dendrimer PAMAM] (Trastuzumab)m, Indonesian Journal of Pharmaceutical Sciences, 12(2), pp. 222-231, 2014.

Humani, T.S., Ramli, M., Rustendi, C.T. & Subur, M., Preparation and Stability Test of 177Lu-DOTA Nimotuzumab as Radiopharmaceutical of Cancer Therapy, Prosiding Seminar Nasional VI SDM Teknologi Nuklir, pp. 663-670, 2010.

Wong, K.J., Baidoo, K.E., Nayak, T.K., Garmestani, K., Brechbiel, M. W. & Milenic, D.E., In Vitro and In Vivo Pre-Clinical Analysis of A F(ab’)2 Fragment of Panitumumab for Molecular Imaging and Therapy of HER1-Positive Cancers, EJNMMI Research, 1(1), pp.1-15, 2011.

Garrido, G., Tikhomirov, I., Rabasa, A., Yang, E., Gracia, E., Iznaga, N., Fernández, L.E., Crombet, T., Kerbel, R.S. & Pérez, R., Bivalent Binding by Intermediate Affinity of Nimotuzumab: A Contribution to Explain Antibody Clinical Profile, Cancer Biology and Therapy, 11(4), pp. 373-382, 2011.

Campos, L.A. & Sancho, J., The Active Site of Pepsin is Formed in the Intermediate Conformation Dominant at Mildly Acidic pH, FEBS Letters, 538(1-3), pp. 89-95, 2003.

Lamoyi, E., Preparation of F(ab’)2 Fragments from Mouse IgG of Various Subclasses, Methods in Enzymology, 121, pp. 652-663, 1986.




DOI: http://dx.doi.org/10.5614%2Fj.math.fund.sci.2017.49.3.4

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