Multiple Tolerization Subtractive Immunization (MTSI) Protocol: Effects on Mice and Monoclonal Antibody Specificity.

Marina De Lima Fontes,Franciny Mara De Lima Neves,Ana Marisa Fusco-Almeida,Sergio Luis Felisbino,Maria José Soares Mendes Giannini,Elenice Deffune,Andrei Moroz,Kelvin Sousa Santos

doi:10.3389/fimmu.2021.760817

Marina De Lima Fontes, Franciny Mara De Lima Neves + Show 6 more

Open Access

https://doi.org/10.3389/fimmu.2021.760817

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Abstract

Monoclonal antibodies (mAbs) have been a valuable tool to elucidate several biological processes, such as stem cell differentiation and cancer, and contributed to virtually all areas of biomedical sciences. Yet, it remains a challenge to obtain mAbs specific to poorly expressed epitopes, or to epitopes that are actually involved in important biological phenomena, such as cell differentiation and metastasis. Drug-induced subtractive immunization, and recently the multiple tolerization subtractive immunization (MTSI) technique, reported by our group, have the potential to level up the field, as they direct the host´s immune response towards these epitopes. However, due to cyclophosphamide (CY) treatment, high mice mortality can be observed, and only a few data are available on how these techniques affect the immune system of mice. Tolerogen and immunogen cells, RWPE-1 and PC-3 cells, respectively, were individually seeded at 2 × 104 cells/cm2, and then adjusted to 2 × 106 cells per mouse before immunization, which was conducted in a subtractive approach (MTSI) with CY. Immunosuppression of mice was recorded via total white blood counting, as well the reactivity of circulating polyclonal antibodies (pAbs). General parameters, including weight, physical appearance, and behavior on mice subjected to three different concentrations of CY were recorded. mAbs were obtained using classical hybridoma techniques, using the spleen of immunized mice. After purification, antibodies were characterized by Western blotting, and Indirect immunofluorescence. In conclusion, all CY dosage were efficient in creating an immunosuppression state, but only the 100 mg/kg body weight was feasible, as the others resulted in extensive mice mortality. pAbs obtained in the peripheral blood of mice showed more reactivity towards tumor cells. MAbs 2-7A50 and 2-5C11 recognized antigens from tumor cells, but not from their non-tumor counterparts, as shown in western blotting and immunofluorescence assays. MTSI technique was successful in generating mAbs that recognize tumor-specific antigens.

Highlights

With the advent of hybridoma technology, postulated by Köhler and Milstein in 1975, and subsequent advances in biotechnology platforms, which includes phage display, mammalian cell antibody display, and transgenic animals, monoclonal antibodies still are prominent in the global market [1,2,3]
Techniques involving radionuclide labeled antibody are extensively used in noninvasive molecular imaging techniques, such as the single photon emission computed tomography (SPECT) and the positron emission tomography (PET)
The effects caused by the administration of 200 mg/kg BW of CY, corresponding to the maximum dose prescribed for BALB/c mice, resulted in the cachectic state of all of them, with remarkable piloerection, hair loss in the dorsal region and, especially, pronounced emaciation, inactivity, evidenced by the hunched posture

Summary

Introduction

With the advent of hybridoma technology, postulated by Köhler and Milstein in 1975, and subsequent advances in biotechnology platforms, which includes phage display, mammalian cell antibody display, and transgenic animals, monoclonal antibodies (mAbs) still are prominent in the global market [1,2,3]. Over the past three years, the number of therapeutic antibodies that entered phase I clinical studies reached 100 new molecules each year [4]. By the end of 2018, 33 new antibodies for cancer treatment were in the final stage of clinical studies, being 80% of which for solid tumors [4]. Techniques involving radionuclide labeled antibody are extensively used in noninvasive molecular imaging techniques, such as the single photon emission computed tomography (SPECT) and the positron emission tomography (PET). These are helpful in order to detect tumor extension and residual tumor lesions that conventional imaging scans are unable to do [7]. There is an enormous gap that needs to be addressed regarding discovery of novel biomarkers, involved in several biological processes, for instance in the triggering and progression of solid tumors [10]

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