Defense Date

2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Chemistry

First Advisor

Professor Jamal Zweit

Second Advisor

Professor Nicholas Farrell

Abstract

Over expression of Sperm Acrosomal SLLP1 Binding protein (SAS1B) on the surface of uterine cancer and the availability of different anti-SAS1B monoclonal antibodies (mAbs) that specifically bind to it, offered an opportunity to label these antibodies with radionuclides for cancer imaging. Although optimizing in vivo stable radiolabeling is challenging, imaging this type of aggressive cancer will improve the treatment strategy. The objective of this work is to develop a radiolabeled anti-SAS1B antibodies with Zircunum-89 (89Zr) and Copper-67 (67Cu) and determine, in vitro and in vivo their uptake and biodistribution. Two anti-SAS1B monoclonal antibodies (SB2, and SB5) were labeled indirectly with 89Zr and 67Cu, using Deferoxamine (DFO) and Para Isothiocyanatobenzyl-1, 4, 7, 10-tetraazacyclododecane tetra acetic acid (p-SCN-Bn- DOTA) respectively. Different molar ratios of anti-SAS1B to the linker were investigated for conjugation, (1:3, 1:5, or 1:10) to determine the optimum conjugation molar ratio to take forward for labeling. The optimized conjugate was used to produce a radiolabeled mAb which was used for both in vitro cell uptake experiments and in vivo tumor imaging and biodistribution study. Results show specific uterine cancer cell uptake of radiolabeled SB2 and SB5 indicating the specificity of anti-SAS1B to its target SAS1B surface antigen expressed on SNU539 uterine cancers cells.

[89Zr]-DFO-SB5 PET imaging revealed uptake in the SNU539 tumor xenograft, albeit with minimal uptake. Uptake was also observed in bones and this is more likely due to detachment of the 89Zr from DFO, and subsequent accumulation in bone. The biodistribution profile from imaging data was reflected in the ex vivo biodistribution of [89Zr]-DFO-SB5.

The second mAb SB2 was radiolabeled indirectly using [67Cu]-DOTA and such radio-conjugate was used to assess its uptake by SNU539 and Sk-OV3 cancer cell lines, which express SAS1B antigen. The [67Cu]-DOTA-SB2 was taken up by both cell lines and this uptake was significantly higher than that of 67CuCl2 or [67Cu]-DOTA, indicating more specific uptake in the case of radiolabeled Mab.

In the absence of radionuclides, the MAb SB2 was conjugated to the near-infrared (NIR) fluorescence dye (IR800CW) to enable the potential for optical imaging. Using fluorescence microscopy, SB2 mAb labeled with the NIR dye showed more binding to SNU539 and Sk-OV3 and no binding to A549, which is consistent with its lack of SAS1B expression. Presence of fluorescence signals in uterine cancer cells that express SAS1B surface antigens compared with cells do not express SAS1B as negative control.

The labeled anti-SAS1B antibodies SB2 and SB5 were evaluated in vitro and in vivo. The results demonstrated the specific binding of both labeled antibodies to the surface uterine cancer receptors (SAS1B) through in vitro cell uptake and in vivo tumor uptake.

Labeled anti-SAS1B mAbs with radionuclides or fluorescent probes could yield an imaging version of the mAb that can be used to determine SAS1B receptor status in such tumors. Such determination could impact more individualized treatment of SAS1B-expressing cancers.

Rights

Elmekharam, Nouri

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

5-5-2021

Included in

Life Sciences Commons

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