Investigation of p-SCN-Bn-DOTA-trastuzumab labeled with radioactive and non-radioactive lutetium and yttrium: a crucial step for future applications

The significance lies in preparing stable, trastuzumab-immunoconjugate through the utilization of non-radioactive LuCl3 and YCl3, via p-SCN-Bn-DOTA. This approach is crucial to determine potential physicochemical alterations in immunoconjugate structure following metal binding. Post-conjugation, employing a 1:20 molar ratio, freeze-drying was performed to obtain stable immunoconjugates for subsequent analysis. Several chemical methods were employed to characterize antibody stability and retained immunoreactivity within the formulated immunoconjugates. Proof of protein integrity came from SDS-PAGE electrophoresis, with uniform fragment intensities (25 kDa for light chain, 50 kDa for heavy chain) indicating antibody non-degradation (1). IR and Raman spectroscopy verified secondary structural changes, with the presence of characteristic amide bands in both spectra indicating the retention of native secondary structure (2). Employing MALDI-TOF-MS, 4.9 p-SCN-Bn-DOTA molecules were determined per antibody molecule. The promising outcomes from non-radioactive labeling provide an opportunity for potential labeling with radioactive lutetium-177 and yttrium-90, each with a specific activity of 200 µCi/mL. Radioisotopes were incubated with p-SCN-Bn-DOTA-trastuzumab for an hour at 40 ºC. Evaluation of radiochemical purity and stability was conducted using the ITLC-SG system. Optimal mobile phases, specifically 0.4 M methanol:sodium acetate (1:1) for yttrium-90 and 0.9% NaCl for lutetium-177, facilitated thorough examination. Remarkable radiolabeling efficiency was achieved, >96% for yttrium-90 and >99% for lutetium-177. Stability assessments after 72 hours demonstrated greater stability in 177Lu-p-SCN-Bn-DOTA-trastuzumab (<1.5% lutetium-177 release) compared to the 90Y-labeled counterpart (<17% yttrium-90 release). This study demonstrates the successful development of radioimmunoconjugates, positioning this agent for potential application in vivo investigations

Possibilities for formulation of Trastuzumab - radioimunoconjugates

Trastuzumab (Herceptin®) is a commercially approved humanized IgG1 monoclonal antibody for treatment of HER2 positive breast cancer. Monoclonal antibodies are selective and specific anticancer therapy which are used for conjugation with another dugs, toxins and radioisotopes. In recent years, with the development of radiopharmacy, are synthetized many stable conjugates with various bifunctional chelators (DOTA, DTPA, HYNIC, 1B4M-DTPA, TCMC), for further labeling with radioisotopes. In order to increase the selectivity of the drugs and toxins and the good clinical results of trastuzumab encouraged many scientists to try to formulate stable conjugates. All radioimmunoconjugates of trastuzumab are in phase of preclinical and clinical examination and allows significant improvement in the general conditions of the patients. For imaging and identification of HER2 positive lesions are developed immunoconjugates labeled with γ and β+ emitters (99mTc-HYNIC-trastuzumab, 111In-DOTA-trastuzumab, 64Ga-DOTA-trastuzumab, 68Ga-DOTA-trastuzumab). In vitro and in vivo investigations shows that conjugates labeled with pure β and α emitters are new promising drugs in treatment of HER2 postitive cancers which allows selective uptake of radioimmunoconjugates by the tumor cells and minimal localizaton in healthy organs. Till now, 177Lu-DOTA-trastuzumab, 90Y-DTPA-trastuzumab, 212Pb-TCMC-trastuzumab, 227Th-DOTA-p-benzyl-trastuzumab and 225Ac-trastuzumab, given good in vivo results and selective internalization in HER2 positive breast, prostate and ovarian cancer cells. The goal of this examination is to formulate stable freeze dried kit trastuzumab-conjugate with bifunctional chelators (DOTA, DTPA, HYNIC and 1B4M-DTPA) and in vitro chemical characterization and identification. The most stable conjugate will be used for obtaining radioimmunogonjugates with 68Ga and 177Lu and their preclinical in vitro and in vivo biodistribution using animal models

Bifunctional chelators for trastuzumab conjugation and successful labeling with radioisotopes

Bifunctional chelators (BFC) are molecules important for formulation of stable metal complex with targeting biomolecules such as antibodies, peptides or proteins. The chelators are covalently attached to the biomolecules on the one side and on the other coordinates to the radioisotopes. The goal is to produce a radiopharmaceuticals with pharmacokinetic and pharmacodynamic stability, without degradation of the complex in the physiological conditions and releasing of metal ion. The choice of the chelators depends of the type of radioactive isotope that will be used. Many efforts have been made in order to formulate a stable immunoconjugates of anti-HER2 monoclonal antibody trastuzumab with different types of BFC for further labeling with radioisotopes for imaging or radiotherapy of HER2 positive metastatic breast cancer. Succinimidyl-hydrazinonicotinamide (HYNIC) is important for trastuzumab conjugation and labeling with 99mTc and 188Re. For formulation of radioimmunoconjugates with 212Pb 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraaza-cyclododecane (TCMC) have been used as a chelator. The most commonly used BFC are 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and diethylene triamine pentaacetic acid (DTPA). Preclinical characterization was made of 111In radiolabeled trastuzumab, previously conjugated with DTPA as a chelator. DTPA was used for trastuzuamab conjugation in a case of labeling with β emitters 86Y and 90Y. Chelating agent DOTA is significant for formulation of trastuzumab-radioimunoconjugates with gamma emitter 67Ga, and potent therapeutic agents with β- emitter 177Lu and α emitter 225Ac. The aim of our study is to formulate a stable immunoconjugates of trastuzumab with DTPA, DOTA and DTPA derivate 1B4M-DTPA (2-(4-isothiocyanatobenzyl)-6-methyl-diethylene-triaminepentaacetic acid) for further labeling with 90Y and 177Lu

Achievements and perspectives in formulation of stable immunoconjugate of the HER2-targeting trastuzumab – potential for rapid labelling with Gallium-68

Trastuzumab (Herceptin®) is a humanized IgG1 monoclonal antibody which is approved for therapy of HER2 positive breast cancer. Good clinical results and improvement of the patient’s general condition makes it interesting for further conjugation, in order to increase the therapeutic effect of trastuzumab. With the development of radiopharmacy many efforts are made for formulation of stable conjugates with various bifunctional chelators, further labelled with radioisotopes (α, β and γ emitters). According to the literature, successful conjugation of trastuzumab is achieved with DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DTPA (diethylenetriaminepentaacetic acid), TCMC (1,4,7,10-tetra-(2-carbamoyl methyl)-cyclododecane), HYNIC (succinimidyl-6-hydrazino-nicotinamide) and DTPA derivate 1B4M-DTPA (2-(4-isothiocyanatobenzyl)-6-methyl-diethylene-triaminepentaacetic acid). Significant radiopharmaceuticals based on peptide and antibody for diagnostic and therapeutical purpose used different radioisotopes (99mTc/188Re, 67Ga, 177Lu, 90Y, 131I). Several studies have shown that already existing radiolabeled formulations, using beta and alfa emitters (90Y-DTPA-trastuzumab, 86Y-DTPA-trastuzumab, 177Lu-DOTA-trastuzumab, 227Th-DOTA-p-benzil-trastuzumab, 225Ac-trastuzumab) are potent against HER2 positive breast cancer. Our already obtained and published results related tothe method for ready to use production of freeze dried kit formulation of Rituximab imunnoconjugates for labeling with Lu-177 and Y-90 including preclinical assessment of three imunnoconjugates (p-SCN-Bn-DOTA, p-SCN-Bn-DTPA and 1B4M-DTPA ) including chemical characterization, animal imaging, biodistribution and toxicological studies, were good reason to have an indication to introduce the same approach for labeling HER2-targeting trastuzumab using 68Ga3+ under mild conditions without subsequent purification for PET imaging. Work progress towards objectives and achievements Progress results and research outcomes critical to work progress and achievements are listed under our project milestones, and referenced to project aims. Our objectives are to synthesize and evaluate the immunoconjugate of the HER2-targeting trastuzumab with Galium – 68 using already established method of freeze dried kit formulation. Milestone 1: Standardize previously established method used for freeze dried kit formulation of Rituximab imunnoconjugates for HER2-targeting trastuzumabmimunnoconjugates. Milestone 2: Conjugation of bifunctional chelators to targeting for HER2-targeting trastuzumab, and radiolabeling with Ga-68 (define the most appropriate immunoconjugate). Milestone 3: In vitro characterization and in vivo biodistribution of 68Ga-labeled conjugates. Significance: Already established method for freeze dried kit formulation of conjugated Rituximab antibody ready for labeling with Lutetium-177, Yttrium-90 for therapeutical purpose that is under the final phase of the evaluation provide good information to introduce the same method for labeling of for HER2-targeting trastuzumab. The simplicity and efficiency of labelling and the possibility of kit-based 68Ga tracer production without complex automated synthesis typical of multistep PET radiochemistry will greatly increase 68Ga PET access to hospitals, expanding the use of the 68Ga generator. In the same time give opportunity to work on the same or similar kit formulation using with Lu-177 for therap

Trastuzumab and its radioimmunoconjugates in treatment of cancer

Monoclonal antibodies are new type of targeted anticancer therapy, which achieve specificity, selectivity and localization in tumor cells. There are many naked antibodies and immunoconjugates commercially approved for different types of cancer (Mehren et al., 2003). In order to improve specificity and selectivity of cytotoxic drugs and toxins, monoclonal antibodies are used for formulation of immunoconjugates. Many efforts are done to develop stable immunoconjugates of trastuzumab with various drugs, toxins and radioisotopes to improve the general conditions of the patients (Sharkey and Goldenberg, 2006). The aim of this paper is to focus on current achievements in the formulation of radioimmunoconjugates of HER2-targeting trastuzumab

Cold labeled trastuzumab-p-SCN-Bn-DTPA and trastuzumab-p-SCN-Bn-1B4M-DTPA conjugates– preparation and spectroscopic analysis

Introduction and aim: The importance of immunoconjugates in treatment of various cancers was motivation for us to formulate a stable cold labeled trastuzumab conjugates with two bifunctional chelators (BFCAs) (p-SCN-Bn-1B4M-DTPA (2-(4-isothiocyanatobenzyl)-6-methyl-diethylene-triaminepentaacetic acid and p-SCN-Bn-DTPA (2-(4-izothiocyanatobenzyl)-diethylenetriaminepentaacetic acid)). The labeling with non-radioactive LuCl3 and YCl3 is important to determine the possible physicochemical changes in the structure of immunoconjugates after metal binding. ATR-IR (Attenuated total reflectance-infrared) and Raman spectroscopy as powerful and non-destructive techniques are appropriate for verification of possible secondary structure chances of trastuzumab after conjugation and labeling. Methodology: Anti-HER2/neu monoclonal antibody trastuzumab was conjugated with p-SCN-Bn-DTPA, p-SCN-Bn-1B4M-DTPA in ratio of 1:10 and 1:50 and lyophilized to solid state. The freeze dried conjugates were labeled with cold LuCl3 and YCl3. The retained secondary structure of the antibody was proven by spectroscopic analysis with ATR-IR and Raman spectroscopy and compared with purified trastuzumab from commercial product Herceptin®. Results and discussion: The ATR-IR and Raman spectra of four samples have shown the presence of characteristic amide bands and retained native IgG1 structure of the antibody principally composed of β-sheets. Characteristic amide I band at ~1670 cm-1 and amide III band (1230-1300 cm-1) were detected in Raman spectra. IR spectra also contain the amide I (1700-1600 cm-1), amide II (1480-1575 cm-1) and amide III bands (1255-1244 cm-1) specific for secondary structure of the proteins. Conclusion: No significant changes in antibody structure after cold labeling gives us a hope for further radiolabeling of immunoconjugates with 177LuCl3 and 90YCl3 and development of radioimmunotherapeutics and diagnostic products active against HER2 positive breast tumors

p-SCN-Bn-DOTA-Trastuzumab immunoconjugates and vibrational spectroscopy examination

Introduction: Attenuated total reflectance-infrared (ATR-IR) and Raman spectroscopy, as vibrational techniques, provide reliable molecular structure information and are significant for determination of any of changes in the secondary structure. The aim of this study is structural examination of non-labeled conjugated antibody with p-SCN-Bn-DOTA 2-(4-izothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) in differ molar ratio by applying of these two techniques. Material and methods: Freeze dried kit of immunoconjugates were produced after conjugation of antibody with the 10- and 50-fold molar excess of p-SCN-Bn-DOTA. The lyophilized non-labeled samples were used for further physical-chemical characterization with ATR-IR and Raman spectroscopy, by directly applying under the laser beam of the instrument. The spectra of naked antibody purified from commercial product Herceptin® was used for comparison with the collected spectra of conjugated samples. Results: The ATR-IR and Raman spectra of conjugated antibody correspond with the spectra of naked antibody. The presence of characteristic amide I band at ~1670 cm-1 and amide III band (1230-1300 cm-1) in Raman spectra and amide I (1700-1600 cm-1), amide II (1480-1575 cm-1) and amide III bands (1255-1244 cm-1) in ATR-IR spectra have showed retained IgG1 structure of the antibody principally composed of β-sheets. Conclusion: The minor difference in the ATR-IR and Raman spectra between immunoconjugates and native trastuzumab showed that only insignificant changes occurred in the protein’s structure as a result of chelator binding. The retained β-sheet structure of the antibody supports the opportunity for further development of anti-HER2 positive breast cancer radioimmunotherapeutics and diagnostic products

Freeze dried kit formulation of trastuzumab-immunoconjugates

Trastuzumab is a humanized anti-HER2 monoclonal antibody used for therapy of metastatic breast cancer. Conjugated antibodies with toxins, drugs and radionuclides, provide high hope for development of cancer-specific cytotoxic reagents. The formulation of stable immunoconjugates with a bifunctional chelators (BFCA) is required in order to obtain successful radiolabeling. The aim of this examination was to development a stable freeze dried trastuzumab immunoconjugates with BFCA p-SCN-Bn-DTPA, p-SCN-Bn-DOTA, 1B4M-DTPA

ATR-IR and Raman spectoscropic characterization of p-SCN-Bn-DTPA and p-SCN-Bn-1B4M-DTPA trastuzumab conjugates

Introduction: As powerful and non-destructive techniques, Attenuated total reflectance-infrared (ATR-IR) and Raman spectroscopy provide valuable molecular structure information and are convenient for verification of any changes in the native state of proteins. The purpose of this experimental work is to determine the secondary structure of trastuzumab after conjugation with (p-SCN-Bn-1B4M-DTPA (2-(4-isothiocyanatobenzyl)-6-methyl-diethylene-triaminepentaacetic acid) and p-SCN-Bn-DTPA (2-(4-izothiocyanatobenzyl)-diethylenetriaminepentaacetic acid)) by applying of these two techniques. Material and methods: The 10- and 50-fold molar excess of chelators were conjugated with pure trastuzumab and freeze dried to solid state. Vibrational spectroscopy (Raman and ATR-IR) was applied for further physico-chemical examinations of lyophilized non-labeled samples by directly applying under the laser beam of the instrument. The spectra of conjugated samples were compared with naked trastuzumab purified from commercial product Herceptin®. Results: The presence of characteristic amide bands (amide I, II and III bands) in ATR-IR and Raman spectra have indicated of retained native IgG1 structure of the antibody principally composed of β-sheets. The same as pure antibody, Raman spectra of conjugates contain the characteristic amide I band at ~1670 cm-1 and amide III band (1230-1300 cm-1). IR spectra of the samples also correspond with naked antibody and contain the amide I (1700-1600 cm-1), amide II (1480-1575 cm-1) and amide III bands (1255-1244 cm-1) specific for secondary structure of the proteins. Conclusion: No integrity changes, physicochemical and structural modifications of trastuzumab after conjugation approve retained β-sheet structure of the antibody and support the opportunity for further development of radioimmunotherapeutics and diagnostic products active against HER2 positive breast tumors

Electrophoresis and Raman spectroscopy characterization of integrity and secondary structure of p-SCN-Bn-DTPA- and p-SCN-Bn-1B4M-DTPA-conjugated trastuzumab

Trastuzumab is a humanized monoclonal antibody approved for treatment of HER2 positive breast cancer. Conjugation of trastuzumab offers a promising strategy of selective anticancer therapy. Trastuzumab-emtansin is a new generation of cytotoxic drug conjugated antibody with higher tumor selectivity and less toxicity of emtansin. Conjugates of trastuzumab with bifunctional chelators (BFCs) for further radiolabeling are a step ahead in the field of radiopharmacy for therapy and imaging of aggressive HER2 positive cancers. The purpose of this study is characterization of integrity and secondary structure of antibody in already formulated lyophilized conjugates with p-SCN-Bn-DTPA- and p-SCN-Bn-1B4M-DTPA- by applying SDS-PAGE electrophoresis and Raman spectroscopy. The results are positive and give an opportunity for further radiolabeling of freeze dried conjugates