Towards tumour targeting with copper-radiolabelled macrocycle–antibody conjugates: synthesis, antibody linkage, and complexation behaviour

Abstract

A set of four tetra-azamacrocyclic ligands bearing aminomethylphenyl substituents have been prepared and may be attached to a monoclonal antibody via an intermediate thiol-specific vinylpyridine linker molecule. The resultant conjugates may be efficiently radiolabelled with %u or 67Cu at pH 4 to minimise non-specific protein binding, and the copper labelled antibody-conjugate is stable with respect to copper loss in vivo. The forward rate of copper binding has been optimised through a kinetic analysis using stopped-flow spectrophotometry. In succinate buffer, anionic copper species CU(SUCC),~-(log p = 4.35) and HCu(succ),-(log p = 9.64) are the kinetically significant copper species in the pH range 3.6-5.6.A challenging aspect of current clinical oncology involves the development of effective methods of radioimmunoimaging and radioimmunotherapy.2 The two techniques are complementary: in uiuo imaging provides not only useful information about the size, morphology and location of tumour deposits but it is also vital for prognosis and for monitoring the effects of therapeutic radiation on the tumour following treatment. Large and accessible primary tumours are often best removed by surgery but smaller-more widely distributed-secondary metastases may be cleared following radioimmunotherapy. Most radioimaging applications use y-ray emission from the nuclear decay of a given radioisotope (e.g. 99mTc, 1311 or "'In). Notwithstanding the development of single photon-emitter computerised tomography, vastly superior high resolution images may be obtained with positron emission tomography (down to 3 mm).3 The most common positron emitters "Rb and 68Ga have half-lives (1.4 and 1.1 min) that are far too short to permit localisation of a radiolabelled antibody to the tumour. In this respect 64Cu is more suitable ( t , = 12.8 h) and it was prepared for these studies 'carrier-free' from 64Zn0 by an (n,p) reaction followed by electrochemical extraction.There is a particular interest in those monoclonal antibodies which bind specifically to tumour associated antigens for the treatment of the more common malignancies e.g. carcinoma of the breast, lung, or colon. The antibody B72.3 binds specifically to tumour associated glycoprotein (TAG-72) found with a high incidence in colon carcinoma t i ~s u e . ~ By irreversibly attaching a Paper 9/03466H

A novel tetrabranched neurotensin(8–13) cyclam derivative: Synthesis, 64Cu-labeling and biological evaluation

“…After 14 h, the 64 Cu-labeling yield was lower than 5% in ammonium acetate buffer solution at ambient temperature. Such slow complexation kinetics were also obtained for the labeling of cyclam-antibody conjugates under comparable conditions [72]. Replacement of acetate by the non-coordinating 2-[N-morpholino]ethanesulfonate anion causes a significant acceleration.…”

New Heterocyclic Mono‐ and Bis(α‐hydroxymethyl)phosphinic Acids: Synthesis and CuIIBinding Abilities

“…These compounds (especially the pyridine derivatives) can be viewed as good chelating agents with a wide application in biological screening and NMR imaging. [12,13] Therefore, we have modified and extended our method used for the synthesis of (α-hydroxymethyl)phosphinic acids 2a-d to the synthesis of the corresponding heterocyclic bis(α-hydroxymethyl)phosphinic acids 3a-d, as illustrated in Scheme 2. The structures and yields of products 3a-d are given in Table 2.…”

A Facile, Water-Soluble Method for Modification of Proteins with DOTA. Use of Elevated Temperature and Optimized pH To Achieve High Specific Activity and High Chelate Stability in Radiolabeled Immunoconjugates

“…Bifunctional chelating agents which form physiologically stable complexes with metal ions are desirable for radioimmunoscintigraphy and radioimmunotherapy in order to reduce radiation damage and toxicity to normal organs and tissues (7). One class of chelating agents that has shown great promise for monoclonal antibody applications are the polyazamacrocyclic polycarboxylate ligands (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23), and one of these compounds, 1,4,7,10-tetraazacyclododecane N,N""',N"'-tetraacetic acid (DOTAl), has become a subject of widespread interest because it forms extremely stable complexes with a wide variety of metals (24)(25)(26)(27)(28)(29).…”