ABSTRACT
Three types of metal-chelating polymers (MCPs) with hydrazide end groups were synthesized. (1) The first set of polymers (the F-series) was synthesized with a furan end group, and all of the pendant groups along the chain carried only a diethylenetriaminepentaacetic acid (DTPA) metal-chelating functionality. The hydrazide was introduced via a Diels-Alder reaction between the furan and 3,3'-N-[ε-maleimidocaproic acid] hydrazide (EMCH). (2) The P-series polymers was designed to carry several copies of a nuclear-localization peptide sequence (NLS peptides, CGYGPKKKRKVGG, harboring the NLS from the simian virus 40 large T-antigen) in addition to the DTPA metal-chelating groups. (3) The third type of polymer (the P-Py series) was a variation of the P-series polymers but with the introduction of a small number of pyrene chromophores along the backbone to allow for UV measurement of the incorporation of the MCPs into trastuzumab (tmab). These hydrazide-terminated polymers were site-specifically conjugated to aldehyde groups generated by NaIO4 oxidation of the pendant glycan in the Fc domain of tmab. The immunoconjugates were radiolabeled with (111)In and analyzed by SE-HPLC to confirm the attachment of the polymer to the antibody. HER2 binding assays demonstrated that neither the MCPs nor the presence of the NLS peptides interfered with specific antigen recognition on SK-Br-3 cells, although nonspecific binding was increased by polymer conjugation. Our results suggest that MCPs can be site-specifically attached to antibodies via oxidized glycans in the Fc domain and labeled with (111)In to construct radioimmunoconjugates with preserved immunoreactivity.
Subject(s)
Antibodies, Monoclonal, Humanized , Antineoplastic Agents/chemical synthesis , Chelating Agents/chemical synthesis , Electrons , Polyglutamic Acid/chemical synthesis , Radioimmunotherapy/methods , Antibodies, Monoclonal, Humanized/metabolism , Antibodies, Monoclonal, Humanized/therapeutic use , Antineoplastic Agents/metabolism , Antineoplastic Agents/therapeutic use , Binding Sites/physiology , Breast Neoplasms/drug therapy , Breast Neoplasms/mortality , Cell Line, Tumor , Chelating Agents/metabolism , Chelating Agents/therapeutic use , Electrons/therapeutic use , Female , Humans , Polyglutamic Acid/metabolism , Polymers/chemical synthesis , Polymers/metabolism , Polymers/therapeutic use , Receptor, ErbB-2/metabolism , TrastuzumabABSTRACT
We describe the synthesis and characterization of a family of poly(N-alkylacrylamide) polymers carrying 2-6 fluorescent dye molecules, â¼70 pendant DTPA (diethylenetriaminepentaacetic acid) groups, and an orthogonal maleimide end-group for covalent attachment to an antibody (Ab). These dual-purpose labels were designed for use in multiplexed immunoassays based on both mass cytometry and fluorescent flow cytometry. A challenge in the polymer synthesis was finding conditions for attaching a sufficient number of dye molecules to each polymer chain. Although attachment of a terminal maleimide to the polymers was not as efficient as anticipated, the end-functional polymers were still effective in labeling Abs. Secondary goat antimouse IgG was labeled with the four dual-label polymers as well as a control polymer, and while the resultant antibody-polymer conjugates showed positive performance in mass cytometric and fluorescent assays, some trials showed problems such as low signal and nonspecific adsorption. Four primary antibody conjugates were prepared and used to stain cells in 4-plex assays. The results of both primary assays are bittersweet in that the CD3-FITC and CD45-DyLight 649 conjugates performed well, while the CD13-DyLight 405 and the CD38-DyLight 549 conjugates did not.
Subject(s)
Acrylic Resins/chemistry , Biological Assay , Fluorescent Dyes/chemistry , Maleimides/chemistry , Pentetic Acid/chemistry , Adsorption , Animals , Antibodies/chemistry , Antibodies/immunology , Flow Cytometry , Goats , Image Cytometry/methods , Immunoconjugates/chemistry , Immunoconjugates/immunology , Immunoglobulin G/chemistry , Immunoglobulin G/immunology , Mice , Molecular Weight , Samarium/chemistryABSTRACT
PURPOSE: To study the effects of backbone composition and charge of biotin-functionalized metal-chelating polymers (Bi-MCPs) for (111)In complexed to streptavidin (SAv)-trastuzumab Fab fragments on tumor and normal tissue localization. METHODS: Bi-MCPs were synthesized with a polyacrylamide [Bi-PAm(DTPA)(40)], polyaspartamide [Bi-PAsp(DTPA)(33)] or polyglutamide [Bi-PGlu(DTPA)(28)] backbone and harboured diethylenetriaminepentaacetic acid (DTPA) chelators for (111)In. Bi-PAm(DTPA)(40) had a net negative charge; Bi-PAsp(DTPA)(33) and Bi-PGlu(DTPA)(28) were zwitterionic with a net neutral charge. Binding to HER2+ SKOV-3 human ovarian carcinoma cells was determined. Tissue uptake was studied in Balb/c mice by MicroSPECT/CT imaging and biodistribution studies. Tumor and normal tissue uptake of (111)In-labeled Bi-PAsp(DTPA)(33) or Bi-PGlu(DTPA)(28) complexed to SAv-Fab was evaluated 48 h post-injection in athymic mice with subcutaneous SKOV-3 xenografts. RESULTS: SAv-Fab complexed to MCPs bound specifically to SKOV-3 cells; but specific binding was decreased 2-fold. Liver uptake was 5-13 fold higher for Bi-PAm(DTPA)(40) than Bi-PAsp(DTPA)(33) and Bi-PGlu(DTPA)(28) but was reduced by decreasing negative charges by saturation with indium. (111)In-Bi-PAsp(DTPA)(33) complexed to SAv-Fab accumulated in SKOV-3 tumors; low tumor uptake was found for (111)In-Bi-PGlu(DTPA)(28)-SAv-Fab. CONCLUSIONS: Zwitterionic MCPs composed of polyaspartamide with a net neutral charge are most desirable for constructing radioimmunoconjugates.
Subject(s)
Antibodies, Monoclonal, Humanized/pharmacokinetics , Antineoplastic Agents/pharmacokinetics , Chelating Agents/chemistry , Immunoconjugates/pharmacokinetics , Indium Radioisotopes/pharmacokinetics , Polymers/chemistry , Animals , Antibodies, Monoclonal, Humanized/chemistry , Antineoplastic Agents/chemistry , Biotin/chemistry , Cell Line, Tumor , Female , Humans , Immunoconjugates/chemistry , Immunoglobulin Fab Fragments/chemistry , Indium Radioisotopes/chemistry , Mice , Mice, Inbred BALB C , Mice, Nude , Ovarian Neoplasms/diagnosis , Ovarian Neoplasms/drug therapy , Pentetic Acid/analogs & derivatives , Streptavidin/chemistry , TrastuzumabABSTRACT
We describe the synthesis of metal chelating polymers based on polyaspartamide and polyglutamide backbones as carriers for (111)In in radioimmunoconjugates. These polymers [PAsp(DTPA), PGlu(DTPA)] have a biotin end group and diethylenetriaminepentaacetic acid (DTPA) chelators attached to the primary amines of the diethylenetriamine (DET) pendant groups of biotin-poly{N'-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} [PAsp(DET)] and of biotin-poly{N'-[N-(2-aminoethyl)-2-aminoethyl]glutamide} [PGlu(DET)]. Like Asn-containing proteins and polypeptides, polyaspartamides undergo uncatalyzed degradation under model physiological conditions (10 mM phosphate buffer, pH 7.4, 150 mM NaCl). We studied the uncatalyzed degradation of the polyaspartamide polymers by size exclusion chromatography and found that the degradation rate was sensitive to the nature of the pendant groups. The metal-free polymer underwent somewhat slower degradation than the corresponding polymers in which the DTPA groups were saturated with Eu(3+) or In(3+), but even after 14 days, substantial fractions of the polymers survived. We conclude that these polymers undergo negligible degradation on the time scale (24-48 h) of radioimmunotherapy treatment of tumors with (111)In. From a mechanistic perspective, we note that these degradation rates are on the order of the deamidation rates reported [J. Peptide Res. 2004, 63, 426] for Asn-containing pentapeptides, with half-times on the order of 10 days, but much slower than the rapid decay (hours) reported recently [Biomaterials 2010, 31, 3707] for poly{N'-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} itself. This variation in degradation rate can be explained in terms of the influence of positive charges on the pendant group enhancing the acidity of the side-chain amide nitrogen of the aspartamide repeat unit. The DET pendant group is positively charged at pH 7, but in indium-loaded PAsp(DTPA) this charge is offset by the net negative charge of the DTPA-In complex.
Subject(s)
Coordination Complexes/chemistry , Peptides/chemistry , Chemistry, Physical , Coordination Complexes/chemical synthesis , Hydrolysis , Molecular Structure , Peptides/chemical synthesisABSTRACT
A mild, fast and flexible method for photoimmobilization of biomolecules based on the light-initiated thiol-ene reaction has been developed. After investigation and optimization of various surface materials, surface chemistries and reaction parameters, microstructures and microarrays of biotin, oligonucleotides, peptides, and MUC1 tandem repeat glycopeptides were prepared with this photoimmobilization method. Furthermore, MUC1 tandem repeat glycopeptide microarrays were successfully used to probe antibodies in mouse serum obtained from vaccinated mice. Dimensions of biomolecule microstructures were shown to be freely controllable through photolithographic techniques, and features down to 5 microm in size covering an area of up to 75x25 mm were created. Use of a confocal laser microscope with a UV laser as UV-light source enabled further reduction of biotin feature size opening access to nanostructured biochips.
Subject(s)
Biotin/chemistry , Microarray Analysis , Mucin-1/chemistry , Oligonucleotides/chemistry , Sulfhydryl Compounds/chemistry , Animals , Antibodies/immunology , Antibodies/metabolism , Glycopeptides/chemistry , Glycopeptides/metabolism , Immobilized Proteins/chemistry , Immobilized Proteins/metabolism , Light , Mice , Mucin-1/metabolism , Photochemical Processes , Ultraviolet RaysABSTRACT
Progress in the development of protein-immobilization strategies and methods has made protein biochips increasingly accessible. The integration of these assay and analysis platforms into biomedical and biotechnological research has substantially expanded the repertoire of methods available for proteomics and biomarker research and for drug development. This Minireview highlights selected developments in the application of protein biochips in these fields.
Subject(s)
Biomarkers , Biotechnology , Drug Discovery , Protein Array Analysis/statistics & numerical data , Proteomics , Animals , Biomedical Research , HumansABSTRACT
Protein biochips are at the heart of many medical and bioanalytical applications. Increasing interest has been focused on surface activation and subsequent functionalization strategies for immobilizing these biomolecules. Different approaches using covalent and noncovalent chemistry are reviewed; particular emphasis is placed on the chemical specificity of protein attachment and on retention of protein function. Strategies for creating protein patterns (as opposed to protein arrays) are also outlined. An outlook on promising and challenging future directions for protein biochip research and applications is also offered.