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Polylysine (PL) was used to prepare derivatives with chelating properties. For this purpose PL was modified with cyclic or mixed anhydride of diethylene triamine pentaacetic acid (DTPA) or with deferoxamine (DFA). The modification permits to introduce into single polylysine molecule up to 100 DTPA residues (for 55 kDa polylysine), capable of firm binding metals like In or Gd, which are widely used in radioscintigraphy and NMR-tomography. To make polymeric chelates specific towards target areas, they were conjugated with specific monoclonal antibodies. Special technique of antibody modification with chelating polymer has been developed, permitting to bind whole antibody or its Fab with the polymer via minimal number of chemical bonds and with minimal loss in immunoreactivity. Very high specific radioactivity was achieved for antibodies labeled with 111 In: ca. 100 μCi per 1 μg of protein. In vivo studies were performed in rabbits with experimental myocardial infarction, utilizing monoclonal antibodies against cardiac myosin, labeled with 111In via DTPA-containing PL-based chelating polymers. Good accumulation of antibody-polymer conjugates in infarcted areas was demonstrated (target-to-normal ratio can reach several dozens).

Polylysine-based chelating polymers were used for site-specific modification of anti-CEA mAb Fab' fragments via their SH group distal to the antigen-binding site of the antibody molecule. Conjugation was performed using chain-terminal (pyridyldithio)propionate or 4-(p-maleimidophenyl)butyrate moieties to form reducible (S-S) or stable (S-C) bonds between a polymer and Fab' molecule, respectively. One S-S conjugate (S-S9) and two different S-C conjugates (S-C3 and S-C9) were prepared using 3- and 9-kDa molecular weight polymers. No significant loss of immunoreactivity was observed in solid-phase immunoassay, 90-95% of 111In-labeled conjugates being bound to CEA-coated Sepharose beads. After labeling with 111In, the conjugates had a specific radioactivity of 90-120 microCi/micrograms. Injected in nude mice bearing LS 174T carcinoma, the conjugates produced different biodistribution patterns. S-S9 was practically unable to accumulate in tumor and produced very rapid blood clearance of radioactivity and high uptake of radioactivity in liver, spleen, and especially kidneys (225% ID/g 24 h postinjection). S-C3 and S-C9 produced practically the same blood clearances (much slower than that of S-S9) and significant tumor uptake (9-10% ID/g at 24 h). S-C3 gave significantly lower radioactivity in spleen, skin, and bones, and cleared more rapidly from liver and kidneys. Renal uptake for S-C3 and S-C9 was rather high (45% ID/g at 24 h), but much lower than for S-S9.

Modification of antibodies with chelating polymers may be helpful for radioimmunoimaging, radioimmunotherapy, and NMR tomography. Succinylated polylysine was activated with carbodiimide/N-hydroxysulfosuccinimide in dimethyl sulfoxide and isolated as a dry solid. Sulfosuccinimide-esterified polymer was used for the two-stage coupling of an amino-containing chelating agent (deferoxamine) to monoclonal R11D10 (IgG) or its Fab fragment. Conjugates were separated from free components by using gel-chromatography and anion-exchange chromatography. Antibody-coupling efficiency and the loss of its immunoreactivity upon modification have been studied for polymers with different deferoxamine content. Specific binding of 67Ga to the corresponding antigen via the conjugate has been demonstrated.

F(ab′) 2 fragments of anti-carcinoembryonic antigen (CEA) monoclonal antibody (mAb) were modified with three chain-terminal polylysine-based chelating polymers so as to carry different electron charges. Immunoreactive conjugates labeled with 111 In up to a specific radioactivity of 120–140 μCi/μg were injected into nude mice bearing human colorectal carcinoma, and the biodistribution patterns were compared with each other and with that of an anti-CEA F(ab′) 2 -DTPA control. Immunoconjugate modified with positively-charged polymer produced the highest tumor uptake [up to 20% injected dose per gram (ID/g)], with very significant non-specific radioactivity in normal organs (particularly kidneys). When modified with a polymer carrying only a slight negative charge, the immunoconjugate also produced fairly high tumor uptake (up to 18% ID/g), with much lower non-specific radioactivity in normal organs. Highly negatively-charged conjugate produced the lowest tumor uptake (up to 8% ID/g), whereas blood and whole-body clearances were the fastest but slower than those of conventionally labeled F(ab′) 2 mAb. The possible mechanisms for the effects described are discussed.

A method is suggested for the preparation of chelating polymers containing a single terminal reactive group capable of interaction with proteins. These polymers were synthesized from N-CBZ-polylysine and DTPA and contain a terminal SH or pyridyldisulfide group. A polymer molecule with MW 13,500 is able to carry up to 40 DTPA residues. Polymers easily and quantitatively bind with antibodies (Fab fragments of antimyosin antibodies R11D10) with minimal effect on antibody immunoreactivity as revealed in ELISA assay and in direct immunoanalysis. Conjugates prepared can chelate radioactive metal ions reaching very high specific radioactivity (greater than 1 mCi 111In/10 micrograms of protein). Perspectives for their application are discussed.

Antimyosin Fab has been modified to carry highly negatively charged synthetic polymers containing DTPAs (DTPA-PL) as chelating agents, of starting molecular weights 3.3 and 17 kD. The immunoreactivities of the modified antibodies were unaffected by the modification procedure. The isoelectric points (PI) of unmodified antimyosin (AM) Fab (PI range 7-9, Mr = 52kD) were changed to PIs predominantly between 4 and 5 (Mr = 59 kD for DTPA-PL3.3kD-AM-Fab and 67kD for DTPA-PL17kD-AM-Fab). These AM-Fab preparations were tested for specific target localization and visualization in vivo in an experimental canine model of acute myocardial infarction. The charge-modified 111In-labeled AM-Fab preparations showed enhanced target (necrotic myocardium) visualization within 30 min of intravenous infusion and decreased background activity in normal myocardium (mean %ID/g +/- s.e.m., 0.0076 +/- 0.0006, n = 164, and 0.0056 +/- 0.0004, n = 92, for 111In-DTPA-PL3.3kD- and DTPA-PL17kD-AM-Fab respectively) relative to conventional 111In-DTPA-AM-Fab (0.0263 +/- 0.0037, n = 135) (p less than 0.001) or radioiodinated AM-Fab (0.0098 +/- 0.0006, n = 256) (p less than or equal to 0.001). Furthermore, the concentration of negatively charged 111In-labeled antimyosin Fab decreased in non-target organs such as the liver and kidneys. In diagnostic and therapeutic applications, charge-modified macromolecules may improve target localization and reduce non-target organ activity.

Monoclonal antibodies to heavy chains of the heart myosin were chemically modified by chelate polymers containing EDTA or DTPA residues. The modification allows binding up to 90 atoms of heavy metals (e.g. 111In, Mn2+, Cd3+) per protein globule, i.e. an increase in the specific activity of labeling 20 to 50-fold in comparison with previous methods. Specific affinity of the modified protein is preserved. Conjugates obtained may be useful for in vivo immuno-imaging.

Polyethyleneimine or polylysines of differing molecular sizes were substituted with either EDTA or DTPA and then with succinic acid groups. These polymers were then reacted with the amino groups on myosin-specific monoclonal antibody or its Fab using a water soluble carbodiimide. The polymer-antibody complexes were capable of binding up to 150 di- or trivalent ions per mole (Mn++, Gd , or 111In ) without attendant loss of antigen binding. The polylysine derivatives of the intact antibody were rapidly cleared and sequestered in the liver, whereas the polylysine 14-kilodalton (kd) derivative of Fab was cleared from the circulation with minimal hepatic and kidney sequestration. This differed from the biodistribution of intact antimyosin or its Fab labeled with 111In via direct attachment of DTPA to the epsilon amino group of the lysyl residues. Applications in magnetic resonance and nuclear imaging are envisioned.