Tumor targeting of radiometal labeled anti-CEA recombinant T84.66 diabody and t84.66 minibody: comparison to radioiodinated fragments

Recombinant antibody fragments offer potential advantages over intact monoclonal antibodies in the radioimmunoscintigraphy (RIS) of solid tumors. Due to their smaller molecular size, antibody fragments have shown rapid tumor targeting and blood clearance, a more uniform tumor distribution and a lower potential to elicit a human immune response. Previously, we have expressed two genetically engineered antibody fragments, the T84.66 diabody (scFv dimer) and the T84.66 minibody (scFv-CH3 dimer), specific to carcinoembryonic antigen (CEA). When radioiodinated, both antibody fragments exhibited rapid tumor targeting and rapid blood clearance in xenografted mice. To extend and optimize their future clinical RIS utility with radiometals, these antibody fragments were conjugated with the macrocycle 1,4,7,10-tetraazacyclododecane N,N',N' ',N' "-tetraacetic acid (DOTA) and labeled with 111In. Tumor targeting and biodistribution studies were carried out in athymic mice xenografted with a human colorectal tumor cell line, LS174T. The [111In]T84.66 diabody (55 kDa) exhibited very rapid tumor targeting with 12.5 +/- 0.4% injected dose per gram (% ID g(-1) +/- standard error) at 2 h and reached a maximum of 13.3 +/- 0.9% ID g(-1) at 6 h. However, kidney uptake was observed to reached a peak of 183.5 +/- 21.0% ID g(-1) at 6 h, a result similar to that reported by others for other low molecular weight fragments labeled with radiometals. Preadministration of an oral dose of D-lysine resulted in a 59% lowering of the renal accumulation at 6 h, but was accompanied by a 31% reduction of tumor uptake to 9.2 +/- 1.2% ID g(-1). The second recombinant antibody fragment, the [111In]T84.66 minibody (80 kDa), displayed rapid tumor targeting of 14.2 +/- 6.1% ID g(-1) at 2 h, and reached a maximum activity of 24.5 +/- 6.1% ID g(-1) by 12 h. Renal uptake achieved a plateau of 12-13% ID g(-1) which cleared to 7.2% ID g(-1) at 72 h. However, hepatic uptake was elevated and reached a maximum of 26.0 +/- 1.0% ID g(-1) at 12 h in these xenograft-bearing mice. Experiments in nontumor bearing mice showed a reduction of hepatic activity at 12 h to 16.6 +/- 1.5% ID g(-1), indicative of an intrinsic hepatic accumulation of the [111In]DOTA-T84.66 minibody or metabolites. While the anti-CEA [111In]DOTA-T84.66 diabody and T84.66 minibody retain the rapid tumor targeting properties of the radioiodinated form, the normal organ accumulation (kidneys and liver, respectively) of the [111In]DOTA forms appeared problematic for RIS and RIT applications. Development of alternative blocking strategies or new metabolizable chelates are under investigation to enhance the utility of the radiometal form of these and other promising recombinant antibody fragments.     

Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index

Antibody-drug conjugates enhance the antitumor effects of antibodies and reduce adverse systemic effects of potent cytotoxic drugs. However, conventional drug conjugation strategies yield heterogenous conjugates with relatively narrow therapeutic index (maximum tolerated dose/curative dose). Using leads from our previously described phage display-based method to predict suitable conjugation sites, we engineered cysteine substitutions at positions on light and heavy chains that provide reactive thiol groups and do not perturb immunoglobulin folding and assembly, or alter antigen binding. When conjugated to monomethyl auristatin E, an antibody against the ovarian cancer antigen MUC16 is as efficacious as a conventional conjugate in mouse xenograft models. Moreover, it is tolerated at higher doses in rats and cynomolgus monkeys than the same conjugate prepared by conventional approaches. The favorable in vivo properties of the near-homogenous composition of this conjugate suggest that our strategy offers a general approach to retaining the antitumor efficacy of antibody-drug conjugates, while minimizing their systemic toxicity.     

Site-specific expression of single-stranded antibody fragments in Nicotiana tabacum

Antibody expression and immunomodulation are modern molecular techniques to produce pharmaceuticals and to interfere with cellular metabolism or pathogen infectivity in plants. Nonetheless, there is still no generally applicable strategy to express correctly folded active antibodies or antibody fragments in different cell compartments. To facilitate expression, single-chain antibody fragments (scFvs) were made of mouse monoclonal antibodies, J2 and P6 that specifically recognize double-stranded RNA (dsRNA). Stabilizing double-stranded replication intermediates could modulate the biological activity of dsRNAs in plants, especially to influence virus replication. Along with cytoplasmic expression, scFvs were anchored to the plasma membrane; targeted to the apoplast for secretion and made ER-resident. Expression levels were analysed and transgenic plants were evaluated for resistance or tolerance to potato virus Y infection. We have established strategies for expression of correctly assembled antibodies or antibody fragments in different plant cell compartments.     

Site-specific immobilization of recombinant antibody fragments through material-binding peptides for the sensitive detection of antigens in enzyme immunoassays

The immobilization of an antibody is one of the key technologies that are used to enhance the sensitivity and efficiency of the detection of target molecules in immunodiagnosis and immunoseparation. Recombinant antibody fragments such as VHH, scFv and Fabs produced by microorganisms are the next generation of ligand antibodies as an alternative to conventional whole Abs due to a smaller size and the possibility of site-directed immobilization with uniform orientation and higher antigen-binding activity in the adsorptive state. For the achievement of site-directed immobilization, affinity peptides for a certain ligand molecule or solid support must be introduced to the recombinant antibody fragments. In this mini-review, immobilization technologies for the whole antibodies (whole Abs) and recombinant antibody fragments onto the surfaces of plastics are introduced. In particular, the focus here is on immobilization technologies of recombinant antibody fragments utilizing affinity peptide tags, which possesses strong binding affinity towards the ligand molecules. Furthermore, I introduced the material-binding peptides that are capable of direct recognition of the target materials. Preparation and immobilization strategies for recombinant antibody fragments linked to material-binding peptides (polystyrene-binding peptides (PS-tags) and poly (methyl methacrylate)-binding peptide (PMMA-tag)) are the focus here, and are based on the enhancement of sensitivity and a reduction in the production costs of ligand antibodies. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.     

Site-specific conjugation of metal carbonyl dendrimer to antibody and its use as detection reagent in immunoassay

We describe here the conjugation of polyclonal goat anti-rabbit antibody to generation 4 polyamidoamine (G4-PAMAM) dendrimers carrying (i) (η⁵-cyclopentadienyl) iron dicarbonyl succinimidato complexes as infrared (IR) probes, (ii) nitroaniline entities as nuclear magnetic resonance (NMR) probes, (iii) acetamide groups for surface neutralization, and (iv) hydrazide-terminated spacer arms for the reaction with aldehyde. To preserve a high binding affinity, the conjugation was performed on the carbohydrate moieties located on the Fc fragment. The resulting conjugates were characterized by Fourier transform-IR, ultraviolet (UV), and high-mass matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. On the basis of relative concentration ratios of IR probes and antibody, an average labeling of 30 IR probes per antibody was reached (i.e., more than twice the value obtained with our previous strategy that generated no spacer arm). Immunoassays revealed that the antibody-dendrimer conjugates retained 55.1% of immunoreactivity on average with respect to underivatized antibody. Finally, the conjugates were used to quantify their antigen by solid-phase carbonyl metallo immunoassay (CMIA). Results showed a significant enhancement of the IR signal, demonstrating the efficiency of the new conjugation strategy and the potential of the new antibody-dendrimer conjugates as universal immunoanalytical reagents.     

Fusogenics: a recombinant immunotoxin-based screening platform to select internalizing tumor-specific antibody fragments

Antibody-based therapeutics play a vital role in the treatment of certain cancers; however, despite commercial success, various strategies are being pursued to increase their potency and hence improve patient outcomes. The use of antibodies to deliver a cytotoxic payload offers a promising alternative for more efficacious therapies. Immunotoxins are composed of an internalizing antibody fragment linked to a bacterial or plant toxin. Once internalized, the payload, such as Pseudomonas exotoxin A (PE), blocks protein synthesis and induces apoptosis. Typically, immunotoxins are developed by first isolating a tumor-specific antibody, which is then either chemically linked to a toxin or reengineered as a fusion protein. Here, the authors describe the development of Fusogenics, an immunotoxin-based screening method that selects internalizing tumor-specific antibodies using a functional assay. Selected immune library clones were characterized and shown to be selective against normal tissues and specific to tumor tissues. In summary, the Fusogenics immunotoxin platform represents a unique, single-step selection approach combining specificity and functionality to isolate novel internalizing tumor-specific antibody fragments with potential for direct clinical application in the treatment of cancer.     

Site-specific conjugation of monodispersed DOTA-PEGn to a thiolated diabody reveals the effect of increasing peg size on kidney clearance and tumor uptake with improved 64-copper PET imaging

Optimal PET imaging of tumors with radiolabeled engineered antibodies requires, among other parameters, matching blood clearance and tumor uptake with the half-life of the engineered antibody. Although diabodies have favorable molecular sizes (50 kDa) for rapid blood clearance (t(1/2) = 30-60 min) and are bivalent, thereby increasing tumor uptake, they exhibit substantial kidney uptake as their major route of clearance, which is especially evident when they are labeled with the PET isotope (64)Cu (t(1/2) = 12 h). To overcome this drawback, diabodies may be conjugated to PEG, a modification that increases the apparent molecular size of the diabody and reduces kidney uptake without adversely affecting tumor uptake or the tumor to blood ratio. We show here that site-specific attachment of monodispersed PEGn of increasing molecular size (n = 12, 24, and 48) can uniformly increase the apparent molecular size of the PEG-diabody conjugate, decrease kidney uptake, and increase tumor uptake, the latter due to the increased residence time of the conjugate in the blood. Since the monodispersed PEGs were preconjugated to the chelator DOTA, the conjugates were able to bind radiometals such as (111)In and (64)Cu that can be used for SPECT and PET imaging, respectively. To allow conjugation of the DOTA-PEG to the diabody, the DOTA-PEG incorporated a terminal cysteine conjugated to a vinyl sulfone moiety. In order to control the conjugation chemistry, we have engineered a surface thiolated diabody that incorporates two cysteines per monomer (four per diabody). The thiolated diabody was expressed and purified from bacterial fermentation and only needs to be reduced prior to conjugation to the DOTA-PEGn-Cys-VS. This novel imaging agent (a diabody with DOTA-PEG48-Cys-VS attached to introduced thiols) gave up to 80%ID/g of tumor uptake with a tumor to blood ratio (T/B) of 8 at 24 h when radiolabeled with (111)In and 37.9% ID/g of tumor uptake (T/B = 8) at 44 h when radiolabeled with (64)Cu in PET imaging in an animal model. Tumor uptake was significantly improved from the 50% ID/g at 24 h observed with diabodies that were pegylated on surface lysine residues. Importantly, there was no loss of immunoreactivity of the site-specific Cys-conjugated diabody to its antigen (TAG-72) compared to the parent, unconjugated diabody. We propose that thiolated diabodies conjugated to DOTAylated monodisperse PEGs have the potential for superior SPECT and PET imaging in a clinical setting.     

Technetium-99m labeled 50H.19 antibody fragments: Interaction of the antibody with platelets

The monoclonal antibody 50H.19 recognized three antigens (M_r = 31-, 40-, 45-K) on normal and thromboasthenic platelets, but only one (M_r = 31-K) on Bernard-Soulier platelets. The intact antibody and its F(ab′)₂ fragments, had direct platelet-aggregating activity, and induced the platelet release reaction. The intact antibody potentiated platelet aggregation induced by platelet-activating factor or thrombin. Additions of indomethacin did not inhibit aggregation: addition of PGI₂, or a calcium channel blocker completely inhibited aggregation. A reduced amount of platelet-aggregating activity was observed with antibody fragments prepared for labeling with ^99mTc by pre-exposure to stannous ions, and herein used in biodistribution studies and elsewhere in thrombus imagining studies (J. Nucl. Med. 27: 1315; 1986). Antibody fragments radiolabeled with ^99mTc bound to isolated platelets and to clots containing platelets.     

Development of novel linkers to conjugate pharmacophores to a carrier antibody

We have developed modified maleimide novel linkers with improved chemical stability that could potentially be used in conjugating various pharmacophores such as oligo nucleotides, peptides, and proteins to antibodies to afford novel biologics with well-defined therapeutic benefits and improved pharmacokinetic properties. These linkers expand the array of tools available for bioconjugation of pharmacophores to antibodies.     

Isolation and characterization of recombinant antibody fragments against CDC2a from Arabidopsis thaliana.

In order to obtain recombinant antibody fragments that bind the cell-cycle protein CDC2a from Arabidopsis thaliana (CDC2aAt), two phage display libraries of single-chain variable (scFv) fragments were constructed. One library was derived from mice immunized with recombinant CDC2aAt N-terminally fused to a His6-tag (His-CDC2aAt) and the other was made out of an anti-PSTAIRE hybridoma cell line. Six specific His-CDC2aAt-binding phage clones (3D1, 3D2, 3D10, 3D25, 4D21 and 4D47) were isolated by panning. The isolated monoclonal phage clones, as well as the soluble scFv fragments produced in the periplasm of Escherichia coli, bind His-CDC2aAt in ELISA and on Western blots. Moreover, four clones (3D1, 3D2, 3D10 and 4D21) detect specifically CDC2aAt from Arabidopsis cell suspensions on Western blots. Clone 4D21 binds the PSTAIRE epitope, whereas the 3D1, 3D2 and 3D10 clones bind, as yet unidentified, epitopes of CDC2aAt. Furthermore, the accumulation and antigen-binding activity of these scFv fragments in a reducing environment were assessed. No interaction could be shown between the scFv fragments and CDC2aAt in a yeast two-hybrid assay. However, after transient expression of the scFv fragments in the cytosol of tobacco leaves, three of six scFv fragments (3D1, 3D2 and 3D10) accumulated in the plant cytosol and ELISA results indicate that these scFv fragments retained antigen-binding activity.     

Differential conjugation of tat peptide to superparamagnetic nanoparticles and its effect on cellular uptake

Surface modification of superparamagnetic contrast agents with HIV-1 tat peptide has emerged as a promising means for intracellular magnetic labeling and noninvasive tracking of a large number of cell types with MRI. To achieve efficient intracellular delivery of the nanoparticles, we investigated the effect on cellular uptake of superparamagnetic iron oxide particles by varying the number of attached tat peptides. First, we report here a modified P2T method in measuring the numbers of surface attachments per particle through disulfide linkage. The method was shown to have desirable simplicity and reproducibility. With the P2T method as a tool, conjugates with progressively higher ratios of peptide-to-particle were synthesized. We were able to demonstrate that higher numbers of tat peptide facilitate the cellular uptake of iron oxide nanoparticles in a nonlinear fashion. Cells labeled with these optimized preparations were readily detectable by MR imaging. The increase in sensitivity could allow in vivo tracking of 100-fold lower cell concentration than currently described.     

Trifunctional conjugation reagents. Reagents that contain a biotin and a radiometal chelation moiety for application to extracorporeal affinity adsorption of radiolabeled antibodies

A method of removing radiolabeled monoclonal antibodies (mAbs) from blood using a device external to the body, termed extracorporeal affinity-adsorption (EAA), is being evaluated as a means of decreasing irradiation of noncancerous tissues in therapy protocols. The EAA device uses an avidin column to capture biotinylated-radiolabeled mAbs from circulated blood. In this investigation, three trifunctional reagents have been developed to minimize the potential deleterious effect on antigen binding brought about by the combination of radiolabeling and biotinylation of mAbs required in the EAA approach. The studies focused on radiolabeling with (111)In and (90)Y, so the chelates CHX-A' '-DTPA and DOTA, which form stable attachments to these radionuclides, were incorporated in the trifunctional reagents. The first trifunctional reagent prepared did not incorporate a group to block the biotin cleaving enzyme biotinidase, but the two subsequent reagents coupled aspartic acid to the biotin carboxylate for that purpose. All three reagents used 4,7,10-trioxa-1,13-tridecanediamine as water-soluble spacers between an aminoisophthalate core and the biotin or chelation group. The mAb conjugates were radioiodinated to evaluate cell binding as a function of substitution. Radioiodination was used so that a direct comparison with unmodified mAb could be made. Evaluation of the number of conjugates per antibody versus cell binding immunoreactivities indicated that minimizing the number of conjugates was best. Interestingly, a decrease of radioiodination yield as a function of the number of isothiocyanate containing conjugates per mAb was noted. The decreased yields were presumably due to the presence of thiourea functionality formed in the conjugation reaction. Radiolabeling with (111)In and (90)Y was facile at room temperature for conjugates containing the CHX-A' ', but elevated temperature (e.g., 45 degrees C) was required to obtain good yields with the DOTA chelate. Stability of (90)Y labeled mAb in serum, and when challenged with 10 mM EDTA, was high. However, challenging the (90)Y labeled mAb with 10 mM DTPA demonstrated high stability for the DOTA containing conjugate, but low stability for the CHX-A' ' containing conjugate. Thus, the choice between these two chelating moieties might be made on requirements for facile and gentle labeling versus very high in vivo stability. Application of the trifunctional biotinylation reagents to the blood clearance of labeled antibodies in EAA is under investigation. The new reagents may also be useful for other applications.     

Quantitation of the tumor-targeting properties of antibody fragments conjugated to cell-permeating HIV-1 TAT peptides

Human monoclonal antibodies are promising agents for the development of more selective anticancer therapeutics. However, the tumor-targeting efficiency of most anticancer antibodies is severely limited by their poor penetration into the tumor mass. Recent studies have shown that a peptide derived from the HIV TAT protein could improve the distribution of cytoplasmic reporter proteins when administered systemically as fusion proteins or cross-linked chimeras. In this article, we tested by quantitative biodistribtution analysis whether conjugation to TAT peptides could improve the tumor targeting properties of scFv(L19)-Cys: an engineered human antibody fragment specific for the ED-B domain of fibronectin, a marker located in the modified extracellular matrix surrounding tumor neovasculature. Our results show that TAT peptides, consisting either of L-amino acids or D-amino acids, can efficiently transduce target cells when conjugated to fluorophores and/or antibody fragments, suggesting a receptor-independent cell entry mechanism. However, conjugation of scFv(L19)-Cys to TAT peptides resulted in a severely reduced tumor targeting performance compared to the unconjugated antibody, as measured in murine F9 teratocarcinoma-bearing mice, after intravenous injection of the radiolabeled antibody preparations. Our results outline the usefulness of TAT peptides for the efficient in vitro transduction of cells with globular proteins. In particular, the use of TAT peptides composed of D-amino acids may significantly reduce proteolytic degradation. At the same time, the poor biodistribution properties of antibody-TAT conjugates cast doubts over the applicability of this methodology for the delivery of biopharmaceuticals in vivo.     

Applications of mesenchymal stem cells labeled with Tat peptide conjugated quantum dots to cell tracking in mouse body

Fluorescent quantum dots have great potential in cellular labeling and tracking. Here, PEG encapsulated CdSe/ZnS quantum dots have been conjugated with Tat peptide, and introduced into living mesenchymal stem cells. The Tat peptide conjugated quantum dots in mesenchymal stem cells were assessed by fluorescent microscopy, laser confocal microscope and. flow cytometry. The result shows that Tat peptide conjugated quantum dots could enter mesenchymal stem cells efficiently. The Tat-quantum dots labeled stem cells were further injected into the tail veins of NOD/SCID beta2 M null mice, and the tissue distribution of these labeled cells in nude mice were examined with fluorescence microscope. The result shows that characteristic fluorescence of quantum dots was observed primarily in the liver, the lung and the spleen, with little or no quantum dots accumulation in the brain, the heart, or the kidney.     

Improved biological activity of antisense oligonucleotides conjugated to a fusogenic peptide.

Recently several groups reported a dramatic improvement of reporter gene transfection efficiency using a fusogenic peptide, derived from the Influenza hemagglutinin envelop protein. This peptide changes conformation at acidic pH and destabilizes the endosomal membranes thus resulting in an increased cytoplasmic gene delivery. We describe the use of a similar fusogenic peptide in order to improve the antiviral potency of antisense oligodeoxynucleotides (anti TAT) and oligophosphorothioates (S-dC28) on de novo HIV infected CEM-SS lymphocytes in serum-free medium. We observed as 5 to 10 fold improvement of the anti HIV activities of the phosphodiester antisense oligonucleotides after chemical coupling to the peptide in a one to one ratio by a disulfide or thioether bond. No toxicities were observed at the effective doses (0.1-1 microM). No sequence specificity was obtained and the fusogenic peptide possessed some antiviral activities on its own (IC50: 6 microM). A S-dC28-peptide disulfide linked conjugate and a streptavidin-peptide-biotinylated S-dC28 adduct showed similar activities as the free S-dC28 oligonucleotide (IC50: 0.1-1 nM). As expected, all the compounds were less potent in the presence of serum but the relative contribution of peptide coupling was maintained.     

Structure-function relationship of myotoxin a using peptide fragments.

Myotoxin a, a small basic polypeptide isolated from the venom of prairie rattlesnake (Crotalus viridis viridis), has been shown to bind to sarcoplasmic reticulum (SR) Ca(2+)-ATPase. The attachment of myotoxin a to Ca(2+)-ATPase is believed to cause uncoupling of the calcium pump. In order to further elucidate which portion of myotoxin a is important for the uncoupling action, five peptides were synthesized and two peptide fragments were obtained by chemical cleavage. These peptides correspond to discrete portions of the primary sequence of myotoxin a. The peptides are equivalent to the primary sequence of myotoxin a from 1 to 16 residues, 7 to 22 residues, 13 to 28 residues, 19 to 34 residues, and 25 to 42 residues. Chemically produced fragments are equivalent to 1 to 28 residues and 29 to 42 residues of myotoxin a. Peptides of the sequences "YKQCHKKGGHCFPKEK" and "LGKMDCRWKWKCCKKGSG" of myotoxin a inhibited 45Ca uptake into isolated SR and bound to Ca(2+)-ATPase. The same peptides caused weak skeletal muscle vacuolization similar to that caused by native myotoxin a and increased serum creatine kinase activity. The active peptides correspond to the N-terminal and C-terminal portions of myotoxin a. The inactive or less active peptides have sequences which correspond to the middle sequence of myotoxin a. From this study, both the N-terminal and the C-terminal regions of primary sequence of myotoxin a are required to express myotoxin a's biological activity.     

Site-specific modification of a single-chain antibody using a novel glyoxylyl-based labeling reagent.

A novel, highly specific protein modification approach is described. By using conventional molecular cloning techniques, a protein can be constructed and expressed such that the N-terminal residue is replaced by cysteine. Its 1,2-aminothiol structure reacts very specifically with a glyoxylyl group at pH 7 or below, forming a relatively stable thiazolidine bridge. Therefore, a glyoxylyl-based labeling agent (e.g., radioactive tags, fluorescent probes, biotin) can be used to specifically modify a protein at its N-terminus. To highlight this novel approach, a recombinant anti-insulin single chain antibody (scFv) was specifically biotinylated at its N-terminus even in the presence of other proteins in the total cell lysate. The glyoxylyl-biotinylated scFv retained binding activity similar to unmodified scFv.     

High-yield production of recombinant antibody fragments in HEK-293 cells using sodium butyrate

To develop new recombinant monoclonal antibody fragments for therapy and imaging, it is indispensable to have a simple and easy procedure to handle the eukaryotic expression system for production of proteins in high amounts. Gene amplification techniques such as the dehydrofolate reductase (DHFR) system in Chinese hamster ovary cells or the glutamine synthase system in myeloma cells have a couple of disadvantages. The selection procedure is complex, time-consuming, and not fruitful in all cases. The toxic drug methotrexate (for the DHFR system) can increase the production rate but decreases the specific growth rate of the cells. The production rate is not always stable over a long-term cultivation period. To overcome these problems, we are using stably transfected human embryonic kidney (HEK-293) cells in combination with an efficient screening method. Sodium butyrate can increase the expression of recombinant antibody fragments in the transfectomas up to 500 micrograms/4.2 x 10(7) cells/24 h corresponding to 175 micrograms/mL culture medium. This strategy allows a rapid development of new recombinant monoclonal antibody fragments and allows one to proceed rapidly to in vivo testing.     

Chemical modification to reduce renal uptake of disulfide-bonded variable region fragment of anti-Tac monoclonal antibody labeled with 99mTc.

The anti-Tac disulfide-bonded variable region fragment (dsFv) is a genetically engineered, 25 kDa, murine monoclonal antibody fragment that recognizes the alpha subunit of the interleukin-2 receptor (IL-2Ralpha). The dsFv radiolabeled with the tetrafluorophenyl ester (TFP) of [99mTc]mercaptoacetyltriglycine ([99mTc]MAG3-TFP) showed rapid tumor uptake and fast blood clearance in mice, resulting in high tumor-to-nontumor background ratios. However, its high renal uptake was a problem. In this study, we tested the effect of lowering the isoelectric point (pI) of dsFv to <9.3 on renal and tumor uptake. To lower the pI, dsFv was acylated simultaneously with both [99mTc]MAG3-TFP and TFP-glycolate. The acylation of dsFv decreased its pI and its immunoreactivity inversely proportional to the molar ratio of TFP-glycolate to dsFv, whereas the conjugation of [99mTc]MAG3-TFP alone did not. When biodistribution studies were performed in nude mice, the effect of the lowered pI was reflected primarily in decreased kidney uptake and whole-body retention, with its highest effect seen at the earliest time point (15 min) after injection. In tumor-bearing nude mice, glycolated [99mTc]MAG3-dsFv with a pI range of 4.9 to 6.5 accumulated selectively into IL-2 receptor-positive SP2/Tac tumor similar to that of the control [125I]dsFv labeled by the Iodo-Gen method, whereas its renal uptake was 25% of [125I]dsFv at 15 min. At 90 min, the ratios of tumor to receptor-negative SP2/0 tumor, liver, kidney, stomach, and blood had peaked at 10.9, 8.5, 0.3, 5.0, and 6.2, respectively, for the glycolated [99mTc]MAG3-dsFv. The corresponding ratios for [125I]dsFv were 3.7, 5.0, 0.1, 1.5, and 2.1, respectively.     

Structural effect of a recombinant monoclonal antibody on hinge region peptide bond hydrolysis

IgG hinge region peptide bonds are susceptible to degradation by hydrolysis. To study the effect of Fab and Fc on hinge region peptide bond hydrolysis, a recombinant humanized monoclonal IgG1 antibody, its F(ab')2 fragment, and a model peptide with amino acid sequence corresponding to the hinge region were incubated at 40 degrees C in formulation buffer including complete protease inhibitor and EDTA for 0, 2, 4, 6 and 8 weeks. Two major cleavage sites were identified in the hinge region of the intact recombinant humanized monoclonal antibody and its F(ab')2 fragment, but only one major cleavage site of the model peptide was identified. Hinge region peptide bond hydrolysis of the intact antibody and its F(ab')2 fragment degraded at comparable rates, while the model peptide degraded much faster. It was concluded that Fab region of the IgG, but not Fc portion had significant effect on preventing peptide bond cleavage by direct hydrolysis. Hydrolysis of hinge region peptide bonds was accelerated under both acidic and basic conditions.