Altered gene expression and spongiotrophoblast differentiation in placenta from a mouse model of diabetes in pregnancy.

AIMS/HYPOTHESIS: Pregnancies complicated by diabetes have a higher risk of adverse outcomes for mothers and children, including predisposition to disease later in life, e.g. metabolic syndrome and hypertension. We hypothesised that adverse outcomes from diabetic pregnancies may be linked to compromised placental function, and sought to identify cellular and molecular abnormalities in diabetic placenta. METHODS: Using a mouse model of diabetic pregnancy, placental gene expression was assayed at mid-gestation and cellular composition analysed at various stages. Genome-wide expression profiling was validated by quantitative PCR and tissue localisation studies were performed to identify cellular correlates of altered gene expression in diabetic placenta. RESULTS: We detected significantly altered gene expression in diabetic placenta for genes expressed in the maternal and those expressed in the embryonic compartments. We also found altered cellular composition of the decidual compartment. In addition, the junctional and labyrinth layers were reduced in diabetic placenta, accompanied by aberrant differentiation of spongiotrophoblast cells. CONCLUSIONS/INTERPRETATION: Diabetes during pregnancy alters transcriptional profiles in the murine placenta, affecting cells of embryonic and maternal origin, and involving several genes not previously implicated in diabetic pregnancies. The molecular changes and abnormal differentiation of multiple cell types precede impaired growth of junctional zone and labyrinth, and of placenta overall. Regardless of whether these changes represent direct responses to hyperglycaemia or are physiological adaptations, they are likely to play a role in pregnancy complications and outcomes, and to have implications for developmental origins of adult disease.

Effects of humanization and gene shuffling on immunogenicity and antigen binding of anti-TAG-72 single-chain Fvs.

One major constraint in the clinical application of murine monoclonal antibodies (MAbs) is the development of a human antimurine antibody response. The immunogenicity of MAbs can be minimized by replacing nonhuman regions with corresponding human sequences. The studies reported in our article were undertaken to analyze the immunoreactivity and the immunogenicity of the CC49 single-chain antibody fragments (scFvs): (i) an scFv construct comprised of mouse CC49 VL and VH (m/m scFv), (ii) a light chain shuffled scFv with human VL (Hum4 VL) and mouse CC49 VH (h/m scFv), and (iii) a humanized scFv assembled from Hum4 VL and CC49 VH complementary determining regions (CDRs) grafted onto a VH framework of MAb 21/28' CL (h/CDR scFv). The CC49 scFvs competed for an antigen binding site with CC49 IgG in a similar fashion in a competition radioimmunoassay and were able to inhibit the binding of CC49 IgG to the antigen completely. The immunogenicity of CC49 scFvs was tested using sera with antiidiotypic antibodies to MAb CC49 obtained from patients treated by CC49 IgG in clinical trials. All tested sera exhibited the highest reactivity to the m/m scFv. However, the sera demonstrated differential reactivities to h/CDR scFv and h/m scFv. Replacement of the mouse chain in h/m scFv and h/CDR scFv decreased or completely averted serum reactivity. Our studies compared for the first time the antigen binding and immunogenicity of different scFv constructs containing the mouse, CDR grafted or human variable chains. These results indicate that the humanized CC49 scFv is potentially an important agent for imaging and therapeutic applications with TAG-72-positive tumors.

99mTc-labeled divalent and tetravalent CC49 single-chain Fv's: novel imaging agents for rapid in vivo localization of human colon carcinoma.

UNLABELLED: Radioimmunopharmaceutical agents enabling rapid high-resolution imaging, high tumor-to-background ratios, and minimal immunogenicity are being sought for cancer diagnosis and imaging. Genetic engineering techniques have allowed the design of single-chain Fv's (scFv's) of monoclonal antibodies (mAbs) recognizing tumor-associated antigens. These scFv's show good tumor targeting and biodistribution properties in vivo, indicating their potential as imaging agents when labeled with a suitable radionuclide. METHODS: Divalent (sc(Fv)(2)) and tetravalent ([sc(Fv)(2)](2)) scFv's of mAb CC49 were evaluated for radioimmunolocalization of LS-174T colon carcinoma xenografts in athymic mice. scFv's were radiolabeled with (99m)Tc by way of the bifunctional chelator succinimidyl-6-hydrazinonicotinate hydrochloride using tricine as the transchelator. The immunoreactivity and in vitro stability of the scFv's were analyzed after radiolabeling. Biodistribution and pharmacokinetic studies were performed to determine the tumor-targeting potential of the radiolabeled scFv's. Whole-mouse autoradiography illustrated the possible application of these (99m)Tc-labeled multivalent scFv's for imaging. RESULTS: The radiolabeling procedure gave > or =95% radiometal incorporation, with a specific activity of >74 MBq/mg scFv. In solid-phase radioimmunoassay, both sc(Fv)(2) and [sc(Fv)(2)](2) exhibited 75%-85% immunoreactivity, with nonspecific binding between 0.8% and 1.2%. Size-exclusion high-performance liquid chromatography showed sc(Fv)(2) as a 60-kDa protein and [sc(Fv)(2)](2) as a 120-kDa protein. Blood clearance studies showed the elimination half-life of (99m)Tc-labeled sc(Fv)(2) as 144 min and that of [sc(Fv)(2)](2) as 307 min. Whole-body clearance studies confirmed the rapid elimination of scFv's, with half-lives of 184 +/- 19 min for sc(Fv)(2) and 265 +/- 39 min for [sc(Fv)(2)](2) (P < 0.001). At 6 h after administration, the tumor localization was 7.2 +/- 0.7 percentage injected dose per gram of tumor (%ID/g) for (99m)Tc-sc(Fv)(2). (99m)Tc-[sc(Fv)(2)](2) showed a tumor uptake of 19.1 +/- 1.1 %ID/g at the same time; the amount of radioactivity in the tumors was 4-fold higher than in the spleen and kidneys and 2-fold higher than in the liver. Macroautoradiography performed at 6 and 16 h after administration clearly detected the tumor with both scFv's. CONCLUSION: (99m)Tc-labeled multivalent scFv's show good tumor-targeting characteristics and high radiolocalization indices (tumor-to-background ratio). These reagents, therefore, have the potential for use in clinical imaging studies of cancer in the field of nuclear medicine.

Single-Dose versus fractionated radioimmunotherapy of human colon carcinoma xenografts using 131I-labeled multivalent CC49 single-chain fvs.

The prospects of radiolabeled antibodies in cancer detection and therapy remain promising. However, efforts to achieve cures, especially of solid tumors, with the systemic administration of radiolabeled monoclonal antibodies (MAbs) have met with limited success. Using genetic engineering techniques, MAbs have been tailored to improve the therapeutic index (tumor:normal tissue ratio) in clinical radioimmunotherapy. In the present study, we investigated the potential of tetravalent ([sc(Fv)2]2) and divalent [sc(Fv)2] single chain Fvs of MAb CC49 for therapy in athymic mice bearing s.c. LS-174T human colon carcinoma xenografts. Mice received 1,000 microCi of 131I-labeled [sc(Fv)2]2 or 131I-labeled sc(Fv)2, either as a single injection on day 6 or as four injections (250 microCi each) on days 6, 7, 8, and 9; the day of tumor implantation was taken as day 0. The median survival for the control group was 26 days. Comparisons of single and fractionated therapeutic regimens showed median survival as 32 (P < 0.001) and 53 days (P < 0.0001), respectively for [sc(Fv)2]2 and 26 (P > 0.5) and 38 days (P < 0.0001), respectively for sc(Fv)2 when compared with the control groups. The time for the quadrupling of tumor volume for single and fractionated therapeutic treatments were: 9.0 +/- 0.8 and 21.1 +/- 2.9 days respectively for sc(Fv)2; 16.6 +/- 1.9 and 32.9 +/- 2.7 days respectively for [sc(Fv)2]2; and 8.3 +/- 0.7 and 8.4 +/- 0.6 days respectively for the control group. No 131I-labeled systemic toxicity was observed in any treatment groups. The results show that radioimmunotherapy delivery for sc(Fv)2 and [sc(Fv)2]2 in a fractionated schedule clearly presented a therapeutic advantage over single administration. The treatment group receiving tetravalent scFv showed a statistically significant prolonged survival with both single and fractionated administrations suggesting a promising prospect of this reagent for cancer therapy and diagnosis in MAb-based radiopharmaceuticals.

Relative position of the hexahistidine tag effects binding properties of a tumor-associated single-chain Fv construct.

Hexahistidine tag (His-tag) is the most widely used tag for affinity purification of recombinant proteins for their structural and functional analysis. In the present study, single chain Fv (scFv) constructs were engineered form the monoclonal antibody (MAb) CC49 which is among the most extensively studied MAb for cancer therapy. For achieving efficient purification of scFvs by immobilized metal-ion affinity chromatography (IMAC), a His-tag was placed either at the C-terminal (scFv-His6) or N-terminal (His6-scFv) of the coding sequence. Solid-phase radioimmunoassay for scFv-His6 showed only 20-25% binding whereas both His6-scFv and scFv (no His-tag) showed 60-65% binding. Surface plasmon resonance studies by BIAcore revealed the binding affinity constant (KA) for His6-scFv and scFv as 1.19 x 10(6) M(-1) and 3.27 x 10(6) M(-1), respectively. No K(A) value could be calculated for scFv-His6 due to poor binding kinetics (kon and koff). Comparative homology modeling for scFv and scFv-His6 showed that the C-terminal position of the His-tag partially covered the antigen-binding site of the protein. The study demonstrates that the C-terminal position of His-tag on the CC49 scFv adversely affects the binding properties of the construct. The results emphasize the importance of functional characterization of recombinant proteins expressed with purification tags.

Site-specific photobiotinylation of antibodies, light chains, and immunoglobulin fragments.

The high affinity of biotin for avidin has been exploited for many antibody-based assays. This requires that biotin is covalently conjugated to the antibody molecule. Several chemically reactive biotinylation reagents are commercially available. Except for the attachment via sulfhydryl groups in the immunoglobulin (Ig) molecule, these reagents attach biotin randomly to various amino acid side chains. Although non-site-specific modification of antibodies does not interfere in most immunoassays, specific application and sensitive antibodies would benefit from site-specific biotinylation. Here we describe an affinity biotinylation technique based on a photoreactive biotin reagent. The design of this reaction was possible from the discovery of a conserved binding site in the variable Ig domain for nucleotides and nucleosides. The described photoaffinity biotinylation offers the advantages of ease, convenience, and production of a reproducible and defined biotinylated antibody preparation.

Pharmacokinetics and biodistribution of a light-chain-shuffled CC49 single-chain Fv antibody construct.

Murine monoclonal antibodies to tumor-associated glycoprotein 72 (anti-TAG-72 mAb B72.3 and CC49) are among the most extensively studied mAb for immunotherapy of adenocarcinomas. They have been used clinically to localize primary and metastatic tumor sites; however, murine mAb generally induce potent human anti-(mouse antibody) responses. The immunogenicity of murine mAb can be minimized by genetic humanization of these antibodies, where non-human regions are replaced by the corresponding human sequences or complementary determining regions are grafted into the human framework regions. We have developed a humanized CC49 single-chain antibody construct (hu/muCC49 scFv) by replacing the murine CC49 variable light chain with the human subgroup IV germline variable light chain (Hum4 VL). The major advantages of scFv molecules are their excellent penetration into the tumor tissue, rapid clearance rate, and much lower exposure to normal organs, especially bone marrow, than occur with intact antibody. The biochemical properties of hu/muCC49 scFv were compared to those of the murine CC49 scFv (muCC49 scFv). The association constants (Ka) for hu/muCC49 and muCC49 constructs were 1.1 x 10(6) M(-1) and 1.4 x 10(6) M(-1) respectively. Pharmacokinetic studies in mice showed similar rapid blood and whole-body clearance with a half-life of 6 min for both scFv. The biodistribution studies demonstrated equivalent tumor targeting to human colon carcinoma xenografts for muCC49 and hu/ muCC49 scFv. These results indicate that the human variable light-chain subgroup IV can be used for the development of humanized or human immunoglobulin molecules potentially useful in both diagnostic and therapeutic applications with TAG-72-positive tumors.

Divalent forms of CC49 single-chain antibody constructs in Pichia pastoris: expression, purification, and characterization.

Single-chain variable fragments (scFvs) are tumor-recognition units that hold enormous potential in antibody-based therapeutics. Their clinical applications, however, require the large scale production and purification of biologically active recombinant scFvs. In the present study, we engineered and expressed divalent non-covalent [(scFv)(2)-His(6)] and covalent [sc(Fv)(2)-His(6)] scFvs of a tumor-associated monoclonal antibody (MAb) CC49 in Pichia pastoris. The purity and immunoreactivity of the scFvs were analyzed by SDS-PAGE, HPLC, and competitive ELISA. The binding affinity constant (K(A)), determined by surface plasmon resonance analysis using BIAcore, was 4.28 x 10(7), 2.75 x 10(7), and 1.14 x 10(8) M(-1) for (scFv)(2)-His(6), sc(Fv)(2)-His(6), and CC49 IgG, respectively. The expression of scFvs in P. pastoris was 30 to 40-fold higher than in Escherichia coli. Biodistribution studies in athymic mice bearing LS-174T human colon carcinoma xenografts showed equivalent tumor-targeting of CC49 dimers generated in yeast (scFv)(2)-His(6) and bacteria (scFv)(2) with 12.52% injected dose/gram (%ID/g) and 11. 42%ID/g, respectively, at 6 h post-injection. Interestingly, the pharmacokinetic pattern of dimeric scFvs in xenografted mice exhibited a slower clearance of His-tagged scFvs from the blood pool than scFvs lacking the His-tag (0.1 >/= p >/= 0.05). In conclusion, improved yields of divalent scFvs were achieved using the P. pastoris expression/secretion system. The in vitro and in vivo properties of these scFvs suggest possible therapeutic applications.

Genetically engineered tetravalent single-chain Fv of the pancarcinoma monoclonal antibody CC49: improved biodistribution and potential for therapeutic application.

Failure of radiolabeled monoclonal antibodies (MAbs) in the treatment of solid tumors, for the most part, is a result of undesirable pharmacokinetics that lead to significant radiation exposure of normal tissues and an inadequate delivery of radiation doses to tumors. Using genetic engineering, antitumor MAbs can be optimized for desirable clinical applications. In the present study, we report the generation of a tetravalent single-chain Fv [[sc(Fv)2]2] of the murine MAb CC49 that recognizes the tumor-associated glycoprotein, TAG-72. [Sc(Fv)2]2 was expressed as a secreted soluble protein in Pichia pastoris under the regulation of alcohol oxidase 1 promoter. The in vitro binding properties of the tetravalent construct were analyzed by solid-phase RIA and surface plasmon resonance studies using BIAcore. The binding affinity constant (K(A)) for the [sc(Fv)2]2 and CC49 IgG were similar, i.e., 1.02 x 10(8) M(-1) and 1.14 x 10(8) M(-1), respectively, and were 4-fold higher than its divalent scFv [sc(Fv)2; 2.75 x 10(7) M(-1)]. At 6 h postadministration, the percentage of injected dose accumulated/g of LS-174T colon carcinoma xenografts was 21.3+/-1.3, 9.8+/-1.3, and 17.3+/-1.1 for radioiodinated [sc(Fv)2]2, sc(Fv)2, and IgG, respectively. Pharmacokinetic analysis of blood clearance studies showed the elimination half-life for [sc(Fv)2]2, sc(Fv)2, and IgG as 170, 80, and 330 min, respectively. The gain in avidity resulting from multivalency along with an improved biological half-life makes the tetravalent construct an important reagent for cancer therapy and diagnosis in MAb-based radiopharmaceuticals.

Pharmacokinetics and biodistribution of engineered single-chain antibody constructs of MAb CC49 in colon carcinoma xenografts.

UNLABELLED: Monoclonal antibodies (MAbs) have been proven useful in clinical studies for both diagnostic and therapeutic applications. The single-chain Fv (scFv) construct made from MAbs has potential applications for improved cancer diagnosis and therapy. A new CC49 scFv construct recognizing a tumor-associated mucin, TAG-72, was engineered and evaluated by immunological, pharmacokinetic and biodistribution analysis. METHODS: The CC49 scFv construct was generated in which the V(L) and V(H) variable region genes were joined together with a 25-amino acid helical linker (205C). The new CC49 scFv(205C) was expressed as a monomer as well as a stable noncovalent dimer ([scFv]2). The pharmacokinetic, biodistribution and tumor targeting characteristics of radiolabeled CC49 scFv were compared with CC49 IgG and enzymatically derived fragments F(ab')2 and Fab', using the athymic mice bearing human colon cancer xenografts. RESULTS: The association constant (K(A)) for the intact CC49, dimeric scFv (scFv)2 and monomeric scFv were 1.7 x 10(9), 1.99 x 10(9) and 0.52 x 10(9) M(-1) by Scatchard analysis and 1.14 x 10(8), 4.46 x 10(7) and 1.5 x 10(7) M(-1), respectively, by BIAcore analysis. Pharmacokinetic studies showed that more than 50% of monomeric scFv (approximately 27 kDa) was cleared from the blood in less than 10 min. The CC49 Fab' generated enzymatically from the parent murine Mab' (50 kDa) had a blood clearance that was faster than that of the (scFv)2 (60 kDa) with half of the activity cleared from the serum within 30 and 50 min, respectively. The CC49 dimeric scFv(205C) showed a two-fold higher tumor uptake (than scFv or Fab') reaching 10 %ID/g at 60 min after injection. The scFv dimer also showed an excellent stability and increased avidity in vivo compared with the monomer, as demonstrated by the longer retention in tumor with 3%ID/g remaining at 48 h. CONCLUSION: The rapid clearance from the blood, higher tumor uptake and longer retention of the stable dimer of CC49 scFv make it an important agent for potential imaging and therapeutic applications.

Radioimmunotherapy of human colon cancer xenografts using a dimeric single-chain Fv antibody construct.

Progress in the use of monoclonal antibodies (MAbs) for the treatment of solid tumors is limited by a number of factors, including poor penetration of the labeled IgG molecule into the tumors, their inability to reach the tumor in sufficient quantities without significant normal tissue toxicity, and the development of a human antimouse antibody response to the injected MAb. One possible way to alter the pharmacology of antibodies is via the use of smaller molecular weight antibody fragments called single-chain Fvs (scFvs). A divalent construct of MAb CC49, CC49 (scFv)2, composed of two noncovalently associated scFvs, was generated and shown to bind a tumor-associated antigen (TAG-72) epitope with a similar binding affinity to that of the murine IgG. The therapeutic potential of this construct after labeling with 131I was examined in athymic mice bearing established s.c. human colon carcinoma (LS-174T) xenografts. Treatment groups (n = 10) received a single dose of 131I-labeled CC49 (scFv)2 (500-2000 microCi) or 131I-labeled CC49 IgG (250 and 500 microCi). The group of mice treated with the lowest dose of 131I-(scFv)2 (500 microCi) showed statistically significant prolonged survival, compared with controls (P = 0.036). Complete tumor regression was observed in 20% of mice given 1500 microCi of labeled (scFv)2 and 30 and 60% of mice treated with 250 and 500 microCi of labeled IgG, respectively. In conclusion, the CC49 (scFv)2 construct provides a promising delivery vehicle for therapeutic applications.

Effects of genetic engineering on the pharmacokinetics of antibodies.

Monoclonal antibodies (MAbs) may be considered 'magic bullets' due to their ability to recognize and eradicate malignant cells. MAbs, however, have practical limitations for their rapid application in the clinics. The structure of antibody molecules can be engineered to modify functional domains such as antigen-binding sites and/or effector functions. Advances in genetic engineering have provided rapid progress in the development of new immunoglobulin constructs of MAbs with defined research and therapeutic application. Recombinant antibody constructs are being engineered, such as human-mouse chimeric, domain-dispositioned, domain-deleted, humanized and single-chain Fv fragments. Genetically-engineered antibodies differ in size and rate of catabolism. Pharmacokinetic studies show that the intact IgG (150 kD), enzymatically derived fragments Fab' (50 kD) and single chain Fv (28 kD) have different clearance rates. These antibody forms clear 50% from the blood pool in 2.1 days, 30 minutes and 10 minutes, respectively. Genetically-engineered antibodies make a new class of immunotherapeutic tracers for cancer treatment.

Single-chain antibodies in pancreatic cancer.

Pancreatic cancer is a therapeutic challenge for surgical and medical oncology. Development of specific molecular tracers for the diagnosis and treatment of this lethal cancer has been one of our major goals. Monoclonal antibodies (MAbs) have been successfully used as selective carriers for delivering radionuclides, toxins or cytotoxic drugs to malignant cell populations; therefore, monoclonal antibody technology has led to a significant amount of research into optimizing targeted therapy. This targeted therapy results in the selective concentration of cytotoxic agents or radionuclides in tumors and should lessen the toxicity to normal tissues, which would normally limit the dosage and effectiveness of systemically administered drugs. The MAb CC49 reacts with a unique disaccharide, Sialyl-Tn, present on tumor-associated mucin (TAG-72) expressed by a majority of human adenocarcinomas. The unique Sialyl-Tn epitope has provided a potential target for immunotherapy of cancer. A single chain Fv (scFv) recombinant protein from CC49 MAb was prepared by engineering the DNA fragments for coding heavy-chain and light-chain variable regions with an appropriate oligonucleotide linker. scFv molecules, when compared to intact MAbs and the more conventional enzymatically derived F(ab')2 and Fab' fragments, offer several advantages as carriers for the selective delivery of radionuclides to tumors. The divalent antibody fragments (sc(Fv)2 or (scFv)2) display an affinity constant similar to that of the intact CC49 IgG and are stable with storage, and after radiolabeling. In preclinical studies, both the covalent and the non-covalent dimeric scFvs exhibit excellent tumor targeting properties with characteristics similar to those of the monomer, e.g., the rapid blood clearance, low kidney uptake and small size suitable for rapid penetration through tumor tissue. Increased tumor targeting of the dimers are probably due to their increased functional affinity attributable to valency, coupled with their higher molecular weight and fewer interactions with normal organs. These properties make these constructs superior to monovalent CC49 scFv. The relatively high tumor uptake, the in vitro and in vivo targeting specificity, and the stability in storage demonstrated by the dimeric CC49 sc(Fv)2 makes it a promising delivery vehicle for therapeutic applications in pancreatic cancer.

Binding characteristics and tumor targeting of a covalently linked divalent CC49 single-chain antibody.

Multivalency is a recognized means of increasing the functional affinity of single-chain Fvs (scFvs) for optimizing tumor uptake. A unique divalent single-chain Fv protein [sc(Fv)2], based on the variable regions of the monoclonal antibody (MAb) CC49, has been generated that differs from other dimeric single-chain constructs in that a linker sequence (L) is encoded between the repeated V(L) and V(H) domains (V(L)-L-V(H)-L-V(L)-L-V(H)). This construct was expressed in soluble form in Escherichia coli and purified by ion-exchange and gel-filtration chromatography. Purity and immunoreactivity were determined by SDS-PAGE, HPLC and competitive RIA. sc(Fv)2 exhibited a relative K(A) (3.34 x 10(7) M(-1)) similar to that of the native IgG (1.14 x 10(8) M(-1)) as determined by BIAcore analysis. Pharmacokinetic studies showed rapid blood clearance for sc(Fv)2, with a T(1/2) less than 40 min. Whole-body clearance analysis also revealed rapid clearance, suggesting no significant retention in the extravascular space or normal tissues. Biodistribution studies of radiolabeled sc(Fv)2 showed tumor uptake greater than 6% ID/g after 30 min, which remained at this level for 6 hr. High tumor uptake and retention of sc(Fv)2 coupled with rapid blood and whole-body clearance makes this dimeric scFv of MAb CC49 a strong candidate for imaging and therapeutic applications.

Charge-modified single chain antibody constructs of monoclonal antibody CC49: generation, characterization, pharmacokinetics, and biodistribution analysis.

A novel strategy was developed in which an antibody scFv fragment of the monoclonal antibody (MAb) CC49 was modified by engineering DNA coding sequences to lower its isoelectric point. Negatively charged amino acids were added to the carboxy terminus of the CC49 VH region by adding nucleotide sequences in a polymerase chain reaction (PCR) amplification of the coding sequence of CC49 scFv. Two new DNA constructs coding for CC49 scFv with lower isoelectric points of 5.8 and 5.2 were engineered. These novel strategy-generated, charge-modified antibody constructs were compared for their immunological, pharmacokinetic, and biodistribution properties in athymic mice bearing LS-174T human colon carcinoma xenografts.

Pharmacokinetics and biodistribution of genetically-engineered antibodies.

Monoclonal antibodies (MAbs), because of their inherent specificity, are ideal targeting agents. They can be used to deliver radionuclides, toxins or cytotoxic drugs to a specific tissue or malignant cell populations. Intact immunoglobulin (IgG) molecules have several practical limitations of their pharmacology; their relatively large size of approximately 150,000 daltons leads to a slow clearance from the blood pool and the body resulting in significant exposure to normal organs with limited quantities delivered to tumors. The IgG molecule shows a relatively poor diffusion from the vasculature into and through the tumor. Attempts to modify the pharmacology of the Ig molecule have classically involved the use of proteases to generate F(ab')2 and Fab' fragments with molecular weights of approximately 100,000 and 50,000 daltons, respectively. Fv fragments of IgG are one of the smallest size functional modules of antibodies that retain high affinity binding of an antigen. Their smaller size, approximately 25,000 daltons, enables better tumor penetration and makes them potentially more useful than a whole antibody molecule for clinical applications. Molecular cloning and expression of the variable region genes of IgG has greatly facilitated the generation of engineered antibodies. A single-chain Fv (scFv) recombinant protein, prepared by connecting genes encoding for heavy-chain and light-chain variable regions at the DNA level by an appropriate oligonucleotide linker, clears from the blood at much faster rate than intact IgG. The scFv fragment can retain an antigen-binding affinity similar to that of a monovalent Fab' fragment; this however, represents a relative decrease in binding affinity when compared to intact antibodies. The scFv with its faster clearance and lower affinity results in a lower percent-injected dose localizing in tumors when compared to the divalent IgG molecule. This may be adequate for imaging but probably not for therapy. The valency of the MAb fragment is critical for the functional affinity of an antibody to a cell surface or a polymeric antigen. In attempts to generate multivalent forms of scFv molecules, non-covalently linked scFv dimeric and trimeric molecules, disulfide linked dimeric scFvs, as well as covalently linked chimeric scFvs have been studied. These multivalent scFvs generally have a higher functional affinity than the monovalent form resulting in better in vivo targeting. Another way to alter the pharmacology of the scFvs is to modify its net charge. Charge-modified scFvs with desired isoelectric points (pI), have been prepared by inserting negatively charged amino acids on the template of the variable region genes. This can help to overcome undesirable elevations in renal uptake seen with most antibody fragments. In conclusion, genetic manipulations of the immunoglobulin molecules are effective means of altering stability, functional affinity, pharmacokinetics, and biodistribution of the antibodies required for the generation of the "magic bullet".

Site-specific photobiotinylation of immunoglobulins, fragments and light chain dimers.

Herein we report a new method to rapidly photoinsert biotin into a specific and highly conserved site on the Ig structure using a mild photochemical activation step. This site resides in the Fv fragment and involves invariant residues which provide base stacking interactions to the purine ring of ATP (Rajagopalan et al. (1996) Proc. Natl. Acad. Sci. USA 93, 6019-6024). Biotin was coupled to either the phosphate or the ribose of the 8-azidopurine nucleotide or nucleoside photoaffinity probe and shown to insert into the affinity site efficiently. Several monoclonal and polyclonal antibodies, as well as enzymatic and recombinant antibody fragments and light chain dimers were photoaffinity biotinylated and used in ELISA, FACS and Western blots. The selectivity of this site-specific biotinylation method also allows for biotinylation of antibodies in culture supernatants and immune sera without prior purification. Because the biotinylation takes place under physiological conditions and within a short time period, photobiotinylation would be the preferred method for antibodies which are easily damaged by classical non-site specific random biotinylation chemistry.

Antibody-mediated gene delivery for B-cell lymphoma in vitro.

The targeted introduction of therapeutic genes into malignant cells based on receptor mediated endocytosis of ligand-DNA conjugates recently was established as a transfection system and provides a promising strategy for cancer therapy. Antiidiotype antibodies could be of particular interest for this approach because their immunoglobulin receptor idiotypes represent highly specific tumor markers. Their safe and specific applicability in vivo, alone or as immunotoxins, has been proven in clinical trials for passive immunotherapy and vaccination strategies. For these reasons we have explored the utility of antiidiotype antibodies for gene delivery systems using the reporter genes beta-galactosidase and luciferase. Two monoclonal antibodies, SIC5 and 5D10, specific for B-lymphoma cell lines, which represent models for murine plasmacytoma (38C13) and human non-Hodgkin's lymphoma (SU-DHL-4) have been covalently linked to polylysine via the heterobifunctional cross-linker SPDP. Highly efficient uptake and internalization of the immunoconjugates have been shown by fluorescence microscopy and fluorescence-activated cell sorting (FACS) analysis. Successful transfections have been shown at the RNA and the reporter gene level (beta-galactosidase, luciferase) using different promoter/enhancer systems. Beta-galactosidase activity was detected by flow cytometry (FACS-gal) analysis for both cell lines, and SU-DHL-4 cells showed significant luciferase activity.

Novel unconventional binding site in the variable region of immunoglobulins.

The variable immunoglobulin (Ig) domains contain hypervariable regions that are involved in the formation of the antigen binding site. Besides the canonical antigen binding site, so-called unconventional sites also reside in the variable region that bind bacterial and viral proteins. Docking to these unconventional sites does not typically interfere with antigen binding, which suggests that these sites may be a part of the biological functions of Igs. Herein, a novel unconventional binding site is described. The site is detected with 8-azidopurine nucleotide photoaffinity probes that label antibodies efficiently and under mild conditions. Tryptic peptides were isolated from photolabeled monoclonal antibodies and aligned with the variable antibody domains of heavy and light chains. The structure of a variable Ig fragment was used to model the binding of the purine nucleotide to invariant residues in a hydrophobic pocket of the Ig molecule at a location distant from the antigen binding site. Monoclonal and polyclonal antibodies were biotinylated with the photoaffinity linker and used in fluorescence-activated cell sorter and ELISA analyses. The data support the utility of this site for tethering diagnostic and therapeutic agents to the variable Ig fragment region without impairing the structural and functional integrity of antibodies.

Serum- and bradykinin-induced calcium transients in familial Alzheimer's fibroblasts.

The calcium-sensitive photoprotein, aequorin, was used to examine serum- and bradykinin-induced transient increases in free cytosolic calcium ions in skin fibroblasts from 10 individuals with early onset familial AD (FAD), including four who were biopsied before their clinical symptoms would allow a diagnosis of AD, 2 individuals with late onset FAD, 8 at-risk but nonsymptomatic individuals, and 13 controls. The data show that (a) among controls, the peaks of the calcium transients increase in height as a function of donor age; (b) transients induced by 10% serum, 10 nM bradykinin (BK) or 100 nM BK were generally lower in FAD fibroblasts, including those from donors in the early stages of the disease, than in age-matched control cells; (c) such transients are reduced in cells from a proportion of the nonsymptomatic, at-risk individuals. Thus, serum- and BK-induced calcium transients are reduced in fibroblasts from both early and more advanced stage FAD donors and perhaps even from donors who are presymptomatic carriers of the defective gene. The data also suggest that changes in calcium transients in FAD fibroblasts neither mimic nor exaggerate the effects of normal aging.