Conjugation Of Fab Research Articles

A single-chain fab derived drug conjugate for HER2 specific delivery

Despite the development of antibody-drug conjugates, the fragment Fab-based drug conjugates offer some unique capabilities in terms of safety, clearance, penetration and others. Current methods for preparing Fab drug conjugates are limited by the availability and stability of Fab proteins, leaving reports on this rare. Here, we found that a single-chain scaffold of Fab enables stabilization of the paired structure and supports high-yield expression in bacteria cytoplasm. Furthermore, we conjugated anti-neoplastic agent SN38 to the C-terminus by sortase A ligation and generated a homogenous Fab conjugate with the drug-to-Fab ratio of 1. The resulting anti-HER2 Fab-SN38 conjugate demonstrated potent and antigen-dependent cell-killing ability with the aid of its special cathepsin-triggered cyclization-promoted release mechanism. In vivo, Fab-SN38 can prevent growths of HER2-positive tumors in athymic mice and be well tolerated to the treatment at 7 mg/kg per dose. Anti-tumor activity, high dose tolerance and penetration advantage observed in this study would merit Fab conjugate investigation in target chemotherapy.

Bispecific FpFs: a versatile tool for preclinical antibody development.

We previously described FpFs 1̲ (Fab-PEG-Fab) as binding mimetics of IgGs. FpFs are prepared with di(bis-sulfone) conjugation reagents 3̲ that undergo disulfide rebridging conjugation with the accessible disulfide of each Fab (Scheme 1). We have now prepared bispecific FpFs 2̲ (bsFpF and Fab1-PEG-Fab2) as potential bispecific antibody mimetics with the intent that bsFpFs could be used in preclinical antibody development since sourcing bispecific antibodies may be challenging during preclinical research. The di(bis-sulfone) reagent 3̲ was first used to prepare a bsFpF 2̲ by the sequential conjugation of a first Fab and then a second Fab to another target (Scheme 2). Seeking to improve bsFpF synthesis, the asymmetric conjugation reagent, bis-sulfone bis-sulfide 1̲6̲, with different thiol conjugation reactivities at each terminus (Scheme 4) was examined and the bsFpFs appeared to be formed at similar conversion to the di(bis-sulfone) reagent 3̲. To explore the advantages of using common intermediates in the preparation of bsFpF families, we investigated bsFpF synthesis with a protein conjugation-ligation approach (Scheme 5). Reagents with a bis-sulfone moiety for conjugation on one PEG terminus and a ligation moiety on the other terminus were examined. Bis-sulfone PEG trans-cyclooctene (TCO) 2̲8̲ and bis-sulfone PEG tetrazine (Tz) 3̲0̲ were used to prepare several bsFpFs targeting various therapeutic targets (TNF-α, IL6R, IL17, and VEGF) and tissue affinity targets (hyaluronic acid and collagen II). Surface plasmon resonance (SPR) binding studies indicated that there was little difference between the dissociation rate constant (k d) for the unmodified Fab, mono-conjugated PEG-Fab and the corresponding Fab in a bsFpF. The Fab association rate (k a) in the bsFpF was slower than for PEG-Fab, which may be because of mass differences that influence SPR results. These observations suggest that each Fab will bind to its target independently of the other Fab and that bsFpF binding profiles can be estimated using the corresponding PEG-Fab conjugates.

Abstract 2005: FSHR-mediated targeted delivery of paclitaxel immunoliposomes in receptor overexpressing ovarian cancer cells

Abstract The purpose of this study was to explore the Fabʹ fragment of anti-FSHR (Follicle Stimulating Hormone Receptor) antibody as a targeting ligand to graft on the surface of paclitaxel (PTX) liposomes (PLs) for site-specific drug delivery in ovarian cancer (OC) cells. Anti-FSHR antibody Fab’ fragment conjugated PTX encapsulated immunoliposomes (ILs) were prepared, characterized, and evaluated to determine their in vitro efficacy in FSHR receptor positive (Caov3) and negative (SKOV3) OC cells. FSHR expression was determined by confocal microscopy and flow cytometry. The results confirmed that FSHR expression levels of Caov3 were higher and no expression was detected in SKOV3. ILs were prepared by covalent conjugation of Fab’ fragments to functionalized PLs via thioether linkage which was confirmed by SDS-PAGE analysis. Immunoreactivity of the prepared ILs was determined against Fabʹ in Caov3 cells using flow cytometry and it was observed that after conjugation, Fabʹ remained intact and also maintained the binding characteristic to the receptor. In MTT assay at the end of 48 h, the IC50 value for ILs was found to be 10.8 and 3.5 folds lower than Taxol® and PLs respectively in Caov3. However, being receptor negative, there was no significant difference noticed for IC50 when PLs and ILs were compared for SKOV3. To prove, whether immune tagging improves cell-specific uptake of ILs in FSHR-expressing cells, cell uptake studies were carried out using confocal microscopy and flow cytometry which showed higher uptake of ILs in Caov3 but not in SKOV3. For assessment of anti-angiogenic activity, a wound scratch assay was performed on Caov3 cells. At an equimolar concentration of treatment given to the cells by PLs and ILs, the % recovery of the wound was 1.3 and 2.3 times lower than Taxol® respectively indicating the superior performance of ILs in inhibiting cell proliferation. The quantitation of cell death after treatment was carried out by flow cytometry after staining with propidium iodide and the highest amount of cell death was seen in ILs group for Caov3 cells. Evaluation of cell cycle in the Caov3 indicated superior results of arrest of cells in G2/M phase. Further, an increase in cell death was observed as indicated by the increase in % of cells in subG1 stage. Performance of PLs was similar in Caov3 and SKOV3 leading to almost around 22% of arrest in G2/M stage, however, for ILs, the number of cells in G2/M was 28% for Caov3 cells at the end of 24 h indicating that the targeted formulation was able to better get internalized in the cells due to receptor-mediated uptake. The results highlight the importance of FSHR as a prominent target for OC therapy and the potential of anti-FSHR Antibody Fab' conjugated nanocarriers as a site-specific delivery system to reduce the limitations associated with OC chemotherapy. Citation Format: Priyanka Bhatt. FSHR-mediated targeted delivery of paclitaxel immunoliposomes in receptor overexpressing ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2005.

Direct Tie2 Agonists Stabilize Vasculature for the Treatment of Diabetic Macular Edema.

PurposeDiabetic macular edema (DME) is the leading cause of vision loss and blindness among working-age adults. Although current intravitreal anti-vascular endothelial growth factor (VEGF) therapies improve vision for many patients with DME, approximately half do not achieve the visual acuity required to drive. We therefore sought additional approaches to resolve edema and improve vision for these patients.MethodsWe explored direct agonists of Tie2, a receptor known to stabilize vasculature and prevent leakage. We identified a multivalent PEG–Fab conjugate, Tie2.1-hexamer, that oligomerizes Tie2 and drives receptor activation and characterized its activities in vitro and in vivo.ResultsTie2.1-hexamer normalized and stabilized intercellular junctions of stressed endothelial cell monolayers in vitro, suppressed vascular leak in mice under conditions where anti-VEGF alone was ineffective, and demonstrated extended ocular exposure and robust pharmacodynamic responses in non-human primates.ConclusionsTie2.1-hexamer directly activates the Tie2 pathway, reduces vascular leak, and is persistent within the vitreal humor.Translational RelevanceOur study presents a promising potential therapeutic for the treatment of DME.

Extending the in vivo Half-Life of Adalimumab Fab via Sortase A-Mediated Conjugation of Adalimumab Fab with Modified Fatty Acids

AbstractAdalimumab, a full-length monoclonal antibody, is widely used as an anti-tumor necrosis factor-α (anti-TNF-α) agent. In this article, we aimed to prolong the in vivo half-life of adalimumab antigen-binding fragment (Fab) through Sortase A (SrtA)-mediated conjugation of its Fab with fatty acid (FA). In our study, adalimumab Fab analog was prepared by adding an SrtA recognition sequence (LPETGG) and His6 tag to the heavy chain C-terminal of the Fab via (G4S)3 linker. Four FA motifs with different linkers were designed and synthesized by solid-phase methodology, then conjugated with the Fab analog using SrtA to produce Fab bioconjugates. The successful generation of four Fab bioconjugates (Fab–FA1, Fab–FA2, Fab–FA3, and Fab–FA4) was confirmed by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and mass spectrometry. Then, the bioactivities and half-life of these Fab bioconjugates were examined using TNF-α-/human serum albumin (HSA)-binding enzyme-linked immunosorbent assay, cytotoxicity assay, and pharmacokinetic study, respectively. All Fab bioconjugates exhibited similar TNF-α-neutralizing activities when compared with the Fab analog, even in the presence of albumin, indicating that there were no apparent influences on the functional site of Fab after FA modification. However, different degrees of affinity for HSA were observed among these Fab–FA bioconjugates, with Fab–FA3 exhibiting the maximal affinity. An in vivo study in mice further revealed remarkably improved pharmacokinetics of Fab– FA3 with a 15.2-fold longer plasma half-life (19.86 hours) compared with that of the Fab analog (1.31 hours). In summary, we have developed a novel long-acting adalimumab Fab bioconjugate, Fab–FA3, with more sustained in vivo activity, which can be used for drug development targeting TNF-α-mediated inflammatory diseases.

Site-Specific Immuno-PET Tracer to Image PD-L1.

The rapid ascension of immune checkpoint blockade treatments has placed an emphasis on the need for viable, robust, and noninvasive imaging methods for immune checkpoint proteins, which could be of diagnostic value. Immunoconjugate-based positron emission tomography (immuno-PET) allows for sensitive and quantitative imaging of target levels and has promising potential for the noninvasive evaluation of immune checkpoint proteins. However, the advancement of immuno-PET is currently limited by available imaging tools, which heavily rely on full-length IgGs with Fc-mediated effects and are heterogeneous mixtures upon random conjugation with chelators for imaging. Herein, we have developed a site-specific αPD-L1 Fab conjugate with the chelator 1,4,7-triazacyclononane- N, N', N″-triacetic acid (NOTA), enabling radiolabeling for PET imaging, using the amber suppression-mediated genetic incorporation of unnatural amino acid (UAA), p-azidophenylalanine. This Fab conjugate is homogeneous and demonstrated tight binding toward the PD-L1 antigen in vitro. The radiolabeled version, 64Cu-NOTA-αPD-L1, has been employed in PET imaging to allow for effective visualization and mapping of the biodistribution of PD-L1 in two normal mouse models, including the capturing of different PD-L1 expression levels in the spleens of the different mouse types. Follow-up in vivo blocking studies and ex vivo fluorescent staining further validated specific tissue uptakes of the imaging agent. This approach illustrates the utility of UAA-based site-specific Fab conjugation as a general strategy for making sensitive PET imaging probes, which could facilitate the elucidation of the roles of a wide variety of immune checkpoint proteins in immunotherapy.

Construction and evaluation of pH-sensitive immunoliposomes for enhanced delivery of anticancer drug to ErbB2 over-expressing breast cancer cells

1,5-Dihexadecyl N,N-diglutamyl-lysyl-L-glutamate (GGLG) liposomes were previously developed to enhance drug delivery efficiency in tumor cells owing to its pH-responsive properties. Herein, we report the modification of GGLG liposomes by conjugating a Fab′ fragment of an ErbB2 antibody to the terminus of PEG (polyethylene glycol)-lipid (Fab′-GGLG liposomes). The conjugation of Fab′ fragments did not affect the antibody activity, drug (doxorubicin, DOX) encapsulation efficiency, stability during storage or pH-sensitivity. However, the binding affinity of Fab′-GGLG liposomes was enhanced to ErbB2-overexpressing HCC1954 cells specifically, and the cell association increased 10-fold in comparison to GGLG liposomes. Consequently, intracellular DOX delivery was enhanced, with an increased cytotoxicity in HCC1954 cells (i.e., IC50 of 1.17 and 3.08 μg/mL for Fab′-GGLG-DOX and GGLG-DOX liposomes, respectively). Further, a significantly enhanced tumor growth inhibition was obtained in an ErbB2-overexpressing breast cancer-bearing mouse model. Therefore, a potent anticancer drug delivery system was constructed by the immunological modification of pH-sensitive liposomes.

Abstract 2065: Anti-FSHR antibody Fab’ fragment conjugated immunoliposomes loaded with cyclodextrin-paclitaxel complex for improved in vitro efficacy on ovarian cancer cells

Abstract Purpose of the study was to prepare and characterize cyclodextrin-paclitaxel (CD-PTX) complex encapsulated immunoliposomes (ILs) conjugated with Fab’ fragment of anti-FSHR antibody and to determine their in vitro efficacy for cellular uptake, cytotoxicity, anti-metastatic activity and cell apoptosis on ovarian cancer (OC) cells. CD-PTX complex was formulated using Heptakis(2,6-di-O-methyl)-beta-cyclodextrin (DMBCD) to improve aqueous solubility and liposomal loading of PTX. The complex was encapsulated in PEGylated liposomes (PLs) using reverse phase evaporation technique. ILs were prepared by covalent conjugation of Fab’ fragments of anti-FSHR antibody to functionalized PLs via thioether linkage which was confirmed by SDS-PAGE analysis. The ILs were characterized for size, zeta potential, entrapment, loading efficiency and in vitro drug release profile. ILs were also evaluated for uptake efficiency and in vitro cytotoxicity in FSHR-expressing Caov-3 OC cells. To establish anti-metastatic effect, the ILs, PLs and equal amounts of PTX solution were studied on Caov-3 cells by in vitro wound scratch assay. The effect of PTX solution, PLs and ILs on Caov-3 cell apoptosis and cell cycle was examined using FACS technique. Increased aqueous solubility of 11 mg/ml was achieved for PTX using DMBCD at molar ratio of 20 and the cytotoxic properties of CD-PTX complex was also retained similar to PTX as studied in Caov-3 cells. The PLs and ILs were found to be nanosized with optimum entrapment and improved loading efficiency of PTX. The in vitro release profiles exhibited controlled release of PTX from PLs (21.235±0.423%) and ILs (18.219±0.603%) after completion of 48 h. The IC50 values from MTT assay suggested that ILs was 3.7 and 8.1 times more cytotoxic than PLs and PTX solution respectively for Caov-3 cells after 24 h period. The ILs showed more anti-migration effect and less covered wound (39.66±5.2%) at a concentration of 3nM as compared with PTX solution (70.45±7.12%) and PLs (61.38±4.41%). After 24 h treatment no major distinction in% apoptosis was observed between PTX solution, PLs and ILs. However, after 48 h treatment, PLs and ILs respectively showed about 5.36% and 9.12% higher apoptosis when compared to PTX solution. In Caov-3 cells, after 48 h the ILs at 3nM concentration showed increased G2 phase cell arrest as compared to PLs which indicates the superiority of ILs. To conclude, ILs displayed improved intracellular uptake and enhanced cytotoxic as well as anti-metastatic effects compared to PTX solution and PLs in OC cells. The results clearly highlight the importance of FSHR as one of the prominent targets for OC therapy and also the potential of anti-FSHR Ab Fab’ conjugated nanocarriers to reduce the limitations associated with OC chemotherapy. Citation Format: Priyanka Bhatt, Rohan Lalani, Rajashree Mashru, Ambikanandan Misra. Anti-FSHR antibody Fab’ fragment conjugated immunoliposomes loaded with cyclodextrin-paclitaxel complex for improved in vitro efficacy on ovarian cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2065.

The tumor-inhibitory effectiveness of a novel anti-Trop2 Fab conjugate in pancreatic cancer.

Human trophoblastic cell surface antigen 2 (Trop2) has been reported to act oncogenically. In this study, one-step quantitative real-time polymerase chain reaction (qPCR) test and immunohistochemistry (IHC) analysis with were employed to evaluate the relationship between Trop2 expression and the clinicopathological features of patients with PC. Then a novel anti-Trop2 Fab antibody was conjugated with Doxorubicin (DOX) to form Trop2Fab-DOX, an antibody-drug conjugate. This Trop2Fab-DOX conjugate was characterized by cell ELISA and immunofluorescence assay. MTT and wound healing analyses were used to evaluate the inhibitory effect of Trop2Fab-DOX on PC cell growth in vitro, while xenograft nude mice model was established to examine the tumor-inhibitory effects of PC in vivo. High Trop2 expression was observed in PC tissues and Trop2 expression was associated with several malignant attributes of PC patients, including overall survival. Trop2Fab-DOX can bind to the Trop2-expressing PC cells and provide an improved releasing type of DOX. In addition, Trop2Fab-DOX inhibited the proliferation and suppressed the migration of PC cells in a dose-dependent manner in vitro, while inhibited the growth of PC xenografts in vivo. Trop2 is a specific marker for PC, and a novel Trop2Fab-DOX ADC has a potent antitumor activity

Enhanced Tumor-targeted Gene Delivery by Immunolipoplexes Conjugated with the Humanized Anti-TAG-72 Fab' Fragments

Cationic immunoliposomes were prepared by conjugation of Fab' fragments of the recombinant humanized monoclonal antibody (HuCC49) against tumor-associated glycoprotein (TAG)-72 to sterically unilamella liposomes. The cationic immunoliposomes are composed of cationic lipid (O,O'-dimyristyl-N-lysyl aspartate, DMKD), cholesterol, and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)2000] (DPPE-PEG-maleimide) with a molar ratio of 0.5:0.47:0.03. Plasmid DNA was effectively condensed by addition of transferrin (Tf) during the formation of anti-TAG-72 PEG-immunolipoplexes (PILPs). These anti-TAG-72 PILPs were able to adhere to the surface of TAG-72-overexepressing LS174T human colon cancer cells more effectively than conventional liposomes, thereby facilitating gene delivery in vitro. Furthermore, intravenous administration of the anti-TAG-72 PILPs into the tumor-carrying mice exhibited efficient localization of the reporter gene in the tumor tissues.

Delivering Drugs Into the Brain: Barriers and Possibilities

Delivering Drugs Into the Brain: Barriers and Possibilities

Abstract 4553: Novel bispecific 111In-labeled immunoconjugates (bsICs) for imaging HER2/HER3 heterodimerization in breast cancer

Abstract Objective: Our objective was to construct novel bsICs capable of binding HER2 and HER3 labeled with 111In for imaging HER2-HER3 heterodimerization in breast cancer (BC) possibly through higher avidity for dimeric vs. monomeric complexes. Methods: bsICs were composed of trastuzumab Fab recognizing HER2 linked to a 7.5 kDa HER3-binding peptide of heregulin-β1 (pHRG). Fab were produced by digestion of trastuzumab IgG with papain. Fab were modified with Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) or related analogues with polyethyleneglycol (PEG) spacers (SM-PEG12-NHS and SM-PEG24-NHS) to introduce maleimide groups for cross-linking to pHRG thiolated with Traut's reagent. bsICs were derivatized with diethylenetriaminepentaacetic acid (DTPA) for labeling with 111In. The ability to independently bind HER2 or HER3 was determined in competition assays using 111In-bsICs on cells expressing HER2, HER3 or both receptors competed with a 25-fold excess of Fab, pHRG or combined ligands. Results: Conjugation of Fab to pHRG was confirmed by SDS-PAGE, Western blot and size-exclusion HPLC. bsICs were labeled with 111In to a radiochemical purity of 98.9 ± 0.2% (specific activity 375 ± 14 MBq/mg). Insertion of a PEG spacer between Fab and pHRG was required for displacement of binding of 111In-bsICs to HER3 but not HER2. For 111In-bsICs with the longest spacer (PEG24), the binding to SKBR-3 cells (HER2+/HER3+) was reduced to 32.7 ± 0.7%, 85.9 ± 1.0% or 30.4 ± 1.6%, respectively by Fab, pHRG or both ligands. The binding to SKOV-3 cells (HER2+/HER3-) was reduced to 16.7 ± 0.8%, 88.8 ± 5.3% or 13.4 ± 0.1% by Fab, pHRG or both ligands. The binding to MDA-MB-468 cells (HER2-/HER3+) was reduced to 98.5 ± 1.5%, 68.8 ± 0.3% or 64.8 ± 4.5% by Fab, pHRG or both ligands. Less efficient competition for HER3 was observed for the PEG12 spacer and no competition for 111In-bsICs without a spacer. Conclusion: 111In-bsICs composed of trastuzumab Fab and pHRG bound specifically to HER2 and HER3 when an appropriate length PEG spacer was inserted between these two binding moieies. These 111In-bsICs may be useful for imaging HER2-HER3 heterodimerization in BC. Supported by the Ontario Institute of Cancer Research (1 mm Challenge) and the Canadian Breast Cancer Foundation (Ontario Branch). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4553.

Targeted gene therapy of LS174 T human colon carcinoma by anti-TAG-72 immunoliposomes

Anti-tumor-associated glycoprotein (TAG)-72 PEG-immunoliposomes (PILs) were prepared by conjugation of Fab' fragments of recombinant humanized monoclonal antibody, HuCC49, to sterically stabilize unilamellar liposomes (90-110 nm in diameter) to target TAG-72-overexpressing cancer cells. The liposomes consisted of 1-palmitonyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), 92 mol percent, O,O'-dymyrisyl-N-lysyl aspartate (DMKD cationic lipid), 4 mol percent, distearoyl-phosphatidyl-ethanolamine-polyethylene glycol 2000 (DSPE-PEG(2000)), 3 mol percent and DSPE-maleimide (DSPE-PEG(2000)-Mal), 1 mol percent. These anti-TAG-72 PILs were able to adhere to the surface of TAG-72-overexpressing LS174 T human colon cancer cells more effectively than conventional liposomes. Also, in vitro gene transfection of the LS174 T cells by the anti-TAG-72 PILs in the presence of a high concentration of fetal bovine serum (up to 60%) was greater than that by conventional cationic lipoplexes. Intravenously administered anti-TAG-72 PILs efficiently localized in the LS174 T tumor tissues, while the non-targeted conventional liposomes did not. Intravenous administration of the anti-TAG-72 PILs containing plasmids encoding antiangiogenic proteins, such as angiostatin K1/3, endostatin and saxatilin, significantly inhibited in vivo growth of LS174 T tumors and angiogenesis in the tumor tissues. These results demonstrated the potential of TAG-72-mediated targeting of immunoliposomes as a modality for systemic gene delivery to human colon cancer cells.

Design of targeted lipid nanocapsules by conjugation of whole antibodies and antibody Fab’ fragments

Immunonanocapsules were synthesized by conjugation to lipid nanocapsules (LNC) of whole OX26 monoclonal antibodies (OX26 MAb) directed against the transferrin receptor (TfR). The TfR is overexpressed on the cerebral endothelium and mediates the transcytosis mechanism. Fab’ fragments, known for their reduced interaction with the reticuloendothelial system, were also conjugated to LNC. This coupling was facilitated by the incorporation of lipid PEG 2000 functionalized with reactive-sulfhydryl maleimide groups (DSPE–PEG 2000–maleimide) into LNC shells by a post-insertion procedure, developed initially for liposome pegylation. An interfacial model using the dynamic rising drop technique helped determine the parameters influencing the DSPE–PEG 2000–maleimide insertion and the quality of the anchorage. Heat was essential to promote both an important and stable adsorption of DSPE–PEG 2000–maleimide onto LNC. OX26 MAb were thiolated to react with maleimide functions whereas thiol residues on Fab’ fragments were used directly. The number of ligands per nanocapsule was adjusted according to their initial quantity in the coupling reaction mixture, with densities from 16 to183 whole antibodies and between 42 and 173 Fab’ fragments per LNC. The specific association of immunonanocapsules to cells overexpressing TfR was thus demonstrated, suggesting their ability to deliver drugs to the brain.

Abrogation of allergic reactions by a bispecific antibody fragment linking IgE to CD300a

Initiated and regulated by mast cells, allergic responses are balanced through an intricate network of positive and negative signals. We have recently shown that the inhibitory receptor CD300a is expressed on human mast cells and modulates a large array of their functions. We sought to evaluate CD300a as a negative regulator of allergic inflammation in vivo by means of a bispecific antibody linking CD300a with IgE. Bispecific antibody fragments were generated by chemical conjugation of Fab' fragments of anti-human IgE and CD300a (IE1H) and anti-mouse IgE and CD300a (IE1M). IgE-sensitized human mast cells were activated simultaneously with anti-IgE and IE1H. Phosphorylation of signaling proteins and calcium influx were evaluated by using fluorescence-activated cell sorting. Degranulation was assessed on the basis of tryptase and IL-4 release. IE1M was administered simultaneously with allergen challenge in 2 murine models of allergic disease. Passive cutaneous anaphylaxis was assessed by means of dye exudation. Experimental airway inflammation was assessed on the basis of tryptase and cytokine content, eosinophilic infiltration, and lung histology (hematoxylin and eosin and periodic acid-Schiff stain). IE1H potently inhibited IgE-mediated mast cell degranulation in a dose-dependent manner by inhibiting the signaling events induced by FcvarepsilonRI. IE1M completely abolished dye exudation in passive cutaneous anaphylaxis. IE1M abrogated allergic airway inflammation. Our results demonstrate that specific targeting of CD300a on mast cells is a potent strategy for inhibiting allergic reactions. This work demonstrates a potent approach for the therapy of allergic diseases.

Site-specific conjugation of bifunctional chelator BAT to mouse IgG1 Fab1 fragment

To perform a site-specific conjugation of Fab' fragments of a mouse monoclonal antibody(MoAb) B43(of IgG1 subtype) to a bifunctional chelator 6-[p-(bromoacetamido)benzyl]-1,4,8,11-tetraazacyclotetradecane-N,N',N' 'N' ' '-tetraacetic acid (BAT) via the thiol groups in the hinge distal to the antigen-binding site of the Fab'. B43 was cleaved using a simple 2-step method. First, stable F(ab')(2) was produced by pepsin treatment. Fab' with free thiol in the hinge region was then obtained by cysteine reduction of F(ab')2. Second, a site-specific conjugation of Fab' to thiol-specific BAT was performed in a one-step reaction. The Fab' fragment had approximately 1.8 free thiol groups per molecule after cysteine reduction. The conjugation efficiency and the chemical yield were approximately 1.28 moles chelator/Fab' and 74% of the initial concentration of Fab', respectively. The F(ab')2, Fab' and Fab'-BAT all maintained reasonable antigen-binding properties. (67)Cu labeling of the conjugate under standard conditions did not impair the immunoreactivity of Fab'-BAT. This is a simple and efficient method for producing immunoreactive conjugates of Fab'-BAT, which can be used to make radiometal-labeled conjugates for further diagnostic and therapeutic applications.

728. Anti-TAG-72 Immunoliposomes for Efficient Tumor-Targeted Gene Therapy

One of the most promising strategies for cancer gene therapy is achievement of tumor-specific expression of tumor-killing genes without any related cytotoxicity. A tumor-recognizing vehicle with monoclonal antibodies or their fragments specific to tumor surface antigens, so call immunoliposomes (ILs), is a modality for tumor-targeted delivery. However, currently there are very limited data on this type of gene delivery vehicle. Hence, we have developed novel immunoliposomes specific to tumor cells overexpressing tumor-associated glycoprotein 72 (TAG-72) for drug or gene delivery. The anti-TAG-72 ILs were prepared by conjugation of Fab' fragments of recombinant humanized monoclonal antibody HzCC49 to sterically stabilized unilamellar liposomes (90-110 nm in diameter) to create targetability to TAG-72-overexpressing cancer cells. The liposomes consisted of POPC (1-palmitonyl-2-oleoyl-sn-glycerol-3-phosphocholine, 92 mol %), DMKD cationic lipid (O, O'-dymyrisyl-N-lysyl aspartate, 4 mol %), DSPE-PEG2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol) 2000], 3 mol %), and DSPE-PEG2000-MAL (DSPE-[maleimide(polyethylene glycol) 2000], 1 mol %). The anti-TAG-72 ILs were able to adhere to the surface of TAG-72-overexpressing LS174T human colon cancer cells more effectively than conventional liposomes. Also, in vitro gene transfection to the LS174T cells by the anti-TAG-72 ILs in the presence of a high concentration of fetal bovine serum (up to 60%) were greater than that by the conventional cationic lipoplexes. The intravenously administered anti-TAG-72 ILs were efficiently localized in the LS174T tumor tissues while the non-targetable conventional liposomes were not. Intravenous administration of the anti-TAG-72 ILs containing plasmids encoding anti-angiogenic proteins, such as angiostatin K1/3, endostatin, and saxatilin, significantly inhibited in vivo growth of the LS174T tumors and angiogenesis in the tumor tissues. These results demonstrated that the TAG-72-mediated targeting of immunoliposomes would be a possible modality for systemic gene delivery to human colon cancer cells.

Pegylated polyethylenimine-Fab' antibody fragment conjugates for targeted gene delivery to human ovarian carcinoma cells.

Specific targeting of ovarian carcinoma cells using pegylated polyethylenimine (PEG-PEI) conjugated to the antigen binding fragment (Fab') of the OV-TL16 antibody, which is directed to the OA3 surface antigen, was the objective of this study. OA3 is expressed by a majority of human ovarian carcinoma cell lines. To demonstrate the ability of the PEG-PEI-Fab' to efficiently complex DNA, an ethidium bromide exclusion assay was performed. Comparison with PEG-PEI or PEI 25 kDa showed only minor differences in the ability to condense DNA. Since conjugation of Fab' to PEG-PEI might influence complex stability, this issue was addressed by incubating the complexes with increasing amounts of heparin. This assay revealed stability similar to that of unmodified PEG-PEI/DNA or PEI 25 kDa/DNA complexes. Complexes displayed a size of approximately 150 nm with a zeta potential close to neutral. The latter property is of particular interest for potential in vivo use, since a neutral surface charge reduces nonspecific interactions. Binding studies using flow cytometry and fluorescently labeled DNA revealed a more than 6-fold higher degree of binding of PEG-PEI-Fab'/DNA complexes to epitope-expressing cell lines compared to unmodified PEG-PEI/DNA complexes. In OA3-expressing OVCAR-3 cells, luciferase reporter gene expression was elevated up to 80-fold compared to PEG-PEI and was even higher than that of PEI 25 kDa. The advantage of this system is its specificity, which was demonstrated by competition experiments with free Fab' in the cell culture media during transfection experiments and by using OA3-negative cells. In the latter case, only a low level of reporter gene expression could be achieved with PEG-PEI-Fab'.

Detection of the factor V Leiden mutation by direct allele-specific hybridization of PCR amplicons to photoimmobilized locked nucleic acids.

Individuals carrying the factor V Leiden mutation have been shown to have an increased risk of developing venous thromboembolism. Our aim was to develop an ELISA-like assay to detect the mutation in PCR-amplified genomic DNA using novel, high-affinity DNA analogs, termed locked nucleic acids (LNAs). LNA octamer probes complementary to the factor V wild-type or mutated sequence were covalently attached to individual wells of a microtiter plate. Biotinylated factor V amplicons were added, and hybridization to the immobilized LNA probes was scored colorimetrically using a horseradish peroxidase-anti-biotin Fab conjugate and tetramethylbenzidine substrate. In a prospective study of 53 patients, the assay reproducibly scored both factor V homozygotes and heterozygotes with excellent sensitivity and specificity. All results were in complete agreement with the results obtained with the conventional PCR-restriction fragment length polymorphism technique. The simplicity of the assay and its procedural relatedness to the widely used ELISA format should make it useful for routine factor V testing in the clinical laboratory.

Simplified preformed chelate protein radiolabeling with technetium-99m mercaptoacetamidoadipoylglycylglycine (N3S-adipate).

A simplified kiet has been developed for 99mTc protein radiolabeling using an N3S triamide mercaptide bifunctional chelating agent and the preformed chelate approach. The process combined N3S chelating agent, gluconate intermediate transfer agent, stannous reducing agent, and gentisic acid stabilizer into a lyophilized formulation. With sulfur donor atom hemithioacetal protection of the ligand, delta-2,3,5,6-tetrafluorothiophenyl alpha-S-(1-ethoxyethyl)mercaptoacetamido-L-adipoylglycylglycine , optimum 99mTc chelation was achieved in a single step. Subsequent reaction with NR-LU-10 antibody Fab fragment followed by purification via QAE Sephadex anion exchange resin filter afforded 99mTc-N3S-NR-LU-10 Fab conjugate with retained immunoreactivity and effective tumor targeting properties.