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Investigator(s) |
Rudnick SI, Adams GP. Affinity and avidity in antibody-based tumor targeting. Cancer Biother Radiopharm. 2009 Apr;24(2):155-61.
Many factors contribute to successful tumor targeting by antibodies. Besides properties of the tumor tissue and general antibody pharmacology, a relationship exists between an antibody and its antigen that can shape penetration, catabolism, specificity, and efficacy. The affinity and avidity of the binding interactions play critical roles in these dynamics. In this work, we review the principles that guide models predicting tumor penetration and cellular internalization while providing a critical overview of studies aimed at experimentally determining the specific role of affinity and avidity in these processes. One should gain the perspective that binding affinity can, in part, dictate the localization of antibodies in tumors, leading to high concentrations in the perivascular space or low concentrations diffused throughout the tumor. These patterns can be simply due to the diminution of available dose by binding antigen and are complicated by internalization and degradation stemming from slow rates of dissociation. As opposed to the trend of simply increasing affinity to increase efficacy, novel strategies that increase avidity and broaden specificity have made significant progress in tumor targeting.
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Adams
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Robinson MK, Shaller C, Garmestani K, Plascjak PS, Hodge KM, Yuan QA, Marks JD, Waldmann TA, Brechbiel MW, Adams GP. Effective treatment of established human breast tumor xenografts in immunodeficient mice with a single dose of the alpha-emitting radioisotope astatine-211 conjugated to anti-HER2/neu diabodies. Clin Cancer Res. 2008 Feb;14(3):875-82.
Purpose: Successful radioimmunotherapy strategies depend on selecting radioisotopes with physical properties complementary to the biological properties of the targeting vehicle. Small, engineered antitumor antibody fragments are capable of rapid, highly specific tumor targeting in immunodeficient mouse models. We hypothesized that the C6.5 diabody, a noncovalent anti-HER2 single-chain Fv dimer, would be an ideal radioisotope carrier for the radioimmunotherapy of established tumors using the short-lived a-emitting radioisotope At-211. Experimental Design: Immunodeficient nude mice bearing established HER2/neu-positive MDA-MB-361/DYT2 tumors treated with N-succinimidyl N-(4-[At-211]astatophenethyl)succinamate (At-211-SAPS)-C6.5 diabody. Additional cohorts of mice were treated with At-211-SAPS T84.66 diabody targeting the carcinoembryonic antigen or At-211-SAPS on a diabody specific for the Mullerian inhibiting substance type II receptor, which is minimally expressed on this tumor cell line. Results: A single i.v. injection of At-211-SAPS C6.5 diabody led to a 30-day delay in tumor growth when a 20 mu Ci dose was administered and a 57-day delay in tumor growth (60% tumor-free after 1 year) when a 45 mu Ci dose was used. Treatment of mice bearing the same tumors with At-211-SAPS T84.66 diabody at the same doses led to a delay in tumor growth, but no complete responses, likely due to substantially lower expression of this antigen on the MDA-MB-361/DYT2 tumors. In contrast, a dose of 20 mu Ci of At-211-SAPS on the anti-Mullerian-inhibiting substance type II receptor diabody did not affect tumor growth rate, demonstrating specificity of the therapeutic effect. Conclusions: These findings indicate that diabody molecules can be effective agents for targeted radioimmunotherapy of solid tumors using powerful, short-lived alpha-emitting radioisotopes.
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Adams
Robinson
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Capobianco JA, Shih WY, Yuan QA, Adams GP, Shih WH. Label-free, all-electrical, in situ human epidermal growth receptor 2 detection. Rev Sci Instrum. 2008 Jul;79(7):076101.
Using 3-mercaptopropyltrimethoxysilane (MPS)-coated (PbMg(13)Nb(23)O(3))(0.63)-(PbTiO(3))(0.37) (PMN-PT)/tin and lead zirconate titanate/glass piezoelectric microcantilever sensors (PEMSs) with single-chain variable fragment (scFv) immobilized on the MPS surface, we have demonstrated real-time, label-free detection of human epidermal growth factor receptor 2 (Her2) in a background of 1 mgml bovine serum albumin. Coupled with a scFv with a K(D) of 3.4x10(-8)M, the MPS-insulated PMN-PT/tin PEMS 560 mum long and 720 mum wide exhibited a Her2 concentration sensitivity of 5 ngml in a background of 1 mgml BSA.
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Adams
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Yuan QA, Robinson MK, Simmons HH, Russeva M, Adams GP. Isolation of anti-MISIIR scFv molecules from a phage display library by cell sorter biopanning. Cancer Immunology Immunotherapy. 2008 Mar;57(3):367-78.
While cell surface antigens represent the most common targets for antibody-based cancer therapy, isolation of new antibodies specific for these targets from single-chain Fv phage display libraries has been hindered by limitations associated with traditional selection techniques. Solid phase panning is often associated with conformational changes to the target protein due to its immobilization on plastic tubes that can limit the ability of the isolated scFv to bind to conformational epitopes and solution panning methods require the use of secondary tags that often mask desired sequences and create unintended epitopes. Commonly utilized cell-based panning methods typically yield a panel of single-chain Fv (scFv) molecules that are specific for numerous cell surface antigens, often obscuring the desired clones. Here, we describe a novel cell sorter-based system to isolate single-chain Fv molecules specific for defined antigen targets expressed on stably-transformed mammalian cells. We employed these methods to isolate promising scFv clones that bind specifically to the Mullerian inhibiting substance type II receptor, a cell surface ovarian cancer antigen that has proven to be a difficult target for selection strategies.
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Adams
Robinson
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Sui JH, Aird DR, Tamin A, Murakami A, Yan MY, Yammanuru A, Jing HQ, Kan B, Liu X, Zhu Q, Yuan QA, Adams GP, Bellini WJ, Xu JG, Anderson LJ, Marasco WA. Broadening of Neutralization Activity to Directly Block a Dominant Antibody-Driven SARS-Coronavirus Evolution Pathway. PLoS Pathogens. 2008 Nov;4(11).
Phylogenetic analyses have provided strong evidence that amino acid changes in spike ( S) protein of animal and human SARS coronaviruses (SARS-CoVs) during and between two zoonotic transfers (2002/03 and 2003/04) are the result of positive selection. While several studies support that some amino acid changes between animal and human viruses are the result of inter-species adaptation, the role of neutralizing antibodies (nAbs) in driving SARS-CoV evolution, particularly during intra-species transmission, is unknown. A detailed examination of SARS-CoV infected animal and human convalescent sera could provide evidence of nAb pressure which, if found, may lead to strategies to effectively block virus evolution pathways by broadening the activity of nAbs. Here we show, by focusing on a dominant neutralization epitope, that contemporaneous-and cross-strain nAb responses against SARS-CoV spike protein exist during natural infection. In vitro immune pressure on this epitope using 2002/03 strain-specific nAb 80R recapitulated a dominant escape mutation that was present in all 2003/04 animal and human viruses. Strategies to block this nAb escape/naturally occurring evolution pathway by generating broad nAbs (BnAbs) with activity against 80R escape mutants and both 2002/03 and 2003/04 strains were explored. Structure-based amino acid changes in an activation-induced cytidine deaminase ( AID) "hot spot'' in a light chain CDR ( complementarity determining region) alone, introduced through shuffling of naturally occurring non-immune human VL chain repertoire or by targeted mutagenesis, were successful in generating these BnAbs. These results demonstrate that nAb-mediated immune pressure is likely a driving force for positive selection during intra-species transmission of SARS-CoV. Somatic hypermutation (SHM) of a single VL CDR can markedly broaden the activity of a strain-specific nAb. The strategies investigated in this study, in particular the use of structural information in combination of chain-shuffling as well as hot-spot CDR mutagenesis, can be exploited to broaden neutralization activity, to improve anti-viral nAb therapies, and directly manipulate virus evolution.
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Adams
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Robinson MK, Hodge KM, Horak E, Sundberg AL, Russeva M, Shaller CC, von Mehren M, Shchaveleva I, Simmons HH, Marks JD, Adams GP. Targeting ErbB2 and ErbB3 with a bispecific single-chain Fv enhances targeting selectivity and induces a therapeutic effect in vitro. Br J Cancer. 2008 Nov 4;99(9):1415-25.
Inappropriate signalling through the EGFR and ErbB2/HER2 members of the epidermal growth factor family of receptor tyrosine kinases is well recognised as being causally linked to a variety of cancers. Consequently, monoclonal antibodies specific for these receptors have become increasingly important components of effective treatment strategies for cancer. Increasing evidence suggests that ErbB3 plays a critical role in cancer progression and resistance to therapy. We hypothesised that co-targeting the preferred ErbB2/ErbB3 heterodimer with a bispecific single-chain Fv (bs-scFv) antibody would promote increased targeting selectivity over antibodies specific for a single tumour-associated antigen (TAA). In addition, we hypothesised that targeting this important heterodimer could induce a therapeutic effect. Here, we describe the construction and evaluation of the A5-linker-ML3.9 bs-scFv (ALM), an anti-ErbB3/ErbB2 bs-scFv. The A5-linker-ML3.9 bs-scFv exhibits selective targeting of tumour cells in vitro and in vivo that co-express the two target antigens over tumour cells that express only one target antigen or normal cells that express low levels of both antigens. The A5-linker-ML3.9 bs-scFv also exhibits significantly greater in vivo targeting of ErbB2'+'/ErbB3'+' tumours than derivative molecules that contain only one functional arm targeting ErbB2 or ErbB3. Binding of ALM to ErbB2'+'/ErbB3'+' cells mediates inhibition of tumour cell growth in vitro by effectively targeting the therapeutic anti-ErbB3 A5 scFv. This suggests both that ALM could provide the basis for an effective therapeutic agent and that engineered antibodies selected to co-target critical functional pairs of TAAs can enhance the targeting specificity and efficacy of antibody-based cancer therapeutics.
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Adams
von Mehren
Robinson
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Robinson MK, Shaller C, Garmestani K, Plascjak PS, Hodge KM, Yuan QA, Marks JD, Waldmann TA, Brechbiel MW, Adams GP. Effective treatment of established human breast tumor xenografts in immunodeficient mice with a single dose of the alpha-emitting radioisotope astatine-211 conjugated to anti-HER2/neu diabodies. Clin Cancer Res. 2008 Feb 1;14(3):875-82.
PURPOSE: Successful radioimmunotherapy strategies depend on selecting radioisotopes with physical properties complementary to the biological properties of the targeting vehicle. Small, engineered antitumor antibody fragments are capable of rapid, highly specific tumor targeting in immunodeficient mouse models. We hypothesized that the C6.5 diabody, a noncovalent anti-HER2 single-chain Fv dimer, would be an ideal radioisotope carrier for the radioimmunotherapy of established tumors using the short-lived alpha-emitting radioisotope (211)At. EXPERIMENTAL DESIGN: Immunodeficient nude mice bearing established HER2/neu-positive MDA-MB-361/DYT2 tumors treated with N-succinimidyl N-(4-[(211)At]astatophenethyl)succinamate ((211)At-SAPS)-C6.5 diabody. Additional cohorts of mice were treated with (211)At-SAPS T84.66 diabody targeting the carcinoembryonic antigen or (211)At-SAPS on a diabody specific for the Mullerian inhibiting substance type II receptor, which is minimally expressed on this tumor cell line. RESULTS: A single i.v. injection of (211)At-SAPS C6.5 diabody led to a 30-day delay in tumor growth when a 20 muCi dose was administered and a 57-day delay in tumor growth (60% tumor-free after 1 year) when a 45 muCi dose was used. Treatment of mice bearing the same tumors with (211)At-SAPS T84.66 diabody at the same doses led to a delay in tumor growth, but no complete responses, likely due to substantially lower expression of this antigen on the MDA-MB-361/DYT2 tumors. In contrast, a dose of 20 muCi of (211)At-SAPS on the anti-Mullerian-inhibiting substance type II receptor diabody did not affect tumor growth rate, demonstrating specificity of the therapeutic effect. CONCLUSIONS: These findings indicate that diabody molecules can be effective agents for targeted radioimmunotherapy of solid tumors using powerful, short-lived alpha-emitting radioisotopes.
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Adams
Robinson
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Yuan QA, Robinson MK, Simmons HH, Russeva M, Adams GP. Isolation of anti-MISIIR scFv molecules from a phage display library by cell sorter biopanning. Cancer Immunol Immunother. 2008 Mar;57(3):367-78.
While cell surface antigens represent the most common targets for antibody-based cancer therapy, isolation of new antibodies specific for these targets from single-chain Fv phage display libraries has been hindered by limitations associated with traditional selection techniques. Solid phase panning is often associated with conformational changes to the target protein due to its immobilization on plastic tubes that can limit the ability of the isolated scFv to bind to conformational epitopes and solution panning methods require the use of secondary tags that often mask desired sequences and create unintended epitopes. Commonly utilized cell-based panning methods typically yield a panel of single-chain Fv (scFv) molecules that are specific for numerous cell surface antigens, often obscuring the desired clones. Here, we describe a novel cell sorter-based system to isolate single-chain Fv molecules specific for defined antigen targets expressed on stably-transformed mammalian cells. We employed these methods to isolate promising scFv clones that bind specifically to the Mullerian inhibiting substance type II receptor, a cell surface ovarian cancer antigen that has proven to be a difficult target for selection strategies.
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Adams
Robinson
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Robinson MK, Hodge KM, Horak E, Sundberg AL, Russeva M, Shaller CC, von Mehren M, Shchaveleva I, Simmons HH, Marks JD, Adams GP. Targeting ErbB2 and ErbB3 with a bispecific single-chain Fv enhances targeting selectivity and induces a therapeutic effect in vitro. Br J Cancer. 2008 Oct;99(9):1415-25.
Inappropriate signalling through the EGFR and ErbB2/HER2 members of the epidermal growth factor family of receptor tyrosine kinases is well recognised as being causally linked to a variety of cancers. Consequently, monoclonal antibodies specific for these receptors have become increasingly important components of effective treatment strategies for cancer. Increasing evidence suggests that ErbB3 plays a critical role in cancer progression and resistance to therapy. We hypothesised that co-targeting the preferred ErbB2/ErbB3 heterodimer with a bispecific single-chain Fv (bs-scFv) antibody would promote increased targeting selectivity over antibodies specific for a single tumour-associated antigen (TAA). In addition, we hypothesised that targeting this important heterodimer could induce a therapeutic effect. Here, we describe the construction and evaluation of the A5-linker-ML3.9 bs-scFv (ALM), an anti-ErbB3/ErbB2 bs-scFv. The A5-linker-ML3.9 bs-scFv exhibits selective targeting of tumour cells in vitro and in vivo that co-express the two target antigens over tumour cells that express only one target antigen or normal cells that express low levels of both antigens. The A5-linker-ML3.9 bs-scFv also exhibits significantly greater in vivo targeting of ErbB2'+'/ErbB3'+' tumours than derivative molecules that contain only one functional arm targeting ErbB2 or ErbB3. Binding of ALM to ErbB2'+'/ ErbB'+3' cells mediates inhibition of tumour cell growth in vitro by effectively targeting the therapeutic anti-ErbB3 A5 scFv. This suggests both that ALM could provide the basis for an effective therapeutic agent and that engineered antibodies selected to co-target critical functional pairs of TAAs can enhance the targeting specificity and efficacy of antibody-based cancer therapeutics.
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Adams
von Mehren
Robinson
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Loo L, Robinson MK, Adams GP. Antibody engineering principles and applications. Cancer J. 2008 May-Jun;14(3):149-53.
Antibodies have emerged as significant agents for the treatment of a number of diseases including cancer and autoimmunity. However, most of the antibodies currently used in clinical practice were developed from humanized or chimeric molecules based on mouse monoclonal antibodies. Recent advances in antibody selection and engineering techniques have led to the development of antibodies specific for highly conserved targets, the creation of novel antibody-based structures, significant improvements in affinity for target antigens, enhanced ability to engage immune effector functions, and the creation of fusion proteins with direct cytotoxic properties. This review provides an overview of the techniques that we expect will have the greatest impact on the field of antibody engineering.
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Adams
Robinson
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Adams
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Adams
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Nordberg E, Friedman M, Gostring L, Adams GP, Brismar H, Nilsson FY, Stahl S, Glimelius B, Carlsson J. Cellular studies of binding, internalization and retention of a radiolabeled EGFR-binding affibody molecule. Nucl Med Biol. 2007 Aug;34(6):609-18.
Introduction: The cellular binding and processing of an epidermal growth factor receptor (EGFR) targeting affibody molecule, (Z(EGFR:955))(2), was studied. This new and small molecule is aimed for applications in nuclear medicine. The natural ligand epidermal growth factor (EGF) and the antibody cetuximab were studied for comparison. Methods: All experiments were made with cultured A431 squamous carcinoma cells. Receptor specificity, binding time patterns, retention and preliminary receptor binding site localization studies were all made after (125) I, labeling. Internalization was studied using Oregon Green 488, Alexa Fluor 488 and CypHer5E markers. Results: [(125) I](Z(EGFR:955))(2) and [(125) I]cetuximab gave a maximum cellular uptake of I-125 within 4 to 8 h of incubation, while [(125) I]EGF gave a maximum uptake already after 2 h. The retention studies showed that the cell-associated fraction of 125 1 after 48 It of incubation was similar to 20% when delivered as [(125) I](Z(FGFR:955))(2) and similar to 25% when delivered as [I-125] cetuximab. [(125) I]EGF-mediated delivery gave a faster (125) I release, where almost all cell-associated radioactivity had disappeared within 24 It. All three substances were internalized as demonstrated with confocal microscopy. Competitive binding studies showed that both EGF and cetuximab inhibited binding Of (Z(EGFR:955))(2) and indicated that the three substances competed for an overlapping binding site. Conclusion: The results gave information on cellular processing of radionuclides when delivered with (Z(EGFR:955))(2) in comparison to delivery with EGF and cetuximab. Competition assays suggested that [I-125](Z(EGFR:955))(2) bind to Domain III of EGFR. The affibody molecule (Z(EGFR:955))(2) can be a candidate for EGFR imaging applications in nuclear medicine. (c) 2007 Elsevier Inc. All rights reserved.
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Adams
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Yuan QA, Robinson MK, Simmons HH, Russeva M, Adams GP. Isolation of anti-MISIIR scFv molecules from a phage display library by cell sorter biopanning. Cancer Immunol Immunother. 2007;.
While cell surface antigens represent the most common targets for antibody-based cancer therapy, isolation of new antibodies specific for these targets from single-chain Fv phage display libraries has been hindered by limitations associated with traditional selection techniques. Solid phase panning is often associated with conformational changes to the target protein due to its immobilization on plastic tubes that can limit the ability of the isolated scFv to bind to conformational epitopes and solution panning methods require the use of secondary tags that often mask desired sequences and create unintended epitopes. Commonly utilized cell-based panning methods typically yield a panel of single-chain Fv (scFv) molecules that are specific for numerous cell surface antigens, often obscuring the desired clones. Here, we describe a novel cell sorter-based system to isolate single-chain Fv molecules specific for defined antigen targets expressed on stably-transformed mammalian cells. We employed these methods to isolate promising scFv clones that bind specifically to the Mullerian inhibiting substance type II receptor, a cell surface ovarian cancer antigen that has proven to be a difficult target for selection strategies.
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Adams
Robinson
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Zhou Y, Daryl C, Zou H, Hayes ME, Adams GP, Kirpotin DB, Marks JD. Impact of single-chain fv antibody fragment affinity on nanoparticle targeting of epidermal growth factor receptor-expressing tumor cells. J Mol Biol. 2007 Aug;371(4):934-47.
To determine the importance of single-chain Fv (scFv) affinity on binding, uptake, and cytotoxicity of tumor-targeting nanoparticles, the affinity of the epidermal growth factor receptor (EGFR) scFv antibody C10 was increased using molecular evolution and yeast display. A library containing scFv mutants was created by error-prone PCR, displayed on the surface of yeast, and higher affinity clones selected by fluorescence activated cell sorting. Ten mutant scFv were identified that had a 3-18-fold improvement in affinity (K-D=15-88 nM) for EGFR-expressing A431 tumor cells compared to C10 scFv (K-D=264 nM). By combining mutations, higher affinity scFv were generated with K-D ranging from 0.9 nM to 10 nM. The highest affinity scFv had a 280-fold higher affinity compared to that of the parental C10 scFv. Immunoliposome nanoparticles (ILs) were prepared using EGFR scFv with a 280-fold range of affinities, and their binding and uptake into EGFR-expressing tumor cells was quantified. At scFv densities greater than 148 scFv/IL, there was no effect of scFv affinity on IL binding and uptake into tumor cells, or on cytotoxicity. At lower scFv densities, there was less uptake and binding for ILs constructed from the very low affinity C10 scFv. The results show the importance of antibody fragment density on nanoparticle uptake, and suggest that engineering ultrahigh affinity scFv may be unnecessary for optimal nanoparticle targeting. (c) 2007 Elsevier Ltd. All rights reserved.
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Adams
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Friedman M, Nordberg E, Hoiden-Guthenberg I, Brisimar H, Adams GP, Nilsson FY, Carlss J, Stahl S. Phage display selection of Affibody molecules with specific binding to the extracellular domain of the epidermal growth factor receptor. Protein Engineering Design & Selection. 2007 Apr;20(4):189-99.
Affibody molecules specific for the epidermal growth factor receptor (EGFR) have been selected by Phage display technology from a combinatorial protein library based on the 58-residue, protein A-derived Z domain. EGFR is overexpressed in various malignancies and is frequently associated with poor patient prognosis, and the information provided by targeting this receptor could facilitate both patient diagnostics and treatment. Three selected Affibody variants were shown to selectively bind to the extracellular domain of EGFR (EGFR-ECD). Kinetic biosensor analysis revealed that the three monomeric Affibody molecules bound with similar affinity, ranging from 130 to 185 nM. Head-to-tail dimers of the Affibody molecules were compared for their binding to recombinant EGFR-ECD in biosensor analysis and in human epithelial cancer A431 cells. Although the dimeric Affibody variants were found to bind in a range of 2550 nM affinities in biosensor analysis, they were found to be low !
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Adams
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Steffen AC, Orlova A, Wikman M, Nilsson FY, Stahl S, Adams GP, Tolmachev V, Carlsson J. Affibody-mediated tumour targeting of HER-2 expressing xenografts in mice. EUROPEAN JOURNAL OF NUCLEAR MEDICINE AND MOLECULAR IMAGING. 2006 Jun;33(6):631-8.
Purpose: Targeted delivery of radionuclides for diagnostic and therapeutic applications has until recently largely been limited to receptor ligands, antibodies and antibody-derived molecules. Here, we present a new type of molecule, a 15-kDa bivalent affibody called (Z(HER2:4))(2), with potential for such applications. The (Z(HER2:4))(2) affibody showed high apparent affinity (K (D)=3 nM) towards the oncogene product HER-2 (also called p185/neu or c-erbB-2), which is often overexpressed in breast and ovarian cancers. The purpose of this study was to investigate the in vivo properties of the new targeting agent. Methods: The biodistribution and tumour uptake of the radioiodinated (Z(HER2:4))(2) affibody was studied in nude mice carrying tumours from xenografted HER-2 overexpressing SKOV-3 cells. Results: The radioiodinated (Z(HER2:4))(2) affibody was primarily excreted through the kidneys, and significant amounts of radioactivity were specifically targeted to the tumours. The blood-borne radioactivity was, at all times, mainly in the macromolecular fraction. A tumour-to-blood ratio of about 10:1 was obtained 8 h post injection, and the tumours could be easily visualised with a gamma camera at this time point. Conclusion: The results indicate that the (Z(HER2:4))(2) affibody is an interesting candidate for applications in nuclear medicine, such as radionuclide-based tumour imaging and therapy.
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Adams
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Yuan Q, Simmons HH, Robinson MK, Russeva M, Marasco WA, Adams GP. Development of engineered antibodies specific for the Muellerian inhibiting substance type II receptor: a promising candidate for targeted therapy of ovarian cancer. Molecular Cancer Therapeutics. 2006;5(8):2096-105.
The Muellerian inhibiting substance type II receptor (MISIIR) is involved in Muellerian duct regression as part of the development of the male reproductive system. In adult females, MISIIR is present on ovarian surface epithelium and is frequently expressed on human epithelial ovarian cancer cells. Muellerian inhibiting substance has been found to be capable of inhibiting the growth of primary human ovarian cancer cells derived from ascites and ovarian cancer cell lines. This suggested to us that MISIIR could be an attractive target for antibody-based tumor targeting and growth inhibition strategies. Here, we describe the prodn. of recombinant human MISIIR extracellular domain-human Ig Fc domain fusion proteins and their use as targets for the selection of MISIIR-specific human single-chain variable fragments (scFv) mols. from a human nonimmune scFv phage display library. The binding kinetics of the resulting anti-MISIIR scFv clones were characterized and two were employed as the basis for the construction of bivalent scFv:Fc antibody-based mols. Both bound specifically to human ovarian carcinoma cells in flow cytometry assays and cross-reacted with mouse MISIIR. These results indicate that antibody-based constructs may provide a highly specific means of targeting MISIIR on human ovarian carcinoma cells for the purpose of diagnosing and treating this disease. [on SciFinder (R)]
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Adams
Robinson
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Adams
Ridge
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Adams
Weiner
Robinson
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Adams GP, Tai M, McCartney JE, Marks JD, Stafford WF, Houston LL, Huston JS, Weiner LM. Avidity-Mediated Enhancement of In vivo Tumor Targeting by Single-Chain Fv Dimers. Clin Cancer Res. 2006;12(5):1599-605.
Radiolabeled single-chain Fv (sFv) mols. display highly specific tumor retention in the severe combined immunodeficient (SCID) mouse model; however, the abs. quantity of sFv retained in the tumors is diminished by the rapid renal elimination resulting from the small size of the sFv mols. (Mr 27,000) and by dissocn. of the monovalent sFv from tumor-assocd. antigen. We previously reported significant improvement in tumor retention without a loss of targeting specificity on converting monovalent sFv into divalent [(sFv')2] dimers, linked by a disulfide bond between COOH-terminal cysteinyl peptides engineered into the sFv'. However, our data for enhanced dimer localization in tumors could not distinguish between the contributions of enhanced avidity and increased systemic retention assocd. with the larger size of 54 kDa [(sFv')2] dimers relative to 27-kDa sFv. In this investigation, we have compared tumor targeting of divalent anti-c-erbB-2/HER2/neu 741F8-1 (sFv')2 homodimers with monovalent 741F8/26-10 (sFv')2 heterodimers (Mr 54,000) and 741F8 sFv monomers (741F8 sFv has binding specificity for erbB-2/HER2/neu and 26-10 sFv specificity for digoxin and related cardiac glycosides). These studies allowed us to distinguish the dominant effect of valency over mol. wt. in accounting for the superior tumor retention of 741F8-1 (sFv')2 homodimers. Each of the radioiodinated species was administered i.v. to SCID mice bearing SK-OV-3 human tumor xenografts and tumor localization at 24 h post i.v. injection was detd. for 125I-741F8-1 (sFv')2 (3.57 %ID/g), 125I-741F8/26-10 (sFv')2 (1.13 %ID/g), and 125I-741F8-1 sFv (1.25 %ID/g). These findings substantiate that the improved tumor retention of (sFv')2 homodimers over sFv monomers results from the availability of dual binding sites rather than from the slower systemic clearance of homodimers. [on SciFinder (R)]
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Adams
Weiner
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Adams
Weiner
Litwin
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Robinson MK, Doss M, Shaller C, Narayanan D, Marks JD, Adler LP, Trotter DE, Adams GP. Quantitative immuno-positron emission tomography imaging of HER2-positive tumor xenografts with an iodine-124 labeled anti-HER2 diabody. Cancer Res. 2005;65(4):1471-8.
Positron emission tomog. (PET) provides an effective means of both diagnosing/staging several types of cancer and evaluating efficacy of treatment. To date, the only U.S. Food and Drug Administration-approved radiotracer for oncol. PET is 18F-fluoro-deoxyglucose, which measures glucose accumulation as a surrogate for malignant activity. Engineered antibody fragments have been developed with the appropriate targeting specificity and systemic elimination properties predicted to allow for effective imaging of cancer based on expression of tumor assocd. antigens. We evaluated a small engineered antibody fragment specific for the HER2 receptor tyrosine kinase (C6.5 diabody) for its ability to function as a PET radiotracer when labeled with iodine-124. Our studies revealed HER2-dependent imaging of mouse tumor xenografts with a time-dependent increase in tumor-to-background signal over the course of the expts. Radioiodination via an indirect method attenuated uptake of radioiodine in tissues that express the Na/I symporter without affecting the ability to image the tumor xenografts. In addn., we validated a method for using a clin. PET/computed tomog. scanner to quantify tumor uptake in small-animal model systems; quantitation of the tumor targeting by PET correlated with traditional necropsy-based anal. at all time points analyzed. Thus, diabodies may represent an effective mol. structure for development of novel PET radiotracers. [on SciFinder (R)]
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Adams
Robinson
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Robinson MK, Russeva M, Horak EM, Heitner T, Simmons HH, Shaller CC, Reid M, Hodge K, Garrison J, Tesfaye A, Marks JD, Weiner LM, Adams GP. A bispecific single-chain Fv molecule targeting HER2 and HER3 exhibits tumor-targeting specificity in vivo and a therapeutic effect in vitro. Clin Cancer Res. 2005 Part;11(24):8983S-8983S.
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Adams
Weiner
Robinson
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Horak E, Heitner T, Robinson MK, Simmons HH, Garrison J, Russeva M, Furmanova P, Lou J, Zhou Y, Yuan Q, Weiner LM, Adams GP, Marks JD. Isolation of scFvs to In Vitro Produced Extracellular Domains of EGFR Family Members. Cancer Biotherapy & Radiopharmaceuticals. 2005;20(6):603-13.
The members of the epidermal growth factor receptor (EGFR) family are over expressed in a variety of malignancies and are frequently linked to aggressive disease and a poor prognosis. Although clin. effective monoclonal antibodies (MAbs) have been developed to target HER2 and EGFR, the remaining two family members, HER3 and HER4, have not been the subject of significant efforts. In this paper, we have taken the initial steps required to generate antibodies with potential clin. utility that target the members of the EGFR family. The genes for the extracellular domains (ECDs) of all four members of the EGFR family were cloned and used to stably transfect 293 (HEK) cells. Milligram quantities of each ECD were produced and characterized. The HER3, HER4, and EGFR ECDs were then employed as targets for the selection of antibodies from naive human scFv (single-chain Fv) phage display libraries. Six unique scFv clones were isolated that bound specifically to HER3, 13 unique clones were isolated with specificity for HER4 and 52 unique anti-EGFR clones were isolated. These scFvs provide a valuable and potentially clin. relevant panel of agents to target the members of the EGFR family. [on SciFinder (R)]
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Adams
Weiner
Robinson
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Steffen AC, Wikman M, Tolmachev V, Adams GP, Nilsson FY, Stahl S, Carlsson J. In vitro characterization of a bivalent anti-HER-2 affibody with potential for radionuclide-based diagnostics. Cancer Biotherapy and Radiopharmaceuticals. 2005 Jun;20(3):239-48.
The 185 kDa transmembrane glycoprotein human epidermal growth factor receptor 2 (HER-2) (p185/neu, c-ErbB-2) is overexpressed in breast and ovarian cancers. Overexpression in breast cancer correlates with poor patient prognosis, and visualization of HER-2 expression might provide valuable diagnostic information influencing patient management. We have previously described the generation of a new type of affinity ligand, a 58-amino-acid affibody (Z(HER2:4)) with specific binding to HER-2. In order to benefit from avidity effects, we have created a bivalent form of the affibody ligand, (Z(HER2:4))(2). The monovalent and bivalent ligands were compared in various assays. The new bivalent affibody has a molecular weight of 15.6 kDa and an apparent affinity (K-D) against HER-2 of 3 W After radioiodination, using the linker molecule N-succinimidyl p-(trimethylstannyl) benzoate (SPMB), in vitro binding assays showed specific binding to HER-2 overexpressing cells. Internalization of I -125 was shown after delivery with both the monovalent and the bivalent affibody. The cellular retention of I-125 was longer after delivery with the bivalent affibody when,compared to delivery with the monovalent affibody. With approximately the same affinity as the monoclonal antibody trastuzumab (Herceptin (TM)) but only one tenth of the size, this new bivalent molecule is a promising candidate for radionuclide-based detection of HER-2 expression in tumors. I-125 was used in this study as a surrogate marker for the diagnostically relevant radioisotopes I-123 for single photon emission computed tomography (SPECT)/gamma-camera imaging and I-124 for positron emission tomography (PET).
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Adams
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Adams
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Adams GP, Weiner LM. Monoclonal antibody therapy of cancer. Nat Biotechnol. 2005 Sep;23(9):1147-57.
The most significant recent advances in the application of monoclonal antibodies (mAbs) to oncology have been the introduction and approval of bevacizumab (Avastin), an anti-vascular endothelial growth factor antibody, and of cetuximab (Erbitux), an anti-epidermal growth factor antibody. In combination with standard chemotherapy regimens, bevacizumab significantly prolongs the survival of patients with metastatic cancers of the colorectum, breast and lung. Cetuximab, used alone or with salvage chemotherapy, produces clinically meaningful anti-tumor responses in patients with chemotherapy-refractory cancers of the colon and rectum. In addition, the anti-HER2/neu antibody trastuzumab ( Herceptin), in combination with standard adjuvant chemotherapy, has been shown to reduce relapses and prolong disease-free and overall survival in high-risk patients after definitive local therapy for breast cancer. These exciting recent results provide optimism for the development of mAbs that bind novel targets, exploit novel mechanisms of action or possess improved tumor targeting. Progress in the clinical use of radioimmunoconjugates remains hindered by complexity of administration, toxicity concerns and insufficiently selective tumor targeting.
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Adams
Weiner
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Adams GP, Horak EM, Weiner LM, Marks JD. Bispecific scFv antibody molecules capable of binding to different epitopes of EGFR protein family for diagnosis and treatment of cancer. 2004 20030404.;:57 pp.
Bispecific single chain antibody mols. are disclosed which may be used to advantage to treat various forms of cancer assocd. with the overexpression of members of the EGFR protein family. The cancer is breast, colon, ovarian, endometrial, gastric, pancreatic, prostate and salivary gland cancer. The EGFR protein family consists EGFR, HER2/neu, HER3 and HER4. [on SciFinder (R)]
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Adams
Weiner
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Adams GP, Shaller CC, Dadachova E, Simmons HH, Horak EM, Tesfaye A, Klein-Szanto AJ, Marks JD, Brechbiel MW, Weiner LM. A single treatment of yttrium-90-labeled CHX-A"-C6.5 diabody inhibits the growth of established human tumor xenografts in immunodeficient mice.[see comment]. Cancer Res. 2004 Sep 1;64(17):6200-6.
Antitumor diabody molecules are noncovalent single-chain Fv dimers that recapitulate the divalent binding properties of native IgG antibodies. Diabodies are capable of substantial accumulation in tumor xenografts expressing relevant antigens in immunodeficient mouse models. With a Mr of approximately 55,000, diabodies are rapidly cleared from the circulation, resulting in tumor-to-blood ratios that significantly exceed those achieved early after the administration of monoclonal antibodies. We have evaluated the therapeutic potential of the beta-emitting isotope yttrium-90 (t1/2, 64 hours) conjugated to the C6.5K-A diabody that specifically targets the HER2/neu human tumor-associated antigen. We have found that a single intravenous dose of 150 microCi (200 microg) 90Y-CHX-A"-C6.5K-A diabody substantially inhibits the growth rates of established MDA-361/DYT2 human breast tumor xenografts in athymic nude mice. In contrast, 300 microCi (300 microg) 90Y-CHX-A"-C6.5K-A diabody resulted in only a minor delay in the growth of SK-OV-3 human ovarian cancer xenografts. The maximum tolerated dose was also dependent on the tumor xenograft model used. These studies indicate that genetically engineered antitumor diabody molecules can be used as effective vehicles for radioimmunotherapy.
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Adams
Weiner
Klein-Szanto
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