Improved Bivalent Miniantibodies, with Identical Avidity as Whole Antibodies, Produced by High Cell Density Fermentation of Escherichia coli

Pack, Peter; Kujau, Marian J.; Schroeckh, Volker; Knüpfer, Uwe; Wenderoth, Rolf; Riesenberg, D.; Plückthun, Andreas

doi:10.1038/nbt1193-1271

Cited by 67 publications

(52 citation statements)

References 31 publications

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“…Cells were grown in a bioreactor in a defined medium using the fedbatch technique (14,15). The production level of the anti-testosterone Fab fragment was about 50 mg/liter.…”

Section: Methodsmentioning

confidence: 99%

Structural Insights into Steroid Hormone Binding

Valjakka¹,

Takkinenz²,

Teerinen³

et al. 2002

Journal of Biological Chemistry

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The monoclonal anti-testosterone antibody (3-C 4 F 5 ) has a relatively high affinity (3 ؋ 10 8 M ؊1 ) with an overall good specificity profile. However, the earlier characterized binding properties have shown that both the affinity and specificity of this antibody must be improved if it is intended for use in clinical immunoassays. In this paper, the crystal structures of the recombinant antitestosterone (3-C 4 F 5 ) Fab fragment have been determined in the testosterone-bound and free form at resolutions of 2.60 and 2.72 Å, respectively. The high affinity binding of the (3-C 4 F 5 ) Fab is mainly determined by shape complementarity between the protein and testosterone. Only one direct hydrogen bond is formed between the hydroxyl group of the testosterone D-ring and the main-chain oxygen of Gly100 J H. The testosterone is deeply bound in a hydrophobic pocket, and the close shape complementarity is mainly formed by the third complementarity-determining regions ( The number of different, closely related steroid hormones in human serum is high, and their relative concentrations, which usually are very low, can vary greatly even between normal healthy individuals. Testosterone, the main male sex hormone, is one of the structurally similar steroid hormones. Measurement of serum testosterone levels is important in the evaluation of conditions associated with hyperandrogenism in women (1, 2) and to diagnose hypogonadism, impotence, and problems in spermatogenesis and pubertical development in men (3, 4). Development of monoclonal antibodies that would fulfill the high affinity and selectivity requirements needed for the accurate measurement of testosterone levels in serum samples has not been successful with the use of hybridoma technology. Rabbit polyclonal antibodies are used in most current immunoassays of steroid hormones. However, the supply of polyclonal reagents with consistent quality is a severe problem for the immunodiagnostic industry and requires continuous immunization of many animals. The inherent batch-to-batch variation of the polyclonal sera makes it necessary to optimize the assay parameters for each new serum batch.The three-dimensional structures of steroid binding Fab fragments are important for a general understanding of the molecular basis of specific interactions between antibodies and hydrophobic molecules such as steroids. The molecular basis of steroid antibody interactions has been studied in detail by determining the structures of a progesteronebinding antibody (DB3) in its free and bound form and with different high affinity cross-reactive progesterone derivatives (5-7). The binding site of the DB3 antibody is a hydrophobic pocket, and the binding affinity and specificity are determined by van der Waals interactions and a few hydrogen bonds formed with the progesterone hapten (5). The high affinity binding mode of a number of progesterone derivatives is due to two alternate docking orientations for the steroid skeleton (6). In the cases of the anti-digoxin 26-10 and 40-50 antibodies, the h...

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“…Cells were grown in a bioreactor in a defined medium using the fedbatch technique (14,15). The production level of the anti-testosterone Fab fragment was about 50 mg/liter.…”

Section: Methodsmentioning

confidence: 99%

Structural Insights into Steroid Hormone Binding

Valjakka¹,

Takkinenz²,

Teerinen³

et al. 2002

Journal of Biological Chemistry

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“…Recently, attention has focused upon the generation of divalent scFv-based molecules with molecular weights in the range of the renal threshold for first-pass clearance. These include 50-kDa diabodies (Holliger, 1993), 55-kDa (scFv')2 , 60 to 65-kDa amphipathic helix-based scFv dimers (Pack et al, 1992(Pack et al, , 1993 and 80-kDa (scFv-CH3)2 LD minibodies and Flex minibodies (Hu Shi-zhen et al, 1996). While each of these proteins is capable of binding two antigen molecules, they differ in the orientation, flexibility and span of their binding sites.…”

mentioning

confidence: 99%

Prolonged in vivo tumour retention of a human diabody targeting the extracellular domain of human HER2/neu

Adams

Schier²,

Am³

et al. 1998

Br J Cancer

184

110

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Summary Single-chain Fv (scFv) molecules exhibit highly specific tumour-targeting properties in tumour-bearing mice. However, because of their smaller size and monovalent binding, the quantities of radiolabelled scFv retained in tumours limit their therapeutic applications. Diabodies are dimeric antibody-based molecules composed of two non-covalently associated scFv that bind to antigen in a divalent manner. In vitro, diabodies produced from the anti-HER2/neu (c-erbB-2) scFv C6.5 displayed approximately 40-fold greater affinity for HER2Ineu by surface plasmon resonance biosensor measurements and significantly prolonged association with antigen on the surface of SK-OV-3 cells (tl,2 cell surface retention of > 5 h vs 5 min) compared with C6.5 scFv. In SK-OV-3 tumour-bearing scid mice, radioiodinated C6.5 diabody displayed a highly favourable balance of quantitative tumour retention and specificity. By as early as 4 h after i.v. administration, significantly more diabody was retained in tumour (10 %ID g-1) than in blood (6.7 %ID ml-') or normal tissue (liver, 2.8 %ID g-'; lung, 7.1 %ID g-1; kidney, 5.2 %ID g-1). Over the next 20 h, the quantity present in blood and most tissues dropped approximately tenfold, while the tumour retained 6.5 %ID g-1 or about two-thirds of its 4-h value. In contrast, the 24-h tumour retention of radioiodinated C6.5 scFv monomer was only 1 %ID g-1. When diabody retentions were examined over the course of a 72-h study and cumulative area under the curve (AUC) values were determined, the resulting tumor-organ AUC ratios were found to be superior to those previously reported for other monovalent or divalent scFv molecules. In conclusion, the diabody format provides the C6.5 molecule with a distinct in vitro and in vivo targeting advantage and has promise as a delivery vehicle for therapeutic agents.

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“…While chemical linkage to prepare bsAbs is convenient, it is a tedious, low-yield process involving multiple steps of modification and purification. Thus, recombinant engineering is preferred, with notable examples including 2 different scFv's joined with a flexible linker 14 ; minibodies that contain 2 different scFv's joined at the C-termini via a hinge that is further fused to a helix-turn-helix 15 or leucine zipper motif 16 to facilitate heterodimerization; and diabodies that form heterodimers with a short linker between the V L and V H domains. 17,18 However, these bsAbs have a relatively short half-life in vivo compared with IgG, because of their smaller size and lack of an Fc fragment.…”

Section: Introductionmentioning

confidence: 99%

Bispecific anti-CD20/22 antibodies inhibit B-cell lymphoma proliferation by a unique mechanism of action

Goldenberg

Cardillo

et al. 2008

Blood

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Combination immunotherapy with anti-CD20 and anti-CD22 mAbs shows promising activity in non-Hodgkin lymphoma. Therefore, bispecific mAbs (bsAbs) were recombinantly constructed from veltuzumab (humanized anti-CD20) and epratuzumab (humanized anti-CD22) and evaluated in vitro and in vivo. While none of the parental mAbs alone or mixed had notable antiproliferative activity against Burkitt lymphoma cells when not crosslinked, the bsAbs [eg, anti-CD20 IgG-anti-CD22 (scFv) 2 ] were inhibitory without cross-linking and synergistic with B-cell antigen (BCR)-mediated inhibition. The bsAbs demonstrated higher antibodydependent cellulary cytoxicity (ADCC) activity than the parental mAbs, but not complement-dependent cytoxicity (CDC) of the parental CD20 mAb. Cross-linking both CD20 and CD22 with the bsAbs resulted in the prominent redistribution of not only CD20 but also CD22 and BCR into lipid rafts. Surprisingly, appreciable translocation of CD22 into lipid rafts was also observed after treatment with epratuzumab. Finally, the bsAbs inhibited Daudi lymphoma transplant growth, but showed a significant advantage over the parental anti-CD20 mAb only at the highest dose tested. These results suggest that recombinantly fused, complementary, bispecific, anti-CD20/22 antibodies exhibit functional features distinct from their parental antibodies, perhaps representing new candidate therapeutic molecules. IntroductionCD20 is the principal target in the immunotherapy of B-cell lymphomas, 1,2 while a mAb against CD22, epratuzumab, also has promising activity as a single agent. [3][4][5] Despite encouraging results with these mAbs, there are ongoing efforts to improve immunotherapy, because durable responses are only achieved in a portion of patients. 5 One emerging approach is combination therapy with different biologic agents, 6,7 such as 2 mAbs directed against distinct cell-surface antigens. Ideally, the 2 mAbs would have distinct mechanisms of action so that the therapeutic outcome would be additive or synergistic, and the combined antitumor effects would mitigate resistance to either of the mAbs.In vitro cell-based studies have shown that the combination of epratuzumab and rituximab has a greater antiproliferative effect than either mAb alone. 8 The results of 3 phase 2 clinical trials showed that epratuzumab combined with rituximab was well tolerated and suggested an improved activity in non-Hodgkin lymphoma (NHL), compared with historical reports of rituximab in similar patients. 9-11 However, combination mAb therapy involves sequential administration, thus requiring lengthy infusion times and potentially higher costs. The objectives of this study were to generate a single agent, namely a bsAb that targets both CD20 and CD22 antigens, and to evaluate its properties.A bsAb can be prepared by chemical conjugation, and this was used to prepare a bsAb Fab'x Fab' from hA20 (veltuzumab), a humanized anti-CD20 mAb, 12 and hLL2 (epratuzumab), a humanized anti-CD22 mAb, 13 in earlier studies. While chemical linkage to prepare bsAbs ...

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Improved Bivalent Miniantibodies, with Identical Avidity as Whole Antibodies, Produced by High Cell Density Fermentation of Escherichia coli

Cited by 67 publications

References 31 publications

Structural Insights into Steroid Hormone Binding

Structural Insights into Steroid Hormone Binding

Prolonged in vivo tumour retention of a human diabody targeting the extracellular domain of human HER2/neu

Bispecific anti-CD20/22 antibodies inhibit B-cell lymphoma proliferation by a unique mechanism of action

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