An improved linker for single-chain Fv with reduced aggregation and enhanced proteolytic stability

Abstract

The effects of linker length on binding affinity and degree of aggregation have been examined in the antifluorescein 4-4-20 and anticarcinoma CC49 single-chain Fvs. Longer linkers in the antifluorescein sFvs have higher affinities for fluorescein and aggregate less. A proteolytically susceptible site between Lys8 and Ser9, in the previously reported 212 linker has been identified. A new linker sequence, 218 (GSTSGSGKPGSGEGSTKG) was designed in which a proline was placed at the C-terminal side of the proteolytic clip site in the 212 linker. The CC49 sFv containing the 218 linker showed reduced aggregation and was found to be more stable to proteolysis in vitro, when compared to the CC49/212 sFv. The CC49 sFv with the longer 218 linker had higher affinity than CC49/212 sFv. An aggregated CC49/212 sFv sample had higher affinity than CC49/218 sFv. The CC49/218 and CC49/212 sFvs had similar blood clearances in mice, while the aggregated CC49/212 sFv remained in circulation significantly longer. In mice bearing LS-174T human colon carcinoma xenografts, the CC49/218 sFv showed higher tumor uptake than the CC49/212 sFv and lower tumor uptake than the aggregated CC49/212 sFv. The higher tumor uptake of the CC49/218 is most likely a result of its higher resistance to proteolysis. The higher affinity and higher tumor uptake of the aggregated CC49/212 sFv are most likely due to the repetitive nature of the TAG-72 antigen and the higher avidity of multivalent aggregates. When the sFvs were radiolabeled with a lutetium-chelate the CC49/218 sFv showed a lower accumulation in the liver and spleen compared to the aggregated CC49/212 sFv.

Production of bispecific antibodies in “knobs-into-holes” using a cell-free expression system

“…For example, diL2K scFv is constructed as V H -V L and 5-10xdiL2K BiTE as 5-10(V L -V H )xdiL2K(V H -V L ). However, other factors should also be taken into consideration during novel KIH design such as 1) the orientation within the scFv (V L -V H vs. V H -V L ), 59,60 2) the order of 2 scFvs on BiTE construct, 61,62 and 3) the length and composition of the linkers (between 2 scFv as well as between V L and V H ), 63,64 all of which likely contribute to KIH folding and assembly.…”

Human natural killer cell line modified with a chimeric immunoglobulin T-cell receptor gene leads to tumor growth inhibition in vivo

“…The V L region was amplified from pUC19 -LC431 by polymerase chain reaction (PCR ) using primer BW431-V L -1A (5 0 -CGAGTCTAGA ACCATGGACA TCCAGATGACC-3 0 ) and BW431 -V L -2 (5 0 -CACCAC-TCCC GGGCTTTCCT GAACCGGAAG TGGATCC-TTT GATTTC CACC TTGGTCCCTT G -3 0 ) and the V H region was amplified from pAB -431/ 26-V H -humÁ3 25,26 using primer BW431 -V H -1 (5 0 -AAGCCCGGGA GTG-GTGAAGG TAGCACTAAA GGCCAGGTCC AGCTG-CAGGA GAGC-3 0 ) and BW431-V H -2 ( 5 0 -TTGGATCCGG CCGCACCTGA GGAGACGGTG ACCGT-3 0 ). The V L fragment digested with NcoI and SmaI and the V H fragment digested with SmaI and NotI (restriction enzymes from New England Biolabs, Frankfurt am Main, Germany ) were cloned into the NcoI and NotI sites of the E. coli periplasmatic expression vector pOPE51 33 (kindly provided by S Dübel, Molecular Genetics, University Heidelberg, Germany ) to obtain the scFv scBW431/26 consisting of the N terminal V L region followed by the Linker-218 ( -GSTSGSGKPGSGEGSTKG -) 4 and the V H region (pOPE51 -scBW431 /26).…”

“…Horseradish peroxidase -conjugated goat anti -human -IgG -Fc F( ab 0 ) 2 (50 L /well; Dianova, Hamburg, Germany ) was used diluted 1:1000 in blocking buffer as secondary antibody and incubated for 1 hour at room temperature. The enzyme reaction was performed using 100 L /well 2 mg / mL o -phenyldiamine (Sigma ) in citrate -phosphate buffer (10 g /L citric acid, 14 g /L Na 2 HPO 4 [Serva ], pH5.0; add 20 L 30% H 2 O 2 [ Sigma ] per 100 mL before use ). The reaction was stopped by adding 50 L / well 3 N NaOH ( Roth, Karlsruhe, Germany ).…”

“…Immunoglobulin -based chimeric T-cell receptors (cIgTCRs ), also called ''T bodies'', 2 are from particular interest. They consist of a single-chain Fv (scFv ) fragment derived from the variable ( V ) domains of the light (V L ) and heavy chain ( V H ) of monoclonal antibodies ( mAb ) joined by a flexible peptide linker 3,4 fused to a signal-transducing chain like the chain of the Fc"RI receptor or the CD3 chain; thus, they combine the specificity of an antibody -based MHC -unrestricted recognition with the T-cell function. 2,5 The development of efficient retroviral gene delivery systems for activated primary T cells and tumor-infiltrating lymphocytes ( TILs ) 6,7 permitted the possibility to generate large numbers of virus - 8 or tumor-specific T cells.…”

Method of Detecting and Targeting Mutations in Cancer

“…This is an area with much potential room for optimization, and may differ based on the separation of the two DNA binding sites. However, in general flexible linkers consisting of serine, glycine, and threonine have been shown to work well and are an appropriate starting point for investigation [30,31].…”