Proteases Associated With Gynecological Tumors

Ruppert, Clemens; Ehrenforth, S.; Tutschek, B.; Vering, A.; Mw, Beckmann; Scharrer, I.; Bender, H. G.

doi:10.3892/ijo.4.3.717

Cited by 2 publications

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“…The introduction of sequences into the connector which are recognized by MMP2/9 or uPA provides the option to produce bispecific dsFv-containing entities whose second binding entity is inactive until it encounters these proteases. MMPs and uPA are expressed in some disease tissues, for example in the environment of tumors or inflamed tissues ( Liotta et al ., 1980 ; Stetler-Stevenson, 1994 ; Ruppert et al ., 1997 ; van't Veer et al ., 2002 ; Minn et al ., 2005 ; Cudic and Fields, 2009 ; Bao et al ., 2010 ; Mukhopadhyay et al ., 2010 ; Scott and Taggart, 2010 ). The sequences GPLGMLSQ, GPLGLWAQ and GPLGIAGQ are substrates for MMP2 and MMP9 ( Netzel-Arnett et al ., 1991 , 1993 ), and the peptide sequence GGGRR has been shown to be a substrate for uPA ( Chung and Kratz, 2006 ).…”

Section: Resultsmentioning

confidence: 99%

Bispecific antibody derivatives with restricted binding functionalities that are activated by proteolytic processing

Metz¹,

Panke

Haas³

et al. 2012

Protein Engineering Design and Selection

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We have designed bispecific antibodies that bind one target (anti-Her3) in a bivalent IgG-like manner and contain one additional binding entity (anti-cMet) composed of one VH and one VL domain connected by a disulfide bond. The molecules are assembled by fusing a VH,Cys44 domain via flexible connector peptides to the C-terminus of one H-chain (heavy chain), and a VL,Cys100 to another H-chain. To ensure heterodimerization during expression in mammalian cells, we introduced complementary knobs-into-holes mutations into the different H-chains. The IgG-shaped trivalent molecules carry as third binding entity one disulfide-stabilized Fv (dsFv) without a linker between VH and VL. Tethering the VH and VL domains at the C-terminus of the CH3 domain decreases the on-rates of the dsFv to target antigens without affecting off-rates. Steric hindrance resolves upon removal of one side of the double connection by proteolysis: this improves flexibility and accessibility of the dsFv and fully restores antigen access and affinity. This technology has multiple applications: (i) in cases where single-chain linkers are not desired, dsFvs without linkers can be generated by addition of furin site(s) in the connector that are processed during expression within mammalian cells; (ii) highly active (toxic) entities which affect expression can be produced as inactive dsFvs and subsequently be activated (e.g. via PreScission cleavage) during purification; (iii) entities can be generated which are targeted by the unrestricted binding entity and can be activated by proteases in target tissues. For example, Her3-binding molecules containing linkers with recognition sequences for matrix metalloproteases or urokinase, whose inactivated cMet binding site is activated by proteolytic processing.

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Section: Resultsmentioning

confidence: 99%

Bispecific antibody derivatives with restricted binding functionalities that are activated by proteolytic processing

Metz¹,

Panke

Haas³

et al. 2012

Protein Engineering Design and Selection

View full text Add to dashboard Cite

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Noorden

Jonges²,

Marle

et al. 1997

Clinical and Experimental Metastasis

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Metastatic rat colon cancer cells but not normal rat hepatocytes showed activity of cathepsin B on their plasma membranes. Activity was visualized in living cells with a new fluorogenic substrate, [Z-Arg]2-cresyl violet, and confocal microscopy. When these cancer cells were injected into the portal vein of rats, the animals developed tumors in the liver in a heterogeneous fashion. Three- to four-fold more tumors were found in the small caudate lobe than in the other three large lobes of the liver. Oral treatment with a selective water-soluble inhibitor of extracellular cathepsin B, Mu-Phe-homoPhe-fluoromethylketone, resulted in 60% reduction of the number of tumors and 80% reduction of the volume of tumors in the three large lobes whereas tumor development was not affected in the small caudate lobe. This study supports the conclusions that (a) extracellular cathepsin B plays a crucial but complex role in liver colonisation by rat colon carcinoma cells in vivo, (b) its selective inhibition suppresses tumor growth heterogeneously in the liver and (c) the caudate lobe of the liver is a relatively large risk factor for tumor development.

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Proteases Associated With Gynecological Tumors

Cited by 2 publications

References 0 publications

Bispecific antibody derivatives with restricted binding functionalities that are activated by proteolytic processing

Bispecific antibody derivatives with restricted binding functionalities that are activated by proteolytic processing

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