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Recombinant FAP binding proteins and uses thereof

A technology for binding proteins and ankyrins, which can be applied in drug combinations, immunoglobulins, peptide/protein components, etc., and can solve problems such as incomplete understanding of potential effects, complex effects, and incomplete understanding

Pending Publication Date: 2022-04-01
MOLECULAR PARTNERS AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The role of FAP in tumor biology is complex and not fully understood, and thus the potential effects of FAP inhibition in tumor biology are also not fully understood

Method used

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  • Recombinant FAP binding proteins and uses thereof
  • Recombinant FAP binding proteins and uses thereof
  • Recombinant FAP binding proteins and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0236] Example 1: Selection of binding proteins comprising an ankyrin repeat domain with binding specificity for FAP

[0237] Using ribosome display (Hanes, J. and Plückthun, A., PNAS 94, 4937-42, 1997), similarly from A number of ankyrin repeat proteins with binding specificity for human FAP (hFAP) were selected from the library. Binding of selected clones to recombinant human FAP targets was assessed by homogeneous time-resolved fluorescence (HTRF) of crude extracts, demonstrating the successful selection of hundreds of hFAP-specific binding proteins. For example, the ankyrin repeat domain of SEQ ID NO: 1 to 33 constitutes the amino acid sequence of a selected binding protein comprising an ankyrin repeat domain with binding specificity for hFAP. Individual ankyrin repeat modules from such ankyrin repeat domains with binding specificity for hFAP are provided in SEQ ID NOs: 48 to 134.

[0238] Selection of FAP-specific ankyrin repeat proteins by ribosome display

[023...

Embodiment 2

[0293] Example 2: Determination of Ankyrins with Binding Specificity for FAP by Surface Plasmon Resonance (SPR) Analysis The dissociation constant (K D )

[0294] The binding affinity of the purified ankyrin repeat protein to the human FAP target was analyzed using the ProteOn instrument (BioRad) and measured according to standard procedures known to those skilled in the art.

[0295] Briefly, human FAP was diluted in 10 mM sodium acetate pH 5.3 buffer and covalently immobilized on a GLC chip (BioRad) to a level of approximately 2000 resonance units (RU). Then by injecting 200 μl of running buffer (PBS, pH 7.4, containing 0.005% Tween ) (on-rate measurement) followed by injection of running buffer flow at a constant flow rate of 100 μl / min for at least 25 minutes (off-rate measurement) to measure the interaction of ankyrin repeat protein and hFAP. Use 15 μl of 10 mM glycine (pH 2), followed by 15 μl of 124 mM H 3 P0 4 to regenerate. The signal (i.e., RU value) betwee...

Embodiment 3

[0300] Example 3: Determination of FAP-specific ankyrin repeat protein binding on FAP+ cells by FACS

[0301] Binding of purified FAP-specific ankyrin repeat protein to FAP-expressing cells was analyzed by FACS.

[0302] Using a multi-well plate, add 20,000 WI38 cells per well in 50 μl PBS. 50 μl of appropriate dilutions of ankyrin repeat protein (2x dilution steps) were then added to the cells and incubated on ice for 30 minutes. After the ankyrin repeat protein binding reaction, the cells were washed twice with PBS. Direct immunofluorescence detection of His-tagged ankyrin repeat protein using Penta-His AF647 conjugate (QIAgen) and direct immunofluorescence detection using LIVE / DEAD were then performed together in one step. TM Live cell assay with Fixable Green Dead Cell Stain Kit (ThermoFisher). Combine Penta-His AF647 conjugate with LIVE / DEAD TM Fixable green dye was co-applied to cells at 1 / 200 (Penta-His) and 1 / 1000 (L / D Fixable) final dilutions and incubated on ic...

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Abstract

The present invention relates to recombinant binding proteins comprising a designed ankyrin repeat domain having binding specificity for fibroblast activation protein (FAP). Furthermore, the invention relates to nucleic acids encoding such binding proteins, pharmaceutical compositions comprising such binding proteins or nucleic acids and such binding proteins, use of nucleic acids or pharmaceutical compositions in methods for localization or delivery of bioactive molecules in FAP-expressing tissues, such as tumor tissues, and for treatment, diagnosis or imaging of diseases, such as cancer, in mammals, including humans.

Description

[0001] Cross references to related patent applications [0002] This application claims the benefit and priority of European Patent Application EP19178277 filed with the European Patent Office on 04.06.2019. The content of European Patent Application EP19178277 is hereby incorporated by reference in its entirety, including all tables, figures and claims. technical field [0003] The present invention relates to recombinant binding proteins comprising a designer ankyrin repeat domain with binding specificity for fibroblast activation protein (FAP). Furthermore, the present invention relates to nucleic acids encoding such binding proteins, pharmaceutical compositions comprising such binding proteins or nucleic acids, and the use of such binding proteins, nucleic acids or pharmaceutical compositions for localization or Delivery of biomolecules or bioactive compounds and use in methods of treatment, diagnosis or imaging of diseases, such as cancer, in mammals, including humans. ...

Claims

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Application Information

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IPC IPC(8): A61K38/00
CPCC07K2319/00C07K2319/70A61K38/00C07K16/40C07K2318/20C07K14/435C07K2317/92C07K2317/73A61P35/00A61K2039/505A61K51/08A61K47/6871A61K51/1075C07K14/47C07K2317/74C07K2319/31
Inventor U·菲德勒C·梅茨M·R·穆勒D·斯内尔
Owner MOLECULAR PARTNERS AG
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