Multi-specific proteins
A protein-specific technology, applied in the field of multi-specific proteins
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Embodiment 1
[0308] Example 1 - Design of Multispecific Binding Proteins
[0309] Generation of various forms of multispecific binding proteins (see Figure 5A and Figure 5B ), and their ability to enhance NF-κB activation in h4-1BB-HT1080 reporter cells was assessed.
[0310] NF-κB reporter cells (h4-1BB-HT1080) transfected with human 4-1BB were used to assess the presence of the FAP binding domain shown in SEQ ID NO: 2 and SEQ ID NO: 2 in the presence of CHO cells expressing human FAP. Functional activity of various multispecific binding proteins from one to three 4-1BB binding domains shown in 3.
[0311] In vitro NF-κB activation assay in the presence of CHO cells expressing human FAP: HT1080 human fibrosarcoma cells were stably transfected with cDNA encoding full-length human 4-1BB and the pNIFTY-Lucia NF-κB reporter gene as follows . In the same manner, CHO cells were stably transfected with cDNA encoding human FAP. Using a 96-well plate, plate 40,000 h4-1BB-HT1080 reporter c...
Embodiment 2
[0314] Example 2 - Binding affinity of multispecific molecules
[0315] The following examples describe experiments performed to determine the species cross-reactivity of different domains in the multispecific binding protein A (MpA) comprising SEQ ID NO:6. The interaction of MpA with serum albumin, 4-1BB and FAP was analyzed for three species (human, cynomolgus monkey and mouse).
[0316] Materials and methods
[0317] ProteOn setup for binding of MpA to 4-1BB of different species: SPR measurements were performed using a ProteOn XPR36 instrument (BioRad). The running buffer contains PBS pH7.4 (PBST). bio.h4-1BB, bio.c4-1BB and bio.m4-1BB were immobilized on NLC chips (BioRad) to a level of 320RU. Binding of MpA to 4-1BB was measured by injecting MpA at serial dilutions of 30 nM, 10 nM, 3.3 nM, 1.1 nM and 0.3 nM with 180 s of association and 1800 s of dissociation using a constant flow rate of 100 μl / min (see table 3). Measurements were repeated three times and 10 m...
Embodiment 3
[0338] Example 3 - Analysis of Simultaneous Binding of MpA to 4-1BB, FAP and Human Serum Albumin by Surface Plasmon Resonance combine
[0339] The following experiment describes the performance of a surface plasmon resonance experiment analyzing the simultaneous binding of a multispecific binding protein A (MpA) comprising SEQ ID NO: 6 to human 4-1BB, human FAP and human serum albumin, respectively.
[0340] The analysis was performed using the following setup: Biotinylated human 4-1BB was immobilized on the surface of a neutralizing avidin-coated NLC chip before starting the binding measurements. In the first step, MpA is added, which has a slow off-rate of 4-1BB. Second, hFAP was applied as a secondary target, and then HSA was added as a tertiary and final target.
[0341] SPR measurements were performed using a ProteOn XPR36 instrument (BioRad). PBS pH 7.4 containing 0.005% Tween 20 was used as running buffer. 360 RU of 1OnM biotinylated human 4-1BB (bio.h4-1BB-Fc) w...
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