Bispecific antibodies comprising an antigen-binding site binding to LAG3
A multispecific antibody, bispecific technology for engineered immunoglobulin domains to address immune exhaustion, tumor escape, infection and increased mortality
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example 1
[0241] Generation of a Universal Autonomous Human Heavy Chain Variable Domain (aVH) Library
[0242] Based on the sequence B1ab (a Herceptin derived template) to generate a universal aVH library. In B1ab, four (4) hydrophobic residues that became surface exposed in the absence of the light chain interface were replaced by more hydrophilic residues identified by phage display. These mutations were found to be compatible with structures having a VH domain fold. They increase the hydrophilicity and thus the stability of the scaffold and allow the expression of stable and soluble aVH domains in the absence of light chain partners ( figure 1 A).
[0243] To generate an aVH phage display library based on the B1ab sequence and randomized in the CDR3 region, 2 fragments were assembled by "Splitting by Overlap Extension" (SOE) PCR. Fragment 1 comprises the 5' end of the aVH encoding gene comprising framework 3, while fragment 2 comprises the end of framework 3, framework 4 of the a...
example 2
[0251] Identification of aVH domains containing stabilized disulfide bridges
[0252] To further stabilize the aVH scaffold, the introduction of additional disulfide bridges that constrain the flexibility of the protein chains was tested. Positions that allow disulfide bridge formation when mutated to cysteines were identified by 1) structural modeling, or 2) searching for Ig-like V-type sequences with additional stabilizing disulfide bonds in nature.
[0253] In the first approach, the crystal structure of the molecule with the closest structural homology to the aVH used was identified. (www.pdb.org, entry ID 3B9V). Using a computer algorithm, a Cα / Cα pair with a distance of less than 63 pairs of amino acids. From this 63 pairs, amino acid pairs that had a strong effect on core packing or clearly violated Cβ / Cβ geometry were excluded. Therefore, 8 pairs of different residues were selected.
[0254] In the second approach, manual database screening was performed to ident...
example 3
[0259] Novel library template for generation of a stabilized universal autonomous human heavy chain variable domain (aVH) library
[0260] Based on SEQ ID NO:30 and SEQ ID NO:37, a new aVH library template was designed for generating aVH library with higher stability. The following optional modifications were made in the template sequence: (1) The mutation K94S was introduced. (2) Introduce the mutation L108T, which is a common sequence variant found in antibody J elements. However, the above mutations had no specific effect. image 3 An overview of all library templates is given in .
[0261] Generation of a new universal autonomous human heavy chain variable domain (aVH) library with stabilized disulfide bridge 52a / 71
[0262] To generate new aVH libraries based on additional stabilizing disulfide bridges at positions 52a and 71, four new templates were designed (SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO: 45). Three of the four templates had additional sequenc...
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