Common light chains and methods of use
a light chain and common technology, applied in the field of bispecific antigen binding molecules, can solve the problems of igg-like formats, unable to activate the effector mechanism mediated by the fc domain, and suffer from the toxicity of the native effector functions inherent in igg molecules,
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example 1
Purification of Biotinylated Folate Receptor-Fc Fusions
[0284]To generate new antibodies against human FolR1 the following antigens and screening tools were generated as monovalent Fc fusion proteins (the extracellular domain of the antigen linked to the hinge region of Fc-knob which is co-expressed with an Fc-hole molecule). The antigen genes were synthesized (Geneart, Regensburg, Germany) based on sequences obtained from GenBank or SwissProt and inserted into expression vectors to generate fusion proteins with Fc-knob with a C-terminal Avi-tag for in vivo or in vitro biotinylation. In vivo biotinylation was achieved by co-expression of the bacterial birA gene encoding a bacterial biotin ligase during production. Expression of all genes was under control of a chimeric MPSV promoter on a plasmid containing an oriP element for stable maintenance of the plasmids in EBNA containing cell lines.
[0285]For preparation of the biotinylated monomeric antigen / Fc fusion molecules, exponentially ...
example 2
Generation of Common Light Chain with CD3ε Specificity
[0291]The T cell activating bispecific molecules described herein comprise at least one CD3 binding moiety. This moiety can be generated by immunizing laboratory animals, screening phage library or using known anti-CD3 antibodies. The common light chain with CD3c specificity was generated by humanizing the light chain of a murine parental anti-CD3c antibody (CH2527). For humanization of an antibody of non-human origin, the CDR residues from the non-human antibody (donor) have to be transplanted onto the framework of a human (acceptor) antibody. Generally, acceptor framework sequences are selected by aligning the sequence of the donor to a collection of potential acceptor sequences and choosing one that has either reasonable homology to the donor, or shows similar amino acids at some positions critical for structure and activity. In the present case, the search for the antibody acceptor framework was performed by aligning the mous...
example 3
SPR Assessment of Humanized Variants with CD3ε Specificity
[0293]Humanized VL variants were assessed as chimera in a 2+1 classical format (FIG. 1D), i.e. humanized light chain V-domains were paired with murine heavy chain V-domains. SPR assessment was carried out on a ProteOn XPR36 instrument (Bio-Rad). More precisely, the variants were captured directly from the culture supernatant on an anti-Fab derivatized GLM sensorchip (Goat Anti-Human IgG, F(ab′)2 Fragment Specific, Jackson ImmunoResearch) in vertical orientation. The following analytes were subsequently injected horizontally as single concentrations to assess binding to human and cynomolgus CD3ε: 3 μM hu CD3ε(−1-26)-Fc(knob)-avi (ID807) and 2.5 μM cy CD3ε-(−1-26)-Fc(knob)-Avi-Fc(hole) (ID873), respectively. Binding responses were qualitatively compared to binding of the murine control construct and graded+(comparable binding observed), + / − (reduced binding observed) and − (no binding observed). The capture antibody was regener...
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