The 3′-primer was also designed to add a V5-tag and a stop codon. The Fab HC constructs were prepared by PCR using primers complementary to the 5′ sequence and to the junction between the C H1 domain and the hinge region. Additional mutations introduced by site-directed mutagenesis included C127S in the A33 Fab fragment C H1 domain and P151A in the A33 HC C H1 domain as indicated. –ESKYGPPCPSCP– to –ESKYGPPCPPCP–), which reduces the amount of HC : LC dimers formed. All IgG4 HCs contain an S225P mutation (i.e. Variable domains were synthesised by DNA 2.0. The variable domain from an in-house humanised mouse antibody (277) was also used. All HCs contained either the human IgG1 or IgG4 C H1,2,3 domains fused to either mouse or humanised V H domains with specificity for A33, TNF-α, CD20, CD25 (Zenapax), CD25 (Simulect) and CD11a. CHO-S suspension cells (Life Technologies) were grown in CD CHO chemically defined medium. However, the fidelity of this retention has not been analysed for engineered antibodies as it has been assumed that the retention is efficient, at least during the expression of commonly expressed IgG formats.ĬHO adherent cells (CHO) L761H were grown in Dulbecco's Modified Eagle Medium (DMEM) containing 10% foetal calf serum. The quality control system also functions in Chinese hamster ovary (CHO) cells used for commercial antibody production with BiP retaining unassembled HC dimers. This cellular mechanism ensures that only correctly folded and assembled molecules are secreted from an antibody-producing cell. This interaction is reversed upon LC assembly with the C L domain acting as a template to allow correct folding of the C H1 domain. The HC dimer is retained due to a lack of folding of the C H1 domain in the absence of the cognate LC, resulting in a high-affinity interaction with the intracellular chaperone, immunoglobulin HC-binding protein (BiP). Crucially, heavy chains (HCs) are retained in the cell as dimeric intermediates until two light chains (LCs) assemble with the HC dimer to form a tetramer, which is then released from this retention and secreted from the cell. The correct folding and assembly of antibodies requires an elegant cellular quality control process. This discovery will have an impact on the efficient production of both humanised antibodies and the design of novel antibody formats. This subversion of the normal quality control process is dependent on the HC variable domain, is prevalent with engineered antibodies and can occur when only the Fab fragments are expressed. We show that the secreted heavy-chain dimers contain folded C H1 domains and contribute to the heterogeneity of antibody species secreted during the expression of therapeutic antibodies. This domain acts as a switch for secretion, only folding upon interaction with the LC C L domain.

Quality control of recombinant antibody assembly in vivo is thought to rely upon folding of the HC C H1 domain. Here, we show that some humanised heavy chains (HCs) can fold, form dimers and be secreted even in the absence of a light chain (LC). This process involves the creation of molecules, which retain the specificity of the rodent antibody but contain predominantly human coding sequence.

Rodent monoclonal antibodies with specificity towards important biological targets are developed for therapeutic use by a process of humanisation.

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