Ensuring correct chain pairing between each heavy chain (HC) and its intended light chain (LC) is a major challenge when creating multispecific antibodies. Incorrect pairing reduces product purity and complicates downstream processing. To address this, Adimab scientists designed and validated orthogonal CH1-Cκ interfaces that drive correct HC–LC pairing in bi- and multispecific antibody formats. 

Approach and outcomes 

• CH1-Cκ interface designs were generated computationally, and candidates with the greatest computed ΔΔG were tested experimentally across five bispecific antibody pairs. Two orthogonal sets of mutations were identified: Set 1 with three CH1 and three Cκ mutations, and Set 2 with two CH1 and three Cκ mutations. 

• When put on opposite Fab arms, Set 1 and Set 2 drive 100% correct HC–LC pairing, as measured by LC-MS on ProA-purified material. These results are confirmed by analytical ion exchange chromatography. Importantly, the CH1-Cκ mutations do not alter target binding or biophysical properties. 

• CH3 heterodimerization mutations enables a chain exchange approach for rapid production of large multispecific panels. The bispecific output retention times match the average of the two input antibodies, confirming correct assembly.  

• In the past two years, Adimab has delivered more than 1,200 samples across more than 20 formats to partners, with screening formats leading directly to production in single transient transfections. More than 100 multispecific antibodies incorporating CH1-Cκ mutations have been delivered across 10 programs for 8 partners, demonstrating the broad applicability of the CH1-Cκ interface designs across partner programs. 

Clinical translation 

This CH1-Cκ heterodimerization technology has been applied in clinical-stage programs. Partner Biotheus advanced PM-1080, an EGFR × cMET bispecific antibody incorporating this technology, into Phase III trials for colorectal cancer and non-small cell lung cancer. PM-1080 achieved a stable cell line titer of 6 g/L, high purity after ProA column purification, and no HC–LC mispair. 

Why it matters 

Adimab’s computationally designed CH1-Cκ heterodimerization solution provides a robust, clinically validated approach to correct HC–LC pairing in multispecific antibody development, enabling high-purity manufacturing without compromising antibody biophysical properties or target binding.

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