X-ray data and refinement statistics.

Human epidermal growth factor receptor-2 (HER2) is a well-recognised biomarker associated with 25% of breast cancers. In most cases, early detection and/or treatment correlates with an increased chance of survival. This study, has identified and characterised a highly specific anti-HER2 single-domain antibody (sdAb), NM-02, as a potential theranostic tool. Complete structural description by X-ray crystallography has revealed a non-overlapping epitope with current anti-HER2 antibodies. To reduce the immunogenicity risk, NM-02 underwent a humanisation process and retained wild type-like binding properties. To further de-risk the progression towards chemistry, manufacturing and control (CMC) we performed full developability profiling revealing favourable thermal and physical biochemical ‘drug-like’ properties. Finally, the application of the lead humanised NM-02 candidate (variant K) for HER2-specific imaging purposes was demonstrated using breast cancer HER2+/BT474 xenograft mice.</div

SPR sensorgrams for NM-02 (parent) and humanised variants (B, D and E).

SPR sensorgrams for NM-02 (parent) and humanised variants (B, D and E).</p

Co-crystal structure of NM-02 in complex with HER2-ECD.

(a) Composite figure of NM-02 (grey) in complex with HER2-ECD (green) overlaid with co-crystal structures comprising Trastuzumab (yellow; PDB ID 1N8Z) and Pertuzumab (orange; PDB ID 1S78). (b) Stick view of interface between NM-02 (grey) and HER2-ECD (green). (c) Residue contact map, highlighting the number of interactions made by CDR3. Structural figures rendered in PyMOL.</p

Biochemical characterisation of anti-HER2 sdAb, NM-02.

(a) Cross-reactivity testing of NM-02, demonstrating specific binding to recombinant human HER2-ECD (blue) but not recombinant murine HER2-ECD (red). (b) Cell surface adherence of NM-02 to HER2+ (BT474-Red and SKOV3-Orange) but not HER2- (MDA-MB-231-Blue) cell lines using flow cytometry. (c) and (d) Competition testing by surface plasmon resonance, showing that NM-02 (green) binds HER2-ECD following the sequential binding of either Trastuzumab (c) or Pertuzumab (d). Therefore, the NM-02 epitope is non-competitive with frontline therapeutics.</p

SPECT/CT imaging of humanised anti-HER2 sdAb in HER2+ breast cancer BT474 xenograft model.

(a) Representative SPECT/CT images of HER2+ BT474 xenograft mice injected with either [99mTc]-NM-02 or [99mTc] humanised NM-02 (variant K) 90-mins post injection. In both cases, this signal can be specifically ‘blocked’ by co-injection of excess unlabelled sdAb. Red arrows denote location of subcutaneous HER2 positive BT474 xenograft. (b) Histogram depicting signal intensity on a per organ basis. Specific labelling of the xenograft tumor is clearly detected above baseline (as determined by non-targeted/non-associated organs). Strong signals arising from the urinary tract (bladder and kidneys) are consistent with renal clearance, as expected for small (< 69 kDa) biologics.</p

SPR sensorgrams for NM-02 humanised variants (E-I).

SPR sensorgrams for NM-02 humanised variants (E-I).</p