Table 1.
Strategies to promote bispecificity by heterodimer formation.
| Strategy/format | Mutation | Target | Bispecificity analysis/yield (%) | Protein expression/purification/yield (g/L) | Remarks | Reference |
|---|---|---|---|---|---|---|
| Quadroma | NA | EpCAM × CD3, HER3 × CD3, CD20 × CD3 | Ion-exchange chromatography and SEC/12.5% | Hybridoma/protein A | Associated human anti-mouse antibody response | (26, 46) |
| Knobs-in-holes | T366W/T366Y (knobs) and T366S/L368A/T394W/F405A/Y407V(T) (holes) | CD3 × CD4, c-MPL × HER3, VEGF-A × Ang2, CD20 × hL243γ1, EGFR × IGF1R, HER3 × cMET, CD3 × EpCAM, CD3 × HER2, EGFR × HER2, CD4 × CD70, MET × EGFR | Electroblotting, SLD, SEC, MS-TOF (73–100%) | HEK293, Escherichia coli, and cell-free expression system/protein A, DEAE, and Mab Select Sure/0.004–1.0 g/L | Faulty light and heavy chain pairing | (19, 27–29, 47–53) |
| Biochemical optimization | S364H/F405A (CH3A) andY349T/T394F (CH3B) | CD16 × HER2 and CD3 × HER2 | HPLC/SEC (89%) | HEK293F/protein A, nickel affinity chromatography | (54) | |
| Biochemical optimization | L368E/K409R (CH3), R221E/R228E (IgG1-hinge), and R223E/R225E/R228E (IgG2-hinge) | CD3 × CD20, EGFR × ErbB2 | Ion-exchange chromatography and LCMS (65–100%) | HEK293/protein A/in vitro cell-free assembly | Cognate chain pairing | (55) |
| Biochemical optimization | P228S (IgG1-hinge) and F405L/K409R (CH3) | CD20 × EGFR | ESI-MS (95.7%) | HEK293/protein A/SEC/473.4 g/L | High yield and efficiency | (56) |
| Biochemical optimization | H435R and Y436F (IgG1-CH3) | CD20 × CD3 | SEC (100%) | Stable CHO-K1/protein A/0.2–0.3 g/L | Cognate chain pairing | (57, 58) |
| Biochemical optimization | S354C (CH3A), Y394C (CH3B), F126C (CH1), S121C (LC), C44 (VH), and C100 (VL) | EGFR × IGF1R, CD20 × hL243γ1, HER3 × cMET | MS-TOF, SEC (73%) | HEK293/protein A/0.004–0.03 g/L | Prevent homodimer formation | (49, 52, 53) |
| Biochemical optimization | F241R, F243S, F241S, F243R (CH2) and C226S, C229S (hinge) | NA | MS-TOF (90%) | E. coli/protein A | Avoid covalent bonding of heterodimers | (50) |
| Electrostatic optimization | K409D (CH3A), D399R (CH3B), K409E (CH3A), D399K (CH3B), and K409E (CH3A), D399R (CH3B) | CD3 × TARTK | LC/MS (98%) | HEK293/Select Sure column and SEC | Prevent homodimer formation | (59) |
| Electrostatic optimization | K409W, K360E, K370E (CH3A) and D399V, F405T, Q347R, E357N, S364B (CH3B) | VEGFR-2 × MET | SEC (80–90%) | HEK293/protein A | Prevent homodimer formation | (60, 61) |
| Electrostatic optimization | Q39K, Q105K (VH), S183D (CH1), Q38D (VL), S176D (CL), K392D, K409D (CH3A), and E356K, D399K (CH3B) | HER2 × EGFR | SEC (100%) | Stable CHO/protein A/0.2–0.3 g/L | Cognate chain pairing | (57) |
| Electrostatic optimization | T350V, L351Y, F405A, Y407V (CH3A) and T350V, T366L, K393L, T394W (CH3B) | HER2 × ErbB2 | SEC (95%) | CHO/protein A/0.25 g/L | Improved biophysical properties | (62) |
| κλ-body | NA | CD19 × CD47, CD47 × EpCAM | Isoelectric focusing/41.5% | PEAK cells/protein A, CaptureSelect immunoglobulin gamma (IgG)-CH1, KappaSelect, and LambdaFabSelect affinity chromatography/1.5 g/L | Exploits variable light chains for generating bispecifics | (63) |
| Bispecific T-cell engagers | NA | CD3 × 17-1A, CD3 × CD19 | Cytofluorometry, ELISA, and SDS-PAGE | CHO/nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography | Recruitment of T-cells via CD3 ligation | (64, 65) |
| Diabody | NA | HEL × phOx | FPLC | E. coli/affinity chromatography/0.3–1.0 mg/L | Easy construction and expression in bacteria | (64–66) |
| Tandem diabody | NA | CD16 × CD30, CD3 × CD19 | SEC | E. coli/immobilized-metal chelating chromatography (IMAC)/0.48–0.6 mg/L | Increased valence, stability and activity | (66–68) |
| Dual-affinity-retargeting | NA | CD16 × CD32B | SEC | HEK293, CHO-S/affinity chromatography | Increased valence and affinity | (67–69) |
| Bispecific/trispecific killer cell engager | NA | CD16xCD19, CD16xCD19xCD22 | SEC | E. coli/ion-exchange chromatography | Natural killer cell activation | (69–72) |
CH3A and CH3B mean that these mutations are located on the partner chain.
Abbreviations: SLD, scanning laser densitometry; SEC, size exclusion chromatography; MS-TOF, time-of-flight mass spectrometry; FPLC, fast protein liquid chromatography; LCMS, liquid chromatography–mass spectrometry; ESI-MS, electrospray ionization mass spectrometry; DEAE, diethylaminoethanol ion-exchange resin; NA, information not available.