TABLE 3.
New variants to improve the cognate LC-HC pairings by negative design principle
Hetero-IgG1 variants in the absence of Fn3 and Fn3-FLAG-His6 tags were produced by transient transfection of 2936E cells. The tolerance of LC-HC pairings were assessed by Western blotting. Most variants take the negative design principle by introducing opposite charged residues at the same positions of opposing chain. Asterisk on V02 indicates the disulfide bond instead of charge pairs was introduced in the respective antibody.
| Variant | Anti-HER2 humAb4D5–8 HC1 (K392D + K409D) |
Anti-HER2 humAb4D5–8 LC1 |
Anti-EGFR E7.6.3 HC2 (E356K + D399K) |
Anti-EGFR 7.6.3 LC2 |
||||
|---|---|---|---|---|---|---|---|---|
| VH1 | CH1 | VL1 | CL | VH2 | CH1 | VL2 | CL | |
| V01 | Q39K + Q105K | K147D | Q38D + A43D | T180K | G44D + Q105D | K147K | G100K + A43K | S131D |
| V02* | Q39K + Q105C | K147D | Q38D + A43C | T180K | G44C + Q105D | K147K | G100C + A43K | S131D |
| V03 | Q39K + Q105K | P171D | Q38D + A43D | S162K | G44D + Q105D | A141K | G100K + A43K | F116D |
| V04 | Q39K+Q105K | P171D | Q38D + A43D | S162K | Q105D | A141K | A43K | F116D |
| V05 | Q39K + Q105K | P171D | Q38D + A43D | S162K | Q39D + Q105D | A141K | Q38K + A43K | F116D |
| V06 | Q39K + Q105K | K147D | Q38D + A43D | T180K | G44D + Q105D | G100K + A43K | ||
| V07 | Q39K | P171D | Q38D | S162K | Q39D | A141K | Q38K | F116D |
| V08 | Q39K | A141D | Q38D | F116K | Q39D | P171K | Q38K | S162D |
| V09 | Q39K | A141D | Q38D | F116K | Q39D | A141K | Q38K | F116D |
| V10 | Q39K | P171D | Q38D | S162K | Q39D | P171K | Q38K | S162D |
| V11 | Q105K | A141D | A43D | F116K | Q105D | A141K | A43K | F116D |
| V12 | Q105K | P171D | A43D | S162K | Q105D | P171K | A43K | S162D |
| V13 | Q105K | A141D | A43D | F116K | Q105D | P171K | A43K | S162D |
| V14 | Q105K | P171D | A43D | S162K | Q105D | A141K | A43K | F116D |
| V15 | Q39K | S183D | Q38D | S176K | Q39D | S183K | Q38K | S176D |
| V16 | Q105K | S183D | A43D | S176K | Q105D | S183K | A43K | S176D |
| V17 | Q39K | S183D | Q38D | S176K | Q105D | S183K | A43K | S176D |
| V18 | Q105K | S183D | A43D | S176K | Q39D | S183K | Q38K | S176D |
| V19 | Q39K | A141D + S183K | Q38D | F116K + S176D | Q39D | A141K + S183D | Q38K | F116D + S176K |
| V20 | Q39K | A141D + P171K | Q38D | F116K + S162D | Q39D | A141K + P171D | Q38K | F116D + S162K |
| V21 | Q39K+Q105K | K147D | Q38D + A43D | T180K | Q39D + Q105D | K147K | Q38K + A43K | T180D |
| V22 | Q39K+Q105K | K147D | Q38D + A43D | T180K | Q39D + Q105D | K147K | Q38K + A43K | S131D |
| V23 | Q39K+Q105K | S183D | Q38D + A43D | S176K | Q39D + Q105D | S183K | Q38K + A43K | S176D |
| V24 | Q39K+Q105K | P171D | Q38D + A43D | S162K | Q39D + Q105D | P171K | Q38K + A43K | S162D |
| V25 | G44K + Q105K | S183D | Q100D + A43D | S176K | G44D + Q105D | S183K | G100K + A43K | S176D |