Binding symmetry and surface flexibility mediate antibody self-association

(1)

Supporting Information

for

Binding symmetry and surface flexibility mediate antibody

self-association

Joseph D. Schrag

1,#

, Marie-Ève Picard

2,#

, Francis Gaudreault

1,#

, Louis-Patrick Gagnon

1

, Jason

Baardsnes

1

, Mahder S. Manenda

2

, Joey Sheff

3

, Christophe Deprez

1

, Cassio Baptista

1

, Hervé

Hogues

1

, John F. Kelly

3

, Enrico O. Purisima

1

, Rong Shi

2

, and Traian Sulea

1,

*

1

_{Human Health Therapeutics Research Centre, National Research Council Canada, 6100}

Royalmount Ave., Montreal, QC H4P 2R2, Canada

2

_{Département de Biochimie, de Microbiologie et de Bio-informatique, PROTEO, and Institut de}

Biologie Intégrative et des Systèmes (IBIS), Université Laval, Pavillon

Charles-Eugène-Marchand, Québec City, QC G1V 0A6, Canada

3

_{Human Health Therapeutics Research Centre, National Research Council Canada, 100 Sussex}

Dr., Ottawa, ON K1A 0R6, Canada

* Corresponding author: 514-496-1924; [email protected]

(2)

!

2!

Table&S1.!Diffusion!data!from!DLS!experiments!for!full5length!IgG1!variants.!

4°C! ! After!25weeks!storage!at!40°C! ! mAb&variant& Conc& mg/ml& Dmean&

1087_cm2_/s&₁₀stdev&87_cm2_/s& ₁₀D87median&_cm2_/s& N& ₁₀87D_cm0&2_/s& _ml/mgkD& &

&

&& Conc&

mg/ml&

Dmean&

1087_cm2_/s& ₁₀stdev&87_cm2_/s& ₁₀D87median&_cm2_/s& N& ₁₀87D_cm0&2_/s& _ml/mgkD& & & & Parental! 1.0! 4.56! !! 4.56! 1! 3.82! 0.000! !! 1.0! 4.48! 0.09! 4.50! 10! 4.62! 50.012! ! (monomer)! !! !! !! !! !! !! !! !! 2.0! 4.49! 0.07! 4.52! 10! !! !! ! !! !! !! !! !! !! !! !! !! 4.0! 4.38! 0.05! 4.39! 10! !! !! ! !! 8.0! 3.63! !! 3.63! 1! !! !! !! 8.0! 4.20! 0.02! 4.20! 10! !! !! ! !! 16.0! 3.83! 0.01! 3.83! 10! !! !! !! 16.0! 3.73! 0.02! 3.73! 10! !! !! ! WH_335MH_985MH_99! _1.0! _2.19! _0.14! _{2.16! 10!} _{1.98! 50.032! !!} _1.0! _1.42! _0.01! _{1.43! 5!} _{1.03! 50.041!} ! !(multimer)! 2.0! 1.88! 0.05! 1.89! 5! !! !! !! 2.0! 1.17! 0.01! 1.17! 5! !! !! ! !! 4.0! 1.62! 0.02! 1.62! 10! !! !! !! 4.0! 0.90! 0.02! 0.89! 5! !! !! ! !! !! !! !! !! !! !! !! !! 8.1! 0.49! 0.06! 0.46! 5! !! !! ! !! 16.1! 0.95! 0.02! 0.95! 10! !! !! !! 16.1! 0.40! 0.01! 0.40! 5! !! !! ! WH335FH985MH99! 1.0! 2.11! 0.05! 2.10! 10! 1.90! 50.031! !! 1.0! 2.16! 0.11! 2.19! 10! 1.85! 50.029! ! !(multimer)! 1.9! 1.85! 0.03! 1.86! 10! !! !! !! 1.9! 1.91! 0.01! 1.90! 10! !! !! ! !! 3.9! 1.56! 0.02! 1.56! 10! !! !! !! 3.9! 1.60! 0.02! 1.59! 10! !! !! ! !! !! !! !! !! !! !! !! !! 7.7! 1.30! 0.01! 1.30! 10! !! !! ! !! 15.5! 1.01! 0.02! 1.01! 10! !! !! !! 15.5! 1.04! 0.01! 1.04! 10! !! !! ! WH_335MH_985DH_99! _1.0! _4.50! _0.01! _{4.50! 5!} _{4.51! 50.017! !!} _1.0! _4.47! _0.06! _{4.48! 10!} _{4.49! 50.016!} ! !(monomer)! 2.0! 4.44! 0.03! 4.45! 5! !! !! !! 2.0! 4.22! 0.29! 4.34! 10! !! !! ! !! 4.0! 4.14! 0.12! 4.17! 10! !! !! !! 4.0! 4.22! 0.02! 4.22! 5! !! !! ! !! 8.0! 3.90! 0.01! 3.90! 10! !! !! !! 8.0! 3.90! 0.04! 3.90! 10! !! !! ! !! 15.9! 3.32! 0.01! 3.32! 5! !! !! !! 15.9! 3.34! 0.03! 3.35! 10! !! !! ! WH335FH985DH99! 1.0! 4.32! 0.22! 4.34! 4! 4.53! 50.014! !! 1.0! 4.53! 0.01! 4.53! 5! 4.55! 50.017! ! !(monomer)! 2.1! 4.44! 0.08! 4.46! 10! !! !! !! 2.1! 4.36! 0.07! 4.40! 10! !! !! ! !! 4.1! 4.29! 0.01! 4.29! 10! !! !! !! 4.1! 4.25! 0.02! 4.25! 10! !! !! ! !! 8.2! 3.97! 0.04! 3.97! 10! !! !! !! 8.2! 3.90! 0.03! 3.91! 10! !! !! ! !! 16.5! 3.45! 0.01! 3.45! 10! !! !! !! 16.5! 3.30! 0.01! 3.31! 10! !! !! ! WH_335MH_985MH_995RL_30b! _1.0! _2.15! _0.01! _{2.16! 5!} _{1.78! 50.030! !!} _1.0! _2.24! _0.04! _{2.25! 10!} _{2.00! 50.040!} ! !(multimer)! 2.0! 1.87! 0.02! 1.87! 10! !! !! !! 2.0! 1.94! 0.02! 1.94! 10! !! !! ! !! 4.0! 1.52! 0.05! 1.51! 10! !! !! !! 4.0! 1.61! 0.02! 1.62! 10! !! !! ! !! 7.9! 1.24! 0.03! 1.23! 10! !! !! !! 7.9! 1.31! 0.01! 1.30! 10! !! !! ! !! 15.8! 0.99! 0.01! 0.98! 10! !! !! !! 15.8! 0.75! 0.28! 0.75! 10! !! !! ! WH335FH985MH995RL30b! !! !! !! !! !! 1.32! 50.026! !! 1.0! 1.92! 0.02! 1.92! 5! 1.65! 50.034! ! (multimer)! 1.9! 1.39! 0.09! 1.40! 4! !! !! !! 1.9! 1.70! 0.02! 1.71! 5! !! !! ! !! !! !! !! !! !! !! !! !! 3.9! 1.40! 0.00! 1.40! 5! !! !! ! !! 7.7! 0.97! 0.01! 0.97! 5! !! !! !! 7.7! 1.10! 0.01! 1.09! 5! !! !! ! !! 15.5! 0.80! 0.01! 0.80! 5! !! !! !! 15.5! 0.82! 0.01! 0.81! 5! !! !! ! WH_335MH_985DH_995RL_30b! _1.1! _{4.51! !!} _{4.51! 1!} _{4.38! 50.014! !!} _1.1! _4.51! _0.04! _{4.52! 10!} _{4.52! 50.016!} ! !(monomer)! 2.1! 4.23! 0.10! 4.22! 9! !! !! !! 2.1! 4.34! 0.13! 4.38! 10! !! !! ! !! 4.3! 4.07! 0.08! 4.06! 3! !! !! !! 4.3! 4.21! 0.03! 4.22! 10! !! !! ! !! 8.5! 3.84! 0.06! 3.85! 7! !! !! !! 8.5! 3.88! 0.02! 3.88! 10! !! !! ! !! 17.0! 3.32! 0.03! 3.32! 10! !! !! !! 17.0! 3.29! 0.04! 3.30! 10! !! !! ! WH335FH985DH995RL30b! !! !! !! !! !! 3.83! 50.005! !! 1.0! 4.26! 0.53! 4.47! 10! 4.54! 50.016! ! !(monomer)! 2.0! 4.32! 0.04! 4.32! 2! !! !! !! 2.0! 4.39! 0.09! 4.43! 10! !! !! ! !! 3.9! 3.45! !! 3.45! 1! !! !! !! 3.9! 4.27! 0.02! 4.27! 10! !! !! ! !! !! !! !! !! !! !! !! !! 7.8! 3.93! 0.04! 3.94! 10! !! !! ! !! 15.7! 3.51! 0.07! 3.53! 10! !! !! !! 15.7! 3.39! 0.01! 3.40! 10! !! !!

Self5diffusion!coefficients!(D

0

)!and!diffusion!interaction!parameters!(k

D

)!were!derived!from!the!intercept!and!

slope,!respectively,!of!the!linear!relationship!between!diffusion!coefficients!(D)!and!concentration!(Conc):!

D!=!D

0

!+!D

0

Xk

D

XConc,!as!described!in!the!Materials!and!Methods!section.!

(3)

3 Table S2. Diffusion data from DLS experiments for the Fab variants in the SnAggR set (Table 2 in the main paper).

4°C After 2-weeks storage at 40°C

Fab variant Conc

mg/ml Dmean 10-7_cm2_/s stdev 10-7_cm2_/s Dmedian 10-7_cm2_/s N D0 10-7_cm2_/s kD ml/mg Conc mg/ml Dmean 10-7_cm2_/s stdev 10-7_cm2_/s Dmedian 10-7_cm2_/s N D0 10-7_cm2_/s kD ml/mg Parental 0.5 7.65 0.37 7.80 35 7.71 -0.004 0.5 7.69 0.66 7.94 41 7.86 -0.010 1.0 7.77 0.32 7.86 46 1.0 7.67 0.31 7.78 56 2.0 7.65 0.39 7.73 54 2.0 7.68 0.16 7.74 61 4.0 7.63 0.07 7.64 24 4.0 7.45 0.38 7.64 21 4.1 7.54 0.27 7.69 22 4.1 7.44 0.26 7.56 39 8.0 7.48 0.13 7.49 27 8.0 7.10 0.76 7.39 21 8.1 7.46 0.14 7.48 21 8.1 7.08 0.49 7.23 33 16.0 7.42 0.06 7.44 6 16.0 7.42 0.03 7.41 6 WH₃₃ _1.0 _7.42 _0.65 _7.71 ₄ _{7.59 -0.004} _1.0 _7.70 _0.04 _7.70 ₃ _{7.79 -0.008} 2.0 7.49 0.20 7.58 5 2.0 7.57 0.21 7.65 6 4.0 7.36 0.43 7.54 6 4.0 7.55 0.04 7.53 3 8.0 7.44 0.07 7.45 6 8.0 7.34 0.04 7.33 6 16.0 7.12 0.02 7.12 6 16.0 6.81 0.03 6.82 6 MH₉₈ _1.0 _7.80 _0.07 _7.82 ₆ _{7.59 -0.004} _1.0 _7.66 _0.04 _7.68 ₃ _{7.81 -0.010} 2.0 7.53 0.36 7.71 6 2.0 7.56 0.05 7.56 6 4.0 7.33 0.17 7.43 5 4.0 7.48 0.06 7.49 6 8.0 7.35 0.04 7.34 6 8.0 7.26 0.02 7.26 6 16.0 7.04 0.06 7.04 6 16.0 6.59 0.02 6.59 6 FH₉₈ _0.5 _7.44 _0.18 _7.46 ₆ _{7.65 -0.014} _0.5 _7.39 _0.12 _7.43 ₅ _{7.58 -0.022} 1.0 7.66 0.01 7.67 6 1.0 7.55 0.19 7.61 6 2.0 7.48 0.08 7.51 6 2.0 7.35 0.21 7.43 5 4.0 7.16 0.06 7.17 6 4.0 6.81 0.26 6.79 3 8.0 6.80 0.04 6.81 6 8.0 6.27 0.30 6.34 4 WH₉₈ _{7.34 -0.007} _0.4 _6.86 _0.67 _7.09 ₄ _6.88 _0.002 0.9 6.14 0.42 6.10 3 1.7 6.97 0.39 7.15 8 1.7 7.06 0.70 7.43 7 3.5 7.24 0.14 7.25 8 3.5 6.74 0.24 6.81 9 7.0 6.92 0.15 6.98 10 7.0 6.95 0.04 6.96 10 MH₉₉ _0.5 _7.47 _0.33 _7.55 ₅ _{7.81 -0.009} _0.5 _7.55 _0.42 _7.78 ₃ _{8.08 -0.025} 1.0 7.75 0.24 7.75 6 1.0 7.49 0.46 7.67 6 2.0 7.73 0.10 7.73 6 2.0 7.71 0.03 7.71 6 4.0 7.54 0.02 7.53 6 4.0 7.45 0.10 7.46 6 8.0 7.24 0.01 7.24 6 8.0 6.42 0.15 6.44 6 RL_30b _1.2 _7.70 _0.07 _7.70 ₅ _{7.66 -0.006} 2.4 7.45 0.11 7.44 5 4.9 7.43 0.08 7.46 10 19.4 6.73 0.03 6.73 5 WH_33-MH₉₈ _1.0 _7.61 _0.17 _7.62 ₆ _{7.81 -0.008} _1.0 _7.58 _0.08 _7.59 ₆ _{7.79 -0.010} 2.0 7.61 0.07 7.62 6 2.0 7.63 0.04 7.63 6 4.0 7.49 0.10 7.50 6 4.0 7.41 0.04 7.43 3 8.0 7.41 0.08 7.40 4 8.0 7.16 0.03 7.16 6 16.0 6.86 0.05 6.87 6 16.0 6.47 0.06 6.46 6 WH_33-FH₉₈ _{6.49 0.013} _0.5 _6.41 _0.79 _6.51 ₇ _{7.41 -0.003} 1.0 6.33 0.26 6.27 3 2.0 7.48 0.04 7.50 5 4.0 6.93 0.33 6.95 5 4.0 7.42 0.03 7.42 10 8.0 7.12 0.02 7.12 10 8.0 7.19 0.03 7.20 10 WH_33-WH₉₈ _0.4 _5.61 _0.61 _5.78 ₅ _{6.90 -0.002} _0.4 _7.55 _0.34 _7.75 ₅ _{7.65 -0.015} 0.9 6.63 0.21 6.63 4 0.9 7.40 0.25 7.38 9 1.8 7.10 0.08 7.11 10 1.8 7.47 0.07 7.49 10 3.6 7.02 0.04 7.05 10 3.6 7.24 0.04 7.24 10 7.2 6.72 0.05 6.74 10 7.2 6.83 0.04 6.83 10 MH_98-MH₉₉ _1.0 _7.49 _0.32 _7.60 ₆ _{7.71 -0.007} _1.0 _7.55 _0.01 _7.55 ₂ _{7.77 -0.013} 2.0 7.58 0.14 7.64 6 2.0 7.43 0.12 7.50 6

(4)

4

4.0 7.48 0.04 7.48 6 4.0 7.25 0.11 7.25 6 8.0 7.29 0.03 7.28 6 8.0 7.07 0.05 7.08 6 16.0 6.81 0.05 6.80 6 16.0 6.14 0.04 6.15 6 FH_98-MH₉₉ _1.0 _7.39 _0.29 _7.40 ₆ _{7.57 -0.011} _1.0 _7.55 _0.03 _7.55 ₃ _{7.50 -0.013} 2.0 7.48 0.11 7.50 6 2.0 7.15 0.23 7.28 3 4.0 7.26 0.06 7.25 4 4.0 6.97 0.16 7.03 6 8.0 6.88 0.03 6.89 6 8.0 6.77 0.02 6.76 6 16.0 6.28 0.04 6.28 6 16.0 5.91 0.04 5.90 6 WH_98-MH₉₉ _0.5 _7.32 _0.63 _7.32 ₂ _{7.53 -0.020} 1.0 7.57 0.12 7.60 6 1.9 7.29 0.20 7.37 3 3.9 6.86 0.23 6.75 3 7.8 6.27 0.40 6.45 6 WH_33-MH₉₉ _1.0 _7.79 _0.04 _7.81 ₆ _{7.87 -0.009} _1.0 _7.76 _0.06 _7.77 ₆ _{7.81 -0.011} 2.0 7.69 0.04 7.69 6 2.0 7.58 0.07 7.60 6 4.0 7.57 0.05 7.56 6 4.0 7.40 0.06 7.40 6 8.0 7.31 0.17 7.36 6 8.0 7.09 0.26 7.19 6 16.0 6.86 0.01 6.87 3 16.0 6.42 0.04 6.41 6 FH_98-RL_30b _1.0 _7.33 _0.24 _7.38 ₆ _{7.54 -0.007} _1.0 _7.68 _0.10 _7.73 ₅ _{7.71 -0.010} 2.0 7.29 0.29 7.31 4 2.0 7.65 0.06 7.67 6 4.0 7.45 0.03 7.44 3 4.0 7.30 0.08 7.31 6 8.0 7.22 0.03 7.22 6 8.0 7.10 0.02 7.11 6 16.0 6.74 0.08 6.74 6 16.0 6.39 0.18 6.43 6 WH_33-MH_98-MH₉₉ _1.0 _6.04 _0.04 _6.05 ₆ _{5.94 -0.004} _1.0 _5.97 _0.03 _5.97 ₆ _{5.91 -0.024} 1.4 5.96 0.14 6.02 10 1.4 5.51 0.33 5.60 5 2.0 5.95 0.04 5.96 6 2.0 5.74 0.04 5.73 6 2.7 5.79 0.21 5.88 5 2.7 5.82 0.07 5.82 10 4.0 5.88 0.01 5.89 6 4.0 5.19 0.08 5.20 6 5.4 5.69 0.05 5.70 8 5.4 5.58 0.04 5.59 10 8.0 5.77 0.04 5.77 6 8.0 4.24 0.06 4.26 6 10.9 5.53 0.07 5.55 7 10.9 5.30 0.05 5.31 10 16.0 5.56 0.02 5.55 6 16.0 2.95 0.05 2.95 6 21.8 5.46 0.02 5.47 10 21.8 4.70 0.08 4.71 10 WH_33-FH_98-MH₉₉ _1.0 _5.87 _0.03 _5.88 ₆ _{5.81 -0.004} _1.0 _5.19 _0.22 _{5.13 16} _{5.00 -0.031} 2.0 5.76 0.04 5.77 6 2.0 4.99 0.12 5.01 16 4.0 5.69 0.03 5.70 6 4.0 4.24 0.20 4.29 16 8.0 5.63 0.02 5.63 6 8.0 3.56 0.35 3.51 16 16.0 5.45 0.01 5.45 6 16.0 2.40 0.25 2.31 16 WH_33-WH_98-MH₉₉ _1.0 _6.53 _0.03 _6.52 ₆ _{6.31 -0.006} _1.0 _6.55 _0.13 _6.55 ₆ _{6.47 -0.008} 1.1 6.13 0.16 6.14 3 1.1 6.05 0.86 6.53 5 2.0 6.40 0.03 6.40 6 2.0 6.55 0.06 6.57 6 2.3 6.13 0.20 6.18 4 2.3 6.34 0.13 6.36 10 4.0 6.19 0.03 6.20 6 4.0 6.31 0.05 6.30 6 4.5 5.86 0.16 5.83 4 4.5 6.11 0.06 6.14 10 8.0 6.03 0.02 6.03 6 8.0 5.98 0.06 5.98 6 9.0 5.84 0.02 5.84 5 16.0 5.67 0.03 5.67 6 16.0 5.63 0.02 5.62 6 18.0 5.64 0.03 5.65 10 18.0 5.50 0.03 5.51 5 WH_33-MH_98-DH₉₉ _1.0 _7.61 _0.07 _7.62 ₆ _{7.60 -0.007} _1.0 _7.61 _0.09 _7.63 ₆ _{7.84 -0.016} 1.1 7.15 0.56 7.39 7 1.1 7.33 0.30 7.35 10 2.0 7.51 0.07 7.54 6 2.0 7.57 0.04 7.56 3 2.2 7.37 0.31 7.48 10 2.2 7.41 0.17 7.46 10 4.0 7.38 0.08 7.36 6 4.0 7.24 0.09 7.21 6 4.3 7.26 0.11 7.28 10 4.3 7.23 0.04 7.23 5 8.0 7.22 0.03 7.21 6 8.0 7.00 0.14 7.02 6 8.6 7.05 0.04 7.04 9 8.6 6.87 0.03 6.88 10 16.0 6.72 0.02 6.71 6 16.0 5.63 0.24 5.62 6 17.2 6.65 0.03 6.65 10 17.2 6.20 0.07 6.20 5 WH_33-FH_98-DH₉₉ _1.0 _7.62 _0.22 _7.69 ₆ _{7.68 -0.007} _1.0 _7.74 _0.02 _7.75 ₆ _{7.82 -0.014}

(5)

5

1.2 7.52 0.31 7.59 5 1.2 7.54 0.02 7.55 4 2.0 7.60 0.07 7.61 6 2.0 7.58 0.09 7.59 6 2.3 7.59 0.05 7.61 4 2.3 7.54 0.03 7.54 5 4.0 7.48 0.02 7.48 6 4.0 7.37 0.03 7.36 3 4.6 7.34 0.17 7.41 10 4.6 7.23 0.01 7.23 5 8.0 7.30 0.02 7.29 6 8.0 7.05 0.09 7.05 6 9.2 7.09 0.04 7.09 10 9.2 6.71 0.06 6.73 10 16.0 6.85 0.03 6.86 6 16.0 6.07 0.07 6.07 6 18.4 6.67 0.02 6.68 10 18.4 5.79 0.04 5.79 4 WH_33-WH_98-DH₉₉ _1.0 _7.48 _0.12 _7.52 ₅ _{7.67 -0.007} _1.0 _7.69 _0.05 _7.68 ₃ _{7.64 -0.009} 1.4 7.69 0.03 7.70 4 2.0 7.58 0.02 7.59 6 2.0 7.56 0.02 7.57 3 2.8 7.39 0.17 7.42 10 2.8 7.30 0.07 7.32 7 4.0 7.44 0.03 7.44 6 4.0 7.35 0.02 7.34 3 5.5 7.32 0.01 7.32 5 5.5 7.25 0.05 7.24 10 8.0 7.31 0.04 7.31 6 8.0 7.32 0.02 7.32 3 11.0 6.96 0.03 6.96 10 11.0 6.81 0.07 6.81 10 16.0 6.75 0.03 6.76 6 16.0 6.65 0.01 6.65 3 22.0 6.39 0.03 6.39 10 22.0 6.14 0.04 6.14 10 MH_98-MH_99-RL_30b _1.1 _6.42 _1.45 _7.15 ₇ _{7.60 -0.008} _1.1 _7.46 _0.08 _{7.48 10} _{7.27 -0.035} 2.3 7.54 0.09 7.56 9 2.3 6.98 0.05 6.99 10 4.6 7.38 0.06 7.40 10 4.6 6.14 0.16 6.11 10 9.1 7.06 0.04 7.06 10 9.1 4.65 0.28 4.66 10 18.2 6.50 0.09 6.52 10 18.2 2.64 0.30 2.64 10 MH_98-DH_99-RL_30b _1.0 _7.34 _0.49 _7.56 ₄ _{7.63 -0.011} _{7.81 -0.014} 1.1 7.26 0.28 7.33 5 2.0 7.61 0.03 7.61 5 2.2 7.51 0.12 7.47 5 4.0 7.29 0.10 7.31 6 4.4 7.34 0.19 7.45 10 8.0 6.97 0.23 6.97 6 8.9 6.82 0.04 6.82 10 16.0 6.33 0.08 6.33 6 17.8 5.79 0.07 5.79 5 WH_98-DH_99-RL_30b _{6.83 -0.011} _1.0 _7.53 _0.05 _7.52 ₃ _{7.63 -0.018} 1.1 6.89 0.16 6.87 4 2.0 7.34 0.14 7.28 3 2.2 6.70 0.03 6.69 5 4.0 7.13 0.05 7.13 3 4.3 6.41 0.07 6.43 5 8.0 6.53 0.04 6.54 3 8.7 6.18 0.04 6.18 10 16.0 5.46 0.02 5.46 3 WH_98-RH_99-RL_30b _0.5 _5.92 _1.12 _6.07 ₉ _{6.74 -0.009} 1.1 6.61 0.27 6.61 10 2.2 6.72 0.14 6.78 10 4.3 6.50 0.06 6.53 10 8.7 6.19 0.02 6.20 5 WH_33-MH_98-RL_30b _1.1 _6.21 _1.24 _6.31 ₈ _{6.95 -0.003} _1.1 _7.40 _0.32 _{7.55 10} _{7.61 -0.015} 2.2 5.84 1.73 5.77 4 2.2 7.35 0.09 7.34 10 4.4 7.31 0.15 7.38 5 4.4 7.05 0.11 7.04 10 8.8 7.05 0.07 7.06 10 8.8 6.71 0.05 6.70 10 17.6 6.50 0.15 6.55 10 17.6 5.63 0.13 5.65 10 WH_33-FH_98-RL_30b _{7.63 -0.011} _0.5 _7.93 _0.12 _7.96 ₅ _{7.86 -0.026} 1.0 7.69 0.16 7.70 5 1.0 7.50 0.04 7.52 3 2.0 7.20 0.44 7.20 2 2.0 7.30 0.24 7.34 6 4.0 7.25 0.21 7.32 6 4.0 7.13 0.10 7.16 3 8.0 6.67 6.67 1 8.0 6.27 0.23 6.30 5 WH_33-MH_98-MH_99-RL_30b _0.5 _6.20 _0.17 _6.18 ₆ _{6.05 -0.016} _0.5 _6.08 _0.10 _6.12 ₅ _{6.12 -0.038}

(6)

6

1.0 6.04 0.05 6.04 6 1.0 5.78 0.08 5.78 6 1.2 5.73 0.12 5.74 10 2.0 5.89 0.06 5.91 3 2.0 5.72 0.03 5.72 6 2.4 5.59 0.06 5.58 10 4.0 5.57 0.05 5.59 3 4.0 5.17 0.10 5.21 6 4.7 5.32 0.10 5.34 10 8.0 5.31 0.14 5.38 3 8.0 3.77 0.15 3.76 6 9.5 5.03 0.05 5.03 10 18.9 4.51 0.03 4.52 10 WH_33-FH_98-MH_99-RL_30b _0.5 _5.92 _0.05 _5.91 ₆ _{5.43 -0.019} _0.5 _5.61 _0.08 _5.61 ₆ _{5.77 -0.066} 0.7 5.34 0.42 5.63 9 1.0 5.61 0.25 5.71 6 1.0 5.49 0.06 5.51 6 1.5 5.42 0.06 5.44 10 2.0 5.58 0.12 5.60 5 2.0 4.94 0.11 4.94 6 2.9 5.58 5.58 1 2.9 5.03 0.03 5.04 10 4.0 5.39 5.39 1 4.0 3.98 0.16 4.03 6 5.8 4.24 0.08 4.24 2 5.8 4.38 0.04 4.38 10 11.7 5.47 0.03 5.48 10 11.7 3.72 0.06 3.72 10 WH_33-WH_98-MH_99-RL_30b _{4.50 0.012} _1.2 _5.77 _0.56 _5.81 ₃ _{6.11 -0.005} 2.3 6.16 0.02 6.15 5 4.6 5.30 0.32 5.23 5 4.6 6.03 0.05 6.04 10 9.3 4.77 0.36 4.66 5 9.3 5.78 0.03 5.79 10 18.5 5.58 0.06 5.59 10 18.5 5.53 0.04 5.55 10 WH_33-MH_98-DH_99-RL_30b _0.5 _7.65 _0.13 _7.64 ₄ _{7.51 -0.005} _0.5 _7.55 _0.06 _7.55 ₆ _{7.68 -0.034} 1.0 7.64 0.09 7.66 4 1.0 7.55 0.17 7.54 6 1.1 7.39 0.45 7.53 10 2.0 7.76 0.04 7.76 2 2.0 7.12 0.38 7.26 6 2.2 7.54 0.09 7.58 10 4.0 7.29 0.35 7.41 3 4.0 6.03 0.29 5.96 6 4.4 7.37 0.02 7.38 5 8.0 7.23 0.20 7.19 4 8.0 5.00 0.28 5.04 4 8.9 6.92 0.05 6.91 5 17.7 6.55 0.08 6.56 10 WH_33-FH_98-DH_99-RL_30b _0.5 _7.43 _7.43 ₁ _{7.62 -0.019} _0.5 _8.03 _0.05 _8.04 ₆ _{8.07 -0.031} 1.0 7.57 7.57 1 1.0 7.81 0.02 7.81 6 2.0 7.32 7.32 1 2.0 7.57 0.07 7.58 4 2.2 7.50 0.06 7.47 5 4.0 7.17 0.19 7.22 7 4.4 7.24 0.07 7.22 10 8.0 5.55 0.51 5.50 6 17.7 6.54 0.03 6.55 10 WH_33-WH_98-DH_99-RL_30b _1.0 _7.65 _0.10 _7.63 ₆ _{7.78 -0.009} _1.0 _7.52 _0.06 _7.52 ₂ _{7.57 -0.011} 1.1 7.23 0.16 7.19 5 2.0 7.65 0.04 7.64 4 2.0 7.53 0.02 7.53 3 2.2 7.15 0.28 7.26 4 4.0 7.46 0.03 7.47 6 4.0 6.97 0.64 7.22 6 4.5 7.23 0.05 7.21 5 8.0 7.24 0.04 7.22 6 8.0 6.97 0.02 6.98 3 9.0 6.89 0.03 6.89 10 16.0 6.72 0.03 6.71 3 16.0 6.14 0.07 6.14 6 18.0 6.32 0.02 6.32 5

Self-diffusion coefficients (D

0

) and diffusion interaction parameters (k

D

) were derived from the intercept and

slope, respectively, of the linear relationship between diffusion coefficients (D) and concentration (Conc):

D = D

0

+ D

0

·k

D

·Conc, as described in the Materials and Methods section.

(7)

!

7!

(8)

!

8!

Table&S4.!Tm

!data!from!DSF!experiments!on!Fab!variants!(see!also!Table!2!in!the!main!paper).!

!

&

T

m

&(°C)&by&DSF&

Mean&

stdev&

N&

Parent!

81.1!

0.3!

23!

W

H

33!

80.8!

0.0!

3!

M

H

98!

80.9!

0.1!

3!

F

H

98!

80.6!

0.1!

3!

M

H

99!

79.9!

0.5!

3!

W

H

33GM

H

98!

80.6!

0.1!

3!

M

H

98GM

H

99!

80.6!

0.1!

3!

F

H

98GM

H

99!

80.2!

0.2!

3!

W

H

98GM

H

99!

79.8!

0.1!

3!

W

H

33GM

H

99!

80.4!

0.1!

3!

F

H

98GR

L

30b!

80.0!

0.1!

4!

W

H

33GM

H

98GM

H

99!

79.7!

0.5!

3!

W

H

33GF

H

98GM

H

99!

79.7!

0.5!

3!

W

H

33GW

H

98GM

H

99!

79.5!

0.4!

3!

W

H

33GM

H

98GD

H

99!

80.5!

0.2!

3!

W

H

33GF

H

98GD

H

99!

79.8!

0.2!

3!

W

H

33GW

H

98GD

H

99!

79.6!

0.2!

3!

M

H

98GM

H

99GR

L

30b!

80.1!

0.1!

5!

M

H

98GD

H

99GR

L

30b!

80.7!

0.1!

5!

W

H

98GD

H

99GR

L

30b!

79.3!

0.1!

3!

W

H

33GF

H

98GR

L

30b!

80.4!

0.0!

3!

W

H

33GM

H

98GM

H

99GR

L

30b!

79.6!

0.1!

3!

W

H

33GF

H

98GM

H

99GR

L

30b!

79.2!

0.5!

3!

W

H

33GW

H

98GM

H

99GR

L

30b!

80.0!

0.1!

4!

W

H

33GM

H

98GD

H

99GR

L

30b!

79.9!

0.2!

3!

W

H

33GF

H

98GD

H

99GR

L

30b!

80.0!

0.4!

4!

W

H

33GW

H

98GD

H

99GR

L

30b!

79.0!

0.0!

3!

!

(9)

!

9!

Table&S5.!Predictions!of!aggregation!propensity!in!the!SnAggR!set!with!various!methods.!

!

! Fab!variant! AUC!dataa! in#silico!predictionsb# Structure?based!methods!

(folded!monomers)! Sequence?based!methods!(unfolding!pathway)! Dimer!

%! LogK(µM)!!d SAP! DI?F! A3D?S! A3D?D! CS?SC! AGS! CS! WLZ! TNG! PST! Parent! 2.8! 4.20! 45.5! ?0.16! ?311.4! ?229.3! ?1.889! ?1.6! 0.884! 576.7! 1860! 125.3! WH_33! _1.2! _6.17! _45.4! _{?0.16! ?311.7! ?221.5! ?1.890!} _?1.7! _0.903! _576.7! _1853! _126.7! MH_98! _1.4! _5.25! _50.8! _{0.17! ?306.3! ?227.6! ?1.862!} _?1.0! _0.949! _482.4! _1861! _125.3! FH98! 1.0! 3.11! 56.5! 0.27! ?308.4! ?215.0! ?1.835! ?0.8! 0.997! 580.1! 1889! 125.3! WH_98! _2.8! _3.76! _59.1! _{0.35! ?303.8! ?212.3! ?1.836!} _?1.0! _0.992! _482.4! _1878! _125.3! MH_99! _2.4! _5.34! _47.7! _{?0.10! ?308.9! ?223.4! ?1.890!} _?1.3! _0.915! _575.0! _1864! _125.3! RL_30b! _2.6! _3.95! _48.7! _{0.01! ?318.1! ?219.8! ?1.901!} _?1.8! _0.854! _576.7! _1848! _125.3! WH_33?MH_98! _1.8! _6.71! _52.3! _{0.17! ?306.7! ?238.2! ?1.866!} _?1.0! _0.969! _482.4! _1854! _126.7! WH_33?FH_98! _3.3! _3.51! _54.7! _{0.27! ?307.2! ?233.7! ?1.840!} _?0.8! _1.016! _580.1! _1881! _126.7! WH_33?WH_98! _1.4! _3.43! _54.3! _{0.25! ?314.2! ?229.2! ?1.840!} _?1.0! _1.012! _482.4! _1870! _126.7! MH_98?MH_99! _2.1! _4.30! _55.2! _{0.27! ?304.2! ?211.9! ?1.829!} _?0.7! _1.009! _482.4! _1906! _125.3! FH_98?MH_99! _8.1! _2.70! _61.3! _{0.38! ?299.7! ?237.1! ?1.802!} _?0.5! _1.061! _579.4! _2133! _125.3! WH_98?MH_99! _1.1! _2.86! _57.5! _{0.35! ?304.6! ?219.2! ?1.804!} _?0.7! _1.056! _482.4! _2067! _125.3! WH_33?MH_99! _2.5! _4.55! _50.6! _{?0.10! ?318.8! ?214.3! ?1.891!} _?1.3! _0.935! _575.0! _1856! _126.7! FH_98?RL_30b! _0.6! _4.11! _57.1! _{0.47! ?309.7! ?196.6! ?1.846!} _?1.0! _0.967! _580.1! _1877! _125.3! WH_33?MH_98?MH_99! _86.1! _1.43! _56.4! _{0.27! ?308.2! ?218.5! ?1.831!} _?0.7! _1.029! _482.4! _1898! _126.7! WH_33?FH_98?MH_99! _93.0! _1.18! _60.8! _{0.38! ?313.8! ?218.5! ?1.808!} _?0.5! _1.081! _579.4! _2126! _126.7! WH_33?WH_98?MH_99! _78.6! _1.87! _59.5! _{0.35! ?300.3! ?216.3! ?1.811!} _?0.7! _1.076! _482.4! _2059! _126.7! WH33?MH98?DH99! 1.8! 3.46! 46.9! ?0.13! ?319.1! ?216.2! ?1.892! ?1.3! 0.911! 482.4! 1853! 126.7! WH_33?FH_98?DH_99! _0.9! _4.19! _51.4! _{?0.04! ?309.3! ?234.6! ?1.878!} _?1.1! _0.948! _482.4! _1853! _126.7! WH_33?WH_98?DH_99! _2.7! _3.51! _48.7! _{?0.06! ?309.6! ?219.7! ?1.880!} _?1.3! _0.945! _482.4! _1853! _126.7! MH_98?MH_99?RL_30b! _0.3! _6.08! _53.9! _{0.46! ?299.7! ?220.7! ?1.839!} _?0.9! _0.979! _482.4! _1893! _125.3! MH_98?DH_99?RL_30b! _0.6! _5.27! _48.3! _{0.03! ?319.1! ?212.5! ?1.905!} _?1.5! _0.862! _482.4! _1848! _125.3! WH_98?DH_99?RL_30b! _4.1! _3.66! _51.9! _{0.11! ?318.3! ?217.5! ?1.890!} _?1.5! _0.895! _482.4! _1848! _125.3! WH_98?RH_99?RL_30b! _46.7! _2.12! _51.3! _{0.53! ?309.0! ?226.8! ?1.887!} _?1.4! _0.899! _482.4! _1850! _125.3! WH_33?MH_98?RL_30b! _1.6! _3.75! _51.0! _{0.37! ?312.5! ?205.0! ?1.877!} _?1.3! _0.939! _482.4! _1841! _126.7! WH_33?FH_98?RL_30b! _1.0! _4.86! _58.2! _{0.47! ?311.5! ?209.0! ?1.851!} _?1.1! _0.986! _580.1! _1869! _126.7! WH_33?MH_98?MH_99?RL_30b! _89.2! _1.11! _53.7! _{0.46! ?311.1! ?187.8! ?1.845!} _?0.9! _0.999! _482.4! _1886! _126.7! WH_33?FH_98?MH_99?RL_30b! _93.5! _1.11! _59.7! _{0.57! ?311.3! ?226.3! ?1.820!} _?0.7! _1.051! _579.4! _2113! _126.7! WH_33?WH_98?MH_99?RL_30b! _86.7! _1.56! _52.5! _{0.54! ?312.8! ?231.6! ?1.821!} _?0.9! _1.046! _482.4! _2047! _126.7! WH_33?MH_98?DH_99?RL_30b! _0.9! _3.60! _47.3! _{0.03! ?317.1! ?230.9! ?1.903!} _?1.5! _0.881! _482.4! _1840! _126.7! WH_33?FH_98?DH_99?RL_30b! _1.1! _4.90! _48.2! _{0.13! ?314.2! ?234.7! ?1.887!} _?1.4! _0.919! _482.4! _1840! _126.7! WH_33?WH_98?DH_99?RL_30b! _1.2! _3.12! _48.0! _{0.11! ?316.5! ?227.7! ?1.891!} _?1.5! _0.915! _482.4! _1840! _126.7! a!

_{Taken!from!Table!2!in!the!main!paper.!}

b!

_{Methods!abbreviations:}

!

SAP:!Spatial!Aggregation!Propensity;!

DI?F:!Developability!Index!fitted!on!%!dimer!data!from!the!SnAggR!set!(Table!2!in!the!main!paper);!

A3D?S:!AGGRESCAN3D!Static!mode;!

A3D?D:!AGGRESCAN3D!Dynamic!mode;!

CS?SC:!CamSol!Structurally?Corrected;!

AGS:!AGGRESCAN;!

CS:!CamSol;!

WLZ:!Waltz;!

TNG;!TANGO;!

PST:!PASTA.!

(10)

!

10!

Figure'S1.'Size!exclusion!of!affinity3matured!full3length!IgG1!antibody!variants.'Parental!bH1!

Y

H

_333D

H

_983G

H

_{99!(black),!Y}

H

_33W3D

H

_98M3G

H

_{99D!(magenta),!Y}

H

_33W3D

H

_98F3G

H

_{99D!(blue),!Y}

H

_33W3

(11)

!

11!

Figure'S2.!Superposition!of!the!four!crystal!structures!of!dimeric!affinity3matured!Fab!mutants!

of!the!parental!bH1!Fab!determined!in!this!work.!Green:!Y

H

33W3D

H

98M3G

H

99M!mutant.!Black:!

Y

H

33W3D

H

98F3G

H

99M!mutant.!Blue:!Y

H

33W3D

H

98M3G

H

99M3S

L

30bR!mutant.!Cyan:!Y

H

33W3

D

H

98F3G

H

99M3S

L

30bR!mutant.!!

!

(12)

!

12!

!

'

Figure'S3.'Differential!summed!deuteration!(Monomer!–!Dimer,!ΔDs

)!is!shown!in!(A)!and!(B)!for!

heavy!and!light!chains,!respectively.!The!amino!acid!sequence!is!plotted!on!the!x3axis,!and!each!

peptide!is!shown!as!a!grey!rectangle.!CDR!regions!are!highlighted!in!red!boxes.!Red!dashed!lines!

represent!the!95%!confidence!limits!for!the!summed!differences!in!deuteration!at!all!labeling!

times!(±0.5!Da).!(C)!ΔD

s

!superimposed!on!the!proposed!3D!dimer!structure.!Significantly!

protected!regions!upon!dimerization/mutation!are!shown!in!blue,!unchanged!regions!in!grey,!

and!missing!coverage!in!green.!Data!collected!in!triplicate!from!independent!HX!experiments.!

!

(13)

!

13!

!

Figure'S4.'(A)'Deuteration!kinetic!plots!for!all!57!heavy3chain!peptides!in!the!monomeric!

(W

H

333F

H

983D

H

99,!open!squares)!and!dimeric!(W

H

333F

H

983M

H

99,!filled!circles)!mutants.!Total!

coverage!was!80%.!Data!collected!in!triplicate,!and!errors!bars!represent!±1!x!SD.!(B)'

Deuteration!kinetic!plots!for!all!57!light3chain!peptides!in!the!monomeric!(W

H

333F

H

983D

H

99,!

open!squares)!and!dimeric!(W

H

333F

H

983M

H

99,!filled!circles)!mutants.!Total!coverage!was!99%.!

Data!collected!in!triplicate,!and!errors!bars!represent!±1!x!SD.!

!

A

B

(14)

!

14!

!

'

Figure'S5.!Sequence!details!of!the!variable!domains!of!the!affinity3matured!Fab!W

H

333F

H

983 M

H

99!variant!of!the!parental!bH1!Fab.!Amino3acid!residues!are!numbered!using!the!Chothia!

scheme,!whereas!the!CDR!loops!are!delineated!according!to!Kabat!scheme!except!CDR3H1!

which!is!according!to!the!union!of!Kabat!and!Chothia!definitions.!

!

(15)

!

15!

!

Figure'S6.!Internal!water3filled!cavity!at!the!dimeric!interface!of!the!Fab!variant!W

H

333F

H

983 M

H

99.!(A)!Overall!position!of!the!water3filled!cavity!relative!to!the!structure!of!the!dimer.!Z3axis!

slicing!through!the!molecular!surface!reveals!the!shape!of!the!cavity.!(B)!Close3up!view!of!the!

intermolecular!H3bonds!(yellow!dashed!sticks)!mediated!by!these!internal!water!molecules!(red!

spheres).!Two!nearby!chlorine!atoms!(green!spheres)!were!also!modeled!in!the!electron!density!

maps.!Color!coding!of!the!chains!is!the!same!as!in!Figure!4!from!the!main!paper.!!

!

A

B

Water3filled!cavity

(16)

!

16!

!

Figure'S7.!Specificity!of!CDR!conformations!for!their!cognate!complexes.!(A)'The!Fab!in!the!

dimerization!conformation!(determined!experimentally!in!this!work)!clashes!with!the!antigen!

molecule!(VEGF,!translucent!surface)!after!superposition!onto!the!antigen3bound!Fab!structure!

(PDB!code!3BDY).!(B)!Two!Fab!molecules!in!the!antigen3bound!conformation!clash!after!

superposition!on!the!dimeric!Fab!structure!(one!Fab!molecule!shown!as!ribbon,!the!other!Fab!

molecule!shown!as!translucent!surface).!

!

A

B

Y

H

100a

F

H

100 Y

L

32 Antigen

Dimerization'Conformation

F

H

98 M

H

99

Antigen=bound'Conformation

Fab

(17)

!

17!

!

Figure'S8.!Overlay!of!Fab!variants!bearing!W

H

_{33!(minimized!crystal!structure!–!beige!C!atoms)!}

and!Y

H

_{33!(minimized!modeled!structure!–!light!blue!C!atoms).!Intermolecular!H3bonds!to!the!}

other!monomer!in!the!dimeric!complex!are!shown!as!yellow!dashed!sticks.!Energetic!

contributions!of!mutations!at!position!

H

_{33!calculated!with!the!SIE!method!as!presented!in!the!}

main!paper.!

!

(18)

!

18!

!

Figure'S9.'Performance!of!structure3based!in#silico!aggregation!prediction!methods!for!the!

SnAggR!data!set!(Table!2)!based!on!the!homo3dimeric!conformation!of!the!CDR!determined!in!

this!work!by!X3ray!crystallography.!See!the!Methods!section!and!Figure!7!in!the!main!paper!for!

more!details.!

!

(19)

!

19!

'

Figure'S10.!Performance!of!sequence3based!in#silico!aggregation!prediction!methods!for!the!

SnAggR!data!set!(Table!2).!See!the!Methods!section!and!Figure!7!in!the!main!paper!for!more!

details.!Predicted!values!for!all!variants!are!listed!in!Table!S5.!

!

(20)