RosettaAntibodyDesign (RAbD): A general framework for computational antibody design

Abstract

A structural-bioinformatics-based computational methodology and framework have been developed for the design of antibodies to targets of interest. RosettaAntibodyDesign (RAbD) samples the diverse sequence, structure, and binding space of an antibody to an antigen in highly customizable protocols for the design of antibodies in a broad range of applications. The program samples antibody sequences and structures by grafting structures from a widely accepted set of the canonical clusters of CDRs (North et al., J. Mol. Biol., 406:228-256, 2011). It then performs sequence design according to amino acid sequence profiles of each cluster, and samples CDR backbones using a flexible-backbone design protocol incorporating cluster-based CDR constraints. Starting from an existing experimental or computationally modeled antigen-antibody structure, RAbD can be used to redesign a single CDR or multiple CDRs with loops of different length, conformation, and sequence. We rigorously benchmarked RAbD on a set of 60 diverse antibody-antigen complexes, using two design strategies-optimizing total Rosetta energy and optimizing interface energy alone. We utilized two novel metrics for measuring success in computational protein design. The design risk ratio (DRR) is equal to the frequency of recovery of native CDR lengths and clusters divided by the frequency of sampling of those features during the Monte Carlo design procedure. Ratios greater than 1.0 indicate that the design process is picking out the native more frequently than expected from their sampled rate. We achieved DRRs for the non-H3 CDRs of between 2.4 and 4.0. The antigen risk ratio (ARR) is the ratio of frequencies of the native amino acid types, CDR lengths, and clusters in the output decoys for simulations performed in the presence and absence of the antigen. For CDRs, we achieved cluster ARRs as high as 2.5 for L1 and 1.5 for H2. For sequence design simulations without CDR grafting, the overall recovery for the native amino acid types for residues that contact the antigen in the native structures was 72% in simulations performed in the presence of the antigen and 48% in simulations performed without the antigen, for an ARR of 1.5. For the non-contacting residues, the ARR was 1.08. This shows that the sequence profiles are able to maintain the amino acid types of these conserved, buried sites, while recovery of the exposed, contacting residues requires the presence of the antigen-antibody interface. We tested RAbD experimentally on both a lambda and kappa antibody-antigen complex, successfully improving their affinities 10 to 50 fold by replacing individual CDRs of the native antibody with new CDR lengths and clusters.

Fig 1. Schematic diagrams of RosettaAntibodyDesign.

A. The outer loop: The protocol starts by (1) Choosing a CDR from those that are set to design [L1, L2, etc.] randomly according to set weights (default is equal weighting) and (2) grafting a random structure for that CDR from the CDRSet, a set of CDR structures from the PDB that satisfy user-defined input rules. (3) Regional Sequence Design is then setup for all designable regions and (4) structural constraints on the CDRs and SiteConstraints on the antibody-antigen orientation, if any, are set. (5) N Inner cycles are then completed, followed by (6) the application of the Monte Carlo criterion to either accept or reject the preliminary designs. (7) Finally, the lowest energy designs are output. B. The inner loop: (1) The antigen-antibody interface is first optionally optimized by running N cycles of RosettaDock [45]. Interface residues set to undergo sequence design will be designed. (2) The inner Monte Carlo criterion is then applied. The conformations of the CDR, its stem, and surrounding residues, and CDRs are then optimized according to the instruction file. (3) Residues from neighboring regions are designed if enabled (Fig 2 shows this packing/design shell). (4) The inner Monte Carlo criterion is then applied again and (5) the lowest energy decoy found in the inner loop is returned to the outer loop.

Fig 2. Packing shell setup.

During the inner optimization cycle, a packing shell is created (cyan) around the chosen CDR (in this case, L1 in yellow), and its neighbors (in this case, L3 and the DE loop (L4) in blue). By default, 6 Å is used as the packing shell distance. During the inner loop, all side chains are optimized and amino acid changes are made to any CDRs or regions set to sequence. The chosen CDR and its neighbors additionally undergo backbone optimization during this stage according to the minimization type chosen.

Fig 3. Example CDR instruction file used for successful 2J88 antibody design L14_7.

Lines beginning with # are comments and are ignored by the program. Further details are provided in Methods.

Fig 4. Computational benchmarking of the opt-E protocol.

Recovery metrics on 60 antibodies for the opt-E protocol (optimization of total Rosetta energy) for each CDR that underwent GraftDesign in the RAbD design protocol. (A) %Recovered and %Sampled for each CDR length and cluster for the opt-E simulations. (B) Design risk ratios (DRR) for recovery of CDR length and cluster for the opt-E simulations. 95% confidence intervals for the Risk Ratio statistics are calculated as described in Methods.

Fig 5. Antigen risk ratios for the opt-E protocol.

Risk Ratios of benchmarks showing the enrichment in the recovery of native lengths and clusters in the presence of the native antigen compared to simulations performed in its absence. (A) %Recovered length and cluster for the simulations in the presence and absence of antigen. (B) Length and Cluster Antigen Risk Ratios (ARRs) A risk ratio greater than 1.0 indicates enrichment of the native length and cluster in the presence of the antigen over simulations performed in the absence of the antigen.

Fig 6. Computational benchmarking of the opt-dG antibody design protocol.

Recovery metrics on 60 antibodies for the opt-dG protocol (optimization of Rosetta interface energy) for each CDR that underwent GraftDesign in the RAbD design protocol. (A) %Recovered and %Sampled for each CDR length and cluster for the opt-E simulations. (B) Design risk ratios (DRR) for recovery of CDR length and cluster for the opt-dG simulations. 95% confidence intervals for the Risk Ratio statistics are calculated as described in Methods.

Fig 7. Sequence design with the opt-E protocol on the 60 antibody benchmark.

(A) Sequence recovery for amino acids in contact with the antigen and those not in contact with the antigen from the antigen-present and antigen-absent simulations. (B). Antigen risk ratios (ARRs) for the contacting and non-contacting residues. Values greater than 1.0 indicate that the native residue types were present in the design simulations in the presence of the antigen more often than they were present in the design simulations in the absence of the antigen.

Fig 8. Designed antibodies against bee hyaluronidase.

(A) Apparent Binding Affinity (K_D) of expressed and tested antibody designs for bee hyaluronidase (PDB: 2J88), grouped by designed CDR cluster, as determined using Surface Plasmon Resonance (SPR) on a Biacore 4000. The dotted blue line represents the binding affinity of the native antibody on the Biacore machine (1.57x10^-8 nM). Binding affinity is shown for the 26 designs that had detectable binding affinity (out of 30 tested). The native CDRs are L1-11-2 and H2-9-1. (B) Kinetic sensorgrams of WT 2J88 Antibody to Bee Hyaluronidase. Two repeats of XPR (left); Biacore 4000 (right). (C) Kinetic sensorgrams of design L14_7 to Bee Hyaluronidase Two repeats of XPR (left); Biacore 4000 (right). (D) Model of the interface changes in design L14-7, with designed L1 cluster L1-11-2 (cyan), superimposed onto the WT antibody from PDB ID 2J88 (gray). (E) Designed Sequences vs WT.

Fig 9. Binding of designed antibodies to HIV gp120.

(A) Apparent binding affinity (KD) of WT CH103 antibody and designed antibodies to a panel of gp120 antigens. Here, 30 designs were expressed and tested, where 7 had detectable binding to these gp120s. (B) Binding affinity (KD) of the designed antibody, H2-6, versus the wild-type antibody CH103. (C) Kinetic sensorgrams of CH103 WT and design H2-6 to two select GP120s, Core Bal and PVO as determined through a Biacore 4000. (D) Model of the interface changes in design H2-6, with designed H2 cluster H2-10-1 (cyan), superimposed onto the WT antibody from PDB ID 4JAN (gray) (E) Alignment of H2-6 and the WT antibody CH103 from PDB ID 4JAN.