Therapeutic antibody engineering and optimization, lead-to-clinical-candidate. Covers sequence humanization (germline alignment, framework retention), affinity maturation, developability (aggregation, stability, PTMs), structure modeling (AlphaFold/PDB CDR analysis), immunogenicity prediction, and m

Antibody Engineering & Optimization AI guided antibody optimization pipeline from preclinical lead to clinical candidate. Covers sequence humanization, structure modeling, affinity optimization, developability assessment, immunogenicity prediction, and manufacturing feasibility. KEY PRINCIPLES : 1. Report first approach Create optimization report before analysis 2. Evidence graded humanization Score based on germline alignment and framework retention 3. Developability focused Assess aggregation, stability, PTMs, immunogenicity 4. Structure guided Use AlphaFold/PDB structures for CDR analysis 5. Clinical precedent Reference approved antibodies for validation 6. Quantitative scoring Developability score (0 100) combining multiple factors 7. English first queries Always use English terms in tool calls, even if user writes in another language. Respond in user's language LOOK UP, DON'T GUESS When uncertain about any scientific fact, SEARCH databases first (PubMed, UniProt, ChEMBL, ClinVar, etc.) rather than reasoning from memory. A database verified answer is always more reliable than a guess. When to Use Apply when user asks: "Humanize this mouse antibody sequence" "Optimize antibody affinity for [target]" "Assess developability of this antibody" "Predict immunogenicity risk for [sequence]" "Engineer bispecific antibody against [targets]" "Reduce aggregation in antibody formulation" "Design pH dependent binding antibody" "Analyze CDR sequences and suggest mutations" Critical Workflow Requirements 1. Report First Approach (MANDATORY) 1. Create the report file FIRST : antibody optimization report.md 2. Progressively update as analysis completes 3. Output separate files : optimized sequences.fasta All optimized variants humanization comparison.csv Before/after comparison developability assessment.csv Detailed scores See REPORT TEMPLATE.md for the full report template with section formats. 2. Documentation Standards (MANDATORY) Every optimization MUST include per variant documentation with: Original and optimized sequences Humanization score (% human framework) CDR preservation confirmation Metrics table (humanness, aggregation risk, predicted KD, immunogenicity) Data source citations Phase 0: Tool Verification Required Tools Tool Purpose Category IMGT search genes Germline gene identification Humanization IMGT get sequence Human framework sequences Humanization SAbDab search structures Antibody structure precedents Structure TheraSAbDab search by target Clinical antibody benchmarks Validation alphafold get prediction Structure modeling Structure iedb search epitopes Epitope identification Immunogenicity iedb search bcell B cell epitope prediction Immunogenicity UniProt get entry by accession Target antigen information Target STRING get interaction partners Protein interaction network Bispecifics PubMed search articles Literature precedents Validation CRITICAL : SOAP tools (IMGT, SAbDab, TheraSAbDab) require an operation parameter. See QUICK START.md for correct usage. Workflow Overview Phase 1: Input Analysis & Characterization Goal : Annotate sequences, identify species/germline, find clinical precedents. Key steps : 1. Annotate CDRs using IMGT numbering (CDR H1: 27 38, CDR H2: 56 65, CDR H3: 105 117) 2. Identify closest human germline genes via IMGT search genes 3. Search clinical precedents via TheraSAbDab search by target 4. Get target antigen info via UniProt get entry by accession Output : Sequence information table, CDR annotation, target info, clinical precedent list. See WORKFLOW DETAILS.md Phase 1 for code examples. Phase 2: Humanization Strategy Goal : Select human framework, design CDR grafting, identify backmutations. Key steps : 1. Search IMGT for IGHV/IGKV human germline genes 2. Score candidate frameworks by identity, CDR compatibility, and clinical use 3. Design CDR grafting onto selected framework 4. Identify Vernier zone residues that may need backmutation (positions 2, 27 30, 47 48, 67, 69, 71, 78, 93 94) 5. Generate at least 2 variants: full humanization and with key backmutations 6. Calculate humanization score (framework humanness, CDR preservation, T cell epitopes, aggregation risk) Output : Framework selection rationale, grafting design, backmutation analysis, humanized sequences. See WORKFLOW DETAILS.md Phase 2 for code examples. Phase 3: Structure Modeling & Analysis Goal : Predict structure, analyze CDR conformations, map epitope. Key steps : 1. Predict Fv structure via alphafold get prediction (VH:VL) 2. Assess pLDDT scores by region (framework, CDRs, interface) 3. Classify CDR canonical structures and calculate RMSD 4. Search known epitopes via iedb search epitopes 5. Compare with clinical antibody structures via SAbDab search structures Output : Structure quality table, CDR conformation analysis, epitope mapping, structural comparison. See WORKFLOW DETAILS.md Phase 3 for code examples. Phase 4: Affinity Optimization Goal : Design affinity improving mutations via computational screening. Key steps : 1. Identify interface residues (distance cutoff 4.5 A) 2. Screen all amino acid substitutions at CDR interface positions 3. Rank by predicted binding energy change (ddG < 0.5 kcal/mol = favorable) 4. Design combination strategy: single double triple mutants 5. Consider CDR H3 extension, tyrosine enrichment, salt bridge formation 6. Optional: pH dependent binding via histidine substitutions Output : Ranked mutation list, combination strategy, expected affinity improvements. See WORKFLOW DETAILS.md Phase 4 for code examples. Phase 5: Developability Assessment Goal : Comprehensive developability scoring (0 100) across five dimensions. Key steps : 1. Aggregation, pI, hydrophobic patches (sequence computable): run scripts/developability.py seq <VH/VL . It returns AGGRESCAN aggregation prone regions (real per residue propensity), isoelectric point, and Kyte Doolittle hydrophobic patches. 2. PTM liability : Scan for deamidation (NG/NS), isomerization (DG/DS), oxidation (Met/Trp), N glycosylation (N X S/T) 3. Stability (Tm) and 4. Expression titer : NOT computable from sequence alone — use external predictors (sequence→Tm/titer ML tools; report only if actually run). Do not fabricate Tm/titer numbers. 5. Solubility : external solubility predictor (e.g. NetSolP) Scoring : Weighted average (aggregation 0.30, PTM 0.25, stability 0.20, expression 0.15, solubility 0.10). Tiers: T1 ( 75), T2 (60 75), T3 (<60). Output : Component scores, overall score, tier classification, mitigation recommendations. See WORKFLOW DETAILS.md Phase 5 and CHECKLISTS.md for scoring details. Phase 6: Immunogenicity Prediction Goal : Predict immunogenicity risk and design deimmunization strategy. Key steps : 1. Scan 9 mer peptides against IEDB for MHC II binding epitopes 2. Count non human residues in framework regions 3. Assess aggregation related immunogenicity 4. Calculate total risk score (0 100, lower is better): Low <30, Medium 30 60, High 60 5. Propose deimmunization mutations (remove T cell epitopes while preserving CDRs) 6. Compare with clinical precedent ADA rates Output : T cell epitope list, risk score breakdown, deimmunization strategy, clinical comparison. See WORKFLOW DETAILS.md Phase 6 for code examples. Phase 7: Manufacturing Feasibility Goal : Assess expression, purification, formulation, and CMC feasibility. Key steps : 1. Assess codon optimization for CHO, identify rare codons 2. Design signal peptide 3. Plan 3 step purification: Protein A capture cation exchange polishing viral nanofiltration 4. Recommend formulation (buffer, pH, stabilizer, tonicity) 5. Define analytical characterization panel (SEC MALS, CEX, CE SDS, SPR, DSF) 6. Estimate CMC timeline and costs (typically 18 24 months, $1.65 2.65M to IND) Output : Expression assessment, purification strategy, formulation recommendation, CMC timeline. See MANUFACTURING.md for detailed manufacturing content and WORKFLOW DETAILS.md Phase 7 for code. Phase 8: Final Report & Recommendations Goal : Compile all findings into a ranked recommendation with validation plan. Key outputs : 1. Top candidate with key metrics (humanness, affinity, developability, immunogenicity, stability, expression) 2. Key improvements table comparing original vs. optimized 3. Experimental validation plan : In vitro (3 4 months) Lead optimization (2 3 months) Preclinical (6 12 months) 4. Backup variants with profiles and recommendations 5. IP considerations : FTO analysis, CDR novelty, patentability 6. Next steps : Immediate (month 1 3), short term (4 6), long term (7 24) See REPORT TEMPLATE.md for the full report template. Tool Reference IMGT Tools IMGT search genes : Search germline genes (IGHV, IGKV, etc.) IMGT get sequence : Get germline sequences IMGT get gene info : Database information Antibody Databases SAbDab search structures : Search antibody structures SAbDab get structure : Get structure details TheraSAbDab search therapeutics : Search by name TheraSAbDab search by target : Search by target antigen Immunogenicity iedb search epitopes : Search epitopes iedb search bcell : B cell epitopes iedb search mhc : MHC II epitopes iedb get epitope references : Citations Structure & Target alphafold get prediction : Structure prediction UniProt get entry by accession : Target info RCSBData get entry : Experimental structures Systems Biology (for Bispecifics) STRING get interaction partners : Protein interactions STRING get enrichment : Pathway analysis Reference Files File Contents QUICK START.md Getting started guide, SOAP tool parameters, Python SDK and MCP usage WORKFLOW DETAILS.md Code examples for all 8 phases REPORT TEMPLATE.md Full report template with section formats and example tables MANUFACTURING.md Detailed manufacturing content (expression, purification, formulation, CMC) EXAMPLES.md Complete clinical scenario examples (humanization, affinity, bispecific) CHECKLISTS.md Evidence grading, completeness checklists, scoring details, special considerations

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