Imported from zja2004/BGI-CLI (data/skills/tooluniverse-antibody-engineering/SKILL.md). Install upstream with npx skills add zja2004/BGI-CLI --skill tooluniverse-antibody-engineering. Copyright stays with the author.

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

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:

   • File name: antibody_optimization_report.md
   • Initialize with section headers
   • Add placeholder: [Analyzing...]

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

2. Documentation Standards (MANDATORY)

Every optimization MUST include:

### Optimized Variant: VH_Humanized_v1

**Original Sequence**: EVQLVESGGGLVQPGG... (mouse)
**Humanized Sequence**: EVQLVQSGAEVKKPGA... (human framework)
**Humanization Score**: 87% human framework
**CDR Preservation**: 100% (all CDR residues retained)

**Metrics**:
| Metric | Original | Optimized | Change |
|--------|----------|-----------|--------|
| Humanness | 62% | 87% | +25% |
| Aggregation risk | 0.58 | 0.32 | -45% |
| Predicted KD | 5.2 nM | 3.8 nM | +27% affinity |
| Immunogenicity | High | Low | -65% |

*Source: IMGT germline analysis, IEDB predictions*

Phase 0: Tool Verification

Required Tools

Workflow Overview

Phase 1: Input Analysis & Characterization
├── Sequence annotation (CDRs, framework)
├── Species identification
├── Target antigen identification
├── Clinical precedent search
└── OUTPUT: Input characterization
    ↓
Phase 2: Humanization Strategy
├── Germline gene alignment (IMGT)
├── Framework selection
├── CDR grafting design
├── Backmutation identification
└── OUTPUT: Humanization plan
    ↓
Phase 3: Structure Modeling & Analysis
├── AlphaFold prediction
├── CDR conformation analysis
├── Epitope mapping
├── Interface analysis
└── OUTPUT: Structural assessment
    ↓
Phase 4: Affinity Optimization
├── In silico mutation screening
├── CDR optimization strategies
├── Interface improvement
└── OUTPUT: Affinity variants
    ↓
Phase 5: Developability Assessment
├── Aggregation propensity
├── PTM site identification
├── Stability prediction
├── Expression prediction
└── OUTPUT: Developability score
    ↓
Phase 6: Immunogenicity Prediction
├── MHC-II epitope prediction (IEDB)
├── T-cell epitope risk
├── Aggregation-related immunogenicity
└── OUTPUT: Immunogenicity risk score
    ↓
Phase 7: Manufacturing Feasibility
├── Expression level prediction
├── Purification considerations
├── Formulation stability
└── OUTPUT: Manufacturing assessment
    ↓
Phase 8: Final Report & Recommendations
├── Ranked variant list
├── Experimental validation plan
├── Next steps
└── OUTPUT: Comprehensive report

Phase 1: Input Analysis & Characterization

1.1 Sequence Annotation

def annotate_antibody_sequence(sequence):
    """Annotate antibody sequence with CDRs and framework regions."""

    # Use IMGT numbering scheme (standard for antibodies)
    # CDR definitions (IMGT):
    # CDR-H1: 27-38, CDR-H2: 56-65, CDR-H3: 105-117
    # CDR-L1: 27-38, CDR-L2: 56-65, CDR-L3: 105-117

    annotation = {
        'sequence': sequence,
        'length': len(sequence),
        'regions': {
            'FR1': sequence[0:26],
            'CDR1': sequence[26:38],
            'FR2': sequence[38:55],
            'CDR2': sequence[55:65],
            'FR3': sequence[65:104],
            'CDR3': sequence[104:117],
            'FR4': sequence[117:]
        }
    }

    return annotation

1.2 Species & Germline Identification

def identify_germline(tu, vh_sequence, vl_sequence):
    """Identify germline genes for VH and VL chains using IMGT."""

    # Search for human germline genes
    vh_germlines = tu.tools.IMGT_search_genes(
        gene_type="IGHV",
        species="Homo sapiens"
    )

    vl_germlines = tu.tools.IMGT_search_genes(
        gene_type="IGKV",  # or IGLV for lambda
        species="Homo sapiens"
    )

    # Get sequences for top matches
    # Calculate identity % for each germline
    # Return closest matches

    return {
        'vh_germline': 'IGHV1-69*01',
        'vh_identity': 87.2,
        'vl_germline': 'IGKV1-39*01',
        'vl_identity': 89.5
    }

1.3 Clinical Precedent Search

def search_clinical_precedents(tu, target_antigen):
    """Find approved/clinical antibodies against same target."""

    # Search Thera-SAbDab for clinical antibodies
    therapeutics = tu.tools.TheraSAbDab_search_by_target(
        target=target_antigen
    )

    approved = [ab for ab in therapeutics if ab['phase'] == 'Approved']
    clinical = [ab for ab in therapeutics if 'Phase' in ab['phase']]

    return {
        'approved_count': len(approved),
        'clinical_count': len(clinical),
        'examples': approved[:3],
        'insights': extract_design_patterns(approved)
    }

1.4 Output for Report

## 1. Input Characterization

### 1.1 Sequence Information

| Property | Heavy Chain (VH) | Light Chain (VL) |
|----------|------------------|------------------|
| **Length** | 118 aa | 107 aa |
| **Species** | Mouse (Mus musculus) | Mouse (Mus musculus) |
| **Humanness** | 62% | 68% |
| **Closest human germline** | IGHV1-69*01 (87% identity) | IGKV1-39*01 (90% identity) |

### 1.2 CDR Annotation (IMGT Numbering)

**Heavy Chain**:
- FR1: 1-26, CDR-H1: 27-38, FR2: 39-55, CDR-H2: 56-65, FR3: 66-104, CDR-H3: 105-117, FR4: 118-128

**CDR Sequences**:
| CDR | Sequence | Length | Canonical Class |
|-----|----------|--------|-----------------|
| CDR-H1 | GYTFTSYYMH | 10 | H1-13-1 |
| CDR-H2 | GIIPIFGTANY | 11 | H2-10-1 |
| CDR-H3 | ARDDGSYSPFDYWG | 14 | - (unique) |
| CDR-L1 | RASQSISSYLN | 11 | L1-11-1 |
| CDR-L2 | AASSLQS | 7 | L2-8-1 |
| CDR-L3 | QQSYSTPLT | 9 | L3-9-cis7-1 |

### 1.3 Target Information

| Property | Value |
|----------|-------|
| **Target** | PD-L1 (Programmed death-ligand 1) |
| **UniProt** | Q9NZQ7 |
| **Function** | Immune checkpoint, inhibits T-cell activation |
| **Disease relevance** | Cancer immunotherapy target |

### 1.4 Clinical Precedents

**Approved antibodies targeting PD-L1**:
1. **Atezolizumab** (Tecentriq) - IgG1, approved 2016
2. **Durvalumab** (Imfinzi) - IgG1, approved 2017
3. **Avelumab** (Bavencio) - IgG1, approved 2017

**Key insights**: All approved anti-PD-L1 antibodies use human IgG1 scaffolds with effector function modifications.

*Source: TheraSAbDab, UniProt*

Phase 2: Humanization Strategy

2.1 Framework Selection

def select_human_framework(tu, mouse_sequence, cdr_sequences):
    """Select optimal human framework for CDR grafting."""

    # Search IMGT for human germline genes
    vh_genes = tu.tools.IMGT_search_genes(
        gene_type="IGHV",
        species="Homo sapiens"
    )

    # For each candidate framework:
    # 1. Calculate sequence identity to mouse FR
    # 2. Check CDR canonical class compatibility
    # 3. Assess structural compatibility
    # 4. Consider clinical precedents

    candidates = []
    for gene in vh_genes[:20]:  # Top 20 human germlines
        gene_seq = tu.tools.IMGT_get_sequence(
            accession=gene['accession'],
            format='fasta'
        )

        score = calculate_framework_score(
            mouse_fr=extract_framework(mouse_sequence),
            human_fr=extract_framework(gene_seq),
            cdr_compatibility=check_cdr_compatibility(cdr_sequences, gene_seq)
        )

        candidates.append({
            'germline': gene['name'],
            'identity': score['identity'],
            'cdr_compatibility': score['cdr_compatibility'],
            'clinical_use': count_clinical_uses(gene['name']),
            'overall_score': score['total']
        })

    # Sort by overall score
    return sorted(candidates, key=lambda x: x['overall_score'], reverse=True)

2.2 CDR Grafting Design

def design_cdr_grafting(mouse_sequence, human_framework, cdr_sequences):
    """Design CDR grafting with backmutation identification."""

    # Graft mouse CDRs onto human framework
    grafted_sequence = graft_cdrs(
        human_framework=human_framework,
        mouse_cdrs=cdr_sequences
    )

    # Identify Vernier zone residues (affect CDR conformation)
    vernier_residues = [2, 27, 28, 29, 30, 47, 48, 67, 69, 71, 78, 93, 94]

    # Identify potential backmutations
    backmutations = []
    for pos in vernier_residues:
        if mouse_sequence[pos] != human_framework[pos]:
            backmutations.append({
                'position': pos,
                'human_aa': human_framework[pos],
                'mouse_aa': mouse_sequence[pos],
                'reason': 'Vernier zone - may affect CDR conformation',
                'priority': 'High' if pos in [27, 29, 30, 48] else 'Medium'
            })

    return {
        'grafted_sequence': grafted_sequence,
        'backmutations': backmutations,
        'humanness_score': calculate_humanness(grafted_sequence)
    }

2.3 Humanization Scoring

def calculate_humanization_score(sequence, human_germline):
    """Calculate comprehensive humanization score."""

    # Framework humanness (% identity to human germline)
    fr_identity = calculate_framework_identity(sequence, human_germline)

    # T-cell epitope content (lower is better)
    tcell_epitope_count = predict_tcell_epitopes(sequence)

    # Unusual residues in human context
    unusual_residues = count_unusual_residues(sequence)

    # Aggregation hotspots
    aggregation_motifs = find_aggregation_motifs(sequence)

    score = {
        'framework_humanness': fr_identity,  # 0-100%
        'cdr_preservation': 100,  # Always 100% initially
        'tcell_epitopes': tcell_epitope_count,
        'unusual_residues': unusual_residues,
        'aggregation_risk': len(aggregation_motifs),
        'overall_score': calculate_weighted_score(
            fr_identity, tcell_epitope_count, unusual_residues, aggregation_motifs
        )
    }

    return score

2.4 Output for Report

## 2. Humanization Strategy

### 2.1 Framework Selection

**Selected Human Frameworks**:

| Chain | Germline | Identity | CDR Compatibility | Clinical Use | Score |
|-------|----------|----------|-------------------|--------------|-------|
| **VH** | IGHV1-69*01 | 87.2% | Excellent | 127 antibodies | 94/100 |
| **VL** | IGKV1-39*01 | 89.5% | Excellent | 89 antibodies | 92/100 |

**Rationale**:
- IGHV1-69*01: Most frequently used human germline in therapeutic antibodies
- High sequence identity minimizes risk of affinity loss
- Excellent CDR canonical class compatibility
- Proven clinical track record

### 2.2 CDR Grafting Design

**Grafting Strategy**: Direct CDR transfer with Vernier zone optimization

| Region | Source | Sequence | Rationale |
|--------|--------|----------|-----------|
| FR1 | IGHV1-69*01 | EVQLVQSGAEVKKPGA... | Human framework |
| CDR-H1 | Mouse | GYTFTSYYMH | Retain binding |
| FR2 | IGHV1-69*01 | VKWVRQAPGQGLE... | Human framework |
| CDR-H2 | Mouse | GIIPIFGTANY | Retain binding |
| FR3 | IGHV1-69*01 | RVTMTTDTSTSTYME... | Human framework |
| CDR-H3 | Mouse | ARDDGSYSPFDYWG | Retain binding |
| FR4 | IGHJ4*01 | WGQGTLVTVSS | Human framework |

### 2.3 Backmutation Analysis

**Identified Vernier Zone Residues** (may require backmutation):

| Position | Human | Mouse | Region | Impact | Priority |
|----------|-------|-------|--------|--------|----------|
| 27 | T | A | CDR-H1 boundary | CDR conformation | High |
| 48 | I | V | FR2 | VH-VL interface | High |
| 67 | A | S | FR3 | CDR-H2 support | Medium |
| 71 | R | K | FR3 | CDR-H2 support | Medium |
| 93 | A | T | FR3 | CDR-H3 base | Medium |

**Recommendation**: Test versions with/without backmutations at positions 27 and 48

### 2.4 Humanized Sequences

**Version 1: Full humanization** (no backmutations)

VH_Humanized_v1 | 87% human framework EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGGIIPIFGTANY AQKFQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARARDDGSYSPFDYWGQGTLVTVSS

**Version 2: With key backmutations** (positions 27, 48)

VH_Humanized_v2 | 85% human framework + backmutations EVQLVQSGAEVKKPGASVKVSCKASGYAFTSYYMHWVRQAPGQGLEWMVGIIPIFGTANY AQKFQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARARDDGSYSPFDYWGQGTLVTVSS

**Humanization Metrics**:
| Metric | Original (Mouse) | v1 (Full) | v2 (Backmut) |
|--------|------------------|-----------|--------------|
| Framework humanness | 62% | 87% | 85% |
| CDR preservation | 100% | 100% | 100% |
| Vernier zone match | Mouse | Human | Mixed |
| Predicted affinity | Baseline | 60-80% | 80-100% |

*Source: IMGT germline database, CDR analysis*

Phase 3: Structure Modeling & Analysis

3.1 AlphaFold Structure Prediction

def predict_antibody_structure(tu, vh_sequence, vl_sequence):
    """Predict antibody Fv structure using AlphaFold."""

    # Combine VH and VL with linker
    fv_sequence = vh_sequence + ":" + vl_sequence  # AlphaFold uses : for chain separator

    # Predict structure
    prediction = tu.tools.AlphaFold_get_prediction(
        sequence=fv_sequence,
        return_format='pdb'
    )

    # Extract pLDDT scores
    plddt_scores = extract_plddt(prediction)

    # Analyze by region
    regions = {
        'VH_FR': np.mean([plddt_scores[i] for i in range(0, 26)]),
        'CDR_H1': np.mean([plddt_scores[i] for i in range(26, 38)]),
        'CDR_H2': np.mean([plddt_scores[i] for i in range(55, 65)]),
        'CDR_H3': np.mean([plddt_scores[i] for i in range(104, 117)]),
        'VL_FR': np.mean([plddt_scores[i] for i in range(len(vh_sequence), len(vh_sequence)+26)]),
        'CDR_L1': np.mean([plddt_scores[i] for i in range(len(vh_sequence)+26, len(vh_sequence)+38)]),
    }

    return {
        'structure': prediction,
        'mean_plddt': np.mean(plddt_scores),
        'regional_plddt': regions,
        'cdr_confidence': np.mean([regions['CDR_H1'], regions['CDR_H2'], regions['CDR_H3']])
    }

3.2 CDR Conformation Analysis

def analyze_cdr_conformation(structure):
    """Analyze CDR loop conformations and canonical classes."""

    # Extract CDR coordinates
    cdr_coords = extract_cdr_regions(structure)

    # Classify canonical structures
    cdr_classes = {
        'CDR-H1': classify_canonical_structure(cdr_coords['H1']),
        'CDR-H2': classify_canonical_structure(cdr_coords['H2']),
        'CDR-H3': 'Non-canonical (14 aa)',  # Usually unique
        'CDR-L1': classify_canonical_structure(cdr_coords['L1']),
        'CDR-L2': classify_canonical_structure(cdr_coords['L2']),
        'CDR-L3': classify_canonical_structure(cdr_coords['L3'])
    }

    # Calculate RMSD to known canonical structures
    rmsd_values = calculate_canonical_rmsd(cdr_coords, cdr_classes)

    return {
        'classes': cdr_classes,
        'rmsd': rmsd_values,
        'confidence': assess_conformation_confidence(rmsd_values)
    }

3.3 Epitope Mapping

def map_epitope(tu, target_protein, antibody_structure):
    """Identify epitope on target protein."""

    # Get target structure or predict
    target_info = tu.tools.UniProt_get_protein_by_accession(
        accession=target_protein
    )

    # Search for known epitopes
    epitopes = tu.tools.iedb_search_epitopes(
        sequence_contains=target_protein,
        structure_type="Linear peptide",
        limit=20
    )

    # Search for structural antibody complexes
    sabdab_results = tu.tools.SAbDab_search_structures(
        query=target_info['protein_name']
    )

    # Analyze binding interface
    interface = {
        'epitope_candidates': epitopes,
        'structural_precedents': sabdab_results,
        'predicted_interface': predict_binding_interface(antibody_structure)
    }

    return interface

3.4 Output for Report

## 3. Structure Modeling & Analysis

### 3.1 AlphaFold Predictions

**Structure Quality**:

| Variant | Mean pLDDT | VH pLDDT | VL pLDDT | CDR pLDDT | Confidence |
|---------|------------|----------|----------|-----------|------------|
| Original (Mouse) | 89.2 | 91.4 | 88.7 | 85.3 | High |
| VH_Humanized_v1 | 87.8 | 89.6 | 88.2 | 83.1 | High |
| VH_Humanized_v2 | 88.9 | 90.8 | 88.5 | 84.8 | High |

**Regional Confidence (v2)**:
- Framework regions: 92.3 (very high)
- CDR-H1, H2, L1, L2: 87-91 (high)
- CDR-H3: 78.4 (moderate - expected for unique CDR-H3)
- VH-VL interface: 90.1 (high)

### 3.2 CDR Conformation Analysis

**Canonical Classes** (Humanized v2):

| CDR | Length | Canonical Class | RMSD to Class | Status |
|-----|--------|-----------------|---------------|--------|
| CDR-H1 | 10 | H1-13-1 | 0.8 Å | ✓ Maintained |
| CDR-H2 | 11 | H2-10-1 | 1.1 Å | ✓ Maintained |
| CDR-H3 | 14 | Non-canonical | N/A | Unique structure |
| CDR-L1 | 11 | L1-11-1 | 0.9 Å | ✓ Maintained |
| CDR-L2 | 7 | L2-8-1 | 0.7 Å | ✓ Maintained |
| CDR-L3 | 9 | L3-9-cis7-1 | 1.0 Å | ✓ Maintained |

**Assessment**: All CDR conformations well-preserved in humanized variants. Low RMSD values indicate minimal structural perturbation from humanization.

### 3.3 Epitope Analysis

**Known PD-L1 Epitopes** (IEDB):

| Epitope | Sequence | Position | Binding Antibodies | Conservation |
|---------|----------|----------|-------------------|--------------|
| Epitope 1 | LQDAG...VPEPP | 19-113 | Durvalumab, Avelumab | 98% |
| Epitope 2 | FTVT...PGPN | 54-68 | Atezolizumab | 100% |
| Epitope 3 | RLEDL...NVSI | 115-127 | Research Abs | 95% |

**Predicted Binding Interface**:
- Primary contact residues: CDR-H3 (70%), CDR-H1 (15%), CDR-H2 (10%)
- Secondary contacts: CDR-L3 (5%)
- Estimated buried surface area: 820 Ų

### 3.4 Structural Comparison

**Superposition with Clinical Antibodies** (SAbDab):

| Reference | PDB ID | VH RMSD | VL RMSD | CDR-H3 RMSD | Notes |
|-----------|--------|---------|---------|-------------|-------|
| Atezolizumab | 5X8L | 1.2 Å | 1.4 Å | 2.8 Å | Similar approach angle |
| Durvalumab | 5X8M | 1.8 Å | 1.5 Å | 3.4 Å | Different epitope |
| Research Ab | 5C3T | 0.9 Å | 1.1 Å | 1.5 Å | Very similar |

*Source: AlphaFold, IEDB, SAbDab*

Phase 4: Affinity Optimization

4.1 In Silico Mutation Screening

def design_affinity_variants(antibody_structure, target_structure):
    """Design affinity maturation variants using computational screening."""

    # Identify interface residues
    interface_residues = identify_interface_residues(
        antibody_structure,
        target_structure,
        distance_cutoff=4.5  # Angstroms
    )

    # Focus on CDR residues
    cdr_interface = [res for res in interface_residues if is_cdr_residue(res)]

    # Design mutations for each position
    variants = []
    for position in cdr_interface:
        # Try all amino acids except original
        for aa in 'ACDEFGHIKLMNPQRSTVWY':
            if aa != antibody_structure.sequence[position]:
                predicted_ddg = predict_binding_energy_change(
                    structure=antibody_structure,
                    mutation=f"{antibody_structure.sequence[position]}{position}{aa}"
                )

                if predicted_ddg < -0.5:  # Favorable change (more negative = better)
                    variants.append({
                        'position': position,
                        'original': antibody_structure.sequence[position],
                        'mutant': aa,
                        'predicted_ddg': predicted_ddg,
                        'predicted_kd_fold': calculate_kd_change(predicted_ddg)
                    })

    # Rank by predicted improvement
    return sorted(variants, key=lambda x: x['predicted_ddg'])

4.2 CDR Optimization Strategies

def cdr_optimization_strategies(cdr_sequence, cdr_name):
    """Identify CDR optimization strategies based on sequence and structure."""

    strategies = []

    # Strategy 1: Extend CDR for increased contact area
    if len(cdr_sequence) < 12 and cdr_name == 'CDR-H3':
        strategies.append({
            'strategy': 'CDR-H3 extension',
            'rationale': 'Add 1-2 residues to increase contact surface',
            'expected_impact': '+2-5x affinity improvement',
            'examples': ['Extension with Gly-Tyr', 'Extension with Ser-Asp']
        })

    # Strategy 2: Tyrosine enrichment
    tyr_count = cdr_sequence.count('Y')
    if tyr_count < 2:
        strategies.append({
            'strategy': 'Tyrosine enrichment',
            'rationale': 'Tyr provides pi-stacking and H-bonds',
            'expected_impact': '+2-3x affinity improvement',
            'targets': suggest_tyr_positions(cdr_sequence)
        })

    # Strategy 3: Charged residue optimization
    if 'PD' in cdr_sequence or 'EP' in cdr_sequence:
        strategies.append({
            'strategy': 'Salt bridge formation',
            'rationale': 'Add charged residues for electrostatic interactions',
            'expected_impact': '+1-2x affinity and pH sensitivity',
            'targets': identify_salt_bridge_opportunities(cdr_sequence)
        })

    return strategies

4.3 Output for Report

## 4. Affinity Optimization

### 4.1 Current Affinity Assessment

| Property | Value | Method |
|----------|-------|--------|
| **Predicted KD** | 5.2 nM | Structure-based prediction |
| **Buried surface area** | 820 Ų | AlphaFold model |
| **Interface hotspots** | 6 residues | Energy decomposition |

**Target**: Single-digit nM affinity (KD < 5 nM)

### 4.2 Proposed Affinity Mutations

**High-Priority Mutations** (predicted >2x improvement):

| Position | Original | Mutant | Region | Predicted ΔΔG | KD Fold Improvement | Rationale |
|----------|----------|--------|--------|---------------|---------------------|-----------|
| H100a | S | Y | CDR-H3 | -1.2 kcal/mol | 7.4x | Pi-stacking with target Phe |
| H52 | I | W | CDR-H2 | -0.9 kcal/mol | 4.8x | Increased hydrophobic contact |
| L91 | Q | E | CDR-L3 | -0.7 kcal/mol | 3.3x | Salt bridge with target Arg |
| H58 | G | S | CDR-H2 | -0.6 kcal/mol | 2.7x | H-bond to target backbone |

**Medium-Priority Mutations** (predicted 1.5-2x improvement):

| Position | Original | Mutant | Region | Predicted ΔΔG | KD Fold Improvement | Rationale |
|----------|----------|--------|--------|---------------|---------------------|-----------|
| H33 | Y | F | CDR-H1 | -0.5 kcal/mol | 2.3x | Optimize stacking geometry |
| L50 | A | T | CDR-L2 | -0.4 kcal/mol | 2.0x | Additional H-bond |

### 4.3 Combination Strategy

**Recommended Testing Order**:

1. **Single mutants**: H100aY, H52W, L91E (test individually)
2. **Double mutants**: H100aY+H52W, H100aY+L91E (best combinations)
3. **Triple mutant**: H100aY+H52W+L91E (if additivity observed)

**Expected Outcome**:
- Single mutants: KD 1.5-2.5 nM (3-7x improvement)
- Best double mutant: KD 0.7-1.2 nM (7-15x improvement)
- Triple mutant: KD 0.3-0.6 nM (15-30x improvement) if additive

### 4.4 CDR Optimization Strategies

**Strategy 1: CDR-H3 Extension**
- Current length: 14 aa
- Proposed: Add Gly-Tyr at C-terminus (16 aa total)
- Rationale: Fill gap in binding interface, Tyr provides pi-stacking
- Expected impact: +2-3x affinity

**Strategy 2: Tyrosine Enrichment**
- Current Tyr count: 3 in CDRs
- Target positions: H33, H52a, L96
- Rationale: Tyr provides both hydrophobic and H-bond contacts
- Expected impact: +2-4x affinity

**Strategy 3: pH-Dependent Binding (Optional)**
- For tumor-selective uptake
- Add His residues at interface: H100a, L91
- pKa ~6.0: Bind at pH 7.4, release at pH 6.0
- Expected impact: Tumor selectivity, faster recycling

*Source: In silico modeling, structural analysis*

Phase 5: Developability Assessment

5.1 Aggregation Propensity

def assess_aggregation(sequence):
    """Comprehensive aggregation risk assessment."""

    # Identify aggregation-prone regions (APR)
    aprs = find_aggregation_motifs(sequence)

    # Hydrophobic patches on surface
    hydrophobic_patches = identify_surface_hydrophobic(sequence)

    # Charge patches (extreme pI regions)
    charge_patches = identify_charge_clusters(sequence)

    # Sequence-based prediction scores
    tango_score = predict_tango_score(sequence)  # Beta-aggregation
    aggrescan_score = predict_aggrescan(sequence)  # General aggregation

    # Isoelectric point
    pi = calculate_isoelectric_point(sequence)

    return {
        'apr_count': len(aprs),
        'apr_regions': aprs,
        'hydrophobic_patches': hydrophobic_patches,
        'charge_patches': charge_patches,
        'tango_score': tango_score,
        'aggrescan_score': aggrescan_score,
        'pi': pi,
        'overall_risk': categorize_risk(tango_score, aggrescan_score, len(aprs))
    }

5.2 PTM Site Identification

def identify_ptm_sites(sequence):
    """Identify post-translational modification liability sites."""

    ptm_sites = {
        'deamidation': [],
        'isomerization': [],
        'oxidation': [],
        'glycosylation': []
    }

    # Deamidation: Asn followed by Gly or Ser (NG, NS motifs)
    for i, aa in enumerate(sequence[:-1]):
        if aa == 'N' and sequence[i+1] in ['G', 'S']:
            ptm_sites['deamidation'].append({
                'position': i,
                'motif': sequence[i:i+2],
                'risk': 'High' if sequence[i+1] == 'G' else 'Medium',
                'region': identify_region(i)
            })

    # Isomerization: Asp followed by Gly or Ser (DG, DS motifs)
    for i, aa in enumerate(sequence[:-1]):
        if aa == 'D' and sequence[i+1] in ['G', 'S']:
            ptm_sites['isomerization'].append({
                'position': i,
                'motif': sequence[i:i+2],
                'risk': 'High',
                'region': identify_region(i)
            })

    # Oxidation: Met and Trp residues
    for i, aa in enumerate(sequence):
        if aa in ['M', 'W']:
            ptm_sites['oxidation'].append({
                'position': i,
                'residue': aa,
                'risk': 'Medium',
                'region': identify_region(i)
            })

    # N-glycosylation: N-X-S/T motif (X != P)
    for i in range(len(sequence)-2):
        if sequence[i] == 'N' and sequence[i+1] != 'P' and sequence[i+2] in ['S', 'T']:
            ptm_sites['glycosylation'].append({
                'position': i,
                'motif': sequence[i:i+3],
                'region': identify_region(i)
            })

    return ptm_sites

5.3 Developability Scoring

def calculate_developability_score(sequence, structure):
    """Calculate comprehensive developability score (0-100)."""

    # Component scores
    aggregation = assess_aggregation(sequence)
    ptm = identify_ptm_sites(sequence)
    stability = predict_thermal_stability(structure)
    expression = predict_expression_level(sequence)
    solubility = predict_solubility(sequence)

    # Scoring rubric (0-100 for each)
    scores = {
        'aggregation': score_aggregation(aggregation),  # 100 = low risk
        'ptm_liability': score_ptm_risk(ptm),  # 100 = no PTM sites
        'stability': score_stability(stability),  # 100 = Tm > 70°C
        'expression': score_expression(expression),  # 100 = >1 g/L
        'solubility': score_solubility(solubility)  # 100 = >100 mg/mL
    }

    # Weighted average
    weights = {
        'aggregation': 0.30,  # Most critical
        'ptm_liability': 0.25,
        'stability': 0.20,
        'expression': 0.15,
        'solubility': 0.10
    }

    overall = sum(scores[k] * weights[k] for k in scores.keys())

    return {
        'component_scores': scores,
        'overall_score': overall,
        'tier': categorize_developability(overall)
    }

5.4 Output for Report

## 5. Developability Assessment

### 5.1 Overall Developability Score

| Variant | Aggregation | PTM Liability | Stability | Expression | Solubility | **Overall** | Tier |
|---------|-------------|---------------|-----------|------------|------------|-------------|------|
| Original (Mouse) | 58 | 45 | 72 | 65 | 70 | **62** | T3 |
| VH_Humanized_v1 | 72 | 55 | 75 | 78 | 75 | **71** | T2 |
| VH_Humanized_v2 | 68 | 58 | 74 | 75 | 73 | **69** | T2 |
| Affinity_opt | 85 | 72 | 78 | 80 | 82 | **79** | T1 |

**Scoring**: 0-100 scale (higher is better), Tiers: T1 (>75), T2 (60-75), T3 (<60)

### 5.2 Aggregation Analysis

**Aggregation-Prone Regions** (APR) in VH:

| Position | Sequence | Region | TANGO Score | Risk | Recommendation |
|----------|----------|--------|-------------|------|----------------|
| 85-92 | STSTAYMEL | FR3 | 42 | Medium | Consider T86S mutation |
| 108-112 | DDGSY | CDR-H3 | 28 | Low | Monitor in formulation |

**Overall Aggregation Risk**:
- VH: Low (TANGO: 15, AGGRESCAN: -12)
- VL: Very Low (TANGO: 8, AGGRESCAN: -18)
- pI: VH 7.2, VL 5.8 (favorable for purification)

**Recommendations**:
- Formulate at pH 6.0-6.5 (below pI of VH)
- Add arginine-glutamate (20-50 mM) to reduce aggregation
- Target concentration: >100 mg/mL achievable

### 5.3 PTM Liability Sites

**High-Risk PTM Sites** (require mitigation):

| Position | Motif | PTM Type | Risk | Region | Mitigation Strategy |
|----------|-------|----------|------|--------|---------------------|
| H54-55 | NG | Deamidation | High | CDR-H2 | Mutate to NQ or QG |
| H84-85 | DS | Isomerization | High | FR3 | Mutate to ES or DA |
| L28 | M | Oxidation | Medium | CDR-L1 | Mutate to Leu or Ile |

**Medium-Risk Sites**:
- H89: Trp (oxidation) - Monitor but likely stable in framework
- L97: Asn (deamidation, NS motif) - Low risk in CDR-L3

**Mitigation Priority**:
1. H54-55 (NG → NQ): Removes high-risk deamidation, retains H-bond capability
2. H84-85 (DS → ES): Removes isomerization, maintains charge
3. L28 (M → L): Reduces oxidation risk, maintains hydrophobicity

**Expected Impact**: Mitigation improves PTM score from 72 → 92

### 5.4 Stability Predictions

**Thermal Stability**:

| Variant | Predicted Tm (°C) | ΔTm vs Original | Aggregation Tonset | Stability Tier |
|---------|-------------------|-----------------|-------------------|----------------|
| Original | 68 | - | 62°C | T3 (Marginal) |
| Humanized_v2 | 71 | +3°C | 64°C | T2 (Good) |
| Affinity_opt | 73 | +5°C | 67°C | T2 (Good) |
| PTM_mitigated | 74 | +6°C | 69°C | T1 (Excellent) |

**Target**: Tm >70°C, Tonset >65°C for long-term stability

**Stability Optimization**:
- Framework humanization improved Tm by +3°C
- Removal of destabilizing motifs: +2°C
- Further optimization possible: Proline introduction in loops

### 5.5 Expression & Manufacturing

**Expression Prediction** (CHO cells):

| Variant | Predicted Titer (g/L) | Soluble Fraction | His-tag Purification | Overall |
|---------|----------------------|------------------|---------------------|---------|
| Original | 1.2 | 75% | Good | T2 |
| Humanized_v2 | 1.8 | 85% | Excellent | T1 |
| Affinity_opt | 2.1 | 88% | Excellent | T1 |

**Manufacturing Considerations**:
- No unusual codons → Good for CHO expression
- No free cysteines → No misfolding risk
- Neutral pI → Easy purification by ion exchange
- Low aggregation → High formulation concentration possible

**Predicted Manufacturing Profile**:
- Expression: 2.0 g/L (CHO fed-batch)
- Purification yield: 75-80%
- Final formulation: >150 mg/mL achievable
- Shelf life: >2 years at 4°C (estimated)

*Source: In silico predictions, sequence analysis*

Phase 6: Immunogenicity Prediction

6.1 T-Cell Epitope Prediction

def predict_tcell_epitopes(tu, sequence):
    """Predict T-cell epitopes using IEDB tools."""

    # MHC-II binding prediction (immunogenicity risk)
    # Query IEDB for predicted epitopes
    predicted_epitopes = []

    # Scan sequence with 9-mer sliding window
    for i in range(len(sequence) - 8):
        peptide = sequence[i:i+9]

        # Search IEDB for similar epitopes
        iedb_results = tu.tools.iedb_search_epitopes(
            sequence_contains=peptide[:5],  # Core sequence
            limit=10
        )

        # If found in IEDB → higher risk
        if len(iedb_results) > 0:
            predicted_epitopes.append({
                'position': i,
                'peptide': peptide,
                'risk': 'High',
                'evidence': f"{len(iedb_results)} similar epitopes in IEDB"
            })

    # Score overall immunogenicity risk
    risk_score = calculate_immunogenicity_risk(predicted_epitopes, sequence)

    return {
        'epitope_count': len(predicted_epitopes),
        'high_risk_epitopes': [e for e in predicted_epitopes if e['risk'] == 'High'],
        'risk_score': risk_score,
        'recommendation': recommend_deimmunization(predicted_epitopes)
    }

6.2 Immunogenicity Risk Scoring

def calculate_immunogenicity_risk(epitopes, sequence):
    """Calculate comprehensive immunogenicity risk score."""

    # Component 1: T-cell epitope count (IEDB-based)
    tcell_score = len(epitopes) * 10  # Each epitope adds 10 points

    # Component 2: Non-human residues in framework
    non_human_residues = count_non_human_residues(sequence)
    non_human_score = non_human_residues * 5

    # Component 3: Aggregation-related immunogenicity
    aggregation_score = assess_aggregation(sequence)['overall_risk'] * 20

    # Total risk (0-100, lower is better)
    total_risk = min(100, tcell_score + non_human_score + aggregation_score)

    return {
        'tcell_risk': tcell_score,
        'non_human_risk': non_human_score,
        'aggregation_risk': aggregation_score,
        'total_risk': total_risk,
        'category': 'Low' if total_risk < 30 else 'Medium' if total_risk < 60 else 'High'
    }

6.3 Output for Report

## 6. Immunogenicity Prediction

### 6.1 T-Cell Epitope Analysis

**Predicted MHC-II Binding Epitopes** (IEDB):

| Position | Peptide | MHC Alleles | IEDB Matches | Risk Level | Region |
|----------|---------|-------------|--------------|------------|--------|
| VH 48-56 | QGLEWMGGI | HLA-DR1, DR4 | 3 | Medium | FR2 |
| VH 78-86 | TDTSTSTA | HLA-DR1 | 5 | High | FR3 (mouse residues) |
| VL 52-60 | LLIYSASSL | HLA-DR1, DR15 | 2 | Medium | FR2 |

**High-Risk Epitope Details**:
- **VH 78-86 (TDTSTSTA)**: Contains mouse-derived residues T84, S85
  - Found in 5 immunogenic peptides in IEDB
  - Recommendation: Backmutate to human consensus (TSTSSAYL)

### 6.2 Immunogenicity Risk Score

| Variant | T-Cell Epitopes | Non-Human Residues | Aggregation Risk | **Total Risk** | Category |
|---------|-----------------|-------------------|------------------|----------------|----------|
| Original (Mouse) | 12 | 38 | High (40) | **118** | High |
| VH_Humanized_v1 | 5 | 13 | Medium (20) | **60** | Medium |
| VH_Humanized_v2 | 4 | 15 | Medium (18) | **53** | Medium |
| Deimmunized | 2 | 10 | Low (12) | **32** | **Low** |

**Risk Scoring**: 0-100 (lower is better)
- Low risk: <30 (clinical candidate ready)
- Medium risk: 30-60 (acceptable with monitoring)
- High risk: >60 (requires optimization)

### 6.3 Deimmunization Strategy

**Recommended Mutations** (to achieve low risk):

| Position | Original | Mutant | Region | Rationale | Impact |
|----------|----------|--------|--------|-----------|--------|
| VH 78 | T | A | FR3 | Human consensus, removes epitope | -15 risk |
| VH 84 | T | S | FR3 | Human consensus, removes epitope | -12 risk |
| VL 55 | S | A | FR2 | Removes MHC-II binding | -8 risk |

**Expected Outcome**:
- Deimmunization reduces risk score: 53 → 32 (Low)
- T-cell epitopes reduced: 4 → 2
- Maintains CDR sequences (no affinity impact)

### 6.4 Clinical Precedent Comparison

**Approved Antibodies - Immunogenicity Rates**:

| Antibody | Target | % ADA (Anti-Drug Antibodies) | Humanization |
|----------|--------|------------------------------|--------------|
| Atezolizumab | PD-L1 | 30% | Fully human |
| Durvalumab | PD-L1 | 6% | Fully human |
| Trastuzumab | HER2 | 13% | Humanized (93%) |
| Rituximab | CD20 | 11% | Chimeric (66%) |

**Our Candidate**:
- Humanization: 85-87% (similar to trastuzumab)
- Predicted ADA risk: 10-15% (after deimmunization)
- Acceptable for clinical development

*Source: IEDB, TheraSAbDab, clinical trial data*

Phase 7: Manufacturing Feasibility

7.1 Expression Optimization

def assess_manufacturing_feasibility(sequence):
    """Assess manufacturing and CMC feasibility."""

    # Codon optimization for CHO
    cho_optimized = optimize_codons(sequence, host='CHO')
    rare_codons = count_rare_codons(sequence, host='CHO')

    # Signal peptide design
    signal_peptide = design_signal_peptide(sequence)

    # Purification considerations
    purification = {
        'protein_a_binding': check_protein_a_binding(sequence),
        'ion_exchange': suggest_ion_exchange_conditions(sequence),
        'hydrophobic': suggest_hic_conditions(sequence)
    }

    # Formulation
    formulation = {
        'target_concentration': predict_max_concentration(sequence),
        'buffer': suggest_buffer_conditions(sequence),
        'stabilizers': suggest_stabilizers(sequence),
        'shelf_life': predict_shelf_life(sequence)
    }

    return {
        'expression': {'cho_optimized': cho_optimized, 'rare_codons': rare_codons},
        'purification': purification,
        'formulation': formulation
    }

7.2 Output for Report

## 7. Manufacturing Feasibility

### 7.1 Expression Assessment

**Expression System**: CHO (Chinese Hamster Ovary) cells

| Parameter | Assessment | Details |
|-----------|------------|---------|
| **Codon optimization** | Good | 5% rare codons (CHO) |
| **Signal peptide** | Native IgG leader | METDTLLLWVLLLWVPGSTG |
| **Predicted titer** | 2.0 g/L | Fed-batch, 14-day culture |
| **Soluble fraction** | 88% | High solubility predicted |

**Recommendations**:
- Use standard CHO expression system (CHO-K1 or CHO-S)
- Express as full IgG1 (not Fab) for Protein A purification
- Standard fed-batch process (no special requirements)

### 7.2 Purification Strategy

**Recommended 3-Step Purification**:

| Step | Method | Purpose | Expected Yield | Purity |
|------|--------|---------|----------------|--------|
| 1. Capture | Protein A affinity | IgG capture | >95% | >90% |
| 2. Polishing | Cation exchange (SP) | Aggregate/variant removal | >90% | >98% |
| 3. Viral | Nanofiltration (20 nm) | Viral clearance | >95% | >99% |

**Overall Process Yield**: 75-80% (from clarified harvest to final product)

**Purification Conditions**:
- Protein A: Standard pH 3.5 elution
- Cation exchange: pH 5.0-5.5 binding, salt gradient elution
- No special requirements (standard IgG process)

### 7.3 Formulation Development

**Recommended Formulation**:

| Component | Concentration | Purpose |
|-----------|---------------|---------|
| **Antibody** | 150 mg/mL | High concentration for SC delivery |
| **Buffer** | 20 mM Histidine-HCl | pH buffering, stability |
| **pH** | 6.0 | Minimizes aggregation (below pI) |
| **Stabilizer** | 0.02% Polysorbate 80 | Reduces surface adsorption |
| **Tonicity** | 240 mM Sucrose | Isotonic, cryoprotectant |

**Formulation Characteristics**:
- Viscosity: <15 cP (suitable for SC injection)
- Osmolality: 300 mOsm/kg (isotonic)
- Stability: >2 years at 2-8°C (predicted)
- Freeze/thaw: Stable for 5 cycles

**Alternative Formulations** (if needed):
- Lower concentration (100 mg/mL) for IV delivery
- Add arginine-glutamate (50 mM) if aggregation observed
- Trehalose (5%) as alternative stabilizer

### 7.4 Analytical Characterization

**Required Assays** (ICH guidelines):

| Assay | Purpose | Specification |
|-------|---------|---------------|
| **SEC-MALS** | Monomer content | >95% monomer |
| **CEX** | Charge variants | Main peak >70% |
| **CE-SDS** | Purity (reduced/non-reduced) | >95% main peak |
| **IEF/cIEF** | Isoelectric point | pI 7.0-7.5 |
| **SPR/ELISA** | Binding affinity | KD <5 nM |
| **DSF** | Thermal stability | Tm >65°C |
| **Cell-based** | Bioactivity | EC50 <10 nM |

### 7.5 CMC Timeline & Costs

**Estimated Development Timeline**:

| Phase | Duration | Activities | Cost Estimate |
|-------|----------|------------|---------------|
| **Cell line development** | 4-6 months | Transfection, selection, cloning | $150K |
| **Process development** | 6-9 months | Optimization, scale-up | $300K |
| **Analytical development** | 3-6 months | Method development, validation | $200K |
| **GMP manufacturing** | 9-12 months | Tech transfer, clinical batches | $1-2M |
| **Total to IND** | 18-24 months | - | **$1.65-2.65M** |

**Manufacturing Scale**:
- Phase 1: 5-10g (small scale, 50L bioreactor)
- Phase 2: 50-100g (pilot scale, 200L)
- Phase 3: 500g-1kg (commercial scale, 2000L)

### 7.6 Risk Assessment

**Manufacturing Risks**:

| Risk | Probability | Impact | Mitigation |
|------|------------|--------|------------|
| Low expression | Low | Medium | Codon optimization, promoter engineering |
| Aggregation | Low | High | Optimized formulation, process controls |
| Glycosylation heterogeneity | Medium | Low | CHO cell line selection, process optimization |
| Charge variants | Medium | Low | Process pH control, storage conditions |

**Overall Manufacturing Risk**: Low (standard IgG process)

*Source: CMC assessment, manufacturing predictions*

Phase 8: Final Report & Recommendations

Report Template

# Antibody Optimization Report: [ANTIBODY_NAME]

**Generated**: [Date] | **Target**: [Target Antigen] | **Status**: Complete

---

## Executive Summary

[Summary of optimization strategy, key improvements, and recommendations...]

**Top Candidate**: [Variant name]
- Humanization: 87% (from 62%)
- Affinity: 1.2 nM (7x improvement)
- Developability score: 82/100 (Tier 1)
- Immunogenicity: Low risk
- Manufacturing: Standard process

**Recommendation**: Advance to preclinical development

---

## 1. Input Characterization
[Section from Phase 1...]

## 2. Humanization Strategy
[Section from Phase 2...]

## 3. Structure Modeling & Analysis
[Section from Phase 3...]

## 4. Affinity Optimization
[Section from Phase 4...]

## 5. Developability Assessment
[Section from Phase 5...]

## 6. Immunogenicity Prediction
[Section from Phase 6...]

## 7. Manufacturing Feasibility
[Section from Phase 7...]

---

## 8. Final Recommendations

### 8.1 Recommended Candidate

**Variant**: VH_Humanized_Affinity_Optimized_v3

**Sequence**:

VH_v3 | Humanized 87%, Affinity optimized, Deimmunized EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMWGIIPIFGTANY AQKFQGRVTMTTDTSTSSAYMELRSLRSDDTAVYYCARARDDGSYSPFDYWGQGTLVTVSS

VL_v3 | Humanized 90% DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKVEIK

### 8.2 Key Improvements

| Metric | Original | Optimized | Improvement |
|--------|----------|-----------|-------------|
| **Humanness** | 62% | 87% | +40% |
| **Affinity (KD)** | 5.2 nM | 0.8 nM | 6.5x |
| **Developability** | 62/100 | 82/100 | +32% |
| **Immunogenicity risk** | High | Low | -70% |
| **Stability (Tm)** | 68°C | 74°C | +6°C |
| **Expression** | 1.2 g/L | 2.0 g/L | +67% |

### 8.3 Experimental Validation Plan

**Phase 1: In Vitro Characterization** (3-4 months)

| Assay | Purpose | Timeline |
|-------|---------|----------|
| Affinity (SPR/BLI) | Confirm KD | Week 1-2 |
| Cell-based binding | Target engagement | Week 2-3 |
| Thermal stability (DSF) | Tm measurement | Week 3 |
| Aggregation (SEC) | Monomer content | Week 3-4 |
| Expression (CHO) | Titer confirmation | Week 4-8 |
| Immunogenicity (in silico + PBMC) | ADA prediction | Week 8-12 |

**Phase 2: Lead Optimization** (2-3 months)
- Test backup variants if needed
- Formulation development
- Scale-up to 100mg

**Phase 3: Preclinical Studies** (6-12 months)
- In vivo efficacy (tumor models)
- PK/PD studies
- Toxicology (GLP)

### 8.4 Alternative Variants (Backup)

| Variant | Profile | Recommendation |
|---------|---------|----------------|
| VH_v2 | Higher humanness (90%) but lower affinity (1.8 nM) | Backup if immunogenicity issues |
| VH_v4 | Highest affinity (0.5 nM) but lower developability (72/100) | Research tool only |
| VH_v1 | Balanced (affinity 2.1 nM, dev 78/100) | Second backup |

### 8.5 Intellectual Property Considerations

**FTO Analysis Required**:
- Check existing patents on anti-[target] antibodies
- CDR sequence novelty assessment
- Humanization method IP landscape

**Patentability**:
- Novel CDR-H3 sequence (14 aa, unique)
- Specific humanization with affinity improvement
- Combination of mutations (H100aY+H52W+L91E)

### 8.6 Next Steps

**Immediate (Month 1-3)**:
1. Synthesize genes for VH_v3, VL_v3, and 2 backups
2. Express in CHO cells (transient and stable)
3. Purify and characterize (affinity, stability, aggregation)
4. Confirm developability predictions

**Short-term (Month 4-6)**:
1. Develop stable CHO cell line (top candidate)
2. Scale up to 500mg for in vivo studies
3. Formulation development and stability studies
4. Initiate in vivo efficacy studies

**Long-term (Month 7-24)**:
1. GMP manufacturing readiness
2. IND-enabling studies (tox, CMC)
3. File IND
4. Phase 1 clinical trial

---

## 9. Data Sources & Tools Used

| Tool | Purpose | Queries |
|------|---------|---------|
| IMGT | Germline identification | IGHV, IGKV genes |
| TheraSAbDab | Clinical precedents | Anti-[target] antibodies |
| AlphaFold | Structure prediction | VH-VL complex |
| IEDB | Immunogenicity | Epitope prediction |
| SAbDab | Structural analysis | PDB structures |
| UniProt | Target information | [Target accession] |

Evidence Grading System

Completeness Checklist

Phase 1: Input Analysis

• Sequence annotated (CDRs, frameworks)
• Species identified
• Target antigen characterized
• Clinical precedents identified

Phase 2: Humanization

• Germline genes identified (IMGT)
• Framework selected
• CDR grafting designed
• Backmutations analyzed
• ≥2 humanized variants designed

Phase 3: Structure

• AlphaFold structure predicted
• CDR conformations analyzed
• Epitope mapped
• Structural quality assessed

Phase 4: Affinity

• Current affinity estimated
• Affinity mutations proposed
• CDR optimization strategies identified
• Testing plan outlined

Phase 5: Developability

• Aggregation assessed
• PTM sites identified
• Stability predicted
• Expression predicted
• Overall score calculated (0-100)

Phase 6: Immunogenicity

• T-cell epitopes predicted (IEDB)
• Immunogenicity score calculated
• Deimmunization strategy proposed
• Clinical precedent comparison

Phase 7: Manufacturing

• Expression system assessed
• Purification strategy outlined
• Formulation recommended
• CMC timeline estimated

Phase 8: Final Report

• Ranked variant list
• Top candidate recommended
• Experimental validation plan
• Backup variants identified
• Next steps outlined

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_protein_by_accession: Target info
• PDB_get_structure: Experimental structures

Systems Biology (for Bispecifics)

• STRING_get_interactions: Protein interactions
• STRING_get_enrichment: Pathway analysis

Special Considerations

Bispecific Antibody Engineering

• Use STRING tools to identify co-expressed targets
• Design separate binding arms for each target
• Consider asymmetric formats (e.g., CrossMAb, DuoBody)
• Assess aggregation risk (higher for bispecifics)

pH-Dependent Binding

• Add His residues at interface (pKa ~6.0)
• Target: Bind at pH 7.4, release at pH 6.0
• Improves PK via FcRn recycling
• Useful for tumor targeting (acidic microenvironment)

Affinity Ceiling

• Most therapeutic antibodies: KD 0.1-10 nM
• <0.1 nM: May cause target-mediated clearance
• 1-5 nM: Sweet spot for most targets
• Balance affinity vs. developability