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Antibody domain exchange is an

immunological solution to carbohydrate cluster

recognition.

Daniel Calarese … Ian A. Wilson(16 authors from

Scripps, Oxford, FSU, U Ag Vienna)Science June 27, 2003 (300:2065)

Burton, Wilson, and Calarese

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What is “domain exchange”?

• 2 Ordinary Fabs

• VH domains exchangedForming a Fab dimer with

two ordinary VH –VL paratopes

+ two new VH –VH paratopes

H L

Variable Domains

Constant Domains

Paratope

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“Toober” models

Two

Conven-

tional

Fab’s

2G12

Fab’s

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What is 2G12?

• Human monoclonal IgG1 from patient B cells.• Neutralizes unusually broad range of known HIV

strains (25-50%).• Binds to dense cluster of carbohydrate epitopes

on the “silent” face of gp120.• Unusually high affinity among anti-carbohydrate

antibodies.• Antibodies to the 2G12 epitope are unusual in

sera from HIV+ patients (by competition).

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The “Silent” face of gp120

• Is an unusual epitope among HIV+ patient antibodies.• Potential protein epitopes beneath the carbohydrate are

shielded.• Carbohydrate is made by host (not virus enzymes)

hence may be “self” (though the dense cluster of oligomannose residues has not been described on mammalian glycoproteins).

• Has multiple glycoforms diluting any one epitope.• Antibodies to carbohydrate epitopes usually have low

affinities (Kd micromolar; but 2G12 Kd is nanomolar).

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Why is the structure of 2G12 important?

• Paradigm shift: functional framework changes via somatic hypermutation.

• Paradigm shift: VH-VH paratope.• Exception to the rule that Fab:antigen

interactions are monovalent.• Domain-swapped dimer is unprecedented

among >250 published Fab structures.• Explains high affinity and ability to bind closely-

spaced epitopes.

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Protein Crystallography

• Accounts for 85% of published structures.

• Has an overall success rate of ~3%.

• <1% of the human proteome has crystallographically known structure.

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Methods

• Express full IgG1 in CHO cells.• Papain Fab (completeness confirmed by SDS-PAGE).• Purif. Prot. A/G columns, 30 mg/ml.• 1 μl (ligand 5 to 1 molar ratio) + 1 μl cryst. sol’n

(ammonium/sulfate/phosphate, PEG, imidazole malate, pH 6/7), sitting drop crystallization.

• Diffract Stanford Synchrotron cryopres. in Liq. N2

• Phase solved by molecular replacement with best-fitting existing Fab structure (1fvd).

• Model of known amino acid sequence built into electron density map and refined (with restraints) for best fit.

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Crystallographic Results

Unliganded 2G12 Fab*

+Man2 +Man9GlcNAc2

Resolution 2.2 Å 1.75 Å 3.0 Å

Number of Reflections

60,000 126,000 30,000

R 0.22 0.23 0.25

Rfree (5%) 0.27 0.25 0.33

PDB ID 1OM3 1OP3 1OP5

* ~6,600 atoms

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Framework Somatic MutationsFacilitate Domain Exchange

1. Weaken VH-VL

2. Alter H Chain ElbowConserved ball and socket is not mutated but is dislocated.

Mutated residues add stabilization.

3. Strengthen VH-VH’

See details in Protein Explorer

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Is this bizarre Fab dimer for real?It is not observed in >250 published Fab crystal structures.

Fab: about 80% dimer + 20% monomer in solution by• Sedimentation equilibrium analytical ultracentrifugation.• Gel filtration.

Intact IgG1 has compact conformation by• Velocity sedimentation coefficient.• Negative staining electron microscopy.

– With and without gp140 ligand

Apparently the dimer is for real.

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Fig. 2c: Electron microscopy of intact 2G12 IgG1 shows a compact form (neither Y nor T) with or without bound

antigen.

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Mutants of 2G12reducing gp120 binding affinity ~100-1,000 fold

• Primary combining site (VH-VL) 5/11

• Secondary binding site (VH-VH) 4/5

• Domain exchange-facilitating residues– VH-VH 4/4

– Elbow 3/3

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Function of HIV oligomannose

• DC-SIGN is a human lectin.(Dendritic Cell-Specific ICAM-3 Grabbing Nonintegrin)

• Facilitates infection of CD4+ cells by binding HIV carbohydrate.

• Speculation: HIV-1 evolved oligomannose to utilize DC-SIGN.

• 2G12 exploits this “Achilles heel” of HIV-1.

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Conclusions

• 2G12 forms a novel domain-swapped dimer (in 3 crystals and in solution) in both Fab and intact IgG1.

• A novel form of paratope occurs at the VH-VH’ interface.

• The novel conformation is enabled by somatic mutations to framework residues.

• The resulting 4-paratope array accommodates closely spaced carbohydrate epitopes (35 Å, vs. 50-140 Å for Y or T) achieving nanomolar avidity. An epitope on gp120 is predicted.

• Vaccines mimicking oligomannose cluster may elicit protective Abs.• 2IG2: scaffold for designing Abs to other epitope clusters?

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Alternative Antibody Lifestyles

Naturally Occurring

• Camel H chain dimer (no L; “VHH”)• (Muyldermans et al., TiBS 26:230, 2001)

• VH domain-swapped dimer• (Calarese et al., Science 300:2065, 2003)

Engineered

• Fv (VH-VL with no C domains)

• Fv domain-swapped “triabody” (trimer ring)• (Pei et al. PNAS 95:9637, 1997)

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Some High-Impact Crystal Structures

• Myoglobin (1960): first example of protein structure.• Lysozyme (1965): first enzyme.• Fab (1973): first immunoglobulin fold (shared in unrelated proteins,

first SOD).• tRNA (1975): first RNA structure.

• MHC (1987): explained restriction of T cell recognition.• Ribosome (2000): peptidyl transferase is a ribozyme.• 2G12 (2003): unprecedented dimeric paratope with framework

somatic mutations.

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First crystal structureswithout big surprises (less impact)

• DNA double helix (1973-80): predicted correctly in 1953.

• T cell antigen receptor (, 1996).

• TCR (2001)

• CD1 with phospholipid ligand (2002)

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Fig. 3D

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Fig. 3B

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Fig. 6: Model of 2G12 + gp120