Monoclonal antibodies inhibiting HIV PR

Introduction:
The protease of HIV plays a critical role in the maturation of the infectious particles of the virus. The enzyme has therefore been extensively studied with the objective of developing therapeutics that inhibit viral proliferation.
We have produced monoclonal antibodies specific for the HIV-1 protease, and selected those that inhibit enzyme function for use as probes to study the enzyme’s activity and as an eventual aid for the development of potential inhibitors targeted to regions other than the active site. We have characterised two such mAbs, F11.2.32 and 1696, which have inhibition constants in the low nanomolar range and which recognise epitopes from different regions of the protease. The crystal structures of the two antibodies, both in the free state as well as complexes with peptide fragments corresponding to their respective epitopes, have been solved. The structural analyses, taken together with other functional data on the antibodies, suggest mechanisms
of protease inhibition by these antibodies.

Anti-HIV-1 protease monoclonal antibodies:
The anti-protease mAbs were produced in BALB/c mice by the hybridoma technique using recombinant HIV-1 protease as immunogen.
Inhibition constants of the antibodies for proteolysis were determined by measuring the cleavage rate of the substrate KARVNleEF(NO2)EANle using reverse-phase HPLC. Screening of anti-HIV-1 protease monoclonal antibodies for inhibition
of proteolysis by this method led to the selection of two promising candidates, F11.2.32 and 1696, with inhibition constants of 35 nM and 1.0 nM, respectively.
Epitope mapping studies showed that these two antibodies recognise different regions of the protease. F11.2.32 cross-reacts with a peptide corresponding to the segment 36-46 of the HIV-1 protease, which forms an exposed loop at the N-terminal end of the flap region.
Monoclonal antibody 1696 binds to peptides corresponding to the segment 1-7 or to longer sequences such as 1-13, but not the peptide 2-8, showing a critical contribution by the N-terminal proline. Although this region contributes to the b-pleated sheet at the dimer interface, both N-termini form the outer strands and are thus exposed. The N-terminal region is very conserved between the different stains of HIV1. Furthermore, 1696 cross-reacts with the HIV-2 protease and the corresponding peptide 1-7 from this enzyme. For the HIV-2 species, the N-terminal is likewise very conserved, and is also very similar to the HIV-1 protease. Only two conservative changes occur: Ile3->Phe and Thr4->Ser.

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Figure 1:
The main-chain trace of the HIV-1 protease homodimer, showing the location of the epitopes recognized by mAb 1696 (residues 1 to 7 in red) and mAb F11.2.32 (residues 36 to 46 in yellow).

Structural study of monoclonal antibody F11.2.32
The structure of the Fab fragment of F11.2.32 has been determined in the non-complexed state as well as a complex with HIV-1 protease peptide derived from the segment 36-46 (P36-P46).

PBD entry:	Description:
1mf2	free Fab F11.2.32
2hrp	Fab F11.2.32 in complex with HIV-1 PR epitope peptide (36-46)

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Figure 2:
Overall structure of the Fab F11 in complex with HIV-1 epitope peptide (36-46). The Fab F11. is represented by ribbons: light chain constant (CL) and variable (VL) domains in green and heavy chain constant (CH) and variable (VH) domains in red. CDR L1 - L3 and H1 - H3 are marked.
All hypervariable loops, except CDR-L2, make contact with the antigenic peptide. Being at the periphery of the antigen-binding site, CDR-L2 is shielded from contacting the antigen by the long CDR-L1 and CDR-H3 hypervariable loops.
Ten of the eleven residues of the bound peptide, P36-P46, make direct contact with the antibody and are clearly visible in the electron density. The peptide adopts a compact b hairpin conformation that is stabilised by several intramolecular polar interactions, with a type II b turn.

Structural study of monoclonal antibody 1696:
The structure of 1696 has also been studied in the free and complexed state, both with the peptide segment 1-7 from the HIV-1 protease as well as the cross-reacting HIV-2 enzyme. Although the free state is as the Fab fragment, both peptide complexes were formed with the recombinant Fv fragment.

PBD entry:	Description:
1cl7	free Fab 1696
1n4x	free scFv1696
1jp5	scFv1696 in complex with HIV-1 PR epitope peptide (1-7)
1svz	scFv1696 in complex with HIV-2 PR epitope peptide (1-7)

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Figure 3:
An overhead view of the topology of the antigen-binding site. Peptide is represented as stick model, scFv1696 by its solvent accessible surface.
Left: The surfaces are color coded for electrostatic potential: red for negative and blue for positive.
Right: The surfaces are color coded for each of six CDRs.
The topology of the antigen-binding site of 1696 can be described as a shallow groove running between the two domains, but partially blocked off at one end by CDR-H1 and CDR-H3. It thus contrasts with the deep cavity characterising F11.2.32.
Only the first six N-terminal residues of the peptide are visible in the electron density maps of both 1696 complexes. These adopt an extended in conformation, with the segment P2-P4 occupying the b region of the Ramachandran plot. The open conformation of the peptide corresponds roughly to that of the corresponding segment of the native protease

Results:
The protease peptide P36-P46, when bound to F11.2.32, adopts a beta-hairpin turn structure, thus differing significantly from that of the corresponding segment in the native structure of the protease itself. If the interaction of F11.2.32 with the protease is closely mimicked by the peptide complex, then the viral enzyme cannot be in its native conformation, and might even be extensively denatured. This region of the native protease is located at the N-terminal flank of the flap region, which is flexible and plays a crucial role in the proteolytic activity of the enzyme. Structural distortion of the protease upon binding by F11.2.32 would thus be sufficient to account for inhibition of the enzyme by the antibody.
The conformation of the peptide P1-P6, when bound to 1696, is extended as in the native protease. It is not possible, however, to dock the protease into the binding site of the antibody without engendering steric hindrance with the adjacent monomer in the active dimeric form of the enzyme. This would imply that if the 1696 interacts with the protease in the same way as with the peptide, the monomer-monomer interface could be significantly distorted from the conformation present in the active homodimer, even to the extent that the enzyme might be completely dissociated.

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Figure 4:
Comparison of structures adopted by the epitope peptides in Fab F11 and scFv1696 peptide complexes with the corresponding part of unliganded HIV PR.
Left: Comparison of peptide 36-46 from Fab F11-peptide complex (yellow) with the native part of HIV-1 PR (red).
Right: Comparison of peptide 1-7 from scFv1696-peptide complex (yellow) with the N-terminus of HIV-1 PR (red).

References:

mAb F11.2.32:
Lescar J, Stouracova R, Riottot MM, Chitarra V, Brynda J, Fabry M, Horejsi M, Sedlacek J, Bentley GA.: Three-dimensional structure of an Fab-peptide complex: structural basis of HIV-1 protease inhibition by a monoclonal antibody. J Mol Biol. 1997 Apr 18;267(5):1207-22.[MEDLINE]

Lescar J, Stouracova R, Riottot MM, Chitarra V, Brynda J, Fabry M, Horejsi M, Sedlacek J, Bentley GA.:
Preliminary crystallographic studies of an anti-HIV-1 protease antibody that inhibits enzyme activity. Protein Sci. 1996 May;5(5):966-8. [MEDLINE]

Stouracova R, Lescar J, Brynda J, Riottot MM, Chitarra V, Fabry M, Horejsi M, Rezacova P, Bentley G, Sedlacek J.:
Anti-HIV proteinase monoclonal antibody F11.2.32 that inhibits enzyme activity. Gen Physiol Biophys. 1998 Jun;17 Suppl 1:6-8. [MEDLINE]

Lescar J, Stouracova R, Riottot MM, Chitarra V, Brynda J, Fabry M, Horejsi M, Sedlacek J, Bentley GA.: Structural studies of HIV-1 protease-inhibiting antibodies. Gen Physiol Biophys. 1998 Jun;17 Suppl 1:3-6. [MEDLINE]

mAb1696
Rezacova P., Brynda J., Lescar J., Fabry M., Horejsi M., Sieglova I., Sedlacek J., Bentley G.A.: Crystal structure of a cross-reaction complex between an anti-HIV-1 protease antibody and an HIV-2 protease peptide. J. Struct. Biol. (2005): in press
Lescar J, Brynda J, Rezacova P, Stouracova R, Riottot MM, Chitarra V, Fabry M, Horejsi M, Sedlacek J, Bentley GA.: Inhibition of the HIV-1 and HIV-2 proteases by a monoclonal antibody. Protein Sci. 1999 Dec;8(12):2686-96. [MEDLINE]

Rezacova P, Lescar J, Brynda J, Fabry M, Horejsi M, Sedlacek J, Bentley GA. Structural basis of HIV-1 and HIV-2 protease inhibition by a monoclonal antibody. Structure (Camb). 2001 Oct;9(10):887-95.[MEDLINE]

J. Lescar, J. Brynda, M. Fabry, M. Horejsi, P. Rezacova, J. Sedlacek and G. A. Bentley: Structure of a single-chain Fv fragment of an antibody that inhibits the HIV-1 and HIV-2 proteases. Acta Cryst. (2003). D59, 955-957 [MEDLINE]

Review on both mAbs:
Rezacova P, Brynda J, Fabry M, Horejsi M, Stouracova R, Lescar J, Chitarra V, Riottot MM, Sedlacek J, Bentley GA.: Inhibition of HIV protease by monoclonal antibodies. J Mol Recognit. 2002 Sep-Oct;15(5):272-6. [MEDLINE]