doi:10.1016/j.jmb.2009.10.008
`
`J. Mol. Biol. (2009) 394, 905–921
`
`Available online at www.sciencedirect.com
`
`Structure of IL-17A in Complex with a Potent, Fully
`Human Neutralizing Antibody
`Stefan Gerhardt1, W. Mark Abbott1⁎, David Hargreaves1,
`Richard A. Pauptit1, Rick A. Davies1, Maurice R. C. Needham1,
`Caroline Langham1, Wendy Barker1, Azad Aziz1, Melanie J. Snow1,2,3,
`Sarah Dawson1, Fraser Welsh2, Trevor Wilkinson2, Tris Vaugan2,
`Gerald Beste2, Sarah Bishop2, Bojana Popovic2, Gareth Rees2,
`Matthew Sleeman2, Steven J. Tuske3, Stephen J. Coales3,
`Yoshitomo Hamuro3 and Caroline Russell2
`
`1AstraZeneca, Alderley Park,
`Macclesfield, Cheshire SK10
`4TG, UK
`2MedImmune, Milstein
`Building, Granta Park,
`Cambridge CB21 6GH, UK
`3ExSAR Corporation, 11 Deer
`Park Drive, Suite 103,
`Monmouth Junction, NJ 08852,
`USA
`Received 16 July 2009;
`received in revised form
`5 October 2009;
`accepted 5 October 2009
`Available online
`14 October 2009
`
`Edited by I. Wilson
`
`IL-17A is a pro-inflammatory cytokine produced by the newly identified
`Th17 subset of T-cells. We have isolated a human monoclonal antibody to IL-
`17A (CAT-2200) that can potently neutralize the effects of recombinant and
`native human IL-17A. We determined the crystal structure of IL-17A in
`complex with the CAT-2200 Fab at 2.6 Å resolution in order to provide a
`definitive characterization of the epitope and paratope regions. Approxi-
`mately a third of the IL-17A dimer is disordered in this crystal structure. The
`disorder occurs in both independent copies of the complex in the asymmetric
`unit and does not appear to be influenced by crystal packing. The complex
`contains one IL-17A dimer sandwiched between two CAT-2200 Fab
`fragments. The IL-17A is a disulfide-linked homodimer that is similar in
`structure to IL-17F, adopting a cystine-knot fold. The structure is not
`inconsistent with the previous prediction of a receptor binding cavity on IL-
`17 family members. The epitope recognized by CAT-2200 is shown to
`involve 12 amino acid residues from the quaternary structure of IL-17A, with
`each Fab contacting both monomers in the dimer. All complementarity-
`determining regions (CDRs) in the Fab contribute to a total of 16 amino acid
`residues in the antibody paratope. In vitro affinity optimization was used to
`generate CAT-2200 from a parental lead antibody using random muta-
`genesis of CDR3 loops. This resulted in seven amino acid changes (three in
`VL-CDR3 and four in VH-CDR3) and gave an approximate 30-fold increase
`in potency in a cell-based neutralization assay. Two of the seven amino acids
`form part of the CAT-2200 paratope. The observed interaction site between
`CAT-2200 and IL-17A is consistent with data from hydrogen/deuterium
`exchange mass spectrometry and mutagenesis approaches.
`© 2009 Elsevier Ltd. All rights reserved.
`Keywords: interleukin-17; cytokine; antibody; epitope; crystal structure
`
`*Corresponding author. E-mail address: mark.abbott@astrazeneca.com.
`Abbreviations used: CDR, complementarity-determining region; scFv, single-chain variable fragment; VH, variable
`heavy; VL, variable light; SPR, surface plasmon resonance; HTRF, homogeneous time-resolved fluorescence; PDB, Protein
`Data Bank; H/D, hydrogen/deuterium; PBS, phosphate-buffered saline; FRET, fluorescence resonance energy transfer;
`DMEM, Dulbecco's modified Eagle's medium; MEM, minimal essential medium; NEAA, nonessential amino acids; MR,
`molecular replacement.
`
`0022-2836/$ - see front matter © 2009 Elsevier Ltd. All rights reserved.
`
`Lassen - Exhibit 1049, p. 1
`
`

`

`906
`
`Introduction
`
`IL-17A is one of six known members of the IL-17
`cytokine family (IL-17A-F).1 It is a secreted homo-
`dimeric glycoprotein with a molecular mass of
`∼35 kDa.2 IL-17 family members play important
`and distinct roles in adaptive immune responses.
`They mediate their effects through the IL-17 receptor
`family, of which there are five related members (IL-
`17RA–IL-17RE; reviewed by Shen and Gaffen3 and
`Gaffen4). Both IL-17A and IL-17F can bind to either
`IL-17RA or IL-17RC, and it has been proposed that
`these colocalize at the cell surface and function as
`heterodimeric receptors.5 It has also been shown
`that IL-17A and IL-17-F can form functional hetero-
`dimers in human T-cells and can induce neutro-
`philia in a murine adoptive transfer model.6,7
`Recent studies have identified Th17 cells as a
`unique and distinct CD4+ T-cell
`lineage that is
`defined by the production of IL-17A, IL-17F, IL-6,
`tumor necrosis factor, granulocyte–macrophage
`colony-stimulating factor, IL-21, IL-22, and IL-26
`(reviewed by Shen and Gaffen3 and Bettelli et al.8).
`Th17 cells are believed to have evolved as an arm of
`the adaptive immune system and have a critical role
`in maintaining inflammatory responses, a role
`previously ascribed to Th1 cells. Th17 cells are
`therefore emerging as strong candidates for drivers
`of autoimmune disease.9
`IL-17A is not widely expressed in humans and is
`only found at very low concentrations, specifically
`in areas populated by Th17 cells. Interestingly, IL-
`17A is expressed in disease compartments in a range
`of autoimmune diseases (reviewed by Witowski
`et al.10) such as rheumatoid arthritis,11–13 multiple
`sclerosis,14,15 psoriasis,16 and inflammatory bowel
`disease.17 In vivo studies have shown that IL-17A
`has a distinct and critical role in driving both the
`early initiation phase and the late progression phase
`of disease in a number of preclinical models of
`rheumatoid arthritis.18
`Given these recent findings, it is not surprising
`that Th17 cells and members of the IL-17/IL-17
`receptor family have become the focus of intense
`investigation and have been viewed as potential
`targets for therapeutic intervention. One group has
`recently developed an anti-IL-17 antibody that is
`currently in early clinical studies.19
`The reported crystal structure of IL-17F (which
`has a 50% sequence identity to IL-17A) presents a
`disulfide-linked homodimeric glycoprotein that
`adopts a classical cystine-knot fold found in the
`transforming growth factor β, bone morphogenetic
`protein, and nerve growth factor superfamilies;
`however,
`it
`lacks the classical disulfide bond
`responsible for the canonical knot20 and instead
`has two serines replacing the cysteine residues. All
`members of
`the IL-17 family lack the cysteine
`residues required to form the knot, but instead
`have conserved serines.
`IL-17A is the most intensively studied member of
`the IL-17 cytokine family, yet no experimentally
`determined structure has been published to date.
`
`IL-17A in Complex with a Neutralizing Antibody
`
`Here, we describe the generation of CAT-2200, a
`potent, fully human neutralizing monoclonal anti-
`body to IL-17A, and reveal the crystal structure of
`IL-17A in complex with a Fab fragment of this
`antibody. This reveals the definitive epitope and
`paratope of the antibody–antigen complex, fully
`satisfying the experimental intention. It is interesting
`to examine the structural context of the mutations
`that result in the improved potency of the CAT-2200
`antibody in relation to the parental clone and to
`speculate which parts of the IL-17A structure might
`be involved in receptor binding.
`
`Results
`
`Isolation of the anti-IL-17A antibody CAT-2200
`
`IL-17A binding antibodies were isolated from a
`large phage library displaying human single-chain
`variable fragments (scFv)21 by panning selections on
`recombinant human IL-17A. A panel of scFv
`isolated from these selections was identified by
`their ability to neutralize the binding of recombinant
`IL-17A to purified IL-17RA·Fc fusion (receptor–
`ligand binding assay), with IC50 values ranging
`from 4 nM to N1000 nM (data not shown). These
`scFv were reformatted as full-length IgG1 mole-
`cules and tested for neutralization of human IL-17A
`in a functional cell assay measuring the release of
`IL-6 from HT1080 cells in response to IL-17A. The
`most potent
`lead antibody identified from the
`cytokine release assay, TINA12, neutralized the
`activity of IL-17A with an IC50 of 23 nM.
`TINA12 was optimized for affinity by a random-
`ized mutagenesis of the variable heavy (VH) and the
`variable light
`(VL) complementarity-determining
`region (CDR) 3. VH CDR3 and VL CDR3 were
`mutated separately to generate a number of li-
`braries. scFv phage libraries containing CDR3
`variants of the lead antibody were subjected to
`multiple rounds of affinity-based solution-phase
`phage display selections. A panel of optimized
`scFv was isolated from these selections through
`their improved ability to neutralize the binding of
`IL-17A to IL-17RA relative to the parental TINA12
`antibody. The optimization process identified scFv
`antibodies with IC50 values of 0.6–12 nM in the
`receptor–ligand binding assay (data not shown).
`These optimized scFv were reformatted as IgG1 (λ
`light chain) and tested for neutralization of human
`IL-17A on HT1080/IL-6 release assay. The VH and
`VL chains from several of the most potent antibodies
`were recombined, and the most potent recombined
`antibody was then reverted by mutagenesis to the
`closest human germline sequence (genes VH3-23
`and VL6-6a) in the VBASE database,22 generating
`the anti-IL-17A antibody CAT-2200. Any frame-
`work residue that was reverted back to germline
`was assayed to ensure that it did not affect antibody
`affinity. CAT-2200 neutralized IL-17A with an IC50
`of 0.8 nM in the HT1080/IL-6 assay, representing a
`
`Lassen - Exhibit 1049, p. 2
`
`

`

`IL-17A in Complex with a Neutralizing Antibody
`
`907
`
`Table 1. A comparison of lead antibody (TINA12) to optimized antibody (CAT-2200)
`
`Heavy chain CDR3
`
`Affinity Kd (nM)
`
`Light chain CDR3
`
`Potency IC50 (nM)
`IL-17A
`IL-17A
`(E. coli)
`(mammalian)
`IgG
`89 90 91 92 93 94 95 96 97 95 96 97 98 99 100 100a 101 102
`23
`ND
`13.5
`Q S Y D D S
`S V V D L
`I W G V
`A
`G
`S
`TINA12
`CAT-2200 Q T Y D P Y S V V D L
`I H G V
`T
`R N
`0.8
`0.13
`2.1
`The numbering of amino acids is performed in accordance with Kabat et al.23 Affinities were measured with BIAcore by immobilizing the
`antibody and using standard procedures in accordance with the manufacturer's instruction. Potency was determined by the ability to
`inhibit the production of IL-6 from HT1080 cells stimulated with recombinant IL-17A.
`
`∼30-fold improvement over TINA12. The seven
`CDR3 amino acid changes (three in VL and four in
`VH) that result in the increased potency of CAT-2200
`compared that of the TINA12 parental clone are
`shown in Table 1.
`The binding affinity of both TINA12 and CAT-
`2200 for IL-17A was measured using surface
`plasmon resonance (SPR). The increase in apparent
`affinity upon optimization of TINA12 to CAT-2200
`was approximately 6-fold for the IgG1 molecule,
`with almost all of the improvements caused by a
`decrease in off-rate.
`
`Functional characterization of CAT-2200
`
`The functional activity of CAT-2200 was assessed
`against a variety of sources of IL-17A on a bio-
`chemical assay and acting on different cell types.
`The antibody potently inhibited the binding of IL-
`17A to IL-17RA-Fc in homogeneous time-resolved
`fluorescence (HTRF) format (Fig. 1a). To assess
`antibody functional activity in a disease-relevant
`cell system, we investigated the effects of CAT-2200
`on recombinant IL-17A-induced IL-8 responses in
`human primary chondrocytes. Escherichia-coli-
`
`Fig. 1. IL-17A-induced responses and their inhibition by CAT-2200. (a) Inhibition of FLAG-tagged IL-17A binding to
`IL-17RA-Fc using HTRF. Triangles, untagged E.-coli-derived IL-17A; circles, CAT-2200; squares, isotype control antibody.
`(b) Recombinant E.-coli-derived IL-17A induced IL-8 release from primary human chondrocytes. Mean ± SD for one donor
`(n= 3). EC50 =0.46 nM. (c) CAT-2200-mediated inhibition of E. coli IL-17A (2 nM) induced IL-8 release from primary
`human chondrocytes. Mean ± SEM for three donors. Mean IC50 =1.56 nM. (d) CAT-2200-mediated inhibition of T-cell-
`derived IL-17A induced IL-6 release from HT1080 cells compared to isotype control. Mean ± SEM for experiments using
`different T-cell supernatants.
`
`Lassen - Exhibit 1049, p. 3
`
`

`

`908
`
`IL-17A in Complex with a Neutralizing Antibody
`
`derived IL-17A generated a dose-dependent increase
`in IL-8 production from primary human chondro-
`cytes with an EC50 of 0.46 nM. CAT-2200 inhibited
`this response with an IC50 of 1.56 nM (Fig. 1b and c).
`In a second system, recombinant IL-17A derived
`from a mammalian cell line was shown to induce the
`production of IL-6 from HT1080 cells with an EC50
`of approximately 0.3 nM. This effect could be
`inhibited by CAT-2200 with an IC50 of 8 nM when
`using 1 nM IL-17A.
`The neutralizing activity of CAT-2200 against
`native IL-17A derived from human T-cells was
`also analyzed (Fig. 1d). T-cells were cultured under
`conditions enhancing IL-17A production.24 Super-
`natants contained IL-17A plus other mediators,
`including IL-6 and tumor necrosis factor α, which
`may synergize with IL-17A. T-cell supernatants
`from three donors induced IL-6 release from
`HT1080 cells, and the effect of CAT-2200 on IL-6
`levels was assessed. The maximum inhibition with
`CAT-2200 was 30%, which was maintained at con-
`centrations of 8 nM and above. Isotype control IgG
`showed no effect on IL-6 levels at this concentration.
`Partial inhibition almost certainly reflects the pres-
`ence of IL-6 in the T-cell-conditioned medium, as
`well as other cytokines that would have been
`produced under the conditioned medium of stimu-
`lated T-cells. Thus, partial inhibition is likely to
`represent that portion that is a result of the IL-17A
`component. This suggestion is also supported by the
`observation that the potency of CAT-2200 in this
`assay is very similar to that in Fig. 1c when recom-
`binant IL-17A is used. Thus, CAT-2200 is able to
`neutralize the activity of a native T-cell-derived
`source of IL-17A.
`The cross-reactivity of CAT-2200 to different IL-17
`family and species variants was assessed by the
`ability of these proteins to inhibit the binding of
`antibody to recombinant human IL-17A derived
`from the mammalian HEK293/EBNA cell line. The
`rank order of the binding of CAT-2200 to different
`species variants was human N cynomolgus N canine,
`with no observed binding to murine IL-17A. In
`addition, CAT-2200 showed no binding to human
`IL-17 family members B–E. Some weak binding
`(20% inhibition at 1 μM) to IL-17F was observed at
`the highest concentration of IL-17F tested.
`In summary, we have isolated an antibody that
`can potently neutralize the effects of recombinant
`and native human IL-17A on a number of cell
`systems. Furthermore, the antibody does not cross-
`react with the other IL-17 family members B–E;
`however, it does recognize IL-17F, albeit with a low
`potency.
`
`Crystal structure of the IL-17A/CAT-2200 complex
`
`Overall complex structure
`The structure of IL-17A/CAT-2200 was refined to
`2.6 Å resolution with R/Rfree of 21.2%/26.4%. The
`asymmetric unit in the crystal contains two complex
`molecules, each with two Fab fragments bound to the
`
`IL-17A dimer. Hence, the final model of six molecules
`present in the asymmetric unit comprises 2043 amino
`acid residues. Of these, 1720 residues are located
`within four molecules of the antibody Fab fragment
`(heavy chains H, I, J, and K, and light chains L, M, N,
`and O). The remaining 323 residues are found in the
`two IL-17A homodimers (chains A/B and C/D).
`More than 99% of all residues of the complex struc-
`ture were found in the most favored and additionally
`allowed regions of the Ramachandran plot.25 Of the
`remaining residues, 0.2% fall into the generously
`allowed regions and 0.5% fall into the disallowed
`regions.
`The crystal structure shows that, in the antibody
`complex, each IL-17A dimer is sandwiched between
`two Fab fragments (Fig. 2), generating two equiva-
`lent IL-17A/Fab interaction sites related by the IL-
`17A dimer symmetry. The buried surface area per
`interface is around 760 Å2. To our surprise, in the
`complex structure, as illustrated in Fig. 2, the lower
`portion of the IL-17A dimer is disordered, indicating
`that it is flexible and adopts different orientations
`throughout the crystal such that electron density is
`averaged out and is not visible. Thus,
`it is not
`possible to build a model for this part of the IL-17A
`dimer with the current data. Two polypeptide
`segments are affected: 34 or 35 amino acid residues
`at the N-termini and 9 or 11 amino acids starting at
`residue 100 or 101. The two independent copies
`reveal the same disorder, differing in extent by just a
`single residue. In the crystal, lattice interactions are
`mediated through the Fab molecules only. There is
`ample room in the lattice for the entire IL-17A
`molecule to be present in a conformation equivalent
`to that seen in IL-17F. Hymowitz et al. described IL-
`17F as a ‘garment’ with a ‘collar,’ ‘sleeves,’ a ‘body,’
`and a ‘skirt’.20 In the IL-17A structure presented
`here, it is the skirt that is disordered. The epitope
`interaction sites are at the collar and sleeves of the
`structure.
`An overlay of CAT-2200 Fab with Protein Data
`Bank (PDB) entry 1AQK, demonstrating that the
`CDRs are in canonical conformation (with the
`exception of VH-CDR3), is shown in Fig. 3.
`
`Structure of IL-17A and comparison to IL-17F
`The structure of IL-17A and structural alignment
`with IL-17F are shown in Fig. 4a and b. Within the
`IL-17 cytokine family, IL-17A is the closest homo-
`logue to IL-17F, with a 50% sequence identity. The
`structure of IL-17F was solved by Hymowitz et al.20
`(PDB entry 1JPY), unexpectedly revealing a cystine-
`knot fold.26 The IL-17A dimer can be superposed
`onto the IL-17F dimer with a root-mean-square
`deviation (r.m.s.d.) of 1.1 Å for 132 Cα positions. The
`sequence identity for the overlaid portion of the
`polypeptides is 64%, higher than the overall
`sequence identity between the two molecules. This
`is not surprising; apparently, the ordered part of the
`molecule is the more conserved part. Each of the
`protomers of IL-17A present in the asymmetric unit
`of the crystal lattice can be superimposed onto each
`
`Lassen - Exhibit 1049, p. 4
`
`

`

`IL-17A in Complex with a Neutralizing Antibody
`
`909
`
`Fig. 2. Overall structure of the IL-17A/Fab complex. This and all other molecular illustrations in this work were
`prepared using PyMOL (http://www.pymol.org). The IL-17A homodimer is shown with the two molecules of the dimer
`in pale and dark yellow. The Fab fragments are shown with the light chain in blue and with the heavy chain in green. The
`constant and VH and VL domains are labeled. The two interaction sites are equivalent, related by IL-17A dimer symmetry.
`The lower portion of the IL-17A dimer is not visible on the electron density map and does not form part of this model. The
`N-termini and C-termini of monomers A and B are indicated.
`
`other using 74–79 α-carbon atoms, giving an r.m.s.d.
`of between 0.32 Å and 0.48 Å.
`IL-17A has a homodimeric assembly. Each sub-
`unit is formed by a set of two pairs of anti-parallel β-
`strands (β1/β2 and β3/β4). A short helix from
`Asp42 to Arg46 is the only helical feature. The IL-
`17A monomer has the same cystine-knot architec-
`ture identified in the crystal structure of IL-17F. The
`
`is formed by the unique
`classical cystine knot
`arrangement of six cysteine residues. In the structure
`of IL-17A, Cys71 and Cys121, as well as Cys76 and
`Cys123, connect β-strands 2 and 4 to form one part
`of
`the knot. A true cystine knot requires an
`additional disulfide to penetrate the ring formed,
`but cysteine-to-serine replacements at positions 49
`and 89 of
`the amino acid sequence of IL-17A
`
`Fig. 3. Overlay of CAT-2200 Fab with PDB entry 1AQK. The constant domains are shown in white. PDB entry 1AQK is
`shown with the light chain in black and with the heavy chain in red. CAT-2200 is shown with the light chain in blue and
`with the heavy chain in green.
`
`Lassen - Exhibit 1049, p. 5
`
`

`

`910
`
`IL-17A in Complex with a Neutralizing Antibody
`
`Fig. 4 (legend on next page)
`
`Lassen - Exhibit 1049, p. 6
`
`

`

`IL-17A in Complex with a Neutralizing Antibody
`
`911
`
`Fig. 5. Wall-eyed stereo representation of the IL-17A epitope recognized by CAT-2200. Residues from the IL-17A
`homodimer are shown in pale or dark yellow. Residues from the Fab fragments are shown with the light chain in blue and
`with the heavy chain in green. Labelled residues allow the interactions listed in Table 2 to be readily located. Residues in
`brackets are the equivalent residue in IL-17F. When no residue in brackets is indicated, then it is identical between IL-17A
`and IL-17F.
`
`preclude final knot formation. This is exactly the
`same situation seen in IL-17F: Ser49 (IL-17A) adopts
`the same rotamer conformation found at Ser50 in the
`IL-17F dimer, similarly for Ser89 (IL-17A) and Ser90
`(IL-17F).
`intermolecular disulfide bond,
`An additional
`corresponding to that between Cys17 and Cys107
`in IL-17F, must also be present in the structure of IL-
`17A (between Cys10 and Cys106), as the recombi-
`nant protein migrated with a molecular mass of
`17 kDa and 35 kDa when analyzed by SDS-PAGE
`under reducing and nonreducing conditions, respec-
`tively. However, this disulfide is not visible, as it
`occurs in the disordered part of the IL-17A structure.
`
`The IL-17A/CAT-2200 interface
`
`The crystal structure allows epitope interactions
`between IL-17A and CAT-2200 Fab to be examined
`in atomic detail. These are shown in Fig. 5 and
`captured in Table 2. It is only necessary to describe
`one of the two interaction sites, since they are
`equivalent. Epitope–paratope interactions involve
`all CDRs from both heavy and light chains, and
`
`amino acid residues from both monomers of the IL-
`17A dimer. The heavy chain interacts with both
`chains A and B in the IL-17A dimer, while the light
`chain interacts only with monomer B.
`Twelve amino acids from IL17A form the epitope
`that interacts with 16 amino acid residues in the
`antibody paratope. The interactions include nine
`hydrogen bonds and nonpolar van der Waals inter-
`actions. The amino acid residues in IL-17A that form
`part of
`the epitope are Ser40-Tyr43 (inclusive);
`Arg46 in chain A; and Leu74, Pro91, Tyr85-Asn88,
`and Pro126-Ile127 in chain B of the dimer. Residues
`contributed from the light chain are Ala29-Tyr32
`(inclusive;
`from CDR1), Phe49 (FW2), Gln53
`(CDR2), and Tyr91 and Pro93 (CDR3). Residues
`from the heavy chain are Thr28 (FW1), Ser30 (FW1)
`to Tyr32 (CDR1), Tyr58 (CDR2), and Leu96-His98
`(CDR3).
`CAT-2200 binds to IL-17F with an affinity that is
`approximately 3 orders of magnitude weaker than
`that for IL-17A. Of the 12 residues in IL-17A that
`form the epitope, five are different in IL-17F. These
`differences are shown in red and in brackets in
`Fig. 5. Several of the changes are nonconservative
`
`Fig. 4. (a–g) have the same orientation. (a) Wall-eyed stereo figure of the IL-17A homodimer, with disulfide bonds
`shown in cyan. The β-strands are labeled for one of the monomers. The N-termini and C-termini of the ordered portion of
`the IL-17A model are indicated for the other monomer. (b) Structural overlay with IL-17F (shown in green; IL-17A is shown
`in two shades of yellow, as described previously). This clarifies the extent of the missing disordered portion of the IL-17A
`dimer. The N-termini and C-termini here are the termini of IL-17F. (c) A surface representation of the IL-17F homodimer
`structure showing residues Asn35-Met40 in red. This peptide has been described as the “right-hand wall” of the cavity that
`is visible just to the left of Arg37, and the cavity is suggested to be a receptor binding pocket.20 (d) Surface representation of
`the IL-17A homodimer. The peptide Asn36-Tyr44 is shown in red: unlike IL-17F, no cavity is seen in IL-17A, may be
`because the antibody induces a conformation change in the peptide, which is also shown in red in (a) and (b); it occurs at the
`N-terminus of the ordered portion of the molecule, suggesting perhaps that the disorder might even be a consequence of
`antibody binding. (e) Modeling of the IL-17F conformation of Asn36-Tyr44 (highlighted in red) into the IL-17A structure,
`demonstrating that a cavity appears (although there are steric clashes with the antibody). (f) A superposition of IL-17A as
`ball-and-stick with a surface representation of IL-17F. Epitope residues are shown in red. This shows that the epitope
`region would be immediately adjacent to the IL-17F cavity if it were preserved in IL-17A. (g) A close-up of the cavity region
`from (f) demonstrating how equivalent residues fill the cavity.
`
`Lassen - Exhibit 1049, p. 7
`
`

`

`912
`
`IL-17A in Complex with a Neutralizing Antibody
`
`Table 2. IL-17A/Fab direct interactions
`
`Fab
`
`Distance (Å)
`
`IL-17A
`Hydrogen bonds
`Ser A40 O
`Asp A42 N
`Asp A42 OD1
`Asp A42 OD1
`Arg A46 NH2
`Arg A46 NH1
`Tyr B85 O
`His B86 NE2
`Asn B88 OD1
`
`Ser H30 OG
`Ser H31 OG
`Thr H28 OG
`Tyr H32 OH
`Leu H96 O
`Ser H31 O
`Tyr L91 OH
`Ala L29 O
`His H98 N
`
`2.9
`3.2
`2.9
`2.7
`2.6
`2.8
`2.5
`3.0
`2.8
`
`3.5
`3.7
`3.6
`3.4
`3.4
`3.5
`3.8
`3.7
`3.4
`3.3
`3.4
`3.2
`3.7
`3.6
`3.6
`3.6
`3.4
`3.8
`3.8
`3.9
`
`Nonpolar (distance corresponds to the closest atom pair)
`Ser A40
`Thr H28
`Ser A41
`Thr H28
`Tyr A43
`Ser H31
`Ser A41
`Ser H31
`Arg A46
`Tyr H32
`His B86
`Asn L30
`His B86
`Tyr L31
`Tyr B85
`Tyr L31
`His B86
`Tyr L32
`Pro B126
`Tyr L32
`Pro B126
`Phe L49
`Ile B127
`Gln L53
`Tyr B85
`Pro L93
`Leu B74
`Pro L93
`Leu B74
`Tyr H58
`Pro B126
`Leu H96
`Asn B88
`Ile H97
`Met B87
`Ile H97
`His B86
`Ile H97
`Leu B74
`His H98
`
`The residue number contains a chain indicator (H: Fab heavy
`chain; L: Fab light chain; A: monomer A in IL-17A; B: monomer B
`in IL-17A). The distance cutoff used for hydrogen bonds is 3.2 Å,
`and that for nonpolar interactions is 4.0 Å.
`
`and therefore provide a potential rationale for the
`much weaker binding to IL-17F.
`The crystal structure contains the unglycosylated
`cytokine, raising the question of whether glyco-
`
`sylation might affect the antibody binding mode
`revealed. There is a single N-linked glycosylation
`site in IL-17A at Asn45. Although this is adjacent to
`Arg46 (which is part of the epitope) and hence close
`to the antibody binding site, the side chain of Asn45
`is oriented towards the solvent and away from the
`bound antibody, and glycosylation is unlikely to
`prevent antibody binding in the manner shown or to
`contribute to antibody binding. In addition, there is
`no significant difference in antibody recognition
`between E.-coli-derived IL-17A (unglycosylated)
`and mammalian-cell-derived IL-17A (partially gly-
`cosylated; data not shown).
`
`Structural context of sequence differences
`between TINA12 and CAT-2200
`
`A total of seven amino acid changes were identi-
`fied in CDR3 between the optimized antibody CAT-
`2200 and the parent TINA12. The crystal structure of
`the IL-17A/Fab complex allows us to examine the
`structural context of these changes and to speculate
`on how they might have improved affinity. Two of
`the changes, D93P (in VL-CDR3) and W98H (in VH-
`CDR3), are part of the paratope (Fig. 6). Pro93 in VL-
`CDR3 forms a stacking interaction with Tyr85 of IL-
`17A, probably providing a significant improvement
`in interface surface complementarity. Interestingly,
`the D93H mutation also appeared in other opti-
`mized constructs, consistent with the notion that
`side-chain stacking may be beneficial. The D93P
`mutation would restrict the psi main-chain torsion
`angle for the proline residue to ∼−60°, which would
`help rigidify CDR3, although comparison is difficult
`without the structural details of TINA12. The second
`mutation in the paratope is W98H. In the structure,
`this residue has a main-chain amide hydrogen bond
`to the side chain of Asn88 in IL-17A. Since it is a
`main-chain hydrogen bond, it is also presumably
`present in TINA12.
`
`Fig. 6. Wall-eyed stereo representation of the CAT-2200 paratope. Residues from the IL-17A homodimer are shown in
`pale or dark yellow. Residues from the Fab fragments are shown with the light chain in blue and with the heavy chain in
`green. The seven amino acids that are different between CAT-2200 and the lead antibody TINA-12 have been labeled.
`
`Lassen - Exhibit 1049, p. 8
`
`

`

`IL-17A in Complex with a Neutralizing Antibody
`
`913
`
`Fig. 7. H/D exchange pattern of IL-17A. Each horizontal color block represents an analyzed peptic peptide, and each
`block contains a number of time points. The N-terminal 21 residues constitute an Avi tag sequence. Numbering starts at
`the first IL-17A residue. Deuterium build-up patterns of IL-17A in solution (from top: 15 s, 50 s, 150 s, 500 s, and 1500 s).
`The deuteration level of each peptide at each time point is color coded (see bottom right).
`
`The other five substitutions occur away from the
`interface, and any effect on the paratope would have
`to be indirect via either a stabilizing effect on VH–VL
`interactions or intrachain contacts. Two of these
`mutations (S90T and S94Y) form a hydrogen bond
`between CDR3 and the backbone amide of Tyr32
`(VL-CDR1) and Tyr59 (VH-CDR2), respectively,
`which may help stabilize loop conformations that
`affect the shape of the paratope. The effects of these
`distant changes are unlikely to improve binding
`directly, although they may improve the stability of
`the antibody fragment, thereby lowering binding
`energy and indirectly promoting antigen binding.
`The three remaining amino acid substitutions in
`the C-terminal part of VH-CDR3 are A100aT,
`G101R, and S102N. They are located away from
`the paratope, but close to VL residues Tyr36 and
`Ile46. Thr100a forms a hydrogen bond with VL
`Tyr36 that is not possible in TINA12. Perhaps these
`mutations help stabilize VH–VL contacts in the
`antibody, leading to improved binding.
`
`Analysis of IL-17A/CAT-2200 interaction via
`hydrogen/deuterium exchange and mutagenesis
`
`Two additional approaches were taken in order to
`analyze the interaction between IL-17A and CAT-
`2200: hydrogen/deuterium (H/D) exchange coupled
`to mass spectrometry and mutagenesis.
`The principles and methods behind epitope
`mapping by H/D exchange mass spectrometry are
`that the rate of exchange of the antigen is measured
`in the presence and in the absence of antibody and
`the two are compared.27 Following exchange, the
`protein is digested with pepsin, and those peptides
`that exchange more slowly in the presence of
`antibody are the H/D-exchange-defined epitope.
`Figure 7 shows a map of the rate of exchange across
`IL-17A in the absence of antibody. Of particular note
`
`is that the first 42 amino acids of IL-17A exchange
`almost completely even at the shortest time of 15 s,
`suggesting that this part of the protein is extremely
`dynamic. The averages of the differences in the rates
`of exchange in the absence and in the presence of
`antibody are shown in Table 3. Two peptides whose
`exchange properties altered significantly upon
`binding to CAT-2200 (residues 45–53 and 71–87)
`were identified. A third peptide encompassing
`residues 119–132, whose exchange properties were
`slightly altered, was identified. When comparing
`these regions to the crystal structure, it is clear why
`the exchange is altered when the IL-17A is bound to
`CAT-2200. Peptide 45–53 contains Arg46, which
`makes several interactions with the heavy chain.
`Peptide 71–87 contains Tyr85 and His86, both of
`which form multiple interactions with the light
`
`Table 3. Difference in the H/D exchange rates of IL-17A
`in the presence and in the absence of CAT-2200
`
`Start
`−19
`−12
`−2
`26
`45
`56
`71
`90
`100
`113
`119
`
`End
`−15
`−5
`23
`42
`53
`68
`87
`97
`110
`116
`132
`
`Difference (%)
`2
`2
`3
`1
`11
`1
`18
`2
`3
`1
`6
`
`IL-17A was deuterated and exchanged back to hydrogen in the
`presence and in the absence of antibody, as described in Materials
`and Methods. The average difference in the deuteration levels of
`different peptic fragments of the protein was determined by
`digestion with pepsin and analysis by mass spectrometry.
`Residue 1 is Gly24, as described in SwissProt accession number
`Q16552. Residues with negative numbers are in Avi tag.
`
`Lassen - Exhibit 1049, p. 9
`
`

`

`914
`
`IL-17A in Complex with a Neutralizing Antibody
`
`chain, and also Leu74 and Met87, which are
`involved in the binding interface. Peptide 119–132
`contains Pro126 and Ile127, which interact with the
`light chain.
`In summary,
`the data from H/D
`exchange mass spectrometry are consistent with
`the crystal structure and demonstrate the value of
`this approach.
`A mutant of IL-17A was made in order to further
`understand the interaction with CAT-2200. This
`mutant took advantage of the observation that CAT-
`2200 binds to human IL-17A with high affinity, but
`does not bind to murine IL-17A (Table 1). The
`mutant
`IL-17A was designed from the H/D
`exchange experiments covering the region 71–89,
`where perturbation in H/D exchange was most
`pronounced. Every amino acid in that region that
`varied between the human sequence and the mouse
`sequence was changed from human residue to
`murine residue (L74Q, G75R, I77V, D80E, N82K,
`V83L, and Y85H).
`The mutant and wild-type proteins were ex-
`pressed in mammalian cells (HEK293/EBNA), puri-
`fied, and analyzed for their binding to CAT-2200 by
`SPR. The functional ability of the mutants to induce
`IL-6 production from