Specification
FIELD OF THE INVENTION
The present invention relates to IL-17 Receptor A (IL-17RA or 1L-17R) antigen binding
proteins, such as antibodies, polynucleotide sequences encoding said antigen binding proteins,
and compositions and methods for diagnosing and treating diseases mediated by IL-17 Receptor
A activation by one or more IL-17 ligands. The present invention relates to the identification of
neutralizing determinants on IL-17 Receptor A (IL-17RA or IL-17R) and antibodies that bind
thereto. Aspects of the invention also include antibodies that compete for binding with the IL-
17RA neutralizing antibodies described herein.
BACKGROUND
IL-17A is an inflammatory cytokine initially identified as a transcript selectively
expressed by activated T cells. IL-I7RA is a ubiquitously expressed and shown to bind IL-17A
with an affinity of approximately 0.5 nM (Yao et al, 1995, Immunity 3:811-821). Five additional
IL-17-like ligands (IL-17B-IL-17F) and four additional IL-17RA-like receptors (IL-17RB-1L-
17RE) have been identified (Kolls and Linden, 2004, Immunity 2\ :467-476).
IL-17RC has been shown to bind IL-I7A and IL-17F. The observations that IL-17RA
deficiency and IL-17RA antibody neutralization ablate both IL-17A and IL-17F function suggest
that IL-17RC cannot deliver an IL-17A or IL-17F signal in the absence of IL-17RA (Toy et al.,
2006, J. Immunol. 177:36-39; McAllister et al, 2005, J. Immunol. 175:404-412). Additionally,
forced expression of IL-17RC in IL-17RA deficient cells does not restore IL-17A or IL-17F
function (Toy e?fl/., 2006, J. Immunol. 177:36-39).
IL-17A and IL-17F are predominantly expressed by activated CD4* memory T cells
(Kolls and Linden, 2004, supra). It has been proposed that an IL-17A-producing pathogenic
CD4+ T cell subset, ThIL-17, is expanded in the presence of IL-23 (Langrish et al, 2005, J. Exp.
Med. 201:233-240). Additionally, both IL-15 and the TNF superfamily member OX40L have
been shown to induce the expression of IL-17A (Nakae et al, 2003b, Proc. Natl Acad. Sci.
U.S.A. \00:59S6-5990; Zio\kov^ska etal, 2000, J. Immunol 164:2832-2838). IL-6 and TGF-beta
also induce the expression of IL-17A.
1L-17A and IL-17F bind and activate IL-17RA. IL-17RA has been shown to be
important in regulating immune responses. Activation of the IL-17RA leads to production of
cytokines, chemokines, growth factors, and other proteins that contribute to the symptoms and/or
pathology of numerous diseases. IL-17A is an inflammatory cytokine that induces the production
of cytokines and other mediators leading to diseases and physiological effects such as
inflammation, cartilage degradation, and bone resorption. IL-17A also plays a role in a number
of inflammatory conditions including arthritis (rheumatoid arthritis), psoriasis, inflammatory
bowel disease, multiple sclerosis, and asthma. (Li et al., 2004, Huazhong Univ. Sci. Technolog.
Med. Sci. 24:294-296; Fujino et al, 2003, Gut. 52:65-70; Kauffman et al., 2004, J. Invest.
Dermatol. 123:1037-1044; Mannon et ai, 2004, A^. Engl. J Med. 351:2069-2079; Matusevicius et
al., 1999, MultScler 5, 101-104; Linden etal., Eur RespirJ. 2000 May;15(5):973-7; Molet et al.,
2001, J. Allergy Clin. Immunol. 108:430-438). Recent studies have suggested that IL-17F plays a
role in the induction of inflammatory responses (Oda et al, 2006, American J. Resp. Crit. Care
Medicine, Jan. 15, 2006; Numasaki et al, 2004, Immunol Lett. 95:97-104).
Aspects of the invention provide antigen binding proteins that specifically bind IL-I7RA
and inhibit IL-17RA activation mediated by IL-17 family members, such as, but not limited to,
IL-17A and/or IL-17F, as described more fully herein.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGURE I shows a phylogenetic dentogram analysis of the CDRs (complementarity
determining regions) of the variable heavy (VH) and variable light (VL) domains of various IL-
17R antigen binding proteins (antibodies).
FIGURE 2 depicts an alignment of the amino acid sequences of the CDRs of the variable
heavy (VH) domains of various IL-17R antigen binding proteins (antibodies). The CDRl, CDR2,
and CDR3 regions are highlighted.
FIGURE 3 depicts an alignment of the amino acid sequences of the CDRs of the variable
light (VL) domains of various IL-17R antigen binding proteins (antibodies). The CDRl, CDR2,
and CDR3 regions are highlighted.
FIGURE 4 shows that the mean clinical scores of IL-17RA-/- mice (knockout mice or KG
mice) are much lower than that of wild-type (WT) mice in a CIA model of arthritis.
FIGURE 5 shows the delay in experimental autoimmune encephalomyelitis (EAE) onset
for IL-17RA knockout mice compared to wild-type mice in a myelin oligodendrocyte
glycoprotein (MOG)-induced model.
FIGURE 6 shows reduced clinical scores in IL-17RA knockout mice as compared to
wild-type mice in a MOG-induced model.
FIGURE 7 shows IL-17RA knockout mice have reduced total numbers of inflammatory
cells in BAL fluid compared to wild-type in an ovalbumin-induced model of asthma.
FIGURE 8 shows IL-17RA knockout mice have reduced numbers of esoinophils
(FIGURE 8A), neutrophils (FIGURE 88) and lymphocytes (FIGURE 8C) in bronchoalveolar
lavage (BAL) fluid as compared to wild-type mice in an ovalbumin-induced model of asthma.
FIGURE 8D shows no changes in BAL fluid macrophage observed in either WT or IL-17RA
knockout mice (naive and OVA challenged).
FIGURE 9 shows dose-dependent inhibition by an IL-17RA mAb in a wild-type (WT)
collagen-induced arthritis (CIA) model. A P<0.05 was seen when comparing IL-17RA mAb at
100 |xg and 300 |ig treatment groups versus control treatment group (days 13, 15 and 16).
FIGURE 10 shows the results of therapeutic treatment with IL-17RA mAb. The data shows
stabilized mean clinical scores in wild-type mice in a standard CIA model of arthritis. These data
demonstrate that IL-17RA inhibition by an IL-17RA antigen binding protein may be
therapeutically useful in treating rheumatoid arthritis (RA), especially in the preservation of joint
bone and cartilage.
FIGURE 11 shows that therapeutic treatment with anti-IL-I7RA mAb stabilized mean
clinical scores in TNFR p55/p75 knockout mice in a standard CIA model of arthritis. These data
show that 1L-17RA inhibition by an IL-17RA antigen binding protein may be therapeutically
useful in treating RA, especially in the preservation of joint bone and cartilage. Notably, IL-
17RA inhibition was able to stabilize disease in a model independent of TNF signaling.
FIGURE 12 shows exemplary IL-17RA human mAbs (AMH14/AML14, AMH22/AML22,
AMH19/AMLI9, and AMH18/AML18) were able to inhibit cynomologous IL-I7-induced IL-6
production from JTC-12 cells (cynomologous kidney cell line). The (—) line depicts the
positive control value of cynomologous IL-17 in combination with TNF-alpha. The (-.-.-) line
depicts the positive control value of cynomologous TNF-alpha. The (....) line depicts the media
control value.
FIGURE 13 shows sequence variation in the framework regions of SEQ ID NO:40
(AML14) in relation to germline residues and the effect on IC50 values.
FIGURE 14 shows that the two variants having residues returned to germline (see
FIGURE 13) had reduced IL-17A inhibitory activity in relation to AMH14/AML14, indicating that
some variation in the framework regions was tolerated but that some residues may influence
activity. The (-—) line indicates the positive control value of IL-17 stimulation in the absence of
antibody (approximately 4062 pg/ml).
FIGURE 15 shows that the two variants having residues returned to germline (see
FIGURE 13) had reduced IL-17F (in combination with TNF-alpha) inhibitory activity in relation
toAMHM/AMtM.
FIGURES 16A and 16B show the results of multiplexed binning of IL-17RA antibodies.
Shaded values indicate antibody pairs that can bind to 1L-I7RA simultaneously, suggesting that
these antibodies bind to different neutralizing determinants. Boxed values indicate antibodies
paired against themselves.
FIGURE 17 shows mouse IL-17RA (SEQ ID NO:432) and the 5 domains, A, B, C, D, E,
and F that replaced the counterpart domains in the human IL-17RA sequence.
FIGURES 18A-18D shows the amino acid sequences for human and mouse IL-17RA and
human/mouse chimeric IL-17RA proteins.
FIGURE 19 is a table summarizing the IL-17RA mAbs capacity to bind the various
chimeric proteins. Shaded values denote where the IL-I7RA mAbs lost binding to that particular
chimera (n.d. means not determined).
FIGURE 20 depicts the amino acid residues that were replaced with an arginine residue in
SEQIDNO:431.
FIGURE 21 illustrates titration curves of various IL-17RA mAbs binding to the D152R
IL-17RA mutant.
FIGURE 22 is a summary of the arginine scan, binning, and chimera data for various IL-
17RAmAbs.
DETAILED DESCRIPTION OF THE INVENTION
The section headings used herein are for organizational purposes only and are not to be
construed as limiting the subject matter described.
Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, tissue
culture and transformation, protein purification etc. Enzymatic reactions and purification
techniques may be performed according to the manufacturer's specifications or as commonly
accomplished in the art or as described herein. The following procedures and techniques may be
generally performed according to conventional methods well known in the art and as described in
various general and more specific references that are cited and discussed throughout the
specification. See, e.g., Sambrook et al, 2001, Molecular Cloning: A Laboratory Manual, V^
ed.. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., which is incorporated
herein by reference for any purpose. Unless specific definitions are provided, the nomenclature
used in connection with, and the laboratory procedures and techniques of, analytical chemistry,
organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well
known and commonly used in the art. Standard techniques may be used for chemical synthesis,
chemical analyses, pharmaceutical preparation, formulation, and delivery and treatment of
patients.
IL-17A, IL-17F, and IL-17RA
The biologic activities of IL-17A and IL-17F are dependent upon IL-17RA, as shown
herein using both cells and mice that are genetically deficient in IL-17RA and with neutralizing
mAbs (monoclonal antibodies) directed against IL-I7RA {see Examples below).
"IL-17 receptor A" or "IL-17RA" (interchangeably used herein, as well as lL-17 receptor
and IL-17R to refer to the same receptor) as used herein is meant the cell surface receptor and
receptor complexes (such as but not limited to IL-17RA-1L-17RC complex), that bind IL-17A
and IL-17F and as a result initiates a signal transduction pathway within the cell. IL-17RA
proteins may also include variants. IL-17RA proteins may also include fragments, such as the
extracellular domain that don't have all or part of the transmembrane and/or the intracellular
domain, as well as fragments of the extracellular domain. The cloning, characterization, and
preparation of IL-17RA are described, for example, in U.S. Pat. No. 6,072,033, which is
incorporated herein by reference in its entirety. The amino acid sequence of the human IL-17RA
is shown in SEQ ID NO:430. Soluble forms of huIL-17RA useful in the methods of the present
invention include the extracellular domain or the mature form lacking the signal peptide or a
fragment of the extracellular domain that retains the capacity to bind IL-17A and/or IL-17F, or a
heteromeric version of IL-17A and/or IL-17F. Other forms of IL-17RA include muteins and
variants that are at least between 70% and 99% homologous to the native IL-17RA of SEQ ID
NO:430 and as described in U.S. Pat. No. 6,072,033, so long as the IL-I7RA retains the capacity
to bind IL-17A and/or IL-17F, or a heteromeric version of IL-17A and/or IL-I7F. The term "IL-
I7RA" also includes post-translational modifications of the IL-17RA amino acid sequence. Post-
translational modifications include, but is not limited to, N-and 0-1 inked glycosylation.
IL-17RA Antigen Binding Proteins
The present invention provides antigen binding proteins that specifically bind IL-17RA.
Embodiments of antigen binding proteins comprise peptides and/or polypeptides (that optionally
include post-translational modifications) that specifically bind IL-17RA. Embodiments of
antigen binding proteins comprise antibodies and fragments thereof as variously defined herein,
that specifically bind IL-17RA. Aspects of the invention include antibodies that specifically bind
to human IL-17RA and inhibit IL-I7A and/or IL-I7F from binding and activating 1L-I7RA, or a
heteromeric complex of IL-I7RA and IL-17RC. Aspects of the invention include antibodies that
specifically bind to human IL-I7RA and inhibit an IL-17A/IL-I7F heteromer from binding and
acfivating IL-17RA, or a heteromeric complex of IL-I7RA and IL-I7RC. Throughout the
specification, when reference is made to inhibiting IL-17A and/or IL-17F, it is understood that
this also includes inhibiting heteromers of IL-17A and IL-17F. Aspects of the invention include
antibodies that specifically bind to human IL-17RA and partially or fully inhibit IL-I7RA from
forming either a homomeric or heteromeric functional receptor complex, such as, but not limited
to, an IL-17RA-IL-17RC complex. Aspects of the invention include antibodies that specifically
bind to human IL-17RA and partially or fully inhibit IL-I7RA from forming either a homomeric
or heteromeric functional receptor complex, such as, but not limited to IL-17RA/IL-17RC
complex and do not necessarily inhibit IL-17A and/or IL-I7F or an 1L-17A/IL-17F heteromer
from binding to IL-17RA or a IL-I7RA heteromeric receptor complex.
The antigen binding proteins of the invention specifically bind to IL-I7RA. "Specifically
binds" as used herein means that the antigen binding protein preferentially binds IL-17RA over
other proteins. In some embodiments "specifically binds" means that the IL-17RA antigen
binding proteins have a higher affinity for IL-17RA than for other proteins. For example, the
equilibrium dissociation constant is < 10"^ to 10"" M, or <10'^ to
