Title of Invention

INTERLEUKIN-10 ANTIBODIES

Abstract The methods and compositions provided herein relate generally to IL-10 specific antibodies and uses thereof. More specifically, compositions of humanized IL-10 specific antibodies and methods to use such antibodies in modulating the biological activity of IL-10, particularly in autoimmune disorders and pathogen-mediated immunopathology.
Full Text

INTERLEUKIN-10 ANTIBODIES
The present application claims the benefit of U.S. Provisional Patent Application No. 60/518,999; filed November 10,2003, which is herein incorporated by reference in its entirety.
Field of the Invention
[0001] The present invention relates generally to interleukin-10 (IL-10) specific
antibodies and uses thereof. More specifically, the invention relates to humanized antibodies that recognize human IL-10 and modulate its activity, particularly in autoimmune disorders.
Background of the Invention
[0002] Initially known as cytokine synthesis inhibitor factor or CSIF, interleukin-10
(IL-10) is a potent immunomodulator of hematopoietic cells, particularly immune cells.
Cells such as activated Th2 cells, B cells, keratinocytes, monocytes and macrophages
produce IL-10. See. e.g., Moore et aL., Anna. Rev. ImmunoL 11:165 (1993). IL-lOinhibits
activation and effector functions of a number of cells that include T cells, monocytes and
macrophages. In particular, IL-10 inhibits cytokine synthesis, including that of IL-1, IFN-y,
and TNF, by cells such as Thl cells natural killer cells, monocytes, and macrophages. Seef
e.g., Fiorentino et aL., J Exp. med. 170:2081-2095 (1989); Fiorentino et al., J. Immunol
146:3444 (1991); Hsu et al., bxt. BOmmunol. 4:563 (1992); Hsu et aL, Int. Immunol 4:563
(1992); D'Andrea et aL, J. EXP. Med. 178:1041 (1993); de Waal Malefyt et al., J. Exp. Med.
174:915 (1991); Fiarentmo et aL., J Immunol 147:3815 (1991).
[0003] Multiple podbogens, particularly intracellular pathogens, elicit IL-10
production to slow or completely stall the effective removal of the pathogen by the immune response. Moore et aL, ANNU. Rev. Immunol jj_:165 (1993). For example, in blood lymphocytes from patients with HIV, leprosy, or tuberculosis, peripheral blood lymphocytes are typically anergic or uonrespotBive in vitro when challenged with the pathogen. However, the neutralization of IL-10 in these demonstrated that an active effector response,

i.e., Thl reactivity, was present in these cells. Thus, it is believed that IL-10 is effectively commandeered by the pathogen to facilitate its infective state.
[0004J IL-10 is also associated with autoimmunity in vivo. Autoimmunity results
from the development from autoaniibodies, autoreactive T cells, or some combination
thereof that target normal tissue. Gas example of autoimmune disease is systemic lupus
erythematosus (SLE), a chronic rbetanabc disease in which connective tissue throughout the
body becomes inflamed. Autoamfbodies feat attack normal body tissue as if it were an
outside invade result in the characteristic inflammation. While the precise cause is not fully
understood, researchers believe it has feofii genetic and environmental components.
Specifically, B~cell hyperactivity and the presence of various autoantibodies characterize
SLE. Typically, IgG autoantibodies reactive to double stranded DNA (IgG anti-dsDNA
abs) are elevated in patients with SUE- Between 60 and 70% of SLE patients produce IgG
anti-dsDNA abs, some of which are nephrotoxic. SLE is ten times more prevalent in
women than men, with symptoms ranging from facial rashes to disabling and potentially
life-threatening organ dysfunction. It can develop at any age, but is most common in young
adults.
100051 Numerous starties Kitnnort a role for IT^10 in the natholoev associated with
SLE. For example, whOe JL-10 is tjfacafly not produced by cells without appropriate stimulation, both B cells and macrapbages from SLE patients spontaneously produce high levels of IL-10 in vitro. LlarsnEL etaL, Arthritis Rheum. 40:249-60(1997). In several studies, researchers demcais&assd a correlation between serum levels of EL-10 and disease activity. Moreover, baft in vivo aodin vitro studies dononstrated that the blockade of IL-10 production can alleviate the dimca! manifestations of SLE. See, e.g.9 Gonzalez-Amaro, et al. J. Autoimmunity 11:395-402 (1998).
[0006] To date, ooe of Ifae manifestations of SLE, lupus nephritis, has been treated
with through the use of immunosuppressive therapies, e.g., corticosteriods and
cyclophosphamides. Although effective, these therapies are non-specific and substantial
toxicities exist which prevent long term therapy. Thus, specific neutralizing antibodies may
be effective antagonists of IL-10, permitting the removal of the suppressive effects of IL-10
while leaving the remainder of tbc rrmnnne response network intact
(0007) The most significant Inmtaboo in using antibodies as a therapeutic agent in
vivo is the immunogenicity of the antibodies. As most monoclonal antibodies are derived

from rodents, repeated use in humans results in the generation of an immune response against the therapeutic antibody. Such an immune response results in a loss of therapeutic efficacy at a minimum and a potential fatal anaphylactic response at a maximum. Initial efforts to reduce the immunogenicity of rodent antibodies involved the production of chimeric antibodies, in which moose variable regions were fused with human constant regions. Liu et aL, Proa Nad. Arad ScL USA 84:3439 (1987). However, mice injected with hybrids of bumsa variable redoes aad mouse constant regions develop a strong anti-antibody response directed against the human variable region, suggesting that the retention of the entire rodent Fv region in such domeric antibodies may still result in unwanted immunogenicity in patients.
[0008] It is generally believed that complementarity determining region (CDR)
loops of variable domains comprise the binding site of antibody molecules. Therefore, the grafting of rodent CDR loops onto human frameworks (i.e., humanization) was attempted to further minimize rodent sequences. Jones et al., Nature 321:522 (1986); Verhoeyen et al., Science 239:1534 (1988). However, CDR loop exchanges still do not uniformly result in an antibody with the same binding properties as the antibody of origin. Changes in framework residues (FR), residues involved in CDR loop support, in humanized antibodies also are required to preserve antigen binding affinity. Kabat et al., J. Immunol. 147:1709 (1991). While the use of CDR grafting sai framework residue preservation in a number of humanized antibody constructs has been reported, it is difficult to predict if a particular sequence will result in the antibody with the desired binding, and sometimes biological, properties. See, eg., Queen et al., Proa Nail. Acad ScL USA 86:10029 (1989), Goiman et al., Proc. Natl Acad. Sci.USA85:418 (1991), and Hodgson, Bio/Technology 9:421 (1991). Moreover, most prior studies used different human sequences for animal light and heavy variable sequences, rendering tbs predictive nature of such studies questionable. Sequences of known antibodies have been used or, more typically, those of antibodies having known X-ray structures, antibodies NEW and KOL. See, e.go Jones et aL, supra; Verhoeyen et al., supra; and Gorman et aL, supra. Exact sequence information has been reported for only a few humanized constructs.
(0009) The present invention provides humanized monoclonal antibodies which
recognize human IL-10 and mocWate its activity, is particular with regard to autoimmune

disorders. The humanized antibody should provide an alternative therapy choice without the toxicity and non-specificity associated with current treatments.
Brief Summary of the Invention
[0010] Provided herein is a iannaaized recombinant antibody molecule that binds
IL-10, or binding fragment thereof composing: at least one antibody light chain variable region, or binding fragment ibereo£ omiprising a polypeptide having at least one amino acid sequence selected firm the soap consisting of SEQ ID NO: 1 at CDR1, SEQ ID NO:2 at CDR2, and SEQ ID NO3 at CDR3; and a framework region, wherein the amino acid sequence of framework region is all or substantially all of a human immunoglobin amino acid sequence; and at least one antibody heavy chain variable region, or binding fragment thereof, comprising a polypeptide having at least one amino acid sequence selected from the group of SEQ ID NO:6 at CDR1, SEQ ID NO:7 at CDR2, and SEQ ID NO:8 at CDR3; and a framework region, wherein the amino acid sequence of framework region is all or substantially all of a human immunoglobin amino acid sequence. Also provided herein is an antibody, wherein the antibody light chain, or binding fragment thereof, comprises a poiypepiide having a variable rcgkxi of SEQ ID NO:4. la one specific embodiment, the antibody light chain, or binding fragment thereof, comprises a polypeptide having a variable region and a constant region of SEQ ID NO:5. In one specific embodiment, the antibody heavy chain, or binding fragment thereof comprises a polypeptide having a variable region of SEQ ID NO:9. In another specific embodiment, the antibody heavy chain, or binding fragment thereof comprises a poivpeptide having a variable region and a constant region of SEQ ID NO: 10.
[0011] Further provided herein is a chimeric recombinant antibody molecule that
binds IL-10 or binding fragment thereof, comprising: at least one antibody light chain variable region, or binding fragment thereof comprising a polypeptide having at least one amino acid sequence selected from the group consisting of SEQ ID NO:1 at CDR1, SEQ ID NCh2 al CDR2, and SEQ ID NO3 at CDR3; and at least one antibody heavy chain variable region, or binding fragment thereof, comprising a polypeptide having at least one amino acid sequence selected from the group comisting of SEQ ID NO:6 at CDR1, SEQ ID NO:7 at CDR2, and SEQ ID NO.8 at CDR3.

[0012] Also provided herein is a humanized recombinant antibody molecule that
binds IL-10, or binding fragment thereof, comprising: at least one antibody light chain, or binding fragment thereof, comprising a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO;11 at CDR1, SEQ ID NO:12 at CDR2, and SEQ ID NO:13 at CDR3; and 2 framewcsk regkm, wherein the amino acid sequence of framework region is afl or substantially aB of a human immunoglobin amiso acid sequence; and at least one antibody heavy chain, or binding fragment thereof comprising a polypeptide having an anriao acid sequence selected from the group consisting of SEQ ID NO:15atCDRl,SEQIDNO:16at CDR2,andSEQIDNO:17atCDR3;andaframework region, wherein the amino acid sequence of framework region is all or substantially all of a human immunoglobin amino acid sequence. In one specific embodiment, the antibody light chain, or binding fragment thereof^ comprises a polypeptide having a variable region and a constant region of SEQ ID NO: 14. In yet another specific embodiment, the antibody heavy chain, or binding fragment thereof comprises a polypeptide having a variable region and a constant region of SEQ ID NO: 18.
[0013] Further provided herein is a chimeric recombinant antibody molecule that
binds EL-10, or binding fragment thereof, comprising: at least one antibody light chain, or
binding fragment thereof, comprising a polypeptide having at least one amino acid sequence
selected from the group consstag of SEQ ID NO: 11 at CDR1, SEQ ID NO: 12 at CDR2,
and SEQ ID NO:13 at CDR3; and at least one antibody heavy chain, or binding fragment
thereof, comprising a potypep&de saving si least one amino acid sequence selected from the
group consisting of SEQ IDNO:15 at CDR1, SEQ ID NO:16 at CDR2, and SEQ ID NO:17
atCDR3.
10014] In one embodiznesL, the antibodies described supra further comprise a heavy
chain constant regioa. wherein tfae heavy chain constant region comprises a yl, y2, y3, or y4 human heavy chain constant regioa or a variant thereof. In one embodiment, the antibodies described above farther comprise a light chain constant region, wherein the light chain constant region comprises a lambda or a kappa human light chain constant region. In some embodiments, the binding fragment of these antibodies is an antibody fragment selected from the groop consisting of Fab, Fab\ Faib'-SH, Fv, scFv, F(abT>25 and a diabody.

[0015] Further provided herein is a method of suppressing an immune response in a
human subject comprising administering to a subject in need thereof an antibody specific for
IL-10, or a binding fragment thereof in an amount effective to block the biological activity
of IL-10, wherein the antibody is an antibody disclosed herein. The immune response
suppressed by this method is a humoral or a cellular response. In one embodiment, the
subject treated by this method has systemic lapos erythematosus. In another embodiment,
the subject has immune ihrombocytopemc purpura (ITC). In yet another embodiment, the
subject has lupus nephritis. In a farther embodiment, the subject has HIV. In another
embodiment, the subject has hepatitis C. Jn one specific embodiment, the method of
suppressing an immune response m a human subject comprising administering to a subject
in need thereof (1) an antibody specific for IL-10, or a binding fragment thereof, in an
amount effective to block the biological activity of IL-10? wherein the antibody is an
antibody disclosed herein, and (2) an immunosuppressive agent.
[0016] Provided herein is a composition comprising an antibody, or binding
fragment thereof, in combination with a pharmaceutically acceptable carrier or diluent, wherein the antibody is one of the antibodies disclosed supra.
[0017] Further provided heron is an isolated nucleic acid encoding the polypeptide
of the antibodies disclosed supra. Also provided herein is an expression vector comprising the isolated nucleic acid sequence operably linked to control sequences recognized by a host cell transfected with the vector. Provided herein is a host cell comprising the vector comprising the isolated nucleic acid sequence. Further provided herein is a method of producing a polypeptide, coznpdsisg catering the host cell comprising the vector under conditions wherein the nucleic acid sequence is expressed, thereby producing the polypeptide, and recovering the poLypeptide from the host cell.
[0018] Provided herein is aa isolated nucleic acid sequence encoding an antibody
specific for DL-10 comprising a fight chain having the nucleic acid sequence of SEQ ID
NO: 19 and a heavy chain having the nucleic acid sequence of SEQ ID NO;20. In further
embodiments, the light chain has aa American Type Culture Collection (ATCC) deposit
number of PTA-S923 and the heavy chain has an ATCC deposit number of PTA-5922.
[0019] Provided herein is an isolated nucleic acid sequence encoding an antibody
specific for IL-10 comprising a light chain having tbe nucleic acid sequence of SEQ ID NO:21 and a heavy chain having fee nucleic acid sequence of SEQ ID NO:22. In a further

embodiment, the light chain has an ATCC deposit number of PTA-5927 and the heavy chain has an ATCC deposit number of PTA-5926.
[0020J Further provided herein is an isolated nucleic acid sequence encoding a
binding fragment of tbe antibody encoded by the above nucleic acid sequences. In one embodiment, the binding fragment is an antibody fragment selected from the group consisting of FAB,, Fab', Fab'-SH, Fv, scFv, and F(ab%
[0021] Provided berem is a method to identify an acceptor gennline sequence for a
humanized antibody, which method conqsrises the steps of: a) identifying a non-human
antibody that has the desired biological activity; b) determining the amino acid sequence of
a non-human antibody VH and VL domains; and c) comparing the nonhuman antibody
sequence to a group of human gennline sequences, wherein the comparison comprises the
substeps of: 1) assigning the sequence of non-human VH and VL domain sequences residue
numbers; 2) delineating the CDR and FR regions in the sequence; 3) assigning a
predetermined numerical score at each residue position for which the non-human and human
gennline sequences are identical; and 4) totaling all of the residue scores to generate a total
score for each human gennline sequence; and d) identifying the human germline sequence
with the highest total residue score as the acceptor gennline sequence. In one embodiment,
the method further comprises tbe ssbstsps of: 5) assigning a numerical score of 1 for each
residue position for which the non-feiman and human gennline sequences are identical that
was not scored in substep (3) to gsnaline sequences with identical total residue scores after
substep (4); 6) totaling all of ibe r-skiue scores to generate a total score for each human
gennline sequence. In a specific embodiment, the non-human antibody is specific for IL-10
and inhibits the biological actrasy oflL-10. In a specific embodiment, the numerical scores
are assigned to the residues as m Tables 2 and 3 for VH and VL regions, respectively.
[0022] Further provided herein is am antibody generated by the above method.
Brief Description of the Drawings
[00231 Figure 1A shows tbe assignment of residue numbers and numerical scores to
tbe potential acceptor germline sexjaence relative to tbe variable light chain of the anti-human EL-10 antibody, 12G8.

[0024] Figure IB shows the assignment of residue numbers and numerical scores to
the potential acceptor gennline sequence relative to the variable heavy chain of the anti-human DL-10 antibody, 12G8.
[0025] Figure 1C shows the assignment of residue numbers and numerical scores to
the potential acceptor gennline sequence relative to the variable light chain of the anti-human IL-10 antibody, 1ID8.
[0026] Figure ID shows fee assignment of residue numbers and numerical scores to
the potential acceptor gennlme seqaeace relative to the variable heavy chain of the anti-human EL-10 antibody, 12D8.
[0027] Figure 2A is a concentration-time profile for 12G8 antibody administered i.v.
as described in Example IDL
[0028] Figure 2B is a concentration-time profile for 12G8 administered s.c. as
described in Example HI.
[0029] Figure 3 A shows that administration of the humanized anti-IL-10 antibody,
SCH7O898O, confers resistance to Leishmania major infection in IL-10 transgenic mice.
Infection was determined by measuring footpad swelling with a caliper at the times
indicated. 12GS antibody was administreedas described in Example VI.
[0030] Figure 3B shows that adminiration of the rat anti-IL-10 antibody, 12G8,
confers resistance to Leishmania major infection in EL-10 transgenic mice. Infection was determined by measuring footpad swefimg wife a caliper at the times indicated. 12G8 antibody was administered as described in Example VI.
Detailed Description of the Invention
[0031] For clarity of disdosme, and not by way of limitation, the detailed
description of fee invention is divided mto fee subsections feat follow.
A. Definitions
(0032] Unless defined otherwise, all technical and scientific terms used herein have
fee same meaning as is commonly imderslood by one of ordinary skill in fee art to which this invention belongs. All patents, jppikabODs, published applications and other publications referred to herein are incorporated by reference in their entirety. If a definition

set forth in this section is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the defestkm set forth in this section prevails over the definition that is incorporated herein by reference.
[0033] As used herein, "a" or "mF means "at least one" or "one or more."
[0034] As used herein, tbe ferm "antibody" refers to any form of antibody or
fragment thereof that exhibits the desired biological activity. Thus, it is used in the broadest
sense and specifically covers monoclonal antibodies (including full length monoclonal
antibodies), polyclonal antibodies, smltispecifjc antibodies (e.g., bispecific antibodies), and
antibody fragments so long as they exhibit the desired biological activity.
[0035] As used herein, the term 'IL-10 binding fragment" or "binding fragment
thereof encompasses a fragment or a derivative of an antibody that still substantially retain its biological activity of inhibiting IL-10 activity. Therefore, the term "antibody fragment" or IL-10 binding fragment refers to a portion of a full length antibody, generally the antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab!, F(ab!)2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, e.g., sc-Fv; and multispecific antibodies formed from antibody fragments. Typically, a binding fragment or derivative lesaing at least 50% of its IL-10 inhibitory activity. Preferably, a binding fragment or derivative retains at least 60%, 70%, 80%, 90%, 95%, 99% or 100% of its IL-10 inhibitory activity. It is also intended that a IL-10 binding fragment can include conservative amao acid substitutions that do not substantially alter its biologic activity.
[0036] The tenn "maoockmal antibody", as used herein, refers to an antibody
obtained from a population of subskailMhy homogeneous antibodies, le.9 the individual antibodies comprising the population are identical except for possible naturally occurring mutations feat may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically include a multitude of antibodies directed against (or specific for) different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring prodocban of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention

may be made by the hybridoma method first described by Kohler et al., Nature 256: 495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Gackson et aL, Nature 352: 624-628 (1991) and Maries et al., J. Mot BioL 222: 581-597 (1991), for example.
[0037] The monoclonal antibodies herein specifically include "emmeric* antibodies
(immunoglobulins) in winch a portion of tbe heavy and/or light chain is identical with or
homologous to correspooding soqueaces m antibodies derived from a particular species or
belonging to a particular antibody das or subclass, while the remainder of the chain(s) is
identical with or homologous to exsresponding sequences in antibodies derived from another
species or belonging to another antibody class or subclass, as well as fragments of such
antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567;
and Morrison et al., Proc. Nail AcadSci. USA 81: 6851-6855 (1984)).
[0038] As used herein, the term "single-chain Fv" or "scFv" antibody refers to
antibody fragments comprising the VH and V]_ domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a
polypeptide linker between the vh and vl domains which cnables the sfv to from the
desired structure for antigen binding. For a review of sFv, see Pluckthun, THE PHARMACOLOGY OF MONOCLONAL ANTSOWES, vol. 113, Rosenburg and Moore eds. Springer-Veriag, New Yoik, pp. 269-315 (1994).
[0039] As used herein, the term "diabodies" refers to small antibody fragments with
two antigen-binding sites, which fragments comprise a heavy chain vsiable domain (VH)
connected to a tight chain variable domain (V1) in the same polypeptide chain (VH-VL). By
using a linker that is too short to aflaw pairing between the two domains on the same chain,
the domains are forced to pair with the complementary domains of another chain and create
two antigen-binding sites. Diabodies are described more fully in, e.g~, EP 404.097; WO
93/11161; and Hollinger et aL, Proa NatL Acad Set USA 90: 6444-6448 (1993).
[0040] As used herein, the tenn "humanized antibody" refers to forms of antibodies
that contain sequences from non-hnman (eg., murine) antibodies as well as human antibodies. Such antibodies are human antibodies which contain minimal sequence derived from non-human immunoglobulin. In general, tbe humanized antibody will comprise substantially all of at least ooe, and typically two, variable domains, in which all

or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human irnmunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
[0041 ] As used here in , the term "hypervariable region" refers to the ammo acid
residues of an antibody which are responsible for antigen-binding. The hypervariable region comprises amino acid residues from a *complementarity determining region" or "CDR" (Le. residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and residues 31-35 (HI), 50-65 (H2) ami 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and/or those residues from a "hypervariable loop" (Le. residues 26-32 (LI), 50-52 (12) and 91-96 (L3) in the light chain variable domain and 26-32 (HI), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk, J. Mol BioL 196: 901-917 (1987)). As used herein, the term "framework" or "FRn residues refers to those variable domain residues other than the hypervariable region residues defined herein as CDR residues.
[0042] As used herein, the term "conservative substitution'* refers to substitutions of
amino acids are known to those of skill in this art and may be made generally without altering the biological activity of tbe resulting molecule. Those of skill in this art recognize that, in general, single amino acid substitutiondRS in non-essential regions of a polypeptide do not substantially alter biological acxrvky (see, e.g., Watson, et aL., Molecular Biology of the Gene, The Benjamin/Cummmings Pub. Co., p. 224 (4th Edition 1987)). Such exemplary substitutions are preferably made in accordance with those set forth in TABLE 1 as follows:


[0043] Other substitutions are also permissible and may be determined empirically
or in accord with known cot*5€?v»tiv? substitutions.
[0044] As used herein, the fesa "solaced nucleic acid molecule" refers to a nucleic
acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the antibody nucleic acid. An isolated nucleic add mr>kxgfr is otter than in the form or setting in which it is found in nature. Isolated nndoc add molecules therefore are distinguished from the nucleic acid molecule as it exists in natoral ctSs. However, an isolated nucleic acid molecule includes a nucleic acid molecule ocaxased in cells that ordinarily express the antibody where, for example, the nackk acid molecule is in a chromosomal location different from that of natural cells.
[0045] The expression "control sequences" refers to DMA sequences necessary for
the expression of an opexably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylabon sigah, md enhancers.

[0046] Nucleic acid is "operably linked11 when it is placed into a functional
relationship with another nucleic acid sequence. For example, DNA for a presequence or
secretory leader is operably linked to DNA for a polypeptide if it is expressed as a
preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is
operably linked to a coding sequence if it affects the transcription of fee sequence; or a
ribosome binding she is operably farad to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operabiy linked" means that the DNA sequences being
linked are contiguous, and, in the case of a secretory leader, contiguous and in reading
phase. However, enhancers do not lave to be contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not exist, the synthetic
oligonucleotide adaptors or linkers are used in accordance with conventional practice.
[0047] As used herein, the expressions "cell," "cell line," and "cell culture" are used
interchangeably and all such designations include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Wbexc distinct designations are intended, it will be clear from the context.
[0048] As used herein, "tpolyterase chain reaction" or "PCR" refers to a procedure
or technique in which minute amomms of a specific piece of nucleic acid, RNA and/or DNA, are amplified as described in, e.g., U.S. Pat No. 4,683,195. Generally, sequence information from the ends of the region of interest or beyond needs to be available, such that oligonucleotide primers can be designed; these primers will be identical or similar in sequence to opposite strands of the template to be amplified. The 51 terminal nucleotides of the two primers can coincide with fee ends of the amplified material. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA, bacteriophage or plasmid sequences, etc. See generally Mullis et aL, Cold Spring Harbor Symp. QuanL BioL 51.263 (1987); Erlich, ed_, PCR TECHNOLOGY (Stockton Press, N.Y., 1989). As used herein, PCR is considered to be one, but not fee only, example of a ancldc acid polymcrase reaction method for amplifying

a nucleic acid test sample comprising the use of a known nucleic acid as a primer and a
nucleic acid polymerase to amplify or generate a specific piece of nucleic acid.
[0049] As used herein, the term "gennline sequence" refers to a sequence of
unrearranged immunoglobulin DNA sequeiices. Any suitable source of unrearranged immunoglobulin may be used.
[0050] As used herein, the term immanrosuppressive agent"' refers to natural or
synthetic agents that suppress or modulatean immune response. The immune response can be a humoral or cellular response.
B. IL-10 specific antibodies
[0051] The compositions and methods disclosed herein relate to the modulation of
IL-10 activity, particularly in immune responses. Specifically, the compositions and methods herein employ antibodies specific for the cytokine, DL-10. EL-10 is a potent cytokine that modulates T and B cell responses through the regulation of growth, differentiation, and cytokine synthesis of a variety of cell types involved in immune responses. Notably, IL-10 production is frequently associated with autoimmune diseases
and pathogen-induced immunopathokelygy. therefore, a composition and methodsthereof,
that modulates and inhibits DL-10 actmsy can after the development and sustainment of autoimmune disease and related symptoms and ameliorate or reduce pathogen-associated immunopathology.
[0052] Targeting IL-10 activity with humanized antibodies offers several uniquet
advantages. First, targeting IL-10 with antibody pennits a specific suppression of IL-10 activity while leaving the remainder of the immune response intact. In many cases of pathogen-induced immunopathology, the reduction or elimination of IL-10 activity should permit the desired effector immune response to eliminate with pathogen without further pathology. For the aukniaimme patko£, the redaction or elimination of IL-10 activity should reduce or eliminate tbe disease and/or its symptoms while maintaining the patient's immune competence. Second, humanized IL-10 antibodies circumvents the limitation associated with mnmmogemc rodent antibodies- The use of himan sequences eliminates the immunogenicity of tbe exogcnoosly administered antibodies, allowing therapeutic administration.

[0053] Humanized antibodies contain sequences from non-human as well as human
antibodies. Typically, the process ofhumanization begins with the generation of a non-human antibody that has the desired biological activity, i.e., inhibits IL-10 activity. Once a non-human antibody with the appropriate characteristics is identified, recombinant means are then employed to create a bybnd sequeuce using non-human and human sejuences.
C Generation of IL-10 Specific Antibodies
[0054] Any suitable method for gquqaiiiig monoclonal antibodies may be used. For
example, a recipient may be immunized with IL-10 or a fragment thereof. Any suitable method of immunization can be used. Such methods can include adjuvants, other immunostimulants, repeated booster immunizations, and the use of one or more immunization routes.
[0055] Any suitable source of IL-10 can be used as the immunogen for the
generation of the non-human antibody of the compositions and methods disclosed herein. Such forms include, but are not limited whole protein, peptide(s), and epitopes, generated through recombinant, synthetic, chemical or enzymatic degradation means known in the art. IL-10 is an acid-sensitive, mrocovakat homodnner of two interpenetrating polypeptide chain. The cytokine is 160 ammo acids m length with well conserved sequences that include an a-helical bundle structure similar to interferons and hemopoietic cytokines. Human and murine IL-10 have 73% sskio acid homology, with human IL-10 being active on murine and human cells. IL-10 is commercially available or can be produced using well known molecular biology tectrnkjaes. Geobank cDNA sequences are available for the human, pig-tailed macaque, masgabey, ifcesas, and owl monkeys, lenrar, mouse, rat, guinea pig, Syrian hamster, rabbit, cat, dog, as well as others. Recombinant hitman IL-10 is a 17-18 kDa polypeptide that is not N-gijcosylated.
J0056] Any fonn of tbe antigen can be used to generate the antibody that is
sufficient to generate a biologically active antibody. Thus, the eliciting antigen may be a single epitope, multiple epitopes, or tbe entire protein alone or in combination with one or more imnranogenicity enhancing agents known in the art. Tbe eliciting antigen may be an isolated fuU-kngth protein, a cell surface protein (e.g., immunizing with cells transfected with at least a portion of the antigen), or a soluble protein (e.g., immunizing with only the

extracellular domain portion of the protein). The antigen may be produced in a genetically
modified cell. The DNA encoding the antigen may genomic or non-genomic (e.g., cDNA)
and encodes at least a portion of the extracellular domain. As used herein, the term
"portion" refers to the minimal number of amino acids or nucleic acids, as appropriate, to
constitute an immunogenic epitope of the antiigen of interest. Any genetic vectors suitable
for transformation of the cells of inieresrmay be employed, including but not limited to
adenoviral vectors, plasmids, and rxst-rkai vectors, such as cationic lipids,
[0057] Any suitable method cast be used to elicit an antibody with the desired
biologic properties to inhibit IL-10. It is desirable to prepare monoclonal antibodies (mAbs) from various mammalian hosts, such & mice, rodents, primates, humans, etc. Description of techniques for preparing such monoclonal antibodies may be found in, e.g., Stites, et al. (eds.) BASIC AND CLINICAL IMMUNOLOGY (4th ed.) Lange Medical Publications, Los Altos, CA, and references cited therein; Harlow and Lane (1988) ANTIBODIES: A LABORATORY MANUAL CSH Press; Coding (1986) MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (2d ed.) Academic Press, New York, NY. Thus, monoclonal antibodies may be obtained by a variety of techniques familiar to researchers skilled in the art. Typically, spleen cells from 3i! snimal immunized with a desired, ssnti^ss srs innnortsilizc^ commonW bv ftision with 2L myeloma cell. See Kohler and IvHstera (1976) Ear. J. Immunol 6:511-519. Alternative methods of immortalization include transabnnation with Epstein Barr Virus, oncogenes, or retro viruses, or other methods known m the art See, e.g., Doyle, et al. (eds. 1994 and periodic supplements) CELL AND TISSUE COLTURE: LABORATORY PROCEDURES, John Wiley and Sons, New Yoric, NY. Colonies ansag from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by succ cells may be enhanced by various techniques, including injection into fee peritonea] cavity of a vertebrate host. Alternatively, one may isolate DNA sequences which encode a monoclonal antibody or a binding fragment thereof by screening a DNA library from humai B cells according, e.g., to the general protocol outlined by Huse, et aL (1989) Science 246:1275-1281.
|0058] Other suitable techniques involve selection of libraries of antibodies in phage
or similar vectors. See, e.g_, Huse et al., Science 246:1275-1281 (1989); and Ward et aL, Nature 341:544-546 (1989). The potypqotkfes and antibodies of the present invention may be used with or without modification, including chimeric or humanized antibodies.

Frequently, the polypeptides and antibodies will be labeled by joining, either covalently or
non-covalently, a substance which provides for a detectable signal. A wide variety of labels
and conjugation techniques are known and are reported extensively in both the scientific and
patent literature. Suitable labels include radionuclides, enzymes, substrates, cofectors,
inhibitors, fluorescent moieties, chemilumiiiescent moieties, magnetic particles, and the like.
Patents teaching the use of such labels incisSe U.S. Patent Nos. 3,817,837; 3,850,752;
3,939,350; 3,996345; 4,277,437; 4,275J49; and 4,366,241. Also, recombinani
immunoglobulins may be produced, ss£ CabsHy U.S. Patent No. 4,816,567; and Queen et al.
(1989) Proc. Nat'lAcad. Sci. USA 86:10029-10033; or made in transgenic mice, see Mendez
et al. (1997) Nature Genetics 15:146-156; also see Abgenix and Medarex technologies.
[0059] Antibodies or binding compositions against predetermined fragments of IL-
10 can be raised by immunization of animals with conjugates of the polypeptide, fragments, peptides, or epitopes with carrier proteins. Monoclonal antibodies are prepared from cells secreting the desired antibody. These antibodies can be screened for binding to normal or defective IL-10. These monoclonal antibodies will usually bind with at least a Kd of about 1
1 µM, more usually at least about 300 nM, typically at least about 30 nM5 preferably at least about 10 nM, more preferably at least about 3 nM or better, usually determined by ELIS A. Suitable non-human antibodies may also be identified using the biologic assays described in Section D infra.
C. HumanizatioB of IL-10 Specific Antibodies
[0060J Any suitable noc-iranac atstibody can be used as a source for the
hypervariable region. Sources far noo-imman antibodies include, but are not limited to, murine, lupine, bovine, and primates. For the most part, humanized antibodies are human iirtmimoglobulins (recipient antibody) in which hypervariable region residues of the recipient are replaced by hypexvariabie region residues from a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuxnan primate having the desired specificity, affinity, and capacity. In some instances, Fv framewoik region (FR) residues of the human immunoglobulin arc replaced by coKrcspandnig non-human residues. Furthermore, humanized antibodies may comprise residues which are not found in the recipient antibody or in the donor antibody. These modifications are made to farther refine antibody

performance of the desired biological activity. For further details, see Jones et aL, Nature 321: 522-525 (1986); Reichmann et aL, Nature 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol 2: 593-596 (1992).
[0061] Methods for recombinanUy engineering antibodies have been described, e.g.,
by Boss et al. (U.S. Pat No. 4J16397), cabilityy et al. (U.S. Pat. No. 4,816,567), Law et al. (European Patent Application Publication No. 438 310) and Winter (European Patent Application Publication No. 239 400).
[0062] Amino acid sequence varsags ofisEsnanized anti-IL-10 antibody are prepared
by introducing appropriate nucleotide changes into the humanized anti-EL-10 antibody DN A, or by peptide synthesis. Such variants include, for example, deletions from, and/or insertions into and/or substitutions o£ residues within the amino acid sequences shown for the humanized anti-IL-10 F(ab) (e.g. as in SEQ ID NO!s 5 and 10). Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics. The amino acid changes also may alter post-translational processes of the humanized anti-IL-10 antibody, such as changing the number or position of glycosylation sites.
[0063] A useful method for idsSdiicaiioii of certain residues or regions of the
humanized anti-IL-10 antibody polypepode feat are preferred locations for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells, Science 244: 1081-1085 (1989). Here, a residue or group erf target residues are identified (e.g., charged residues such as arg, asp, his, lys, and ghi) and replaced by a neutral or negatively charged amino acid (most preferably ala^ee or polyalanine) to affect the interaction of the amino acids with IL-10 antigen- The amiDo add residues demonstrating functional sensitivity to the substitutions then sse refined by introducing further or other variants at, or for, the sites of substitttikxL Thos, wink the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to analyze the performance of a mutation at a given site, ala scanning or random mutagenesis is conducted at the target codon or region and the expressed humanized anti-IL-10 antibody variants are screened for the desired activity.
[0064] Amino acid sequence insertions include amino- and/or carboxyl-tenninal
fusions ranging in length from one reskhe to potypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.

Examples of terminal insertions include humanized anti-IL-10 antibody with an N-terminal methionyl residue or the antibody fused to an epitope tag. Other insertional variants of the humanized anti-IL-10 antibody molecule include the fusion to the N- or C-terminus of humanized anti-EL-10 antibody of an enzyme or apolypeptide which increases the serum half-life of the antibody.
[0065] Another type of variant is SB aimao acid substitution variant These variants
have at least one amino acid residue in the humanized anti-IL-10 antibody molecole removed and a different residue inseited is its place. The sites of greatest interest for substitutional mutagenesis include the hypervariable loops, but FR alterations are also contemplated. Tables 2 and 3 in the method described below provides guidance as to hypervariable region residues which can be altered. Hypervariable region residues or FR residues involved in antigen binding are generally substituted in a relatively conservative manner.
[0066] Another type of amino acid variant of the antibody alters the original
glycosylation pattern of the antibody. By altering is meant deleting one or more
carbohydrate moieties found in the antibody, and/or adding one or more glycosylation sites
that are not present in the antibody. Glvcosyiation of antibodies is typically either N-linked
or O-linked. N-linked refers to the wttahmeut of the carbohydrate moiety to the side chain
of an asparagine residue. The tripeptide sequeoces asparagine-X-serine and asparagine-X-
threonine, where X is any amino acid except profine, are the recognition sequences for
enzymatic attachment of the cartnbMkaEe moiety to the asparagine side chain. Thus, the
presence of either of these tripeptide seqaeoces in a polypeptide creates a potential
glycosylation site. O-Hnked glycosylation refers to the attachment of one of the sugars N-
aceylgalactosamine, galactose, or xyfase to a hytfroxyamino acid, most commonly serine or
threonine, although S-hydroxjprbfine or 5-hydroxylysine may also be used.
[0067] Addition of gjycosyiatkra sites to the antibody is conveniently accomplished
by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequeoces (for N-linked giycosyiatkm sites). The alteration may also be made by the addition of, or substitution by, one or more serine or threomne residues to the sequence of fee original antibody (for O-Hnked gfycosyiatkm sites).
[0068] Nucleic acid molecules eauodiug ammo acid sequence variants of humanized
IL-10 specific antibody are prepared by a variety of methods known in the art. These

methods include, but are not limited to, isolation from a natural source (in the case of
naturally occurring amino acid sequence variants) or preparation by oligonucleotide-
rnediated (or site-directed) muiagenesis,PCR mutagenesis, and cassette mutagenesis of an
earlier prepared variant or a noo-variant version of humanized anti-DL-10 antibody.
[0069] Ordinarily, amino acid sequence variants of the humanized anti-IL-10
antibody will have an ammo acid scqwrane havmg at least 75% amino acid sequence identity with the original humanized antibody ammo acid sequences of either the heavy or the light chain (e.g. as in SEQ ID NCh5 aad 10), more preferably at least 80%, more preferably at least 85%, more preferably ai least 90%, and most preferably at least 95%. Identity or homology with respect to this sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the humanized anti-DL-10 residues, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. None of N-tenninal, C-terminal, or internal extensions, deletions, or insertions into the antibody sequence shall be construed as affecting sequence identity or homology.
the humanizod antibody can be selected from any class of immmunoglobulins,

(AATA1
[vu / VJ
including IgM, IgG, IgD, IgA, and IgE. preferably the antibody is a IgG antibody. Any isotype of IgG can be used, including IgG2, IgG2, IgG3, and IgG4. Variants of the IgG isotypes are also contemplated the hnmanized antibody may comprise sequences from more than one class or isotype. Optimization of fee necessary constant domain sequences to generate the desired biologic activity is rsadgy achieved by screening the antibodies in the biological assays described below.
[0071] Likewise, either class of fight chain can be used in the compositions and
methods herein. Specifically, iappa, lambda, or variants thereof are useful in the present compositions and methods.
[0072] Any suitable portion of the CDR sequences from the non-human antibody
can be used. The CDR sftqneaccs can be mntageoized by substitution, insertion or deletion of at least one residue such that the CDR seqoeoce is distinct from the human and non-human antibody sequence employed. It is contemplated that such mutations would be minimal. Typically, at least 75% of the immanizedantibody residues will correspond to

those of the non-human CDR residues, more often 90%, and most preferably greater than 95%.
[0073] Any suitable portion of the FR sequences from the human antibody can be
used. The FR sequences can be mutagenized by substitution, insertion or deletion of at least
one residue such that the FR sequence is distinct from fee human and non-human antibody
sequence employed. It is contemplated THST sadi mutations would be minimaL Typically, at
least 75% of the humanized antibody resdoes will correspond to those of the human FR
residues, more often 90%, and most prssaoisly greater than 95%.
[0074] CDR and FR residues are determined according to fee standard sequence
definition of Kabat. Kabat et aL, Sequences of Proteins of Immunological Interest, National Institutes of Health, Befeesda Md. (1987).
[0075] Provided herein is a method to identify an acceptor germline sequence for a
humanized antibody, which method comprises the steps of: a) identifying a non-human
antibody feat has the desired biological activity; b) determining fee amino acid sequence of
a non-human antibody VH and VL domains; and c) comparing fee nonhuman antibody
sequence to a group of human germline sequences, wherein the comparison comprises fee
substeps of: 1) assigning the non-human V sequences residue numbers according to Kabat
supra; 2) delineating fee CDR and FRiepoDS ia the sequence according to Kabat supra; 3)
assigning a predetermined numerical score at specific residue position for which fee non-
human and human antibody gennlme secfacnces are identical; and 4) totaling all of the
residue scores to generate a total score far each human germline sequence; and d)
identifying the human germline sequence-with the highest total residue score as the acceptor
germline sequence. In one embodmeac tbe method further comprises the substeps of: 5)
assigning a numerical score of 1 for each FR residue position for which fee non-famnan and
human antibody gennlme sequences are identical that was not scored in substep (3) to
germline sequences with identical total residue scores after substep (4); 6) totaling all of fee
residue scores to generate a total score for each human germKne sequence. In a specific
embodiment, fee non-bmnan antibody is specific for IL-10 and inhibits the biological
activity of IL-10. Also provided heresa is an antibody generated by the above method.
[0076] In one embodiment, tbe IL-10 antibody is humanized using the following
method. First, the non-human Vl and VH domains of tbe IL-10 antibody are cloned and sequenced, and tbe amino acid sequence determined. Then, the non-human VH sequence are

compared to a group of five human VH gennHne amino acid sequences. The five groups contain one representative from the subgroups IGHV1 and IGHV4 and three representatives from subgroup IGHV3. The VH subgroups are Hsted in M.-P. Lefranc, Exp. Clin. Lnmunogenetics, .18:100-116 (2001). Specifically, the comparison with the five gennline sequences begins with the assignment of resadoe numbers to the non-human VH sequence according to the Kabat numbering system. See Kabat, et al., U. S. Department of Health and Human Services, NIH Pub. 91-3242 (5th Ed., 1991). The non-human VH sequence are then aligned with each of the five human genome seqoences. Since the V genes orfy comprise VH residues 1-94, only these residues are considered in the alignment. Next, the complementarity-determining (CDR) and framework (FR) regions in the sequence are delineated. CDR and FR are delineated according to the combination of the definitions provided in Kabat, et al., U. S. Department of Health and Human Services, NIH Pub. 91-3242 (5th Ed., 1991), and C Chothia & A.M. Lesk, J. Mol Biol, 196:901-917 (1987). Therefore, the CDR definition used is residues 26-35 for CDR1, residues 50-65 for CDR2, and CDR3 is residues 95-102 for CDR3 of the VH domain. The next step involves assigning a numerical score at identified residue position where the non-human and human sequences are identical. One example of this scoring B shown in Table 2 below.



* Noted as affecting CDR conformation in C Chotfcaa et aL, Nature 342:877-883, (1989).
[0077] After the residue positions are assigned a numerical score, all of the residue
scores are totaled. The acceptor germline sequence is the one with the highest total score. In a case where two or more germline sequences have identical scores, then add 1 to the total for each position where the non-human and human sequences are IDENTICAL for the following residues: 1,3,5-23, 25,36, 38,4G-43,46,66, 68, 70, 72, 74, 75, 77, 79-90, and 92 (max 49). The residue scores are totaled again, and the acceptor germline sequence is the one with the highest total score, if two or more geamHne sequences still have identical scores, either one can be used as fee acceptor gamfine sequence.
[0078] If the VL sequence is a member of the kappa subclass of VL, the non-human
VL sequence from the IL-10 specific antibody is compared to a group of four human VL kappa germline amino acid sequences. The four sequences are comprised of one representative from each of four estabfeted human VL subgroups Ksted in V. Barbie & M.-P. Lefranc, Exp. Cliru Jmmunogenetics 15:171-183 (1998) and M.-P. Lefranc, Exp. din. Immunogenetics IS:161 -174 (2001 \ The fora* sequences also correspond to tbe four subgroups listed in Kabal et aL, U. S. Department of Health and Human Services, NTH Pub. -91-3242, pp. 103-130 (5th Ed-, 1991). Tbe comparison of the non-human sequence to the four germline sequences begins with tbe assignment of residue numbers to the non-human VL sequence residues according to Kabal et aL, U. S. Department of Health and Human Services* NIH Pub. 91-3242 (5th Ed., 1991). The noo-human VL sequences are then aligned with each of the four human gennline sequences. Since tbe V genes only comprise VL

residues 1-95, only these residues are considered in the alignment. Next, the complementarity-determining (CDR) and framework (FR) regions are delineated in the sequence. CDR and FR are delineated according to the combination of the definitions provided in Kabat et aL, U. S. Department of Health and Human Services, NH Pub. 91-3242 (5th Ed. 1991), and C. Chotfaia & AM. Lesk, J. Mol BioL9196:901-917 (1987). Therefore, the CDR definition used is reskiaes 24-34 for CDR1, residues 50-56 for CDR2, and residues 89-97 for CDR3 of the VL dama&L Hie next step involves assigning a numerical score at identified residue posatkae "wiiere tie non-human and human sequences are identical. One example of this scoring is shown in Table 3 below.

* Noted as affectm^ CDR copfonnBboo m C Owifci et aL Nature 342:877-SS3. (1989).
10079) After tbe residue positions are assigned a numerical score, all of the residue
scores are totaled. The acceptor germline sequence is the one with fee highest total score.

In a case where two or more germline sequences have identical scores, then add 1 to the total for each position where the non-human and human sequences are IDENTICAL for the following residues: 1,3, 5-23,35,37,39-42,57, 59-61,63, 65-70,72-86, and 88. Tie residue scores are totaled again, and the acceptor germline sequence is the one with fee highest total score. If two or more germline sequences still have identical scores, ekher one can be used as the acceptor gennline sequrary
[0080J For recombmam prodactioe of the embody, the nucleic acid encoding it is
isolated and inserted into a replicable vector for farther cloning (amplification of the DNA)
or for expression. DNA encoding the mooockmal antibody is readily isolated and sequenced
using conventional procedures (e.g., by using oligonucleotide probes that are capable of
binding specifically to genes encoding the heavy and light chains of the antibody). Many
vectors are available. The vector components generally include, but are not limited to, one
or more of the following: a signal sequence, an origin of replication, one or more marker
genes, an enhancer element, a promoter, and a transcription termination sequence.
[0081] In one embodiment, the antibody is a humanized recombinant antibody
molecule that binds IL-10, or binding fragment thereof, comprising: at least one antibody . light chain variable region, or binding fragment thereof, comprising a polypeptide having at least one amino acid sequence selected JOTS the group consisting of SEQ ID NO:1 (KTSQNIFENLA) at CDR1, SEQ ID NCh2 (NASPLQA) at CDR2, and SEQ ID NO:3 (HQYYSGYT) at CDR3; and a framewxkiegkHL wherein the amino acid sequence of framework region is all or substantially a3 of a human immunoglobin amino acid sequence; and at least one antibody heavy chain variable regkm, or binding fragment thereof comprising a polypeptide having at feast one ammo acid sequence selected from the group consisting of SEQ ID NCfc6 (GFTFSDYmiA) at CDR1, SEQ ID NO:7 (SITLDATYTYYRDSVRG) at CDR2, SEQ ID NO:8 (HRGFSVWLDY) at CDR3; and a framework region, wherein the ammo acid sequence of framework region is all or substantially all of a human immunoglobm amino acid sequence. In a specific embodiment, the antibody light chain, or binding fragment thereof comprises a polypeptide having a variable region of SEQ ID NCh4 PIQMIT^PSSLSASVGDRVTITC NASPLQAGWSRreGSGS(jroFl YTFGPGTKLELXRT). In one specific embodiment, the antibody light chain, or binding

fragment thereof, comprises a polypeptide having a variable region and a constant region of
SEQ ID NO:5. See Table 4. In one specific embodiment, the antibody heavy chain, or
binding fragment thereof, comprises a polypeptide having a variable region of SEQ ID
NO:9 (QVQLVESGGGVVQPGRSIJU^CAASGFrFSDYHMAWV
RQAPGKGLEWVASnXID
YYCAimRGFSVWlI)YWGQGTLVTVSSL in another specific embodiment, the
antibody heavy chain, or binding fragment ifasreof, comprises a polypeptide having a
variable region and a constant region of SEQ ID NCfclG. See Table 5.
[0082] Plasmids containing the nucleic adds encoding the humanized 12G8 light
and heavy chains were deposited with fee American Type Culture Collection (ATCC) as
deposit numbers PTA-5923 and PTA-5922, respectively.






[0083] In one embodiment, the antibody is a humanized recombinant antibody
molecule that binds IL-10, or binding fragment thereof, comprising: at least one antibody light chain, or binding fragment thereof, comprising a polypeptide having at least one amino acid sequence selected from the group consisting of SEQ ED NO: 11 (RASESVDDYGHSFMH)at CDR1, SEQ ID NO:12 (RASTLES) at CDR2, and SEQ ID NO: 13 (QQGNEDPWT) at CDR3; and a franework region, wherein the amino acid sequence of framework region is all or substantially all of a human immunoglobm amino acid sequence; and at least one antibody heavy chain, or binding fragment thereof, comprising a polypeptide having at least o&e amino acid sequence selected from the group consisting of SEQ ID NO:15 (GFSLTNYGVH) at CDR1, SEQ ID NO:16 (VIWSGGSTDYNAAFIS) at CDR2, and SBQ ID NO:17 (NRGYDVYFDY) at CDR3; and a framework region, wherein the amino add sequence of framework region is all or substantially all of a human imnmnogiobm aaano acid sequence. In one specific embodiment, the antibody Kght chain, or binding fragment thereof comprises a polypeptide having a variable region and a constant regicB of SBQ ID NO:14. See Table 6. fa yet another specific embodiment, Hie antibody heavy chain, or binding fragment thereof comprises a polypeptide having a variable region and a constant region of SEQ ID NO:18. See Table 7.
{0084] Plasmids containing the bocmnozed 1 IDS heavy aid light chains were
deposited with the ATCC as deposit numbers PTA-5926 and PTA-5927, respectively.





[0085] In one embodiment, the antibodies described supra further comprise a heavy
chain constant region, wherein the heavy chain constant region comprises a yl, y2, y3, or y4
human heavy chain constant region or a variant thereof. In one embodiment, the antibodies
described above farther comprise a light chain constant region, wherein the light chain
constant region comprises a lambda or a kappa hrarran tight chain constant region. In some
embodiments, the binding fragment of these antibodies k aa antibody fragment selected
from the group consisting of Fab, Fab*, Fab'-SH, Fv. scFv, F(ab')2, and a diabody.
[0086] Also provided herein is a chnBenc xscoanbmant antibody molecule that binds
IL-10 or binding fragment thereof comprising: 3L feast one antibody light chain variable
region, or binding fragment thereof^ comprising a polypeptide having at least one amino
acid sequence selected from the group consisting of SEQ ID NO:1 at CDR1, SEQ ID NO:2
at CDR2, and SEQ ID NO:3 at CDR3; and at least one antibody heavy chain variable
region, or binding fragment thereof, comprising a polypeptide having at least one amino
acid sequence selected from the group consisting of SEQ ID NO:6 at CDR1, SEQ ID NO:7
at CDR2, and SEQ ID NO:8 at CDR3.
[0087] In a specific embodiment, the chimeric recombinant antibody molecule has a
light chain as set forth in SRO TD NO- 71 anri a braw chain as art fnrfh in RF.O TF> NO?4
k-* ~ — — ■^ ~ . — . _ — -. j _ . _- — — — ^ — _ . _ .-
See Table 8. Nucleic acids encoding the 12G8 dsiaeric antibody light and heavy chains were deposited at the ATCC as deposit numbers PTA-5925 and PTA-5924, respectively.





[0088] Further provided herein is a chimeric recombinant antibody molecule that
binds IL-10, or binding fragment thereof, comprising: at least one antibody light chain, or binding fragment thereof, comprising a polypeptide having at least one amino acid sequence selected from the group consisting of SEQ ID NO:11 at CDR1, SEQ E> NO:12 at CDR25 and SEQ ID NO: 13 at CDR3; and at least one antibody heavy chain, or binding fragment thereof, comprising a polypepbde having at ieast one ammo acid sequence selected from the group consisting of SEQ ID NO:15 at CDR1, SEQ ID NO:16 at CDR2, and SEQ ID NO:17 atCDR3.
[0089] Further provided herein is an isoisesd nucleic acid encoding the polypeptide
of the antibodies disclosed supra. Also provided herein is an expression vector comprising the isolated nucleic acid sequence operabty fiaksd to coitfrol sequences recognized by a host cell transfected with the vector. Provided heron is a host cell comprising the vector comprising the isolated nucldc acid sequence. Farther provided herein is a method of producing a polypeptide, comprising cu&uring the host cell comprising the vector under conditions wherein the nucldc acid sequence is expressed, thereby producing the polypeptide, and recovering the polypeptide fnxn the host cell.
[0090] Provided heron is an isolated nucleic acid sequence encoding an antibody
specific for IL-10 comprising a light chain having the nockic add sequence of SEQ ID NO: 19 and a heavy chain having the nucleic acid sequence of SEQ ID NO.20. See Tables 4 and 5.

[0091] Provided herein is an isolated nucleic acid sequence encoding an antibody
specific for IL-10 comprising a light chain having the nucleic acid sequence of SEQ ID NO:21 and a heavy chain having the nucleic acid sequence of SEQ ID NO;22. See Tables 6 and 7.
[0092] Further provided herein is an isolated nucleic acid sequence encoding a
binding fragment of the antibody encoded by t&e move nocleic acid sequences, in one
embodiment, the binding fragment is an antibody fragment selected from the group
consisting of Fab, Fab\ Fab'-SR, Fv, scFv, F(afe'V,aodadiabody.
[0093] Bispecific antibodies are also usefel m the present methods and
compositions. As used herein, the term "bispecific antibody" refers to an antibody, typically a monoclonal antibody, having binding specificities for at least two different antigenic epitopes. In one embodiment, the epitopes are from the same antigen. In another embodiment, the epitopes are from two different antigens. Methods for making bispecific antibodies are known in the art. For example, bispecific antibodies can be produced recombinantly using the co-expression of two immunoglobulin heavy chain/light chain pairs. See, e.g.t Milsttin etal. Nature 305: 537-39(1983). Alternatively, bispecific antibodies can be prepared using chemical linkage. See, e*g., Brennan, et al, Science 229: 81 (1985). Bispecific antibodies irolude bispecifc antibody fragments. See, e.g., Hollinger, et al, Proa Natl Acad. ScL USA. 90: 6444^*8 (1993), Gruber, et alt J. Immunol 152: 5368(1994).
D. Biological Activity of Humanized Aoti4L-19 Antibodies
[0094] Antibodies having the characteristics identified herein as being desirable in a
humanized anti-IL-10 antibody can be screened fer inhibitory biologic activity in vitro or suitable binding affinity. To sciecn fer antibodies which bind to the epitope on human IL-10 bound by an antibody of interest (e.g^ those which block binding of the cytokine to its receptor), a routine cross-blocking assay such as that described in ANTIBODIES, A IJ^BORATORY MANUAL, COW Spring Harbor Laboratory, Ed Harlow and David Lane (1938), can be performed. Alternatively, epitope mappmg, e.g^ as described in Champe et al., J. Biol Chem. 270:1388-1394 (1995), can be performed to determine whetber the antibody binds an ephope of interest. Antibody affinities (eg. for human EL-10) may be

determined using standard Scatchard analysis- Preferred humanized antibodies are those which bind human IL-10 with a Kj value of no more than about lx10-7; preferably no more than about lxl 0'8; more preferably no more than about lxlO"9; and most preferably no more than about 2xlO'10.
{0095] The antibodies and fragments thereof usefcl in the present compositions and
methods are biologically active antibodies md fragments. As used herein, the tenn "biologically active" refers to an antibody or antibody fragment that is capable of binding the desired the antigenic epitope and directly or iadksctly exerting a biologic effect. Typically, these effects result from the failure of IL-10 to bind its receptor. As used herein, the term "specific" refers to the selective binding of the antibody to the target antigen epitope. Antibodies can be tested for specificity of binding by comparing binding to EL-10 to binding to irrelevant antigen or antigen mixture under a given set of conditions. If the antibody binds to EL-10 at least 10, and preferably 50 times more than to irrelevant antigen or antigen mixture then it is considered to be specific.
[0096] The inhibitory EL-10 specific antibody can inhibit its biological activity in
any manner, including but not limited to production of IL-1, IFN-y, PGE2, EL-12, TMF, CC
— j rvv/-* «i i-; J *i if r —: -.fxrur** «i««« TT .»_+:*»,«•*,» /^J^CA
CD80, and CD86. The biologic activity of an IL-10 specific antibody can be determined by any useful method. See, e.g., U.S. Patent Nos: 6^239,260 and 6,207,154. In one example, the biologic activity is assessed in cell pFofifex^ks assay jxsmg the murine mast cell line, MC9/2. See Thompson-Snipes et aL. J. Exp. Med. 173:507-10 (1991). IL-10 stimulates the proliferation of this cell line, and therefore an rafajbaory antibody can be identified by its ability to reduce proliferation. The ED50 fcrpsofifentfioB of the MC9/2 cell line is typically 0.5-1.0ng/mL. An antibody is inhibitory for proliferation if it inhibits the proliferation of cells relative to the proliferation of cells m fee abseace of the antibody or in the presence of a non-binding antibody. Proliferation may be quantified using any suitable methods. Typically, the proliferation is determined by assessing the incorporation of radioactive-labeled nucleotides into DNA (&£., ^-thymidine). In another embodiment, proliferation is determined by ATP luminescence. Preferably, the antibody useful in the present compositions inhibits 80% of IHO's biologic activity, more preferably 95%, most preferably 99%.

E. Uses of Humanized Anti-IL-10 Antibodies
[0097] Provided herein is a method of suppressing an immune response in a human
subject comprising administering to a subject in need thereof an antibody specific for EL-10, or a binding fragment thereof, in an amount effective to block fee biological activity of EL-10, wherein the antibody is a humanized recombinani antibody molecule that binds IL-10,, or binding fragment thereof, comprising: at least oae axtibody Eght chain variable region, or binding fragment thereof, comprising a poiypep&de having at least one amino acid sequence selected from the group consisting of SEQ ID NO:1 at CDR1, SEQ ID NO:2 at CDR2, and SEQ ID NO.3 at CDR3; and a framework region, wherein the amino acid sequence of framework region is all or substantially all of a human immunoglobin amino acid sequence; and at least one antibody heavy chain variable region, or binding fragment thereof, comprising a polypeptide having at least one amino acid sequence selected from the group consisting of SEQ ID NO:6 at CDR1, SEQ ID NO:7 at CDR2, and SEQ ID NO:8 at CDR3; and a framework region, wherein the amino acid sequence of framework region is all or substantially all of a human immunoglobin amino acid sequence.
[0098] Further provided herein is a method of suppressing an immune response in a
human subject comprising administering to a safeject in need thereof an antibody specific for EL-10, or a binding fragment thereof in an amoQni: effective to block the biological activity of IL-10, wherein the antibody is a humanized nxombinant antibody molecule that binds IL-10, or binding fragment thereof comprising: at feast one antibody light chain, or binding fragment thereof comprising a polypeptide having at least one amino acid sequence selected from the group consisting of SEQ ID NO:! 1 * CDR1, SEQ ID NO:12 at CDR2, and SEQ ID NO:13 at CDR3; and a framewcak region, wfeidu the amino acid sequence of framework region is all or substantially all of a human immunoglobin amino acid sequence; and at least one antibody heavy chain, or binding fragment thereof comprising a polypeptide having at least one amino acid sapience selected from fee group consisting of SEQ ID NO:15 at CDR1, SEQ ID NO:16 at CDR2, and SEQ ID NO.17 at CDR3; and a framework region, wherein the amino acid sequence of framework region is all or substantially all of a human immunoglobin ammo acid sequence.
[0100] The immune response suppressed by these methods is a humoral or a cellular
response. The suppression of the humoral and cellular responses can be determined using

well known methods in the art. For example, in diseases associated with high levels of
autoreactive antibodies, e.g., SLE, a reduction in fee serum levels of these antibodies
relative to the pre-treatment serum levels is an indication of the suppression of the humoral
response. Likewise, the suppression of the cellular immune response can be determined
using well known in vitro analyses, e.g., proliferatiaB and cytotoxicity assays or
characterization of activation phenotypes of peripheral blood lymphocytes by, e.g^ flow
cytometric analysis. iSee CURRENT PROTOCX>LS IN IMMUNOLOGY, most recent edition- In
one embodiment, the subject treated by this method has systemic lupus erythematosus. in
another embodiment, the subject has immune thrombocytoperric purpura (TTC). In yet
another embodiment, the subject has lupus nephritis. In a further embodiment, the subject
has HIV. In another embodiment, the subject has hepatitis C.
[0101] Provided herein is a composition comprising an antibody, or binding
fragment thereof, in combination with a pharmaceutically acceptable carrier or diluent, wherein the antibody is one of the antibodies disclosed herein.
[0102] Any subject that would benefit from treatment with IL-10 specific antibodies
can be treated using the compositions and methods provided herein. Any subject can be treated with the methods and compositions provided IKS cm. Such a subject is a mammal, preferably a human, with an autoimmune disease or symptom or pathogen-induced immunopathology. In one specific embodiment, the subject has SLE, lupus nephritis, rheumatoid arthritis, ETC, HIV or hepatitis C Vetczasy uses of the disclosed methods and compositions are also contemplated.
[0103] As used herein, "inhibit" or "treat" or ^Treatment" includes a postponement
of development of the symptoms associated with aaofinmune disease or pathogen-induced
immunopathology and/or a reduction in the severity of such symptoms that will or are
expected to develop. The terms farther include ameliorating existing uncontrolled or
unwanted autoimmune-related or patbogea-incfaiced immunopathology symptoms,
preventing additional symptoms, and ameliorating or preventing the underlying causes of
such symptoms. Thus, the teams denote thai a beneficial result has been conferred on a
vertebrate subject with an autoimmune or pa£hogc&-*nducod hnmunopalhology disease or
symptom, or with the potential to develop such a disease or symptom.
[0104] As used herein, the term "tberapevtically effective amount" or "effective
amount" refers to an amount of an IL-10 specific antibody that when administered alone or

in combination with an additional therapeutic agent to a cell, tissue, or subject is effective to prevent or ameliorate the autoimmune disease or pathogen-induced itnmunopathology associated disease or condition or the progression of ibe disease. A therapeutically effective dose further refers to that amount of the compound sufficient to result in amelioration of symptoms, e.g., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment beafas. prevention or amelioration of such conditions. When applied to an indivicfcaal active isgredSenf administered alone, a therapeutically effective dose refers to that ingredient akme. When applied to a combination, a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.
[0105] An antibody useful in the present methods (from whatever source derived,
including without limitation from recombinant and non-recombinant sources) may be
administered to a subject in need, by itself, or in pharmaceutical compositions where it is
mixed with suitable carriers or excipient(s) at doses to treat or ameliorate a variety of
disorders. Such a composition may also contain (in addition to protein and a carrier)
diluents, fillers, salts, buffers, stabilizers, solubilisers, and other materials well known in the
art The term "pharmaceutically acceptable" xoesss a aon-toxic material that does not
interfere with the effectiveness of the biological activity of the active ingredient(s). The
characteristics of the canier will depend on fee roafe of administration.
[0106] The pharmaceutical composition of ibe invention may also contain other
immunosuppressive or immimomodulating agraifr Any suitable immunosuppressive agent can be employed, including but not limited to anri-inflarnmatory agents, corticostexoids, cyclosporine, tacrolimus (te., FK-506), siroHinns^ interferons, soluble cytokine receptors (e.g., sTNRF and sIL-lR), agents tbai oeitixafize cytokine activity (e.g., inflixmab, etanercept), mycophenolate roofetiX 15-deoxyspergnaKn, thalidomide, glatiramer, azathioprine, leflunomide, cyciophosphamide, metbotrexate, and the like. The pharmaceutical composition can also be employed with other therapeutic modalities such as phototherapy and radiation.
[0107] In another embodiment, kits are provided that contain the necessary reagents
to cany out the assays of tbe methods provided harm. Specifically provided herein is a compartment kit comprising one or more containers, wherein a first container comprises one

or more antibodies specific to EL-10, and one or more other containers comprising one or more of the following: reconstitution reagents, administration reagents. The containers can be glass, plastic, or strips of plastic or paper. In one embodiment, the kit also contain written instructions.
[0108] In practicing the methods of treaimes or use provided herein, a
thexapeuticaHy effective amount of antibody piovafed basia is administered to a mammal having a condition suitable for treafmert wife IL-10. Hie antibody may be administered in accordance with the methods herein either alone or m cosnhmaiion with other therapies such as treatments employing other immuiKjmodulatingfa£#af3 (eg., cytokines), immunosuppressive agents, and the like. Whoa c^adnmristered with one or more biologically active agents, the antibody provided herein may be administered either simultaneously with the biologically active agenl(s)s or sequentially. If administered sequentially, the attending physician will decide on the appropriate sequence of administering protein of the present invention in combination with the biologically active agent(s).
[0109] Toxicity and therapeutic efficacy of the antibody compositions, administered
slone or in combination with sn imTninnp»«**«jyvj vg gopnt ran be determined bv stsndard pharmaceutical procedures in cell cultures or escpermental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and fee ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as &e rari© between LD50 and ED50. Antibodies exhibiting high therapeutic indices are prefcaitxL The data obtained from these cell culture assays and animal studies can be used is formulating a range of dosage for use in human. Hie dosage of such compounds lies preferably within a range of circulating concentrations that include the ED* wifli Stfle or BO toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
[0110] Techniques for formulation and acfaninistration of the antibodies of the instant
methods may be found in REMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing CO., Easton, Pa., latest edition. The mode of administration is not particularly important Suitable routes of administration may, for example, include oral, rectal, transnracosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous,

intramedullary injections, as well as intrathecal. direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. Administration of antibody used in fee pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intrapemoneal, parergezaL, ioiraarterial or intravenous injection. Intravenous administration to fhe patient is prclhitai
(0111] Alternately, one may aArrmisxpr fbe asiibody in a local rather than systemic
manner, for example, via injection of the antiboey 3&zc&y into an arthritic joint or
pathogen-induced lesion characterized by immunopathology, often in a depot or sustained
release formulation. Furthermore, one may administer the antibody in a targeted drug
delivery system, for example, in a liposome coated with a tissue-specific antibody, targeting,
for example, arthritic joint or pathogen-induced lesion characterized by immunopathology.
The liposomes will be targeted to and taken up selectively by the afflicted tissue.
[0112] Pharmaceutical compositions for use.in accordance with the present methods
thus may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into preparations that can be used pharmaceutically. These pharmaceutical compositions may be rnaunfectorcd m a manner that is itself known, e.g., by means of conventional mixing, dissolving, gramrtating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyopiaiSziBg processes. Proper formulation is dependent upon the route of administration chosen. When administered in liquid form, a liquid carrier such as water, petroleum, oils of sisal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added. The liquid form of the pharmaceutical composition may fisfeer contain physiological saline solution, dextrose or other saccharide solution, or gtycols such as efeylene glycol, propylene glycol or polyethylene glycol. When administered in Ikpzid form, fee pharmaceutical composition contains from about 0.5 to 90% by weight of protein of the present invention, and preferably from about 1 to 50% protein of fee present invention.
[01131 When a feerapcobcaDy effective amount of antibody of fee methods herein is
administered by intravenous, cutaneous or subcutaneous injection, protein of fee present invention will be in the form of a pyrogen-free, parcrteraDy acceptable aqueous solution. The preparation of such parenterally acceptable protein solutions, having due regard to pH,

isotonicity, stability, and the like, is within the skill in the art A preferred pharmaceutical
composition for intravenous, cutaneous, or subcutaneous injection should contain, in
addition to protein of the present invention, an isotomc vehicle such as Sodium Chloride
Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection,
Lactated Ringer's Injection, or other vehicle as knows m tfee art The pharmaceutical
composition of the present invention may also contam stab&zsrs, preservatives, buffers,
antioxidants, or other additives known to those of sklfi m the art. For injection, the agents of
the invention may be formulated in aqueous sokttwES. preferably in physiologically
compatible buffers such as Hanks1 solution, Ringer's solution, or physiological saline buffer.
For transmucosal administration, penetrants appropdaie to fee barrier to be permeated are
used in the formulation. Such penetrants are generally known in the art.
[0114] For administration by inhalation, fee antibodies for use according to fee
present methods are conveniently delivered in the form of an aerosol spray presentation
from pressurized packs or a nebuliser, wife fee use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromefeane, dichlorotetrafluoroefeane, carbon dioxide
or other suitable gas. In fee case of a pressurized aerosol fee dosage unit may be determined
by providing a valve to deliver a metered amount Capsules and cartridges of. e.g.. gelatin
for use in an inhaler or insufflator may be formulated grnframmg a powder mix of fee
compound and a suitable powder base such as lactose or starch. The compounds may be
formulated for parenteral administration by injectk^ eg:, by bolus injection or continuous
infusion. Formulations for injection may be preseotedia rant dosage form, e.g., in ampules
or in multi-dose containers, wife an added prcsenratree. The compositions may take such
forms as suspensions, solutions or emulsions is oOy or aqueous vehicles, and may contain
fonnulatory agents such as suspending, stabilizing ami/or dispersing agents.
[0115] Pharmaceutical ibnxsulatioDS for paraieral administration include aqueous
solutions of fee active compounds in water-soluble form. Additionally, suspensions of fee active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils soch as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or tiposuaes. Aqueous injection suspensions may contain substances feat increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. OpbaeaDy, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the

preparation of highly concentrated solutions. Alternatively, the active ingredient may be m powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
[0116] The amount of antibody useful in the disclosed methods in the
pharmaceutical composition of the present invention wOl depend upon the nature and severity of the condition being treated, and OB tiie satire of prior treatments that the patient has undergone. Ultimately, fee attending physician will decide toe amount of protein of the present invention with which to treat each individual pafisz& It is to be expected that the dosage will vary according to the age, weight and response of the individual patient. Initially, the attending physician will administer low doses of antibodies of the present methods and observe the patient's response. Larger doses of antibodies of the present invention may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further. It is contemplated that the various pharmaceutical compositions used to practice the methods herein should contain about 0.01 fig to about 100 mg (preferably about 0.1 \xg to about 10 mg, more preferably about 0.1 |ig to about 1 mg) of antibody of the present invention per kg body weight. When administered, the therapeutic composition far use in this invention is, of course, in a pyrogen-free, physiologically acceptable form. Therapeatieally useful agents other than an antibody of the present methods that may also optionally be included in the composition as described above, may alternatively or additionally, be administered simultaneously or sequentially with the composition in the methods of the invention. The exact formulation, route of administration and dosage can be chose& by tiie individual physician in view of the patient's condition. See, e.g., Fingl et aL^ TfcE Pa^JEMACOLOGiCAL BASIS OF THERAPEUTICS (latest edition). Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety sufficient to marrtain 1fce desired therapeutic effects, or minimal effective concentration (MEC). The MEC will vmy for each compound but can be estimated from in vitro data; for example, fee concentration necessary to achieve 50-90% inhibition of biologic activity using the assays described herein-
|0117] The antibody provided herein can be administered alone or in combination
with other therapeutic modalities. The antibody provided herein also can be administered

alone or in combination with other antibodies identified as inhibitors of EL-10 activity or other immunosuppressive agents.
{0118] Any disease where autoimmunity is implicated can be treated with the
present methods. Preferably, autoimmune diseases targeted for treatment with IL-10
specific antibodies are characterized by abnormal IL-10 expression levels and/or a lack of
appropriate cellular, i.e., Thl-mediated, responses. Socn disease include, but are not Hunted
to systemic lupus erythematosus (SLE), immune thxombocytopenic puipura (TFC), lupus
nephritis, diabetes, insulin-dependent diabetes mell^s (IDDM), rheumatoid arthritis (RA).
[0119] Any disease where pathogen-induced inmiunqpathology is implicated can be
treated with the present methods. Preferably, pathogen-induced immunopathologies targeted for treatment with EL-10 specific antibodies are characterized by abnormal IL-10 expression levels and/or a lack of appropriate cellular, le.9 Thl-mediated, responses. Such diseases include, but are not limited to HIV, hepatitis C, visceral leishmaniasis, malaria, filariasis, leprosy, tuberculosis, candidiasis, and M. avium infections. [0120] . The broad scope of this invention is best understood with reference to the following examples, which are not intended to limit the inventions to the specific
F. Examples
Example!. General Methods
[0121] Some of the standard me&ods are described or referenced, e.g., in Maniatis,
et al. (1982) MOLECULAR CLONING, A LABORATORY MANUAL, Cold Spring Harbor Laboratory, Cold Spring Haibor Press; Sambrook, et aL (1989) MOLECULAR CLONING: A LABORATORY MANUAL, (2d ed.), vote. 1-3, CSH Press, NY; Ausubd, et aL, BIOLOGY, Greene Publishing Associates, Brooklyn, NY; or Ansabd, et aL (1987 and Supplements) CURRENT PROTOCOLS IN MOLECUIARBKHJOGY, &ccacAVfley,NewYcHfc Methods for protein purification include such methods as ammonium satiate precipitation, column chromatography, electrophoresis, centrifiigation, crystallizatkm, and others. See, e.g.,

Ausubel, et al. (1987 and periodic supplements); Deutsche (1990) "Guide to Protein Purification" in METH. ENZYMOL., vol. 182, and other volumes in this series; and manufacturer's literature on use of protein purification products, e.g., Pharmacia, Piscataway, N J.3 or Bio-Rad, Richmond, CA. Combination with recombinant techniques allow fusion to appropriate segments. e.g» to a FLAG sequence or an equivalent which can be fused via a protease-removable sequence. See. e^^ Hodraii (1990) "Purification of Recombinant Proteins with Metal Chelate Absorbed* in Setiow (ed.) GENETIC ENGINEERING, PRINCIPLE AND METHODS 12:87-9^ Pfcamn Press, N.Y.; and Crowe, et aL (1992) QIAEXPRESS: THE HIGH LEVEL EXPRESSION & PROTEIN PURIFICATION SYSTEM, Qiagen, Inc., Chatsworth, CA.
[0122] Computer sequence analysis is performed, e.g., using available software
programs, including those from the GCG (U. Wisconsin) and GenBank sources. Public sequence databases were also used, e.g., from GenBank and others.
Example II. Humanization of anti-human IL-10 antibodies
[0123] The humanization of the rat anti-human EL-10 antibody, 12G8, was
performed as described in Section C supra, Fignre 1 shows the assignment of the assignment of residue numbers and corresponding numerical scores for residue positions that are identical to the gennline sequences being examined Calculations are shown for the 12G8 variable regions of the light (Figure 1A) and heasry (Figure IB) of the 12G8 anti-human IL-10 antibody and for the variable regions of the light (Figure 1C) and heavy (Figure ID) of the 11D8 anti-human IL-10 antibody.
Example HL Phaimacokinetics of 12G&, an aaj-fegraan IL-10 antibody
[0124] Objective: To obtain estimates of in-vivo terminal half-lives and
subcutaneous bioavailability for the 12FS antibody in a murine model.
[0125] Antibody: The antibody is administered m a vehicle of 10 mM Na
acetate, 8% sucrose, pH 5.25.
[0126] Mice: CrI:CD-l*(ICR)BRfemakimce were purchased from Charles River
Laboratories.
[0127] Experimental Design: Mice received * single bolus injection of antibody
either intravenously (i.v. in lateral tail vein) or subcataneoosiy (sx. at nape of neck or mid-

scapular or lateral flank). Antibody doses included 0.03,0.3,3.0, and 30 mg/kg per mouse. The mice were observed for up to 28 days post-injection. During this time period, mice were weighed and serum samples taken. Senrai samples for the 12G8 (SCH 708980) groups (Groups 1-8) were taken at 0.5,1,3, 6,10,16 hrs, Day 1,2, 3,5,7,10,14, 21, and 28 post-injection using 5 mice/time point. In fee vehicle group (Group 9), serum samples were taken at pre-injection, lhr post-injection, 14 day ca: 21 azy only using 5 mice/time point Serum EL-10 levels and serum 12G8 antibody levels were determined using specific ELISAs.
[0128] Pharmacokinetic Parameter determinations. AD parameters were estimated
or calculated using WinNonlin Pro v 4.0. For mncompartmental analyses, Model 200 (SC) or Model 201 (IV) was used. Input data were dose-noimalized group arithmetic mean concentration-time data. Input doses were nominal doses for Groups 2-4 and 6-8. Input dose for Groups 1 and 5 was 0.014 mg/kg. For compartmental analyses, Model 3 (SC) or Model 7 (IV) was used. Input data were dose-normalized individual animal concentration-time data. All fits used uniform weighting (wt=l) for individual data points. Input doses were nominal doses for Groups 2-4 and 6-8. Input dose for Groups 1 and 5 was 0.014
estimated/calculated parameters, residuals, and AIC & SBC criteria.






[0131] Concentration-time profiles are shown for 12F8 antibody using various
dosages and routes in Figure 2.
[0132] Conclusions: The doses were within 20% of nominal for all groups except
lowest dose level. Lower than expected concentratk®s5 probably due to presence of anti-SCH708980 (humanized 12G8) antibodies were observed from Day 10 post-injection in groups 7 or 8. (A) IV Bolus Pharmacokmetics, Half-lives, clearance and distribution volumes are typical of those seen for other IgGl mcmockmal antibodies. Distribution volume is approximately equal to or slightly larger than serum volume suggesting minimal extravascular distribution. The terminal half-lives ranged from 10 to 17 days. The increase in AUC was generally dose-proportional suggesting Imear PK over the dose range tested. (B) SC Bolus Pharmacokinetics. The maximum concentrations were generally dose-proportional and were reached by 1-2 days po&dose suggesting consistent rates and extents of absorption over the dose range tested- The increase in AUC was generally dose-proportional suggesting linear PK. The terminal elimination half-lives ranged from 8-14 days, similar to other IgGl monoclonal antibodies. The absolute bioavailability was high, nmge= 70-100%, although the estimates > 90% may be high doe to underestimation of IV AUC.

Example IV. Determining the Equilibrium Dissociation Constant (KcD for humanized anti- human IL-10 antibody SCH 708980 (12G8) using; KmV* A technology.
f 0133] The equilibrium dissociation constant (Kd) fear humanized antibody SCH
708980 was determined using KinExA 3000 (Sapidyne Instruments hie). KinExA uses the principle of the Kinetic Exclusion Assay method based 1. The binding follows the reversible binding equation for equilibrium:
Kon[Ab][Ag]=Kofi{AbAg]
2. Antibody and antigen bind 1:1, and total antibody equals antigen- antibody
complex plus free antibody
3. Instrument signal is linearly related to free antibody concentration
[0134] Materials used: Monoclonal IIWHWIJAMI antibody SCH 708980 to
recombinant human IL-10 (hl2G8); recombinant hmnanIL-10 (hIL-10); recombinant
mouse IL-10 (mIL-10), recombinant cyno IL-10 (cyno EL-10); FMMA particles, 98 micron
(Sapidyne, Cat No. 440198); Neutravidin (Pierce, Ca£>s«L 31000); EZ- link TFP PEO-
Biotin (Pierce, Cat No. 21219); Biotmyiaied ibIL~I0; and Cy5 conjugated Goat anti- HulgG
(H + L) (Jackson Immunoresearch Laboratories CaL No 109-175-088, lot 49069 ).
[0135] PMMA particles were coated with bkftsyiated rhIL5 according to
manufacturer's protocols. For biotinylatiao of ifaIL5 EZ-Iink TFP PEO-hiotin was used according to manufacturer's recommendations (Pierce bulletin 0874). All experimental procedures were done according to the KinExA 3000 manual.
[0136] Experimental conditions: AD runs were done in duplicate. For UL-10 runs
the following conditions were used:
Sample volume: 1.5 ml
Sample flow rale: 0.25 ml/min
Label volume: 0 J> ml
Label flow rate: 025 ml/min




Example V. Application of competitive electrochemitoriraesceace assay (ECLA) to measure binding of anti-hEL-10 monoclonal antibodies aod hIL-IO-Ra to recombinant IL-10 of different origin.
[0143] Summary of technology. Electrochemiluminescence technology was
developed by IGEN, Inc (Gaithersburg, MD) and is employed in the M-series M8/384
analyzer used in this work. Electrochemiluminescence technology utilizes a stable
ruthenium metal chelate (Ori-TAG) which, in the presence of tripropylamine (TPA),
generates electrochemiluminescence upon voltage application. Paramagnetic beads,
microns in diameter, act as the solid phase and facilitate rapid assay kinetics. The
bead/complex is channeled through a flow ceil and captared at an electrode by magnetic
application. Voltage is applied and resulting electeocheamiasiinesrence is measured.
[0144] Materials used. 96 well Polypropylene plates (Costax, Cat. No. 3365,
Fisher Sci. Cat. No. 07200697); assay buffer of 0.1 %BSA50.05%tween20,PBSpH7.5; paramagnetic beads (Streptavidin-Dynabeads, Igea, fcc. Cal .No. 110029); recombinant human DL-10 dimer (hIL-10-dimer); recombinant bnnmIL-10-monoirier (hIL-10-mono); recombinant mouse IL-10 (mIL-10); recombmaat cyao IL-10 (cyno EL-10); and recombinant hIL-1 ORa ( hIL-1 ORa): FLAG4agged protest Ori-Tag labeled anti-FLAG M2 monoclonal antibodies were prepared using Ori-Tag-NH3 ester (Igen, Inc. Cat No. 110034 ) according to manufacturer's protocol (OriTag label: IgG challenge ratio 8:1). Anti-Rag M2 monoclonal antibodies were purchased fiomSigma(CatNo.F3165). Ori-Tag labeled anti hlgG 1A2 monoclonal antibodies were prepared as above using rat anti hlgG monoclonal antibodies. Ori-Tag labeled aod rat IgG aodbodies wore prepared as above from polyclonal Goal anti rat IgG (H+L) antibodies ( Jacksoo hnnamorescaffrh Laboratories, Inc. PA, CaL No. 112-005-143). Biotinyfatol recombinant human IL-10 (

hIL-10-biotin) was prepared using TFP-PEO-biotin (Pierce, Cat. No. 21219) according to manufacturer's recommendations (Pierce bulletin 0874). The rat anti hIL-10 mAb 12G8 (rl2G8): JES3.12G8 and humanized anti hIL-10 mAb 12G8 ( hl2G8-l) were prepared as described herein.
[0145] Protocol. 1/3 serial dilutions in 50 TTOToHters of the assay buffer were
made in 96-well microtiter plate for all unlabeted IL-10 piqmjddoQS ( mIL-10, cyno IL-10,
hXL-10 dimer, WDL-10 mono ) starting with 3 figrml in the first weE. All samples were ran in
duplicates. 50 jil of hIL-10-biotin at 25 ng/ml was added to each well, followed by the
addition of either hBL-lORa ( 50 pi at 100 ng/ml) or aoti ML-10 mAb ( 50 pi at 10 ng/ml).
50 microliters of Ori-Tag conjugated secondary antibodies was added to each well at 500
ng/ml cone. For hIL-1 ORa 5 rl2G8 and hl2G8 the following Ori-Tag conjugated were used
accordingly: anti-FLAG M2-OriTag, anti-rat IgG-OriTag and anti hlgG 1 A2-OriTag.
Finally to each well 50 pi of Streptavidin-Dynabeads at 0.1 mg/ml was added. After a one
hour incubation at room temperature the plate was processed by the M-series M8/384
analyzer. Percent inhibition of the signal by unlabeled IL-10 preparations was calculated
relative to the control samples. To plot the data and calculate IC50 the GraphPad Prism
Software was used.
[01461 Results are shown in the Table bek>w.



10147] The results of the characterization of the im 12G8 antibody and the
humanized 12G8 antibody (SCH708980) are summarized m fee Table below.

Example VI. Neutralizing effects of humanized aflj-temap IL-10 antibody in vivo
[0148] In vivo neutralizing efficacy of SCH 708960 ad JES.12G8 was evaluated in
the Leishmania major model in mice. In this modeL CB6F1 mice normally resistant to parasite infection were rendered susceptible by heterozysosity for a human IL-10 transgene under the control of the MHC class II promoter. CB6F1 or CB6F1 -huIL-1 OTg mice were injected s.c. with SCH 708980 or JES-12G8 weddy beginning three days before s.c. footpad challenge with 15x106 stationary phase L. major parasites. Disease progression was monitored by weddy measurements of footpad swelling- Fig. 3 shows that both SCH708980 (the humanized 12G8) and tbe parental rat 12GS neutralized the protective effect of IL-10 in a dose-dependent manner.

10149] Many modifications and variations of this invention can be made without
departing from its spirit and scope, as will be apparent to those skilled in the art. The
specific embodiments described herein are offered by way of example only, and the
invention is to be limited by the terms of the appeinled claims, along with the foil scope of
equivalents to which such claims are entitled; and the invasion is not to be Hunted by the
specific embodiments that have been presented herein by ^way of example.
[0150] Citation of the above publications or documents is not intended as an
admission that any of the foregoing is pertmest prior &L aor does it constitute any admission as to the contents or date of these publications or documents. U.S. patents and other publications referenced herein are hereby incorporated by reference.







CLAIMS
1. A humanized recombinant antibody molecule that binds IL-1O or binding
fragment thereof, comprising:
at least one antibody light chain variable region, or biading fragment thereof, comprising a polypeptide having at least one amino acid seqnence selected from the group consisting of SEQ ID NO: 1 at CDR1, SEQ ID NO:2 at CDR2, and SEQ ID NO:3 atCDR3; and a framework region, wherein the amino add sequence of framework region is all or substantially all of a human irnmunoglobin amino acid sequence; and
at least one antibody heavy chain variable region, or binding fragment thereof, comprising a polypeptide having at least one amino acid sequence selected from the group consisting of SEQ ID NO:6 at CDR1, SEQ ID NO:7 at CDR2, and SEQ ID NO:8 at CDR3; and a framework region, wherein the amino acid sequence of framework region is all or substantially all of a human immunoglobin amino acid sequence.
2. The antibody of claim 1, further comprising a heavy chain constant region,
wherein the heavy chain constant region comprises a yl3 y2, y3, or y4 human heavy chain
constant region or a variant thereof.
3. The antibody of claim 1, further comprising a light chain constant region,
wherein the light chain constant region comprises a lambda or a kappa human light chain
constant region.
4. The antibody of claim 1, wherein the brndisg fragment is an antibody
fragment selected from the group consisting of Fab, Fab, Fab'-SH, Fv, scFv, F(ab')2, and a
diabody.
5. The antibody of claim 1, wherein the antibody light chain, or binding
fragment thereof, comprises a polypeptide having a variable region of SEQ ID NO:4.

6. The antibody of claim 1, wherein the antibody light chain, or binding
fragment thereof, comprises a polypeptide having a variable region and a constant region of
SEQ ID NO:5.
7. The antibody of claim 1, wherein the antibody heavy chain, or binding
fragment thereof comprises a polypeptide having a variale region of SEQ ID NO:9.
8. Hie antibody of claim 1, wherein the antibody heavy chain, or binding
fragment thereof, comprises a polypeptide having a variable region and a constant region of
SEQ ID NO: 10.
9. A chimeric recombinant antibody molecule that binds IL-10 or binding
fragment thereof, comprising:
at least one antibody light chain variable region, or binding fragment thereof, comprising a polypeptide having at least one amino acid sequence selected from the group of SEQ ID NO:1 at CDR1, SEQ ID NO:2 at CDR2, and SEQ ID NO:3 at CDR3; and
at least one antibody heavy chain variable region, or binding fragment thereof, comprising a polypeptide having at least one ammo acid seqeence selected from the group of SEQ ID NO:6 at CDR1, SEQ ID NO:7 at CDR2, and SQ ID NO:8 at CDR3.
10. A humanized recombinant antibody molecme that binds BL-10, or binding
fragment thereof, comprising:
at least one antibody light chain, or binding fiagment thereof, comprising a polypeptide having at least one amino arid sequence selected from the group consisting of SEQ ID NO:11 at CDR1, SEQ ID NO:12 at CDR2, and SEQ ID NO:13 at CDR3; and a framework region, wherein the amino acid sequence of frameewoik region is all or substantially all of a human immunoglobin ammo acid sequence; and
at least erne antibody heavy chain, or binding fragment thereof, comprising a polypeptide having at least one amino acid sequence selected of SEQ ID NO:15 at CDR1, SEQ ID NO:16 at CDR2, and SEQ ID NO:17 at CDR3; and a framework region, wherein the amino acid sequence of framework region is all or substantially aD of a human immunoglobin amino acid sequence.

11. The antibody of claim 10, further comprising a heavy chain constant region,
wherein the heavy chain constant region comprises a γl, γ2, γ3, or γ4 human heavy chain
constant region or a variant thereof.
12. The antibody of claim 10, farther comprising a lightchain constant region,
wherein fee light chain constant region comprises a lambda or a kappa human light chain
constant region.
13. The antibody of claim 10, wherein fee binding fragment is an antibody
fragment selected from the group consisting of Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2> and
diabody.
14. The antibody of claim 10, wherein fee antibody light chain, or binding
fragment thereof, comprises a polypeptide having a variable region and a constant region of
SEQIDNO:14.
15. The antibody of claim 10, wherein fee antibody heavy chain, or binding
fragment thereof comprises a polypeptide having a variable region and a constant region of
SEQ1DNO:18.
16. A chimeric recombinant antibody molecule that binds IL-10, or binding
fragment thereof comprising:
at least one antibody light chain, or binding fragment thereof, comprising a polypeptide having at least one amino acid sequence of SBQ ID NChl 1 at CDR1, SEQ ID NO:12 at CDR2, and SEQ ID NO:13 at CDR3; and
at least one antibody heavy chain, or binding fragment thereof, comprising a polypeptide having at least one amino add sequence of SEQ ID NO: 15 at CDR1, SEQ ID NO:16 at CDR2, and SEQ ID NO:17 at CDR3.

17. A method of suppressing an immune response in a human subject comprising
administering to a subject in need thereof an antibody specific for IL-10, or a binding
fragment thereof in an amount effective to block the biological activity of IL-10, wherein
the antibody is the antibody of claim 1 or 9.
18. The method of claim 17, wherein the innmsoe response is a humoral
response.
19. The method of claim 17, wherein the subject has systemic lupus
erythematosus.
20. The method of claim 17, wherein the subject has immune thrombocytopenic
purpura (ITC).
21. The method of claim 17, wherein the subject has lupus nephritis.
22. The method of claim 17, wherein the subject has HIV.
23. The method of claim 17, wherein the subject has hepatitis C.
24. The method of claim 17, further comprising acfenmisSering an
iiranunosuppressive agent.
25. A method of suppressing an immune response in a human subject comprising
administering to a subject in need thereof an antibody specific for IL-10, or a binding
fragment thereof, in an amount effective to block tbe biological activity of IL-10, wherein
the antibody is the antibody of claim 10 or 16.
26. The method of claim 25, wherein the immane response is a humoral
response.

27. The method of claim 25, wherein the subject has systemic lupus
erythematosus.
28. The method of claim 25? wherein the subject has immune thrombocytopenic
purpura (TTC).
29. The method of claim 25, wherein fee subject has hapos aepbritis.
30. The method of claim 25, wherein the subject has HIV.
31. The method of claim 25, wherein the subject has hepatitis C.
32. The method of claim 25, further comprising administering an
immunosuppressive agent.
33. A composition comprising an antibody, or binding fragment thereof, in
combination with a phaxxnaceutically acceptable carrier or ditueat, wherein the antibody is
the antibody of claim 1 or 9.
34. The composition of claim 33, further comprising an iinmunosuppressive
agent
35. A composition comprising an antibody, or bmrimg fragment thereof, in
combmatkm with a pharmaceutically acceptable canier or dfiuent, wherein the antibody is
the antibody of claim 10 or 16.
36. The composition of claim 35, further comprising an immunosuppressive
agent,
37. An isolated nucleic acid encoding the polypepttde of claim 1 or 9.
38. An isolated nucleic acid encoding the polypeptide of claim 10 or 16.

39. An expression vector comprising the nucleic acid sequence of claim 37
operably linked to control sequences recognized by a host cell transfected with the vector.
40. A host cell comprising the vector of claim 38.
41. A method of producing a polypepiide, corcijyising culturing the host cell of
claim 40 under conditions wherein the nucleic acid sequence is expressed, thereby
producing the polypeptide, and recovering the polypeptide from the host cell.
42. An expression vector comprising the nucleic acid sequence of claim 38
operably linked to control sequences recognized by a host cell transfected with the vector.
43. A host cell comprising the vector of claim 42.
44. A method of producing a polypeptide, comprising culturing the host cell of
claim 43 under conditions wherein the nucleic acid sequence Is expressed, thereby
producing the polypeptide, and recovering the polypeptide from fee host cell.
45. An isolated nucleic acid sequence encoding an antibody specific for IL-10
comprising a light chain having the nucleic acid seqnrary of 3SEQ ID NO:21 and a heavy
drain having the nucleic acid sequence of SEQ ID NO:22
46. The nucleic acid of claim 45, wherein fee lisfat chain has an American Type
Culture Collection (ATCC) deposit number of FTA-5923 and the heavy chain has an
ATCC deposit number of PTA-5922.
47. An isolated nucleic acid sequence encoding an antibody specific for IL-10
comprising a light chain having fee nucleic add sequence of SEQ ID NO:23 and a heavy
chain having the nucleic acid sequence of SEQ ID NO:24.

48. The nucleic acid of claim 47, wherein light chain has an ATCC deposit
number of PTA-5927 and the heavy chain has an ATCC deposit number of PTA-5926.
49. An isolated nucleic acid sequence encoding a binding fragment of the
antibody encoded by the nucleic acid sequence of claim 45.
50. An isolated nucleic acid sequence encoding a binding fragment of the
antibody encoded by the nucleic acid sequence of claim 47.
51. The nucleic acid of claim 49 or 50, wherein the binding fragment is an
antibody fragment selected from the group consisting of Fab, Fab*, Fab'-SH, Fv, scFv,
F(ab')2, and a diabody.
52. A method to identify an acceptor gennline sequence for a humanized
antibody, which method comprises the steps of:
a) identifying a non-human antibody that has the desired biological activity;
b) determining the amino acid sequence of a non-human antibody vh and v1
domains; and
c) comparing the nonhuman antibody sequence to a group of human gennline
sequences, wherein the comparison comprises the substeps of:
1) assigning the sequence of non-human Va aod V1 domain sequences
residue numbers;
2) delineating the CDR and FR regions in the sequence;
3) assigning a predetermined numerical score at each residue position
for which the non-human and human germline sequences are identical; and
4) totaling all of the residue scores to generate a total score for each
human gennline sequence; and
d) identifying the human gennline sequence with the highest total residue score
as the acceptor gennline sequence.
53. The method of claim 52, further comprising the sobstqps of:

a) assigning a numerical score of 1 for each residue position for which the non-
human and human germline sequences are identical that was not scored in substep (3) to
germiine sequences with identical total residue scores after substep (4);
b) totaling all of the residue scores to generate a total score for each human
germline sequence.
54. The method of claim 52, wherein the VH region is scored as in Table 2.
55. The method of claim 52, wherein the VL region is scored as in Table 3.
56. The method of claim 52, wherein the non-human antibody is specific for IL-
10 and inhibits the biological activity of EL-10.
57. An antibody generated by the method of claim 52.


Documents:

1575-CHENP-2006 ABSTRACT.pdf

1575-CHENP-2006 CLAIMS.pdf

1575-CHENP-2006 CORRESPONDENCE OTHERS.pdf

1575-CHENP-2006 CORRESPONDENCE PO.pdf

1575-CHENP-2006 POWER OF ATTORNEY.pdf

1575-chenp-2006-abstract.pdf

1575-chenp-2006-assignement.pdf

1575-chenp-2006-claims.pdf

1575-chenp-2006-correspondnece-others.pdf

1575-chenp-2006-description(complete).pdf

1575-chenp-2006-drawings.pdf

1575-chenp-2006-form 1.pdf

1575-chenp-2006-form 18.pdf

1575-chenp-2006-form 26.pdf

1575-chenp-2006-form 3.pdf

1575-chenp-2006-form 5.pdf

1575-chenp-2006-pct.pdf


Patent Number 244268
Indian Patent Application Number 1575/CHENP/2006
PG Journal Number 49/2010
Publication Date 03-Dec-2010
Grant Date 26-Nov-2010
Date of Filing 06-Mar-2006
Name of Patentee SCHERING CORPORATION
Applicant Address 2000 Galloping Hill Road, Kenilworth, New Jersey 07033
Inventors:
# Inventor's Name Inventor's Address
1 PRESTA, Leonard, G. 1900 Gough Street, No. 206, San Francsico, California 94109
PCT International Classification Number C07K16/00
PCT International Application Number PCT/US2004/037518
PCT International Filing date 2004-11-09
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/518,999 2003-11-10 U.S.A.