Title of Invention

AN ISOLATED ANTI-INTERFERON ALPHA MONOCLONAL ANTIBODY, OR ANTIGEN BINDING PORTION THEREOF

Abstract The present invention provides isolated anti-interferon alpha monoclonal antibodies, particularly human monoclonal antibodies, that inhibit the biological activity of multiple interferon (IFN) alpha subtypes but do not substantially inhibit the biological activity of IFN alpha 21 or the biological activity of either IFN beta or IFN omega (Figures 1A-3B). Immunoconjugates, bispecific molecules and pharmaceutical compositions comprising the antibodies of the invention are also provided. The invention also provides methods for inhibiting the biological activity of IFN alpha using the antibodies of the invention, as well as methods of treating disease or disorders mediated by IFN alpha, such as autoimmune diseases, transplant rejection and graft versus host disease, by administering the antibodies of the invention.
Full Text an Isolated anti-interferon alpha monoclonal antibody, or antigen
binding portion thereof
Cross Reference to Related Applications
This application claims the benefit of U.S. Provisional Application Serial No.
60/528,757, filed December 10,2003, the contents of which are hereby incorporated by
reference in its entirety.
Background of the Invention
Type I interferons (IFN) (IFN-a, IFN-p, IFN-to, IFN-t) are a family of
structurally related cytokines having antiviral, antitumor and immunomodulatory effects
(Hardy et al. (2001) Blood 97:473; Cutrone and Langer (2001) J. Biol. Chem. 276:17140V
The human IFNa locus includes two subfamilies. The first subfamily consists of at least 14
non allelic genes and 4 pseudogenes having at least 75% homology. The second subfamily,
all or omega (©), contains 5 pseudogenes and 1 functional gene which exhibits 70%
homology with the IFNa genes. The subtypes of IFNa have different specific activities but
they possess the same biological spectrum (Streuli et al. (1981) Proc. Natl. Acad. Sci. USA
78:2848) and have the same cellular receptor (Agnet M. et al. (1983) in "Interferon 5" Ed. I.
Gresser p. 1-22, Academic Press, London).
All human type I interferons bind to a cell surface receptor (IFN alpha
receptor, IFNAR) consisting of two transmembrane proteins, IFNAR-1 and IFNAR-2 (Uze
et. al. (1990) Cell 60:225; Novick et al. (1994) Cell77:391; Pestka et al. (1987) Annu Rev.
Biochem. 56:727; Mogensen et al. (1999) J. Interferon Cytokine Res. 19:1069). IFNAR-1 is
essential for high affinity binding and differential specificity of the IFNAR complex (Cutrone
(2001) supra). While functional differences for each of the type I IFN subtypes have not
been identified it is thought that each may exhibit different interactions with the IFNAR
receptor components leading to potentially diverse signaling outcomes (Cook et al. (1996) J.
Biol. Chem. 271:13448). In particular, studies utilizing mutant forms of IFNAR1 and
IFNAR2 suggested that alpha and beta interferons signal differently through the receptor by
interacting differentially with respective chains (Lewerenz et al. (1998) J. Mol. Biol.
282:585).
Early functional studies of type I IFNs focused on innate defense against viral
infections (Haller et al (1981) J. Exp. Med. 154:199; Lindenmann et al. (1981) Methods
Enzymol. 78:181). More recent studies, however, implicate type I IFNs as pptent
immunoregulatory cytokines in the adaptive immune response. Specifically, type I IFNs
have been1 shown to facilitate differentiation of naive T cells along the Thl pathway
(Brinkmann et al. (1993) J. Exp. Med. 178:1655), to enhance antibody production
(Finkelman et al. (1991) J. Exp. Med. 174:1179) and to support the functional activity and
survival of memory T cells (Santini, et al. (2000)./. Exp. Med. 191:1777; Tough et al. (1996]
Science 272:1947).
Recent work by a number of groups suggests that IFN-a may enhance the
maturation or activation of dendritic cells (DCs) (Santini, et al. (2000) /. Exp. Med.
121:1777; Luft et al. (1998) J. Immunol. 161:1947; Luft et al. (2002) Int. Immunol. 14:367;
Radvanyi et al. (1999) Scand. J. Immunol. 50:499; Paquette et al. (1998) J. Leukoc. Biol.
64:358). Furthermore, increased expression of type I interferons has been described in
numerous autoimmune diseases (Foulis et al. (1987) Lancet 2:1423; Hooks et al. (1982)
Arthritis Rheum 25:396; Hertzog et al. (1988) Clin. Immunol. Immunopathol. 48:192;
Hopkins and Meager (1988) Clin. Exp. Immunol. 73:88; Arvin and Miller (1984) Arthritis
Rheum. 27:582). The most studied examples of this are insulin-dependent diabetes mellitus
(BDDM) (Foulis (1987J supra), systemic lupus erythematosus (SLE) (Hooks (1982) supra;
Blanco et al (2001) Science 294:1540; Ytterberg and Schnitzer (1982) Arthritis Rheum.
25:401; Batteux et al. (1999) Eur. Cytokine Netw. _:509), and autoimmune thyroiditis
(Prummel and Laurberg (2003) Thyroid .13:547; Mazziotti et al. (2002) /. Endocrinol. Invesi
25:624; You et al. (1999) Chin. Med. J. U2:61; Koh et al. (1997) Thyroid 7:891),which are
all associated with elevated levels of IFN a, and rheumatoid arthritis (RA) (Hertzog (1988),
Hopkins and Meager (1988), Arvin and Miller (1984), supra) in which IFN-P may play a
more significant role.
Moreover, administration of interferon a has been reported to exacerbate
underlying disease in patients with psoriasis, autoimmune thyroiditis and multiple sclerosis
and to induce an SLE like syndrome in patients without a previous history of autoimmune
disease. Interferon a has also been shown to induce glomerulonephritis in normal mice and 1
accelerate the onset of the spontaneous autoimmune disease of NZB/W mice. Further, IFN-
therapy has been shown in some cases to lead to undesired side effects, including fever and
neurological disorders. Hence, there are pathological situations in which inhibition of IFN-
a activity may be beneficial to the patient and a need exists for agents effective in inhibiting
IFN-aiactivity.
Summary of the Invention
The present invention provides isolated monoclonal antibodies that bind to
IFN alpha and inhibit the biological activity of multiple IFN alpha subtypes, but not
substantially inhibit the biological activity of IFN alpha subtype 21, or of IFN beta or IFN
omega. In preferred embodiments, the antibodies of the invention are capable of inhibiting
surface expression of cell markers induced by IFN alpha, inhibiting IP-10 expression induced
by IFN alpha and/or inhibiting dendritic cell development mediated by plasma from patients
with systemic lupus erythematosus (SLE). These antibodies can be used for therapeutic,
including prophylactic, purposes, for example in situations where the production or
expression of interferon alpha is associated with pathological symptoms. Such antibodies can
also be used for the diagnosis of various diseases or for the study of the evolution of such
diseases.
In one embodiment, the present invention includes an antibody or antibody
fragment that binds to IFN alpha, preferably human IFN alpha (e.g., human IFN alpha 2a,
human IFN alpha 2b), and inhibits the biological activity of multiple IFN alpha subtypes, but
does not substantially inhibit the biological activity of IFN alpha subtype 21, or IFN beta or
IFN omega. In addition, in various embodiments, the antibodies of the invention are capable of
inhibiting surface expression of cell markers induced by IFN alpha, inhibiting IP-10 expression
induced by IFN alpha and/or inhibiting dendritic cell development mediated by plasma from
patients with systemic lupus erythematosus (SLE). The antibody or antibody fragment
preferably is a human antibody or antibody fragment, or alternatively can be a murine, chimeric
or humanized antibody. In certain embodiments, an antibody of the invention functions by a
non-competitive mechanism of action. For example, in preferred embodiments, the antibody:
(i) does not inhibit the binding of an IFN alpha, such as IFN alpha 2a, to cells expressing
interferon alpha receptor (IFNAR) and (ii) binds to cells expressing IFNAR in the presence of
an IFN alpha, such as IFN alpha 2a.
In one aspect, the invention pertains to isolated antibodies, or antigen binding
portions thereof, wherein the antibodies:
(a) comprise a heavy chain variable region of a human VH 1 -18 or 4-61
gene;
(b) comprise a light chain variable region of a human A27 gene; and
(c) inhibit the biological activity of interferon alpha (e.g., inhibits the
biological activity of at least one IFN alpha subtype).
In smother aspect, the invention pertains to isolated monoclonal antibodies, or
antigen binding portions thereof, comprising a heavy chain variable region comprising
CDR1, CDR2, and CDR3 sequences and a light chain variable region comprising CDR1,
CDR2, and CDR3 sequences, wherein:
(a) the heavy chain variable region CDR3 sequence comprises the amino
acid sequence of SEQ ED NO: 7, 8, or 9, or conservative modifications thereof;
(b) the light chain variable region CDR3 sequence comprises the amino
acid sequence of SEQ ID NO: 16,17, or 18, or conservative modifications thereof;
(c) the antibody inhibits the biological activity of multiple IFN alpha
subtypes but does not substantially inhibit the biological activity of IFN alpha 21; and
(d) the antibody exhibits at least one of the following properties:
(i) the antibody does not substantially inhibit the biological
activity of IFN beta or IFN omega;
(ii) the antibody inhibits IFN-induced surface expression of CD38 or
MHC Class I on peripheral blood mononuclear cells;
(iii) me antibody inhibits IFN-induced expression of IP-10 by
peripheral blood mononuclear cells;
(iv) the antibody inhibits dendritic cell development mediated by
systemic lupus erythematosus (SLE) plasma.
In such antibodies, the heavy chain variable region CDR2 sequence can
comprise the amino acid sequence of SEQ ID NO: 4,5, or 6, or conservative modifications
thereof; and the light chain variable region CDR2 sequence can comprise the amino acid
sequence of SEQ ID NO: 13,14, or 15, or conservative modifications thereof. Furthermore,
in such antibodies, the heavy chain variable region CDR1 sequence can comprise the amino
acid sequence of SEQ ID NO: 1,2, or 3, or conservative modifications thereof; and the light
chain variable region CDR1 sequence can comprise the amino acid sequence of SEQ ID NO:
10,11, or 12, or conservative modifications thereof.
In another aspect, the invention pertains to isolated monoclonal antibodies, or
antigen binding portions thereof, comprising a heavy chain variable region and a light chain
variable region, wherein:
(a) the heavy chain variable region comprises an amino acid sequence that
is at least 80% homologous to SEQ ID NO: 19,20, or 21;
(b) the light chain variable region comprises an amino acid sequence that
is at least 80% homologous to SEQ ID NO: 22,23, or 24;
(c) the antibody inhibits the biological activity of multiple IFN alpha
subtypes but does not substantially inhibit the biological activity of IFN alpha 21; and
(d) the antibody exhibits at least one of the following properties:
(i) the antibody does not substantially inhibit the biological
activity of IFN beta or IFN omega;
(ii) the antibody inhibits IFN-induced surface expression of CD38 or
MHC Class I on peripheral blood mononuclear cells;
(iii) the antibody inhibits IFN-induced expression of IP-10 by
peripheral blood mononuclear cells;
(iv) the antibody inhibits dendritic cell development mediated by
systemic lupus erythematosus (SLE) plasma.
In another aspect, the invention pertains to isolated monoclonal antibodies, or
antigen binding portions thereof, comprising a heavy chain variable region and a light chain
variable region, wherein:
(a) the heavy chain variable region comprises an amino acid sequence
comprising the amino acid sequence of SEQ ID NO: 19,20, or 21; and
(b) the light chain variable region comprises an amino acid comprising the
amino acid sequence of SEQ ID NO: 22,23, or 24;
wherein the antibody inhibits the biological activity of interferon alpha (e.g.,
inhibits the biological activity of at least one IFN alpha subtype).
In yet another aspect, the invention pertains to mutated variants of SEQ ID
NO: 19 having increased stability. Preferred embodiments include an isolated monoclonal
antibody- or antigen binding portion thereof comprising:
(a) a heavy chain variable region comprising an amino acid sequence selected
from the group consisting of SEQ ID NOs: 34,35,36 and 37; and
(b) a light chain variable region comprising the amino acid sequence of SEQ ID
NO: 22;
wherein the antibody inhibits the biological activity of at least one interferon alpha
subtype.
In yet another aspect, the invention pertains to an isolated monoclonal
antibody, or antigen binding portion thereof comprising:
(a) a heavy chain variable region CDR1 comprising an amino acid sequence
selected from the group consisting of SEQ ID NOs: 1,2 and 3;
(b) a heavy chain variable region CDR2 comprising an amino acid sequence
selected from the group consisting of SEQ ID NOs: 4,5 and 6;
(c) a heavy chain variable region CDR3 comprising an amino acid sequence
selected from the group consisting of SEQ ID NOs: 7, 8 and 9;
(d) a light chain variable region CDR1 comprising an amino acid sequence
selected from the group consisting of SEQ ID NOs: 10,11 and 12;
(e) a light chain variable region CDR2 comprising an amino acid sequence
selected from the group consisting of SEQ ID NOs: 13,14 and 15; and
(f) a light chain variable region CDR3 comprising an amino acid sequence
selected from the group consisting of SEQ ID NOs: 16,17 and 18;
wherein the antibody inhibits the biological activity of interferon alpha (e.g.,
inhibits the biological activity of at least one IFN alpha subtype).
In yet another aspect, the invention pertains to an isolated monoclonal
antibody, or antigen binding portion thereof, that competes for binding to IFN alpha 2a or
IFN alpha 2b with any of the above mentioned antibodies.
In yet another aspect, the invention pertains to an isolated human antibody, or
antigen-binding portion thereof, that inhibits the biological activity of multiple interferon
(IFN) alpha subtypes, wherein the antibody does not inhibit binding of IFN alpha to
interferon alpha receptor (TFNAR)-expressing cells and wherein the antibody associates with
IFNAR-expressing cells in the presence, but not the absence, of IFN alpha.
The invention also encompasses nucleic acid molecules that encode the
antibodies or antigen-binding portions thereof in any of the above mentioned antibodies.
The antibodies of the invention can be of any isotype. Preferred antibodies are
of the IgGl or IgG4 isotype. The antibodies of the invention can be full-length antibodies
comprising variable and constant regions, or they can be antigen-binding fragments thereof,
such as a single chain antibody or a Fab fragment.
The invention also encompasses immunoconjugates of the antibodies of the
invention, in which the antibody is linked to a therapeutic agent, such as a cytotoxin or a
radioactive isotope. The invention also encompasses bispeciflc molecules comprising an
antibody of the invention, in which the antibody is linked to a second functional moiety
having a different binding specificity than the antibody.
Pharmaceutical compositions comprising an antibody, or antigen binding
portion thereof, or immunoconjugate or bispeciflc molecule thereof, are also provided. Such
pharmaceutical compositions comprise the active agent and a phannaceutically acceptable
carrier.
In another aspect, the present invention includes a method of inhibiting the
biological activity of interferon alpha, either in vivo or in vitro, comprising contacting
interferon alpha with an anti-IFN alpha antibody of the invention, such that the biological
activity of interferon alpha is inhibited.
In another aspect, the present invention includes a method of treating an
interferon alpha-mediated disease or disorder in a subject, comprising administering to the
subject an anti-IFN alpha antibody of the invention, such that the interferon-alpha mediated
disease in the subject is treated. Examples of diseases that can be treated include
autoimmune diseases (e.g., systemic lupus erythematosus, multiple sclerosis, insulin
dependent diabetes mellitus, inflammatory bowel disease, psoriasis, autoimmune thyroiditis,
rheumatoid arthritis and glomerulonephritis), transplant rejection and graft versus host
disease.
Other features and advantages of the instant invention will be apparent from
the following detailed description and examples, which should not be construed as limiting.
The contents of all references, patents and published patent applications cited throughout this
application are expressly incorporated herein by reference.
Brief Description of the Drawings
Figure 1A shows the nucleotide sequence (SEQ ID NO: 25) and amino acid
sequence (SEQ ID NO: 19) of the heavy chain variable region of the 13H5 human
monoclonal antibody. The CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 4) and CDR3 (SEQ
ID NO: 7) regions are delineated and the V, D and J germline derivations are indicated.
Figure IB shows the nucleotide sequence (SEQ ID NO: 28) and amino acid
sequence (SEQ ID NO: 22) of the light chain variable region of the 13H5 human monoclonal
antibody. The CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO: 13) and CDR3 (SEQ ID NO:
16) regions are delineated and the V and J germline derivations are indicated.
Figure 2A shows the nucleotide sequence (SEQ ID NO: 26) and amino acid
sequence (SEQ ID NO: 20) of the heavy chain variable region of the 13H7 human
monoclonal antibody. The CDR1 (SEQ ID NO: 2), CDR2 (SEQ ID NO: 5) and CDR3 (SEQ
ID NO: 8) regions are delineated and the V, D and J germline derivations are indicated.
Figure 2B shows the nucleotide sequence (SEQ ID NO: 29) and amino acid
sequence (SEQ ID NO: 23) of the light chain variable region of the 13H7 human monoclonal
antibody. The CDR1 (SEQ ID NO: 11), CDR2 (SEQ ID NO: 14) and CDR3 (SEQ ID NO:
17) regions are delineated and the V and J germline derivations are indicated.
Figure 3A shows the nucleotide sequence (SEQ ID NO: 27) and amino acid
sequence (SEQ ID NO: 21) of the heavy chain variable region of the 7H9 human monoclonal
antibody. The CDR1 (SEQ ID NO: 3), CDR2 (SEQ ID NO: 6) and CDR3 (SEQ ID NO: 9)
regions are delineated and the V, D and J germline derivations are indicated.
Figure 3B shows the nucleotide sequence (SEQ ID NO: 30) and amino acid
sequence (SEQ ID NO: 24) of the light chain variable region of the 7H9 human monoclonal

antibodyi The CDR1 (SEQ ID NO: 12), CDR2 (SEQ ID NO: 15) and CDR3 (SEQ ID NO:
18) regions are delineated and the V and J germline derivations are indicated
Figure 4 shows the alignment of the amino acid sequence of the heavy chain
variable region of 13H5 (SEQ ID NO: 19) and 7H9 (SEQ ID NO: 21) with the human
germline VH 1-18 amino acid sequence (SEQ ID NO: 31)
Figure 5 shows the alignment of the amino acid sequence of the heavy chain
variable region of 13H7 ,(SEQ ID NO: 20) with the human germline VH 4-61 amino acid
sequence (SEQ ID NO: 32).
Figure 6 shows the alignment of the amino acid sequence of the light chain
variable region of 13H5 ,(SEQ ID NO: 22), 13H7 (SEQ ID NO: 23) and 7H9 (SEQ D3
NO: 24) with the human germline VK A27 amino acid sequence (SEQ ID NO: 33).
Figure 7 is a graph showing competition of binding of U5I-IFNa 2a to IFNAR-
expressing Daudi cells by unlabeled IFNa 2a (•) versus enhancement of I-IFNa 2a
binding by mAb 13H5 ( T). An isotype control antibody had no effect on binding (?).
Figure 8 is a graph showing binding of 125I-13H5 to Daudi cells in the
presence of IFNa 2a (¦) but not in the absence of IFNa 2a (A). Specific IFNa-dependent
binding of 13H5 is represented by circles (•).
Figure 9 is a graph showing the results of ADCC assays of Raji cell lysis by
fresh human mononuclear cells in the presence of 13H5 (¦), 13H5 + IFNa (A), an isotype
control antibody + IFNa (T), or a positive control antibody (o). Lysis was only seen with
the positive control.
Detailed Description of the Invention
The present invention relates to isolated monoclonal antibodies that bind to
IFN alpha|and that are capable of inhibiting the biological activity of multiple IFN alpha
subtypes, but not the biological activity of IFN alpha subtype 21, or IFN beta or IFN omega.
The antibodies of the invention are capable of inhibiting surface expression of cell markers
induced by IFN alpha, inhibiting IP-10 expression induced by IFN alpha and inhibiting
dendritic cell development mediated by plasma from patients with systemic lupus
erythematosus (SLE). The invention provides isolated antibodies, methods of making such
antibodies, immunoconjugates and bispecific molecules comprising such antibodies and
pharmaceutical compositions containing the antibodies, immunconjugates or bispecific
molecules of the invention. The invention also relates to methods of using the antibodies to
inhibit IFN alpha activity, for example in the treatment of autoimmune disorders, or for
inhibiting or preventing transplant rejection or in the treatment of graft versus host disease.
In order that the present invention may be more readily understood, certain
terms are first defined. Additional definitions are set forth throughout the detailed
description.
The terms "interferon alpha" and "IFN alpha" are used interchangeably and
intended to refer to IFN alpha proteins encoded by a functional gene of the interferon alpha
gene locus with 75% or greater sequence identity to IFN alpha 1 (Geribank number
NP_076918 or protein encoded by Genbank number NM_024013). Examples of IFN alpha
subtypes include IFN alpha 1, alpha 2a, alpha 2b, alpha 4, alpha 5, alpha 6, alpha 7, alpha 8,
alpha 10, alpha 13, alpha 14, alpha 16, alpha 17 and alpha 21. The term "interferon alpha" is
intended to encompass recombinant forms of the various IFN alpha subtypes, as well as
naturally occurring preparations that comprise IFN alpha proteins, such as leukocyte IFN and
lymphoblastoid IFN. The term IFN alpha is not intended to encompass IFN omega alone,
although a composition that comprises both IFN alpha and IFN omega is encompassed by the
term IFN alpha.
The term "IFN alpha receptor" as used herein is intended to refer to members
of the IFN alpha receptor family of molecules that are receptors for the ligand IFN alpha.
Examples of IFN alpha receptors are IFN alpha receptor 1 and IFN alpha receptor 2.
The term "immune response" refers to the action of, for example,
lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble
macromplecules produced by the above cells or the liver (including antibodies, cytokines, and
complement) that results in selective damage to, destruction of, or elimination from the
human body of invading pathogens, cells or tissues infected with pathogens, cancerous cells,
or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.
A "signal transduction pathway" refers to the biochemical relationship
betweenta variety of signal transduction molecules that play a role in the transmission of a
signal from one portion of a cell to another portion of a cell. As used herein, the phrase "cell
surface receptor" includes, for example, molecules and complexes of molecules capable of
receiving a signal and the transmission of such a signal across the plasma membrane of a cell.
An example of a "cell surface receptor" of the present invention is the IFN alpha receptor 1 or
IFN alpha receptor 2.
The term "antibody" as referred to herein includes whole antibodies and any
antigen binding fragment (i.e., "antigen-binding portion") or single chains thereof. An -
"antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L)
chains iriter-connected by disulfide bonds, or an antigen binding portion thereof. Each heavy
chain is comprised of a heavy chain variable region (abbreviated herein as Vh) and a heavy
chain constant region. The heavy chain constant region is comprised of three domains, Chi,
Ch2 and Ch3- Each light chain is comprised of a light chain variable region (abbreviated
herein as Vl) and a light chain constant region. The light chain constant region is comprised
of one domain, Cl- The Vh and Vl regions can be further subdivided into regions of
hypervaiiability, termed complementarity determining regions (CDR), interspersed with
regions that are more conserved, termed framework regions (FR). Each Vh and Vl is
composed of three CDRs and four FRs, arranged from ammo-terminus to carboxy-terminus
in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of
the heavy and light chains contain a binding domain that interacts with an antigen. The
constant regions of the antibodies may mediate the binding of the immunoglobulin to host
tissues or factors, including various cells of the immune system (e.g., effector cells) and the
first component (Clq) of the classical complement system.
The term "antigen-binding portion" of an antibody (or simply "antibody
portion"), as used herein, refers to one or more fragments of an antibody that retain the ability
to specifically bind to an antigen {e.g., IFN alpha). It has been shown that the antigen-
binding function of an antibody can be performed by fragments of a full-length antibody.
Examples of binding fragments encompassed within the term "antigen-binding portion" of an
antibody include (i) a Fab fragment, a monovalent fragment consisting of the Vl, Vh, Cl and
Chi domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments
linked by a; disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the Vh and
Chi domains; (iv) a Fv fragment consisting of the Vl and Vh domains of a single aim of an
antibody, (y) a dAb fragment (Ward et al, (1989) Nature 341:544-546), which consists of a
Vh domain; and (vi) an isolated complementarity determining region (CDR). Furthermore,
although the two domains of the Fv fragment, Vl and Vh, are coded for by separate genes,
they can be joined, using recombinant methods, by a synthetic linker that enables them to be
made as a single protein chain in which the Vl and Vh regions pair to form monovalent
molecules (known as single chain Fv (scFv); see e.g„ Bird et al. (1988) Science 242:423-426;
and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883;. Such single chain
antibodies are also intended to be encompassed within the term "antigen-binding portion" of
an antibody. These antibody fragments are obtained using conventional techniques known to
those with skill in the art, and the fragments are screened for utility in the same manner as are
intact antibodies.
An "isolated antibody", as used herein, is intended to refer to an antibody that
is substantially free of other antibodies having different antigenic specificities (eg., an
isolated antibody that specifically binds IFN alpha is substantially free of antibodies that
specifically bind antigens other than IFN alpha). An isolated antibody that specifically binds
IFN alpha may, however, have cross-reactivity to other antigens, such as IFN alpha
molecules from other species. Moreover, an isolated antibody may be substantially free of
other cellular material and/or chemicals.
The terms "monoclonal antibody" or "monoclonal antibody composition" as
used herein refer to a preparation of antibody molecules of single molecular composition. A
monoclonal antibody composition displays a single binding specificity and affinity for a
particular epitope.
The term "human antibody", as used herein, is intended to include antibodies
having variable regions in which both the framework and CDR regions are derived from
human germline immunoglobulin sequences. Furthermore, if the antibody contains a
constant region, the constant region also is derived from human germline immunoglobulin
sequences. The human antibodies of the invention may include amino acid residues not
encoded by human germline immunoglobulin sequences (e.g., mutations introduced by
random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the
term "human antibody", as used herein, is not intended to include antibodies in which CDR
sequences derived from the germline of another mammalian species, such as a mouse, have
been grafted onto human framework sequences.
The term "human monoclonal antibody" refers to antibodies displaying a
single binding specificity which have variable regions in which both the framework and CDR
regions are derived from human gerrnline immunoglobulin sequences. In one embodiment,
the human monoclonal antibodies are produced by a hybridoma which includes a B cell
obtained from a transgenic nonhuman animal, e.g., a transgenic mouse, having a genome
comprising a human heavy chain transgene and a light chain transgene fused to an
immortalized cell.
The term "recombinant human antibody*', as used herein, includes all human
antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a)
antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for
human immunoglobulin genes or a hybridoma prepared therefrom (described further below),
(b) antibodies isolated from a host cell transformed to express the human antibody, e.g., from
a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody
library, and (d) antibodies prepared, expressed, created or isolated by any other means that
involve splicing of human immunoglobulin gene sequences to other DNA sequences. Such
recombinant human antibodies have variable regions in which the framework and CDR
regions are derived from human germline immunoglobulin sequences. In certain
embodiments, however, such recombinant human antibodies can be subjected to in vitro
mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic
mutagenesis) and thus the amino acid sequences of the Vh and Vl regions of the recombinant
antibodies are sequences that, while derived from and related to human germline Vh and Vl
sequences, may not naturally exist within the human antibody gerrnline repertoire in vivo.
As used herein, "isotype" refers to the antibody class (e.g., IgM or IgGl) that is
encoded by the heavy chain constant region genes.
As used herein, an antibody that "inhibits the biological activity" of an IFN
alpha subtype is intended to refer to an antibody that inhibits the activity of that subtype by at
least 10%, more preferably at least 20%, 30%, 40%, 50%, 60%, 70% or 80%, as compared to
the level of activity in the absence of the antibody, for example using a functional assay such
as those described in the Examples, such as the Daudi cell proliferation assay. Alternatively,
an antibody that "inhibits the biological activity" of an IFN alpha subtype can refer to an
antibody that inhibits the activity of that subtype with an EC50 of less: than 200 nM or less,
more preferably 100 nM or less, even more preferably 50 nM or less and even more
preferably 10 nM or less.
As used herein, an antibody that "does not substantially inhibit the biological
activity" of an IFN alpha subtype, of of IFN beta or IFN omega, is intended to refer to an
antibody that inhibits the activity of that subtype by at less than 10%, more preferably by less
than 5% and even more preferably by essentially undetectable amounts. Alternatively, an
antibody that "does not inhibit the biological activity" of an IFN alpha subtype can refer to an
antibody that inhibits the activity of that subtype with an EC50 of 300 nM or greater.
As used herein, "specific binding" refers to antibody binding to a predetermined
antigen. Typically, the antibody binds with a dissociation constant (Kd) of 10"8 M or less,
and binds to the predetermined antigen with a Kd that is at least two-fold less than its Kd for
binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or
a closely-related antigen. The phrases "an antibody recognizing an antigen" and " an
antibody specific for an antigen" are used interchangeably herein with the term "an antibody
which binds specifically to an antigen".
The term "K^o,." or "Ka", as used herein, is intended to refer to the
association rate of a particular antibody-antigen interaction, whereas the term "K^g" or "Kd,"
as used herein, is intended to refer to the dissociation rate of a particular antibody-antigen
interaction. The term "Kd", as used herein, is intended to refer to the dissociation constant,
which is obtained from the ratio of Kd to Ka (i.e,. Kd/Ka) and is expressed as a molar
concentration (M). Kd values for antibodies can be determined using methods well
established in the art A preferred method for determining the Kd of an antibody is by using
surface plasmon resonance, preferably using a biosensor system such as a Biacore® system.
As used herein, the term "high affinity' for an IgG antibody refers to an
antibody having a Kd of 10"8 M or less, more preferably 10"9 M or less and even more
preferably 10"10 M or less. However, "high affinity" binding can vary for other antibody
isotypes. For example, "high affinity" binding for an IgM isotype refers to an antibody
having a KD of 10"7 M or less, more preferably 10"8 M or less.
As used herein, the term "subject" includes any human or nonhuman animal.
The term "nonhuman animal" includes all vertebrates, e.g., mammals and non-mammals,
such as nonhuman primates, sheep, dogs, cats, horses, cows chickens, amphibians, reptiles,
etc.
Various aspects of the invention are described in further detail in the following
subsections.
r*
Anti-IFN alpha Antibodies
The antibodies of the invention are characterized by particular functional
features or properties of the antibodies. For example, in particular embodiments, the
antibodies bind specifically to multiple subtypes of IFN alpha, such as IFN alpha 2a and IFN
alpha 2b. Preferably, an antibody of the invention binds to IFN alpha 2a and/or alpha 2b with
high affinity, for example with a KD of 10"8 M or less or 10'9 M or less or even 10"10 M or
less. In a preferred embodiment, the antibody binds to human IFN alpha 2a and human IFN
alpha 2b. The binding affinity and kinetics of the antibodies of the invention can be
examined by, for example, Biacore analysis as described in the Examples.
Furthermore, in other embodiments, the antibodies of the invention exhibit
various functional properties. For example, the antibodies may be capable of inhibiting the
biological activity of multiple IFN alpha subtypes but may not substantially inhibit the
biological activity of IFN alpha 21. The antibodies also may not substantially inhibit the
biological activity of IFN beta or IFN omega. The antibodies of the invention also may be
capable of inhibiting IFN-induced surface expression of cell markers, such as CD38 or MHC
Class I, on normal human peripheral blood mononuclear cells. The antibodies also may be
capable of inhibiting IFN-induced expression of IP-10 by normal human peripheral blood
mononuclear cells. Inhibition of biological activity of IFN alpha subtypes, IFN beta and/or
IFN omega can be evaluated using functional assays such as those described in the Examples,
such as a Daudi cell proliferation assay.
Still further, the antibodies may be capable of inhibiting dendritic cell development mediated
by plasma of patients with systemic lupus erythematosus (SLE). Dendritic cell development
can be assessed by examining the expression of cell surface markers, such as CD38, MHC
Class I and/or CD123, as described in the Examples.
In certain preferred embodiments, an antibody of the invention inhibits the
biological activity of IFN alpha by a non-competitive mechanism of action, i.e., the antibody
does not compete for binding of IFN alpha to IFNAR. Rather, such an antibody becomes
associated with cell-surface IFNAR in the presence of IFN alpha and1 inhibits cell signaling
through IFNAR. In other preferred embodiments, an antibody having these binding
properties does not exhibit significant ADCC activity. Assays for examining these functional
properties of the antibody are known in the art, such as the assays described in Examples 8
and 9. For example, the ability of the antibody to inhibit binding of radiolabeled IFN alpha to
IFNAR-expressing cells can be examined. The inability of the antibody to inhibit the binding
of radiolabeled IFN alpha to IFNAR is indicative of a non-competitive mechanism of action.
To further examine mis mechanism of action, the binding of radiolabeled antibody, in the
presence or absence of IFN alpha, to IFNAR-expressing cells can be assayed. Binding of the
radiolabeled antibody to IFNAR-expressing cells in the presence, but not the absence, of IFN
alpha is indicative this mechanism of action.
In a preferred embodiment, antibodies of the invention bind to the IFN alpha -
IFNAR complex with a greater affinity (e.g., Kd) than to IFN alpha alone (one or more
subtypes) and/or to IFNAR alone. For example, in certain embodiments, antibodies of the
invention bind the IFN alpha-IFNAR complex with a Kp of 10'8 M or greater affinity, a Kd of
10"9 M or greater affinity, or a Kd of 10"10 M or greater affinity.
In another preferred embodiment, antibodies of the invention are bispecific for
IFN alpha (one or more subtypes) and IFNAR (IFNAR1 and/or IFNAR2), meaning that the
antibodies associate with both IFN alpha and IFNAR (IFNAR1 and/or IFNAR2).
Accordingly, the present invention includes bispecific molecules comprising at least one first
binding specificity for IFN alpha and a second binding specificity for IFNAR1, wherein, for
example, the second binding specificity for IFNAR1 can be formed by the association of the
antibody with IFN alpha. The present invention also includes bispecific molecules
comprising at least one binding specificity for IFN alpha and a second binding specificity for
IFNAR2, wherein, for example, the second binding specificity for INFAR2 can be formed by
association of the antibody with IFN alpha.
Monoclonal Antibodies 13H5.13H7 and 7H9
Preferred antibodies of the invention are the human monoclonal antibodies
13HS, 13H7, and 7H9, isolated and structurally characterized as described in the Examples.
The VH amino acid sequences of 13H5,13H7, and 7H9 are shown in SEQ ID NOs: 19,20,
and 2Irrespectively. The Vl amino acid sequences of 13H5,13H7, and 7H9 are shown in
SEQ ID] NOs: 22,23 and 24, respectively. Given that each of these antibodies can bind to
IFN alpha, the Vh and Vl sequences can be "mixed and matched" to create other anti- IFN
alpha binding molecules of the invention. IFN alpha binding or neutralizing activity of such
"mixed [and matched" antibodies can be tested using the binding assays described above and
in the Examples (e.g., ELISA, Biacore analysis, Daudi cell proliferation assay). Preferably,
the Vh sequences of 13H5 and 7H9 are mixed and matched, since these antibodies use Vh
sequences derived from the same germline sequence (VH 1-18) and thus they exhibit
structural similarity. Additionally or alternatively, the Vl sequences of 13H5,13H7 and 7H9
can be mixed and matched, since these antibodies use Vl sequences derived from the same
germline sequence (Vk A27) and thus they exhibit structural similarity.
Accordingly, in one aspect, the invention provides an isolated monoclonal
antibody, or antigen binding portion thereof, comprising:
(a) a heavy chain variable region comprising an amino acid sequence selected
from the group consisting of SEQ ID NOs: 19,20, and 21; and
(b) a light chain variable region comprising an amino acid sequence selected
from the group consisting of SEQ ID NOs: 22,23, and 24;
wherein the antibody inhibits the biological activity of interferon alpha.
Preferred heavy and light chain combinations include:
(a) a heavy chain variable region comprising the amino acid sequence of SEQ
ID NO: 19; and (b) a light chain variable region comprising the amino acid sequence of SEQ
IDNO:22;or
(a) a heavy chain variable region comprising the amino acid sequence of SEQ
ID NO: 20; and (b) a light chain variable region comprising the amino acid sequence of SEQ
IDNO:23;or
(a) a heavy chain variable region comprising the amino acid sequence of SEQ
ID NO: 21; and (b) a light chain variable region comprising the amino acid sequence of SEQ
IDNO:24.
In another aspect, the invention provides antibodies that comprise the heavy
chain and light chain CDRls, CDR2s, and CDR3s of 13H5,13H7, and 7H9, or combinations
thereof. The amino acid sequences of the Vh CDRls of 13H5,13H7, and 7H9 are shown in
SEQ IN NOs: 1,2, and 3. The amino acid sequences of the VH CDR2s of 13H5,13H7, and
7H9 are shown in SEQ IN NOs: 4, 5, and 6. The amino acid sequences of the VH CDR3s of
13H5,13H7, and 7H9 are shown in SEQ IN NOs: 7,8, and 9. The amino acid sequences of
the VL CDRls of 13H5,13H7, and 7H9 are shown in SEQ IN NOs: 10,11, and 12. The
amino acid sequences of the VL CDR2s of 13H5,13H7, and 7H9 are shown in SEQ IN NOs:
13,14, and 15. The amino acid sequences of the VL CDR3s of 13H5,13H7, and 7H9 are
shown in SEQ IN NOs: 16,17, and 18. The CDR regions are delineated using the Kabat
system (Kabat. E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth
Edition, U.S. Department of Health and Human Services, NIH Pulibcation No. 91-3242).
Given that each of these antibodies was selected based on IFN binding activity and
that antigen-binding specificity is provided primarily by the CDR1,2 and 3 regions, the Vh
CDR1,2 and 3 sequences and Vl CDR1,2 and 3 sequences can be "mixed and matched"
(i.e., CDRs from different antibodies can be mixed and match, although each antibody must
contain a VH CDR1,2 and 3 and a VL CDR1,2 and 3) to create other anti-IFN alpha
molecules of the invention. IFN alpha binding of such "mixed and matched" antibodies can
be tested using the binding assays described in the Examples (e.g., ELISA and/or Biacore).
Preferably, when Vh CDR sequences are mixed and matched, the CDR1, CDR2 and/or
CDR3 sequence from a particular Vh sequence is replaced with a structurally similar CDR
sequence(s). Likewise, when Vl CDR sequences are mixed and matched, the CDR1, CDR2
and/or CDR3 sequence from a particular Vl sequence preferably is replaced with a
structurally similar CDR sequence(s). For example, the Vh CDRls of 13H5 and 7H9 share
some structural similarity and therefore are amenable to mixing and matching. It will be
readily apparent to the ordinarily skilled artisan that novel Vh and Vl sequences can be
created by substituting one or more Vh and/or Vl CDR region sequences with structurally
similar sequences from the CDR sequences disclosed herein for monoclonal antibodies
antibodies 13H5,13H7 and 7H9.
Accordingly, in another aspect, the invention provides an isolated monoclonal
antibody, or antigen binding portion thereof comprising:
(a) a heavy chain variable region CDR1 comprising an amino acid sequence
selected from the group consisting of SEQ ID NOs: 1,2, and 3;
(b) a heavy chain variable region CDR2 comprising an amino acid sequence
selected from the group consisting of SEQ ID NOs: 4, 5, and 6;
(c) a heavy chain variable region CDR3 comprising an amino acid sequence
selectedjfrom the group consisting of SEQ ID NOs: 7, 8, and 9;
(d) a light chain variable region CDR1 comprising an amino acid sequence
selectedjfrom the group consisting of SEQ ID NOs: 10,11, and 12;
(e) a light chain variable region CDR2 comprising an amino acid sequence
selected from the group consisting of SEQ ID NOs: 13,14, and 15; and
(f) a light chain variable region CDR3 comprising an amino acid sequence
selected from the group consisting of SEQ ID NOs: 16,17, and 18;
wherein the antibody the antibody inhibits the biological activity of interferon
alpha.
In a preferred embodiment, the antibody comprises:
(a) a heavy chain variable region CDR1 comprising SEQ ID NO: 1;
(b) a heavy chain variable region CDR2 comprising SEQ ID NO: 4;
(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 7;
(d) a light chain variable region CDR1 comprising SEQ ID NO: 10;
(e) a light chain variable region CDR2 comprising SEQ ID NO: 13; and
(f) a light chain variable region CDR3 comprising SEQ ID NO: 16.
In another preferred embodiment, the antibody comprises:
(a) a heavy chain variable region CDR1 comprising SEQ ID NO: 2;
(b) a heavy chain variable region CDR2 comprising SEQ ID NO: 5;
(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 8;
(d) a light chain variable region CDR1 comprising SEQ ID NO: 11;
(e) a light chain variable region CDR2 comprising SEQ ID NO: 14; and
(i) a light chain variable region CDR3 comprising SEQ ID NO: 17.
In another preferred embodiment, the antibody comprises:
(a) a heavy chain variable region CDR1 comprising SEQ ID NO: 3;
(b) a heavy chain variable region CDR2 comprising SEQ ID NO: 6;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 9;
(d) a light chain variable region CDR1 comprising SEQ ID NO: 12;
(e) a light chain variable region CDR2 comprising SEQ ED NO: 15; and
(f) a light chain variable region CDR3 comprising SEQ ID NO: 18.
Antibodies Having Particular Germline Sequences
In certain embodiments, an antibody of the invention comprises a heavy chain
variable region from a particular germline heavy chain immunoglobulin gene and/or a light
chain variable region from a particular germline light chain immunoglobulin gene.
For example, in a preferred embodiment, the invention provides an isolated
monoclonal antibody, or an antigen-binding portion thereof, therein the antibody:
(a) comprises a heavy chain variable region of a human VH 1-18 or 4-61 gene;
(b) comprises a light chain variable region of a human Vk A27 gene; and
(c) the antibody inhibits the biological activity of interferon alpha.
In one embodiment, the antibody comprises a heavy chain variable region of a
human VH 1-18 gene. Examples of antibodies having a VH and Vk gene sequence of VH 1-
18 and Vk A27, respectively, include 13H5 and 7H9. In another embodiment, me antibody
comprises a heavy chain variable region of a human VH 4-61 gene. An example of an
antibody having a VH and Vk gene sequence of VH 4-61 and Vk A27, respectively, is 13H7.
As used herein, a human antibody comprises heavy or light chain variable
regions "of (i.e., the products of) or "derived from" a particular germline sequence if the
variable regions of the antibody are obtained from a system that uses human germline
immunoglobulin genes. Such systems include immunizing a transgenic mouse carrying
human immunoglobulin genes with the antigen of interest or screening a human
immunoglobulin gene library displayed on phage with the antigen of interest A human
antibody mat is "of (i.e., the product of) or "derived from" a human germline
immunoglobulin sequence can be identified as such by comparing the amino acid sequence of
the human antibody to the amino acid sequences of human germline immunoglobulins and
selecting the human germline immunoglobulin sequence that is closest in sequence (i.e.,
greatest % identity) to the sequence of the human antibody. A human antibody that is "of
(i.e., the product of) or "derived from" a particular human gennline immunoglobulin
sequence may contain amino acid differences as compared to the germline sequence, due to,
for example, naturally-occurring somatic mutations or intentional introduction of site-directed
mutation. However, a selected human antibody typically is at least 90% identical in amino
acids sequence to an amino acid sequence encoded by a human gennline immunoglobulin
gene and contains amino acid residues that identify the human antibody as being human when
compared to the germline immunoglobulin amino acid sequences of other species (e.g.,
murine germline sequences). In certain cases, a human antibody may be at least 95%, or
even at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid
sequence encoded by the germline immunoglobulin gene. Typically, a human antibody
derived from a particular human germline sequence will display no more than 10 amino acid
differences from the amino acid sequence encoded by the human germline immunoglobulin
gene. In certain cases, the human antibody may display no more than 5, or even no more
than 4,3,2, or 1 amino acid difference from the amino acid sequence encoded by the
germline immunoglobulin gene.
Homologous Antibodies
In yet another embodiment, an antibody of the invention comprises heavy and
light chain variable regions comprising amino acid sequences that are homologous to the
amino acid sequences of the preferred antibodies described herein, and wherein the
antibodies retain the desired functional properties of the anti-IFN alpha antibodies of the
invention.
For example, the invention provides an isolated monoclonal antibody, or
antigen binding portion thereof, comprising a heavy chain variable region and a light chain
variable iregion, wherein:
(a) the heavy chain variable region comprises an amino acid sequence that
is at least 80% homologous to an amino acid sequence selected from the group consisting of
SEQ ID NOs: 19,20, and 21;
(b) the light chain variable region comprises an amino acid sequence that
is at least 80% homologous to an amino acid sequence selected from the group consisting of
SEQ ID NOs: 22,23, and 24;
(c) the antibody inhibits the biological activity of multiple IFN alpha
subtypes but does not substantially inhibit the biological activity of IFN alpha
21;
(d) the antibody exhibits at least one of the following properties:
(i) the antibody does not substantially inhibit the biological
activity of IFN beta or IFN omega;
(ii) the antibody inhibits IFN-induced surface expression of CD38 or
MHC Class I on peripheral blood mononuclear cells;
(iii) the antibody inhibits IFN-induced expression of IP-10 by
peripheral blood mononuclear cells;
(iv) the antibody inhibits dendritic cell development mediated by
systemic lupus erythematosus (SLE) plasma.
In other embodiments, the Vh and/or Vl amino acid sequences maybe 85%,
90%, 95%, 96%, 97%, 98% or 99% homologous to the sequences set forth above. An
antibody having Vh and Vl regions having high (z'.e., 80% or greater) homology to the VH
and VL regions of SEQ ID NOs: 19,20, and 21 and 22,23, and 24, respectively, can be
obtained by mutagenesis (e.g., site-directed or PCR-mediated mutagenesis) of nucleic acid
molecules encoding SEQ ID NOs: 19,20, and 21 and/or 22,23, and 24, followed by testing
of the encoded altered antibody for retained function (i.e., the functions set forth in (c) and
(d) above) using the functional assays described herein.
As used herein, the percent homology between two amino acid sequences is
equivalent to the percent identity between the two sequences. The percent identity between
the two sequences is a function of the number of identical positions shared by the sequences
(j.e., % homology = # of identical positions/total # of positions x 100), taking into account the
number of gaps, and the length of each gap, which need to be introduced for optimal
alignment of the two sequences. The comparison of sequences and determination of percent
identity between two sequences can be accomplished using a mathematical algorithm, as
described in the non-limiting examples below.
The percent identity between two amino acid sequences can be determined
i
using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988))
which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight
residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent
identity between two amino acid sequences can be determined using the Needleman and
Wunsch (J. Mol Biol. 48:444-453 (1970)) algorithm which has been incorporated into the
GAP program in the GCG software package (available at http://www.gcg.com), using either a
Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16,14,12,10, 8, 6, or 4 and a
length weight of 1,2,3,4,5, or 6.
Additionally or alternatively, the protein sequences of the present invention
can further be used as a "query sequence" to perform a search against public databases to, for
example, identify related sequences. Such searches can be performed using the XBLAST
program (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST protein
searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain
amino acid sequences homologous to the antibody molecules of the invention. To obtain
gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in
Altschul et al, (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and
Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST
and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.
Antibodies with Conservative Modifications
In certain embodiments, an antibody of the invention comprises a heavy chain
variable region comprising CDR1, CDR2 and CDR3 sequences and a light chain variable
region Comprising CDR1, CDR2 and CDR3 sequences, wherein one or more of these CDR
sequences comprise specified amino acid sequences based on the preferred antibodies
described herein (e.g., 13H5,13H7, or 7H9), or conservative modifications thereof, and
wherein the antibodies retain the desired functional properties of the anti-IFN alpha
antibodies of the invention. For example, preferred antibodies of the invention include those
in which the heavy chain variable region CDR3 sequence comprises the amino acid sequence
of SEQ ID NO: 3, or conservative modifications thereof, and the light chain variable region
CDR3 sequence comprises the amino acid sequence of SEQ ID NO: 6, or conservative
modifications thereof. Accordingly, the invention provides an isolated monoclonal antibody,
or antigen binding portion thereof, comprising a heavy chain variable region comprising
CDR1, CDR2, and CDR3 sequences and a light chain variable region comprising CDR1,
CDR2, and CDR3 sequences, wherein:
(a) the heavy chain variable region CDR3 sequence comprises the amino
# acid sequence selected from the group consisting of SEQ ID NO: 7,8, and 9, and
conservative modifications thereof;
(b) the light chain variable region CDR3 sequence comprises the amino
acid sequence selected from the group consisting of SEQ ID NO: 16,17, and 18, and
conservative modifications thereof;
(c) the antibody inhibits the biological activity of multiple IFN alpha
subtypes but does not substantially inhibit the biological activity of IFN alpha
21;
(d) the antibody exhibits at least one of the following properties:
(i) the antibody does not substantially inhibit the biological
activity of IFN beta or IFN omega;
(ii) the antibody inhibits IFN-induced surface expression of CD3 8 or
MHC Class I on peripheral blood mononuclear cells; •
(iii). the antibody inhibits IFN-induced expression of IP-10 by
peripheral blood mononuclear cells;
(iv) the antibody inhibits dendritic cell development mediated by
systemic lupus erythematosus (SLE) plasma.
In a further embodiment, the heavy chain variable region CDR2 sequence
comprises the amino acid sequence selected from the group consisting of amino acid
sequences of SEQ ID NO: 4,5, and 6, and conservative modifications thereof; and the light
chain variable region CDR2 sequence comprises the amino acid sequence selected from the
group consisting of amino acid sequences SEQ ID NO: 13,14, and 15, and conservative
modifications thereof. In a still further embodiment, the heavy chain variable region CDR1
sequence comprises the amino acid sequence selected from the group consisting of amino
acid sequences of SEQ ID NO: 1,2, and 3, and conservative modifications thereof; and the
light chain variable region CDR1 sequence comprises the amino acid sequence selected from
the group consisting of amino acid sequences of SEQ ID NO: 10,11, and 12, and
conservative modifications thereof.
As used herein, the term "conservative sequence modifications" is intended to
refer to amino acid modifications that do not significantly affect or alter the binding
characteristics ofmeanu^ody containing the amino acid sequence. Such conservative
modifications include amino acid substitutions, additions and deletions. Modifications can be
introduced into an antibody of the invention by standard techniques known in the art, such as
site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid
substitutions are ones in which the amino acid residue is replaced with an ainino acid residue
having a similar side chain. Families of amino acid residues having similar side chains have
been defined in the art. These families include amino acids with basic side chains (e.g.,
lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged
polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine,
tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and
aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more
amino acid residues within the CDR regions of an antibody of the invention can be replaced
with other amino acid residues from the same side chain family and the altered antibody can
be tested for retained function (i.e., the functions set forth in (c) and (d) above) using the
functional assays described herein.
Antibodies that Bind to the Same Epitope as Anti-IFN Alpha Antibodies of the Invention
In another embodiment, the invention provides antibodies that bind to the
same epitope as do the various human IFN alpha antibodies of the invention provided herein,
such as other human antibodies that bind to the same epitope as the 13H5,13H7, and 7H9
antibodies described herein. Such antibodies can be identified based on their ability to cross-
compete (e.g., to competitively inhibit the binding of, in a statistically significant manner)
with other antibodies of the invention, such as 13H5,13H7 or 7H9, in standard IFN alpha
binding assays. For example, as demonstrated in the Examples by Biacore analysis, 13H5
binds with high affinity to IFN alpha 2a and IFN alpha 2b. Accordingly, in one embodiment,
the invention provides antibodies, preferably human antibodies, that compete for binding to
IFN alpha 2a or IFN alpha 2b with another antibody of the invention (e.g., 13H5,13H7 or
7H9). The ability of a test antibody to inhibit the binding of, e.g., 13H5,13H7 or 7H9 to IFN
alpha 2a or IFN alpha 2b demonstrates that the test antibody can compete with that antibody
for binding toIFN alpha 2a or IFN alpha 2b; such an antibody may, according to non-limiting
theory, bind to the same or a related (e.g., a structurally similar or spatially proximal) epitope
on IFN alpha 2a or IFN alpha 2b as the antibody with which it competes. In a preferred
embodiment, the antibody that binds to the same epitope on IFN alpha 2a or IFN alpha 2b as,
e.g., 13H5,13H7, or 7H9, is a human monoclonal antibody. Such human monoclonal
antibodies can be prepared and isolated as described in the Examples.
Engineered and Modified Antibodies
An antibody of the invention further can be prepared using an antibody having
one or more of the Vh and/or Vl sequences disclosed herein as starting material to engineer a
modified antibody, which modified antibody may have altered properties from the starting
antibody. An antibody can be engineered by modifying one or more residues within one or
both variable regions (i.e., Vh and/or Vl), for example within one or more CDR regions
and/or within one or more framework regions. Additionally or alternatively, an antibody can
be engineered by modifying residues within the constant region(s), for example to alter the
effector function(s) of the antibody.
One type of variable region engineering that can be performed is CDR
grafting. Antibodies interact with target antigens predominantly through amino acid residues
that are located in the six heavy and light chain complementarity determining regions
(CDRs). |For this reason, the amino acid sequences within CDRs are more diverse between
individual antibodies than sequences outside of CDRs. Because CDR sequences are
i
responsible for most antibody-antigen interactions, it is possible to express recombinant
antibodies that mimic the properties of specific naturally occurring antibodies by constructing
expression vectors that include CDR sequences from the specific naturally occurring
antibody grafted onto framework sequences from a different antibody with different
properties (see, e.g., Riechmann, L. et al. (1998) Nature 332:323-327; Jones, P. et al. (1986)
Nature 321:522-525: Queen, C. et al. (1989) Proc. Natl Acad. Sci. U.S.A. 86:10029-10033;
U.S. Patent No. 5,225,539 to Winter, and U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762
and 6,180,370 to Queen et al.)
Accordingly, another embodiment of the invention pertains to an isolated
monoclonal antibody, or antigen binding portion thereof, comprising a heavy chain variable
region comprising CDR1, CDR2, and CDR3 sequences comprising the amino acid sequences
selected from the group consisting of SEQ ID NO: 1,2, and 3, SEQ ID NO: 4,5, and 6 and
SEQ ID NO: 7,8, and 9, respectively, and a light chain variable region comprising CDR1,
CDR2, and CDR3 sequences comprising the amino acid sequences selected from the group
consisting of SEQ ID NO:10,11, and 12, SEQ ED NO: 13,14, and 15 and SEQ ID NO: 16,
17, and 18, respectively. Thus, such antibodies contain the Vh and Vl CDR sequences of
monoclonal antibodies 13H5,13H7 or 7H9, yet may contain different framework sequences
from these antibodies.
Such framework sequences can be obtained from public DNA databases or
published references that include germline antibody gene sequences. For example, germline
DNA sequences for human heavy and light chain variable region genes can be found in the
"VBase" human germline sequence database (available on the Internet at www.mrc-
cpe.cam.ac.uk/vbase'). as well as in Kabat, E. A., et al. (1991) Sequences of Proteins of
Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH
Publication No. 91-3242; Tomlinson, I. M., et al (1992) "The Repertoire of Human Germline
Vh Sequences Reveals about Fifty Groups of Vh Segments with Different Hypervariable
Loops" J. Mol. Biol. 227:776-798: and Cox, J. P.. L. et al. (1994) "A Directory of Human
Germ-line Vh Segments Reveals a Strong Bias in their Usage" Eur. J. Immunol. 24:827-836;
the contents of each of which are expressly incorporated herein by reference.
Preferred framework sequences for use in the antibodies of the invention are
those that are structurally similar to the framework sequences used by selected antibodies of
the invention, e.g., similar to the VH 1-18 or 4-61 and VK A27 framework sequences used by
the preferred monoclonal antibodies of the invention. The Vh CDR1,2 and 3 sequences, and
the Vl CDR1,2 and 3 sequences can be grafted onto framework regions that have the same
sequence as that found in the germline immunoglobulin gene from which the framework
sequence derive, or the CDR sequences can be grafted onto framework regions that contain
one or more mutations as compared to the germline sequences. For example, it has been
found that in certain, instances it is beneficial to mutate residues within the framework regions
to maintain or enhance the antigen binding ability of the antibody (see e.g., U.S. Patent Nos.
5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et at).
i
Another type of variable region modification is to mutate amino acid residues
within the[VH and/or Vl CDR1, CDR2 and/or CDR3 regions to thereby improve one or more
binding properties (e.g., affinity) of the antibody of interest. Site-directed mutagenesis or
PCR-mediated mutagenesis can be performed to introduce the mutation(s) and the effect on
antibody binding, or other functional property of interest, can be evaluated in in vitro or in
vivo assays as described herein and provided in the Examples. Preferably conservative
modifications (as discussed above) are introduced. The mutations may be amino acid
substitutions, additions or deletions, but are preferably substitutions. Moreover, typically no
more than one, two, three, four or five residues are altered within a CDR region are altered.
Accordingly, in another embodiment, the invention provides isolated anti-IFN
alpha monoclonal antibodies, or antigen binding portions thereof, comprising a heavy chain
variable region comprising: (a) a Vh CDR1 region comprising an amino acid sequence
selected from the group consisting of SEQ ID NOs: 1,2, and 3, or an amino acid sequence
having one, two, three, four or five amino acid substitutions, deletions or additions as
compared to SEQ ID NOs: 1,2, or 3; (b) a Vh CDR2 region comprising an amino acid
sequence selected from the group consisting of SEQ ID NOs: 4,5, and 6, or an amino acid
sequence having one, two, three, four or five amino acid substitutions, deletions or additions
as compared to SEQ ID NOs: 4,5, or 6; (c) a Vh CDR3 region comprising an amino acid
sequence selected from the group consisting of SEQ ID NOs: 7,8, and 9, or an amino acid
sequence having one, two, three, four or five amino acid substitutions, deletions or additions
as compared to SEQ ID NOs: 7,8, or 9; (d) a Vl CDR1 region comprising an amino acid
sequence selected from the group consisting of SEQ ID NOs: 10,11, and 12, or an amino
acid sequence having one, two, three, four or five amino acid substitutions, deletions or
i
additions as compared to SEQ ID NOs: 10,11, or 12; (e) a Vl CDR2 region comprising an
amino acid sequence selected from the group consisting of SEQ ID NOs: 13,14, and 15, or
an amino acid sequence having one, two, three, four or five amino acid substitutions,
deletions or additions as compared to SEQ ID NOs: 13,14, or 15; and (f) a Vl CDR3 region
comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 16,
17, and 18, or an amino acid sequence having one, two, three, four or five amino acid
substitutions, deletions or additions as compared to SEQ ID NOs: 16,17, or 18.
Engineered antibodies of the invention include those in which modifications
have been made to framework residues within Vh and/or Vl, e.g. to improve the properties of
the antibody. Typically such framework modifications are made to decrease the
immunogenicity of the antibody. For example, one approach is to 4*backmutate" one or more
framework residues to the corresponding gennline sequence. More specifically, an antibody
that has undergone somatic mutation may contain framework residues that differ from the
germline sequence from which the antibody is derived. Such residues can be identified by
comparing the antibody framework sequences to the germline sequences from which the
antibody is derived. For example, for 13H5, amino acid residue #81 (within FR3) of Vh is a
leucine whereas this residue in the corresponding VH 1-18 gennline sequence is a methionine
(see Figure 4). To return the framework region sequences to their germline configuration, the
somatic mutations can be "backmutated" to the germline sequence by, for example, site-
directed mutagenesis or PCR-mediated mutagenesis (e.g., residue 81 of the Vh of 13H5 can
be "backmutated" from leucine to methionine). Such "backmutated" antibodies are also
intended to be encompassed by the invention.
Another type of framework modification involves mutating one or more
residues within the framework region, or even within one or more CDR regions, to remove T
cell epitopes to thereby reduce the potential immunogenicity of the antibody. This approach
is also referred to as "deimmunization" and is described in fiither detail in U.S. Patent
Publication No. 20030153043 by Carr et al.
In addition or alternative to modifications made within the framework or CDR
regions, antibodies of the invention maybe engineered to include modifications within the Fc
region, typically to alter one or more functional properties of the antibody, such as serum
half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular
cytotoxicity. Furthermore, an antibody of the invention may be chemically modified {e.g.,
one or more chemical moieties can be attached to the antibody) or be modified to alter it's
glycosylation, again to alter one or more functional properties of the antibody. Each of these
embodiments is described in further detail below. The numbering of residues in the Fc region
is that of the EU index of Kabat.
In one embodiment, the hinge region of CHI is modified such that the number
of cysteine residues in the hinge region is altered, e.g., increased or decreased. This approach
is described further in U.S. Patent No. 5,677,425 by Bodmer et al. The number of cysteine
residues infthe hinge region of CHI is altered to, for example, facilitate assembly of the light
and heavy chains or to increase or decrease the stability of the antibody.
In another embodiment, the Fc hinge region of an antibody is mutated to
decrease the biological half life of the antibody. More specifically, one or more amino acid
mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment
such that the antibody has impaired Staphylococcyl protein A (SpA) binding relative to
native Fc-hinge domain SpA binding. This approach is described in further detail in U.S.
Patent No. 6,165,745 by Ward et al.
In another embodiment, the antibody is modified to increase its biological half
life. Various approaches are possible. For example, one or more of the following mutations
can be introduced: T252L, T254S, T256F, as described in U.S. Patent No. 6,277,375 to
Ward. Alternatively, to increase the biological half life, the antibody can be altered within
the CHI or CL region to contain a salvage receptor binding epitope taken from two loops of a
CH2 domain of an Fc region of an IgG, as described in U.S. Patent Nos. 5,869,046 and
6,121,022 by Presta et al.
hi yet other embodiments, the Fc region is altered by replacing at least one
amino acid residue with a different amino acid residue to alter the effector function(s) of the
antibody. For example, one or more amino acids selected from amino acid residues 234,235,
236,237,297, 318,320 and 322 can be replaced with a different amino acid residue such that
the antibody has an altered affinity for an effector ligand but retains the antigen-binding
ability of the parent antibody. The effector ligand to which affinity is altered can be, for
example, an Fc receptor or the CI component of complement. This approach is described in
further detail in U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter et al.
In another example, one or more amino acids selected from amino acid
residues 329, 331 and 322 can be replaced with a different amino acid residue such that the
antibody has altered Clq binding and/or reduced or abolished complement dependent
cytotoxicity (CDC). This approach is described in further detail in U.S. Patent Nos.
6,194,551 by Idusogie et al.
In another example, one or more amino acid residues within amino acid
positions 231 and 239 are altered to thereby alter the ability of the antibody to fix
complement. This approach is described further in PCT Publication WO 94/29351 by
Bodmer et al.
In yet another example, the Fc region is modified to increase the ability of the
antibody ko mediate antibody dependent cellular cytotoxicity (ADCC) and/or to increase the
affinity of the antibody for an Fey receptor by modifying one or more amino acids at the
following positions: 238,239,248,249,252,254,255,256, 258,265,267,268,269,270,
272, 216, 278,280,283,285,286,289, 290,292,293,294,295,296,298,301,303, 305,
307,309,312,315,320,322,324,326,327,329,330,331,333,334,335,337,338,340,
360, 373,376,378, 382,388,389,398,414,416,419,430,434,435,437,438 or 439. This
approach is described further in PCT Publication WO 00/42072 by Presta. Moreover, the
binding sites on human IgGl for FcyRl, FcyRH, FcyRIII and FcRn have been mapped and
variants with improved binding have been described (see Shields, RX. et al. (2001) J. Biol.
Chem. 276:6591-6604). Specific mutations at positions 256,290,298,333,334 and 339
were shown to improve binding to FcyRIII. Additionally, the following combination mutants
were shown to improve FcyRIII binding: T256A/S298A, S298A7E333A, S298A/K224A and
S298A/E333A/K334A.
In still another embodiment, the glycosylation of an antibody is modified. For
example, an aglycoslated antibody can be made (i.e., the antibody lacks glycosylation).
Glycosylation can be altered to, for example, increase the affinity of the antibody for antigen.
Such carbohydrate modifications can be accomplished by, for example, altering one or more
sites of glycosylation within the antibody sequence. For example, one or more amino acid
substitutions can be made that result in elimination of one or more variable region framework
glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may
increase the affinity of the antibody for antigen. Such an approach is described in further
detail in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al.
Additionally or alternatively, an antibody can be made that has an altered type
of glycosylation, such as ahypofucosylated antibody having reduced amounts of fucosyl
residues or an antibody having increased bisecting GlcNac structures. Such altered
glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies.
Such carbohydrate modifications can be accomplished by, for example, expressing the
antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation
machinery have been described in the art and can be used as host cells in which to express
recombinant antibodies of the invention to thereby produce an antibody with altered
glycosylation. For example, EP 1,176,195 by Hanai et al. describes a cell line with a
functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies
expressed in such a cell line exhibit hypofucosylation. PCT Publication WO 03/035835 by
Presta describes a variant CHO cell line, Lecl3 cells, with reduced ability to attach fucose to
Asn(297)jlinked carbohydrates, also resulting in hypofucosylation of antibodies expressed in
that host cell (see also Shields, R.L. et al (2002) J. Biol Chem. 277:26733-26740). PCT
Publication WO 99/54342 by Umana et al. describes cell lines engineered to express
i
gjycoprotein-modifying glycosyl transferases (e.g., beta(l,4)-N-
acetylglucosaminyltransferase HI (GnTlil)) such that antibodies expressed in the engineered
cell lines exhibit increased bisecting GlcNac structures which results in increased ADCC
activity of the antibodies (see also Umana et al. (1999) Nat. Biotech. 17:176-180).
Another modification of the antibodies herein that is contemplated by the
invention is pegylation. An antibody can be pegylated to, for example, increase the
biological (e.g., serum) half life of the antibody. To pegylate an antibody, the antibody, or
fragment ^hereof, typically is reacted with polyethylene glycol (PEG), such as a reactive ester
or aldehyde derivative of PEG, under conditions in which one or more PEG groups become
attached to the antibody or antibody fragment. Preferably, the pegylation is carried out via an
acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous
reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended
to encompass any of the forms of PEG that have been used to derivatize other proteins, such
as mono (C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide.
In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods
for pegylating proteins are known in the art and can be applied to the antibodies of the
invention. See for example, EP 0 154 316 by Nishimura et al. and EP 0 401 384 by Ishikawa
et al.
Modified Antibodies with Increased Stability
In another aspect, the invention provides modified forms of the 13H5 antibody
that exhibit increased stability as compared to wild-type 13H5. As described in further detail
in Example 10, the 13H5 antibody contains a deamidation site at Asn-55 within CDR2 of the
Vh chain. The amino acid sequence at this site, from positions 55 to 58) is N G N T (amino
acid residues 55-58 of SEQ ID NO: 19). Accordingly, in certain embodiments, the amino
acid sequence of the 13H5 Vh chain is mutated at position 55 from asparagine to a different

amino acid. Additionally or alternatively, amino acid positions around Asn-55 that influence
deamidation can be mutated. Preferred amino acid substitutions at position 55 include
aspartic acid and glutamine, with glutamine being more preferred. [The amino acid sequence
of 13H5 with a N55D substitution is shown in SEQ ID NO: 34. The amino acid sequence of
13H5 with a N55Q substitution is shown in SEQ ID NO: 35. In another embodiment, Asn-57
of the 13H5 Vh chain is also mutated, together with mutation of Asn-55. A preferred amino
acid substitution at position 57 is glutamine. The amino acid sequence of 13H5 with N55Q
and N57Q substitutions is shown in SEQ ID NO: 36. These three mutated antibodies exhibit
increased stability, under forced deamidation conditions, as compared to wild-type 13H5, as
described further in Example 11.
In another embodiment, the glycine at amino acid position 56 is mutated to an alanine
(G56A), since it has been determined from model peptides that the rate of deamidation is
approximately 20-fold less with an alanine adjacent to the asparagine, rather than a glycine
adjacent to the alanine (see e.g., Ahern, T. and Manning, M.C., eds. Stability of Protein
Pharmaceuticals. Pharmaceutical Biotechnology, volume 2, chapter 1, pages 1-30). Thus, the
G56A mutation represents a balance between decreased reactivity and niinimal structural
change to the wild type sequence, thus increasing stability while mamtaining activity. The
amino acid sequence of 13H5 with a G56A substitution is shown in SEQ ID NO: 37.
Accordingly, in various embodiments, the invention provides an IFN alpha
antibody of the invention having an amino acid substitution at Asn-55, Gly-56 and/or Asn-57
of the CDR2 of the 13H5 Vh chain, the wild type sequence of which is shown set forth in
SEQ ID NO: 19. Preferred mutated antibodies comprise a heavy chain variable region
comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 34,
35,36 and 37. Preferably, the antibody VH chain is paired with the VK chain of 13H5, as set
forth in SEQ ID NO: 22.
Methods of Engineering Antibodies
As discussed above, the anti-IFN alpha antibodies having VH and VL
sequences disclosed herein can be used to create new anti-IFN alpha antibodies by modifying
the VH and/or VL sequences, or the constant region(s) attached thereto. Thus, in another
aspect of the invention, the structural features of an anti-IFN alpha antibody of the invention,
e.g. 13H5,13H7 or 7H9, are used to create structurally related anti-IFN alpha antibodies that
retain at least one functional property of the antibodies of the invention, such as binding to
IFN alpha. For example, one or more CDR regions of 13H5,13H7 or 7H9, or mutations
thereof, can be combined recombinantly with known framework regions and/or other CDRs
to create additional, recombinantly-engineered; anti-IFN alpha antibodies of the invention, as
discussed above. Other types of modifications include those described in the previous
section. The starting material for the engineering method is one or more of the Vh and/or Vl
sequences provided herein, or one or more CDR regions thereof. To create the engineered
antibody, it is not necessary to actually prepare (z.e., express as a protein) an antibody having
one or|more of the Vh and/or Vl sequences provided herein, or one or more CDR regions
thereof. Rather, the information contained in the sequence(s) is used as the starting material
to create a "second generation" sequence(s) derived from the original sequence(s) and then
the "second generation" sequence(s) is prepared and expressed as a protein.
Accordingly, in another embodiment, the invention provides a method for
preparing an anti-IFN alpha antibody comprising:
(a) providing: (i) a heavy chain variable region antibody sequence comprising
a CDR1 amino acid sequence selected from the group consisting of SEQ ID NOs: 1,2 and 3;
and/or a CDR2, amino acid sequence selected from the group consisting of SEQ ID NOs: 4,5,
and 6; and/or a CDR3 amino acid sequence selected from the group consisting of SEQ ID
NOs: 7, 8, and 9; and/or (ii) a light chain variable region antibody sequence comprising a
CDR1 amino acid sequence selected from the group consisting of SEQ ID NOs: 10,11, and
12; and/or a CDR2 amino acid sequence selected from the group consisting of SEQ ID NOs:
13,14, and 15; and/or a CDR3 amino acid sequence selected from the group consisting of
SEQ ED NOs: \&jj and l£;
(b) altering at least one amino acid residue wifliin the heavy chain variable
region antibody sequence and/or the light chain variable region antibody sequence to create at
least one altered antibody sequence; and
(c) expressing the altered antibody sequence as a protein.
Standard molecular biology techniques can be used to prepare and express the
altered antibody sequence.
Preferably, the antibody encoded by the altered antibody sequence(s) is one
that retains one, some or all of the functional properties of the anti-IFN alpha antibodies
described herein, which functional properties include, but are not limited to:
(i) inhibiting the biological activity of interferon alpha;
(ii) inhibiting the biological activity of multiple IFN alpha subtypes but not
substantially inhibiting the biological activity of IFN alpha 21;
(iii) not substantially inhibiting the biological activity of IFN beta or IFN
omega;
(:iv) inhibiting IFN-induced surface expression of CD3 8 or MHC Class I on
peripheral blood mononuclear cells;
(v) inhibiting IFN-induced expression of IP-10 by peripheral blood
mononuclear cells;
(vi) inhibiting dendritic cell development mediated by systemic lupus
erythematosus (SLE) plasma;
(vii) binding to human interferon alpha 2a with high affinity;
(viii) binding to human interferon alpha 2b with high affinity.
The functional properties of the altered antibodies can be assessed using
standard assays available in the art and/or described herein. For example, the ability of the
antibody to bind IFN alpha can be determined using standard binding assays, such as those
set forth in the Examples (e.g., ELISAs and/or Biacores). The abilityof the antibody to
inhibit various functional activities of interferon alpha can be determined using assays such
as those described in the Examples (e.g., Daudi cell proliferation, IFN-induced cell marker
expression, IFN-induced IP-10 expression etc.)
In certain embodiments of the methods of engineering antibodies of the
invention, mutations can be introduced randomly or selectively along all or part of an antir
IFN alpha antibody coding sequence (e.g., 13H5 coding sequence) and the resulting modified
anti-IFN alpha antibodies can be screened for binding activity and/or other functional
properties as described herein. Mutational methods have been described in the art. For
example, PCT Publication WO 02/092780 by Short describes methods for creating and
screening antibody mutations using saturation mutagenesis, synthetic ligation assembly, or a
combination thereof. Alternatively, PCT Publication WO 03/074679 by Lazar et al.
describes methods of using computational screening methods to optimize physiochemical
properties of antibodies.
Nucleic Acid Molecules Encoding Antibodies of the Invention
Another aspect of the invention pertains to nucleic acid molecules that encode
the antibodies of the invention. The nucleic acids may be present in whole cells, in a cell
lysate, or in a partially purified or substantially pure form. A nucleic acid is "isolated" or
"rendered substantially pure" when purified away from other cellular components or other
contaminants, e.g.y other cellular nucleic acids or proteins, by standard techniques, including
alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis
and others well known in the art. See, F. Ausubel, et al, ed. (1987) Current Protocols in
Molecular Biology, Greene Publishing and Wiley Laterscience, New York. A nucleic acid of
the invention can be, for example, DNA or RNA and may or may not contain intronic
sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.
Nucleic acids of the invention can be obtained using standard molecular
biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared
from transgenic mice carrying human immunoglobulin genes as described further below),
cDNAs encoding the light and heavy chains of the antibody made by the hybridoma can be
obtained by standard PCR amplification or cDNA cloning techniques. For antibodies
obtained from an immunoglobulin gene library (e.g., using phage display techniques), nucleic
acid encoding the antibody can be recovered from the library.
Preferred nucleic acids molecules of the invention are those encoding the Vh
and Vl sequences of the 13H5,13H7, or 7H9 monoclonal antibodies. DNA sequences
encoding the VH sequences of 13H5,13H7, and 7H9 are shown in SEQ ID NOs: 25,26, and
27, respectively. DNA sequences encoding the Vl sequences of 13H5,13H7, and 7H9 are
shown in SEQ ID NOs: 28,29, and 30, respectively.
Once DNA fragments encoding Vh and Vl segments are obtained, these DNA
fragments can be further manipulated by standard recombinant DNA techniques, for example
to convert the variable region genes to full-length antibody chain genes, to Fab fragment
genes or to a scFv gene. In these manipulations, a Vl- or VH-encoding DNA fragment is
operatively linked to another DNA fragment encoding another protein, such as an antibody
constant region or a flexible linker. The term "operatively linked", as used in this context, is
intended to mean that the two DNA fragments are joined such that the amino acid sequences
encoded by the two DNA fragments remain in-frame.
The isolated DNA encoding the Vh region can be converted to a full-length
heavy chain gene by operatively linking the VH-encoding DNA to another DNA molecule
encoding heavy chain constant regions (CHI, CH2 and CH3). The sequences of human
heavy chain constant region genes are known in the art (see e.g., Kabat, E. A., el al. (1991)
Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health
and Human Services, NIH Publication No. 91-3242) and DNA fragments encompassing these
regions can be obtained by standard PCR amplification. The heavy chain constant region can
be an IgGl, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably is
an IgGl or IgG4 constant region. For a Fab fragment heavy chain gene, the VH-encoding
DNA can be operatively linked to another DNA molecule encoding only the heavy chain
CHI constant region.
The isolated DNA encoding the Vl region can be converted to a full-length
light chain gene (as well as a Fab light chain gene) by operatively linking the VL-encoding
DNA to another DNA molecule encoding the light chain constant region, CK. The sequences
of human light chain constant region genes are known in the art (see e.g., Kabat, E. A., et al.
(1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of
Health and Human Services, NIH Publication No. 91-3242) and DNA fragments
encompassing these regions can be obtained by standard PCR amplification. The light chain
constant region can be a kappa or lambda constant region, but most preferably is a kappa
constant region.
To create a scFv gene, the Vh- and VL-encoding DNA fragments are
operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino
acid sequence (Gly4 -Ser)3, such that the Vh and Vl sequences can be expressed as a
contiguous single-chain protein, with the Vl and Vh regions joined by the flexible linker (see
e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Set USA
85:5879-5883; McCafferty et al, (1990) Nature 348:552-554).
Production of Monoclonal Antibodies of the Invention


Monoclonal antibodies (mAbs) of the present invention can be produced by a
variety of techniques, including conventional monoclonal antibody methodology e.g., the
standard somatic cell hybridization technique of Kohler and Milstein (1975) Nature 256: 495.
Although somatic cell hybridization procedures are preferred, in principle, other techniques
for producing monoclonal antibody can be employed e.g., viral or oncogenic transformation
of B lymphocytes.
The preferred animal system for preparing hybridomas is the murine system.
Hybridoma production in the mouse is a very well-established procedure. Immunization
protocols and techniques for isolation of immunized splenocytes for fusion are known in the
art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known.
Chimeric or humanized antibodies of the present invention can be prepared
based on the sequence of a murine monoclonal antibody prepared as described above. DNA
encoding the heavy and light chain immunoglobulins can be obtained from the murine
hybridoma of interest and engineered to contain non-murine (e.g.,. human) immunoglobulin
sequences using standard molecular biology techniques. For example, to create a chimeric
antibody, the murine variable regions can be linked to human constant regions using methods
known in the art (see e.g., U.S. Patent No. 4,816,567 to Cabilly et al.). To create a
humanized antibody, the murine CDR regions can be inserted into a human framework using
methods known in the art (see e.g., U.S. Patent No. 5,225,539 to Winter, and U.S. Patent
Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al).
In a preferred embodiment, the antibodies of the invention are human
monoclonal antibodies. Such human monoclonal antibodies directed against IFN alpha can
be generated using transgenic or transchromosomic mice carrying parts of the human immune
system rather than the mouse system. These transgenic and transchromosomic mice include
mice referred to herein as HuMAb mice and KM mice, respectively, and are collectively
referred to herein as "human Ig mice."
The HuMAb mouse® (Medarex, Inc.) contains human immunoglobulin gene
miniloci that encode unrearranged human heavy (u. and y) and k light chain immunoglobulin
sequences, together with targeted mutations that inactivate the endogenous \x. and k chain loci
(see e.g., Lonberg, et al. (1994) Nature 368(6474): 856-859). Accordingly, the mice exhibit
reduced expression of mouse IgM or k, and in response to immunization, the introduced
human heavy and light chain transgenes undergo class switching and somatic mutation to
generate high affinity human IgGK monoclonal (Lonberg, N. et al. (1994), supra; reviewed in
Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. and
Huszar, D. (1995) Intern. Rev. Immunol. 13:65-93, and Harding, F. and Lonberg, N. (1995)
Ann. N.Y. Acad. Set 764:536-546). The preparation and use of HuMab mice, and the
genomic modifications carried by such mice, is further described in Taylor, L. et al. (1992)
Nucleic Acids Research 20:6287-6295; Chen, L et al. (1993) International Immunology 5:
647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA 90:3720-3724; Choi et al. (1993)
Nature Genetics 4:117-123; Chen, J. et al. (1993) EMBO J. 12: 821-830; Tuaillon etal.
(1994) J. Immunol. 152:2912-2920; Taylor, L. et al. (1994) International Immunology 6:
579-591; and Fishwild, D. et al. (1996) Nature Biotechnology 14: 845-851, the contents of all
of which are hereby specifically incorporated by reference in their entirety. See further, U.S.
Patent Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016;
5,814,318; 5,874,299; and 5,770,429; all to Lonberg and Kay; U.S. Patent No. 5,545,807 to
Surani et al; PCT Publication Nos. WO 92/03918, WO 93/12227, WO 94/25585, WO
97/13852, WO 98/24884 and WO 99/45962, all to Lonberg and Kay; and PCT Publication
No. WO 01/14424 to Korman et al.
In another embodiment, human antibodies of the invention can be raised using
a mouse that carries human immunoglobulin sequences on transgenes and transchomosomes,
such as a mouse that carries a human heavy chain transgene and a human light chain
transchromosome. Such mice, referred to herein as "KM mice", are described in detail in
PCT Publication WO 02/43478 to Ishida et al
Still further, alternative transgenic animal systems expressing human
immunoglobulin genes are available in the art and can be used to raise anti-IFN alpha
antibodies of the invention. For example, an alternative transgenic system referred to as the
Xenomouse (Abgenix, Inc.) can be used; such mice are described in, for example, U.S. Patent
Nos. 5,939,598; 6,075,181; 6,114,598; 6,150,584 and 6,162,963 to Kucherlapati et al.
Moreover, alternative transchromosomic animal systems expressing human
immunoglobulin genes are available in the art and can be used to raise anti-IFN alpha
antibodies of the invention. For example, mice carrying both a human heavy chain
transchromosome and a human light chain tranchromosome, referred to as "TC mice" can be
used; such mice are described in Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97:722-
727. Furthermore, cows carrying human heavy and light chain transchromosomes have been
described in the art (Kuroiwa et ah (2002) Nature Biotechnology 20:889-894) and can be
used to raise anti-IFN alpha antibodies of the invention.
Human monoclonal antibodies of the invention can also be prepared using
phage display methods for screening libraries of human immunoglobulin genes. Such phage
display methods for isolating human antibodies are established in the art See for example:
U.S. Patent Nos. 5,223,409; 5,403,484; and 5,571,698 to Ladner et al; U.S. Patent Nos.
5,427,908 and 5,580,717 to Dower et al; U.S. Patent Nos. 5,969,108 and 6,172,197 to
McCafferty et al; and U.S. Patent Nos. 5,885,793; 6,521,404; 6,544,731; 6,555,313;
6,582,915 and 6,593,081 to Griffiths et al
Human monoclonal antibodies of the invention can also be prepared using
SCID mice into which human immune cells have been reconstituted such that a human
antibody response can be generated upon immunization. Such mice are described in, for
example, U.S. Patent Nos. 5,476,996 and 5,698,767 to Wilson et al
Immunization of Human Ig Mice
When human Ig mice are used to raise human antibodies of the invention, such
mice can be immunized with a purified or recombinant preparation of IFN alpha antigen, as
described by Lonberg, N. et al (1994) Nature 368(6474): 856-859; Fishwild, D. et al (1996)
Nature Biotechnology 14: 845-851; and PCT Publication WO 98/24884 and WO 01/14424.
Preferably, the mice will be 6-16 weeks of age upon the first infusion. For example, a
purified preparation of lymphoblastoid IFN (25-100 ug), prepared by treating a
lymphobiastoid cell line with virus such that the cell line produces an IFN alpha preparation
containing multiple IFN alpha subtypes (but not IFN omega) can be used to immunize the
human Ig mice intraperitoneally. Alternatively, mixtures of recombinant forms of IFN alpha
subtypes can be used as the immunogen.
Detailed procedures to generate fully human monoclonal antibodies to IFN
alpha are described in Example 1 below. Cumulative experience with various antigens has
shown that the transgenic mice respond when initially immunized intraperitoneally (IP) with
antigen in complete Freund's adjuvant, followed by every other week IP immunizations (up to
a total of 6) with antigen in incomplete Freund's adjuvant. However, adjuvants other than
Freund's are also found to be effective. In addition, whole cells in the absence of adjuvant are
found to be highly immunogenic. The immune response can be monitored over the course of
the immunization protocol with plasma samples being obtained by retroorbital bleeds. The
plasma can be screened by ELISA (as described below), and mice with sufficient titers of
anti-IFN 'alpha human immunoglobulin can be used for fusions. Mice can be boosted
intravenously with antigen 3 days before sacrifice and removal of the spleen. It is expected
that 2-3 fusions for each immunization may need to be performed. Between 6 and 24 mice
are typically immunized for each antigen. For HuMab mice, usually both HCo7 and HCol2
strains are used. In addition, both HCo7 and HCol2 transgene can be bred together into a
single mouse having two different human heavy chain transgenes (HCo7/HCol2).
Alternatively or additionally, the KM mouse strain can be used, as described in Example 2.
Generation of Hvbridomas Producing Human Monoclonal Antibodies of the
Invention
To generate hybridomas producing human monoclonal antibodies of the
invention, splenocytes and/or lymph node cells from immunized mice can be isolated and
fused to an appropriate immortalized cell line, such as a mouse myeloma cell line. The
resulting hybridomas can be screened for the production of antigen-specific antibodies. For
example, single cell suspensions of splenic lymphocytes from immunized mice can be fused
to one-sixth the number of P3X63-Ag8.653 nonsecreting mouse myeloma cells (ATCC, CRL
1580) with 50% PEG. Cells are plated at approximately 2 x 105 in fiat bottom microliter
plate, followed by a two week incubation in selective medium containing 20% fetal Clone
Serum, 18% "653" conditioned media, 5% origen (IGEN), 4 mM I^glutamine, 1 mM sodium
pyruvate, 5mM HEPES, 0.055 mM 2-mercaptoethanol, 50 units/ml penicillin, 50 mg/ml
streptomycin, 50 mg/ml gentamycin and IX HAT (Sigma; the HAT is added 24 hours after
the fusion). After approximately two weeks, cells can be cultured in medium in which the
HAT is! replaced with HT. Individual wells can then be screened by ELISA for human
monoclonal IgM and IgG antibodies. Once extensive hybridoma growth occurs, medium can
be observed usually after 10-14 days. The antibody secreting hybridomas can be replated,
screened again, and if still positive for human IgG, the monoclonal antibodies can be
subcloned at least twice by limiting dilution. The stable subclones can then be cultured in
vitro to generate small amounts of antibody in tissue culture medium for characterization.
To purify human monoclonal antibodies, selected hybridomas can be grown in
two-liter spinner-flasks for monoclonal antibody purification. Supernatants can be filtered
and concentrated before affinity chromatography with protein A-sepharose (Pharmacia,
Piscataway, N.J.). Eluted IgG can be checked by gel electrophoresis and high performance
liquid chromatography to ensure purity. The buffer solution can be exchanged into PBS, and
the concentration can be determined by OD280 using 1.43 extinction coefficient. The
monoclonal antibodies can be aliquoted and stored at -80' C.
Generation of Transfectomas Producing Monoclonal Antibodies of the
Invention.
Antibodies of the invention also can be produced in a host cell transfectoma
using, for example, a combination of recombinant DNA techniques and gene transfection
methods as is well known in the art (e.g., Morrison, S. (1985) Science 229:1202).
For example, to express the antibodies, or antibody fragments thereof, DNAs
encoding partial or full-length light and heavy chains, can be obtained by standard molecular
biology techniques (e.g., PCR amplification or cDNA cloning using a hybridoma that
expresses the antibody of interest) and the DNAs can be inserted into expression vectors such
that the genes are operatively linked to transcriptional and translational control, sequences. In
this context, the term "operatively linked" is intended to mean that an antibody gene is ligated
into a vector such that transcriptional and translational control sequences within the vector
serve their intended function of regulating the transcription and translation of the antibody
gene. The expression vector and expression control sequences are chosen to be compatible
with the expression host cell used. The antibody light chain gene and the antibody heavy
chain gene can be inserted into separate vector or, more typically, both genes are inserted into
the same expression vector. The antibody genes are inserted into the expression vector by
standard methods (e.g., ligation of complementary restriction sites on the antibody gene
fragment and vector, or blunt end ligation if no restriction sites are present). The light and
heavy chain variable regions of the antibodies described herein can be used to create full-
length antibody genes of any antibody isotype by inserting them into expression vectors
already encoding heavy chain constant and light chain constant regions of the desired isotype
such thatjthe Vh segment is operatively linked to the Ch segment(s) within the vector and the
YL segment is operatively linked to the O, segment within the vector. Additionally or
alternatively, the recombinant expression vector can encode a signal peptide that facilitates
secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into
the vector such that the signal peptide is linked in-frame to the amino terminus of the
antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a
heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).
In addition to the antibody chain genes, the recombinant expression vectors of
the invention carry regulatory sequences that control the expression of the antibody chain
genes in a host cell. The term "regulatory sequence" is intended to include promoters,
enhancers and other expression control elements (e.g., polyadenylation signals) that control
the transcription or translation of the antibody chain genes. Such regulatory sequences are
described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology
185, Academic Press, San Diego, CA (1990)). It will be appreciated by those skilled in the
art that the design of the expression vector, including the selection of regulatory sequences,
may depend on such factors as the choice of the host cell to be transformed, the level of
expression of protein desired, etc. Preferred regulatory sequences for mammalian host cell
expression include viral elements that direct high levels of protein expression in mammalian
cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV), Simian
Virus 40 (SV40), adenovirus, (e.g., the adenovirus major late promoter (AdMLP) and
polyoma. Alternatively, nonviral regulatory sequences may be used, such as the ubiquitin
promoter or p-globin promoter. Still further, regulatory elements composed of sequences
from different sources, such as the SRa promoter system, which contains sequences from the
SV40 early promoter and the long terminal repeat of human T cell leukemia virus type 1
(Takebe, Y. etal. (1988) Mol. Cell. Biol. 8:466-472).
In addition to the antibody chain genes and regulatory sequences, the
recombinant expression vectors of the invention may carry additional sequences, such as
sequences that regulate replication of the vector in host cells (e.g., origins of replication) and
selectable marker genes. The selectable marker gene facilitates selection of host cells into
which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216,4,634,665 and
5,179,017, all by Axel et al.). For example, typically the selectable marker gene confers
resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the
vector has been introduced. Preferred selectable marker genes include the dihydrofolate
reductase (DHFR) gene (for use in dnfr- host cells with methotrexate selection/amplification)
and the neo gene (for G418 selection).
For expression of the light and heavy chains, the expression vectors)
encoding the heavy and light chains is transfected into a host cell by standard techniques.
The various form;; of the term "transfection" are intended to encompass a wide variety of
techniques commonly used for the introduction of exogenous DNA into a prokaryotic or
eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran
transfection and the like. Although it is theoretically possible to express the antibodies of the
invention, in either prokaryotic or eukaryotic host cells, expression of antibodies in eukaryotic
cells, andi most preferably mammalian host cells, is the most preferred because such
eukaryotic cells, and in particular mammalian cells, are more likely than prokaryotic cells to
assemble and secrete a properly folded and immunologically active antibody. Prokaryotic
expression of antibody genes has been reported to be ineffective for production of high yields
of active antibody (Boss, M. A. and Wood, C. R. (1985) Immunology Today 6:12-13).
Preferred mammalian host cells for expressing the recombinant antibodies of
the invention include Chinese Hamster Ovary (CHO cells) (including dhfr- CHO cells,
described in Urlaub and Chasin, (1980) Proc. Natl Acad. Set USA 77:4216-4220, used with
a DHFR selectable marker, e.g., as described in R. J. Kaufman and P. A. Sharp (1982) Mol
Biol 75P:601-621), NSO myeloma cells, COS cells and SP2 cells. In particular, for use with
NSO myeloma cells, another preferred expression system is the GS gene expression system
disclosed in WO 87/04462, WO 89/01036 and EP 338,841. When recombinant expression
vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are
produced by culturing the host cells for a period of time sufficient to allow for expression of
the antibody in the host cells or, more preferably, secretion of the antibody into the culture
medium in which the host cells are grown. Antibodies can be recovered from the culture
medium using standard protein purification methods.
Characterization of Antibody Binding to Antigen
Antibodies of the invention can be tested for binding to IFN alpha by, for
example!, standard ELISA or by Biacore analysis. Briefly, for ELISAs, microliter plates are
j
coated with IFN alpha (e.g., the recombinant form of different IFN alpha subtypes, or
leukocyte or lymphoblastoid IFN) at 0.25 ug/ml in PBS, and then blocked with 5% bovine
serum aibumin in PBS. Dilutions of antibody (e.g., dilutions of plasma from IFN alpha-
immunized mice) are added to each well and incubated for 1-2 hours at 37°C. The plates are

washed with PBS/Tween and then incubated with secondary reagent (e.g., for human
antibodies, a goat-anti-human IgG Fc-specific polyclonal reagent) conjugated to alkaline
phosphatase for 1 hour at 37°C. After washing, the plates are developed with pNPP substrate
(1 mg/ml), and analyzed at OD of 405-650. Preferably, mice which develop the highest titers
will be used for fusions.
An ELISA assay as described above can also be used to screen for hybridomas
that show positive reactivity with IFN alpha immunogen. Hybridomas that bind with high
avidity to IFN alpha are subcloned and further characterized. One clone from each
hybridoma, which retains the reactivity of the parent cells (by ELISA), can be chosen for
making a 5-10 vial cell bank stored at -140 °C, and for antibody purification.
To purify anti-IFN alpha antibodies, selected hybridomas can be grown in
two-liter spinner-flasks for monoclonal antibody purification. Supernatants can be filtered
and concentrated before affinity chromatography with protein A-sepharose (Pharmacia,
Piscataway, NJ). Eluted IgG can be checked by gel electrophoresis and high performance
liquid chromatography to ensure purity. The buffer solution can be exchanged into PBS, and
the concentration can be determined by OD280 using 1.43 extinction coefficient. The
monoclonal antibodies can be aliquoted and stored at -80 °C.
To determine if the selected anti-IFN alpha monoclonal antibodies bind to
unique epitopes, each antibody can be biotinylated using commercially available reagents
(Pierce, Rockford, EL). Competition studies using unlabeled monoclonal antibodies and
biotinylated monoclonal antibodies can be performed using IFN alpha coated-ELISA plates
as described above. Biotinylated mAb binding can be detected with a strep-avidin-alkaline
phosphatase probe.
To determine the isotype of purified antibodies, isotype ELISAs can be
performed using reagents specific for antibodies of a particular isotype. For example, to
determine the isotype of a human monoclonal antibody, wells of microliter plates can be
coated with 1 |j.g/ml of anti-human immunoglobulin overnight at 4° C. After blocking with
1% BSA, the plates are reacted with 1 jig /ml or less of test monoclonal antibodies or purified
isotype controls, at ambient temperature for one to two hours. The wells can then be reacted
with either human IgGl or human IgM-specific alkaline phosphatase-conjugated probes.
Plates are developed and analyzed as described above.
Anti-IFN alpha human IgGs can be further tested for reactivity with IFN alpha
antigen by Western blotting. Briefly, cell extracts from cells expressing IFN alpha can be
prepared and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. After
electrophoresis, the separated antigens are transferred to nitrocellulose membranes, blocked
with 10% fetal calf serum, and probed with the monoclonal antibodies to be tested. Human
IgG binding can be detected using anti-human IgG alkaline phosphatase and developed with
BCUVNBT substrate tablets (Sigma Chem. Co., St. Louis, Mo.).
Immunoconjugates
In another aspect, the present invention features an anti-IFN alpha antibody, or
a fragment thereof, conjugated to a therapeutic moiety, such as a cytotoxin, a drug (e.g, an
immunosuppressant) or a radiotoxin. Such conjugates are referred to herein as
"immunoconjugates". Immunoconjugates that include one or more cytotoxins are referred to
as "immunotoxins." A cytotoxin or cytotoxic agent includes any agent that is detrimental to
(e.g., kills) cells. Examples include taxoL cytochalasin B, gramicidin D, ethidium bromide,
emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin,
daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-
dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and
puromycin and analogs or homologs thereof. Therapeutic agents also include, for example,
antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-
fluorouracil decarbazine), alkylating agents (e.g., mecMorethamine, thioepa chlorambucil,
melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)
cisplatin); anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin),
antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and
anthramyein (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).
Other preferred examples of therapeutic cytotoxins that can be conjugated to
an antibody of the invention include duocarmycins, calicheamicins, maytansines and
auristatins, and derivatives thereof. An example of a calicheamicin antibody conjugate is
commercially available (Mylotarg™; Wyeth-Ayerst).
Cytoxins can be conjugated to antibodies of the invention using linker
technology available in the art. Examples of linker types that have been used to conjugate a
cytotoxin to an antibody include, but are not limited to, hydrazones, thioethers, esters,
disulfides and peptide-containing linkers. A linker can be chosen that is, for example,
susceptible to cleavage by low pH within the lysosomal compartment or susceptible to
cleavage by proteases, such as proteases preferentially expressed in tumor tissue such as
cathepsins (e.g., cathepsins B, C, D).
For further discussion of types of cytotoxins, linkers and methods for
conjugating therapeutic agents to antibodies, see also Saito, G. et al. (2003) Adv. DrugDeliv.
Rev. 55:199-215; Trail, P.A. et al. (2003) Cancer Immunol Immunother. 52:328-337; Payne,
G. (2003) Cancer Cell 3:207-212; Allen, T.M. (2002) Nat. Rev. Cancer 2:750-763; Pastan, I.
andKreitman, R. J. (2002) Curr. Opin. Investig. Drugs 3:1089-1091; Senter, P.D. and
Springer, C.J. (2001)^cfv. DrugDeliv. Rev. 53:247-264.
Antibodies of the present invention also can be conjugated to a radioactive
isotope to generate cytotoxic radiopharmaceuticals, also referred to as
radioimmunoconjugates. Examples of radioactive isotopes that can be conjugated to
antibodies for use diagnostically or therapeutically include, but are not limited to, iodine131,
indium111, yttrium90 and lutetium177. Method for preparing radioimmunconjugates are
established in the art. Examples of radioimmunoconjugates are commercially available,
including Zevalin™ (IDEC Pharmaceuticals) and Bexxar™ (Corixa Pharmaceuticals), and
similar methods can be used to prepare radioimmunoconjugates using the antibodies of the
invention.
The antibody conjugates of the invention can be used to modify a given
biological response, and the drug moiety is not to be construed as limited to classical
chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide
possessing a desired biological activity. Such proteins may include, for example, an
enzymaticaily active toxin, or active fragment thereof, such as abrin, ricin A, pseudomonas
exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor or interferon-y; or,
biological response modifiers such as, for example, lymphokines, interleukin-1 ("DLrl"),
interleukin-2 ("JJL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating
factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth
factors.
Techniques for conjugating such therapeutic moiety to antibodies are well
known, see, e.g., Arnon et al, "Monoclonal Antibodies For Immunotargeting Of Drugs In
Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp.
243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al, "Antibodies For Drug Delivery", in
Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc.
1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in
Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp.
475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of
Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection
And Therapy, Baldwin et al (eds,), pp. 303-16 (Academic Press 1985), and Thorpe et al,
"The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev.,
62:119-58(1982).
Bispecific Molecules
In another aspect, the present invention features bispecific molecules
comprising an anti-IFN alpha antibody, or a fragment thereof, of the invention. An antibody
of the invention, or antigen-binding portions thereof, can be derivatized or linked to another
functional molecule, e.g., another peptide or protein (e.g., another antibody or ligand for a
receptor) to generate a bispecific molecule that binds to at least two different binding sites or
target molecules. The antibody of the invention may in fact be derivatized or linkd to more
than one; other functional molecule to generate multispeciflc molecules that bind to more than
two different binding sites and/or target molecules; such multispecific molecules are also
intended to be encompassed by the term "bispecific molecule" as used herein. To create a
bispecific molecule of the invention, an antibody of the invention can be functionally linked
(e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or
more other binding molecules, such as another antibody, antibody fragment, peptide or
binding mimetic, such that a bispecific molecule results.
Accordingly, the present invention includes bispecific molecules comprising
at least one first binding specificity for IFN alpha and a second binding specificity for a

second target epitope. In a particular embodiment of the invention, the second target epitope
is an Fc receptor, e.g., human FcyRI (CD64) or a human Fccc receptor (CD89). Therefore,
the invention includes bispecific molecules capable of binding both to FcyR, FcocR or FcsR
expressing effector cells (e.g., monocytes, macrophages or polymorphonuclear cells
(PMNs)), and to target cells expressing IFN alpha. These bispecific molecules target IFN
alpha expressing cells to effector cell and trigger Fc receptor-mediated effector cell activities,
such as phagocytosis of an IFN alpha expressing cells, antibody dependent cell-mediated
cytotoxicity (ADCC), cytokine release, or generation of superoxide anion.
In an embodiment of the invention in which the bispecific molecule is
multispecific, the molecule can further include a third binding specificity, in addition to an
anti-Fc binding specificity and an anti-IFN alpha binding specificity. In one embodiment, the
third binding specificity is an anti-enhancement factor (EF) portion, e.g., a molecule which
binds to a surface protein involved in cytotoxic activity and thereby increases the immune
response against the target cell. The "anti-enhancement factor portion" can be an antibody,
functional antibody fragment or a ligand that binds to a given molecule, e.g., an antigen or a
receptor, and thereby results in an enhancement of the effect of the binding determinants for
the Fc receptor or target cell antigen. The "anti-enhancement factor portion" can bind an Fc
receptor or a target cell antigen. Alternatively, the anti-enhancement factor portion can bind
to an entity that is different from the entity to which the first and second binding specificities
bind. For example, the anti-enhancement factor portion can bind a cytotoxic T-cell (e.g. via
CD2, CD3, CD8, CD28, CD4, CD40, ICAM-1 or other immune cell that results in an
increased immune response against the target cell).
In one embodiment, the bispecific molecules of the invention comprise as a
binding specificity at least one antibody, or an antibody fragment thereof, including, e.g., an
Fab, Fab1, F(ab')2> Fv, or a single chain Fv. The antibody may also be a light chain or heavy
chain dimer, or any minimal fragment thereof such as a Fv or a single chain construct as
described in Ladner et al. U.S. Patent No. 4,946,778, the contents of which is expressly
incorporated by reference.
In one embodiment, the binding specificity for an Fey receptor is provided by
a monoclonal antibody, the binding of which is not blocked by human immunoglobulin G
(IgG). As used herein, the term "IgG receptor" refers to any of the eight y-chain genes
located on chromosome 1. These genes encode a total of twelve transmembrane or soluble
receptorjisoforms which are grouped into three Fey receptor classes: FcyRI (CD64), Fey
RU(CD32), and FcyRIQ (CD 16). In one preferred embodiment, the Fey receptor a human
high affinity FcyRI. The human FcyRI is a 72 kDa molecule, which shows high affinity for
j
monomeric IgG (108 - lO'M"1)-
The production and characterization of certain preferred anti-Fey monoclonal
antibodies are described by Fanger et al. in PCT Publication WO 88/00052 and in U.S. Patent
No. 4,954,617, the teachings of which are fully incorporated by reference herein. These
antibodies bind to an epitope of FcyRI, FcyRH or FcyRin at a site which is distinct from the
Fey binding site of the receptor and, thus, their binding is not blocked substantially by
physiological levels of IgG. Specific anti-FcyRI antibodies useful in this invention are mAb
22, mAb 32, mAb 44, mAb 62 and mAb 197. The hybridoma producing mAb 32 is available
from the American Type Culture Collection, ATCC Accession No. HB9469. hi other
embodiments, the anti-Fey receptor antibody is a humanized form of monoclonal antibody 22
(H22). The production and characterization of the H22 antibody is described in Graziano,
RF. et al. (1995; J. Immunol 155 (10): 4996-5002 and PCT Publication WO 94/10332. The
H22 antibody producing cell line was deposited at the American Type Culture Collection
under the designation HA022CL1 and has the accession no. CRL 11177.
In still other preferred embodiments, the binding specificity for an Fc receptor
is provided by an antibody that binds to a human IgA receptor, e.g., an Fc-alpha receptor (Fc
aRI (CD89)), the binding of which is preferably not blocked by human immunoglobulin A
(IgA). The term "IgA receptor" is intended to include the gene product of one ct-gene (Fccc
RI) located on chromosome 19. This gene is known to encode several alternatively spliced
transmembrane isoforms of 55 to 110 kDa. FcaRI (CD89) is constitutively expressed on
monocytes/macrophages, eosinophilic and neutrophilic granulocytes, but not on non-effector
cell populations. FcaRI has medium affinity (* 5 x 107 M-1) for both IgAl and IgA2, which
is increased upon exposure to cytokines such as G-CSF or GM-CSF (Morton, H.C. et al.
(1996) Critical Reviews in Immunology 16:423-440). Four FcaRI-specific monoclonal
antibodies, identified as A3, A59, A62 and A77, which bind FcaRI outside the IgA ligand
binding domain, have been described (Monteiro, R.C. et al. (1992) J. Immunol. 148:1764).
FcaRI and FcyRI are preferred trigger receptors for use in the bispecific
molecules of the invention because they are (1) expressed primarily on immune effector cells,
e.g., monocytes, PMNs, macrophages and dendritic cells; (2) expressed at high levels (e.g.,

5,000-100,000 per cell); (3) mediators of cytotoxic activities (e.g., ADCC, phagocytosis); (4)
mediate enhanced antigen presentation of antigens, including self-antigens, targeted to them.
While human monoclonal antibodies are preferred, other antibodies which can
be employed in the bispeciflc molecules of the invention are murine, chimeric and humanized
monoclonal antibodies.
The bispecific molecules of the present invention can be prepared by
conjugating the constituent binding specificities, e.g., the anti-FcR and anti-EFN alpha
binding specificities, using methods known in the art. For example, each binding specificity
of the bispecific molecule can be generated separately and then conjugated to one another.
When the binding specificities are proteins or peptides, a variety of coupling or cross-linking
agents can be used for covalent conjugation. Examples of cross-linking agents include
protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-
nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-
pyridyldithio)propionate (SPDP), and sulfosuccimmidyl 4-(N-maleimidomethyl)
cyclohaxane-1-carboxylate (sulfo-SMCC) (see e.g., Karpovsky et al. (1984) J. Exp. Med.
160:1686; Liu, MA et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648). Other methods
include those described in Paulus (1985) Behring Ins. Mitt. No. 78,118-132; Brennan et al.
(1985) Science 229:81-83), and Glennie et al. (1987) J. Immunol. J39: 2367-2375). Preferred
conjugating agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co.
(Rockford, IL).
When the binding specificities are antibodies, they can be conjugated via
sulfhydryl bonding of the C-terminus hinge regions of the two heavy chains. In a particularly
preferred embodiment, the hinge region is modified to contain an odd number of sulfhydryl
residues, preferably one, prior to conjugation.
Alternatively, both binding specificities can be encoded in the same vector and
expressed and assembled in the same host cell. This method is particularly useful where the
bispecific molecule is a mAb x mAb, mAb x Fab, Fab x F(ab")2 or ligand x Fab fusion
protein. A bispecific molecule of the invention can be a single chain molecule comprising
one single chain antibody and a binding determinant, or a single chain bispecific molecule
comprising two binding determinants. Bispecific molecules may comprise at least two single
chain molecules. Methods for preparing bispecific molecules are described for example in
U.S. Patent Number 5,260,203; U.S. Patent Number 5,455,030; U.S. Patent Number
4,881,175;; U.S. Patent Number 5,132,405; U.S. Patent Number 5,091,513; U.S. Patent
Number 5^476,786; U.S. Patent Number 5,013,653; U.S. Patent Number 5,258,498; and
U.S. Patent Number 5,482,858.
Binding of the bispecific molecules to their specific targets can be confirmed
by, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA),
FACS analysis, bioassay (e.g., growth inhibition), or Western Blot assay. Each of these
assays generally detects the presence of protein-antibody complexes of particular interest by
employing a labeled reagent (e.g, an antibody) specific for the complex of interest. For
example, the FcR-antibody complexes can be detected using e.g., an enzyme-linked antibody
or antibody fragment which recognizes and specifically binds to the antibody-FcR
complexes. Alternatively, the complexes can be detected using any of a variety of other
immunoassays. For example, the antibody can be radioactively labeled and used in a
radioimmunoassaLy (RIA) (see, for example, Weintraub, B., Principles of
Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The
Endocrine Society, March, 1986, which is incorporated by reference herein). The radioactive
isotope can be detected by such means as the use of a y counter or a scintillation counter or by
autoradiography.
Pharmaceutical Compositions
In another aspect, the present invention provides a composition, e.g., a
pharmaceutical composition, containing one or a combination of monoclonal antibodies, or
antigen-binding portion(s) thereof, of the present invention, formulated together with a
pharmaceutically acceptable carrier. Such compositions may include one or a combination of
(e.g., two or more different) antibodies, or immunoconjugates or bispecific molecules of the
invention. For example, a pharmaceutical composition of the invention can comprise a
combination of antibodies (or immunoconjugates or bispecifics) that bind to different
epitopes on the target antigen or that have complementary activities.
Pharmaceutical compositions of the invention also can be administered in
combination therapy, i.e., combined with other agents. For example, the combination therapy
can include an anti-IFN alpha antibody of the present invention combined with at least one
other anti-IFN alpha agent (e.g., an immunosuppressing agent).
As used herein, "pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like that are physiologically compatible. Preferably, the
carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or
epidermal administration (e.g., by injection or infusion). Depending on the route of
administration, the active compound, i.e., antibody, immunoconjuage, or bispecific molecule,
may be coated in a material to protect the compound from the action of acids and other
natural conditions that may inactivate the compound.
The pharmaceutical compounds of the invention may include one or more
pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" refers to a salt that
retains the desired biological activity of the parent compound and does not impart any
undesired toxicological effects (see e.g., Berge, S.M., et al. (1977) J. Pharrn. Sci. 66:1-19).
Examples of such salts include acid addition salts and base addition salts. Acid addition salts
include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric,
sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic
organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic
acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the
like. Base addition salts include those derived from alkaline earth metals, such as sodium,
potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such
as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline,
diethanolamine, ethylenediamine, procaine and the like.
A pharmaceutical composition of the invention also may include a
pharmaceutically acceptable anti-oxidant. Examples of pharmaceutically acceptable
antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine
hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-
soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal
chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,
tartaric acid, phosphoric acid, and the like.
Examples of suitable aqueous and nonaqueous carriers that may be employed
in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as
glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof^
vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper
fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by
the maintenance of the required particle size in the case of dispersions, and by the use of
surfactants.
These compositions may also contain adjuvants such as preservatives, wetting
agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms
may be ensured both by sterilization procedures, supra, and by the inclusion of various
antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid,
and the like. It may also be desirable to include isotonic agents, such as sugars, sodium
chloride, and the like into the compositions, hi addition, prolonged absorption of the
injectable pharmaceutical form may be brought about by the inclusion of agents which delay
absorption such as aluminum monostearate and gelatin.
Pharmaceutically acceptable carriers include sterile aqueous solutions or
dispersions and sterile powders for the extemporaneous preparation of sterile injectable
solutions or dispersion. The use of such media and agents for pharmaceutically active
substances is known in the art. Except insofar as any conventional media or agent is
incompatible with the active compound, use thereof in the pharmaceutical compositions of
the invention is contemplated. Supplementary active compounds can also be incorporated
into the compositions.
Therapeutic compositions typically must be sterile and stable under the
conditions of manufacture and storage. The composition can be formulated as a solution,
microemulsion, liposome, or other ordered structure suitable to high drug concentration. The
carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol
(fcr example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and
suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a
coating such as lecithin, by the maintenance of the required particle size in the case of
dispersion and by the use of surfactants. In many cases, it will be preferable to include
isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium
chloride in the composition. Prolonged absorption of the injectable compositions can be
brought about by including in the composition an agent that delays absorption, for example,
monostearate salts and gelatin.
Sterile injectable solutions can be prepared by incorporating the active
compound in the required amount in an appropriate solvent with one or a combination of
'ingredients enumerated above, as required, followed by sterilization microfiltration.
Generally, dispersions are prepared by incorporating the active compound into a sterile
vehiclejthat contains a basic dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the preparation of sterile injectable
solutions, the preferred methods of preparation are vacuum drying and freeze-drying
(lyophilization) that yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
The amount of active ingredient which can be combined with a carrier
material to produce a single dosage form will vary depending upon the subject being treated,
and the particular mode of administration. The amount of active ingredient which can be
combined with a carrier material to produce a single dosage form will generally be that
amount of the composition which produces a therapeutic effect. Generally, out of one
hundred per cent, this amount will range from about Q.01 per cent to about ninety-nine
percent of active ingredient, preferably from about 0.1 per cent to about 70 per cent, most
preferably from about 1 per cent to about 30 per cent of active ingredient in combination with
a pharmaceutically acceptable carrier.
Dosage regimens are adjusted to provide the optimum desired response (e.g., a
therapeutic response). For example, a single bolus may be administered, several divided
doses may be administered over time or the dose may be proportionally reduced or increased
as indicated by the exigencies of the therapeutic situation. It is especially advantageous to
formulate parenteral compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to physically discrete units
suited as unitary dosages for the subjects to be treated; each unit contains a predetermined
quantity of active compound calculated to produce the desired therapeutic effect in
association with the required pharmaceutical carrier. The specification for the dosage unit
forms of the invention are dictated by and directly dependent on (a) the unique characteristics
of the active compound and the particular therapeutic effect to be achieved, and (b) the
limitations inherent in the art of compounding such an active compound for the treatment of
sensitivity in individuals.
For administration of the antibody, the dosage ranges from about 0.0001 to
100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight. For example dosages
can be 03 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body
weight or 10 mg/kg body weight or within the range of 1-10 mg/kg. An exemplary treatment
regime entails administration once per week, once every two weeks, once every three weeks,
once every four weeks, once a month, once every 3 months or once every three to 6 months.
Preferred dosage regimens for an anti-IFN alpha antibody of the invention include 1 mg/kg
body weight or 3 mg/kg body weight via intravenous administration, with the antibody being
given using one of the following dosing schedules: (i) every four weeks for six dosages, then
every three months; (ii) every three weeks; (iii) 3 mg/kg body weight once followed by 1
mg/kg body weight every three weeks.
In some methods, two or more monoclonal antibodies with different binding
specificities are administered simultaneously, in which case the dosage of each antibody
administered falls within the ranges indicated. Antibody is usually administered on multiple
occasions. Intervals between single dosages can be, for example, weekly, monthly, every
three monthgs or yearly. Intervals can also be irregular as indicated by measuring blood
levels of antibody to the target antigen in the patient. In some methods, dosage is adjusted to
achieve a plasma antibody concentration of about 1-1000 ug /ml and in some methods about
25-300 ng /ml.
Alternatively, antibody can be administered as a sustained release formulation,
in which case less frequent administration is required. Dosage and frequency vary depending
on the half-life of the antibody in the patient. In general, human antibodies show the longest
half life, followed by humanized antibodies, chimeric antibodies, and nonhuman antibodies.
The dosage and frequency of administration can vary depending on whether the treatment is
prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is
administered at relatively infrequent intervals over a long period of time. Some patients
continue to receive treatment for the rest of their lives. In therapeutic applications, a
relatively high dosage at relatively short intervals is sometimes required until progression of
the disease is reduced or terminated, and preferably until the patient shows partial or
complete amelioration of symptoms of disease. Thereafter, the patient can be administered a
prophylactic regime.
A.ctual dosage levels of the active ingredients in the pharmaceutical
compositions of the present invention may be varied so as to obtain an amount of the active
ingredient which is effective to achieve the desired therapeutic response for a particular
patient, composition, and mode of administration, without being toxic to the patient. The
selected dosage level will depend upon a variety of pharmacokinetic factors including the
activity of the particular compositions of the present invention employed, or the ester, salt or
amide thereof, the route of administration, the time of administration, the rate of excretion of
the particular compound being employed, the duration of the treatment, other drugs,
compounds and/or materials used in combination with the particular compositions employed,
the age, sex, weight, condition, general health and prior medical history of the patient being
treated, and like factors well known in the medical arts.
A "therapeutically effective dosage" of an anti-IFN alpha antibody of the invention
preferably results; in a decrease in severity of disease symptoms, an increase in frequency and
duration of disease symptom-free periods, or a prevention of impairment or disability due to
the disease affliction. For example, in the case of systemic lupus erythematosus (SLE), a
therapeutically effective dose preferably prevents further deterioration of physical symptoms
associated with SLE, such as, for example, pain or fatigue. A therapeutically effective dose
preferably also prevents or delays onset of SLE, such as maybe desired when early or
preliminary signs of the disease are present. Likewise it includes delaying chronic
progression associated with SLE. Laboratory tests utilized in the diagnosis of SLE include
chemistries (including the measurement of IFN alpha levels), hematology, serology and
radiology. Accordingly, any clinical or biochemical assay that monitors any of the foregoing
may be used to determine whether a particular treatment is a therapeutically effective dose for
treating SLE. One of ordinary skill in the art would be able to determine such amounts based
on such factors as the subject's size, the severity of the subject's symptoms, and the particular
composition or route of administration selected.
A composition of the present invention can be adrmnistered via one or more
routes of administration using one or more of a variety of methods known in the art. As will
be appreciated by the skilled artisan, the route and/or mode of administration will vary
depending upon the desired results. Preferred routes of administration for antibodies of the
invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous,
spinal or other parenteral routes of administration, for example by injection or infusion. The
phrase "parenteral administration" as used herein means modes of administration other than
enteral and topical administration, usually by injection, and includes, without limitation,
intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac,
intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular,
subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
Alternatively, an antibody of the invention can be administered via a non-
parenteral route, such as a topical, epidermal or mucosal route of adrninistration, for example,
intranasally, orally, vaginally, rectally, sublingually or topically.
The active compounds can be prepared with carriers that will protect the
compound against rapid release, such as a controlled release formulation, including implants,
transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible
polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such
formulations are patented or generally known to those skilled in the art. See, e.g., Sustained
and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc.,
New York, 1978.
Therapeutic compositions can be administered with medical devices known in
the art. For example, in a preferred embodiment, a therapeutic composition of the invention
can be administered with a needleless hypodermic injection device, such as the devices
disclosed in U.S. Patent Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880;
4,790,824; or 4,596,556. Examples of well-known implants and modules useful in the
present invention include: U.S. Patent No. 4,487,603, which discloses an implantable micro-
infusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4,486,194,
which discloses a therapeutic device for administering medicants through the skin;
U.S. Patent No. 4,447,233, which discloses a medication infusion pump for delivering
medication at a precise infusion rate; U.S. Patent "No. 4,447,224, which discloses a variable
flow implantable infusion apparatus for continuous drug delivery; U.S. Patent No. 4,439,196,
which discloses an osmotic drug delivery system having multi-chamber compartments; and
U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system. These patents
are incorporated herein by reference. Many other such implants, delivery systems, and
modules are known to those skilled in the art.
La certain embodiments, the human monoclonal antibodies of the invention
can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier
(BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic
compounds of the invention cross the BBB (if desired), they can be formulated, for example,
in liposomes. For methods of manufacturing liposomes, see, e.g., U.S. Patents 4,522,811;
5,374,548; and 5,399,331. The liposomes may comprise one or more moieties which are
selectively transported into specific cells or organs, thus enhance targeted drug delivery (see,
e.g., V.V. Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include
folate or biotin (see, e.g., U.S. Patent 5,416,016 to Low et al); mannosides (Umezawa etal,
(1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (P.G. Bloeman et al. (1995)
FEBSLett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180);
surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134^: pl20 (Schreier
et al. (1994) /. Biol. Chem. 269:9090); see also K. Keinanen; MX. Laukkanen (1994) FEBS
Leu. 346:123; J J. Killion; I.J. Fidler (1994; Immunomethods 4:273.
Uses and Methods of the Invention
Monoclonal anti-IFN alpha antibodies and related derivatives/conjugates and
compositions of the present invention have a variety of in vitro and in vivo diagnostic and
therapeutic utilities. For example, the antibodies can be used to detect IFN alpha protein,
either in vitro or in vitro, using standard antibody/antigen binding assays (e.g., ELISA, RIA).
Furthermore, these molecules can be administered to a subject, e.g., in vivo, to treat, prevent
or diagnose a variety of disorders in which IFN alpha plays a role. As used herein, the term
"subject" is intended to include both human and nonhuman animals. Preferred subjects
include human patients exhibiting autoimmune disorders. The term "nonhuman animals" of
the invention includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman
primates, sheep, dog, cat, cow, horse, chickens, amphibians, reptiles, etc.
The antibody compositions of the invention can be used in the treatment of
autoimmune diseases, such as systemic lupus erythematosus (SLE), multiple sclerosis (MS),
inflammatory bowel disease (TBD; including Crohn's Disease, Ulcerative Colitis and Celiac's
Disease), insulin dependent diabetes mellitus (IDDM), psoriasis, autoimmune thyroiditis,
rheumatoid arthritis (RA) and glomerulonephritis. Furthermore, the- antibody compositions
of the invention can be used for inhibiting or preventing transplant rejection or in the
treatment of graft versus host disease (GVHD).
Antibodies of the invention can be initially tested for binding activity associated with
therapeutic use in vitro. For example, compositions of the invention can be tested using
Biacore, ELISA and flow cytometric assays described in the Examples below. Moreover, the
activity of these molecules can be assayed, for example, by a cell proliferation assay
following exposure to IFN alpha, as described in the Examples below. Suitable melliods for
administering antibodies and compositions of the present invention are well known in the art,
and are described further above. Suitable dosages also can be determined within the skill in
the art and will depend on the age and weight of the subject and the particular drug used.
Exemplary dosages are described further above.
Anti-IFN alpha antibodies of the invention also can be co-administered with
other therapeutic agents as described above.
As noted above, for purposes of therapy, a human antibody composition and a
pharmaceutically acceptable carrier are administered to a patient in a therapeutically effective
amount. A combination of an antibody composition and a pharmaceutically acceptable
carrier isisaid to be administered in a "therapeutically effective amount" if the amount
administered is physiologically significant. An agent is "physiologically significant" if its
presence results in a detectable change in the physiology of a recipient patient. A targeted
therapeutic agent is "therapeutically effective" if it delivers a higher proportion of the
administered dose to the intended target than accrues at the target upon systemic
administration of the equivalent untargeted agent.
Also within the scope of the invention are kits comprising the compositions
(e.g., human antibodies, immunoconjugates and bispecific molecules) of the invention and
instructions for use. The kit can further contain a least one additional reagent, such as one or
more additional human antibodies of the invention (e.g., a human antibody having a
complementary activity which inhibits IFN alpha activity but that is distinct from the first
human antibody).
The present invention is further illustrated by the following examples which
should not be construed as further limiting. The contents of all figures and all references,
patents and published patent applications cited throughout this application are expressly
incorporated herein by reference.
Examples
Example 1: Generation of Human Monoclonal Antibodies Against IFN Alpha
Antigen:
Natural human IFNoc containing multiple sub-types purified from a virally-
stimulated human lymphoblastoid cell line, resulting in production of multiple IFN alpha
subtypes but not IFN omega, was used as the antigen.
Transgenic Transchromosomic KM Mice™:
Fully human monoclonal antibodies to IFN alpha were prepared using the KM
strain of transgenic transchromosomic mice, which expresses human antibody genes. In this
mouse strain, the endogenous mouse kappa light chain gene has been homozygously
disrupted as described in Chen et al. (1993) EMBOJ. 12:811-820 and the endogenous mouse
heavy chain gene has been homozygously disrupted as described in Example 1 of PCT
Publication WO 01/09187 for HuMab mice. The mouse carries a human kappa light chain
transgene, KCo5, as described in Fishwild et al (1996) Nature Biotechnology 14:845-851.
The mouse also carries a human heavy chain traoschromosome, SC20, as described in PCT
Publication WO 02/43478.
KM Mouse™ Immunizations:
To generate fully human monoclonal antibodies to IFN alpha, KM mice™
were immunized with natural human IFNa containing multiple sub-types purified from a
virally-stimulated human lymphoblastoid cell line. General immunization schemes are
described in Lonberg, N. et al (1994) Nature 368: 856-859; Fishwild, D. et al. (1996) Nature
Biotechnology lb 845-851 and PCT Publication WO 98/24884. The mice were 6-16 weeks
of age upon the first infusion of antigen. A purified natural preparation (25-100 jig) of IFN
alpha antigen (i.e., purified from virally stimulated lymphoblastoid cells) was used to
immunize the KM mice™ intraperitonealy (IP) or subcutaneously (Sc).
Transgenic transchromosomic mice were immunized intraperitonealy (IP) or
subcutaneously (Sc) with antigen in complete Freund's adjuvant twice, followed by 2-4
weeks IP innnunization (up to a total of 8 immunizations) with the antigen in incomplete
Freund's adjuvant. The immune response was monitored by retroorbital bleeds. The plasma
was screened by ELISA (as described below), and mice with sufficient titers of anti-IFNa
human immunogolobulin were used for fusions. Mice were boosted intravenously with
antigen 3 and 2 days before sacrifice and removal of the spleen.
Selection of KM Mice™ Producing Anti-IFNa Antibodies:
To select KM mice™ producing antibodies that bound IFNa, sera from
immunized mice were tested by ELISA as described by Fishwild, D. et al (1996). Briefly,
microtiter plates were coated with purified natural IFNa from lymphoblastoid cells at 1-2 ug
/ml in PBS, 50 ul/well, incubated 4 °C overnight then blocked with 200 ul/well of 5%
chicken serum in PBS/Tween (0.05%). Dilutions of plasma from IFNa immunized mice were
added to each well and incubated for 1-2 hours at ambient temperature. The plates were
washed with PBS/Tween and then incubated with a goat-anti-human IgG Fc polyclonal
antibody conjugated with horseradish peroxidase (HRP) for 1 hour at room temperature.
After washing, the plates were developed with ABTS substrate (Sigma, A-l 888,0.22 mg/ml)
and optical density for each well was determined using a spectrophotometer set to
wavelength 415nm with a background correction at 495nm. Mice that developed the highest
titers of anti-IFNoL antibodies were used for fusions. Fusions were performed as described
below and hybridoma supematants were tested for anti-IFNa activity by ELISA.
Generation of Hvbridomas Producing Human Monoclonal Antibodies to IFNa:
Splenocytes were isolated from KM mice™ and fused to a mouse myeloma
cell line based upon standard protocols using PEG. The resulting hybridomas were then
screened for the production of antigen-specific antibodies.
Single cell suspensions of splenic lymphocytes from immunized mice were
fused to one-fourth the number of P3X63-Ag8.653 nonsecreting mouse myeloma cells
(ATCC, CRL1580) or SP2/0 nonsecreting mouse myeloma cells (ATCC, CRL1581) using
50% PEG (Sigma). Cells were plated at a density of about 1x10 5/well in flat bottom
microtiter plates and incubated approximately 2 weeks in selective medium containing 10%
fetal bovine serum, 10% P388D1 (ATCC, CRL TIB-63) conditioned medium, 3-5% origen
(IGEN) in DMEM (Mediatech, CRL 10013, with high glucose, I^glutamine and sodium
pyruvate) plus 5 mM HEPES, 0.055 mM 2-mercaptoethanol, 50 mg/ml gentamicin and lx
HAT (Sigma, CRL P-7185). After 1-2 weeks, cells were cultured in medium in which the
HAT was replaced with HT. Individual wells were then screened by ELISA (described
above) for human anti-IFNa IgG antibodies.
Conditioned medium from the antibody secreting hybridomas identified by
ELISA was tested in a Daudi proliferation assay (described below) for the capacity to block
the anti-proliferative effects of IFNa. The hybridomas with highest neutralizing activity in
the Daudi assay screen were subcloned at least twice by limiting dilution. The resulting stable
subclones were then cultured in vitro to generate small amounts of monoclonal antibody in
tissue culture medium. The Daudi proliferation assay screen was repeated to confirm the
activity of the sub-clones. The sub-clones with highest activity in the Daudi assay were
scaled up to produce sufficient conditioned medium (typically 1L) for purification of
monoclonal anti-IFNa for further characterization.
Screen of Hybridomas for Neutralizing anti-IFNa Antibody: Daudi Proliferation Assay:
Interferon alpha inhibits the proliferation of Daudi (Burkitts lymphoma,
ATCC # CCL-213) cells in a dose dependant manner. A neutralizing antibody, which blocks
interferon binding to its receptor, will restore proliferation. Dose response curves for the
anti-proliferative effects of natural lymphoblastoid IFNa on Daudi were determined and a
concentration sufficient to inhibit Daudi growth by 50% (EC50) was calculated.
Hybridoma conditioned medium was mixed with Daudi cells in culture
medium (RPMI1640 supplemented with 10% FCS, lx 2-ME, L-glutamine and penicillin
streptomycin) with and without the addition of IFNa in a 96 well, flat-bottomed cell culture
plate. The final mixture of reagents was as follows: 1 xlO4 Daudi cells + 10% hybridoma
supernate +/- IFNa at EC50 per lOOul/well. The cells were incubated at 37°C, 5% C02,72
hrs. Proliferation was assayed with the addition of MTS (Promega), 20 ul/well and O.D. at
490nm was read following a further 3 hrs incubation. The viable cell number was
proportional to the O.D. reading. Percentage Daudi inhibition was calculated for hybridoma
supernate + IFNa relative to hybridoma supernate alone and compared to a media control
with and without IFNa. Hybridomas were rank ordered according to the potency of IFNa
blockade and the most active neutralizing hybridomas were selected for sub-cloning.
Hybridoma clones 13H5,13H7 and 7H9 were selected for further analysis.
Example 2: Structural Characterization of Human Monoclonal Antibodies 13H5,
13H7 and 7H9
The cDNA sequences encoding the heavy and light chain variable regions of
the 13H5,13H7, and 7H9 monoclonal antibodies were obtained from the 13H5,13H7, and
7H9 hybridomas, respectively, using standard PCR techniques and were sequenced using
standard DNA sequencing techniques.
The nucleotide and amino acid sequences of the heavy chain variable region of
13H5 are shown in Figure 1A and in SEQ ID NO: 25 and 19, respectively.
Hie nucleotide and amino acid sequences of the light chain variable region of
13H5 are shown in Figure IB and in SEQ ID NO: 28 and 22, respectively.
Comparison of the 13H5 heavy chain immunoglobulin sequence to the known
human germline immunoglobulin heavy chain sequences demonstrated that the 13H5 heavy
chain utilizes a Vr segment from human germline VH 1-18, an undetermined D segment, and
a Jh segment from human germline Jh 4b. The alignment of the 13H5 Vh sequence to the
germline VH 1-18 sequence is shown in Figure 4. Further analysis of the 13H5 Vh
sequence using the Kabat system of CDR region determination led to the delineation of the
heavy chain CDR1, CDR2 and CD3 regions as shown in Figures 1A and 4, and in SEQ ID
NOs: 1,4 and 7, respectively.
Comparison of the 13H5 light chain immunoglobulin sequence to the known
human germline immunoglobulin light chain sequences demonstrated that the 13H5 light
chain utilizes a Vl segment from human germline VK A27 and a JK segment from human
germline JK 1. The alignment of the 13H5 Vl sequence to the germline VK A27 sequence is
shown in Figure 6. Further analysis of the 13H5 Vl sequence using the Kabat system of
CDR region determination led to the delineation of the light chain CDR1, CDR2 and CD3
regions as shown in Figures IB and 6, and in SEQ ID NOs: 10,13 and 16, respectively.
The nucleotide and amino acid sequences of the heavy chain variable region of
13H7 are shown in Figure 2A and in SEQ ID NO: 26 and 20, respectively.
The nucleotide and amino acid sequences of the light chain variable region of
13H7 are shown in Figure 2B and in SEQ ID NO: 29 and 23, respectively.
Comparison of the 13H7 heavy chain immunoglobulin sequence to the known
human germline immunoglobulin heavy chain sequences demonstrated that the 13H7 heavy
chain utilizes a Vh segment from human germline VH 4-61, a D segment from human
geimline 3-10, and a Jh segment from human germline JH 4b. The alignment of the 13H7 Vh
sequence to the germline VH 4-61 sequence is shown in Figure 5. Further analysis of the
13H7 Vh sequence using the Kabat system of CDR region detennination led to the
delineation of the heavy chain CDR1, CDR2 and CD3 regions as shown in Figures 2A and 5,
and in SEQ ID NOs: 2, 5 and 8, respectively.
Comparison of the 13H7 light chain irnmunoglobulin sequence to the known
human germline immunoglobulin light chain sequences demonstrated that the 13H7 light
chain utilizes a Vl segment from human germline VK A27 and a JK segment from human
germline JK 2. The alignment of the 13H7 Vl sequence to the germline VK A27 sequence is
shown in Figure 6. Further analysis of the 13H7 Vl sequence using the Kabat system of
CDR region determination led to the delineation of the light chain CDR1, CDR2 and CD3
regions as shown in Figures 2B and 6, and in SEQ ID NOs: 11,14 and 17, respectively.
The nucleotide and amino acid sequences of the heavy chain variable region of
7H9 are shown in Figure 3A and in SEQ ID NO: 27 and 21, respectively.
The nucleotide and amino acid sequences of the light chain variable region of
7H9 are shown in Figure 3B and in SEQ ID NO: 30 and 24, respectively.
Comparison of the 7H9 heavy chain immunoglobulin sequence to the known
human germline immunoglobulin heavy chain sequences demonstrated that the 7H9 heavy
chain utilizes a VH segment from human germline VH 1-18, a D segment from human
germline 6-6, and a Jh segment from human germline Jh 4b. The alignment of the 7H9 Vh
sequence to the germline VH 1-18 sequence is shown in Figure 4. Further analysis of the
7H9 Vh sequence using the Kabat system of CDR region determination led to the delineation
of the heavy chain CDR1, CDR2 and CD3 regions as shown in Figures 3 A and 4, and in SEQ
ID NOs: 3,6 and 9, respectively.
Comparison of the 7H9 light chain irnmunoglobulin sequence to the known
human germline immunoglobulin light chain sequences demonstrated that the 7H9 light chain
utilizes a Vl segment from human germline VK A27 and a JK segment from human germline
JK 1. The alignment of the 7H9 Vl sequence to the germline VK A27 sequence is shown in
Figure 6. Further analysis of the 7H9 Vl sequence using the Kabat system of CDR region
determination led to the delineation of the light chain CDR1, CDR2 and CD3 regions as
shown in Figures 3B and 6, and in SEQ ID NOs: 12,15 and 18, respectively.
Example 3: Anti-IFN Alpha Human Monoclonal Antibodies Inhibit the Biological
Activity of Multiple Interferon Alpha Subtypes
As described in Example 1, interferon alpha inhibits the proliferation of Daudi
(Burkitts lymphoma, ATCC # CCL-213) cells in a dose dependant manner. A neutralizing
antibody, which blocks interferon binding to its receptor, will restore proliferation. Using
this cell proliferation assay, the specificity of the purified human anti-BFN alpha antibodies
was examined by testing for blockade of natural lymphoblastoid IFNa, natural leukocyte
interferon, 13 recombinant IFN alpha subtypes, IFN beta and IFN omega.
Daudi cells were grown in culture medium (RPMI1640 supplemented with 10% FCS,
lx 2-ME, L-glutamine and penicillin streptomycin) with and without the addition of IFNa in
a 96 well, flat-bottomed cell culture plate. Each type I interferon tested was assayed at EC50
and mixed with a 2-fold serial titration of each antibody, typically from 50 ug/ml (312 nM)
through 381 pg/ml (2.4 pM). The antibody/IFN mixture was added to Daudi cells in a 96-
well bottomed plate to a final density of 1 xlO4 Daudi cells/1 OOul/well and incubated at 37°C,
5% C02,72 hrs. Proliferation was assayed with the addition of MTS (Promega), 20 ul/well,
and O.D. at 490mn was measured following a further 3 hour incubation. The viable cell
number was proportional to the O.D. reading. Percentage blockade of interferon was
calculated relative to Daudi proliferation in the absence of IFN (=100% blockade) and in the
presence of IFN alone (=0% blockade). Antibodies were scored according to the degree of
blockade, resulting in a profile of IFNa subtype specificity for each antibody tested. An EC50
was derived with PRISM™ software using non-linear regression; sigmoidal dose response;
variable slope curve fit. The results demonstrated that the human anti-IFN alpha antibody
13H5 inhibits the action of multiple interferon alpha subtypes, particularly, IFNa 6,2b, 2a, 1,
16,10, 8, 5 and 14, but not IFNa 21, IFNp or IFNco. 13H5 is a low level inhibitor of IFN
alpha subtypes 17,7 and 4. The EC50 values and % blockade of interferon are shown in table
1, below.
Table 1: Antibody Inhibition of Multiple IFN Alpha Subtypes
Example 4: Inhibition of IFN Alpha Induction of Cell Surface Markers by Anti-IFN
Alpha Antibodies
The addition of IFN alpha 2b to cell culture media is known to induce the
expression of the cell surface markers CD38 and MHC Class I on normal peripheral blood
mononuclear cells (PBMNC). The activity of human anti-IFN alpha antibody 13H5 was
tested for inhibition of interferon induced cell surface marker expression on cultures of
primary human cells and assayed by FACS analysis.
The anti-IFNa monoclonal antibody 13H5 and isotype controls were diluted
to 20 ug/ml each in PBMNC culture medium (RPMI1640 + 10% FBS + 1% human serum).
Antibody was dispensed 1.5 ml/well into T25 vented cap culture flasks and mixed with an
equal volume of either 400 iu/ml leukocyte IFN, IFN alpha 2b or IFN «>, diluted in culture
medium or with medium alone. PBMNC were isolated from normal human blood using
heparin coated Vacutainer® CPT™ tubes according to manufacturer recommendations
(Becton Dickinson & Co). Cells were resuspended in culture medium (RPMI 1640 + 10%
FBS + 1% human serum) to 2xl06 cells/ml and were added in equal volume to the Ab/IFN
mixtures such that the final assay contains; 6 x 106 PBMNC + 5 ug/ml Ab +/- 100 iu/ml EFN
per 6 ml medium. Flasks were incubated at 37°C, 5% CO2 for 24 or 48 hrs.
Conditioned medium was harvested from each flask and suspension cells were
recovered by centrifugation at 1000 rpm on a Sorvall RTH-750 rotor. The pelleted cells were
retained on ice and supernate was frozen at -80°C for ELISA. Adherent cells were recovered
from the flask with a PBS wash (2ml), followed by 15 minute incubation in versene (3ml).
The flask was scraped at the end of the versene incubation and the flask was finally rinsed
with PBS wash (2ml). Each of the PBS washes and the versene was combined with the cells
recovered from conditioned medium harvest. The pooled cell suspension was centriftiged at
1000 rpm on a Sorvall RTH-750 rotor, the resulting pellet was resuspensed to 300 ul in
staining buffer (PBS + 0.1M EDTA + 2% FBS + 1% HS) and dispensed lOOul/well into a V-
bottom 96-well plate.
The plate was pulse-centrifuged at 2800 rpm on a Sorvall RTH-750 rotor and
pelleted cells were resuspended 25ul/well in flurochrome labeled antibodies as follows: (1)
mouse anti-MHC I-FITC + mouse anti-CD38-PE, and (2) isotype controls, mouse IgG-FTTC
+ mouse IgG-PE. The plate was incubated on ice for 45 minutes, protected from light. The
cells were washed three times with the addition of 200 ul staining buffer followed by pulse-
celtrifugation and finally resuspended in 200ul of 2% paraformaldehyde in PBS. Staining of
monocyte cells was analyzed by flow cytometry with the Becton Dickinson FACScalibur™,
gates were drawn on the Forward Scatter vs. Side Scatter graph to remove contaminating
cells from the analysis. The results demonstrated that the human monoclonal antibody 13H5
inhibits leukocyte IFN and recombinant IFNa 2b induced changes in expression of CD38 and
MHC Class I on normal PBMNC. The human monoclonal antibody 13H5 does not block
IFNco mediated changes in the cell surface marker expression of CD38 and MHC Class I.
These results are shown in Tables 2 and 3 below.
Example 5: Inhibition of IFN-Iuduction expression of IP-10 by Anti-IFN Alpha
Antibodies
The addition of IFN alpha 2b to cell culture media is known to induce IP-10
expression in normal peripheral blood mononuclear cells (PBMNC). The activity of human
anti-IFN alpha antibody 13H5 was tested for inhibition of interferon induced expression of
IP-10 in normal PBMNC cultures by an ELISA binding assay.
A PBMNC culture was prepared as described in Example 4, conditioned with
leukocyte IFN, IFN alpha 2b, or IFN 10/CXCL10 expression using a quantitative sandwich ELISA kit (Quantikine®, R&D
Systems) at a 1:30 dilution according to manufacturer recommendations. The results
demonstrated that the human monoclonal antibody 13H5 inhibits leukocyte IFN and
recombinant IFNa 2b induced expression of IP-10 in normal PBMNC culture but does not
block IFNoo induced IP-10 expression. These results are shown in Table 4.
Table 4: Antibody Inhibition of in IFN-lhduced IP-10 Expression on Normal PBMNC
In this example, the monoclonal antibody 13H5 was examined for binding
affinity of recombinant IFN alpha 2a and IFN alpha 2b using Biacore analysis.
Purified antibodies at 10 ug/ml, were captured on a CM5 chip coated with
Prot-G. Concentrations of antigen from 80 nM to 10 nM in HBS-EP running buffer was
passed over the chip at a rate of 25 ul/min. The association time allowed was 5 minutes,
followed by a 10 minute dissociation period. Background and non-specific binding of
antigen to both the chip and antibodies was eliminated by detecting the binding to surface
with captured isotype control human-IgG (Sigma) and buffer. Regeneration of the chip was
achieved with a flow rate of lOOul/min for 0.4 minutes using 20 mM NaOH + 400mM NaCl.
The association and dissociation curves were fit to a Langmuir binding model using
BIAevaluation software (Biacore AB). The results are shown below in Table 5.
Example 7: Antibody Inhibition of SLE Plasma Mediated Dendritic Cell Development
SLE plasma induces dendritic cell development from normal human
monocytes. In this example, purified monoclonal human anti-IFN alpha antibodies were
tested for inhibition of dendritic cell development, as assessed by the ability of the antibodies
to inhibit the induction of the cell surface markers CD38, MHC Class I and CD123 by SLE
plasma,
A 25 ml buffy coat was diluted four fold with PBS. The sample was
separated into 4 x50ml conical tubes, and 15ml of lymphocyte separation medium (ICN
Biomedicals) was layered underneath. Following a 30-minute spin at 500 x g, the buffy layer
containing the PBMCs was removed and washed with PBS. Cells were resuspended in
culture media at 4x106 cells/ml. Monocytes were isolated by incubating PBMC (2.0 x 107
cells/ 5ml / 25cm2 flask) for 1.5 hrs at 37°C in culture medium and then washing away non-
adherent cells twice. Following the second wash the cells were cultured in media containing
1% heat inactivated human serum. Twenty five percent SLE patient plasma plus/minus
neutralizing antibodies and isotype controls (30ug/ml) were added to the culture flasks; IFN
alpha 2b (100 & 10 iu/ml) plus 25% normal human plasma was used as a positive control for
marker induction. Flasks were incubated at 37°C, 5%C02 for three to seven days. Dendritic
cells were then recovered from conditioned medium, with PBS and versene treatment if
necessary, before being stained as described for blockade of marker induction in PBMNC
culture (as described in Example 4 above). Staining of dendritic cells was analyzed by flow
cytometry with the Becton Dickinson FACScaUbur™. Gates were drawn on the Forward
Scatter vs. Side Scatter graph to remove contaminating cells from the analysis. The anti-IFN
alpha human monoclonal antibody 13H5 inhibits the IFN alpha dependent process of
dendritic cell development, as demonstrated by normalized expression of cell surface markers
MHC Class I, CD38, and CD123 in the presence of 13H5. The results are shown below in
Table 6, wherein (A), (B), (C) & (D) summarize results for four representative SLE donor
samples.
In this example, several binding experiments using radiolabeled cytokine and
antibody with IFNAR expressing cells were conducted in order to determine the mechanism
of action for 13H5.
In the first set of experiments, recombinant IFNa 2a was radio-iodinated with
a specific activity of 29.3 Ci/mmole (Pierce IODO-GEN® tubes) and was determined to
specifically bind Daudi cells with a Kd of approximately 1 nM. To examine competition
binding of this ligand to cells, glass fiber plates were blocked with 200 |il/well milk buffer
overnight at 4° C. Daudi cells were dispensed at 2 x 106 cells/well in RPMI1640 medium
and were mixed with 125I-IFNa (2 nM), plus a 3-fold dilution series of competitor, either
13H5, an isotype control antibody or unlabelled IFNa (30 nM to 14 pM). The plate was
incubated 2 hours at 4° C on a shaker before being washed with RPMI and air-dried. The
filters were transferred to glass tubes and analyzed for radioactivity.
Representative results from several experiments are shown in Figure 7.
Unlabeled ligand was used as a positive control and was observed to specifically block 125I-
IFNoc binding with an IC50 of approximately 0.5 nM. The 13H5 antibody, however, did not
block binding of iodinated ligand but was instead observed to enhance the radioactive signal
associated with treated cells, contrasting with the behavior of the isotype control antibody,
which had no effect on 125I-IFNa binding to cells. This result indicates mat 13H5 has a non-
competitive mechanism of action and neutralizes biological activity by blockade of signaling
but not by blockade of ligand binding.
The above result also suggested that 13H5 may become associated with the
cell surface in the presence of IFNa. Since each 13H5 molecule has the capacity to bind two
IFNa molecules, it is possible that these events would also result in a second ligand being
linked to the cell membrane. This hypothesis is supported by the observation that cell-
associated radioactivity was enhanced approximately 2-fold at concentrations of antibody and
ligand consistent with a 1:1 ratio of IFNa to 13H5 binding sites.
To further examine the mechanism of action of 13H5, the binding of the
antibody to Daudi cells was assayed using radiolabeled antibody in the presence or absence
of IFNa 2a. The cytokine was used at a concentration (10 nM) calculated to saturate IFNAR
binding based upon earlier binding studies. The 13H5 antibody was radio-iodinated with a
specific activity of 414 Ci/mmole (Pierce IODO-GEN® tubes). To examine antibody
binding to cells, glass fiber plates were blocked with 200 nl/well milk buffer overnight at 4°
C. Daudi cells were dispensed at 2 x 106 cells/well in RPMI1640 medium and were mixed
with a 2-fold diluation series of I25-13H5 (20 nM to 20 pM), plus/minus IFNa 2a (10 nM).
The plate was incubated 2 hours at 4° C on a shaker before being washed with RPMI and air-
dried. The filters were transferred to glass tubes and analyzed for radioactivity. CPM values
measured for 125-13H5 binding alone were subtracted from those measured in the presence of
IFNa 2a in order to determine IFNa 2a dependent binding. Representative results from
several experiments are shown in Figure 8. The results showed dose dependant saturable
binding of 125-13H5 to Daudi cells in the presence of IFNa 2a but negligible binding with125-
13H5 alone. The specific IFNoc-dependent binding of 13H5 is represented in Figure 8 by
circles and was calculated by subtracting CPM for antibody alone (representing non-specific
binding) from total CPM for 13H5 binding in the presence of IFNa.
Thus, in summary, the mechanism of action of 13H5 is a non-competitive one
in which the complex of IFNa bound to 13H5 is capable of binding to 1FNAR on the cell
surface and the biological activity of IFNa is neutralized by blockade of signaling through
IFNAR.
Example 9: Antibody Dependent Cell-Mediated Cytotoxicity Assays with 13H5
Since 13H5 can associate with the cell surface in the presence of IFNa,
antibody dependent cell-mediated cytotoxicity (ADCC) was investigated using a 51Cr-release
assay. Raji cells were used as targets for lysis by fresh human mononuclear cells.
Mononuclear cells were purified from heparinized whole blood by Ficoll Hypaque density
centrifugation. Target cells were labeled with 100 jaCi of 51Cr per 106 cells for 1 hour prior to
dispensing into U-bottom microtiter plates, 104 cells per well, and combining with effector
cells (effector:target ratio = 50:1) plus titrations of antibody. Following 4 hours incubation at
37° C, supernatant conditioned medium was collected and analyzed for radioactivity. Release
of radioactivity in the absence of antibody was used as a control for background and
detergent treatment of target cells was used to determine 100% lysis. Cytotoxicity was
calculated by the formula: % lysis = (experimental cpm - target leak cpm)/(detergent lysis
cpm - target leak cpm) x 100%. Specific lysis = % lysis with 13H5 - % lysis without 13H5.
Assays were performed in triplicate.
The results of the ADCC assay, summarized in Figure 9, demonstrate that
13H5 had no significant ADCC activity on Raji cells, either alone or in the presence of IFNoc
2b. Similarly, an isolype matched IgG displayed no activity, whereas the positive control
(Rituximab) exhibited robust dose dependent cytotoxicity. These results indicate that IFNa
mediated association of 13H5 with the cell surface of IFNAR expressing cells is not
sufficient to mediate ADCC.
Example 10: Examination of Stability of 13H5
The 13H5 antibody contains a potential deamidation site at Asn-55 in the
CDR2 region of the heavy chain. Deamidation of asparagines residues is a common
modification of polypeptides and proteins obtained using recombinant DNA technology and
may result in decreased biological activity and/or stability, though deamidation does not
always correlate with loss of biological activity. Deamidation of asparagines to form aspartic
acid (and iso-Asp) results in a change of net charge, which can be detected by charge-based
analytical methods. To examine deamidation of 13H5 under accelerated conditions (basic
pH), methods for detection of deamidated variants of Fab fragment by IEX-HPLC and
capillary isoelectric focusing (cEIF) were used.
To accelerate deamidation of 13H5, the antibody was exposed to buffer at
alkaline pH. For the starting material, a 102 ul aliquot of 13H5 (at 5.9. mg/ml for a total of
600 ug) was added to 498 ul of PBS and 6 ul of 100X sodium azide stock (2% solution). For
the time zero PBS sample, 130 ul of starting material was combined with 30 ul of PBS and
the sample was placed at -20° C until further analysis. For the time zero sample in
deamidation buffers, 130 ul of starting material was combined with 15 ul of 10X deamidation
buffer (10% ammonium bicarbonate, pH 8.5) and 15 ul of pH adjustment buffer (1M MES,
pH 6.0) and placed at -20° C until further analysis. For the Day 2 sample in PBS, 130 ul of
starting material was combined with 30 ul of PBS and incubated at 40° C for 48 hours and
then the sample was placed at -20° C until further analysis. For the Day 2 sample under
deamidation conditions, 130 ul of starting material was combined with 15 ul of 10X
deamidation buffer and incubated at 40° C for 48 hours. After 48 hours, 15 ul of pH
adjustment buffer was added and the sample was placed at -20° C until further analysis.
To prepare the above samples for analysis, papain digestion was performed.
Reaction conditions used were: 160 fal of sample (130 p.g 13H5), 3.2 jj.1 of 50 mM cysteine
and 6.5 ul of papain enzyme at 1.0 mg/ml in solution. The samples were placed at 40° C for
4 hours and the reaction was stopped by addition of 4.8 |4 of 1M iodoacetamide. After
papain digestion, non-reducing SDS-PAGE was performed to confirm the presence of Fab
and Fc fragments.
To perform IEX-HPLC on the samples, all samples were first dialyzed against
water for 3 hours. Then, 50 |il of each sample was applied to HPLC wit the following
chromatography conditions:
Column = Dionex WCX-10 weak cation exchange column
"A" buffer = 10 mM MES, pH 5.5
"B" buffer = 10 mM MES, pH 5.5; 1.0 M NaCl
Elution = 4-25 % "B" over 30 minutes at 0.8 ml/min
Detection = UV absorbance at 280 nM
The results of IEX-HPLC analysis are summarized in Table 7 below, which shows the peak
areas for deamidated Fab for time zero and Day 2 samples under deamidation conditions:
To examine the pH dependence of forced deamidation, the IBX-HPLC data for
the Day 2 sample in PBS (pH 7.0) was compared to the Day 2 sample under deamidation
conditions. The results are summarized in Table 9 below, which shows the peak areas for the
deamidated Fab for Day 2 PBS and Day 2 under deamidation conditions:
This data supports the existing theory of protein degradation, which predicts that deamidation
of polypeptides via beta-aspartyl shift mechanism occurs at an increased rate under basic pH
as compared to neutral pH.
Example 11:Preparation and Characterization of 13H5 Mutants
with Enhanced Stability
In this example, 13H5 mutants were prepared having an amino acid
substitution at Asn-55 and the stability of these mutants was examined, at Day 2 under forced
deamidation conditions, by cIEF analysis as described in Example 10. The mutants were
prepared by standard recombinant DNA mutagenesis techniques. The sequences of the
mutants at amino acid positions 55-58 of Vh, as compared to wild type 13H5, were as
follows:
13H5 wild-type: N GN T (amino acid residues 55-58 of SEQ ID NO: 19)
Mutant # 1: D G N T (SEQ ID NO: 38)
Mutant #2: Q G N T (SEQ ED NO: 39)
Mutant #3: Q G Q T (SEQ ID NO: 40)
The full-length Vh amino acid sequences of mutants #1, #2 and #3 are shown in SEQ ID
NOs: 34, 35 and 36, respectively.
The results of the cIEF analysis are shown below in Table 10, which shows
the pealdareas for deamidated Fab for the wild type and mutants at Day 2 deamidation
conditions:
i
Table 10:
The results demonstrate that each of the three Asn-55 mutants exhibits greater stability under
forced deamidation conditions than the wild-type 13H5 antibody.
WE CLAIM:
1.An isolated anti-interferon alpha monoclonal antibody, or antigen binding portion thereof,
which comprises:
(a) a heavy chain variable region CDR1 comprising SEQ ED NO: 1;
(b) a heavy chain variable region CDR2 comprising SEQ ID NO: 4;
(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 7;
(d) a light chain variable region CDR1 comprising SEQ ID NO: 10;
(e) a light chain variable region CDR2 comprising SEQ ID NO: 13; and
(f) a light chain variable region CDR3 comprising SEQ ID NO: 16.
2.An isolated anti-interferon alpha monoclonal antibody, or antigen binding portion thereof
comprising:
(a)a heavy chain variable region comprising the amino acid sequence of SEQ ID
NO: 19; and
(b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:
22;
wherein the antibody inhibits the biological activity of at least one interferon alpha
subtype.
3.An isolated anti-interferon alpha monoclonal antibody, or antigen binding portion thereof
comprising:
(a)a heavy chain variable region comprising an amino acid sequence selected from
the group consisting of SEQ ID NOs: 34, 35, 36 and 37; and
(b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:
22;
wherein the antibody inhibits the biological activity of at least one interferon alpha
subtype.
4.An isolated anti-interferon alpha monoclonal antibody, or an antigen binding portion
thereof, which binds an epitope on a human interferon alpha polypeptide recognized by an
antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ
ID NO: 19 and a light chain variable region comprising the amino acid sequence of SEQ ED NO:
22.
5.An isolated anti-interferon alpha monoclonal antibody, or antigen binding portion thereof
comprising a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences and a
light chain variable region comprising CDR1, CDR2, and CDR3 sequences, wherein:
(a) the heavy chain variable region CDR3 sequence comprises the amino acid
sequence of SEQ ED NO: 7, and conservative sequence modifications thereof; and
(b) the light chain variable region CDR3 sequence comprises the amino acid
sequence of SEQ ED NO: 16, and conservative sequence modifications thereof.
6. The antibody as claimed in claim 5, wherein the heavy chain variable region CDR2 sequence
comprises the amino acid sequence of SEQ ID NO: 4, and conservative sequence modifications
thereof; and the light chain variable region CDR2 sequence comprises the amino acid sequence
of SEQ ED NO: 13, and conservative sequence modifications thereof.
7. The antibody as claimed in claim 6, wherein the heavy chain variable region CDR1 sequence
comprises the amino acid sequence of SEQ ED NO: 1, and conservative sequence modifications
thereof; and the light chain variable region CDR1 sequence comprises the amino acid sequence
of SEQ ED NO: 10, and conservative sequence modifications thereof.
8.An isolated anti-interferon alpha monoclonal antibody, or antigen binding portion thereof
comprising a heavy chain variable region and a light chain variable region, wherein:
(a) the heavy chain variable region comprises an amino acid sequence that has at least
95% sequence identity to the amino acid sequence of SEQ ED NO: 19; and
(b) the light chain variable region comprises an amino acid sequence that has at least
95% sequence identity to the amino acid sequence of SEQ ED NO: 22.
9.An isolated anti-interferon alpha monoclonal antibody, or antigen binding portion thereof,
comprising a heavy chain variable region and a light chain variable region, wherein:
(a) the heavy chain variable region comprises an amino acid sequence selected from
the group consisting of SEQ ED NO: 19 and has at least 1, 2, or 3 amino acid
substitutions; and
(b) the light chain variable region comprises an amino acid sequence selected from
the group consisting of SEQ ID NO: 22 and has at least 1, 2, or 3 amino acid
substitutions.
10. The antibody as claimed in any one of claims 1-9, wherein the antibody inhibits IFN-induced
surface expression of CD38 or MHC Class I on peripheral blood mononuclear cells.
11. The antibody as claimed in any one of claims 1-9, wherein the antibody inhibits EFN-induced
expression of IP-10 by peripheral blood mononuclear cells.
12. The antibody as claimed in any one of claims 1-9, wherein the antibody inhibits dendritic cell
development mediated by systemic lupus erythematosus (SLE) plasma.
13. The antibody as claimed in any one of claims 1-9, wherein the antibody is a human antibody.
14. The antibody as claimed in claim 1 or 4-9, wherein the antibody is a chimeric antibody.
15. The antibody as claimed in claim 1 or 4-9, wherein the antibody is a humanized antibody.
16. The antibody as claimed in any one of claims 1-9, wherein the antibody is an IgGl or IgG4
antibody.
17. The antibody as claimed in any one of claims 1-9, wherein the antigen binding portion is a
Fab antibody fragment.
18. The antibody as claimed in any one of claims 1-9, wherein the antigen binding portion is a
single chain antibody (scFv).
19. A pharmaceutical composition comprising the antibody as claimed in any one of claims 1-9.
20.The pharmaceutical composition as claimed in claim 19, wherein the pharmaceutical
composition is lyophilized.
21.The (pharmaceutical composition as claimed in claim 19, wherein the pharmaceutical
composition is an aqueous solution or dispersion.

The present invention provides isolated anti-interferon alpha monoclonal antibodies,
particularly human monoclonal antibodies, that inhibit the biological activity of multiple
interferon (IFN) alpha subtypes but do not substantially inhibit the biological activity of IFN
alpha 21 or the biological activity of either IFN beta or IFN omega (Figures 1A-3B).
Immunoconjugates, bispecific molecules and pharmaceutical compositions comprising the
antibodies of the invention are also provided. The invention also provides methods for inhibiting
the biological activity of IFN alpha using the antibodies of the invention, as well as methods of
treating disease or disorders mediated by IFN alpha, such as autoimmune diseases, transplant
rejection and graft versus host disease, by administering the antibodies of the invention.

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01300-kolnp-2006 correspondence others-1.1.pdf

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1300-kolnp-2006-granted-abstract.pdf

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1300-kolnp-2006-granted-form 3.pdf

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1300-KOLNP-2006-SEQUENCE LISTING.pdf


Patent Number 235935
Indian Patent Application Number 1300/KOLNP/2006
PG Journal Number 05/2010
Publication Date 29-Jan-2010
Grant Date 09-Sep-2009
Date of Filing 17-May-2006
Name of Patentee MEDAREX, INC.
Applicant Address 707 STATE ROAD, PRINCETON, NJ
Inventors:
# Inventor's Name Inventor's Address
1 WITTE, ALISON 3505 BEAN CREEK ROAD, SCOTTS VALLEY, CA 95066
2 CARDARELLI, JOSEPHINE M. 126 LESLIE DRIVE, SAN CARLOS, CA 94070
3 KING, DAVID 1744 TERRACE DRIVE, BELMONT, CA 94002
4 PASSMORE, DAVID 98 CHURCH STREET, APT.6, MOUNTAIN VIEW, CA 94041
5 WILLIAMS, DENISE 3511 CHESBRO AVENUE, SAN JOSE, CA 95123
PCT International Classification Number A61K 39/395
PCT International Application Number PCT/US2004/041777
PCT International Filing date 2004-12-10
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/528,757 2003-12-10 U.S.A.