U.S. patent application number 15/281441 was filed with the patent office on 2017-01-19 for cd86 antagonist multi-target binding proteins.
The applicant listed for this patent is APTEVO RESEARCH AND DEVELOPMENT LLC. Invention is credited to Peter Robert Baum, John W. Blankenship, Sateesh Kumar Natarajan, Philip Tan, Peter Armstrong Thompson.
Application Number | 20170015747 15/281441 |
Document ID | / |
Family ID | 41557444 |
Filed Date | 2017-01-19 |
United States Patent
Application |
20170015747 |
Kind Code |
A1 |
Thompson; Peter Armstrong ;
et al. |
January 19, 2017 |
CD86 ANTAGONIST MULTI-TARGET BINDING PROTEINS
Abstract
This disclosure provides a multi-specific fusion protein
composed of a CD86 antagonist binding domain and another binding
domain that is an IL-10 agonist, an HLA-G agonist, an HGF agonist,
an IL-35 agonist, a PD-1 agonist, a BTLA agonist, a LIGHT
antagonist, a GITRL antagonist or a CD40 antagonist. The
multi-specific fusion protein may also include an intervening
domain that separates the other domains. This disclosure also
provides polynucleotides encoding the multi-specific fusion
proteins, compositions of the fusion proteins, and methods of using
the multi-specific fusion proteins and compositions.
Inventors: |
Thompson; Peter Armstrong;
(Bellevue, WA) ; Baum; Peter Robert; (Seattle,
WA) ; Tan; Philip; (Edmonds, WA) ;
Blankenship; John W.; (Seattle, WA) ; Natarajan;
Sateesh Kumar; (Redmond, WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
APTEVO RESEARCH AND DEVELOPMENT LLC |
Seattle |
WA |
US |
|
|
Family ID: |
41557444 |
Appl. No.: |
15/281441 |
Filed: |
September 30, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13122383 |
May 19, 2011 |
9493564 |
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PCT/US2009/059446 |
Oct 2, 2009 |
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15281441 |
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61102297 |
Oct 2, 2008 |
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61102307 |
Oct 2, 2008 |
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61102315 |
Oct 2, 2008 |
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61102327 |
Oct 2, 2008 |
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61102331 |
Oct 2, 2008 |
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61102334 |
Oct 2, 2008 |
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61102336 |
Oct 2, 2008 |
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61102288 |
Oct 2, 2008 |
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61102319 |
Oct 2, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/244 20130101;
C07K 2317/92 20130101; C07K 16/248 20130101; C07K 14/70521
20130101; C07K 2317/24 20130101; C07K 2317/622 20130101; A61P 1/00
20180101; C07K 2317/75 20130101; C07K 2319/70 20130101; A61P 43/00
20180101; C07K 14/5428 20130101; C07K 16/2818 20130101; A61P 29/00
20180101; C07K 2317/53 20130101; C07K 2317/31 20130101; C07K
16/2833 20130101; A61P 19/04 20180101; A61K 39/39558 20130101; A61P
7/06 20180101; A61P 11/06 20180101; A61P 19/02 20180101; A61P 25/00
20180101; A61K 39/00 20130101; C07K 16/2878 20130101; C07K 16/2827
20130101; C07K 16/2875 20130101; C07K 2317/76 20130101; A61P 17/06
20180101; C07K 2317/55 20130101; A61P 37/06 20180101; A61K 2039/505
20130101; A61P 37/02 20180101; A61P 3/10 20180101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; C07K 16/24 20060101 C07K016/24 |
Claims
1. A multi-specific fusion protein, comprising a CD86 binding
domain linked to a heterologous binding domain by an intervening
domain wherein the heterologous binding domain is an IL-10 agonist,
an HLA-G agonist, an HGF agonist, an IL-35 agonist, a PD-1 agonist,
a BTLA agonist, a LIGHT antagonist, a GITRL antagonist or a CD40
antagonist.
2. The multi-specific fusion protein of claim 1 wherein the CD86
binding domain is a CTLA4 ectodomain or a sub-domain of a CTLA4
ectodomain.
3. The multi-specific fusion protein of claim 1 wherein the CD86
binding domain is a Fab, scFv, a domain antibody, or a heavy
chain-only antibody specific for CD86.
4. The multi-specific fusion protein of claim 3 wherein the CD86
binding domain comprises light and heavy chain variable domains of
FUN1 anti-CD86 antibody or a humanized variant thereof.
5. The multi-specific fusion protein of claim 4 wherein the FUN1
binding domain comprises amino acids 1-258 of SEQ ID NO:187 or
237.
6. The multi-specific fusion protein of claim 1 wherein the CD86
binding domain comprises an amino acid sequence as set forth in any
one of SEQ ID NOS:1-6.
7. The multi-specific fusion protein of claim 1 wherein the
heterologous binding domain comprises the amino acid sequence
provided in any one of SEQ ID NOS:7, 14, 15, 18-22, 25, 26, 29, 32,
33, 39, and 40.
8. The multi-specific fusion protein of claim 1 wherein the
heterologous binding domain comprises an IL-10 agonist comprising
the amino acid sequence provided in SEQ ID NO:7 or a variant
thereof comprising a point mutation at position 87.
9. The multi-specific fusion protein of claim 1 wherein the
heterologous binding domain comprises an HLA-G agonist comprising
the amino acid sequence provided in SEQ ID NO:14 or 15.
10. The multi-specific fusion protein of claim 1 wherein the
heterologous binding domain comprises an HGF agonist comprising the
amino acid sequence provided in any one of SEQ ID NO:18-22.
11. The multi-specific fusion protein of claim 1 wherein the
heterologous binding domain comprises an IL-35 agonist comprising
the amino acid sequence provided in SEQ ID NO:25 or 26.
12. The multi-specific fusion protein of claim 1 wherein the
heterologous binding domain comprises a PD-1 agonist comprising the
amino acid sequence provided in SEQ ID NO:32 or 33.
13. The multi-specific fusion protein of claim 1 wherein the
heterologous binding domain comprises a BTLA agonist comprising the
amino acid sequence provided in SEQ ID NO:29.
14. The multi-specific fusion protein of claim 1 wherein the
heterologous binding domain comprises a LIGHT antagonist comprising
the amino acid sequence provided in SEQ ID NO:29.
15. The multi-specific fusion protein of claim 1 wherein the
heterologous binding domain comprises a GITRL antagonist comprising
the amino acid sequence provided in SEQ ID NO:39 or 40.
16. The multi-specific fusion protein of claim 1 wherein the
intervening domain comprises an immunoglobulin constant region or
sub-region disposed between the CD86 binding domain and the
heterologous binding domain.
17. The multi-specific fusion protein of claim 16 wherein the
immunoglobulin constant region or sub-region is IgG1 CH2CH3.
18. The multi-specific fusion protein of claim 1 wherein the
intervening domain comprises an immunoglobulin constant region
disposed between a first and a second linker.
19. The multi-specific fusion protein of claim 18 wherein the first
and second linkers are independently selected from the linkers
provided in SEQ ID NOs:43-166, 244, 307, 320, 355-379 and
383-398.
20. The multi-specific fusion protein of claim 18 wherein the
intervening domain comprises a human immunoglobulin Fc region,
albumin, transferrin, or a scaffold domain that binds a serum
protein.
21. The multi-specific fusion protein of claim 1 wherein the
intervening domain comprises a structure, from amino-terminus to
carboxy-terminus, as follows: -L1-X-L2- wherein: L1 and L2 are each
independently a linker comprising from two to about 150 amino
acids; and X is an immunoglobulin constant region or sub-region,
albumin, transferrin, or another serum protein binding protein.
22. The multi-specific fusion protein of claim 21 wherein the
immunoglobulin constant region or sub-region is an IgG1 CH2CH3.
23. The multi-specific fusion protein of claim 21 wherein L1 is a
human immunoglobulin hinge region, optionally mutated to replace
one or more cysteines with other amino acids.
24. The multi-specific fusion protein of claim 21 wherein X is a
human IgG1 Fc domain or at least one CH domain thereof, optionally
mutated to eliminate Fc.gamma.RI-III interaction while retaining
FcRn interaction.
25. The multi-specific fusion protein of claim 1 wherein the
intervening domain is a dimerization domain.
26. The multi-specific fusion protein of claim 1 having the
following structure: N-BD1-X-L2-BD2-C wherein: BD1 is a CD86
binding domain that is at least about 90% identical to an
ectodomain of CTLA4; -X- is -L1-CH2CH3-, wherein L1 is the first
IgG1 hinge, optionally mutated by substituting the first cysteine
and wherein -CH2CH3- is the CH2CH3 region of an IgG1 Fc domain,
optionally mutated to eliminate Fc.gamma.RI-III interaction while
retaining FcRn interaction; L2 is a linker selected from SEQ ID
NOs:43-166, 244, 307, 320, 355-379 and 383-398; and BD2 is the
heterologous binding domain.
27. The multi-specific fusion protein of claim 1 wherein the fusion
protein comprises the amino acid sequence provided in any one of
SEQ ID NOs: 9, 13, 17, 24, 28, 31, 35, 38, 42, 171, 173, 175, 177,
179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203,
205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 237, 239, 252,
254, 256, 258, 260, 262, 266, 276, 302, 330, 334, 336, 338, 340,
350, 352, and 354.
28. A composition comprising one or more multi-specific fusion
proteins according to claim 1 and a pharmaceutically acceptable
carrier, diluent, or excipient.
29. The composition of claim 28 wherein the multi-specific fusion
protein exists as a dimer or a multimer in the composition.
30. A polynucleotide encoding the multi-specific fusion protein
according to claim 1.
31. The polynucleotide of claim 30 wherein the polynucleotide
comprises the polynucleotide provided in any one of SEQ ID NOs: 8,
12, 16, 23, 27, 30, 34, 37, 41, 170, 172, 174, 176, 178, 180, 182,
184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208,
210, 212, 214, 216, 218, 220, 222, 236, 238, 251, 253, 255, 257,
259, 261, 265, 275, 301, 329, 333, 335, 337, 339, 349, 351 and
353.
32. An expression vector comprising the polynucleotide according to
claim 30 operably linked to an expression control sequence.
33. A host cell comprising the expression vector according to claim
32.
34. A method for treating a subject with a disorder associated with
CD86, IL-10, HLA-G, HGF, IL-35, PD-1, BTLA, LIGHT, GITRL or CD40,
comprising administering a therapeutically effective amount of a
multi-specific fusion protein of claim 1.
35. The method of claim 34 wherein the disorder is rheumatoid
arthritis, juvenile rheumatoid arthritis, systemic lupus
erythematosus or solid organ transplant.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/122,383, filed May 19, 2011, which is the
U.S. National Phase of International Application No.
PCT/US2009/059446, filed Oct. 2, 2009, which claims the priority
benefit of U.S. Provisional Patent Application No. 61/102,288,
filed Oct. 2, 2008; 61/102,297, filed Oct. 2, 2008; 61/102,307,
filed Oct. 2, 2008; 61/102,315, filed Oct. 2, 2008; 61/102,319,
filed Oct. 2, 2008; 61/102,327, filed Oct. 2, 2008; 61/102,331,
filed Oct. 2, 2008; 61/102,334, filed Oct. 2, 2008; and 61/102,336,
filed Oct. 2, 2008. The contents of each application listed above
are incorporated herein by reference in their entireties.
INCORPORATION OF SEQUENCE LISTING
[0002] The contents of the text file submitted electronically
herewith are incorporated herein by reference in their entirety: A
computer readable format copy of the Sequence Listing (filename:
APVO_026_10US_SeqList_ST25.txt, date recorded: Sep. 29, 2016, file
size 721,803 bytes).
BACKGROUND
[0003] Technical Field
[0004] This disclosure relates generally to the field of
multi-specific binding molecules and therapeutic applications
thereof and more specifically to a fusion protein composed of a
CD86 antagonist binding domain, and another binding domain that is
specific for a heterologous target, such as an IL-10 agonist, an
HLA-G agonist, an HGF agonist, an IL-35 agonist, a PD-1 agonist, a
BTLA agonist, a LIGHT antagonist, a GITRL antagonist or a CD40
antagonist, as well as compositions and therapeutic uses
thereof.
[0005] Description of the Related Art
[0006] The human immune system generally protects the body from
damage by foreign substances and pathogens. One way in which the
immune system protects the body is by producing specialized cells,
referred to as T lymphocytes or T-cells. Intercellular interactions
between T-cells and antigen-presenting cells (APCs) generate T-cell
costimulatory signals that in turn lead to T-cell responses to
antigens. Full T cell activation requires both binding of the
T-cell receptor (TCR) to antigen-MHC complex present on
antigen-presenting cells and binding of the receptor CD28 on the
surface of the T-cell to the CD86 and/or CD80 ligands present on
antigen-presenting cells, particularly dendritic cells.
[0007] CD80 (also known as B7-1) was originally described as a
human B-cell associated activation antigen and was subsequently
found to be a receptor for the related T-cell molecules CD28 and
cytotoxic T lymphocyte-associated antigen-4 (CTLA4). In later
studies, another counterreceptor for CTLA4 known as CD86 (also
known as B7-0 or B7-2) was identified. CD86 shares about 25%
sequence identity with CD80 in its extracellular region. While CD80
and CD86 are generally believed to be functionally equivalent in
their ability to initiate and maintain proliferation of CD4(+) T
cells (Vasilevko et al. (2002) DNA Cell Biol. 21:137-49), and
clinical data with a soluble CTLA4 Ig fusion protein that blocks
this activity for both molecules has shown clinical benefit
(Genovese et al. (2005) NEJM 353:114-1123), there is some evidence
that specific inhibition of CD86 might be of benefit. For example,
engagement of CD86 or CD80 has different effects on B cells.
Specifically, CD80 has been shown to provide a negative signal for
the proliferation and IgG secretion of both normal B cells and B
cell lymphomas, while CD86 enhances the activity of B cells (Suvas
et al. (2002) J. Biol. Chem. 277:7766-7775). There is also some
evidence that engagement of CD80 on T cells is immunosuppressive
(Lang et al. (2002) J. Immunol. 168:3786-3792; Taylor et al. (2004)
J. Immunol. 172:34-39; Paust et al. (2004) PNAS 101:10398-10403)
and that it may mediate further immunosuppression through PD-L1
(CD274) signaling on activated APCs or T cells (Butte et al. (2007)
Immunity 27:111-122; Keir (2008) Ann. Rev. Immunol. 26:677-704).
Accordingly, inhibition of CD86 in the absence of CD80 inhibition
may be beneficial in the treatment of autoimmune and inflammatory
disease as well as B cell lymphomas.
[0008] CTLA4 is a type 1 transmembrane glycoprotein of the
immunoglobulin superfamily that is mainly expressed in activated
T-cells, with some expression also being found in the CD4+CD25+
regulatory T-cell (Treg) subset. CD86 and CD80 are believed to be
the only endogenous ligands for CTLA4. CTLA4 has been shown to bind
CD86 and CD80 with greater affinity and avidity compared with CD28
(Linsley et al. (1991) J. Exp. Med. 174:561-69; Linsley et al.
(1994) Immunity 1:793-801), and plays a key role as a negative
regulator of T-cell activation. Specifically, binding of CTLA4 to
CD80/CD86 leads to downregulation of T-cell responses and to the
preservation of T-cell homeostasis and peripheral tolerance. This
is believed to be due to both antagonism of CD28-dependent
costimulation and directive negative signaling through the CTLA4
cytoplasmic tail. For a review of CTLA4 structure and function, see
Teft et al. (2006) Annu. Rev. Immunol. 24:65-97.
[0009] As mentioned above, a productive immune response requires
both engagement of TCR and binding of CD28 to CD80 and/or CD86. TCR
binding in the absence of CD28 binding leads to T cells either
undergoing apoptosis or becoming anergic. In addition, CD28
signaling has been shown to increase cytokine production by T
cells. Specifically, CD28 stimulation has been shown to increase
production of IL-2, TNF.alpha., lymphotoxin, IFN.gamma. and GM-CSF
5- to 50-fold in activated T cells. Furthermore, induction of
lymphokine and/or cytokine gene expression by CD28 has been shown
to occur even in the presence of the immunosuppressant cyclosporine
(Thompson et al. (1989) Proc. Natl. Acad. Sci. USA 86:1333-1337).
CD28 has also been shown to promote T cell survival by inducing
upregulation of the anti-apoptotic BCL-XL (Alegre et al. (2001)
Nature Rev. Immunol. 1:220-228).
[0010] Soluble forms of CTLA4 have been constructed by fusing the
variable-like extracellular domain of CTLA4 to immunoglobulin
constant domains to provide CTLA4-Ig fusion proteins. Soluble
CTLA-4-Ig has been shown to prevent CD28-dependent costimulation by
binding to both CD86 and CD80 (Linsley et al. (1991) J. Exp. Med.,
174:561-69), and to inhibit costimulation of T cells and have
beneficial immunosuppression effects in humans (Bruce & Boyce
(2007) Ann. Pharmacother. 41:1153-1162). The CTLA4-Ig fusion
protein abatacept is currently employed for the treatment of
rheumatoid arthritis in cases of inadequate response to
anti-TNF.alpha. therapy. However, not all patients respond to
CTLA4-Ig and continued response requires frequent drug
administration, perhaps in part because blockage of interaction of
CD28 with CD86/CD80 is a weak inducer of Tregs and insufficient for
blocking activated effector T cell responses in a disease
milieu.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 shows binding to CD80 by various proteins, including
abatacept, a CTLA4-Ig(N2) (SEQ ID NO:11), and a multi-specific
xceptor fusion protein containing a CTLA4 ectodomain fused to an
IL10 (SEQ ID NO:9).
[0012] FIG. 2 shows that CTLA4-Ig(N2) (SEQ ID NO:11) and a
multi-specific xceptor fusion protein containing a CTLA4 ectodomain
fused to an IL10 (SEQ ID NO:9) can bind to soluble IL10Ra
(sIL10Ra).
[0013] FIGS. 3 and 4 show that a multi-specific xceptor fusion
protein containing a CTLA4 ectodomain fused to an IL10 (SEQ ID
NO:9) can induce STAT3 phosphorylation in PBMC.
[0014] FIG. 5 shows that xceptors containing anti-CD86 binding
domains from 3D1 and humanized FUN1 monoclonal antibodies bind to
CD86 on WIL2-S cells.
[0015] FIG. 6 shows that an xceptor containing a CD86 binding
domain and IL10 can simultaneously bind cell surface CD86 and and
sIL10Ra.
[0016] FIG. 7 shows that various different versions of humanized
anti-CD86 FUN1 SMIPs can bind CD86.
[0017] FIG. 8 shows that CTLA4::IL10 xceptor molecules having
various linkers joining IL10 to the carboxy-terminus (BD2) of the
xceptor can bind IL10R1-Ig. .DELTA.-SEQ ID NO:9; .diamond.-SEQ ID
NO: 171; -SEQ ID NO:302; .tangle-solidup.-SEQ ID NO:173.
[0018] FIG. 9 shows that CTLA4::IL10 xceptor molecules having
shorter linkers joining IL10 to the carboxy-terminus (BD2) of the
xceptor can bind IL10R1-Ig. .DELTA.-SEQ ID NO:171; .diamond.-SEQ ID
NO:175; -SEQ ID NO:177; .tangle-solidup.-SEQ ID NO:179.
[0019] FIG. 10 shows that several xceptor proteins bind to
CD80.
[0020] FIG. 11 shows that several xceptor proteins bind to
CD86.
[0021] FIG. 12 shows that several xceptor proteins bind to
sIL10Ra.
[0022] FIG. 13 shows that several xceptor proteins can
simultaneously bind to CD80 and sIL10Ra.
[0023] FIG. 14 shows that several xceptor proteins are
crossreactive with mouse CD80.
[0024] FIG. 15 shows that several xceptor proteins are
crossreactive with mouse CD86.
[0025] FIGS. 16 and 17 show that several xceptor proteins block a
human T cell response in an MLR assay.
[0026] FIGS. 18 to 20 show that several xceptor proteins block a
mouse T cell response in an MLR assay.
[0027] FIGS. 21 and 22 show that several xceptor proteins
containing a variant IL10 (either IL10 with an I87 mutation or
monoIL10) or a variant CTLA4 block a human T cell response in an
MLR assay.
[0028] FIG. 23 shows that several xceptor proteins containing a
variant IL10 (either IL10 with an I87 mutation or monoIL10) are
less immunostimulatory than mouse IL10 in an MC/9 cell
proliferation assay.
[0029] FIG. 24 shows that several xceptor proteins containing a
variant IL10 (either IL10 with an I87 mutation or monoIL10) are
less immunostimulatory than human IL10 in an MC/9 cell
proliferation assay.
DETAILED DESCRIPTION
[0030] The present disclosure makes possible the targeting of
antigen presenting cells (APCs) to alter activity. For example,
T-cell activity can be modulated by providing multi-specific
xceptor fusion proteins that comprise a first binding domain that
preferentially binds a CD86, and a second binding domain (a
heterologous binding domain). In certain embodiments, a
multi-specific xceptor fusion protein comprises a first and second
binding domain, a first and second linker, and an intervening
domain, wherein one end of the intervening domain is fused via a
linker to the first binding domain that is a CD86 binding domain
and the other end is fused via a linker to the second binding
domain that is an IL-10 agonist, an HLA-G agonist, an HGF agonist,
an IL-35 agonist, a PD-1 agonist, a BTLA agonist, a LIGHT
antagonist, a GITRL antagonist or a CD40 antagonist.
[0031] In certain embodiments, the CD86 binding domain is a CTLA4
ectodomain, a CD28 ectodomain, or an immunoglobulin variable region
binding domain (such as a scFv) specific for CD86 (e.g., from
monoclonal antibodies 3D1 or FUN1). In some embodiments, less than
an entire ectodomain is used. For example, domains within the CTLA4
ectodomain that bind CD86 and prevent binding of CD86 to CD28 can
be used. In further embodiments, the IL10 agonist is IL10 or a
functional region thereof. In further embodiments, the HLA-G
agonist is an HLA-G5, an HLA-G1, an HLA-G mutein, or a functional
region thereof; an ectodomain of an HLA-G5, an HLA-G1 or an HLA-G
mutein; or an immunoglobulin variable region binding domain (such
as a scFv) specific for ILT2, ILT4 or KIR2DL4. In still further
embodiments, the heterologous binding domain is an HGF agonist,
such as an HGF or a sub-domain thereof. In another embodiment, the
heterologous binding domain is an IL35 agonist, such as an
immunoglobulin variable region binding domain (such as a scFv)
specific for IL35R or IL35, or a functional region thereof. In
further embodiments, the LIGHT antagonist is an immunoglobulin
variable region binding domain (such as a scFv) specific for LIGHT,
or a HVEM ectodomain or a functional region thereof. In further
embodiments, the PD-1 agonist is an immunoglobulin variable region
binding domain (such as a scFv) specific for PD-1, or a PD-1 ligand
(e.g. PD-L1 or PD-L2) or a functional region thereof. In further
embodiments, the BTLA agonist is an immunoglobulin-like variable
region binding domain (such as a scFv) specific for BTLA, or a HVEM
ectodomain or a functional region thereof. In certain embodiments,
the GITRL antagonist is an immunoglobulin-like variable region
binding domain (such as a scFv) specific for GITRL, or a GITR
ectodomain, soluble GITR, or a functional region thereof. In
certain embodiments, the CD40 antagonist is an immunoglobulin-like
variable region binding domain (such as a scFv) specific for
CD40.
[0032] Exemplary structures of such multi-specific fusion proteins,
referred to herein as Xceptor molecules, include N-BD-ID-ED-C,
N-ED-ID-BD-C, N--BD1-ID-BD2-C, and N-ED-ID-ED-C, wherein N- and -C
refer to the amino- and carboxy terminus, respectively; BD is an
immunoglobulin-like or immunoglobulin variable region binding
domain; ID is an intervening domain; and ED is an extracellular or
ectodomain, such as a receptor ligand binding domain, ligand,
C-type lectin domain, semaphorin or semaphorin-like domain, or the
like. In some constructs, the ID can comprise an immunoglobulin
constant region or sub-region disposed between the first and second
binding domains. In still further constructs, the BD and ED are
each linked to the ID via the same or different linker (e.g., a
linker comprising one to 50 amino acids), such as an immunoglobulin
hinge region (made up of, for example, the upper and core regions)
or functional variant thereof, or a lectin interdomain region or
functional variant thereof, or a cluster of differentiation (CD)
molecule stalk region or functional variant thereof.
[0033] Prior to setting forth this disclosure in more detail, it
may be helpful to an understanding thereof to provide definitions
of certain terms to be used herein. Additional definitions are set
forth throughout this disclosure.
[0034] In the present description, any concentration range,
percentage range, ratio range, or integer range is to be understood
to include the value of any integer within the recited range and,
when appropriate, fractions thereof (such as one tenth and one
hundredth of an integer), unless otherwise indicated. Also, any
number range recited herein relating to any physical feature, such
as polymer subunits, size or thickness, are to be understood to
include any integer within the recited range, unless otherwise
indicated. As used herein, "about" or "consisting essentially of"
mean.+-.20% of the indicated range, value, or structure, unless
otherwise indicated. It should be understood that the terms "a" and
"an" as used herein refer to "one or more" of the enumerated
components. The use of the alternative (e.g., "or") should be
understood to mean either one, both, or any combination thereof of
the alternatives. As used herein, the terms "include" and
"comprise" are used synonymously. In addition, it should be
understood that the individual compounds, or groups of compounds,
derived from the various combinations of the structures and
substituents described herein, are disclosed by the present
application to the same extent as if each compound or group of
compounds was set forth individually. Thus, selection of particular
structures or particular substituents is within the scope of the
present disclosure.
[0035] A "binding domain" or "binding region" according to the
present disclosure may be, for example, any protein, polypeptide,
oligopeptide, or peptide that possesses the ability to specifically
recognize and bind to a biological molecule (e.g., CD86) or a
complex of more than one of the same or different molecule or
assembly or aggregate, whether stable or transient (e.g. CD86/CD28
complex). Such biological molecules include proteins, polypeptides,
oligopeptides, peptides, amino acids, or derivatives thereof,
lipids, fatty acids, or derivatives thereof; carbohydrates,
saccharides, or derivatives thereof; nucleotides, nucleosides,
peptide nucleic acids, nucleic acid molecules, or derivatives
thereof; glycoproteins, glycopeptides, glycolipids, lipoproteins,
proteolipids, or derivatives thereof; other biological molecules
that may be present in, for example, a biological sample; or any
combination thereof. A binding region includes any naturally
occurring, synthetic, semi-synthetic, or recombinantly produced
binding partner for a biological molecule or other target of
interest. A variety of assays are known for identifying binding
domains of the present disclosure that specifically bind with a
particular target, including FACS, Western blot, ELISA, or Biacore
analysis.
[0036] Binding domains and fusion proteins thereof of this
disclosure can be capable of binding to a desired degree, including
"specifically or selectively binding" a target while not
significantly binding other components present in a test sample, if
they bind a target molecule with an affinity or K.sub.a (i.e., an
equilibrium association constant of a particular binding
interaction with units of 1/M) of, for example, greater than or
equal to about 10.sup.5 M.sup.-1, 10.sup.6 M.sup.-1, 10.sup.7
M.sup.-1, 10.sup.8 M.sup.-1, 10.sup.9 M.sup.-1, 10.sup.10 M.sup.-1,
10.sup.11 M.sup.-1, 10.sup.12 M.sup.-1, or 10.sup.13 M.sup.-1.
"High affinity" binding domains refers to those binding domains
with a K.sub.a of at least 10.sup.7 M.sup.-1, at least 108
M.sup.-1, at least 10.sup.9 M.sup.-1, at least 10.sup.10 M.sup.-1,
at least 10.sup.11 M.sup.1, at least 10.sup.12 M.sup.-1, at least
10.sup.13 M.sup.-1, or greater. Alternatively, affinity may be
defined as an equilibrium dissociation constant (K.sub.d) of a
particular binding interaction with units of M (e.g., 10.sup.-5 M
to 10.sup.-13 M). Affinities of binding domain polypeptides and
fusion proteins according to the present disclosure can be readily
determined using conventional techniques (see, e.g., Scatchard et
al. (1949) Ann. N.Y. Acad. Sci. 51:660; and U.S. Pat. Nos.
5,283,173; 5,468,614, or the equivalent).
[0037] Binding domains of this disclosure can be generated as
described herein or by a variety of methods known in the art (see,
e.g., U.S. Pat. Nos. 6,291,161; 6,291,158). Sources include
antibody gene sequences from various species (which can be
formatted as antibodies, sFvs, scFvs or Fabs, such as in a phage
library), including human, camelid (from camels, dromedaries, or
llamas; Hamers-Casterman et al. (1993) Nature, 363:446 and Nguyen
et al. (1998) J. Mol. Biol., 275:413), shark (Roux et al. (1998)
Proc. Nat'l. Acad. Sci. (USA) 95:11804), fish (Nguyen et al. (2002)
Immunogenetics, 54:39), rodent, avian, ovine, sequences that encode
random peptide libraries or sequences that encode an engineered
diversity of amino acids in loop regions of alternative
non-antibody scaffolds, such as fibrinogen domains (see, e.g.,
Weisel et al. (1985) Science 230:1388), Kunitz domains (see, e.g.,
U.S. Pat. No. 6,423,498), lipocalin domains (see, e.g., WO
2006/095164), V-like domains (see, e.g., US Patent Application
Publication No. 2007/0065431), C-type lectin domains (Zelensky and
Gready (2005) FEBS J. 272:6179), mAb.sup.2 or Fcab.TM. (see, e.g.,
PCT Patent Application Publication Nos. WO 2007/098934; WO
2006/072620), or the like. Additionally, traditional strategies for
hybridoma development using, for example, a synthetic single chain
CD86 as an immunogen in convenient systems (e.g., mice, HuMAb
Mouse.RTM., TC Mouse.TM., KM-Mouse.RTM., llamas, chicken, rats,
hamsters, rabbits, etc.) can be used to develop binding domains of
this disclosure.
[0038] Terms understood by those in the art as referring to
antibody technology are each given the meaning acquired in the art,
unless expressly defined herein. For example, the terms "V.sub.L"
and "V.sub.H" refer to the variable binding region derived from an
antibody light and heavy chain, respectively. The variable binding
regions are made up of discrete, well-defined sub-regions known as
"complementarity determining regions" (CDRs) and "framework
regions" (FRs). The terms "C.sub.L" and "C.sub.H" refer to an
"immunoglobulin constant region," i.e., a constant region derived
from an antibody light or heavy chain, respectively, with the
latter region understood to be further divisible into C.sub.H1,
C.sub.H2, C.sub.H3 and C.sub.H4 constant region domains, depending
on the antibody isotype (IgA, IgD, IgE, IgG, IgM) from which the
region was derived. A portion of the constant region domains makes
up the Fc region (the "fragment crystallizable" region), which
contains domains responsible for the effector functions of an
immunoglobulin, such as ADCC (antibody-dependent cell-mediated
cytotoxicity), CDC (complement-dependent cytotoxicity) and
complement fixation, binding to Fc receptors, greater half-life in
vivo relative to a polypeptide lacking an Fc region, protein A
binding, and perhaps even placental transfer (see Capon et al.
(1989) Nature, 337:525). Further, a polypeptide containing an Fc
region allows for dimerization or multimerization of the
polypeptide. A "hinge region," also referred to herein as a
"linker," is an amino acid sequence interposed between and
connecting the variable binding and constant regions of a single
chain of an antibody, which is known in the art as providing
flexibility in the form of a hinge to antibodies or antibody-like
molecules.
[0039] The domain structure of immunoglobulins is amenable to
engineering, in that the antigen binding domains and the domains
conferring effector functions may be exchanged between
immunoglobulin classes and subclasses. Immunoglobulin structure and
function are reviewed, for example, in Harlow et al., Eds.,
Antibodies: A Laboratory Manual, Chapter 14 (Cold Spring Harbor
Laboratory, Cold Spring Harbor, 1988). An extensive introduction as
well as detailed information about all aspects of recombinant
antibody technology can be found in the textbook Recombinant
Antibodies (John Wiley & Sons, N Y, 1999). A comprehensive
collection of detailed antibody engineering lab Protocols can be
found in R. Kontermann and S. Dubel, Eds., The Antibody Engineering
Lab Manual (Springer Verlag, Heidelberg/New York, 2000). Further
related protocols are also available in Current Protocols in
Immunology (August 2009) published by John Wiley & Sons, Inc.,
Boston, Mass.
[0040] "Derivative" as used herein refers to a chemically or
biologically modified version of a compound that is structurally
similar to a parent compound and (actually or theoretically)
derivable from that parent compound. Generally, a "derivative"
differs from an "analogue" in that a parent compound may be the
starting material to generate a "derivative," whereas the parent
compound may not necessarily be used as the starting material to
generate an "analogue." An analogue may have different chemical or
physical properties of the parent compound. For example, a
derivative may be more hydrophilic or it may have altered
reactivity (e.g., a CDR having an amino acid change that alters its
affinity for a target) as compared to the parent compound.
[0041] The term "biological sample" includes a blood sample, biopsy
specimen, tissue explant, organ culture, biological fluid (e.g.,
serum, urine, CSF) or any other tissue or cell or other preparation
from a subject or a biological source. A subject or biological
source may, for example, be a human or non-human animal, a primary
cell culture or culture adapted cell line including genetically
engineered cell lines that may contain chromosomally integrated or
episomal recombinant nucleic acid sequences, somatic cell hybrid
cell lines, immortalized or immortalizable cell lines,
differentiated or differentiatable cell lines, transformed cell
lines, or the like. In further embodiments of this disclosure, a
subject or biological source may be suspected of having or being at
risk for having a disease, disorder or condition, including a
malignant disease, disorder or condition or a B cell disorder. In
certain embodiments, a subject or biological source may be
suspected of having or being at risk for having a
hyperproliferative, inflammatory, or autoimmune disease, and in
certain other embodiments of this disclosure the subject or
biological source may be known to be free of a risk or presence of
such disease, disorder, or condition.
CD86 Binding Domains
[0042] As set forth herein, CD86 comprises a type I membrane
protein that is a member of the immunoglobulin superfamily. CD86 is
expressed by antigen-presenting cells, and is the ligand for the
two T-cell proteins CD28 and CTLA4. Binding of CD28 with CD28 is a
costimulatory signal for activation of the T-cell, while binding of
CD28 with CTLA4 downregulates T-cell activation and reduces the
immune response. Alternative splicing results in two transcript
variants encoding different isoforms (GenBank.TM. Accessions
NP_787058.3 and NP_008820.2).
[0043] A CD86 binding domain of this disclosure can block binding
of CD86 to CD28 and thereby downregulate T-cell activation. CD86
binding domains contemplated include a CTLA4 extracellular domain,
or sub-domain thereof, a CD28 extracellular domain or sub-domain,
or a CD86-specific antibody-derived binding domain (such as derived
from the FUN1 monoclonal antibody (see e.g., J Pathol. 1993 March;
169(3):309-15); or derived from the 3D1 anti-CD86 monoclonal
antibody.
[0044] In some embodiments, a CD86 binding domain is an
extracellular domain ("ectodomain") of a human CTLA4
(GenBank.TM.Accession NP_005205), such as the mature polypeptide
sequence of SEQ ID NO: 1 (signal peptide: amino acids 1-37). The
amino acid sequence of the CTLA4 ectodomain without the signal
peptide is provided in SEQ ID NO: 410. Applicants note that certain
studies have indicated that the mature polypeptide of the CTLA4
ectodomain begins at the methionine at position 38 of SEQ ID NO: 1,
other studies have indicated that the mature polypeptide begins at
the alanine at position 37. In further embodiments, a CD86 binding
domain is an ectodomain of CTLA4 that has been mutated in order to
have a higher avidity for CD86 than wildtype, or non-mutated, CTLA4
as disclosed, for example, in US Patent Publication No. US
2003/0035816. In certain embodiments, the mutated CTLA4 ectodomain
comprises an alanine or tyrosine at amino acid position 29, and/or
a glutamic acid, asparagine, aspartic acid, glutamine, isoleucine,
leucine or threonine at position 104 of SEQ ID NO:410. The amino
acid sequence for the A29Y L104E CTLA 4 ectodomain variant is
provided in SEQ ID NO:411. In certain embodiments, a CD86 binding
domain is a CTLA-4 variable-like domain, such as the sequence
provided in SEQ ID NO:3, or a CDR of a CTLA-4 variable-like domain,
such as SEQ ID NO: 4 (CDR1), SEQ ID NO:5, (CDR2) or SEQ ID NO:6
(CDR3). Such CDRs are described, for example, in U.S. Pat. No.
7,405,288. In alternative embodiments, a CD86 binding domain is an
extracellular domain ("ectodomain") of a CD28 (GenBank.TM.Accession
NP_006130.1), such as the sequence provided in SEQ ID NO:2. Amino
acids 1-18 of SEQ ID NO:2 are the signal peptide. The amino acid
sequence of the ectodomain of CD28 without the signal peptide is
provided in SEQ ID NO:412.
[0045] In yet further embodiments, a CD86 binding domain comprises
a single chain immunoglobulin-like domain, such as a scFv, that is
specific for CD86. In certain embodiments, the CD86 binding domain
contains the light and heavy variable binding domains from
monoclonal antibody FUN1 or 3D1. The sequences for the heavy chain,
light chain, scFv linker, and CDRs from the FUN1 and 3D1 anti-CD86
monoclonal antibodies are set forth in SEQ NOS:305-313 and 318-326,
respectively, which can be used in the xceptor molecules of the
instant disclosure.
[0046] In one aspect, a CD86 binding domain or fusion protein
thereof of this disclosure is specific for CD86 and has an affinity
with a dissociation constant (K.sub.d) of about 10.sup.-3 M to less
than about 10.sup.-8 M. In certain preferred embodiments, the CD86
binding domain or fusion protein thereof binds CD86 with an
affinity of about 0.3 .mu.M.
[0047] In an illustrative example, CD86 binding domains of this
disclosure can be identified using a Fab phage library of fragments
(see, e.g., Hoet et al. (2005) Nature Biotechnol. 23:344) by
screening for binding to a synthetic or recombinant CD86 (using an
amino acid sequence or fragment thereof as set forth in
GenBank.TM.Accession No. NP 787058.3 or NP_008820.2). In certain
embodiments, a CD86 molecule used to generate a CD86 binding domain
can further comprise an intervening domain or a dimerization
domain, as described herein, such as an immunoglobulin Fc domain or
fragment thereof.
[0048] In some embodiments, CD86 binding domains of this disclosure
comprise V.sub.H and V.sub.L domains as described herein (e.g.,
FUN1, 3D1, or humanized derivatives thereof). Other exemplary
V.sub.H and V.sub.L domains include those described in U.S. Pat.
No. 6,827,934. In certain embodiments, the V.sub.H and V.sub.L
domains are human. In further embodiments, there are provided CD86
binding domains of this disclosure that have a sequence that is at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99%, at least 99.5%, or at least 100% identical to the amino acid
sequence of one or more light chain variable regions (V.sub.L) or
to one or more heavy chain variable regions (V.sub.H), or both, of
SEQ NOS:305 and 306, SEQ NOS:318 and 319, or those disclosed in
U.S. Pat. No. 6,827,934 (incorporated herein by reference), wherein
each CDR can have zero, one, two, or three amino acid changes
(i.e., most changes are in the framework region(s)).
[0049] The terms "identical" or "percent identity," in the context
of two or more polypeptide or nucleic acid molecule sequences,
means two or more sequences or subsequences that are the same or
have a specified percentage of amino acid residues or nucleotides
that are the same over a specified region (e.g., 60%, 65%, 70%,
75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or
100% identity), when compared and aligned for maximum
correspondence over a comparison window, or designated region, as
measured using methods known in the art, such as a sequence
comparison algorithm, by manual alignment, or by visual inspection.
For example, preferred algorithms suitable for determining percent
sequence identity and sequence similarity are the BLAST and BLAST
2.0 algorithms, which are described in Altschul et al. (1977)
Nucleic Acids Res. 25:3389 and Altschul et al. (1990) J. Mol. Biol.
215:403, respectively.
[0050] In any of these or other embodiments described herein where
V.sub.L and V.sub.H domains may be desired, the V.sub.L and V.sub.H
domains may be arranged in either orientation and may be separated
by up to about a 30 amino acid linker as disclosed herein or any
amino acid sequence capable of providing a spacer function
compatible with interaction of the two sub-binding domains. In
certain embodiments, a linker joining the V.sub.H and V.sub.L
domains comprises an amino acid sequence as set forth in any one or
more of SEQ ID NOS: 43-166, 244, 307, 320, 355-379 and 383-398,
such as Linker 46 (SEQ ID NO:88), Linker 130 (SEQ ID NO:163),
Linker 131 (SEQ ID NO:164), Linker 115 (SEQ ID NO:148), or the
linker provided in SEQ ID NO:244. Multi-specific binding domains
will have at least two specific sub-binding domains, by analogy to
camelid antibody organization, or at least four specific
sub-binding domains, by analogy to the more conventional mammalian
antibody organization of paired V.sub.H and V.sub.L chains.
[0051] CDRs are defined in various ways in the art, including the
Kabat, Chothia, AbM, and contact definitions. The Kabat definition
is based on sequence variability and is the most commonly used
definition to predict CDR regions (Johnson et al. (2000) Nucleic
Acids Res. 28:214). The Chothia definition is based on the location
of the structural loop regions (Chothia et al. (1986) J. Mol. Biol.
196:901; Chothia et al. (1989) Nature 342:877). The AbM definition,
a compromise between the Kabat and Chothia definitions, is an
integral suite of programs for antibody structure modeling produced
by the Oxford Molecular Group (Martin et al. (1989) Proc. Nat'l.
Acad. Sci. (USA) 86:9268; Rees et al., ABM.TM., a computer program
for modeling variable regions of antibodies, Oxford, UK; Oxford
Molecular, Ltd.). An additional definition, known as the contact
definition, has been recently introduced (see MacCallum et al.
(1996) J. Mol. Biol. 5:732), which is based on analysis of
available complex crystal structures.
[0052] By convention, the CDR domains in the heavy chain are
referred to as H1, H2, and H3, which are numbered sequentially in
order moving from the amino terminus to the carboxy terminus. The
CDR-H1 is about ten to 12 residues in length and starts four
residues after a Cys according to the Chothia and AbM definitions,
or five residues later according to the Kabat definition. The H1
can be followed by a Trp, Trp-Val, Trp-Ile, or Trp-Ala. The length
of H1 is approximately ten to 12 residues according to the AbM
definition, while the Chothia definition excludes the last four
residues. The CDR-H2 starts 15 residues after the end of H1
according to the Kabat and AbM definitions, which is generally
preceded by sequence Leu-Glu-Trp-Ile-Gly (but a number of
variations are known) and is generally followed by sequence
Lys/Arg-Leu/Ile/Val/Phe/Thr/Ala-Thr/Ser/lle/Ala. According to the
Kabat definition, the length of H2 is about 16 to 19 residues,
while the AbM definition predicts the length to be nine to 12
residues. The CDR-H3 usually starts 33 residues after the end of
H2, is generally preceded by the amino acid sequence Cys-Ala-Arg
and followed by the amino acid Gly, and has a length that ranges
from three to about 25 residues.
[0053] By convention, CDR regions in the light chain are referred
to as L1, L2, and L3, which are numbered sequentially in order
moving from the amino terminus to the carboxy terminus. The CDR-L1
(approximately ten to 17 residues in length) generally starts at
about residue 24 and generally follows a Cys. The residue after the
CDR-L1 is always Trp, which begins one of the following sequences:
Trp-Tyr-Gln, Trp-Leu-Gln, Trp-Phe-Gln, or Trp-Tyr-Leu. The CDR-L2
(about seven residues in length) starts about 16 residues after the
end of L1 and will generally follow residues Ile-Tyr, Val-Tyr,
Ile-Lys, or Ile-Phe. The CDR-L3 usually starts 33 residues after
the end of L2 and generally follows a Cys, which is generally
followed by the sequence Phe-Gly-XXX-Gly and has a length of about
seven to 11 residues.
[0054] Thus, a binding domain of this disclosure can comprise a
single CDR from a variable region of an anti-CD86 antibody, or it
can comprise multiple CDRs that can be the same or different. In
certain embodiments, binding domains of this disclosure comprise
V.sub.H and V.sub.L domains specific for a CD86 comprising
framework regions and CDR1, CDR2 and CDR3 regions, wherein (a) the
V.sub.H domain comprises an amino acid sequence of a heavy chain
CDR3; or (b) the V.sub.L domain comprises an amino acid sequence of
a light chain CDR3; or (c) the binding domain comprises a V.sub.H
amino acid sequence of (a) and a V.sub.L amino acid sequence of
(b); or the binding domain comprises a V.sub.H amino acid sequence
of (a) and a V.sub.L amino acid sequence of (b) and wherein the
V.sub.H and V.sub.L are found in the same reference sequence. In
further embodiments, binding domains of this disclosure comprise
V.sub.H and V.sub.L domains specific for an CD86 comprising
framework regions and CDR1, CDR2 and CDR3 regions, wherein (a) the
V.sub.H domain comprises an amino acid sequence of a heavy chain
CDR1, CDR2, and CDR3; or (b) the V.sub.L domain comprises an amino
acid sequence of a light chain CDR1, CDR2, and CDR3; or (c) the
binding domain comprises a V.sub.H amino acid sequence of (a) and a
V.sub.L amino acid sequence of (b); or the binding domain comprises
a V.sub.H amino acid sequence of (a) and a V.sub.L amino acid
sequence of (b), wherein the V.sub.H and V.sub.L amino acid
sequences are from the same reference sequence.
[0055] In any of the embodiments described herein comprising
specific CDRs, a binding domain can comprise (i) a V.sub.H domain
having an amino acid sequence that is at least 80%, 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino
acid sequence of a V.sub.H domain; or (ii) a V.sub.L domain having
an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid
sequence of a V.sub.L domain; or (iii) both a V.sub.H domain of (i)
and a V.sub.L domain of (ii); or both a V.sub.H domain of (i) and a
V.sub.L domain of (ii) wherein the V.sub.H and V.sub.L are from the
same reference sequence, wherein each CDR has up to three amino
acid changes (i.e., many of the changes are in the framework
region(s)).
[0056] A CD86 binding domain in xceptor fusion proteins of this
disclosure may be an immunoglobulin-like domain, such as an
immunoglobulin scaffold. Immunoglobulin scaffolds contemplated by
this disclosure include a scFv, a domain antibody, or a heavy
chain-only antibody. In a scFv, this disclosure contemplates the
heavy and light chain variable regions are joined by any linker
peptide described herein or known in the art to be compatible with
joining domains or regions in a binding molecule. Exemplary linkers
are linkers based on the Gly.sub.4Ser linker motif, such as
(Gly.sub.4Ser).sub.n, where n=1-5. If a binding domain of a fusion
protein of this disclosure is based on a non-human immunoglobulin
or includes non-human CDRs, the binding domain may be "humanized"
according to methods known in the art.
[0057] Alternatively, a CD86 binding domain of fusion proteins of
this disclosure may be a scaffold other than an immunoglobulin
scaffold. Other scaffolds contemplated by this disclosure present
the CD86-specific CDR(s) in a functional conformation. Other
scaffolds contemplated include, but are not limited an A domain
molecule, a fibronectin III domain, an anticalin, an ankyrin-repeat
engineered binding molecule, an adnectin, a Kunitz domain or a
protein AZ domain affibody.
IL10
[0058] As noted above, in certain embodiments the present
disclosure provides polypeptides containing a binding region or
domain that is an IL10 agonist (i.e., can increase IL10 signaling).
In some embodiments, the IL10 agonist binding domain is an IL10 or
a IL10Fc, or a functional sub-domain thereof. In other embodiments,
the IL10 agonist binding domain is a single chain binding protein,
such as an scFv, that specifically binds to IL10R1 or IL10R2. In
some embodiments, the IL10 agonist binding domain is an IL10
containing a point mutation at position 87 of SEQ ID NO:7, such as
from "I" to "A" or "S" (referred to herein as I87A or I87S,
respectively). The I87 variant IL10 molecules are known to be less
immuno-stimulatory compared to wildtype IL10 (Ding et al., J. Exp.
Med. 191:213, 2000). Additionally, IL10 normally forms a homodimer
with the amino terminal domain of each monomer molecule binding to
the carboxy terminal domain of the other monomer). In one
embodiment, the IL10 agonist binding domain is an IL10 molecule
having a short linker (gggsgg SEQ ID NO:379) that separates the two
subdomains of the molecule (amino and carboxy end domains) so that
these subdomains can form an intramolecular dimer was also
examined. These are referred to herein as monoIL10 molecules.
[0059] IL10 (GenBank.TM.Accession no. NP_000563.1; SEQ ID NO:7) is
a member of a cytokine superfamily that share an alpha-helical
structure. Amino acids 1-18 of SEQ ID NO:7 are the signal peptide
of the precursor IL10 protein. The amino acid sequence of the
mature IL10 protein is provided in SEQ ID NO:418. Although no
empirical evidence exists, it has been suggested that all the
family members possess six alpha-helices (Fickenscher, H. et al.,
(2002) Trends Immunol. 23: 89). IL10 has four cysteines, only one
of which is conserved among family members. Since IL10 demonstrates
a V-shaped fold that contributes to its dimerization, it appears
that disulfide bonds are not critical to this structure. Amino acid
identity of family members to IL10 ranges from 20% (IL-19) to 28%
(IL-20) (Dumouter et al., (2002) Eur. Cytokine Netw. 13:5).
[0060] IL10 was first described as a Th2 cytokine in mice that
inhibited IFN-.alpha. and GM-CSF cytokine production by Th1 cells
(Moore et al., 2001, Annu. Rev. Immunol. 19:683; Fiorentino et al.,
(1989) J. Exp. Med. 170: 2081). Human IL10 is 178 amino acids in
length with an 18 amino acid signal sequence and a 160 amino acid
mature segment. Its molecular weight is approximately 18 kDa
(monomer). Human IL10 contains no potential N-linked glycosylation
site and is not glycosylated (Dumouter et al., (2002) Eur. Cytokine
Netw. 13:5; Vieira et al., (1991) Proc. Natl. Acad. Sci. USA
88:1172). It contains four cysteine residues that form two
intrachain disulfide bonds. Helices A-->D of one monomer
noncovalently interact with helices E and F of a second monomer,
forming a noncovalent V-shaped homodimer. Functional areas have
been mapped on the IL10 molecule. In the N-terminus, pre-helix A
residues #1-9 are involved in mast cell proliferation, while in the
C-terminus, helix F residues #152-160 mediate leukocyte secretion
and chemotaxis.
[0061] Cells known to express IL10 include CD8+ T cells, microglia,
CD14+(but not CD16+) monocytes, Th2 CD4+ cells (mice),
keratinocytes, hepatic stellate cells, Th1 and Th2 CD4+ T cells
(human), melanoma cells, activated macrophages, NK cells, dendritic
cells, B cells (CD5+ and CD19+) and eosinophils. On T cells, the
initial observation of IL10 inhibition of IFN-gamma production is
now suggested to be an indirect effect mediated by accessory cells.
Additional effects on T cells, however, include: IL10 induced CD8+
T cell chemotaxis, an inhibition of CD4+ T cell chemotaxis towards
IL-8, suppression of IL-2 production following activation, an
inhibition of T cell apoptosis via Bcl-2 up-regulation, and an
interruption of T cell proliferation following low antigen exposure
accompanied by CD28 costimulation (Akdis et al., (2001) Immunology
103:131).
[0062] On B cells, IL10 has a number of related, yet distinct
functions. In conjunction with TNF-.beta. and CD40L, IL10 induces
IgA production in naive (IgD+) B cells. It is believed that
TGF-.beta./CD40L promotes class switching while IL10 initiates
differentiation and growth. When TGF-.beta. is not present, IL10
cooperates with CD40L in inducing IgG1 and IgG3 (human), and thus
may be a direct switch factor for IgG subtypes. Interestingly, IL10
has divergent effects on IL-4 induced IgE secretion. If IL10 is
present at the time of IL-4 induced class switching, it reverses
the effect; if it is present after IgE commitment, it augments IgE
secretion. Finally, CD27/CD70 interaction in the presence of IL10
promotes plasma cell formation from memory B cells (Agematsu et
al., (1998) Blood 91: 173).
[0063] Mast cells and NK cells are also impacted by IL10. On mast
cells, IL10 induces histamine release while blocking GM-CSF and
TNF-.alpha. release. This effect may be autocrine as IL10 is known
to be released by mast cells in rat. As evidence of its
pleiotrophic nature, IL10 has the opposite effects on NK cells.
Rather than blocking TNF-.alpha. and GM-CSF production, IL10
actually promotes this function on NK cells. In addition, it
potentiates IL-2 induced NK cell proliferation and facilitates
IFN-.gamma. secretion in NK cells primed by IL-18. In concert with
both IL-12 and/or IL-18, IL10 potentiates NK cell cytotoxicity (Cai
et al., 1999, Eur. J. Immunol. 29: 2658).
[0064] IL10 has a pronounced anti-inflammatory impact on
neutrophils. It inhibits the secretion of the chemokines
MIP-1.alpha., MIP-1.beta. and IL-8, and blocks production of the
proinflammatory mediators IL-1.beta. and TNF-.alpha.. In addition,
it decreases the ability of neutrophils to produce superoxide, and
as a result interferes with PMN-mediated antibody-dependent
cellular cytotoxicity. It also blocks IL-8 and fMLP-induced
chemotaxis, possibly via CXCR1 (Vicioso et al., (1998) Eur.
Cytokine Netw. 9:247).
[0065] On dendritic cells (DCs), IL10 generally exhibits
immunosuppressive effects. It would appear to promote CD14+
macrophage differentiation at the expense of DCs. IL10 seems to
decrease the ability of DCs to stimulate T cells, particularly for
Th1 type cells. Relative to MHC-II expression, it can be
down-regulated, unchanged, or up-regulated (Sharma et al., (1999)
J. Immunol. 163:5020). With respect to CD80 and CD86, IL10 will
either up-regulate or down-regulate its expression. B7-2/CD86 plays
a key role in T cell activation. For this molecule, IL10 is
involved in both up-regulation and down-regulation. Perhaps the
most significant modulation, however, occurs with CD40 (IL10 seems
to reduce its expression). At the regional level, IL10 may block
immunostimulation by inhibiting Langerhans cell migration in
response to proinflammatory cytokines. Alternatively, IL10 blocks
an inflammation-induced DC maturation step that normally involves
CCR1, CCR2 and CCR5 down-regulation and CCR7 up-regulation. This
blockage, with retention of CCR1, CCR2 and CCR5, results in a
failure of DCs to migrate to regional nodes. The result is an
immobile DC that will not stimulate T cells but will bind (and
clear) proinflammatory chemokines without responding to them
(D-Amico et al., (2000) Nat. Immunol. 1:387).
[0066] On monocytes, IL10 has a number of documented effects. For
example, IL10 seems to clearly reduce cell surface MHC-II
expression. It also inhibits IL-12 production following
stimulation. While it promotes a monocyte to macrophage transition
in conjunction with M-CSF, the phenotype of the macrophage is not
clear (i.e. CD16+/cytotoxic vs. CD16-). IL10 also reduces monocyte
GM-CSF secretion and IL-8 production, while promoting IL-1ra
release (Gesser et al., (1997) Proc. Natl. Acad. Sci. USA
94:14620). Hyaluronectin, a connective tissue component, is now
known to be secreted by monocytes in response to IL10. This may
have some importance in cell migration, particularly tumor cell
metastases, where hyaluronectin is known to interrupt cell
migration through extracellular space (Gesser et al., (1997) Proc.
Natl. Acad. Sci. USA 94:14620).
[0067] Fusion proteins of IL10 with either murine or macaque Fc
regions (referred to as IL10Fc) have been shown to inhibit
macrophage function and prolong pancreatic islet xenograft survival
(Feng et al. (1999) Transplantation 68:1775; Asiedu et al. (2007)
Cytokine 40:183), as well as reduce septic shock in a murine model
(Zheng et al. (1995) J. Immunol. 154:5590).
[0068] Human IL10R1 is a 90-110 kDa, single-pass type I
transmembrane glycoprotein that is expressed on a limited number of
cell types (Liu et al., 1994, J. Immunol. 152: 1821). Weak
expression being seen in pancreas, skeletal muscle, brain, heart,
and kidney. Placenta, lung, and liver showed intermediate levels.
Monocytes, B-cells, large granular lymphocytes, and T-cells express
high levels (Liu et al., 1994, J. Immunol. 152: 1821). The
expressed protein is a 578 amino acid protein that contains a 21
amino acid signal peptide, a 215 amino acid extracellular region, a
25 amino acid transmembrane segment, and a 317 amino acid
cytoplasmic domain. There are two FNIII motifs within the
extracellular region and a STAT3 docking site plus a JAK1
association region within the cytoplasmic domain (Kotenko et al.,
2000 Oncogene 19: 2557; Kotenko et al., 1997, EMBO J. 16: 5894).
IL10R1 binds human IL10 with a Kd of 200 pM.
[0069] In some embodiments, binding domains of this disclosure
comprise V.sub.L and V.sub.H domains specific for an IL10R1 or
IL10R2 as described herein. In certain embodiments, the V.sub.L and
V.sub.H domains are human. The V.sub.L and V.sub.H domains may be
arranged in either orientation and may be separated by up to about
a 30 amino acid linker as disclosed herein or any other amino acid
sequence capable of providing a spacer function compatible with
interaction of the two sub-binding domains. In certain embodiments,
a linker joining the V.sub.L and V.sub.H domains comprises an amino
acid sequence as set forth in SEQ ID NOs:43-166, 244, 307, 320,
355-379 and 383-398, such as the linker provided in SEQ ID NO:244,
Linker 46 (SEQ ID NO:88), Linker 130 (SEQ ID NO:163), or Linker 131
(SEQ ID NO:164). Multi-specific binding domains can have at least
two specific sub-binding domains, by analogy to camelid antibody
organization, or at least four specific sub-binding domains, by
analogy to the more conventional mammalian antibody organization of
paired V.sub.L and V.sub.H chains. In further embodiments, binding
domains specific for IL10R1 or IL10R2 of this disclosure may
comprise one or more complementarity determining region ("CDR"), or
multiple copies of one or more such CDRs, which have been obtained,
derived, or designed from variable regions of an anti-IL10R1 or
IL10R2 scFv or Fab fragment or from heavy or light chain variable
regions thereof. Thus, a binding domain of this disclosure can
comprise a single CDR from a variable region of an anti-IL10R1 or
IL10R2, or it can comprise multiple CDRs that can be the same or
different. In certain embodiments, binding domains of this
disclosure comprise V.sub.L and V.sub.H domains specific for an
IL10R1 or IL10R2 comprising framework regions and CDR1, CDR2 and
CDR3 regions.
HLA-G
[0070] As noted above, in certain embodiments the present
disclosure provides polypeptides containing a binding region or
domain that is an HLA-G agonist (i.e., can increase HLA-G
signaling). In some embodiments, the HLA-G agonist binding domain
is an HLA-G1 (SEQ ID NO: 14), an HLA-G5 (SEQ ID NO: 15) or an HLA-G
mutein in which the cysteine at position of 147 of the mature
protein has been mutated to an alternative amino acid, for example
a serine. Amino acids 1-24 and 1-23 of HLA-G1 and HLA-G5,
respectively, represent the signal peptides. In other embodiments,
the HLA-G agonist domain is an ectodomain of HLA-G1 or HLA-G5,
either with or without a 0-2 microglobulin attached to the
N-terminus by a flexible linker. Examples of such linkers include
those provided in SEQ ID NOs:43-166, 244, 307, 320, 355-379 and
383-398 and described below. The preparation of soluble HLA-G1 is
described in US Patent Publication no. US 2004/0044182.
[0071] In yet other embodiments, the HLA-G agonist binding domain
is an immunoglobulin variable binding domain, or a derivative
thereof (e.g., an antibody, Fab, scFv, or the like) that
specifically binds to ILT2, ILT4 or KIR2DL4. Antibodies that are
specific for ILT2, ILT4 or KIR2DL4 include, for example, those
described in US Patent Publication no. US 2003/0232051.
[0072] Human leukocyte antigen G (HLA-G) is a nonclassical major
histocompatability complex (MHC) class I molecule that is a
heterodimer consisting of a heavy chain and a light chain (beta-2
microglobulin), with the heavy chain being anchored in the
membrane. HLA-G functions as an immunomodulatory molecule that
protects fetal tissues from the maternal immune system. While
constitutive expression of HLA-G is limited to fetal tissues, adult
thymic medulla, cornea, pancreatic islets and erythroid and
endothelial cell precursors, its expression can be induced in
cancers, transplantation, multiple sclerosis, inflammatory diseases
and viral infections. The HLA-G primary transcript generates seven
alternative mRNAs that encode the membrane-bound protein isoforms
HLA-G1, -G2, -G3 and -G4, and the soluble protein isoforms HLA-G5,
HLA-G6 and HLA-G7, with HLA-G5 being the soluble form of the cell
surface-bound HLA-G1 protein.
[0073] While HLA-G does not seem to have significant immune
stimulatory functions, it has been shown to bind to inhibitory
receptors, namely ILT2, ILT4, KIR2DL4 and CD8, and thereby interact
with B-cells, T-cells, NK cells and antigen-presenting cells.
Dimeric forms of HLA-G have an affinity for ILT2 that is several
orders of magnitude greater than the affinity for ILT4, KIR2DL4 or
CD8. HLA-G1 has been shown to inhibit the cytolytic function of
uterine and peripheral blood NK cells, the antigen-specific
cytolytic function of cytotoxic T lymphocytes, the
alloproliferative response of CD4+ T-cells, the proliferation of
T-cells and peripheral blood NK cells, and the maturation and
function of dendritic cells (see, for example, Wiendl et al. (2003)
Blood, 126:176-185). It has been suggested that HLA-G may be useful
in reducing inflammatory responses in the CNS associated with
multiple sclerosis (Wiendl et al. (2005) Blood, 128:2689-2704), and
as a therapeutic agent in promoting tolerance to grafts in
transplantations (Carosella et al. (2008) Blood 111:4862-4870).
[0074] In some embodiments, binding domains of this disclosure
comprise V.sub.L and V.sub.H domains specific for a ILT2, ILT4 or
KIR2DL4 as described herein. In certain embodiments, the V.sub.L
and V.sub.H domains are human. The V.sub.L and V.sub.H domains may
be arranged in either orientation and may be separated by up to
about a 30 amino acid linker as disclosed herein or any other amino
acid sequence capable of providing a spacer function compatible
with interaction of the two sub-binding domains. In certain
embodiments, a linker joining the V.sub.L and V.sub.H domains
comprises an amino acid sequence as set forth in any one or more of
SEQ ID NOs:43-166, 244, 307, 320, 355-379 and 383-398, such as
Linker 115 (SEQ ID NO:148), the linker provided in SEQ ID NO:244,
Linker 46 (SEQ ID NO:88), Linker 130 (SEQ ID NO:163), or Linker 131
(SEQ ID NO:164). Multi-specific binding domains can have at least
two specific sub-binding domains, by analogy to camelid antibody
organization, or at least four specific sub-binding domains, by
analogy to the more conventional mammalian antibody organization of
paired V.sub.L and V.sub.H chains.
[0075] In further embodiments, binding domains specific for ILT2,
ILT4 or KIR2DL4 of this disclosure may comprise one or more
complementarity determining region ("CDR"), or multiple copies of
one or more such CDRs, which have been obtained, derived, or
designed from variable regions of an anti-ILT2, -ILT4 or -KIR2DL4
scFv or Fab fragment or from heavy or light chain variable regions
thereof. Thus, a binding domain of this disclosure can comprise a
single CDR from a variable region of an anti-ILT2, -ILT4 or
-KIR2DL4, or it can comprise multiple CDRs that can be the same or
different.
HGF
[0076] As noted above, in certain embodiments the present
disclosure provides polypeptides containing a binding region or
domain that is an HGF agonist (i.e., can increase HGF signaling).
In some embodiments, the HGF agonist binding domain is an HGF or a
functional sub-domain thereof.
[0077] Hepatocyte growth factor (HGF) regulates cell growth, cell
motility, and morphogenesis by activating a tyrosine kinase
signaling cascade after binding to the proto-oncogenic c-Met
receptor. HGF influences a number of cell types and regulates
various biological activities including cytokine production, cell
migration, proliferation and survival. HGF is secreted as a single
inactive polypeptide and is cleaved by serine proteases into a
69-kDa alpha-chain and 34-kDa beta-chain. A disulfide bond between
the alpha and beta chains produces the active, heterodimeric
molecule. Alternative splicing of the HGF gene gives rise to five
different isoforms (isoforms 1-5; GenBank.TM. Accession nos.
NP_000592.3, NP_001010931.1, NP_001010932.1, NP_001010933.1 and
NP_001010934.1, respectively; SEQ ID NOs: 18-22; amino acids 1-31
of each of these sequences is the signal peptide).
[0078] HGF is believed to be a key factor in the prevention and
attenuation of disease progression (Ito et al. (2008) Int. Arch.
Allergy Immunol. 146 Suppl 1:82-87). For example, HGF has been
shown to be effective in suppressing collagen-induced arthritis in
mice (Okunishi et al. (2007) Jnl. Immunol. 179:5504-5513), and to
play a protective role in a mouse model of allergic airway
inflammation (Okunishi et al. (2005) Jnl. Immunol. 175:4745-4753;
Ito et al. Am. J. Respir. Cell. Mol. Biol. (2005) 32:268-280).
[0079] In some embodiments, binding domains of this disclosure
comprise V.sub.L and V.sub.H domains specific for HGF as described
herein. In certain embodiments, the V.sub.L and V.sub.H domains are
human. The V.sub.L and V.sub.H domains may be arranged in either
orientation and may be separated by up to about a 30 amino acid
linker as disclosed herein or any other amino acid sequence capable
of providing a spacer function compatible with interaction of the
two sub-binding domains. In certain embodiments, a linker joining
the V.sub.L and V.sub.H domains comprises an amino acid sequence as
set forth in any one or more of SEQ ID NOs:43-166, 244, 307, 320,
355-379 and 383-398, such as the linker provided in SEQ ID NO:244,
Linker 46 (SEQ ID NO:88), Linker 130 (SEQ ID NO:163), or Linker 131
(SEQ ID NO:164). Multi-specific binding domains can have at least
two specific sub-binding domains, by analogy to camelid antibody
organization, or at least four specific sub-binding domains, by
analogy to the more conventional mammalian antibody organization of
paired V.sub.L and V.sub.H chains. In further embodiments, binding
domains specific for HGF of this disclosure may comprise one or
more complementarity determining region ("CDR"), or multiple copies
of one or more such CDRs, which have been obtained, derived, or
designed from variable regions of an anti-HGF scFv or Fab fragment
or from heavy or light chain variable regions thereof. Thus, a
binding domain of this disclosure can comprise a single CDR from a
variable region of an anti-HGF, or it can comprise multiple CDRs
that can be the same or different. In certain embodiments, binding
domains of this disclosure comprise V.sub.L and V.sub.H domains
specific for an HGF comprising framework regions and CDR1, CDR2 and
CDR3 regions.
IL35
[0080] As noted above, in certain embodiments the present
disclosure provides polypeptides containing a binding region or
domain that is an IL35 agonist (i.e., can increase IL35 signaling).
In some embodiments, the IL35 agonist binding domain is an IL35
(e.g. SEQ ID NO: 25 and 26) or a functional sub-domain thereof. In
certain embodiments, the IL35 agonist binding domain is a single
chain polypeptide comprising the sequences of SEQ ID NO: 25 and 26,
or functional sub-domains thereof. Such single chain polypeptides
may include one or more linkers, including linkers as described
herein. In other embodiments, the IL35 agonist binding domain is a
single chain immunoglobulin variable domain, such as a scFv,
specific for IL35R that has IL35 agonist activity.
[0081] IL-35 is a newly described cytokine of the IL-12 cytokine
subfamily. The heterodimeric molecule is comprised of the IL-12 p35
and the IL-27 Ebi3 subunits. It has recently been shown to be a
potent inducer of Treg function and capable of altering a TH17
response in a mouse model of arthritis (Niedbala et al. (2007) Eur.
J. Immunol. 37:3021; Collison et al. (2007) Nature 450:566).
Therefore, combining IL-35 agonism with CD86 inhibition is
predicted to increase the therapeutic benefit of CD28 inhibition
alone.
[0082] Regulatory T-cells (TREGS) are a critical sub-population of
CD4+ T cells that are important for maintaining self tolerance and
preventing autoimmunity, for limiting chronic inflammatory
diseases, such as asthma and inflammatory bowel disease, and for
regulating homeostatic lymphocyte expansion. IL35 is an
anti-inflammatory cytokine that has been shown to suppress immune
responses by stimulating expansion of regulatory T cells and
suppression of Th17 cell development (Collison et al. (2007) Nature
450:566-9). IL35 is a heterodimer formed from Epstein-Barr
virus-induced gene 3 (EBI3; SEQ ID NO: 25; signal peptide: amino
acids 1-20) and the p35subunit of IL12 (SEQ ID NO: 26; signal
peptide: amino acids 1-56) (Devergne et al. (1997) Proc. Natl.
Acad. Sci. USA 94:12041-12046; U.S. Pat. No. 5,830,451; US Patent
Publication no. US 2007/0299026). It has been shown to have a
therapeutic effect equivalent to that of Enbrel.TM. in a murine
collagen-induced arthritis model (Niedbala et al. (2007) Eur. J.
Immunol. 37:3021-3029), and has thus been proposed as a therapeutic
agent against clinical rheumatoid arthritis.
[0083] In some embodiments, binding domains of this disclosure
comprise V.sub.L and V.sub.H domains specific for an IL35R as
described herein. In certain embodiments, the V.sub.L and V.sub.H
domains are human. The V.sub.L and V.sub.H domains may be arranged
in either orientation and may be separated by up to about a 30
amino acid linker as disclosed herein or any other amino acid
sequence capable of providing a spacer function compatible with
interaction of the two sub-binding domains. In certain embodiments,
a linker joining the V.sub.L and V.sub.H domains comprises an amino
acid sequence as set forth in any one or more of SEQ ID NOs:43-166,
244, 307, 320, 355-379 and 383-398, such as the linker provided in
SEQ ID NO:244, Linker 46 (SEQ ID NO:88), Linker 130 (SEQ ID
NO:163), or Linker 131 (SEQ ID NO:164). Multi-specific binding
domains can have at least two specific sub-binding domains, by
analogy to camelid antibody organization, or at least four specific
sub-binding domains, by analogy to the more conventional mammalian
antibody organization of paired V.sub.L and V.sub.H chains. In
further embodiments, binding domains specific for IL35R of this
disclosure may comprise one or more complementarity determining
region ("CDR"), or multiple copies of one or more such CDRs, which
have been obtained, derived, or designed from variable regions of
an anti-IL35R scFv or Fab fragment or from heavy or light chain
variable regions thereof. Thus, a binding domain of this disclosure
can comprise a single CDR from a variable region of an anti-IL35R,
or it can comprise multiple CDRs that can be the same or different.
In certain embodiments, binding domains of this disclosure comprise
V.sub.L and V.sub.H domains specific for an IL-35R comprising
framework regions and CDR1, CDR2 and CDR3 regions.
Light
[0084] As noted above, in certain embodiments the present
disclosure provides polypeptides containing a binding region or
domain that is a LIGHT antagonist (i.e., can inhibit LIGHT
signaling). In some embodiments, the LIGHT antagonist binding
domain is an HVEM ectodomain (also referred to as sHVEM; SEQ ID NO:
29; signal peptide: amino acids 1-38) or a functional sub-domain
thereof. In other embodiments, the LIGHT antagonist binding domain
is a single chain immunoglobulin-like variable domain, such as a
scFv, specific for LIGHT. In certain embodiments, the LIGHT
antagonist domain is a single chain immunoglobulin-like variable
domain comprising V.sub.H and V.sub.L domains as described in PCT
Patent Publication no. WO 08/027338.
[0085] LIGHT is a member of the TNF superfamily that is expressed
on activated T lymphocytes, monocytes, granulocytes and immature
dendritic cells. Two distinct isoforms of LIGHT have been reported
(GenBank.TM. Accession nos. NP_003798.2 and NP_742011.1). LIGHT has
been shown to regulate T cell immune responses by signaling through
the herpes virus entry mediator (HVEM) and the lymphotoxin beta
receptor (LT.beta.R). Both HVEM and LT.beta.R bind LIGHT with high
affinity, with expression of HVEM being detected in T cells, B
cells, natural killer cells and endothelial cells, and LT.beta.R
being expressed in monocytes and stromal cells but not T cells and
B cells. LIGHT has been shown to be a co-stimulatory molecule for
CD28-independent T cell activation and to preferentially induce
IFN-.gamma. and GM-CSF production. Blockade of LIGHT by in vivo
administration of LT.beta.R-Ig fusion protein or anti-LIGHT
antibodies results in decreased T cell-mediated immune responses
and ameliorates graft-versus-host disease in a murine model (Tamada
et al. (2000) Nat. Med. 6:283-9). Constitutive expression of LIGHT
leads to tissue destruction and autoimmune-like disease syndromes
(Granger & Rickert (2003) Cytokine Growth Factor Rev.
14:289-96).
[0086] In some embodiments, binding domains of this disclosure
comprise V.sub.L and V.sub.H domains specific for LIGHT as
described herein. In certain embodiments, the V.sub.L and V.sub.H
domains are human. The V.sub.L and V.sub.H domains may be arranged
in either orientation and may be separated by up to about a 30
amino acid linker as disclosed herein or any other amino acid
sequence capable of providing a spacer function compatible with
interaction of the two sub-binding domains. In certain embodiments,
a linker joining the V.sub.L and V.sub.H domains comprises an amino
acid sequence as set forth in any one or more of SEQ ID NOs:43-166,
244, 307, 320, 355-379 and 383-398, such as the linker provided in
SEQ ID NO:244, Linker 46 (SEQ ID NO:88), Linker 130 (SEQ ID
NO:163), or Linker 131 (SEQ ID NO:164). Multi-specific binding
domains can have at least two specific sub-binding domains, by
analogy to camelid antibody organization, or at least four specific
sub-binding domains, by analogy to the more conventional mammalian
antibody organization of paired V.sub.L and V.sub.H chains.
[0087] In further embodiments, binding domains specific for LIGHT
of this disclosure may comprise one or more complementarity
determining region ("CDR"), or multiple copies of one or more such
CDRs, which have been obtained, derived, or designed from variable
regions of an anti-LIGHT scFv or Fab fragment or from heavy or
light chain variable regions thereof. Thus, a binding domain of
this disclosure can comprise a single CDR from a variable region of
an anti-LIGHT, or it can comprise multiple CDRs that can be the
same or different. In certain embodiments, binding domains of this
disclosure comprise V.sub.L and V.sub.H domains specific for a
LIGHT comprising framework regions and CDR1, CDR2 and CDR3
regions.
PD-1
[0088] As noted above, in certain embodiments the present
disclosure provides polypeptides containing a binding region or
domain that is a PD-1 agonist (i.e., can increase PD-1 signaling).
In some embodiments, the PD-1 agonist binding domain is a PD1-L1
(e.g. SEQ ID NO: 32; signal peptide: amino acids 1-18), a PD1-L2
(e.g. SEQ ID NO: 33; signal peptide: amino acids 1-19), or a
functional sub-domain thereof. In other embodiments, the PD-1
agonist binding domain is a single chain immunoglobulin-like
variable domain, such as a scFv, specific for PD-1. Antibodies
specific for PD-1 include, for example, those described in US
Patent Publication No. US 2006/0210567.
[0089] PD-1 (GenBank.TM. Accession NP_005009.1) is a member of the
CD28/CTLA4 family that is expressed on activated T cells, B cells
and myeloid cells. PD-1 contains an immunoreceptor tyrosine-based
inhibitory motif. PD-1 functions by binding to programmed death-1
ligand 1 (PD1-L1; also known as CD274) and programmed death-1
ligand 2 (PD1-L2). Human PD-L1 and PD-L2 are expressed on both
immature and mature dendritic cells, IFN.gamma.-treated monocytes
and follicular dendritic cells. Mice deficient in PD-1 show a
variety of autoimmune pathologies, demonstrating that PD-1 is a
negative regulator of the immune response (Nishimura & Honjo
(2001) Trends Immunol. 2:265; Nishimura et al. (1999) Immunity
11:141). Binding of PD-1 to PD1-L1 and PD1-L2 has been shown to
result in down-regulation of T cell activation (Freeman et al.
(2000) J. Exp. Med. 192:1027; Latchman et al. (2001) Nat. Immunol.
2:261; Carter et al. (2002) Eur. J. Immunol. 32:634).
[0090] In some embodiments, binding domains of this disclosure
comprise V.sub.L and V.sub.H domains specific for a PD-1 as
described herein. In certain embodiments, the V.sub.L and V.sub.H
domains are human. The V.sub.L and V.sub.H domains may be arranged
in either orientation and may be separated by up to about a 30
amino acid linker as disclosed herein or any other amino acid
sequence capable of providing a spacer function compatible with
interaction of the two sub-binding domains. In certain embodiments,
a linker joining the V.sub.L and V.sub.H domains comprises an amino
acid sequence as set forth in SEQ ID NOs:43-166, 244, 307, 320,
355-379 and 383-398, such as the linker provided in SEQ ID NO:244,
Linker 46 (SEQ ID NO:88), Linker 130 (SEQ ID NO:163), or Linker 131
(SEQ ID NO:164). Multi-specific binding domains can have at least
two specific sub-binding domains, by analogy to camelid antibody
organization, or at least four specific sub-binding domains, by
analogy to the more conventional mammalian antibody organization of
paired V.sub.L and V.sub.H chains.
[0091] In further embodiments, binding domains specific for PD-1 of
this disclosure may comprise one or more complementarity
determining region ("CDR"), or multiple copies of one or more such
CDRs, which have been obtained, derived, or designed from variable
regions of an anti-PD-1 scFv or Fab fragment or from heavy or light
chain variable regions thereof. Thus, a binding domain of this
disclosure can comprise a single CDR from a variable region of an
anti-PD-1, or it can comprise multiple CDRs that can be the same or
different.
BTLA
[0092] As noted above, in certain embodiments the present
disclosure provides polypeptides containing a binding region or
domain that is a BTLA agonist (i.e., can increase BTLA signaling).
In some embodiments, the BTLA agonist binding domain is a HVEM
ectodomain (also referred to as sHVEM; SEQ ID NO: 29; signal
peptide: amino acids 1-38) or a functional sub-domain thereof (e.g.
amino acids 54-78 of SEQ ID NO: 29). In other embodiments, the BTLA
agonist binding domain is a single chain immunoglobulin-like
variable domain, such as a scFv, specific for BTLA. Agonist
antibodies specific for BTLA are described, for example, in Krieg
et al. (2005) J. Immunol. 175:6420-6472.
[0093] BTLA (GenBank.TM. Accession nos. NP_001078826.1 and
NP_861445.3; isoforms 2 and 1, respectively) is a cell surface
protein that is a member of the immunoglobulin family and is
expressed on B-cells, T-cells and antigen presenting cells. The
ligand for BTLA is herpes virus entry mediator (HVEM), which is a
member of the tumor-necrosis factor receptor family and also acts
as a ligand for LIGHT (Sedy et al. (2005) Nat. Immunol. 6:90-98). A
binding site for BTLA has been identified in CRD1 of HVEM (amino
acids 54-78 of SEQ ID NO: 29; PCT Patent Publication no. WO
2006/063067). This site is distinct from that occupied by LIGHT but
overlaps the gD binding site of HVEM. While binding of HVEM to
LIGHT induces a strong immune response, binding of HVEM to BTLA
results in negative regulation of T cell responses (Murphy et al.
(2006) Nat. Rev. Immunol. 6:671-681). It has been indicated that
binding of BTLA to HVEM activates tyrosine phosphorylation of BTLA
thereby inducing association with the protein tyrosine phosphatases
SHP-1 and SHP-2 (Gavrieli et al. (2003) Biochem. Biophys. Res.
Commun. 312:1236), although some data question whether SHP
recruitment is responsible for the negative regulatory activity of
BTLA (Chemnitz et al. (2006) J. Immunol. 176:6603-6614).
[0094] Soluble HVEM has been shown to inhibit anti-CD3-induced
proliferation of CD4+ T cells, with this effect being reversed by
anti-BTLA antibodies (Gonzalez et al. (2005) Proc. Natl. Acad. Sci.
USA 102:1116-1121). Similarly, an agonistic anti-BTLA monoclonal
antibody was shown to inhibit anti-CD3-mediated CD4+ T-cell
proliferation and cytokine production (Krieg et al. (2005) J.
Immunol. 175:6420-6472). Mice lacking an intact BTLA gene show an
increased sensitivity to experimental autoimmune encephalomyelitis,
(Watanabe et al. (2003) Nat. Immunol. 4:670-679) and prolonged
airway inflammation (Deppong et al. (2006) J. Immunol.
176:3909-3913).
[0095] In some embodiments, binding domains of this disclosure
comprise V.sub.L and V.sub.H domains specific for a BTLA as
described herein. In certain embodiments, the V.sub.L and V.sub.H
domains are human. The V.sub.L and V.sub.H domains may be arranged
in either orientation and may be separated by up to about a 30
amino acid linker as disclosed herein or any other amino acid
sequence capable of providing a spacer function compatible with
interaction of the two sub-binding domains. In certain embodiments,
a linker joining the V.sub.L and V.sub.H domains comprises an amino
acid sequence as set forth in SEQ ID NOs:43-166, 244, 307, 320,
355-379 and 383-398, such as the linker provided in SEQ ID NO:244,
Linker 46 (SEQ ID NO:88), Linker 130 (SEQ ID NO:163), or Linker 131
(SEQ ID NO:164). Multi-specific binding domains can have at least
two specific sub-binding domains, by analogy to camelid antibody
organization, or at least four specific sub-binding domains, by
analogy to the more conventional mammalian antibody organization of
paired V.sub.L and V.sub.H chains.
[0096] In further embodiments, binding domains specific for BTLA of
this disclosure may comprise one or more complementarity
determining region ("CDR"), or multiple copies of one or more such
CDRs, which have been obtained, derived, or designed from variable
regions of an anti-BTLA scFv or Fab fragment or from heavy or light
chain variable regions thereof. Thus, a binding domain of this
disclosure can comprise a single CDR from a variable region of an
anti-BTLA, or it can comprise multiple CDRs that can be the same or
different.
GITRL
[0097] As noted above, in certain embodiments the present
disclosure provides polypeptides containing a binding region or
domain that is a GITRL antagonist (i.e., can inhibit GITRL
signaling). In some embodiments, the GITRL antagonist binding
domain is a GITR ectodomain (also referred to as sGITR; SEQ ID NO:
39 and 40; signal peptides: amino acids 1-25 for each of these
sequences) or a functional sub-domain thereof. In other
embodiments, the GITRL antagonist binding domain is a single chain
immunoglobulin-like variable domain, such as a scFv, specific for
GITRL. Antagonistic antibodies against GITRL are described, for
example, in US Patent Publication No. 2005/0014224.
[0098] Glucocorticoid-induced tumor necrosis factor receptor (GITR;
also known as AITR), a type I transmembrane protein, is a member of
the TNF receptor superfamily (Nocentini et al. (2007) Eur. J
Immunol. 37:1165-69). GITR plays an important role in the
regulation of T cell proliferation and TCR-mediated apoptosis. GITR
expression is upregulated on T cells, with a high level of GITR
being constitutive expressed on CD4+CD25.sup.+ regulatory T cells
(Kwon et al. (2003) Exp. Mol. Med. 35:8-16), with expression also
occurring on macrophages, B cells and NK cells (Liu et al. (2008)
J. Biol. Chem. 283:8202-8210). GITR's cognate ligand, GITRL is
constitutively expressed on antigen-presenting cells, such as
dendritic cells and B cells. Binding of GITR to GITRL has been
shown to render CD4.sup.+CD25.sup.- effector T cells resistant to
the inhibitory effects of CD4.sup.+CD25.sup.+ regulatory T cells.
GITR activation by either GITRL or an agonistic antibody has been
shown to increase TCR-induced T cell proliferation and cytokine
production, and to rescue T cells from anti-CD3-induced apoptosis
(Nocentini et al. (1997) Proc. Natl. Acad. Sci. USA 94:6216-6221).
In addition, binding of GITR to GITRL can inhibit T regulatory
cells and/or render effector T cells more resistant to T regulatory
cell-mediated suppression (Kanamaru et al. (2004) J. Immunol.
172:7306-7314).
[0099] Studies have shown that administration of anti-GITR mAb
during the induction phase of experimental autoimmune
encephalomyelitis significantly enhances the severity of clinical
disease as well as increasing CNS inflammation and autoreactive T
cell responses (Kohm et al. (2004) J. Immunol. 172:4686-4690). In
addition, activation of GITR signaling exacerbates both murine
asthma and collagen-induced arthritis (Patel et al. (2005) Eur. J.,
Immunol. 35:3581-90).
[0100] In some embodiments, binding domains of this disclosure
comprise V.sub.L and V.sub.H domains specific for GITRL as
described herein. In certain embodiments, the V.sub.L and V.sub.H
domains are human. The V.sub.L and V.sub.H domains may be arranged
in either orientation and may be separated by up to about a 30
amino acid linker as disclosed herein or any other amino acid
sequence capable of providing a spacer function compatible with
interaction of the two sub-binding domains. In certain embodiments,
a linker joining the V.sub.L and V.sub.H domains comprises an amino
acid sequence as set forth in SEQ ID NOs:43-166, 244, 307, 320,
355-379 and 383-398, such as the linker provided in SEQ ID NO:244,
Linker 46 (SEQ ID NO:88), Linker 130 (SEQ ID NO:163), or Linker 131
(SEQ ID NO:164). Multi-specific binding domains can have at least
two specific sub-binding domains, by analogy to camelid antibody
organization, or at least four specific sub-binding domains, by
analogy to the more conventional mammalian antibody organization of
paired V.sub.L and V.sub.H chains.
[0101] In further embodiments, binding domains specific for GITRL
of this disclosure may comprise one or more complementarity
determining region ("CDR"), or multiple copies of one or more such
CDRs, which have been obtained, derived, or designed from variable
regions of an anti-GITRL scFv or Fab fragment or from heavy or
light chain variable regions thereof. Thus, a binding domain of
this disclosure can comprise a single CDR from a variable region of
an anti-GITRL, or it can comprise multiple CDRs that can be the
same or different. In certain embodiments, binding domains of this
disclosure comprise V.sub.L and V.sub.H domains specific for a
GITRL comprising framework regions and CDR1, CDR2 and CDR3
regions.
CD40
[0102] As noted above, in certain embodiments the present
disclosure provides polypeptides containing a binding region or
domain that is a CD40 antagonist (i.e., can inhibit CD40
signaling). In some embodiments, the CD40 antagonist binding domain
is a single chain immunoglobulin-like variable domain, such as a
scFv, specific for CD40. Antagonistic antibodies against CD40 are
described, for example in US Patent Publication no. US
2008/0057070, and U.S. Pat. Nos. 5,874,082 and 6,838,261.
[0103] CD40 is a 55 kDa cell-surface antigen found on the surface
of normal and neoplastic B cells, dendritic cells, antigen
presenting cells, endothelial cells, monocytic cells and epithelial
cells. CD40 expression on antigen presenting cells plays an
important co-stimulatory role in the action of T-helper and
cytotoxic T lymphocytes. Expression of the CD40 ligand (CD40L, also
known as CD154) is upregulated on T cells during a normal immune
response. Binding of T cell expressed CD40L to B cell expressed
CD40 leads to B cell proliferation and differentiation, antibody
production, isotope switching and B-cell memory generation. A human
anti-CD40 antagonistic antibody has been shown to have antileukemia
activity on human chronic lymphocytic leukemia cells (Luqman et al.
(2008) Blood 112:711-720).
[0104] In some embodiments, binding domains of this disclosure
comprise V.sub.L and V.sub.H domains specific for CD40 as
described, for example, in US Patent Publication no. US
2008/0057070. In certain embodiments, the V.sub.L and V.sub.H
domains are human. The V.sub.L and V.sub.H domains may be arranged
in either orientation and may be separated by up to about a 30
amino acid linker as disclosed herein or any other amino acid
sequence capable of providing a spacer function compatible with
interaction of the two sub-binding domains. In certain embodiments,
a linker joining the V.sub.L and V.sub.H domains comprises an amino
acid sequence as set forth in SEQ ID NOs:43-166, 244, 307, 320,
355-379 and 383-398, such as the linker provided in SEQ ID NO:244,
Linker 46 (SEQ ID NO:88), Linker 130 (SEQ ID NO:163), or Linker 131
(SEQ ID NO:164). Multi-specific binding domains can have at least
two specific sub-binding domains, by analogy to camelid antibody
organization, or at least four specific sub-binding domains, by
analogy to the more conventional mammalian antibody organization of
paired V.sub.L and V.sub.H chains.
[0105] In further embodiments, binding domains specific for CD40 of
this disclosure may comprise one or more complementarity
determining region ("CDR"), or multiple copies of one or more such
CDRs, which have been obtained, derived, or designed from variable
regions of an anti-CD40 scFv or Fab fragment or from heavy or light
chain variable regions thereof. Thus, a binding domain of this
disclosure can comprise a single CDR from a variable region of an
anti-CD40, or it can comprise multiple CDRs that can be the same or
different. In certain embodiments, binding domains of this
disclosure comprise V.sub.L and V.sub.H domains specific for a CD40
comprising framework regions and CDR1, CDR2 and CDR3 regions as
described, for example, in US Patent Publication no. US
2008/0057070.
Multi-Specific Fusion Proteins
[0106] The present disclosure provides multi-specific fusion
proteins comprising a domain that binds to a CD86 ("CD86 binding
domain") and a domain that binds a molecule other than a CD86
("heterologous binding domain"). In certain embodiments, the
heterologous binding domain is an IL-10 agonist, an HLA-G agonist,
an HGF agonist, an IL-35 agonist, a PD-1 agonist, a BTLA agonist, a
LIGHT antagonist, a GITRL antagonist or a CD40 antagonist.
[0107] In certain embodiments, the heterologous binding domain is
an IL10 agonist, such as IL10, IL10Fc or a single chain binding
domain that specifically binds to IL10R1 or IL10R2. In certain
embodiments, the heterologous binding domain is an HLA-G agonist,
such as an HLA-G1, an HLA-G5, an HLA-G mutein, or a functional
region thereof (such as an ectodomain), or a single chain binding
domain that specifically binds to ILT2, ILT4 or KIR2DL4. In certain
embodiments, the heterologous binding domain is an HGF agonist,
such as an HGF or a sub-domain thereof. In certain embodiments, the
heterologous binding domain is an IL35 agonist, such as an IL35 or
a sub-domain thereof, a single chain IL35 or subdomain thereof, or
a single chain immunoglobulin-like variable domain specific for
IL35R and having IL35 agonist activity. In certain embodiments, the
heterologous binding domain is a LIGHT antagonist, such as a HVEM
ectodomain or a sub-domain thereof, or a single chain
immunoglobulin-like variable domain specific for LIGHT. In certain
embodiments, the heterologous binding domain is a PD-1 agonist,
such as a PD1-L1, PD1-L2 or a sub-domain thereof, or a single chain
immunoglobulin-like variable domain specific for PD-1. In certain
embodiments, the heterologous binding domain is a BTLA agonist,
such as a HVEM ectodomain or a sub-domain thereof, or a single
chain immunoglobulin-like variable domain specific for BTLA. In
certain embodiments, the heterologous binding domain is a GITRL
antagonist, such as a GITR ectodomain or a sub-domain thereof, or a
single chain immunoglobulin-like variable domain specific for
GITRL. In certain embodiments, the heterologous binding domain is a
CD40 antagonist, such as a single chain immunoglobulin-like
variable domain specific for CD40.
[0108] Generally, the fusion proteins of the present invention make
use of mature proteins that do not include the leader peptide
(signal peptide). Accordingly, while certain sequences provided
herein for binding domain proteins (such as for CTLA4, CD28, HLA-G1
and HLA-G5 and others described herein) include the leader peptide,
the skilled person would readily understand how to determine the
mature protein sequence from sequences including a signal peptide.
In certain embodiments, it may be useful to include the leader
sequence.
[0109] It is contemplated that a CD86 binding domain may be at the
amino-terminus and the heterologous binding domain at the
carboxy-terminus of a fusion protein. In certain embodiments, the
xceptor molecule is as set forth in SEQ ID NO:9, 13, 17, 24, 28,
31, 35, 42, 171, 173, 175, 177, 179, 181, 187, 189, 191, 193, 219,
221, 223, 237, 262, 302, 330, 336, 338, 340, or 400. It is also
contemplated that the heterologous binding domain may be at the
amino-terminus and the CD86 binding domain may be at the
carboxy-terminus. In certain embodiments, the xceptor molecule is
as set forth in SEQ ID NO: 183, 185, 199, 201, 203, 205, 207, 211,
213, 254, 258, 266, 276, 350, 352, or 354. As set forth herein, the
binding domains of this disclosure may be fused to each end of an
intervening domain (e.g., an immunoglobulin constant region or
sub-region thereof). Furthermore, the two or more binding domains
may be each joined to an intervening domain via a linker, as
described herein.
[0110] As used herein, an "intervening domain" refers to an amino
acid sequence that simply functions as a scaffold for one or more
binding domains so that the fusion protein will exist primarily
(e.g., 50% or more of a population of fusion proteins) or
substantially (e.g., 90% or more of a population of fusion
proteins) as a single chain polypeptide in a composition. For
example, certain intervening domains can have a structural function
(e.g., spacing, flexibility, rigidity) or biological function
(e.g., an increased half-life in plasma, such as in human blood).
Exemplary intervening domains that can increase half-life of the
fusion proteins of this disclosure in plasma include albumin,
transferrin, a scaffold domain that binds a serum protein, or the
like, or fragments thereof.
[0111] In certain embodiments, the intervening domain contained in
a multi-specific fusion protein of this disclosure is a
"dimerization domain," which refers to an amino acid sequence that
is capable of promoting the association of at least two single
chain polypeptides or proteins via non-covalent or covalent
interactions, such as by hydrogen bonding, electrostatic
interactions, Van der Waal's forces, disulfide bonds, hydrophobic
interactions, or the like, or any combination thereof. Exemplary
dimerization domains include immunoglobulin heavy chain constant
regions or sub-regions. It should be understood that a dimerization
domain can promote the formation of dimers or higher order multimer
complexes (such as trimers, tetramers, pentamers, hexamers,
septamers, octamers, etc.).
[0112] A "constant sub-region" is a term defined herein to refer to
a peptide, polypeptide, or protein sequence that corresponds to or
is derived from part or all of one or more constant region domains,
but not all constant region domains of a source antibody. In a
preferred embodiment, the constant sub-region is an IgG CH2CH3,
preferably an IgG1 CH2CH3. In some embodiments, the constant region
domains of a fusion protein of this disclosure may lack or have
minimal effector functions of antibody-dependent cell-mediated
cytotoxicity (ADCC) and complement activation and
complement-dependent cytotoxicity (CDC), while retaining the
ability to bind some Fc receptors (such as FcRn binding) and
retaining a relatively long half life in vivo. In certain
embodiments, a binding domain of this disclosure is fused to a
human IgG1 constant region or sub-region, wherein the IgG1 constant
region or sub-region has one or more of the following amino acids
mutated: leucine at position 234 (L234), leucine at position 235
(L235), glycine at position 237 (G237), glutamate at position 318
(E318), lysine at position 320 (K320), lysine at position 322
(K322), or any combination thereof (numbering according to Kabat).
For example, any one or more of these amino acids can be changed to
alanine. In a further embodiment, an IgG1 Fc domain has each of
L234, L235, G237, E318, K320, and K322 (according to EU numbering)
mutated to an alanine (i.e., L234A, L235A, G237A, E318A, K320A, and
K322A, respectively), and optionally an N297A mutation as well
(i.e., essentially eliminating glycosylation of the CH2
domain).
[0113] Methods are known in the art for making mutations inside or
outside an Fc domain that can alter Fc interactions with Fc
receptors (CD16, CD32, CD64, CD89, Fc.epsilon.R1, FcRn) or with the
complement component C1q (see, e.g., U.S. Pat. No. 5,624,821;
Presta (2002) Curr. Pharma. Biotechnol. 3:237). Particular
embodiments of this disclosure include compositions comprising
immunoglobulin or fusion proteins that have a constant region or
sub-region from human IgG wherein binding to FcRn and protein A are
preserved and wherein the Fc domain no longer interacts or
minimally interacts with other Fc receptors or C1q. For example, a
binding domain of this disclosure can be fused to a human IgG1
constant region or sub-region wherein the asparagine at position
297 (N297 under the Kabat numbering) has been mutated to another
amino acid to reduce or eliminate glycosylation at this site and,
therefore, abrogate efficient Fc binding to Fc.gamma.R and C1q.
Another exemplary mutation is a P331S, which diminishes C1q binding
but does not affect Fc binding.
[0114] In further embodiments, an immunoglobulin Fc region may have
an altered glycosylation pattern relative to an immunoglobulin
reference sequence. For example, any of a variety of genetic
techniques may be employed to alter one or more particular amino
acid residues that form a glycosylation site (see Co et al. (1993)
Mol. Immunol. 30:1361; Jacquemon et al. (2006) J. Thromb. Haemost.
4:1047; Schuster et al. (2005) Cancer Res. 65:7934; Warnock et al.
(2005) Biotechnol. Bioeng. 92:831), such as N297 of the CH2 domain
(EU numbering). Alternatively, the host cells producing fusion
proteins of this disclosure may be engineered to produce an altered
glycosylation pattern. One method known in the art, for example,
provides altered glycosylation in the form of bisected,
non-fucosylated variants that increase ADCC. The variants result
from expression in a host cell containing an
oligosaccharide-modifying enzyme. Alternatively, the Potelligent
technology of BioWa/Kyowa Hakko is contemplated to reduce the
fucose content of glycosylated molecules according to this
disclosure. In one known method, a CHO host cell for recombinant
immunoglobulin production is provided that modifies the
glycosylation pattern of the immunoglobulin Fc region, through
production of GDP-fucose.
[0115] Alternatively, chemical techniques are used to alter the
glycosylation pattern of fusion proteins of this disclosure. For
example, a variety of glycosidase and/or mannosidase inhibitors
provide one or more of desired effects of increasing ADCC activity,
increasing Fc receptor binding, and altering glycosylation pattern.
In certain embodiments, cells expressing a multispecific fusion
protein of the instant disclosure (containing a CD86 antagonist
domain linked to an IL-10 agonist, an HLA-G agonist, an HGF
agonist, an IL-35 agonist, a PD-1 agonist, a BTLA agonist, a LIGHT
antagonist, a GITRL antagonist or a CD40 antagonist) are grown in a
culture medium comprising a carbohydrate modifier at a
concentration that increases the ADCC of immunoglycoprotein
molecules produced by said host cell, wherein said carbohydrate
modifier is at a concentration of less than 800 .mu.M. In a
preferred embodiment, the cells expressing these multispecific
fusion proteins are grown in a culture medium comprising
castanospermine or kifunensine, more preferably castanospermine at
a concentration of 100-800 .mu.M, such as 100 .mu.M, 200 .mu.M, 300
.mu.M, 400 .mu.M, 500 .mu.M, 600 .mu.M, 700 .mu.M, or 800 .mu.M.
Methods for altering glycosylation with a carbohydrate modifier
such as castanospermine are provided in US Patent Application
Publication No. 2009/0041756 or PCT Publication No. WO
2008/052030.
[0116] In another embodiment, the immunoglobulin Fc region may have
amino acid modifications that affect binding to effector cell Fc
receptors. These modifications can be made using any technique
known in the art, such as the approach disclosed in Presta et al.
(2001) Biochem. Soc. Trans. 30:487. In another approach, the Xencor
XmAb.TM. technology is available to engineer constant sub-regions
corresponding to Fc domains to enhance cell killing effector
function (see Lazar et al. (2006) Proc. Nat'l. Acad. Sci. (USA)
103:4005). Using this approach, for example, one can generate
constant sub-regions with improved specificity and binding for
FC.gamma.R, thereby enhancing cell killing effector function.
[0117] In still further embodiments, a constant region or
sub-region can optionally increase plasma half-life or placental
transfer in comparison to a corresponding fusion protein lacking
such an intervening domain. In certain embodiments, the extended
plasma half-life of a fusion protein of this disclosure is at least
two, at least three, at least four, at least five, at least ten, at
least 12, at least 18, at least 20, at least 24, at least 30, at
least 36, at least 40, at least 48 hours, at least several days, at
least a week, at least two weeks, at least several weeks, at least
a month, at least two months, at least several months, or more in a
human.
[0118] A constant sub-region may include part or all of any of the
following domains: a C.sub.H2 domain, a CH.sub.3 domain (IgA, IgD,
IgG, IgE, or IgM), and a C.sub.H4 domain (IgE or IgM). A constant
sub-region as defined herein, therefore, can refer to a polypeptide
that corresponds to a portion of an immunoglobulin constant region.
The constant sub-region may comprise a C.sub.H2 domain and a
C.sub.H3 domain derived from the same, or different,
immunoglobulins, antibody isotypes, or allelic variants. In some
embodiments, the C.sub.H3 domain is truncated and comprises a
carboxy-terminal sequence listed in U.S. patent application Ser.
No. 12/041,590 (which is a CIP of PCT/US2007/071052) as SEQ ID
NOS:366-371. In certain embodiments, a constant sub-region of a
polypeptide of this disclosure has a C.sub.H2 domain and C.sub.H3
domain, which may optionally have an amino-terminal linker, a
carboxy-terminal linker, or a linker at both ends.
[0119] A "linker" is a peptide that joins or links other peptides
or polypeptides, such as a linker of about 2 to about 150 amino
acids. In fusion proteins of this disclosure, a linker can join an
intervening domain (e.g., an immunoglobulin-derived constant
sub-region) to a binding domain or a linker can join two variable
regions of a binding domain, or two regions within a single chain
polypeptide formed from a heterodimeric molecule, such as EBI3 (SEQ
ID NO: 25) and the p35subunit of IL12 (SEQ ID NO: 26) of IL35. For
example, a linker can be an amino acid sequence obtained, derived,
or designed from an antibody hinge region sequence, a sequence
linking a binding domain to a receptor, or a sequence linking a
binding domain to a cell surface transmembrane region or membrane
anchor. In some embodiments, a linker can have at least one
cysteine capable of participating in at least one disulfide bond
under physiological conditions or other standard peptide conditions
(e.g., peptide purification conditions, conditions for peptide
storage). In certain embodiments, a linker corresponding or similar
to an immunoglobulin hinge peptide retains a cysteine that
corresponds to the hinge cysteine disposed toward the
amino-terminus of that hinge. In further embodiments, a linker is
from an IgG1 or IgG2A hinge and has one cysteine or two cysteines
corresponding to hinge cysteines. In certain embodiments, one or
more disulfide bonds are formed as inter-chain disulfide bonds
between intervening domains. In other embodiments, fusion proteins
of this disclosure can have an intervening domain fused directly to
a binding domain (i.e., absent a linker or hinge). In some
embodiments, the intervening domain is a dimerization domain, such
as an IgG1 CH2CH3 Fc portion.
[0120] The intervening or dimerization domain of multi-specific
fusion proteins of this disclosure may be connected to one or more
terminal binding domains by a peptide linker. In addition to
providing a spacing function, a linker can provide flexibility or
rigidity suitable for properly orienting the one or more binding
domains of a fusion protein, both within the fusion protein and
between or among the fusion proteins and their target(s). Further,
a linker can support expression of a full-length fusion protein and
stability of the purified protein both in vitro and in vivo
following administration to a subject in need thereof, such as a
human, and is preferably non-immunogenic or poorly immunogenic in
those same subjects. In certain embodiments, a linker of an
intervening or a dimerization domain of multi-specific fusion
proteins of this disclosure may comprise part or all of a human
immunoglobulin hinge.
[0121] Additionally, a binding domain may comprise a V.sub.H and a
V.sub.L domain, and these variable region domains may be combined
by a linker. Exemplary variable region binding domain linkers
include those belonging to the (Gly.sub.nSer) family, such as
(Gly.sub.3Ser).sub.n(Gly.sub.4Ser).sub.1,
(Gly.sub.3Ser).sub.1(Gly.sub.4Ser).sub.n,
(Gly.sub.3Ser).sub.n(Gly.sub.4Ser).sub.n, or (Gly.sub.4Ser).sub.n,
wherein n is an integer of 1 to 5 (see, e.g., Linkers 22, 29, 46,
89, 90, 116, 130, and 131 corresponding to SEQ ID NOS:64, 71, 88,
131, 132, 149, 163 and 164, respectively). In preferred
embodiments, these (Gly.sub.4Ser)-based linkers are used to link
variable domains and are not used to link a binding domain (e.g.,
scFv) to an intervening domain (e.g., an IgG CH2CH3).
[0122] Exemplary linkers that can be used to join an intervening
domain (e.g., an immunoglobulin-derived constant sub-region) to a
binding domain or a linker that can join two variable regions of a
binding domain are listed in SEQ ID NOS:43-166, 244, 307, 320,
355-379 and 383-398.
[0123] Linkers contemplated in this disclosure include, for
example, peptides derived from any inter-domain region of an
immunoglobulin superfamily member (e.g., an antibody hinge region)
or a stalk region of C-type lectins, a family of type II membrane
proteins. These linkers range in length from about two to about 150
amino acids, or about two to about 40 amino acids, or about eight
to about 20 amino acids, preferably about ten to about 60 amino
acids, more preferably about 10 to about 30 amino acids, and most
preferably about 15 to about 25 amino acids. For example, Linker 1
is two amino acids in length and Linker 116 is 36 amino acids in
length (Linkers 1-133 are provided in SEQ ID NOS:43-166,
respectively; additional exemplary linkers are provided in SEQ ID
NOS:244, 307, 320, 355-379, and 383-398).
[0124] Beyond general length considerations, a linker suitable for
use in the fusion proteins of this disclosure includes an antibody
hinge region selected from an IgG hinge, IgA hinge, IgD hinge, IgE
hinge, or variants thereof. In certain embodiments, a linker may be
an antibody hinge region (upper and core region) selected from
human IgG1, human IgG2, human IgG3, human IgG4, or fragments or
variants thereof. As used herein, a linker that is an
"immunoglobulin hinge region" refers to the amino acids found
between the carboxyl end of CH1 and the amino terminal end of CH2
(for IgG, IgA, and IgD) or the amino terminal end of CH3 (for IgE
and IgM). A "wild type immunoglobulin hinge region," as used
herein, refers to a naturally occurring amino acid sequence
interposed between and connecting the CH1 and CH2 regions (for IgG,
IgA, and IgD) or interposed between and connecting the CH2 and CH3
regions (for IgE and IgM) found in the heavy chain of an antibody.
In preferred embodiments, the wild type immunoglobulin hinge region
sequences are human.
[0125] According to crystallographic studies, an IgG hinge domain
can be functionally and structurally subdivided into three regions:
the upper hinge region, the core or middle hinge region, and the
lower hinge region (Shin et al. (1992) Immunological Reviews
130:87). Exemplary upper hinge regions include EPKSCDKTHT (SEQ ID
NO:383) as found in IgG1, ERKCCVE (SEQ ID NO:384) as found in IgG2,
ELKTPLGDTTHT (SEQ ID NO:385) or EPKSCDTPPP (SEQ ID NO:386) as found
in IgG3, and ESKYGPP (SEQ ID NO:387) as found in IgG4. Exemplary
middle hinge regions include CPPCP (SEQ ID NO:398) as found in IgG1
and IgG2, CPRCP (SEQ ID NO:388) as found in IgG3, and CPSCP (SEQ ID
NO:389) as found in IgG4. While IgG1, IgG2, and IgG4 antibodies
each appear to have a single upper and middle hinge, IgG3 has four
in tandem--one of ELKTPLGDTTHTCPRCP (SEQ ID NO:390) and three of
EPKSCDTPPPCPRCP (SEQ ID NO:391).
[0126] IgA and IgD antibodies appear to lack an IgG-like core
region, and IgD appears to have two upper hinge regions in tandem
(see SEQ ID NOS:392 and 393). Exemplary wild type upper hinge
regions found in IgA1 and IgA2 antibodies are set forth in SEQ ID
NOS: 394 and 395, respectively.
[0127] IgE and IgM antibodies, in contrast, instead of a typical
hinge region have a CH2 region with hinge-like properties.
Exemplary wild-type CH2 upper hinge-like sequences of IgE and IgM
are set forth in SEQ ID NO:396 (VCSRDFTPPT VKILQSSSDG GGHFPPTIQL
LCLVSGYTPG TINITWLEDG QVMDVDLSTA STTQEGELAS TQSELTLSQK HWLSDRTYTC
QVTYQGHTFE DSTKKCA) and SEQ ID NO:397 (VIAELPPKVS VFVPPRDGFF
GNPRKSKLIC QATGFSPRQI QVSWLREGKQ VGSGVTTDQV QAEAKESGPT TYKVTSTLTI
KESDWLGQSM FTCRVDHRGL TFQQNASSMC VP), respectively.
[0128] An "altered wild type immunoglobulin hinge region" or
"altered immunoglobulin hinge region" refers to (a) a wild type
immunoglobulin hinge region with up to 30% amino acid changes
(e.g., up to 25%, 20%, 15%, 10%, or 5% amino acid substitutions or
deletions), (b) a portion of a wild type immunoglobulin hinge
region that is at least 10 amino acids (e.g., at least 12, 13, 14
or 15 amino acids) in length with up to 30% amino acid changes
(e.g., up to 25%, 20%, 15%, 10%, or 5% amino acid substitutions or
deletions), or (c) a portion of a wild type immunoglobulin hinge
region that comprises the core hinge region (which portion may be
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, or at least 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length). In
certain embodiments, one or more cysteine residues in a wild type
immunoglobulin hinge region may be substituted by one or more other
amino acid residues (e.g., one or more serine residues). An altered
immunoglobulin hinge region may alternatively or additionally have
a proline residue of a wild type immunoglobulin hinge region
substituted by another amino acid residue (e.g., a serine
residue).
[0129] Alternative hinge and linker sequences that can be used as
connecting regions may be crafted from portions of cell surface
receptors that connect IgV-like or IgC-like domains. Regions
between IgV-like domains where the cell surface receptor contains
multiple IgV-like domains in tandem and between IgC-like domains
where the cell surface receptor contains multiple tandem IgC-like
regions could also be used as connecting regions or linker
peptides. In certain embodiments, hinge and linker sequences are
from five to 60 amino acids long, and may be primarily flexible,
but may also provide more rigid characteristics, and may contain
primarily an .alpha.-helical structure with minimal .beta.-sheet
structure. Preferably, sequences are stable in plasma and serum and
are resistant to proteolytic cleavage. In some embodiments,
sequences may contain a naturally occurring or added motif such as
CPPC (SEQ ID NO:422) that confers the capacity to form a disulfide
bond or multiple disulfide bonds to stabilize the C-terminus of the
molecule. In other embodiments, sequences may contain one or more
glycosylation sites. Examples of hinge and linker sequences include
interdomain regions between the IgV-like and IgC-like or between
the IgC-like or IgV-like domains of CD2, CD4, CD22, CD33, CD48,
CD58, CD66, CD80, CD86, CD96, CD150, CD166, and CD244. Alternative
hinges may also be crafted from disulfide-containing regions of
Type II receptors from non-immunoglobulin superfamily members such
as CD69, CD72, and CD161.
[0130] In certain embodiments, a linker of the present invention
comprises a scorpion linker. Scorpion linkers include peptides
derived from interdomain regions of an immunoglobulin superfamily
member, e.g., hinge-like peptides derived from immunoglobulin hinge
regions, such as IgG1, IgG2, IgG3, IgG4, IgA, and IgE hinge
regions. In certain embodiments, a hinge-like scorpion linker will
retain at least one cysteine capable of forming an interchain
disulfide bond under physiological conditions. Scorpion linkers
derived from IgG1 may have 1 cysteine or two cysteines, and may
retain the cysteine corresponding to an N-terminal hinge cysteine
of wild-type IgG1. Non-hinge-like peptides are also contemplated as
scorpion linkers, provided that such peptides provide sufficient
spacing and flexibility to provide a single-chain protein capable
of forming two binding domains, one located towards each protein
terminus (N and C) relative to a more centrally located constant
sub-region domain. Exemplary non-hinge-like scorpion linkers
include peptides from the stalk region of C-type lectin stalk
regions of Type II membrane proteins, such as the stalk regions of
CD69, CD72, CD94, NKG2A and NKG2D. In some embodiments, the
scorpion linker comprises a sequence selected from the group
consisting of SEQ ID NOs:355-359 and 365.
[0131] In some embodiments, a linker has a single cysteine residue
for formation of an interchain disulfide bond. In other
embodiments, a linker has two cysteine residues for formation of
interchain disulfide bonds. In further embodiments, a linker is
derived from an immunoglobulin interdomain region (e.g., an
antibody hinge region) or a Type II C-type lectin stalk region
(derived from a Type II membrane protein; see, e.g., exemplary
lectin stalk region sequences set forth in of PCT Application
Publication No. WO 2007/146968, such as SEQ ID NOS:111, 113, 115,
117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 149, 151, 153,
155, 157, 159, 161, 163, 165, 167, 169, 231, 233, 235, 237, 239,
241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265,
267, 269, 271, 273, 275, 277, 279, 281, 287, 289, 297, 305, 307,
309-311, 313-331, 346, 373-377, 380, or 381 from that publication,
which sequences are incorporated herein by reference).
[0132] In one aspect, exemplary multi-specific fusion proteins
containing a CD86 binding domain as described herein will also
contain at least one additional binding region or domain that is
specific for a target other than a CD86 (a "heterologous binding
domain"). For example, a multi-specific fusion protein of this
disclosure has a CD86 binding domain linked by an intervening
domain to a binding domain that is an IL-10 agonist, an HLA-G
agonist, an HGF agonist, an IL-35 agonist, a PD-1 agonist, a BTLA
agonist, a LIGHT antagonist, a GITRL antagonist or a CD40
antagonist. In certain embodiments, a multi-specific fusion protein
comprises a first and second binding domain, a first and second
linker, and an intervening domain, wherein one end of the
intervening domain is fused via the first linker to a first binding
domain that is a CD86 binding domain (e.g., a CTLA4 ectodomain, a
CD28 ectodomain, an anti-CD86) and at the other end is fused via
the second linker to a different binding domain that is an IL-10
agonist, an HLA-G agonist, an HGF agonist, an IL-35 agonist, a PD-1
agonist, a BTLA agonist, a LIGHT antagonist, a GITRL antagonist or
a CD40 antagonist.
[0133] In certain embodiments, the first linker and second linker
of a multi-specific fusion protein of this disclosure are each
independently selected from, for example, Linkers 1-133 as provided
in SEQ ID NOS:43-166 and the linkers provided in SEQ ID NOS:244,
307, 320, 355-379 and 383-398. For example, the first or second
linker can be any one of Linkers 47, 58, 126-131 (SEQ ID NOS:89,
100, and 159-164, respectively), or the linkers provided in SEQ ID
NO:244 or 355-379, or any combination thereof. In further examples,
one linker is Linker 47 (SEQ ID NO:89) or Linker 132 (SEQ ID
NO:165) and the other linker is the linker provided in SEQ ID
NO:355, or Linker 127 (SEQ ID NO:160) or one linker is Linker 58
(SEQ ID NO:100) or Linker 133 (SEQ ID NO:166) and the other linker
is Linker 126 (SEQ ID NO:159), or one linker is Linker 58 (SEQ ID
NO:100) or Linker 133 (SEQ ID NO:166) and the other linker is
Linker 127 (SEQ ID NO:160), or one linker is Linker 58 (SEQ ID
NO:100) or Linker 133 (SEQ ID NO:166) and the other linker is
Linker 128 (SEQ ID NO: 161), or one linker is Linker 58 (SEQ ID NO:
100) or Linker 133 (SEQ ID NO:166) and the other linker is Linker
129 (SEQ ID NO:162). In further examples, binding domains of this
disclosure that comprise V.sub.H and V.sub.L domains, such as those
specific for CD86, can have a further (third) linker between the
V.sub.H and V.sub.L domains, such as the linker provided in SEQ ID
NO:244, SEQ ID NO:89, Linker 46 (SEQ ID NO:88), Linker 130 (SEQ ID
NO:163), or Linker 131 (SEQ ID NO:164). In any of these
embodiments, the linkers may be flanked by one to five additional
amino acids internally (e.g., Linker 131 has an alanine internal to
the (G.sub.4S) core sequence), on either end (e.g., Linker 130 has
a serine on the amino-end of the (G.sub.4S) core sequence) or on
both ends (e.g., Linker 120 has two amino acids
(asparagine-tyrosine) on one end and three amino acids
(glycine-asparagine-serine) one the other end of the (G.sub.4S)
core sequence), which may simply be a result of creating such a
recombinant molecule (e.g., use of a particular restriction enzyme
site to join nucleic acid molecules may result in the insertion of
one to several amino acids), and for purposes of this disclosure
may be considered a part of any particular linker core
sequence.
[0134] In further embodiments, the intervening domain of a
multi-specific fusion protein of this disclosure is comprised of an
immunoglobulin constant region or sub-region, wherein the
intervening domain is disposed between a CD86 binding domain and a
binding domain that is an IL-10 agonist, an HLA-G agonist, an HGF
agonist, an IL-35 agonist, a PD-1 agonist, a BTLA agonist, a LIGHT
antagonist, a GITRL antagonist or a CD40 antagonist. In certain
embodiments, the intervening domain of a multi-specific fusion
protein of this disclosure has a CD86 binding domain at the
amino-terminus and a binding domain that is an IL-10 agonist, an
HLA-G agonist, an HGF agonist, an IL-35 agonist, a PD-1 agonist, a
BTLA agonist, a LIGHT antagonist, a GITRL antagonist or a CD40
antagonist at the carboxy-terminus. In other embodiments, the
intervening domain of a multi-specific fusion protein of this
disclosure has a binding domain that is an IL-10 agonist, an HLA-G
agonist, an HGF agonist, an IL-35 agonist, a PD-1 agonist, a BTLA
agonist, a LIGHT antagonist, a GITRL antagonist or a CD40
antagonist at the amino-terminus and a CD86 binding domain at the
carboxy-terminus.
[0135] In further embodiments, the immunoglobulin constant region
sub-region includes CH2 and CH3 domains of immunoglobulin G1
(IgG1). In related embodiments, the IgG1 CH2 and CH3 domains have
one or more of the following amino acids mutated (i.e., have a
different amino acid at that position): leucine at position 234
(L234), leucine at position 235 (L235), glycine at position 237
(G237), glutamate at position 318 (E318), lysine at position 320
(K320), lysine at position 322 (K322), or any combination thereof
(numbering according to Kabat). For example, any one of these amino
acids can be changed to alanine. In a further embodiment, according
to Kabat numbering, the CH2 domain has each of L234, L235, and G237
mutated to an alanine (i.e., L234A, L235A, and G237A,
respectively), and the IgG1 CH3 domain has each of E318, K320, and
K322 mutated to an alanine (i.e., E318A, K320A, and K322A,
respectively).
[0136] In certain embodiments, a multi-specific fusion protein of
this disclosure may comprise a small modular immunopharmaceutical"
(SMIP.TM.). In this regard, the term SMIP.TM. refers to a highly
modular compound class having enhanced drug properties over
monoclonal and recombinant antibodies. SMIPs comprise a single
polypeptide chain including a target-specific binding domain,
based, for example, upon an antibody variable domain, in
combination with a variable FC region that permits the specific
recruitment of a desired class of effector cells (such as, e.g.,
macrophages and natural killer (NK) cells) and/or recruitment of
complement-mediated killing. Depending upon the choice of target
and hinge regions, SMIPs can signal or block signaling via cell
surface receptors. As used herein, engineered fusion proteins,
termed "small modular immunopharmaceutical" or "SMIP.TM. products",
are as described in US Patent Publication Nos. 2003/133939,
2003/0118592, and 2005/0136049, and International Patent
Publications WO02/056910, WO2005/037989, and WO2005/017148.
[0137] In some embodiments, a multi-specific fusion protein may
comprise a PIMS molecule such as those described in US Patent
Publication No. 2009/0148447 and International Patent Publication
WO2009/023386.
[0138] In certain embodiments, the multi-specific fusion proteins
of the invention can be engineered with different front and back
end affinities in order to target specific cell types. For example,
use of an anti-CD86 binding domain (e.g., 3D1, FUN1, or humanized
variants thereof) that has a higher affinity for CD86 than an
engineered IL10 agonist (e.g., having an I87A or I87S mutation, or
a monoIL10 structure) has for huIL10R1, and combining such
molecules in an xceptor molecule of this disclosure can be used to
favor targeting to a specific cell type of interest, such as
antigen-presenting cells (APCs). In this regard, fusion proteins
can be made that have higher or lower affinity for CD86 or higher
or lower affinity for any of the heterologous target proteins
described herein, depending on the desired cell type to target. In
preferred embodiments, the CD86 antagonist binding domain
preferentially targets the multi-target specific xceptor molecule
to APCs by having a greater affinity for CD86 than the heterologous
binding domain has for its binding partner.
[0139] In some embodiments, a multi-specific fusion protein of this
disclosure has a CD86 binding domain that comprises a CTLA4
extracellular domain or sub-domain, a CD28 extracellular domain or
sub-domain, or a CD86-specific antibody-derived binding domain. In
certain embodiments, a CD86-specific antibody-derived binding
domain is derived from the FUN1 monoclonal antibody (see e.g., J
Pathol. 1993 March; 169(3):309-15); or derived from the 3D1
anti-CD86 monoclonal antibody. In certain embodiments, a CD86
binding domain is a sCTLA4, such as the mature polypeptide sequence
of SEQ ID NO:1. In certain embodiments, the CD86 binding domain is
a sCTLA4, such as the sequence of SEQ ID NO:1 or a variable-like
domain of CTLA4, such as SEQ ID NO:3, or a sub-domain thereof. In
other embodiments, a CD86 binding domain is a sCD28, such as the
mature polypeptide sequence of SEQ ID NO:2 (signal peptide: amino
acids 1-18). In still further embodiments, the CD86 binding domain
comprises light and heavy chain variable domains from FUN1 (e.g.,
SEQ ID NOS:305 and 306) or 3D1 (e.g., SEQ ID NOS:318 and 319),
preferably in the form of an scFv.
[0140] In further embodiments, a multi-specific fusion protein of
this disclosure has a CD86 binding domain and a heterologous
binding domain that is an IL-10 agonist, an HLA-G agonist, an HGF
agonist, an IL-35 agonist, a PD-1 agonist, a BTLA agonist, a LIGHT
antagonist, a GITRL antagonist or a CD40 antagonist (see e.g., the
amino acid sequences of heterologous binding domains provided in
SEQ ID NOS:7, 14, 15, 18-22, 25, 26, 29, 32, 33, 36, 39 and
40).
[0141] Exemplary structures of such multi-specific fusion proteins,
referred to herein as Xceptor molecules, include N-BD1-ID-BD2-C,
N--BD2-ID-BD1-C, wherein N and C represent the amino-terminus and
carboxy-terminus, respectively; BD1 is a CD86 binding domain, such
as an immunoglobulin-like or immunoglobulin variable region binding
domain, or an ectodomain; X is an intervening domain, and BD2 is
binding domain that is an IL-10 agonist, an HLA-G agonist, an HGF
agonist, an IL-35 agonist, a PD-1 agonist, a BTLA agonist, a LIGHT
antagonist, a GITRL antagonist or a CD40 antagonist. In some
constructs, X can comprise an immunoglobulin constant region or
sub-region disposed between the first and second binding domains.
In some embodiments, a multi-specific fusion protein of this
disclosure has an intervening domain (X) comprising, from
amino-terminus to carboxy-terminus, a structure as follows:
-L1-X-L2-, wherein L1 and L2 are each independently a linker
comprising from two to about 150 amino acids; and X is an
immunoglobulin constant region or sub-region. In further
embodiments, the multi-specific fusion protein will have an
intervening domain that is albumin, transferrin, or another serum
protein binding protein, wherein the fusion protein remains
primarily or substantially as a single chain polypeptide in a
composition.
[0142] The amino acid sequences of exemplary Xceptor fusion
proteins are provided in SEQ ID NOS:9, 13, 17, 24, 28, 31, 35, 38,
42, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193,
195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219,
221, 223, 237, 239, 252, 254, 256, 258, 260, 262, 266, 276, 302,
330, 334, 336, 338, 340, 350, 352, and 354; encoded by the
polynucleotide sequences provided in SEQ ID NOS:8, 12, 16, 23, 27,
30, 34, 37, 41, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188,
190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214,
216, 218, 220, 222, 236, 238, 251, 253, 255, 257, 259, 261, 265,
275, 301, 329, 333, 335, 337, 339, 349, 351 and 353
respectively.
[0143] In still further embodiments, a multi-specific fusion
protein of this disclosure has the following structure:
N-BD1-X-L2-BD2-C, wherein BD1 is a CD86 binding domain, such a
binding domain that is at least about 90% identical to a CTLA4
ectodomain; -X- is -L1-CH2CH3-, wherein L1 is a first IgG1 hinge,
optionally mutated by substituting the first or second cysteine and
wherein -CH2CH3- is the CH2CH3 region of an IgG1 Fc domain; L2 is a
linker selected from SEQ ID NOS:43-166, 244, 307, 320, 355-379 and
383-398; and BD2 is a binding domain that is an IL-10 agonist, an
HLA-G agonist, an HGF agonist, an IL-35 agonist, a PD-1 agonist, a
BTLA agonist, a LIGHT antagonist, a GITRL antagonist or a CD40
antagonist, as described herein.
[0144] In particular embodiments, a multi-specific Xceptor fusion
protein has (a) a CD86 binding domain comprising an amino acid
sequence at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or at least
100% identical to a mature polypeptide sequence of SEQ ID NO:1 or
SEQ ID NO: 2, and (b) a binding domain that is an IL-10 agonist, an
HLA-G agonist, an HGF agonist, an IL-35 agonist, a PD-1 agonist, a
BTLA agonist, a LIGHT antagonist, a GITRL antagonist or a CD40
antagonist comprising an amino acid sequence at least 80%, 90%,
95%, 96%, 97%, 98%, 99% or at least 100% identical to a
corresponding mature polypeptide sequence of the aforementioned
heterologous binding proteins as provided in SEQ ID NOS:7, 14, 15,
18-22, 25, 26, 29, 32, 33, 36, 39 and 40), wherein, from
amino-terminus to carboxy-terminus or from carboxy-terminus to
amino-terminus, (i) a CD86 binding domain of (a) or binding domain
of (b) is fused to a first linker, (ii) the first linker is fused
to an immunoglobulin heavy chain constant region of CH2 and CH3 as
set forth in any one of SEQ ID NOS:409 and 415-417, (iii) the
CH2CH3 constant region polypeptide is fused to a second linker, and
(iv) the second linker is fused to a CD86 binding domain of (a) or
a binding domain of (b). In certain embodiments, the first linker
is Linker 47 (SEQ ID NO:89), Linker 132 (SEQ ID NO:165) or Linker
133 (SEQ ID NO:166), the second linker is any one of Linkers
126-129 (SEQ ID NOS:159-162), and a further (third) linker between
the CD86 binding domain V.sub.H and V.sub.L domains is Linker 130
(SEQ ID NO:163) or Linker 131 (SEQ ID NO:164).
[0145] The amino acid sequences of exemplary Xceptor fusion
proteins are provided in SEQ ID NOS:9, 13, 17, 24, 28, 31, 35, 38,
42, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193,
195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219,
221, 223, 237, 239, 252, 254, 256, 258, 260, 262, 266, 276, 302,
330, 334, 336, 338, 340, 350, 352, and 354; encoded by the
polynucleotide sequences provided in SEQ ID Nos:8, 12, 16, 23, 27,
30, 34, 37, 41, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188,
190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214,
216, 218, 220, 222, 236, 238, 251, 253, 255, 257, 259, 261, 265,
275, 301, 329, 333, 335, 337, 339, 349, 351 and 353
respectively.
Making Multi-Specific Fusion Proteins
[0146] To efficiently produce any of the binding domain
polypeptides or fusion proteins described herein, a leader peptide
is used to facilitate secretion of expressed polypeptides and
fusion proteins. Using any of the conventional leader peptides
(signal sequences) is expected to direct nascently expressed
polypeptides or fusion proteins into a secretory pathway and to
result in cleavage of the leader peptide from the mature
polypeptide or fusion protein at or near the junction between the
leader peptide and the polypeptide or fusion protein. A particular
leader peptide will be chosen based on considerations known in the
art, such as using sequences encoded by polynucleotides that allow
the easy inclusion of restriction endonuclease cleavage sites at
the beginning or end of the coding sequence for the leader peptide
to facilitate molecular engineering, provided that such introduced
sequences specify amino acids that either do not interfere
unacceptably with any desired processing of the leader peptide from
the nascently expressed protein or do not interfere unacceptably
with any desired function of a polypeptide or fusion protein
molecule if the leader peptide is not cleaved during maturation of
the polypeptides or fusion proteins. Exemplary leader peptides of
this disclosure include natural leader sequences (i.e., those
expressed with the native protein) or use of heterologous leader
sequences, such as
H.sub.3N-MDFQVQIFSFLLISASVIMSRG(X).sub.n--CO.sub.2H, wherein X is
any amino acid and n is zero to three (SEQ ID NOS:167, 419, 420,
and 421) or H.sub.3N-MEAPAQLLFLLLLWLPDTTG-CO.sub.2H (SEQ ID NO:
168).
[0147] As noted herein, variants and derivatives of binding
domains, such as ectodomains, light and heavy variable regions, and
CDRs described herein, are contemplated. In one example, insertion
variants are provided wherein one or more amino acid residues
supplement a specific binding agent amino acid sequence. Insertions
may be located at either or both termini of the protein, or may be
positioned within internal regions of the specific binding agent
amino acid sequence. Variant products of this disclosure also
include mature specific binding agent products, i.e., specific
binding agent products wherein a leader or signal sequence is
removed, and the resulting protein having additional amino terminal
residues. The additional amino terminal residues may be derived
from another protein, or may include one or more residues that are
not identifiable as being derived from a specific protein.
Polypeptides with an additional methionine residue at position -1
are contemplated, as are polypeptides of this disclosure with
additional methionine and lysine residues at positions -2 and -1.
Variants having additional Met, Met-Lys, or Lys residues (or one or
more basic residues in general) are particularly useful for
enhanced recombinant protein production in bacterial host
cells.
[0148] As used herein, "amino acids" refer to a natural (those
occurring in nature) amino acid, a substituted natural amino acid,
a non-natural amino acid, a substituted non-natural amino acid, or
any combination thereof. The designations for natural amino acids
are herein set forth as either the standard one- or three-letter
code. Natural polar amino acids include asparagine (Asp or N) and
glutamine (Gln or Q); as well as basic amino acids such as arginine
(Arg or R), lysine (Lys or K), histidine (His or H), and
derivatives thereof; and acidic amino acids such as aspartic acid
(Asp or D) and glutamic acid (Glu or E), and derivatives thereof.
Natural hydrophobic amino acids include tryptophan (Trp or W),
phenylalanine (Phe or F), isoleucine (Ile or I), leucine (Leu or
L), methionine (Met or M), valine (Val or V), and derivatives
thereof; as well as other non-polar amino acids such as glycine
(Gly or G), alanine (Ala or A), proline (Pro or P), and derivatives
thereof. Natural amino acids of intermediate polarity include
serine (Ser or S), threonine (Thr or T), tyrosine (Tyr or Y),
cysteine (Cys or C), and derivatives thereof. Unless specified
otherwise, any amino acid described herein may be in either the D-
or L-configuration.
[0149] Substitution variants include those fusion proteins wherein
one or more amino acid residues in an amino acid sequence are
removed and replaced with alternative residues. In some
embodiments, the substitutions are conservative in nature; however,
this disclosure embraces substitutions that are also
non-conservative. Amino acids can be classified according to
physical properties and contribution to secondary and tertiary
protein structure. A conservative substitution is recognized in the
art as a substitution of one amino acid for another amino acid that
has similar properties. Exemplary conservative substitutions are
set out in Table 1 (see WO 97/09433, page 10, published Mar. 13,
1997), immediately below.
TABLE-US-00001 TABLE 1 Conservative Substitutions I Side Chain
Characteristic Amino Acid Aliphatic Non-polar G, A, P, I, L, V
Polar - uncharged S, T, M, N, Q Polar - charged D, E, K, R Aromatic
H, F, W, Y Other N, Q, D, E
[0150] Alternatively, conservative amino acids can be grouped as
described in Lehninger (Biochemistry, Second Edition; Worth
Publishers, Inc. NY:NY (1975), pp. 71-77) as set out in Table 2,
immediately below.
TABLE-US-00002 TABLE 2 Conservative Substitutions II Side Chain
Characteristic Amino Acid Non-polar (hydrophobic) Aliphatic: A, L,
I, V, P Aromatic F, W Sulfur-containing M Borderline G
Uncharged-polar Hydroxyl S, T, Y Amides N, Q Sulfhydryl C
Borderline G Positively Charged (Basic) K, R, H Negatively Charged
D, E (Acidic)
[0151] Variants or derivatives can also have additional amino acid
residues which arise from use of specific expression systems. For
example, use of commercially available vectors that express a
desired polypeptide as part of a glutathione-S-transferase (GST)
fusion product provides the desired polypeptide having an
additional glycine residue at position -1 after cleavage of the GST
component from the desired polypeptide. Variants which result from
expression in other vector systems are also contemplated, including
those wherein histidine tags are incorporated into the amino acid
sequence, generally at the carboxy and/or amino terminus of the
sequence.
[0152] Deletion variants are also contemplated wherein one or more
amino acid residues in a binding domain of this disclosure are
removed. Deletions can be effected at one or both termini of the
fusion protein, or from removal of one or more residues within the
amino acid sequence.
[0153] In certain illustrative embodiments, fusion proteins of this
disclosure are glycosylated, the pattern of glycosylation being
dependent upon a variety of factors including the host cell in
which the protein is expressed (if prepared in recombinant host
cells) and the culture conditions.
[0154] This disclosure also provides derivatives of fusion
proteins. Derivatives include specific binding domain polypeptides
bearing modifications other than insertion, deletion, or
substitution of amino acid residues. In certain embodiments, the
modifications are covalent in nature, and include for example,
chemical bonding with polymers, lipids, other organic, and
inorganic moieties. Derivatives of this disclosure may be prepared
to increase circulating half-life of a specific binding domain
polypeptide, or may be designed to improve targeting capacity for
the polypeptide to desired cells, tissues, or organs.
[0155] This disclosure further embraces fusion proteins that are
covalently modified or derivatized to include one or more
water-soluble polymer attachments such as polyethylene glycol,
polyoxyethylene glycol, or polypropylene glycol, as described U.S.
Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 and
4,179,337. Still other useful polymers known in the art include
monomethoxy-polyethylene glycol, dextran, cellulose, and other
carbohydrate-based polymers, poly-(N-vinyl
pyrrolidone)-polyethylene glycol, propylene glycol homopolymers, a
polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated
polyols (e.g., glycerol) and polyvinyl alcohol, as well as mixtures
of these polymers. Particularly preferred are polyethylene glycol
(PEG)-derivatized proteins. Water-soluble polymers may be bonded at
specific positions, for example at the amino terminus of the
proteins and polypeptides according to this disclosure, or randomly
attached to one or more side chains of the polypeptide. The use of
PEG for improving therapeutic capacities is described in U.S. Pat.
No. 6,133,426.
[0156] A particular embodiment of this disclosure is an
immunoglobulin or an Fc fusion protein. Such a fusion protein can
have a long half-life, e.g., several hours, a day or more, or even
a week or more, especially if the Fc domain is capable of
interacting with FcRn, the neonatal Fc receptor. The binding site
for FcRn in an Fc domain is also the site at which the bacterial
proteins A and G bind. The tight binding between these proteins can
be used as a means to purify antibodies or fusion proteins of this
disclosure by, for example, employing protein A or protein G
affinity chromatography during protein purification.
[0157] Protein purification techniques are well known to those of
skill in the art. These techniques involve, at one level, the crude
fractionation of the polypeptide and non-polypeptide fractions.
Further purification using chromatographic and electrophoretic
techniques to achieve partial or complete purification (or
purification to homogeneity) is frequently desired. Analytical
methods particularly suited to the preparation of a pure fusion
protein are ion-exchange chromatography; exclusion chromatography;
polyacrylamide gel electrophoresis; and isoelectric focusing.
Particularly efficient methods of purifying peptides are fast
protein liquid chromatography and HPLC.
[0158] Certain aspects of the present disclosure concern the
purification, and in particular embodiments, the substantial
purification, of a fusion protein. The term "purified fusion
protein" as used herein, is intended to refer to a composition,
isolatable from other components, wherein the fusion protein is
purified to any degree relative to its naturally obtainable state.
A purified fusion protein therefore also refers to a fusion
protein, free from the environment in which it may naturally
occur.
[0159] Generally, "purified" will refer to a fusion protein
composition that has been subjected to fractionation to remove
various other components, and which composition substantially
retains its expressed biological activity. Where the term
"substantially purified" is used, this designation refers to a
fusion binding protein composition in which the fusion protein
forms the major component of the composition, such as constituting
about 50%, about 60%, about 70%, about 80%, about 90%, about 95%,
about 99% or more of the protein, by weight, in the
composition.
[0160] Various methods for quantifying the degree of purification
are known to those of skill in the art in light of the present
disclosure. These include, for example, determining the specific
binding activity of an active fraction, or assessing the amount of
fusion protein in a fraction by SDS/PAGE analysis. A preferred
method for assessing the purity of a protein fraction is to
calculate the binding activity of the fraction, to compare it to
the binding activity of the initial extract, and to thus calculate
the degree of purification, herein assessed by a "-fold
purification number." The actual units used to represent the amount
of binding activity will, of course, be dependent upon the
particular assay technique chosen to follow the purification and
whether or not the expressed fusion protein exhibits a detectable
binding activity.
[0161] Various techniques suitable for use in protein purification
are well known to those of skill in the art. These include, for
example, precipitation with ammonium sulfate, PEG, antibodies and
the like, or by heat denaturation, followed by centrifugation;
chromatography steps such as ion exchange, gel filtration, reverse
phase, hydroxylapatite, and affinity chromatography; isoelectric
focusing; gel electrophoresis; and combinations of these and other
techniques. As is generally known in the art, it is believed that
the order of conducting the various purification steps may be
changed, or that certain steps may be omitted, and still result in
a suitable method for the preparation of a substantially purified
protein.
[0162] There is no general requirement that the fusion protein
always be provided in its most purified state. Indeed, it is
contemplated that less substantially purified proteins will have
utility in certain embodiments. Partial purification may be
accomplished by using fewer purification steps in combination, or
by utilizing different forms of the same general purification
scheme. For example, it is appreciated that a cation-exchange
column chromatography performed utilizing an HPLC apparatus will
generally result in greater purification than the same technique
utilizing a low pressure chromatography system. Methods exhibiting
a lower degree of relative purification may have advantages in
total recovery of protein product, or in maintaining binding
activity of an expressed protein.
[0163] It is known that the migration of a polypeptide can vary,
sometimes significantly, with different conditions of SDS/PAGE
(Capaldi et al. (1977) Biochem. Biophys. Res. Comm. 76:425). It
will therefore be appreciated that under differing electrophoresis
conditions, the apparent molecular weights of purified or partially
purified fusion protein expression products may vary.
Polynucleotides, Expression Vectors, and Host Cells
[0164] This disclosure provides polynucleotides (isolated or
purified or pure polynucleotides) encoding the multi-specific
fusion protein of this disclosure, vectors (including cloning
vectors and expression vectors) comprising such polynucleotides,
and cells (e.g., host cells) transformed or transfected with a
polynucleotide or vector according to this disclosure.
[0165] In certain embodiments, a polynucleotide (DNA or RNA)
encoding a binding domain of this disclosure, or a multi-specific
fusion protein containing one or more such binding domains is
contemplated. Expression cassettes encoding multi-specific fusion
protein constructs are provided in the examples appended
hereto.
[0166] The present disclosure also relates to vectors that include
a polynucleotide of this disclosure and, in particular, to
recombinant expression constructs. In one embodiment, this
disclosure contemplates a vector comprising a polynucleotide
encoding a multi-specific fusion protein containing a CD86 binding
domain and an IL-10 agonist, an HLA-G agonist, an HGF agonist, an
IL-35 agonist, a PD-1 agonist, a BTLA agonist, a LIGHT antagonist,
a GITRL antagonist or a CD40 antagonist domain of this disclosure,
along with other polynucleotide sequences that cause or facilitate
transcription, translation, and processing of such multi-specific
fusion protein-encoding sequences.
[0167] Appropriate cloning and expression vectors for use with
prokaryotic and eukaryotic hosts are described, for example, in
Sambrook et al., Molecular Cloning: A Laboratory Manual, Second
Edition, Cold Spring Harbor, N.Y., (1989). Exemplary
cloning/expression vectors include cloning vectors, shuttle
vectors, and expression constructs, that may be based on plasmids,
phagemids, phasmids, cosmids, viruses, artificial chromosomes, or
any nucleic acid vehicle known in the art suitable for
amplification, transfer, and/or expression of a polynucleotide
contained therein
[0168] As used herein, "vector" means a nucleic acid molecule
capable of transporting another nucleic acid to which it has been
linked. Exemplary vectors include plasmids, yeast artificial
chromosomes, and viral genomes. Certain vectors can autonomously
replicate in a host cell, while other vectors can be integrated
into the genome of a host cell and thereby are replicated with the
host genome. In addition, certain vectors are referred to herein as
"recombinant expression vectors" (or simply, "expression vectors"),
which contain nucleic acid sequences that are operatively linked to
an expression control sequence and, therefore, are capable of
directing the expression of those sequences.
[0169] In certain embodiments, expression constructs are derived
from plasmid vectors. Illustrative constructs include modified
pNASS vector (Clontech, Palo Alto, Calif.), which has nucleic acid
sequences encoding an ampicillin resistance gene, a polyadenylation
signal and a T7 promoter site; pDEF38 and pNEF38 (CMC ICOS
Biologics, Inc.), which have a CHEF1 promoter; and pD18 (Lonza),
which has a CMV promoter. Other suitable mammalian expression
vectors are well known (see, e.g., Ausubel et al., 1995; Sambrook
et al., supra; see also, e.g., catalogs from Invitrogen, San Diego,
Calif.; Novagen, Madison, Wis.; Pharmacia, Piscataway, N.J.).
Useful constructs may be prepared that include a dihydrofolate
reductase (DHFR)-encoding sequence under suitable regulatory
control, for promoting enhanced production levels of the fusion
proteins, which levels result from gene amplification following
application of an appropriate selection agent (e.g.,
methotrexate).
[0170] Generally, recombinant expression vectors will include
origins of replication and selectable markers permitting
transformation of the host cell, and a promoter derived from a
highly-expressed gene to direct transcription of a downstream
structural sequence, as described above. A vector in operable
linkage with a polynucleotide according to this disclosure yields a
cloning or expression construct. Exemplary cloning/expression
constructs contain at least one expression control element, e.g., a
promoter, operably linked to a polynucleotide of this disclosure.
Additional expression control elements, such as enhancers,
factor-specific binding sites, terminators, and ribosome binding
sites are also contemplated in the vectors and cloning/expression
constructs according to this disclosure. The heterologous
structural sequence of the polynucleotide according to this
disclosure is assembled in appropriate phase with translation
initiation and termination sequences. Thus, for example, the fusion
protein-encoding nucleic acids as provided herein may be included
in any one of a variety of expression vector constructs as a
recombinant expression construct for expressing such a protein in a
host cell.
[0171] The appropriate DNA sequence(s) may be inserted into a
vector, for example, by a variety of procedures. In general, a DNA
sequence is inserted into an appropriate restriction endonuclease
cleavage site(s) by procedures known in the art. Standard
techniques for cloning, DNA isolation, amplification and
purification, for enzymatic reactions involving DNA ligase, DNA
polymerase, restriction endonucleases and the like, and various
separation techniques are contemplated. A number of standard
techniques are described, for example, in Ausubel et al. (Current
Protocols in Molecular Biology, Greene Publ. Assoc. Inc. & John
Wiley & Sons, Inc., Boston, Mass., 1993); Sambrook et al.
(Molecular Cloning, Second Ed., Cold Spring Harbor Laboratory,
Plainview, N.Y., 1989); Maniatis et al. (Molecular Cloning, Cold
Spring Harbor Laboratory, Plainview, N.Y., 1982); Glover (Ed.) (DNA
Cloning Vol. I and II, IRL Press, Oxford, U K, 1985); Hames and
Higgins (Eds.) (Nucleic Acid Hybridization, IRL Press, Oxford, U K,
1985); and elsewhere.
[0172] The DNA sequence in the expression vector is operatively
linked to at least one appropriate expression control sequence
(e.g., a constitutive promoter or a regulated promoter) to direct
mRNA synthesis. Representative examples of such expression control
sequences include promoters of eukaryotic cells or their viruses,
as described above. Promoter regions can be selected from any
desired gene using CAT (chloramphenicol transferase) vectors or
other vectors with selectable markers. Eukaryotic promoters include
CMV immediate early, HSV thymidine kinase, early and late SV40,
LTRs from retrovirus, and mouse metallothionein-I. Selection of the
appropriate vector and promoter is well within the level of
ordinary skill in the art, and preparation of certain particularly
preferred recombinant expression constructs comprising at least one
promoter or regulated promoter operably linked to a nucleic acid
encoding a protein or polypeptide according to this disclosure is
described herein.
[0173] Variants of the polynucleotides of this disclosure are also
contemplated. Variant polynucleotides are at least 90%, and
preferably 95%, 99%, or 99.9% identical to one of the
polynucleotides of defined sequence as described herein, or that
hybridizes to one of those polynucleotides of defined sequence
under stringent hybridization conditions of 0.015M sodium chloride,
0.0015M sodium citrate at about 65-68.degree. C. or 0.015M sodium
chloride, 0.0015M sodium citrate, and 50% formamide at about
42.degree. C. The polynucleotide variants retain the capacity to
encode a binding domain or fusion protein thereof having the
functionality described herein.
[0174] The term "stringent" is used to refer to conditions that are
commonly understood in the art as stringent. Hybridization
stringency is principally determined by temperature, ionic
strength, and the concentration of denaturing agents such as
formamide. Examples of stringent conditions for hybridization and
washing are 0.015M sodium chloride, 0.0015M sodium citrate at about
65-68.degree. C. or 0.015M sodium chloride, 0.0015M sodium citrate,
and 50% formamide at about 42.degree. C. (see Sambrook et al.,
Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y., 1989).
[0175] More stringent conditions (such as higher temperature, lower
ionic strength, higher formamide, or other denaturing agent) may
also be used; however, the rate of hybridization will be affected.
In instances wherein hybridization of deoxyoligonucleotides is
concerned, additional exemplary stringent hybridization conditions
include washing in 6.times.SSC, 0.05% sodium pyrophosphate at
37.degree. C. (for 14-base oligonucleotides), 48.degree. C. (for
17-base oligonucleotides), 55.degree. C. (for 20-base
oligonucleotides), and 60.degree. C. (for 23-base
oligonucleotides).
[0176] A further aspect of this disclosure provides a host cell
transformed or transfected with, or otherwise containing, any of
the polynucleotides or vector/expression constructs of this
disclosure. The polynucleotides or cloning/expression constructs of
this disclosure are introduced into suitable cells using any method
known in the art, including transformation, transfection and
transduction. Host cells include the cells of a subject undergoing
ex vivo cell therapy including, for example, ex vivo gene therapy.
Eukaryotic host cells contemplated as an aspect of this disclosure
when harboring a polynucleotide, vector, or protein according to
this disclosure include, in addition to a subject's own cells
(e.g., a human patient's own cells), VERO cells, HeLa cells,
Chinese hamster ovary (CHO) cell lines (including modified CHO
cells capable of modifying the glycosylation pattern of expressed
multivalent binding molecules, see US Patent Application
Publication No. 2003/0115614), COS cells (such as COS-7), W138,
BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562, HEK293 cells, HepG2
cells, N cells, 3T3 cells, Spodoptera frugiperda cells (e.g., Sf9
cells), Saccharomyces cerevisiae cells, and any other eukaryotic
cell known in the art to be useful in expressing, and optionally
isolating, a protein or peptide according to this disclosure. Also
contemplated are prokaryotic cells, including Escherichia coli,
Bacillus subtilis, Salmonella typhimurium, a Streptomycete, or any
prokaryotic cell known in the art to be suitable for expressing,
and optionally isolating, a protein or peptide according to this
disclosure. In isolating protein or peptide from prokaryotic cells,
in particular, it is contemplated that techniques known in the art
for extracting protein from inclusion bodies may be used. The
selection of an appropriate host is within the scope of those
skilled in the art from the teachings herein. Host cells that
glycosylate the fusion proteins of this disclosure are
contemplated.
[0177] The term "recombinant host cell" (or simply "host cell")
refers to a cell containing a recombinant expression vector. It
should be understood that such terms are intended to refer not only
to the particular subject cell but to the progeny of such a cell.
Because certain modifications may occur in succeeding generations
due to either mutation or environmental influences, such progeny
may not, in fact, be identical to the parent cell, but are still
included within the scope of the term "host cell" as used
herein.
[0178] Recombinant host cells can be cultured in a conventional
nutrient medium modified as appropriate for activating promoters,
selecting transformants, or amplifying particular genes. The
culture conditions for particular host cells selected for
expression, such as temperature, pH and the like, will be readily
apparent to the ordinarily skilled artisan. Various mammalian cell
culture systems can also be employed to express recombinant
protein. Examples of mammalian expression systems include the COS-7
lines of monkey kidney fibroblasts, described by Gluzman (1981)
Cell 23:175, and other cell lines capable of expressing a
compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK
cell lines. Mammalian expression vectors will comprise an origin of
replication, a suitable promoter and, optionally, enhancer, and
also any necessary ribosome binding sites, polyadenylation site,
splice donor and acceptor sites, transcriptional termination
sequences, and 5'-flanking nontranscribed sequences, for example,
as described herein regarding the preparation of multivalent
binding protein expression constructs. DNA sequences derived from
the SV40 splice, and polyadenylation sites may be used to provide
the required nontranscribed genetic elements. Introduction of the
construct into the host cell can be effected by a variety of
methods with which those skilled in the art will be familiar,
including calcium phosphate transfection, DEAE-Dextran-mediated
transfection, or electroporation (Davis et al. (1986) Basic Methods
in Molecular Biology).
[0179] In one embodiment, a host cell is transduced by a
recombinant viral construct directing the expression of a protein
or polypeptide according to this disclosure. The transduced host
cell produces viral particles containing expressed protein or
polypeptide derived from portions of a host cell membrane
incorporated by the viral particles during viral budding.
Compositions and Methods of Use
[0180] To treat human or non-human mammals suffering a disease
state associated with CD86, IL-10, HLA-G, IL-35, PD-1, BTLA, LIGHT,
GITRL or CD40 dysregulation, a multi-specific fusion protein of
this disclosure is administered to the subject in an amount that is
effective to ameliorate symptoms of the disease state following a
course of one or more administrations. Being polypeptides, the
multi-specific fusion proteins of this disclosure can be suspended
or dissolved in a pharmaceutically acceptable diluent, optionally
including a stabilizer of other pharmaceutically acceptable
excipients, which can be used for intravenous administration by
injection or infusion, as more fully discussed below.
[0181] A pharmaceutically effective dose is that dose required to
prevent, inhibit the occurrence of, or treat (alleviate a symptom
to some extent, preferably all symptoms of) a disease state. The
pharmaceutically effective dose depends on the type of disease, the
composition used, the route of administration, the type of subject
being treated, the physical characteristics of the specific subject
under consideration for treatment, concurrent medication, and other
factors that those skilled in the medical arts will recognize. For
example, an amount between 0.1 mg/kg and 100 mg/kg body weight
(which can be administered as a single dose, or in multiple doses
given hourly, daily, weekly, monthly, or any combination thereof
that is an appropriate interval) of active ingredient may be
administered depending on the potency of a binding domain
polypeptide or multi-specific protein fusion of this
disclosure.
[0182] In certain aspects, compositions of fusion proteins are
provided by this disclosure. Pharmaceutical compositions of this
disclosure generally comprise one or more type of binding domain or
fusion protein in combination with a pharmaceutically acceptable
carrier, excipient, or diluent. Such carriers will be nontoxic to
recipients at the dosages and concentrations employed.
Pharmaceutically acceptable carriers for therapeutic use are well
known in the pharmaceutical art, and are described, for example, in
Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R.
Gennaro (Ed.) 1985). For example, sterile saline and phosphate
buffered saline at physiological pH may be used. Preservatives,
stabilizers, dyes and the like may be provided in the
pharmaceutical composition. For example, sodium benzoate, sorbic
acid, or esters of p-hydroxybenzoic acid may be added as
preservatives. Id. at 1449. In addition, antioxidants and
suspending agents may be used. Id. The compounds of the present
invention may be used in either the free base or salt forms, with
both forms being considered as being within the scope of the
present invention.
[0183] Pharmaceutical compositions may also contain diluents such
as buffers; antioxidants such as ascorbic acid, low molecular
weight (less than about 10 residues) polypeptides, proteins, amino
acids, carbohydrates (e.g., glucose, sucrose, or dextrins),
chelating agents (e.g., EDTA), glutathione or other stabilizers or
excipients. Neutral buffered saline or saline mixed with
nonspecific serum albumin are exemplary appropriate diluents.
Preferably, product is formulated as a lyophilizate using
appropriate excipient solutions as diluents.
[0184] Compositions of this disclosure can be used to treat disease
states in human and non-human mammals that are a result of or
associated with CD86, IL-10, HLA-G, IL-35, PD-1, BTLA, LIGHT, GITRL
or CD40 dysregulation. As discussed above, blocking of binding of
CD86 to CD28, for example by administration of CTLA4Ig, has been
shown to be effective in treating autoimmune disorders, such as
rheumatoid arthritis. IL10 is known to have immunosuppressive
properties (Commins et al. (2008) J. Allergy Clin. Immunol.
121:1108-11; Ming et al., (2008) Immunity 28:468-476), and
beneficial responses have been seen following administration of
IL10 to patients with psoriasis (Asadullah et al. (1999) Arch.
Dermatol. 135:187-92) and inflammatory bowel disease (Schreiber et
al. (2000) Gastroenterology 119:1461-72). As noted above, it has
been suggested that HLA-G may be useful in reducing inflammatory
responses in the CNS associated with multiple sclerosis (Wiendl et
al. (2005) Blood, 128:2689-2704), and as a therapeutic agent in
promoting tolerance to grafts in transplantations (Carosella et al.
(2008) Blood 111:4862-4870). HGF has been shown to be effective in
reducing disease both in a mouse model of arthritis and in a mouse
model of asthma. IL35 has been shown to be effective in reducing
disease in a mouse model of arthritis and to suppress T-cell
proliferation. As discussed above, LIGHT antagonists have been
shown to be effective in reducing graft vs. host disease and to
suppress T-cell proliferation. In addition, LIGHT is believed to
play a role in inflammatory bowel disease and Crohn's disease.
PD1-L1 or PD1-L2 to PD-1 has been shown to be effective in reducing
T-cell activation and cytokine production. BTLA has been shown to
be effective in reducing T-cell activation and cytokine production.
Binding of GITRL to GITR has been shown to increase disease
severity in animal models of asthma and arthritis, and is known to
increase T cell inflammatory and immune responses. As discussed
above, CD40 signaling is involved in diseases such as autoimmune
diseases, cancers, and organ and tissue graft rejections.
[0185] Thus, multi-specific fusion proteins of this disclosure are
useful in treating various autoimmune and/or inflammatory
disorders, such as rheumatoid arthritis, juvenile rheumatoid
arthritis, asthma, systemic lupus erythematosus (SLE), inflammatory
bowel disease (including Crohn's disease and ulcerative colitis),
graft versus host disease, psoriasis, multiple sclerosis,
dermatomyositis, polymyositis, pernicious anaemia, primary biliary
cirrhosis, acute disseminated encephalomyelitis (ADEM), Addison's
disease, ankylosing spondylitis, antiphospholipid antibody syndrome
(APS) autoimmune hepatitis, diabetes mellitus type 1, Goodpasture's
syndrome, Graves' disease, Guillain-Barre syndrome (GBS),
Hashimoto's disease, idiopathic thrombocytopenic purpura, lupus
erythematosus, pemphigus vulgaris, Sjogren's syndrome, temporal
arteritis (also known as "giant cell arteritis"), autoimmune
hemolytic anemia, bullous pemphigoid, vasculitis, coeliac disease,
endometriosis, hidradenitis suppurativa, interstitial cystitis,
morphea, scleroderma, narcolepsy, neuromyotonia, vitiligo and
autoimmune inner ear disease. In addition, multi-specific fusion
proteins of this disclosure are useful in suppressing detrimental
immune alloresponse in organ transplant (including solid organ
transplant or allograft), cell transplant, or the like.
[0186] "Pharmaceutically acceptable salt" refers to a salt of a
binding domain polypeptide or fusion protein of this disclosure
that is pharmaceutically acceptable and that possesses the desired
pharmacological activity of the parent compound. Such salts include
the following: (1) acid addition salts, formed with inorganic acids
such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric
acid, phosphoric acid, and the like; or formed with organic acids
such as acetic acid, propionic acid, hexanoic acid,
cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic
acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric
acid, tartaric acid, citric acid, benzoic acid,
3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid,
methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic
acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid,
4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,
4-toluenesulfonic acid, camphorsulfonic acid,
4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic
acid, 3-phenylpropionic acid, trimethylacetic acid, tertiary
butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic
acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic
acid, and the like; or (2) salts formed when an acidic proton
present in the parent compound either is replaced by a metal ion,
e.g., an alkali metal ion, an alkaline earth ion, or an aluminum
ion; or coordinates with an organic base such as ethanolamine,
diethanolamine, triethanolamine, N-methylglucamine, or the
like.
[0187] In particular illustrative embodiments, a polypeptide or
fusion protein of this disclosure is administered intravenously by,
for example, bolus injection or infusion. Routes of administration
in addition to intravenous include oral, topical, parenteral (e.g.,
sublingually or buccally), sublingual, rectal, vaginal, and
intranasal. The term parenteral as used herein includes
subcutaneous injections, intravenous, intramuscular, intrasternal,
intracavernous, intrathecal, intrameatal, intraurethral injection
or infusion techniques. The pharmaceutical composition is
formulated so as to allow the active ingredients contained therein
to be bioavailable upon administration of the composition to a
patient. Compositions that will be administered to a patient take
the form of one or more dosage units, where for example, a tablet
may be a single dosage unit, and a container of one or more
compounds of this disclosure in aerosol form may hold a plurality
of dosage units.
[0188] For oral administration, an excipient and/or binder may be
present, such as sucrose, kaolin, glycerin, starch dextrans,
cyclodextrins, sodium alginate, ethyl cellulose, and carboxy
methylcellulose. Sweetening agents, preservatives, dye/colorant,
flavor enhancer, or any combination thereof may optionally be
present. A coating shell may also optionally be used.
[0189] In a composition intended to be administered by injection,
one or more of a surfactant, preservative, wetting agent,
dispersing agent, suspending agent, buffer, stabilizer, isotonic
agent, or any combination thereof may optionally be included.
[0190] For nucleic acid-based formulations, or for formulations
comprising expression products according to this disclosure, about
0.01 .mu.g/kg to about 100 mg/kg body weight will be administered,
for example, by the intradermal, subcutaneous, intramuscular, or
intravenous route, or by any route known in the art to be suitable
under a given set of circumstances. A preferred dosage, for
example, is about 1 .mu.g/kg to about 20 mg/kg, with about 5
.mu.g/kg to about 10 mg/kg particularly preferred. It will be
evident to those skilled in the art that the number and frequency
of administration will be dependent upon the response of the
host.
[0191] The pharmaceutical compositions of this disclosure may be in
any form that allows for administration to a patient, such as, for
example, in the form of a solid, liquid, or gas (aerosol). The
composition may be in the form of a liquid, e.g., an elixir, syrup,
solution, emulsion or suspension, for administration by any route
described herein.
[0192] A liquid pharmaceutical composition as used herein, whether
in the form of a solution, suspension or other like form, may
include one or more of the following components: sterile diluents
such as water for injection, saline solution (e.g., physiological
saline), Ringer's solution, isotonic sodium chloride, fixed oils
such as synthetic mono- or digylcerides that may serve as the
solvent or suspending medium, polyethylene glycols, glycerin,
propylene glycol or other solvents; antibacterial agents such as
benzyl alcohol or methyl paraben; antioxidants such as ascorbic
acid or sodium bisulfite; buffers such as acetates, citrates or
phosphates; chelating agents such as ethylenediaminetetraacetic
acid; and agents for the adjustment of tonicity such as sodium,
chloride, or dextrose. The parenteral preparation can be enclosed
in ampoules, disposable syringes or multiple dose vials made of
glass or plastic. Physiological saline is a preferred additive. An
injectable pharmaceutical composition is preferably sterile.
[0193] It may also be desirable to include other components in the
preparation, such as delivery vehicles including aluminum salts,
water-in-oil emulsions, biodegradable oil vehicles, oil-in-water
emulsions, biodegradable microcapsules, and liposomes. Examples of
adjuvants for use in such vehicles include
N-acetylmuramyl-L-alanine-D-isoglutamine (MDP), lipopolysaccharides
(LPS), glucan, IL-12, GM-CSF, .gamma.-interferon, and IL-15.
[0194] While any suitable carrier known to those of ordinary skill
in the art may be employed in the pharmaceutical compositions of
this disclosure, the type of carrier will vary depending on the
mode of administration and whether a sustained release is desired.
For parenteral administration, the carrier may comprise water,
saline, alcohol, a fat, a wax, a buffer, or any combination
thereof. For oral administration, any of the above carriers or a
solid carrier, such as mannitol, lactose, starch, magnesium
stearate, sodium saccharine, talcum, cellulose, glucose, sucrose,
magnesium carbonate, or any combination thereof, may be
employed.
[0195] Also contemplated is the administration of multi-specific
fusion protein compositions of this disclosure in combination with
a second agent. A second agent may be one accepted in the art as a
standard treatment for a particular disease state, such as
inflammation, autoimmunity, and cancer. Exemplary second agents
contemplated include cytokines, growth factors, steroids, NSAIDs,
DMARDs, chemotherapeutics, radiotherapeutics, or other active and
ancillary agents.
[0196] This disclosure contemplates a dosage unit comprising a
pharmaceutical composition of this disclosure. Such dosage units
include, for example, a single-dose or a multi-dose vial or
syringe, including a two-compartment vial or syringe, one
comprising the pharmaceutical composition of this disclosure in
lyophilized form and the other a diluent for reconstitution. A
multi-dose dosage unit can also be, e.g., a bag or tube for
connection to an intravenous infusion device.
[0197] This disclosure also contemplates a kit comprising a
pharmaceutical composition in a unit dose or multi-dose container,
e.g., a vial, and a set of instructions for administering the
composition to patients suffering a disorder as described
herein.
[0198] All U.S. patents, U.S. patent application publications, U.S.
patent applications, foreign patents, foreign patent applications,
non-patent publications, tables, sequences, webpages, or the like
referred to in this specification, are incorporated herein by
reference, in their entirety. The following examples are intended
to illustrate, but not limit, this disclosure.
EXAMPLES
Xceptor Sequences
[0199] Nucleotide expression cassettes and amino acid sequences of
exemplary multi-specific fusion proteins having a CTLA4 ectodomain
are provided in SEQ ID NOS:8, 12, 16, 23, 27, 30, 34, 37 and 41 and
SEQ ID NOS:9, 13, 17, 24, 28, 31, 35, 38, and 42, respectively. The
activity of these exemplary multi-specific fusion proteins was
tested as described in Examples 1-6 below. Abbreviations used in
the following examples include the following terms: PBS-T: PBS, pH
7.2-7.4 and 0.1% Tween.RTM.20; Working buffer: PBS-T with 1% BSA;
Blocking buffer: PBS-T with 3% BSA.
Example 1
Anti-CD86 Binding Domains
[0200] Hybridomas 3D1 and FUN1 were used to clone the anti-CD86
variable binding domains of these monoclonal antibodies. The
sequences for the heavy chain, light chain, scFv linker, and CDRs
from the FUN1 and 3D1 anti-CD86 monoclonal antibodies are found in
SEQ NOS:305-313 and 318-326, respectively.
[0201] The following humanized FUN1 anti-CD86 monoclonal antibody
variable binding domains were used to construct SMIP proteins and
xceptors. The FUN1 CDRs were grafted into human germline sequences
as follows: (1) FUN1-11 has Igkv4-1*01 FR for light chain and
IgHV1-F*01 FR for heavy chain; (2) FUN1-21 has Igkv4-1*01 FR for
light chain and IgHV1-2*02 FR for heavy chain; (3) FUN1-31 has
Igkv4-1*01 FR for light chain and IgHV3-11*01 FR for heavy chain;
(4) FUN1-12 has Igkv1-27*01 FR for light chain and IgHV1-F*01 FR
for heavy chain; (5) FUN1-22 has Igkv1-27*01 FR for light chain and
IgHV1-2*02 FR for heavy chain; and (6) FUN1-32 has Igkv1-27*01 FR
for light chain and IgHV3-11*01 FR for heavy chain. The germline
CDRs for the humanized molecules are similar to the original FUN1
molecules.
[0202] Consequently, six versions of humanized FUN1 SMIP proteins
were also generated as follows: (1) FUN1-11 (SEQ ID NO:225); (2)
FUN1-21 (SEQ ID NO:227); (3) FUN1-31 (SEQ ID NO:229); (4) FUN1-12
(SEQ ID NO:231); (5) FUN1-22 (SEQ ID NO:233); and (6) FUN1-32 (SEQ
ID NO:235). Binding activity of these humanized FUN1 SMIP molecules
is shown in FIG. 7. In addition, the FUN1 variable domains (scFv)
and humanized FUN1 scFv were used to make xceptors, such as
IL10::FUN1 (SEQ ID NO:183); FUN1::IL10 (SEQ ID NO:187);
FUN1-21::IL10 (SEQ ID NO:237); IL10::FUN1-21 (SEQ ID NO:254); IL10
I87A::FUN1-21 (SEQ ID NO:258); and monoIL10::FUN1-21 binding domain
was also made using a short A2 hinge (SEQ ID N0:276) (wherein the
A2 hinge amino acid sequence is set forth in SEQ ID NO:364).
[0203] These, and all the other constructs described herein, were
cloned into appropriate mammalian expression vectors and expressed
in various cell lines to produce protein for particular functional
assays.
Example 2
Xceptor Binding to IL10-R1 by BIAcore.TM.
[0204] IL10-R1 binding activity was examined for an Xceptor
including a CTLA4 ectodomain and an IL10 domain (SEQ ID N0:9),
substantially as follows.
[0205] Surface plasmon resonance (SPR) measurements were performed
on a BIAcore.TM. T100 SPR (Pharmacia Biotech AB, Uppsala) using
HBS-P+ (GE Healthcare) as a running buffer. IL-10R1 (25 .mu.g/mL in
10 mM sodium acetate, pH 4.0; R&D Systems) was directly
immobilized onto a CM5 chip using standard amine coupling chemistry
(Biacore Amine Coupling Kit, GE Healthcare), with final
immobilization levels of 867, 2687, and 6719 Ru (resonance units).
IL-10-containing constructs were injected for 300 seconds, at a
flow rate of 50 .mu.l/min, in a series of concentrations from 100
pM to 10 nM. Dissociation was monitored for 1200 seconds, and the
surface was regenerated by injecting 2 M magnesium chloride, pH
7.58, for 60 seconds, followed by injecting 20 mM EDTA (in HBS-P+)
for 60 seconds. Binding interactions with the surface were stable
through at least 30 regeneration cycles. Data were analyzed using
BiaEvaluation for the T100, version 2.0 (GE Healthcare). Binding
kinetics of the CTLA4/IL10 Xceptor to immobilized IL-10R1 could not
be fit to a 1:1 Langmuir binding model, but could be fit with high
accuracy to a bivalent analyte binding model. Equilibrium
dissociation constants (K.sub.D) could be calculated with high
accuracy for each construct by fitting the observed response at
saturation to a steady-state equilibrium model, and are provided
below in Table 3. Inclusion of the CTLA4 ectodomain in the Xceptor
fusion protein had no apparent effect on the IL10/IL10R1
interaction.
TABLE-US-00003 TABLE 3 Immobilized First Site First Site Second
Site Second Site K.sub.D Protein Analyte k.sub.a (M.sup.-1s.sup.-1)
k.sub.d (s.sup.-1) k.sub.a (s.sup.-1) k.sub.d (s.sup.-1) (nM)
IL10-R1 IL10* 5.7 .times. 10.sup.5 2.6 .times. 10.sup.-4 -- -- 0.46
IL10-R1 SEQ ID .sup. 7 .times. 10.sup.5 2.9 .times. 10.sup.-4 18
.times. 10.sup.-3 8.6 .times. 10.sup.-3 0.41** NO: 9 *Literature
value (Yoon et al. (2006) J. Biol. Chem 281: 35088-35096)
**Calculated from k.sub.a1 and k.sub.d1
Example 3
Xceptor Binding to CD80 and Both CD80 and IL10 by ELISA
[0206] CD80 and IL10R binding activity was examined by ELISA for
abatacept, a CTLA4-Ig construct referred to as CTLA4-N2 (SEQ ID NO:
10 and 11), and the CTLA4/IL10 Xceptor of SEQ ID NO: 9,
substantially as follows.
CD80 Binding
[0207] Each well of a 96-well black Maxisorp CD80 plate (Nunc
Catalog #437111) plate was coated with CD80-mIg (Ancell Catalog
#510-020) at 2 .mu.g/ml solution and incubated overnight at
4.degree. C. The plate was then blocked with Blocking Buffer (PBS-T
with 3% non-fat dry milk). Samples of the proteins to be tested
serially diluted Blocking Buffer were added in duplicate wells to
the CD80-mIg coated plate, the plate was covered, and incubated at
room temperature for about 1 hour. After washing, 100 .mu.l per
well horse radish peroxidase goat anti-human IgG (gamma) diluted
1:1,000 in Blocking Buffer was added, the plate was covered, and
incubated at room temperature for 60 minutes, followed by a 10
minute incubation at room temperature in QuantaBlu.TM. Fluorogenic
Peroxidase Substrate (Thermo Scientific Catolog #15169). The
absorbance of each well was read at 420 nm. The unrelated fusion
protein TRU-015 was employed as a negative control.
[0208] The results of these experiments are shown in FIG. 1.
CTLA4-N2 was found to bind as well as abatacept to CD80-m Ig in
this ELISA format, while the CTLA4/IL10 Xceptor appeared to show
weaker CD80 binding.
Xceptor Binding to Both sIL10R1 and sCD80
[0209] Each well of a 96-well black Maxisorp CD80 plate (Nunc
Catalog #437111) plate was coated with sIL10Ra (R&D Systems
Catalog #510-020) at 2 .mu.g/ml solution and incubated overnight at
4.degree. C. The plate was then blocked with Blocking Buffer (PBS-T
with 3% non-fat dry milk). Samples of the proteins to be tested
serially diluted Blocking Buffer were added in duplicate wells to
the sIL10Ra coated plate, the plate was covered, and incubated at
room temperature for about 1 hour. After washing, anti-CD152
antibody (Ancell #359-020) at 10 ng/ul or CD80-mIg (Ancell
#510-020) at 5 ug/ml was added followed by horse radish peroxidase
goat anti-mouse IgG (Fc) (Pierce #31439) diluted 1:10,000 in
Blocking Buffer was added, the plate was covered, and incubated at
room temperature for 60 minutes, followed by a 10 minute incubation
at room temperature in QuantaBlu.TM. Fluorogenic Peroxidase
Substrate (Thermo Scientific Catolog #15169). The absorbance of
each well was read at 420 nm. The unrelated fusion protein TRU-015
was employed as a negative control.
[0210] FIG. 2 shows the results obtained for CTLA4-N2 and the
CTLA4::IL10 xceptor of SEQ ID NO: 9. These results demonstrate that
both the CTLA4 and IL10 domains of the CTLA4-Ig-IL10 Xceptor are
able to bind to their ligand/receptor simultaneously.
Example 4
Xceptor Induced Stat3 Phosphorylation
[0211] Binding of IL10 to IL10-R1 is known to activate Jak-1 and
Tyk which in turn lead to activation of STAT3 (see, for example,
Williams et al. (2007) J. Biol. Chem. 282:6965-6975). In addition,
studies have demonstrated that flow cytometry may be employed to
study the phosphorylation of STAT3 in PBMC (Lafarge et al. (2007)
BMC Mol. Biol. 8:64). The ability of various IL10-containing
constructs, including the CTLA4/IL10 Xceptor of SEQ ID NO: 9, to
induce phosphorylation of STAT3 in human PBMC was examined
substantially as follows.
[0212] PBMCs were isolated from a human donor and cultured
overnight in complete media (RPMI, 10% FBS, pen/strep) at
2.times.10.sup.6 cells/ml. The following morning, the PBMCs were
washed once, resuspended with pre-warmed RPMI 1640 (no supplements)
at 4.times.10.sup.6 cells/ml and incubated at 37.degree. C. for 2.5
hrs. Treatments were prepared at a 2.times. concentration in 0.25
mL of RPMI 1640 and mixed with 1.times.10.sup.6 PBMCs in 0.25 mL of
RPMI 1640. The samples were then incubated for 15 min at 37.degree.
C. Upon completion of the 15 min incubation, 0.5 mL of ice cold BD
Cytofix Buffer (BD Biosciences, cat #554655) was added to each
tube. Cells were incubated on ice for 30 min and then washed with 2
mls of DPBS+2.5% FBS (FACS Buffer). After decanting and vortexing
the samples, 0.5 mL of ice cold BD PERM BUFFER III (BD Biosciences,
cat #558050) was added to each tube and the samples were then
incubated on ice for 30 min. Samples were washed 3.times. with 2 mL
of FACS Buffer, and resuspended in .about.0.2 mL of FACS buffer
after the final wash. 20 uL of FITC conjugated anti-Human STAT3 mAb
(BD Biosciences, clone PY705) was added to each sample. Cells were
incubated in the dark at room temperature for 30 min. Samples were
then washed 3.times. with FACS Buffer to remove any unbound
antibody. Samples were analyzed on a LSRII flow cytometer. A gate
was applied to live lymphocytes based on SSC and FSC profiles and
MFI for FITC was determined.
[0213] As shown in FIG. 3 and FIG. 4, all IL-10 containing
constructs increased STAT3 phosphorylation in a dose dependent
fashion.
Example 5
Xceptor Binding to CD86 and Both CD86 and IL10
[0214] A human B-lymphoblastoid cell line that expresses CD86
(WIL2-S) was used to examine CD86 binding, and a CHO cell line
expressing CD86 (HuCD86-2A2 cells) on the surface was used in
combination with soluble IL10 Receptor1 (IL10R1) fusion protein
linked to a murine IgG Fc or an anti-IL10 antibody to examine the
simultaneous binding of the CD86 antagonist and IL10 agonist found
on xceptor molecules. Briefly, WIL2-S or HuCD86-2A2 cells were
incubated with test molecules containing a CD86 antagonist at
concentrations ranging from saturation to background levels. To the
HuCD86-2A2 cells, an IL10R1-muIg fusion protein or a murine
anti-IL10 antibody was further added to form a complex with the
test molecules that had bound to the cell surface via CD86. After
the incubation, cells were washed and a fluorophore
(R-phycoerythrin) tagged F(Ab').sub.2 antibody specific for the Fc
portion of the xceptor molecule, IL10R-Ig fusion protein, or
anti-IL10 antibody. The tagged cells were then passed through a
flow cytometer and data was analyzed by plotting median
fluorescence intensity of each sample.
[0215] As shown in FIG. 5, binding to CD86 on WIL2-S cells by
xceptor molecules containing an anti-CD86 binding domain (e.g.,
from hybridoma antibodies 3D1 and FUN1) showed higher affinity than
binding of CTLA4 ectodomain containing xceptor molecules. More
specifically, anti-CD86 3D1 containing xceptor 3D1::IL10 (SEQ ID
NO:189) had slightly higher affinity to CD86 than anti-CD86 FUN1
containing xceptor FUN1::IL10 (SEQ ID NO:187), whereas CTLA4-Ig
(SEQ ID NO:11) and a CTLA4 containing xceptor (SEQ ID NO:173) had
much lower affinity for CD86 than the anti-CD86 binding domains
from hybridoma antibodies 3D1 and FUN1.
[0216] As shown in FIG. 6, xceptor molecules containing a CD86
antagonist and IL10 agonist could simultaneously bind CD86 on
HuCD86-2A2 cells (CHO cells expressing CD86 on cell surface
developed in house) and soluble IL10R1. Furthermore, FIG. 6 shows
that the IL10 variants had different binding affinities for IL10R1.
For example, the xceptor molecules CTLA4::monoIL10 (SEQ ID NO:181)
and (CTLA4::IL10)-75 (SEQ ID NO:173) had similar affinities for
IL10R1, but the xceptors containing the viral mutated IL10 form,
CTLA4::IL10187A (SEQ ID NO:191) and CTLA4::IL10187S (SEQ ID
NO:193), showed much lower affinity for IL10R1.
[0217] In another experiment, different versions of humanized FUN1
SMIPs were tested for CD86 binding. Supernatants of HEK293 cells
transiently transfected with the six different versions of
humanized FUN1 SMIPs were examined for CD86 binding using
HuCD86-2A2 cells. FIG. 7 shows that that FUN1-21 (SEQ ID NO:227)
had the best binding affinity to CD86 followed by FUN1-22 (SEQ ID
NO:233) and then FUN1-11 (SEQ ID NO:225); the remaining humanized
FUN1 SMIP proteins (FUN1-12, SEQ ID NO:229; FUN1-31, SEQ ID NO:231;
FUN1-32, SEQ ID NO:235) did not show detectable binding.
Example 6
Varying Binding Domain Linker Lengths in Xceptor Proteins
[0218] Linker stability for CTLA4::IL10 molecules was examined. All
linker variants were stably transfected in CHOK1 SV cells and
stable bulk populations were cultured at 37.degree. C. and shifted
to 34.degree. C. on day 3. Proteins were purified by Protein A
column and followed by second step SEC column. ELISA assays were
performed to measure the IL10 binding to IL10R1-mIg fusion protein.
The results in FIG. 8 showed that xceptor (CTLA4::IL10)-65 (SEQ ID
NO:9) had reduced IL10 binding due to instability of the linker,
but (CTLA4::IL10)-68 (SEQ ID NO:171), (CTLA4::IL10)-69 (SEQ ID
NO:302), and (CTLA4::IL10)-75 (SEQ ID NO:173) were stable in these
culture conditions.
[0219] Shorter linker variants were also tested in CTLA4::IL10
xceptor proteins for binding to IL10R1 by ELISA. The shorter linker
variants were transiently transfected and proteins were Protein A
column purified. ELISA assays were performed to measure the IL10
binding to IL10R1-mIg fusion protein. The results in FIG. 9 showed
that the shorter linker variants (CTLA4::IL10)-77 (SEQ ID NO:175),
Q0033 (CTLA4::IL10)-78 (SEQ ID NO:177), and (CTLA4::IL10)-79 (SEQ
ID NO:179) worked as well as the longer linkers for back end IL10
binding affinity (e.g., compared to (CTLA4::IL10)-68; SEQ ID
NO:171).
Example 7
Serum Stability and Pharmacokinetics of Xceptor Proteins
[0220] The serum stability of (CTLA4::IL10)-75 (SEQ ID NO:173) was
tested. In this experiment, purified protein of SEQ ID NO:173 was
treated in mouse serum at 37.degree. C. for 24 hours, 72 hours and
F/T (freeze/thaw) and spike in at the time of assay (TO). All
stability samples were tested for their binding to CD80 (using CD80
expressing 1F6 CHO cells) as well as for simultaneous binding to
CD80 and anti-IL10 antibody binding to IL10 on the xceptor. The
results showed that SEQ ID NO:173 was very stable and retained
anti-IL10 binding after incubation in mouse serum at the tested
concentrations (from about 0.01 nM to about 10 nM).
[0221] Pharmacokinetic (PK) studies for (CTLA4::IL10)-68 (SEQ ID
NO:171) and (CTLA4::IL10)-75 (SEQ ID NO:173) were conducted in
female Balb/c mice, using 3 mice per time point. Mice were injected
intravenously with 200 .mu.g/mouse. Mouse serum was collected at 15
minutes, 2 hours, 6 hours, 24 hours, 48 hours, 96 hours, 7 days and
14 days after treatment. Samples were tested for CD80 binding
(CTLA4 assay) and simultaneous binding to CD80 and anti-IL10
antibody (IL10 assay). The results are summarized in Table 4
below.
TABLE-US-00004 TABLE 4 PK Study Summary (CTLA4::IL10)-68
(CTLA4::IL10)-75 PK Estimates for Abatacept PK Parameter PK
Parameter Abatacept, (CTLA4::IL10)- Estimates Estimates Estimates
68 and (CTLA4::IL10)-75 CTLA4 CTLA4 IL-10 CTLA4 IL-10 Parameter
Units Assay Assay Assay Assay Assay HL_Lambda_z hr 45.49 32.62
29.36 34.59 31.48 Vz_obs mL/kg 117.30 84.571 158.57 206.48 285.30
Cl_obs mL/hr/kg 1.79 1.797 3.74 4.14 6.28
Example 8
Xceptor Binding to CD80, CD86 and IL10R1
[0222] CD80 and IL10R1 binding was examined by ELISA, and/or CD86
and IL10R1 binding was examined using CD86-expressing CHO cells and
either IL10R1-Ig or anti-IL10. Binding to mouse CD80 and CD86 was
examined by ELISA using biotin-labeled mouse antibodies. The
molecules examined included the control CTLA4-Ig (SEQ ID NO:11)
fusion protein, and the following test xceptor molecules:
(CTLA4::IL10)-65 (SEQ ID NO:9); (CTLA4::IL10)-68 (SEQ ID NO:171);
(CTLA4::IL10)-69 (SEQ ID NO:302), (CTLA4::PDL2)-65 (SEQ ID NO:336),
substantially as described in Examples 3 and 5.
[0223] As shown in FIGS. 10 and 11, the (CTLA4::IL10)-65,
(CTLA4::IL10)-68, and (CTLA4::IL10)-69 xceptor molecules all bound
CD80 by ELISA and CD86 on cells. The results in FIG. 12 show that
CTLA4::IL10 xceptor molecules can interact with huIL10R1. Further,
as shown in FIG. 13, CTLA4::IL10 xceptors can engage both BD1
(amino-terminal binding domain) and BD2 (carboxy-terminal binding
domain) simultaneously to CD80 and IL10R1 by ELISA. Additionally,
FIGS. 14 and 15 show that the (CTLA4::IL10)-65, (CTLA4::IL10)-68,
and (CTLA4::IL10)-69 xceptor molecules, specific for human
molecules, are capable of crossreacting with both mouse CD80 and
mouse CD86.
Example 9
Xceptor Binding to CD80 by BIAcore.TM.
[0224] CD80 binding activity was examined for abatacept
(Orencia.RTM., Bristol-Myers Squibb), a CTLA4-Fc fusion containing
the L104E A29Y mutations, analogous to belatacept (SEQ ID NO:217),
three CTLA4::IL10 xceptor linker variants each having a CTLA4
ectodomain and an IL10 domain: (CTLA4::IL10)-65 (SEQ ID NO:9),
(CTLA4::IL10)-68 (SEQ ID NO:171), and (CTLA4::IL10)-75 (SEQ ID
NO:173); an xceptor including a CTLA4 ectodomain with the L104E
A29Y mutations and an IL10 domain (SEQ ID NO:219), and an xceptor
containing a CTLA4 ectodomain and a PD-L1 domain (SEQ ID NO:13),
substantially as described herein. Examination of binding of
CTLA4-Fc to CD80/86 by BIAcore has been described previously
(Greene et al. (1996) J. Biol. Chem. 271:26762-26771; van der Merwe
et al. (1997) J. Exp. Med. 185:393-403; Collins et al. (2002)
Immunity 17:201-210). Binding of CD80 by CTLA4 is of moderate
affinity (K.sub.d=.about.200 nM), and is characterized by a fast on
rate (4-8.times.10.sup.5 M.sup.-1s.sup.-1) and a moderate off rate
(0.090 s.sup.-1). Binding of dimeric CTLA4-Fc to CD80 is biphasic,
and has been reported with two off-rates (0.004, 0.086 s.sup.-1).
The L104E A29Y mutations on CTLA4-Fc have been reported to increase
the affinity for CD80 two-fold over the wild type CTLA4-Fc (Larsen
et al (2005) Am. J. Transplant. 5:443-453), primarily by decreasing
the initial off-rate (reported as 0.00108 vs 0.00221 s.sup.-1).
[0225] Surface plasmon resonance (SPR) measurements were performed
on a BIAcore.TM. T100 SPR (Pharmacia Biotech AB, Uppsala) using
HBS-EP+ (GE Healthcare) as a running buffer. CD80-m IgG (25
.mu.g/mL in 10 mM sodium acetate, pH 4.0; Ancell, Inc) was directly
immobilized onto a CM5 chip using standard amine coupling chemistry
(Biacore Amine Coupling Kit, GE Healthcare), with final
immobilization levels of 317, 973, and 1678 Ru (resonance units).
CTLA4-containing constructs were injected for 150 seconds, at a
flow rate of 10 .mu.l/min, in a series of concentrations from 5 nM
to 1 .mu.M. Dissociation was monitored for 600 seconds, and the
surface was regenerated by injecting 50 mM sodium citrate, 500 mM
sodium chloride, pH 4.0, for 60 seconds. Binding interactions with
the surface were stable through at least 75 regeneration cycles.
Data were analyzed using BiaEvaluation for the T100 software
(version 2.0.1, GE Healthcare).
[0226] Binding kinetics of the CTLA4 constructs to immobilized CD80
could not be fit to a 1:1 Langmuir binding model, but could be fit
with high accuracy to a bivalent analyte binding model. Increasing
the association phase by increasing the length of injection did not
alter the calculated kinetic parameters. Equilibrium dissociation
constants (K.sub.D) could be calculated with high accuracy for each
construct by fitting the observed response at saturation to a
steady-state equilibrium model. The results for all CD80 binding
molecules are summarized in Table 5 below.
TABLE-US-00005 TABLE 5 Immobilized First Site First Site Second
Site Second Site K.sub.D Protein Analyte k.sub.a (M.sup.-1s.sup.-1)
k.sub.d (s.sup.-1) k.sub.a (s.sup.-1) k.sub.d (s.sup.-1) (nM) CD80
abatacept 5.5 .+-. 0.02 .times. 10.sup.5 0.006 0.00019 9.4 .+-.
0.08 .times. 10.sup.-4 130 .+-. 10 CD80 SEQ ID NO: 9 1.9 .+-. 0.01
.times. 10.sup.5 0.008 0.0045 .+-. 0.0001 0.015 .+-. 0.0003 233
.+-. 27 CD80 SEQ ID NO: 13 0.36 .+-. 0.002 .times. 10.sup.5 7.4
.+-. 0.047 .times. 10.sup.-4 0.0057 0.064 176 .+-. 29 CD80 SEQ ID
NO: 171 9.77 .+-. 5.83 .times. 10.sup.5 0.0145 0.0190 0.0434 37.5
.+-. 9.5 CD80 SEQ ID NO: 173 12.1 .+-. 6.81 .times. 10.sup.5 0.0124
0.0207 0.0431 28.0 .+-. 6.5 CD80 SEQ ID NO: 217 1.55 .+-. 0.203
.times. 10.sup.5 0.00373 0.00102 0.00332 13.3 .+-. 6.2 CD80 SEQ ID
NO: 219 0.908 .+-. 0.307 .times. 10.sup.5 0.00235 0.00406 0.00706
19.1 .+-. 5.3
[0227] Equilibrium affinities for the Xceptors were determined to
resemble that for abatacept and the reported affinity for CTLA4-Fc
(200 nM; Greene et al. 1996, Ibid). Binding kinetics for the
CTLA4::PDL1 xceptor were different from those for abatacept or the
CTLA4::IL10 xceptor, although the on/off rate compensation gives a
similar affinity. This may be due to the fact that PD-L1 binds CD80
with a weaker affinity than CTLA4 (K.sub.D of 2.5 .mu.M). Similar
to previous studies, CTLA4 variants containing the L104E A29Y
mutation (SEQ ID NOS:217 and 219) had a higher affinity for CD80,
with a roughly two fold improvement in initial off rate (0.00373
s.sup.-1 for SEQ ID NO:217 as compared to 0.006 s.sup.-1 for
abatacept).
Example 10
Xceptor Binding to CD86 by BIAcore.TM.
[0228] CD86 binding activity was examined for abatacept, a CTLA4-Fc
fusion containing the L104E A29Y mutations, analogous to belatacept
(SEQ ID NO:217), an xceptor containing a CTLA4 ectodomain and an
IL10 domain (SEQ ID NO: 9), an xceptor containing a CTLA4
ectodomain with the L104E A29Y mutations and an IL10 domain (SEQ ID
NO:219), and different constructs (SMIP, PIMS, and xceptor)
containing antibody variable domains from the 3D1 and FUN1
anti-CD86 antibodies, substantially as described herein. Binding of
CD86 by CTLA4 is of low affinity (K.sub.d=.about.2.2 .mu.M), and is
characterized by a fast on rate (2-13.times.10.sup.5
M.sup.-1s.sup.-1) and a moderate off rate (0.42 s.sup.-1). The
L104E A29Y mutations on CTLA4-Fc have been reported to increase the
affinity for CD86 four-fold over the wild type CTLA4-Fc (Larsen et
al (2005) Am. J. Transplant. 5:443-453), primarily by decreasing
the initial off-rate (reported as 0.00206 vs 0.00816 s.sup.-1).
Apparently, no kinetic or equilibrium affinity data has been
previously published for the 3D1 or FUN1 antibodies, so their
relative affinities were determined.
[0229] Lower-Affinity CD86 Binding Domains Based on the CTLA4
Ectodomain.
[0230] SPR measurements were performed on a BIAcore.TM. T100 SPR
(Pharmacia Biotech AB, Uppsala) using HBS-EP+(GE Healthcare) as a
running buffer. CD86-mIgG (25 .mu.g/mL in 10 mM sodium acetate, pH
4.0, Ancell, Inc) was directly immobilized onto a CM5 chip using
standard amine coupling chemistry (Biacore Amine Coupling Kit, GE
Healthcare), with final immobilization levels of 37, 373, and 903
Ru. CTLA4-containing constructs were injected for 150 seconds, at a
flow rate of 10 .mu.l/min, in a series of concentrations from 4 nM
to 10 Dissociation was monitored for 600 seconds, and the surface
was either regenerated by injecting 50 mM sodium citrate, 500 mM
sodium chloride, pH 5.0, for 60 seconds (wild type CTLA4) or 10 mM
glycine, pH 1.7 (L104E A29Y CTLA4). Binding interactions with the
surface, and immobilization levels, were stable through at least
100 regeneration cycles. Data were analyzed using BiaEvaluation for
the T100 software (version 2.0.1, GE Healthcare). Owing to the low
affinity of CTLA4 to CD86, and the very fast on and off rates
(literature values are 0.2-1.3.times.10.sup.6 M.sup.-1s.sup.-1 for
k.sub.a and 0.42 s.sup.-1 for k.sub.d, at the limit of detection
for the BIAcore.TM. T100 instrument) binding kinetics of abatacept
to immobilized CD86 could not be determined. Binding kinetics of
the constructs with the L104E A29Y CTLA4 domain (SEQ ID NO:217; SEQ
ID NO:219) to immobilized CD86 could be determined with reasonable
accuracy by fitting the observed response to the bivalent analyte
model, however. Equilibrium dissociation constants (K.sub.D) could
be calculated with high accuracy for all constructs by fitting the
observed response at saturation to a steady-state equilibrium
model. The results are shown in Table 6, below.
[0231] Higher-Affinity CD86 Binding Domains Based on the 3D1 and
FUN1 Antibody Variable Domains.
[0232] SPR measurements were performed as listed above, with the
following exceptions: HBS-P+ (GE Healthcare) was used as a running
buffer; dissociation was monitored for 1200 seconds; and the
surface was regenerated by injecting 10 mM glycine, pH 1.7, for 60
seconds. Binding kinetics to immobilized CD86 could be determined
in all cases. However, equilibrium dissociation constants (K.sub.D)
could be calculated with high accuracy for each construct by
fitting the observed response at saturation to a steady-state
equilibrium model. The results are shown in Table 6 below.
TABLE-US-00006 TABLE 6 Immobilized First Site First Site Second
Site Second Site K.sub.D Protein Analyte k.sub.a (M.sup.-1s.sup.-1)
k.sub.d (s.sup.-1) k.sub.a (s.sup.-1) k.sub.d (s.sup.-1) (nM) CD86
abatacept -- -- -- -- 3200 .+-. 1600 CD86 SEQ ID NO: 217 0.847
.times. 10.sup.5 0.01016 0.0298 0.0344 772 .+-. 300 CD86 SEQ ID NO:
219 0.451 .times. 10.sup.5 0.00910 .+-. 0.0001 0.011 0.0221 670
.+-. 180 CD86 3D1 SMIP 3.23 .times. 10.sup.5 4.40 .+-. 0.05 .times.
10.sup.-5 0.0055 0.0276 11.7 .+-. 1.2 CD86 SEQ ID NO: 189 9.74 .+-.
0.066 .times. 10.sup.5 7.06 .+-. 0.04 .times. 10.sup.-5 0.0094
0.0462 26.5 .+-. 2.9 CD86 SEQ ID NO: 328 1.12 .times. 10.sup.5 2.37
.+-. 0.13 .times. 10.sup.-5 0.00077 0.00377 28.0 .+-. 1.9 CD86 SEQ
ID NO: 185 6.17 .+-. 0.12 .times. 10.sup.5 8.30 .+-. 0.12 .times.
10.sup.-5 0.0124 0.102 35.7 .+-. 2.5 CD86 FUN1 mAb 1.29 .+-. 0.69
.times. 10.sup.5 2.28 .+-. 0 .times. 10.sup.-5 0.00278 0.0154 36.0
.+-. 5.5 CD86 SEQ ID NO: 225 0.139 .+-. 0.556 .times. 10.sup.5 35.0
.times. 10.sup.-5 7.5 .times. 10.sup.-5 1.25 .+-. 0.25 .times.
10.sup.-6 119 .+-. 40 CD86 SEQ ID NO: 227 1.86 .+-. 0.951 .times.
10.sup.5 13.9 .times. 10.sup.-5 0.00578 0.0127 26.9 .+-. 5.4 CD86
SEQ ID NO: 402 0.5 .times. 10.sup.5 9.1 .times. 10.sup.-5 0.00113
0.00837 70.9 .+-. 5.9 CD86 muIL 10-Ig 0.941 .times. 10.sup.5 .sup.
18 .times. 10.sup.-5 0.0203 0.0722 50.6 .+-. 7.sup. CD86 SEQ ID NO:
254 1.58 .times. 10.sup.5 12.8 .times. 10.sup.-5 0.0064 0.0387 98.6
.+-. 10 CD86 SEQ ID NO: 258 0.407 .times. 10.sup.5 43.3 .+-. 1.2
.times. 10.sup.-5 0.0198 0.0579 88.5 .+-. 19 CD86 SEQ ID NO: 276
0.126 .times. 10.sup.5 34.4 .times. 10.sup.-5 0.011 0.0111 178 .+-.
18
[0233] Equilibrium affinities for abatacept (3.2 .mu.M) resembled
the reported affinity for CTLA4-Fc (2.2 .mu.M; Greene et al. 1996,
Ibid). Similar to previous studies, CTLA4 variants containing the
L104E A29Y mutation (SEQ ID NO:217; SEQ ID NO:219) had a
four-to-five fold higher affinity for CD86 (670-772 nM). Constructs
containing 3D1 murine single-chain antibody fragments (scFvs) on
the N-terminus (3D1 SMIP, SEQ ID NO: 317; 3D1::IL10, SEQ ID NO:189)
had higher affinities (11.7, 26.5 nM) than the corresponding
constructs with the 3D1 scFv on the C-terminus (3D1 PIMS, SEQ ID
NO: 319; IL10::3D1, SEQ ID NO:185); examining the binding kinetics,
this appeared to arise from both a higher initial on-rate and a
lower initial-off rate, although in all cases, the affinity was at
least 100-fold higher for CD86 than abatacept. For the FUN1
antibody, the parent murine monoclonal antibody was examined along
with SMIP proteins containing two humanized single-chain FUN1
antibody fragments (SEQ ID NOS:225 and 227); the latter of the two
(SEQ ID NO:227) showed similar binding kinetics and overall
affinity to CD86 as the parent FUN1 mAb (26.9, 36 nM,
respectively), which, again, was significantly higher than that of
abatacept. Xceptor or PIMS molecules containing humanized FUN1
antibody fragments at the carboxy-terminus (IL10::FUN1-21, SEQ ID
NO: 254; (IL10 I87A:: FUN1-21)-75, SEQ ID NO:258; (monoIL10-A2
hinge:: FUN1-21)-75, (SEQ ID NO:276); FUN1-21 PIMS, SEQ ID NO:402)
had lower affinities than the xceptor containing the parent
antibody sequence at the amino-terminus (FUN1::IL10, SEQ ID
NO:187)) or the same FUN1-21 binding sequence on a SMIP protein
(SEQ ID NO:227).
Example 11
Xceptor Binding to Murine CD86 by BIAcore.TM.
[0234] Murine CD86 binding activity was examined for abatacept, a
CTLA4-Fc fusion containing the L104E A29Y mutations, analogous to
belatacept (SEQ ID NO:217), two xceptors containing different BD2
linkers but the same CTLA4 and IL10 domains (SEQ ID NOS:171 and
173), an xceptor containing a CTLA4 ectodomain with the L104E A29Y
mutations and an IL10 domain (SEQ ID NO:219), a murine CTLA4 fusion
to human Fc domain (SEQ ID NO:404), and different constructs
containing antibody variable domains from the rat GL1 anti-murine
CD86 antibody, including two xceptors containing a GL1 antibody
fragment and human IL10 domain (GL1::IL10, SEQ ID NO:252; and
IL10::GL1, SEQ ID NO:256), substantially as described below. Human
CTLA4 is known to be cross-reactive to murine CD86, but, to the
best of our knowledge, no kinetic or affinity measurements have
been previously described. Similarly, the rat GL1 antibody has been
described as being specific for murine CD86, but no kinetic or
affinity measurements have been reported. SPR measurements were
performed on a BIAcore.TM. T100 SPR
[0235] (Pharmacia Biotech AB, Uppsala) using HBS-EP+ (GE
Healthcare) as a running buffer. Murine CD86-mIgG (25 .mu.g/mL in
10 mM sodium acetate, pH 4.0, R&D Systems, Inc) was directly
immobilized onto a CM5 chip using standard amine coupling chemistry
(Biacore Amine Coupling Kit, GE Healthcare), with final
immobilization levels of 150, 493, and 746 Ru. CTLA4-containing
constructs were injected for 150 seconds, at a flow rate of 10
.mu.l/min, in a series of concentrations from 4 nM to 8 .mu.M.
Dissociation was monitored for 600 seconds (CTLA4 constructs) or
1200 seconds (GL1 constructs), and the surface was regenerated by
injecting 10 mM glycine, pH 1.7, for 60 seconds. Binding
interactions with the surface, and immobilization levels, were
stable through at least 100 regeneration cycles. Data were analyzed
using BiaEvaluation for the T100 software (version 2.0.1, GE
Healthcare). Binding kinetics to immobilized murine CD86 could be
determined for all constructs, and fit with high accuracy to a
bivalent analyte model (Table 7). Equilibrium dissociation
constants (K.sub.D) could also be calculated with high accuracy for
each construct by fitting the observed response at saturation to a
steady-state equilibrium model. For the CTLA4 variants (SEQ ID
NOS:171, 173, 217, 219, and 404), simultaneous equilibrium fits
across all three flow cells at three different immobilization
densities gave more accurate results than fitting any one flow cell
(a so-called `multiple Rmax` fit), and so those affinities are
listed. The results are shown in Table 7, below.
TABLE-US-00007 TABLE 7 Immobilized First Site First Site Second
Site Second Site K.sub.D Protein Analyte k.sub.a (M.sup.-1s.sup.-1)
k.sub.d (s.sup.-1) k.sub.a (s.sup.-1) k.sub.d (s.sup.-1) (nM)
muCD86 SEQ ID NO: 171 0.104 .times. 10.sup.5 0.0241 0.0398 0.0627
1540 .+-. 210 muCD86 SEQ ID NO: 173 0.132 .times. 10.sup.5 0.0228
0.0405 0.0625 1810 .+-. 230 muCD86 SEQ ID NO: 217 0.278 .times.
10.sup.5 0.0787 0.0728 .+-. 0.0023 0.168 920 .+-. 88 muCD86 SEQ ID
NO: 219 0.321 .times. 10.sup.5 0.0418 0.00026 0.00193 870 .+-. 160
muCD86 muCTLA4-Ig 0.278 .times. 10.sup.5 0.0386 0.000659 0.00311
2250 .+-. 190 muCD86 GL1 mAb 0.789 .+-. 0.346 .times. 10.sup.5 10.6
.times. 10.sup.-5 0.00879 0.00635 37.7 .+-. 4.4 muCD86 GL1 SMIP
1.18 .+-. 0.427 .times. 10.sup.5 6.2 .times. 10.sup.-5 0.00964 .+-.
0.00031 0.0185 26.2 .+-. 5.4 muCD86 GL1 PIMS 1.77 .times. 10.sup.5
1.25 .times. 10.sup.-4 0.0431 0.0977 78.3 .+-. 20 muCD86 SEQ ID NO:
252 2.19 .times. 10.sup.5 1.9 .times. 10.sup.-4 0.00943 0.0182 86.4
.+-. 8.3 muCD86 SEQ ID NO: 256 6.73 .times. 10.sup.4 4.4 .times.
10.sup.-5 0.0271 0.088 95.1 .+-. 13
[0236] Xceptors containing human CTLA4 and human IL10 domains (SEQ
ID NOS:171 and 173) had slightly higher affinities to murine CD86
(1.54, 1.81 .mu.M) than the reported affinity for human CTLA4-Fc
for human CD86 (2.2 .mu.M; Greene et al. 1996). Human CTLA4
variants containing the L104E A29Y mutation (SEQ ID NOS:217 and
219) had only a two fold higher affinity for murine CD86 (870-920
nM); this seems to arise from a combination of a beneficial higher
initial on-rate for murine CD86 and a detrimental higher initial
off-rate. Murine CTLA4 seems to have an analogous, or slightly
lower overall affinity for murine CD86 than human CTLA4. For the
GL1 antibody, the parent rat monoclonal antibody was examined along
with a SMIP containing a single-chain GL1 antibody fragment (GL1
SMIP, SEQ ID NO:239); both showed similar binding kinetics and
overall affinity to murine CD86 (37.7, 26.2 nM, respectively),
which, was significantly higher (.about.50 fold) than that of human
or murine CTLA4-containing constructs. Xceptors containing IL-10
and the GL1 antibody fragment (SEQ ID NOS:252 and 256) showed a
3-fold lower affinity to murine CD86 compared to the parent
antibody or SMIP, although this appeared to arise from either a
reduced initial on-rate or off-rate in each case.
Example 12
Xceptor Binding to PD1 by BIAcore.TM.
[0237] PD1 binding activity was examined for PDL1-Fc (SEQ ID
NO:268) and PDL2-Fc (SEQ ID NO:270), as well as xceptors containing
a CTLA4 ectodomain and either PDL1 (SEQ ID NO:13) or PDL2 (SEQ ID
NO:336) domains, substantially as follows. Binding of PD1 by PDL2
has been generally reported to be higher affinity than the binding
of PD1 by PDL1; a prior kinetic analysis (Youngnak et al, (2003)
Biochem. Biophys. Res. Comm. 307, 672) suggested moderate
affinities (112 nM for PDL1, 37 nM for PDL2), whereas a equilibrium
analysis done in an alternate format (Butte et al, (2007) Immunity
27, 111) suggested weaker affinities (770 nM for PDL1, 590 nM for
PDL2).
[0238] SPR measurements were performed on a BIAcore.TM. T100 SPR
(Pharmacia Biotech AB, Uppsala) using HBS-P+ (GE Healthcare) as a
running buffer. A construct with the PD1 ectodomain fused to a
C-terminal AviTag.TM. (SEQ ID NO:406) was initially biotinylated
using the BirA enzyme (Avidity, Inc., Aurora, Colo.) in 10 mM Tris,
pH 8.0, and buffer exchanged into PBS. Neutravidin (100 .mu.g/mL in
10 mM sodium acetate, pH 4.0, Thermo Scientific, Rockford, Ill.)
was directly immobilized onto a CM5 chip using standard amine
coupling chemistry (Biacore Amine Coupling Kit, GE Healthcare),
with final immobilization levels of 191, 771, and 1522 Ru, and used
to capture biotinylated PD1 at levels of 171, 597, and 1244 Ru,
respectively. PDL1/2-containing constructs were injected for 120
seconds, at a flow rate of 30 .mu.l/min, in a series of
concentrations from 6 nM to 2 .mu.M. Dissociation was monitored for
1200 seconds, and the surface was regenerated by injecting 50 mM
NaOH, 1M NaCl for 30 seconds. Binding interactions with the
surface, and immobilization levels, were stable through at least 50
regeneration cycles.
[0239] Data were analyzed using BiaEvaluation for the T100 software
(version 2.0.1, GE Healthcare). Binding kinetics to immobilized PD1
could be determined for all constructs, and fit either with high
accuracy to a bivalent analyte model (SEQ ID NOS:268 and 270) or a
1:1 binding model (SEQ ID NOS:13 and 336) (Table 8). Equilibrium
dissociation constants (K.sub.D) could also be calculated with high
accuracy for each construct by fitting the observed response at
saturation to a steady-state equilibrium model. The results are
shown in Table 8, below.
TABLE-US-00008 TABLE 8 Immobilized First Site First Site Second
Site Second Site K.sub.D Protein Analyte k.sub.a (M.sup.-1s.sup.-1)
k.sub.d (s.sup.-1) k.sub.a (s.sup.-1) k.sub.d (s.sup.-1) (nM) PD1
SEQ ID NO: 13 0.699 .times. 10.sup.5 0.165 n/a n/a 2360** PD1 SEQ
ID NO: 336 1.16 .times. 10.sup.5 0.0395 n/a n/a 340** PD1 SEQ ID
NO: 268 0.96 .+-. 0.54 .times. 10.sup.5 0.0544 3.18 .times.
10.sup.-6 0.000191 246 .+-. 27 PD1 PDL1-Fc* 1.07 .times. 10.sup.5
0.010 n/a n/a 112** PD1 SEQ ID NO: 270 1.65 .times. 10.sup.5
0.00724 0.0352 0.395 .sup. 42 .+-. 4.4 PD1 PDL2-Fc* 1.22 .times.
10.sup.5 0.0032 n/a n/a 26** *Literature value (Youngnak et al,
(2003) Biochem. Biophys. Res. Comm. 307, 672) **Kinetic K.sub.D
calculated from first site k.sub.a and k.sub.d
[0240] PDL1-Fc (SEQ ID NO:268) and PDL2-Fc (SEQ ID NO:270) showed
similar binding kinetics and overall affinities to those reported
in literature. Xceptors containing CTLA4 with PDL1 or PDL2 domains
fused at the carboxy-terminus (SEQ ID NOS:13 and 336) had
noticeably weaker (.about.10 fold) affinities to PD1 (2.36, 0.34
.mu.M) than the amino-terminal PDL1/PDL2 fusions (246, 42 nM); this
primarily arises from a noticeable increase in the initial off-rate
(0.165, 0.0395 vs 0.0544, 0.00724), indicating the PDL1/2:PD1
complex may be destabilized when the binding domain is at the BD2
(carboxy-terminal) position.
Example 13
Xceptor Fusion Proteins Block Human T Cell Responses
[0241] This example demonstrates that xceptor fusion proteins of
this disclosure can block a human T cell response. A mixed
lymphocyte reaction (MLR) was used to test blocking by xceptor
fusion proteins. In brief, human peripheral blood mononuclear cells
(PBMC) from two donors were isolated using standard methods and
kept separate. Based on previous studies, PBMC from one donor were
designated as the "Responder" population and PBMC from the second
donor were designated as the "Stimulator" population. Both donor
PBMC were labeled with CFSE using standard methods. To prevent cell
division, Stimulator PBMC were treated with mitomycin-C(MMC). MMC
(Sigma #M4287-2 mg) was reconstituted in sterile distilled water
(Gibco #15230) at a concentration of 0.5 mg/ml. Stimulator PBMC
were suspended at a concentration of 1.times.10.sup.6/ml in
complete culture media (CM), (RPMI-1640 containing 10% human B
serum, 100 U/ml penicillin, 100 ug/ml streptomycin, 2 mM
L-glutamine, NEAA, Na-pyruvate, CM 0.2 um filtered) and MMC was
added to a final concentration of 25 .mu.g/ml. The Stimulator
PBMC/MMC mixture was then incubated at 37.degree. C., 5% CO.sub.2,
for 30 minutes after which cells were washed thrice with CM.
Responder and Stimulator cells were suspended at a concentration of
4.times.10.sup.6/ml in CM and 0.05 ml of each cell population was
added per well of a 96 well-flat bottom tissue culture plate for a
final 2.times.10.sup.5 cells/well/donor. All treatments at the
designated concentrations shown in figures were added to the plate
at the same time as the cells (note concentrations shown for
antibodies and fusion proteins are at molar equivalents). MLR
conditions (96 well plate PBMC treatment set-up) were then
incubated at 37.degree. C., 5% CO.sub.2, for the duration of the
experiment. MLR experiments were harvested 7-8 days at which cells
were stained with fluorescently tagged antibodies against CD5
(e-Bioscience) and CD25 (BD Biosciences) and run on a flow
cytometer (LSR II, Becton Dickenson). Data was analyzed using
FlowJo flow cytometry software (TreeStar). The gating strategy was
as follows: cells that fell within a FSC:SSC lymphocyte gate were
analyzed for CD5 expression, cells that then subsequently fell
within the CD5+ gate were analyzed for CFSE dilution and CD25
up-regulation. Cells that were CD5+, CFSE.sup.low and CD25.sup.high
were considered activated T cells.
[0242] FIGS. 16, 17, 21, and 22 show that many different kinds of
xceptor fusion proteins containing a CD86 antagonist in combination
with a heterologous binding domain are capable of blocking a T cell
response to Responder/Stimulator MLR conditions.
Example 14
CD86 Antagonist Xceptors Block a Mouse T Cell Response
[0243] Mice splenocytes from two different mouse strains, C57BL/6
(or B6D2F1) and BALB/c, were isolated utilizing the scalpel/nylon
mesh and RBC lyse method. Based on previous studies, splenocytes
from mouse strain C57Bl/6 (or B6D2F1) were designated as the
"Responder" population and splenocytes from mouse strain BALB/c
were designated as the "Stimulator" population. Both mouse strain
splenocytes were labeled with CFSE as previously described. To
prevent cell division, Stimulator splenocytes were treated with
mitomycin-C (MMC). MMC (Sigma #M4287-2 mg) was reconstituted in
sterile distilled water (Gibco #15230) at a concentration of 0.5
mg/ml. Stimulator splenocytes were suspended at a concentration of
5.times.10.sup.7/ml in complete culture media (CM), (RPMI-1640
containing 10% FBS, 100 U/ml penicillin, 100 .mu.g/ml streptomycin,
2 mM L-glutamine, NEAA, Na-pyruvate, and 0.05 mM 2-mercaptoethanol)
and MMC was added to a final concentration of 50 .mu.g/ml. The
Stimulator splenocyte/MMC mixture was then incubated at 37.degree.
C., 5% CO.sub.2, for 20 minutes after which cells were washed
thrice with CM. Responder and Stimulator cells were suspended at a
concentration of 8.times.10.sup.6/ml in CM and 0.05 ml of each cell
population was added per well of a 96 well-flat bottom tissue
culture plate for a final 4.times.10.sup.5 cells/well/strain. All
treatments at the designated concentrations shown in FIGS. 18-20
were added to the plate at the same time as the cells (note
concentrations shown for antibodies and fusion proteins are at
molar equivalents). MLR conditions (96 well plate with
splenocyte/treatment set-up) were then incubated at 37.degree. C.,
5% CO.sub.2, for the duration of the experiment. MLR experiments
were harvested 4-5 days at which cells were stained with
fluorescently tagged antibodies against CD5 (BD Biosciences) and
CD25 (BD Biosciences) and run on a flow cytometer (LSR II, Becton
Dickenson). Data was analyzed using FlowJo flow cytometry software
(TreeStar). The gating strategy was as follows: cells that fell
within a FSC:SSC lymphocyte gate were analyzed for CD5 expression,
cells that then subsequently fell within the CD5+ gate were
analyzed for CFSE dilution and CD25 up-regulation. Cells that were
CD5+, CFSE.sup.low and CD25.sup.high were considered activated T
cells.
[0244] FIGS. 18-20 show that many different kinds of xceptor fusion
proteins containing a CD86 antagonist in combination with a
heterologous binding domain are capable of blocking a mouse T cell
response to Responder (B6D2F1)/Stimulator (BALB/c) MLR
conditions.
Example 15
Immunostimulatory Activity of Xceptor Molecules Containing IL10
[0245] The immunostimulatory activity of IL10 in various xceptor
fusion proteins was tested in an in vitro cell proliferation assay.
In particular, the MC/9 mouse mast liver cell line was used as
follows: MC/9 cell line (American Type Culture Collection.TM.
#CRL-8306) were grown in DMEM or RPMI plus 10% FBS and 5% Rat
T-STIM (BD #354115). MC/9 cells were washed and rested in media
without Rat T-STIM overnight (incubated at 37.degree. C., 5%
CO.sub.2, in 96 well flat bottom plate at 1.times.10.sup.5
cells/well, 100 .mu.l/well). Various concentrations of IL10 protein
and xceptor fusion proteins were incubated for 24 hours and
proliferation was assessed by [.sup.3H] thymidine incorporation
after 6 hours.
[0246] The I87 variant of IL10 is known to be less
immuno-stimulatory compared to wild-type IL10 (Ding et al., J. Exp.
Med. 191:213, 2000). IL10 normally forms a homodimer with the amino
terminal domain of each monomer molecule binding to the carboxy
terminal domain of the other monomer). The IL10 I87 variant (I87A
and I87S), along with an IL10 molecule having a short linker
(gggsgg; SEQ ID NO:379) that further separates the two subdomains
of IL10 allowing these subdomains to form an intramolecular dimer,
were examined in the xceptor format for immunostimulatory
activity.
[0247] FIG. 23 shows that mouse IL10 is capable of enhancing MC/9
cell proliferation to greater extent than CTLA4::IL10-I87A (SEQ ID
NO:191) xceptor, which contains a single mutation in amino acid 87
of IL10. FIG. 24 shows that both human IL10 and (CTLA4::IL10)-75
(SEQ ID NO:173) are capable of enhancing MC/9 cell proliferation
more than either CTLA4::IL10-I87A (SEQ ID NO:191) or
CTLA4::monoIL-10 (SEQ ID NO:181).
Example 16
Engineering Xceptor Molecules to Target Specific Cell Types
[0248] This Example describes the engineering of Xceptor fusion
proteins to target specific cell types. This is achieved by
engineering BD1 and BD2 affinities. Four Xceptor molecules were
engineered with different affinity for CD86 and huIL10R1. Table 9
shows the different affinity ratios for these four molecules. By
improving affinity for CD86 through the use of, for example, a CD86
binding domain (e.g., 3D1 or humanized FUN1) and an engineered IL10
molecule (I87A) with lower affinity for huIL10R1, then such an
arrangement can be used to favor targeting to a specific cell type
of interest, such as APC.
TABLE-US-00009 TABLE 9 CD86 IL10R1 IL10R1/CD86 BD1 BD2 Affinity
(nM) Affinity (nM) Affinity Ratio CLTA4 IL10 2000* 0.1.sup.&
0.00005 CTLA4 IL10I87A 2000* 10.sup.# .sup. 0.005 anti-CD86 IL10
40* 0.1.sup.& 0.0025 anti-CD86 IL10I87A 40* 10.sup.# .sup. 0.25
*Approximate equilibrium affinity from in-house measurement of
CTLA4, 3D1 and humanized FUN1 binding to CD86 .sup.&Approximate
affinity based on Tan et al. (J. Biol. Chem. 268: 21053, 1993)
.sup.#Approximate affinity based on Ding et al. (J. Exp. Med. 191:
213, 2000)
Example 17
Xceptor Activity in In Vivo Rheumatoid Arthritis Animal Models
Rheumatoid Arthritis
[0249] The therapeutic efficacy of any of the xceptor molecules
disclosed herein is examined in at least one of two murine models
of rheumatoid arthritis (RA), namely the collagen induced arthritis
(CIA) and glucose-6-phosphate isomerase (G6PI) models. Each of
these models has been shown to be useful for predicting efficacy of
certain classes of therapeutic drugs in RA (see Holmdahl (2000)
Arthritis Res. 2:169; Holmdahl (2006) Immunol. Lett. 103:86;
Holmdahl (2007) Methods Mol. Med. 136:185; McDevitt, H. (2000)
Arthritis Res. 2:85; Kamradt and Schubert (2005) Arthritis Res.
Ther. 7:20).
[0250] (a) CIA Model
[0251] The CIA model is the best characterized mouse model of
arthritis in terms of its pathogenesis and immunological basis. In
addition, it is the most widely used model of RA and, although not
perfect for predicting the ability of drugs to inhibit disease in
patients, is considered by many to be the model of choice when
investigating potential new therapeutics for RA (Jirholt et al.
(2001) Arthritis Res. 3:87; Van den Berg, W. B. (2002) Curr.
Rheumatol. Rep. 4:232; Rosloniec (2003) Collagen-Induced Arthritis.
In Current Protocols in Immunology, eds. Coligan et al., John Wiley
& Sons, Inc, Hoboken, N.J.).
[0252] In the CIA model, arthritis is induced by immunization of
male DBA/1 mice with collagen II (CII) in Complete Freund's
Adjuvant (CFA). Specifically, mice are injected
intradermally/subcutaneously with CII in CFA on Day -21 and boosted
with CII in Incomplete Freund's Adjuvant (IFA) on Day 0. Mice
develop clinical signs of arthritis within days of the boost with
CII/IFA. A subset of mice (0% to 10%) immunized with CII/CFA
develop signs of arthritis on or around Day 0 without a boost and
are excluded from the experiments. In some CIA experiments, the
boost is omitted and mice are instead treated with Xceptor or
control starting 21 days after immunization with CII/CFA (i.e. the
day of first treatment is Day 0).
[0253] Mice are treated with Xceptor, vehicle (PBS), or negative or
positive control in a preventative and/or therapeutic regimen.
Preventative treatment starts on Day 0 and continues through the
peak of disease in control (untreated) mice. Therapeutic treatment
starts when the majority of mice show mild signs of arthritis.
Enbrel.RTM., which has been shown to have good efficacy in both the
CIA and G6PI-induced models of arthritis, is used as a positive
control. Data collected in every experiment includes clinical
scores and cumulative incidence of arthritis. Clinical signs of
arthritis in the CIA model are scored using a scale from 0 to 4 as
shown in Table 10 below.
TABLE-US-00010 TABLE 10 Score Observations 0 No apparent swelling
or redness 1 Swelling/redness in one to three digits 2 Redness
and/or swelling in more than three digits, mild swelling extending
into the paw, swollen or red ankle, or mild swelling/redness of
forepaw 3 Swollen paw with mild to moderate redness 4 Extreme
redness and swelling in entire paw
[0254] (b) G6PI Model
[0255] In the G6PI model, arthritis is induced by immunization of
DBA/1 mice with G6PI in adjuvant (Kamradt and Schubert (2005)
Arthritis Res. Ther. 7:20; Schubert et al., (2004) J. Immunol.
172:4503; Bockermann, R. et al. (2005) Arthritis Res. Ther.
7:R1316; Iwanami et al., (2008) Arthritis Rheum. 58:754; Matsumoto
et al., (2008) Arthritis Res. Ther. 10:R66). G6PI is an enzyme
present in virtually all cells in the body and it is not known why
immunization induces a joint specific disease. A number of agents,
such as CTLA4-Ig, TNF antagonists (e.g. Enbrel.RTM.) and anti-IL6
receptor monoclonal antibody, have been shown to inhibit
development of arthritis in the G6PI model.
[0256] Male DBA/1 mice are immunized with G6PI in Complete Freund's
Adjuvant (CFA) in order to induce arthritis. Specifically, mice are
injected intradermally/subcutaneously with G6PI in CFA on Day 0 and
develop clinical signs of arthritis within days of the
immunization. As with the CIA model discussed above, mice are
treated with xceptor, vehicle (PBS), or negative or positive
control in a preventative and/or therapeutic regimen. Preventative
treatment starts on Day 0 and continues through the peak of disease
in control mice. Therapeutic treatment starts when the majority of
mice show mild signs of arthritis. Enbrel.RTM., which has been
shown to have good efficacy in both the CIA and G6PI-induced models
of arthritis, is used as a positive control. Data collected in
every experiment includes clinical scores and cumulative incidence
of arthritis. Clinical signs of arthritis in the G6PI model are
scored using a scale similar to that employed for the CIA
model.
[0257] While this invention has been described in conjunction with
the specific embodiments outlined herein, it is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art. Accordingly, the embodiments of this
disclosure as set forth above are intended to be illustrative, not
limiting. Various changes may be made without departing from the
spirit and scope of this disclosure as defined in the following
claims. All of the patents, patent application publications, patent
applications, and non-patent publications referred to in this
specification and/or listed in the Application Data Sheet, are
incorporated herein by reference, in their entirety.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20170015747A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20170015747A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References