U.S. patent application number 12/459291 was filed with the patent office on 2009-12-24 for soluble ctla4 mutant molecules and uses thereof.
This patent application is currently assigned to Bristol-Myers Squibb Company. Invention is credited to William Brady, Nitin K. Damle, Jeffrey A. Ledbetter, Peter S. Linsley, Philip M. Wallace.
Application Number | 20090317397 12/459291 |
Document ID | / |
Family ID | 34595724 |
Filed Date | 2009-12-24 |
United States Patent
Application |
20090317397 |
Kind Code |
A1 |
Linsley; Peter S. ; et
al. |
December 24, 2009 |
Soluble CTLA4 mutant molecules and uses thereof
Abstract
The invention identifies the CTLA4 receptor as a ligand for the
B7 antigen. The complete amino acid sequence encoding human CTLA4
receptor gene is provided. Methods are provided for expressing
CTLA4 as an immunoglobulin fusion protein, for preparing hybrid
CTLA4 fusion proteins, and for using the soluble fusion proteins,
fragments and derivatives thereof, including monoclonal antibodies
reactive with B7 and CTLA4, to regulate T cell interactions and
immune responses mediated by such interactions.
Inventors: |
Linsley; Peter S.; (Seattle,
WA) ; Ledbetter; Jeffrey A.; (Seattle, WA) ;
Damle; Nitin K.; (Monmouth Junction, NJ) ; Brady;
William; (Bothell, WA) ; Wallace; Philip M.;
(Seattle, WA) |
Correspondence
Address: |
LOUIS J. WILLE;BRISTOL-MYERS SQUIBB COMPANY
PATENT DEPARTMENT, P O BOX 4000
PRINCETON
NJ
08543-4000
US
|
Assignee: |
Bristol-Myers Squibb
Company
|
Family ID: |
34595724 |
Appl. No.: |
12/459291 |
Filed: |
June 30, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11725384 |
Mar 19, 2007 |
7572772 |
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12459291 |
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10461000 |
Jun 13, 2003 |
7311910 |
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11725384 |
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09454651 |
Dec 6, 1999 |
6887471 |
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10461000 |
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08228208 |
Apr 15, 1994 |
6090914 |
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09454651 |
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08008898 |
Jan 22, 1993 |
5770197 |
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08228208 |
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07723617 |
Jun 27, 1991 |
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08008898 |
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Current U.S.
Class: |
424/139.1 ;
435/320.1; 435/375; 435/69.7; 514/1.1; 530/350; 530/387.3;
536/23.4 |
Current CPC
Class: |
Y10S 514/885 20130101;
C07K 2319/00 20130101; C07K 16/2818 20130101; C07K 16/00 20130101;
C07K 2317/73 20130101; A61K 2039/505 20130101; C07K 14/70521
20130101; C07K 16/247 20130101; A61K 38/00 20130101; C07K 2317/34
20130101; C07K 16/2827 20130101; C07K 2319/30 20130101; C07K
14/7155 20130101 |
Class at
Publication: |
424/139.1 ;
530/387.3; 536/23.4; 435/320.1; 435/69.7; 530/350; 435/375;
514/12 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/00 20060101 C07K016/00; C12P 21/04 20060101
C12P021/04; C12N 15/63 20060101 C12N015/63; C07K 14/00 20060101
C07K014/00; C12N 5/02 20060101 C12N005/02; A61K 38/16 20060101
A61K038/16 |
Claims
1. A soluble CTLA4 mutant molecule having the extracellular domain
of CTLA4 which binds B7-1 and/or B7-2.
2. The soluble CTLA4 mutant molecule of claim 1, further comprising
an amino acid sequence which alters the solubility of the soluble
CTLA4 mutant molecule.
3. The soluble CTLA4 mutant molecule of claim 2, wherein the amino
acid sequence comprises a human immunoglobulin constant region.
4. The soluble CTLA4 mutant molecule of claim 1, wherein the
soluble CTLA4 mutant molecule is any of HS2, HS4, HS6, HS7, HS8,
and HS9.
5. The soluble CTLA4 mutant molecule of claim 1, wherein the
soluble CTLA4 mutant molecule comprises an extracellular domain of
CTLA4 having the amino acid motif MYPPPY (SEQ ID NO:35), wherein
the second proline in the amino acid motif MYPPPY (SEQ ID NO:35) is
replaced with alanine.
6. A nucleic acid molecule comprising a nucleotide sequence
encoding the amino acid sequence corresponding to the soluble CTLA4
mutant molecule of claim 1.
7. A vector comprising the nucleotide sequence of claim 6.
8. A host vector system comprising the vector of claim 7 in a
suitable host cell.
9. The host vector system of claim 8, wherein the suitable host
cell is a prokaryotic cell or a eukaryotic cell.
10. A method for producing a soluble CTLA mutant protein comprising
growing the host vector system of claim 8 so as to produce the
protein in the host cell and recovering the protein so
produced.
11. A soluble CTLA mutant protein produced by expressing the mutant
molecule of claim 1 in a host vector system.
12. A method for regulating a T cell interaction with a B7-1 and/or
B7-2 positive cell comprising contacting the B7-1 and/or B7-2
positive cell with the soluble CTLA4 mutant molecule of claim 1 so
as to regulate the T cell interaction.
13. The method of claim 12, wherein the soluble CTLA4 mutant
molecule is any of HS2, HS4, HS6, HS7, HS8, and HS9.
14. The method of claim 12, wherein the soluble CTLA4 mutant
molecule comprises an extracellular domain of CTLA4 having the
amino acid motif MYPPPY, wherein the second proline in the amino
acid motif MYPPPY (SEQ ID NO:35) is replaced with alanine.
15. The method of claim 12, wherein the B7-1 and/or B7-2 positive
cell is an antigen presenting cell.
16. The method of claim 12, wherein the interaction of the
CTLA4-positive T cells with the B7-1 and/or B7-2 positive cells is
inhibited.
17. A method for treating a pathological condition mediated by T
cell interactions with B7-1 and/or B7-2 positive cells comprising
administering to a subject the soluble CTLA4 mutant molecule of
claim 1, in an amount effective to regulate T cell interactions
with said B7-1 and/or B7-2 positive cells.
18. The method of claim 17, wherein said T cell interactions are
inhibited.
19. The method of claim 17, wherein the pathological condition is
graft versus host disease.
20. A method for inhibiting the interaction of CTLA4 positive T
cells with B7-1 and/or B7-2 positive cells in vivo comprising
contacting CTLA4-positive T cells with an antibody or antibody
fragment, reactive with an extracellular domain of CTLA4, wherein
CTLA4 comprises amino acids 1-187 of SEQ ID NO:14, thereby
inhibiting CTLA4-positive T cell interactions with B7-1 and/or B7-2
positive cells.
21. A method for treating a pathological condition in a subject
comprising contacting in the subject CTLA4-positive T cells with an
antibody or antibody fragment, reactive with an extracellular
domain of CTLA4, wherein CTLA4 comprises amino acids 1-187 of SEQ
ID NO:14, thereby treating the pathological condition.
22. A method for blocking CTLA4-B7-1 and/or CTLA4-B7-2 interactions
in the subject comprising contacting in the subject CTLA4-positive
T cells with an antibody or antibody fragment, reactive with an
extracellular domain of CTLA4, wherein CTLA4 comprises amino acids
1-187 of SEQ ID NO:14, thereby blocking CTLA4-B7-1 and/or
CTLA4-B7-2 interactions in the subject.
23. The method of claims 20, 21, or 22, wherein the antibody or
antibody fragment recognizes and binds the extracellular domain of
human CTLA4 comprising amino acids 1-125 of SEQ ID NO:14.
24. The method of claims 20, 21, or 22, wherein the antibody or
antibody fragment recognizes and binds the extracellular domain of
CTLA4 comprising amino acids 1-125 of SEQ ID NO:14, and wherein
said binding prevents the binding of CTLA4 to B7-1 and/or B7-2
antigen.
25. The method of claims 20, or 21, wherein the said antibody or
antibody fragment blocks CTLA4-B7-1 and/or CTLA4-B7-2
interactions.
26. The method of claims 20, 21, or 22, wherein the antibody
fragment is a Fab, F(ab').sub.2, or Fv fragment.
27. The method of claims 20, 21, or 22, wherein the antibody or
antibody fragment is a monoclonal antibody.
Description
[0001] This application is a divisional of U.S. Ser. No.
11/725,384, filed Mar. 19, 2007, which is a divisional 10/461,000,
filed Jun. 13, 2003, which is a divisional of U.S. Ser. No.
09/454,651, filed Dec. 6, 1999, now U.S. Pat. No. 6,887,471, issued
on May 3, 2005, which was a divisional of U.S. Ser. No. 08/228,208,
filed Apr. 15, 1994, now U.S. Pat. No. 6,090,914, issued on Jul.
18, 2000, which was a continuation-in-part of U.S. Ser. No.
08/008,898, filed Jan. 22, 1993, now U.S. Pat. No. 5,770,197,
issued on Jun. 23, 1998, which was a continuation-in-part of U.S.
Ser. No. 07/723,617, filed Jun. 27, 1991, now abandoned, the
contents of all of which are incorporated by reference in their
entirety into the present application.
[0002] Throughout this application various publications are
referenced. The disclosures of these publications in their
entireties are hereby incorporated by reference into this
application in order to more fully describe the state of the art to
which this invention pertains.
[0003] The present invention relates to expression of CTLA4 hybrid
fusion proteins, the CTLA4 receptor gene, identification of the
interaction between the CTLA4 receptor and cells expressing B7
antigen, and to methods for regulating cellular interactions
involving the CTLA4 receptor and the B7 antigen.
BACKGROUND OF THE INVENTION
[0004] The hallmark of a vertebrate immune system is the ability to
discriminate "self" from "non-self" (foreign). This property has
led to the evolution of a system requiring multiple signals to
achieve optimal immune activation (Janeway, Cold Spring Harbor
Symp. Quant. Biol. 54:1-14 (1989)). T cell-B cell interactions are
essential to the immune response. Levels of many cohesive molecules
found on T cells and B cells increase during an immune response
(Springer et al., (1987), supra; Shaw and Shimuzu, Current Opinion
in Immunology, Eds. Kindt and Long, 1:92-97 (1988)); and Hemler
Immunology Today 9:109-113 (1988)). Increased levels of these
molecules may help explain why activated B cells are more effective
at stimulating antigen-specific T cell proliferation than are
resting B cells (Kaiuchi et al., J. Immunol. 131:109-114 (1983);
Kreiger et al., J. Immunol. 135:2937-2945 (1985); McKenzie, J.
Immunol. 141:2907-2911 (1988); and Hawrylowicz and Unanue, J.
Immunol. 141:4083-4088 (1988)).
[0005] The generation of a T lymphocyte ("T cell") immune response
is a complex process involving cell-cell interactions (Springer et
al., A. Rev. Immunol. 5:223-252 (1987)), particularly between T and
accessory cells such as B cells, and production of soluble immune
mediators (cytokines or lymphokines) (Dinarello and Mier, New Engl.
Jour. Med 317:940-945 (1987)). This response is regulated by
several T-cell surface receptors, including the T-cell receptor
complex (Weiss et al., Ann. Rev. Immunol. 4:593-619 (1986)) and
other "accessory" surface molecules (Springer et al., (1987)
supra). Many of these accessory molecules are naturally occurring
cell surface differentiation (CD) antigens defined by the
reactivity of monoclonal antibodies on the surface of cells
(McMichael, Ed., Leukocyte Typing III, Oxford Univ. Press, Oxford,
N.Y. (1987)).
[0006] Antigen-independent intercellular interactions involving
lymphocyte accessory molecules are essential for an immune response
(Springer et al., (1987), supra). For example, binding of the T
cell-associated protein, CD2, to its ligand LFA-3, a widely
expressed glycoprotein (reviewed in Shaw and Shimuzu, supra), is
important for optimizing antigen-specific T cell activation
(Moingeon et al., Nature 339:314 (1988)).
[0007] An important adhesion system involves binding of the LFA-1
glycoprotein found on lymphocytes, macrophages, and granulocytes
(Springer et al., (1987), supra; Shaw and Shimuzu (1988), supra) to
its ligands ICAM-1 (Makgoba et al., Nature 331:86-88 (1988)) and
ICAM-2 (Staunton et al., Nature 339:61-64 (1989)). The T cell
accessory molecules CD8 and CD4 strengthen T cell adhesion by
interaction with MHC class I (Norment et al., Nature 336:79-81
(1988)) and class II (Doyle and Strominger, Nature 330:256-259
(1987)) molecules, respectively. "Homing receptors" are important
for control of lymphocyte migration (Stoolman, Cell 56:907-910
(1989)).
[0008] The VLA glycoproteins are integrins which appear to mediate
lymphocyte functions requiring adhesion to extracellular matrix
components (Hemler, supra). The CD2/LFA-3, LFA-1/ICAM-1 and ICAM-2,
and VLA adhesion systems are distributed on a wide variety of cell
types (Springer et al., (1987), supra; Shaw and Shimuzu, (1988,)
supra and Hemler, (1988), supra).
[0009] Numerous in vitro studies have demonstrated that cytokines
are involved in the generation of alloreactive effector cells. For
example, membrane bound IL-4 and soluble IL-4 receptor were
administered separately to mice and were shown to augment the
lymphoproliferative response (William C. Fanslow et al. "Regulation
of Alloreactivity in vivo by IL-4 and the soluble Il-4 receptor" J.
Immunol. 147:535-540 (1991)). Specifically, administration of IL-4
to BALB/c mice resulted in slight augmentation of the
lymphoproliferative response. In contrast, the soluble IL-4
receptor suppressed this response to allogeneic cells in a dose
dependent manner. Moreover, a neutralizing antibody against IL-4
and another against soluble IL-4 receptor were effective inhibitors
of the lymphoproliferative response.
[0010] It was proposed many years ago that B lymphocyte activation
requires two signals (Bretscher and Cohn, Science 169:1042-1049
(1970)) and now it is believed that all lymphocytes require two
signals for their optimal activation, an antigen specific or clonal
signal, as well as a second, antigen non-specific signal (Janeway,
supra). Freeman et al. (J. Immunol. 143(8):2714-2722 (1989))
isolated and sequenced a cDNA clone encoding a B cell activation
antigen recognized by mAb B7 (Freeman et al., J. Immunol. 138:3260
(1987)). COS cells transfected with this cDNA have been shown to
stain by both labeled mAb B7 and mAb BB-1 (Clark et al., Human
Immunol. 16:100-113 (1986); Yokochi et al., J. Immunol. 128:823
(1981)); Freeman et al., (1989) supra; and Freedman et al., (1987),
supra)). In addition, expression of this antigen has been detected
on cells of other lineages, such as monocytes (Freeman et al.,
supra).
[0011] The signals required for a T helper cell (T.sub.h) antigenic
response are provided by antigen-presenting cells (APC). The first
signal is initiated by interaction of the T cell receptor complex
(Weiss, J. Clin. Invest. 86:1015 (1990)) with antigen presented in
the context of class II major histocompatibility complex (MHC)
molecules on the APC (Allen, Immunol. Today 8:270 (1987)). This
antigen-specific signal is not sufficient to generate a full
response, and in the absence of a second signal may actually lead
to clonal inactivation or anergy (Schwartz, Science 248:1349
(1990)). The requirement for a second "costimulatory" signal
provided by the MHC has been demonstrated in a number of
experimental systems (Schwartz, supra; Weaver and Unanue, Immunol.
Today 11:49 (1990)). The molecular nature of this second signal(s)
is not completely understood, although it is clear in some cases
that both soluble molecules such as interleukin (IL)-1 (Weaver and
Unanue, supra) and membrane receptors involved in intercellular
adhesion (Springer, Nature 346:425 (1990)) can provide
costimulatory signals.
[0012] CD28 antigen, a homodimeric glycoprotein of the
immunoglobulin superfamily (Aruffo and Seed, Proc. Natl. Acad. Sci.
84:8573-8577 (1987)), is an accessory molecule found on most mature
human T cells (Damle et al., J. Immunol. 131:2296-2300 (1983)).
Current evidence suggests that this molecule functions in an
alternative T cell activation pathway distinct from that initiated
by the T-cell receptor complex (June et al., Mol. Cell. Biol.
7:4472-4481 (1987)). Monoclonal antibodies (mAbs) reactive with
CD28 antigen can augment T cell responses initiated by various
polyclonal stimuli (reviewed by June et al., supra). These
stimulatory effects may result from mAb-induced cytokine production
(Thompson et al., Proc. Natl. Acad. Sci. 86:1333-1337 (1989); and
Lindsten et al., Science 244:339-343 (1989)) as a consequence of
increased mRNA stabilization (Lindsten et al., (1989), supra).
Anti-CD28 mAbs can also have inhibitory effects, i.e., they can
block autologous mixed lymphocyte reactions (Damle et al., Proc.
Natl. Acad. Sci. 78:5096-6001 (1981)) and activation of
antigen-specific T cell clones (Lesslauer et al., Eur. J. Immunol.
16:1289-1296 (1986)).
[0013] Studies have shown that CD28 is a counter-receptor for the B
cell activation antigen, B7/BB-1 (Linsley et al, Proc. Natl. Acad.
Sci. USA 87:5031-5035 (1990)). For convenience the B7/BB-1 antigen
is hereafter referred to as the "B7 antigen". The B7 ligands are
also members of the immunoglobulin superfamily but have, in
contrast to CD28 and CTLA4, two Ig domains in their extracellular
region, an N-terminal variable (V)-like domain followed by a
constant (C)-like domain.
[0014] An important non-specific costimulatory signal is delivered
to the T cell when there are at least two homologous B7 family
members found on APC's, B7-1 (also called B7 or CD80) and B7-2,
both of which can deliver costimulatory signals to T cells via
either CD28 or CTLA4. Costimulation through CD28 or CTLA4 is
essential for T cell activation since a soluble Ig fusion protein
of CTLA4 (CTLA4-Ig) has successfully been used to block T cell
activation events in vitro and in vivo. Failure to deliver this
second signal may lead to clonal inactivation or T cell anergy.
[0015] Interactions between CD28 and B7 antigen have been
characterized using genetic fusions of the extracellular portions
of B7 antigen and CD28 receptor, and Immunoglobulin (Ig)
C.hoarfrost.1 (constant region heavy chains) (Linsley et al, J.
Exp. Med. 173:721-730 (1991)). Immobilized B7Ig fusion protein, as
well as B7 positive CHO cells, have been shown to costimulate T
cell proliferation.
[0016] T cell stimulation with B7 positive CHO cells also
specifically stimulates increased levels of transcripts for IL-2.
Additional studies have shown that anti-CD28 mAb inhibited IL-2
production induced in certain T cell leukemia cell lines by
cellular interactions with a B cell leukemia line (Kohno et al.,
Cell. Immunol. 131-1-10 (1990)).
[0017] CD28 has a single extracellular variable region (V)-like
domain (Aruffo and Seed, supra). A homologous molecule, CTLA4 has
been identified by differential screening of a murine cytolytic-T
cell cDNA library (Brunet et al., Nature 328:267-270 (1987)).
[0018] Transcripts of the CTLA4 molecule have been found in T cell
populations having cytotoxic activity, suggesting that CTLA4 might
function in the cytolytic response (Brunet et al., supra; and
Brunet et al., Immunol. Rev. 103-21-36 (1988)). Researchers have
reported the cloning and mapping of a gene for the human
counterpart of CTLA4 (Dariavach et al., Eur. J. Immunol.
18:1901-1905 (1988)) to the same chromosomal region (2q33-34) as
CD28 (Lafage-Pochitaloff et al., Immunogenetics 31:198-201
(1990)).
[0019] An Ig fusion of CTLA4 binds to B7-1 with .about.20 fold
higher avidity than a corresponding Ig fusion of CD28.
[0020] Sequence comparison between this human CTLA4 DNA and that
encoding CD28 proteins reveals significant homology of sequence,
with the greatest degree of homology in the juxtamembrane and
cytoplasmic regions (Brunet et al., 1988, supra; Dariavach et al.,
1988, supra).
[0021] The high degree of homology between CD28 and CTLA4, together
with the co-localization of their genes, raises questions as to
whether these molecules are also functionally related. However,
since the protein product of CTLA4 has not yet been successfully
expressed, these questions remain unanswered.
[0022] Expression of soluble derivatives of cell-surface
glycoproteins in the immunoglobulin gene superfamily has been
achieved for CD4, the receptor for HIV-1, and CD28 and B7
receptors, using hybrid fusion molecules consisting of DNA
sequences encoding amino acids corresponding to portions of the
extracellular domain of CD4 receptor fused to antibody domains
(immunoglobulin.hoarfrost.1 (Capon et al., Nature 337:525-531
(1989) (CD4) and Linsley et al., J. Exp. Med., supra (CD28 and
B7)).
[0023] There is a need for molecules which can identify in vitro B7
positive B cells, i.e., activated B cells, for leukocyte typing and
FAC sorting. Further, there is a need for molecules which may be
used to prevent the rejection of organ transplants and inhibit the
symptoms associated with lupus erythmatosus and other autoimmune
diseases. In the past, major therapies relied on
panimmunosuppressive drugs, such as cyclosporine A or monoclonal
antibodies (MAbs) to CD3 to prevent organ transplants or inhibit
symptoms of lupus. Unfortunately, these drugs must frequently be
taken for the life of the individual, depress the entire immune
system, and often produce secondary health ailments such as
increased frequency of infections and cancer.
SUMMARY OF THE INVENTION
[0024] Accordingly, the present invention provides the complete and
correct DNA sequence encoding the amino acid sequence corresponding
to the CTLA4 receptor protein, and identifies B7 antigen (e.g. B7-1
and B7-2 antigens) as a natural ligand for the CTLA4 receptor. The
invention also provides a method for expressing the DNA as a CTLA4
immunoglobulin (Ig) fusion protein product. Embodiments of the
invention include CTLA4Ig fusion protein, and hybrid fusion
proteins including CD28/CTLA4Ig fusion proteins (which is also
referred to herein as the CTLA4/CD28Ig fusion protein). Also
provided are methods for using the CTLA4 fusion protein, B7Ig
fusion protein, hybrid fusion proteins, and fragments and/or
derivatives thereof, such as monoclonal antibodies reactive with
CTLA4 and the B7 antigen, to regulate cellular interactions and
immune responses.
[0025] The human CTLA receptor protein of the invention is encoded
by 187 amino acids and includes a newly identified N-linked
glycosylation site.
[0026] The CTLA4Ig fusion protein of the invention binds the B7
antigen expressed on activated B cells, and cells of other
lineages, a ligand for CD28 receptor on T cells. The CTLA4 Ig binds
B7 antigen with significantly higher affinity than B7 binding to
the CD28 receptor. The CTLA4Ig construct has a first amino acid
sequence corresponding to the extracellular domain of the CTLA4
receptor fused to a second amino acid sequence corresponding to the
human Ig C.hoarfrost.1 domain. The first amino acid sequence
contains amino acid residues from about position 1 to about
position 125 of the amino acid sequence corresponding to the
extracellular domain of CTLA4 joined to a second amino acid
sequence containing amino acid residues corresponding to the hinge,
CH2 and CH3 regions of human IgC.hoarfrost.1. The fusion protein is
preferably produced in dimeric form. Soluble CTLA4Ig is a potent
inhibitor in vitro of T and B lymphocyte responses.
[0027] Also contemplated in the invention are soluble CTLA4 and
hybrid fusion proteins thereof, e.g., soluble hybrid fusion
proteins, such as CD28/CTLA4Ig fusion proteins. The extracellular
domain of CTLA4 is an example of a soluble CTLA4 molecule.
Alternatively, a molecule having the extracellular domain of CTLA4
attached to a peptide tag is another example of a soluble CTLA4
molecule.
[0028] As an example of a soluble hybrid fusion protein, the
present invention provides CD28/CTLA4Ig fusion proteins having a
first amino acid sequence corresponding to fragments of the
extracellular domain of CD28 joined to a second amino acid sequence
corresponding to fragments of the extracellular domain of CTLA4Ig
and a third amino acid sequence corresponding to the hinge, CH2 and
CH3 regions of human IgC.gamma.1. One embodiment of the hybrid
fusion proteins is a CD28/CTLA4 Ig fusion construct having a first
amino acid sequence containing amino acid residues from about
position 1 to about position 94 of the amino acid sequence
corresponding to the extracellular domain of CD28, joined to a
second amino acid sequence containing amino acid residues from
about position 94 to about position 125 of the amino acid sequence
corresponding to the extracellular domain of CTLA4, joined to a
third amino acid sequence containing amino acids residues
corresponding to the hinge, CH2 and CH3 regions of human
IgC.gamma.1. Other embodiments of the hybrid fusion proteins of the
invention are described in Tables I and II and Example 7.
[0029] Also included in the invention is a method for regulating T
cell interactions with other cells by inhibiting the interaction of
CTLA4-positive T cells with B7 positive cells by reacting the T
cells with ligands for the CTLA4 receptor. The ligands include B7Ig
fusion protein, a monoclonal antibody reactive with CTLA4 receptor,
and antibody fragments.
[0030] The invention also provides a method for regulating T cell
interactions with B7 positive cells, using a ligand for the B7
antigen. Such a ligand is soluble CTLA4 fusion protein, e.g.,
CTLA4Ig fusion protein, of the invention, its fragments or
derivatives, soluble CD28/CTLA4 hybrid fusion protein, e.g., the
CD28/CTLA4Ig hybrid fusion protein, or a monoclonal antibody
reactive with the B7 antigen.
[0031] The invention further includes a method for treating immune
system diseases mediated by T cell interactions with B7 positive
cells by administering a ligand reactive with B7 antigen to
regulate T cell interactions with B7 positive cells. The ligand is
the CTLA4Ig fusion protein, or the CD28/CTLA4Ig fusion protein
hybrid, or a monoclonal antibody reactive with B7 antigen.
[0032] A monoclonal antibody reactive with soluble CTLA4 fusion
protein and a monoclonal antibody reactive with soluble CD28/CTLA4
fusion protein are described for use in regulating cellular
interactions.
[0033] A novel Chinese Hamster Ovary cell line stably expressing
the CTLA4Ig fusion protein is also disclosed.
[0034] Further, the present invention provides a method for
blocking B7 interaction so as to regulate the immune response. This
method comprises contacting lymphocytes with a B7-binding molecule
and an IL4-binding molecule.
[0035] Additionally, the present invention provides a method for
regulating an immune response which comprises contacting
B7-positive lymphocytes with a B7-binding molecule and an
IL4-binding molecule.
[0036] Also, the invention provides method for inhibiting tissue
transplant rejection by a subject, the subject being a recipient of
transplanted tissue. This method comprises administering to the
subject a B7-binding molecule and an IL4-binding molecule.
[0037] The present invention further provides a method for
inhibiting graft versus host disease in a subject which comprises
administering to the subject a B7-binding molecule and an
IL4-binding molecule.
BRIEF DESCRIPTION OF THE FIGURES
[0038] FIG. 1 is a diagrammatic representation of CTLA4Ig fusion
constructs (SEQ ID NO:33; SEQ ID NO:34) as described in Example 2,
infra.
[0039] FIG. 2 is a photograph of a gel obtained from SDS-PAGE
chromatographic purification of CTLA4Ig as described in Example 2,
infra.
[0040] FIG. 3 depicts the complete amino acid sequence encoding
human CTLA4 receptor (SEQ ID NOs: 13 and 14) fused to the
oncostatin M signal peptide (position -25 to -1), and including the
newly identified N-linked glycosylation site (position 109-111), as
described in Example 3, infra.
[0041] FIG. 4 depicts the results of FACS.sup.R analysis of binding
of the B7Ig fusion protein to CD28- and CTLA4-transfected COS cells
as described in Example 4, infra.
[0042] FIG. 5 depicts the results of FACS.sup.R analysis of binding
of purified CTLA4Ig on B7 antigen-positive (B7.sup.+) CHO cells and
on a lymphoblastoid cell line (PM LCL) as described in Example 4,
infra.
[0043] FIG. 6 is a graph illustrating competition binding analysis
of .sup.125I labeled B7Ig to immobilized CTLA4Ig as described in
Example 4, infra.
[0044] FIG. 7 is a graph showing the results of Scatchard analysis
of .sup.125I-labeled B7Ig binding to immobilized CTLA4Ig as
described in Example 4, infra.
[0045] FIG. 8 is a photograph of a gel from SDS-PAGE chromatography
of immunoprecipitation analysis of B7 positive CHO cells and PM LCL
cells surface-labeled with .sup.125I as described in Example 4,
infra.
[0046] FIG. 9 is a graph depicting the effects on proliferation of
T cells of CTLA4Ig as measured by [.sup.3H]-thymidine incorporation
as described in Example 4, infra.
[0047] FIG. 10 is a bar graph illustrating the effects of CTLA4Ig
on helper T cell (T.sub.h)-induced immunoglobulin secretion by
human B cells as determined by enzyme immunoassay (ELISA) as
described in Example 4, infra.
[0048] FIGS. 11A, 11B, and 11C are line graphs showing the survival
of human pancreatic islet xenografts.
[0049] FIGS. 12A, 12B, 12C, and 12D are photographs of
histopathology slides of human islets transplanted under the kidney
capsule of B10 mice.
[0050] FIG. 13 is a line graph showing the prolongation of islet
graft survival with MAb to human B7.
[0051] FIG. 14 is a line graph showing induction of donor-specific
unresponsiveness to islet graft antigens by CTLA4Ig.
[0052] FIG. 15 is a line graph showing antibody serum titer levels
of mice injected with sheep red blood cells (SRBC), mAb L6 and rat
Ig, mAb L6 and anti-IL4, CTLA4Ig and rat Ig, CTLA4Ig and anti-IL4.
The X axis measures the antibody-serum titer. The Y axis measures
time in days. The closed box represents mice injected with SRBC at
day 0 and day 46. The open box represents mice injected with SRBC
at day 46. The closed circle represents mice injected with mAb L6
and rat immunoglobulin. The open circle represents mice injected
with mAb L6 and anti-IL4 antibody. The closed triangle represents
mice injected with CTLA4Ig and rat immunoglobulin. The open
triangle represents mice injected with CTLA4Ig and anti-IL4
antibody.
[0053] FIG. 16 is a line graph showing antibody serum titer levels
of mice injected with KLH, mAb L6 and rat Ig, mAb L6 and anti-IL4,
CTLA4Ig and rat Ig, CTLA4Ig and anti-IL4. The X axis measures the
antibody-serum titer. The Y axis measures time in days. The closed
box represents mice injected with keyhole limpet hemocyanin (KLH)
at day 46. The closed circle represents mice injected with mAb L6
and rat immunoglobulin. The open circle represents mice injected
with mAb L6 and anti-IL4 antibody. The closed triangle represents
mice injected with CTLA4Ig and rat immunoglobulin. The open
triangle represents mice injected with CTLA4Ig and anti-IL4
antibody.
[0054] FIG. 17 is a graph showing the sequencing alignment of CD28
and CTLA4 family members. Sequences of human (H) (SEQ ID NO:21),
mouse (M) (SEQ ID NO:19), rat (R) SEQ ID NO:20, and chicken (Ch)
(SEQ ID NO:22) CD28 are aligned with human and mouse CTLA4 (SEQ ID
NO:17; SEQ ID NO:18). Residues are numbered from the mature protein
N-terminus with the signal peptides and transmembrane domains
underlined and the CDR-analogous regions noted. Dark shaded areas
highlight complete conservation of residues while light shaded
areas highlight conservative amino acid substitutions in all family
members.
[0055] FIG. 18 is a line graph showing CTLA4Ig and CD28Ig mutants
(SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:27; SEQ ID NO:29) bind
B7-1.
[0056] FIG. 19 is a schematic map of CTLA4/CD28Ig hybrid fusion
proteins. Open areas represent CD28 sequence; filled areas
represent CTLA4 sequence; cross-hatched areas represent beginning
of IgG Fc (also refer to Table I).
[0057] FIGS. 20a and 20b. A line graph showing that CTLA4/CD28Ig
hybrid fusion proteins bind with high avidity to B7-1 CHO
cells.
[0058] FIG. 21. Molecular model of monomeric CTLA4Ig v-like
extracellular domain.
DETAILED DESCRIPTION OF THE INVENTION
Definition
[0059] As used in this application, the following words or phrases
have the meanings specified.
[0060] As used herein "blocking B7 interaction" means to interfere
with the binding of the B7 antigen to its ligands such as CD28
and/or CTLA4 thereby obstructing T cell and B cell interaction.
[0061] As used herein a "B7-binding molecule" means any molecule
which will bind the B7 antigen.
[0062] As used herein an "IL4-binding molecule" means any molecule
which will recognize and bind to IL4.
[0063] As used herein a "CTLA4 mutant" means a molecule having
amino acids which are similar to the amino acid sequence of the
extracellular domain of CTLA4 so that the molecule recognizes and
binds a B7 antigen.
[0064] As used herein a "CD28 mutant" means a molecule having amino
acids which are similar to the amino acid sequence of the
extracellular domain of CD28 so that the molecule recognizes and
binds a B7 antigen.
[0065] As used herein a "CTLA4/CD28 hybrid fusion protein" is a
molecule having at least portions of the extracellular domains of
both CTLA4 and CD28 so that the molecule recognizes and binds a B7
antigen.
[0066] In order that the invention herein described may be more
fully understood, the following description is set forth.
[0067] This invention is directed to the isolation and expression
of the human CTLA4 receptor found on T cell surfaces, which binds
to the B7 antigen expressed on activated B cells, and cells of
other lineages, and to expression of soluble fusion protein
products of the CTLA4 receptor gene. The invention also provides
methods for using the expressed CTLA4 receptor to regulate cellular
interactions, including T cell interactions with B7 positive
cells.
[0068] In a preferred embodiment, the complete and correct DNA
sequence encoding the amino acid sequence corresponding to human
CTLA4 receptor protein of the invention is cloned using PCR. The
cDNA containing the complete predicted coding sequence of CTLA4 was
assembled from two PCR fragments amplified from H38 RNA, and
inserted into the expression vector, CDM8 as described in detail in
the Examples, infra. Isolates were transfected into COS cells and
tested for binding of B7Ig, a soluble fusion protein having an
amino acid sequence corresponding to the extracellular domain of B7
and a human immunoglobulin (Ig) C.gamma.1 region, as described by
Linsley et al., J. Exp. Med. 173:721-730 (1991).
[0069] The DNA sequence of one isolate, designated as OMCTLA4, was
then determined and found to correspond exactly to the predicted
human CTLA4 sequence, fused at the N-terminus to the signal peptide
from oncostatin M. The CTLA4 receptor is encoded by 187 amino acids
(exclusive of the signal peptide and stop codons) and includes a
newly identified N-linked glycosylation site at amino acid
positions 109-111 (see FIG. 3, infra). The CTLA4 receptor is
expressed using the oncostatin M signal peptide.
[0070] In another preferred embodiment, soluble forms of the
protein product of the CTLA4 receptor gene (CTLA4Ig) are prepared
using fusion proteins having a first amino acid sequence
corresponding to the extracellular domain of CTLA4 and a second
amino acid sequence corresponding to the human IgC.gamma.1
domain.
[0071] Cloning and expression plasmids (CDM8 and .pi.LN) were
constructed containing cDNAs encoding portions of the amino acid
sequence corresponding to human CTLA4 receptor based on the cDNA
sequence described herein, where the cDNA encoding a first amino
acid sequence corresponding to a fragment of the extracellular
domain of the CTLA4 receptor gene is joined to DNA encoding a
second amino acid sequence corresponding to an IgC region that
permits the expression of the CTLA4 receptor gene by altering the
solubility of the expressed CTLA4 protein.
[0072] Thus, soluble CTLA4Ig fusion protein is encoded by a first
amino acid sequence containing amino acid residues from about
position 1 to about position 125 of the amino acid sequence
corresponding to the extracellular domain of CTLA4 joined to a
second amino acid sequence containing amino acid residues
corresponding to the hinge, CH2 and CH3 regions of human
IgC.gamma.1. The fusion protein is preferably produced in dimeric
form. The construct was then transfected into COS or CHO cells, and
CTLA4Ig was purified and identified as a dimer.
[0073] In accordance with the practice of this invention, CTLA4Ig
and the CTLA4/CD28 fusion protein hybrid may have amino acid
substitutions in the amino acid sequence corresponding to the
external domain of CTLA4 so as to produce molecules which would
retain the functional property of CTLA4, namely, the molecule
having such substitutions will still bind the B7 antigen. These
amino acid substitutions include, but are not necessarily limited
to, amino acid substitutions known in the art as
"conservative".
[0074] For example, it is a well-established principle of protein
chemistry that certain amino acid substitutions, entitled
"conservative amino acid substitutions," can frequently be made in
a protein without altering either the conformation or the function
of the protein.
[0075] Such changes include substituting any of isoleucine (D,
valine (V), and leucine (L) for any other of these hydrophobic
amino acids; aspartic acid (D) for glutamic acid (E) and vice
versa; glutamine (Q) for asparagine (N) and vice versa; and serine
(S) for threonine (T) and vice versa. Other substitutions can also
be considered conservative, depending on the environment of the
particular amino acid and its role in the three-dimensional
structure of the protein. For example, glycine (G) and alanine (A)
can frequently be interchangeable, as can alanine and valine
(V).
[0076] Methionine (M), which is relatively hydrophobic, can
frequently be interchanged with leucine and isoleucine, and
sometimes with valine. Lysine (K) and arginine (R) are frequently
interchangeable in locations in which the significant feature of
the amino acid residue is its charge and the differing pK's of
these two amino acid residues are not significant. Still other
changes can be considered "conservative" in particular
environments.
[0077] In fact, using the methodologies disclosed herein, mutants
of the B7-binding molecule were produced. One mutant comprises (1)
a sequence beginning with the amino acid at position 1 and ending
with the amino acid at position 95 of the CD28 receptor protein;
(2) a sequence beginning with the amino acid at position 95 and
ending with amino acid at position 125 of the extracellular domain
of CTLA4; and (3) a sequence corresponding to the human IgC.gamma.1
domain.
[0078] The second mutant comprises (1) a sequence beginning with
the amino acid at position 1 and ending with the amino acid at
position 95 of the CD28 receptor protein; (2) a sequence beginning
with the amino acid at position 95 and ending with amino acid at
position 120 of the extracellular domain of CTLA4; and (3) a
sequence corresponding to the human IgC.gamma.1 domain.
[0079] The present invention provides a method for blocking B7
interaction so as to regulate the immune response which comprises
contacting lymphocytes with a B7-binding molecule and an
IL4-binding molecule. The lymphocytes may be B7 positive
lymphocytes.
[0080] Further, the present invention provides a method for
regulating an immune response which comprises contacting
B7-positive lymphocytes with a B7-binding molecule and an
IL4-binding molecule.
[0081] The immune response may be a B cell response resulting in
the inhibition of antibody production. Additionally, the immune
response may be a T cell response resulting in inhibition of cell
mediated immunity. Further, the immune response may be an
inhibition of lymphocyte proliferation.
[0082] Also, the present invention provides a method for inhibiting
tissue transplant rejection by a subject, the subject being a
recipient of transplanted tissue. This method can comprise
administering to the subject a B7-binding molecule and an
IL4-binding molecule.
[0083] The invention further provides a method for inhibiting graft
versus host disease in a subject which comprises administering to
the subject a B7-binding molecule and an IL4-binding molecule.
[0084] In accordance with the practice of this invention, the
B7-binding molecule may be a CTLA4Ig fusion protein. For example,
the CTLA4Ig fusion protein may be a fusion protein having a first
amino acid sequence containing amino acid residues from about
position 1 to about position 125 of the amino acid sequence
corresponding to the extracellular domain of CTLA4 and a second
amino acid sequence containing amino acid residues corresponding to
the hinge, CH2 and CH3 regions of human immunoglobulin
C.gamma.1.
[0085] Alternatively, the B7-binding molecule may be a soluble
CD28/CTLA4 hybrid fusion protein. For example, the CD28/CTLA4 Ig
fusion protein hybrid may be a fusion protein hybrid having a first
amino acid sequence corresponding to a portion of the extracellular
domain of CD28 receptor fused to a second amino acid sequence
corresponding to a portion of the extracellular domain of CTLA4
receptor and a third amino acid sequence corresponding to the
hinge, CH2 and CH3 regions of human immunoglobulin C.gamma.1.
[0086] Further, the IL4-binding molecule may be a monoclonal
antibody which specifically recognizes and binds to IL4.
Alternatively, the IL4-binding molecule is a soluble IL4 receptor
which recognizes and binds to IL4 (Fanslow et al. 1991).
[0087] DNA encoding the amino acid sequence corresponding to the
CTLA4Ig fusion protein has been deposited with the American Type
Culture Collection (ATCC) in Rockville, Md., under the provisions
of the Budapest Treaty on May 31, 1991 and has been accorded ATCC
accession number: 68629.
[0088] The present invention provides the first protein product of
CTLA4 transcripts in the form of a soluble fusion protein. The
CTLA4Ig protein forms a disulfide-linked dimer having two subunits,
each of which has an M.sub.r of approximately 50,000 indicating
that native CTLA4 probably exists on the T cell surface as a
disulfide-linked homodimer.
[0089] B7 antigen has been shown to be a ligand for CD28 receptor
on T cells (Linsley et al., Proc. Natl. Acad. Sci. USA, supra). The
CTLA4 receptor molecule appears functionally and structurally
related to the CD28 receptor; both are receptors for the B cell
activation antigen, B7, while CTLA4 appears to have higher affinity
for B7, among the highest yet reported for lymphoid adhesion
systems. However, CTLA4Ig was shown to bind more strongly to B7
positive (B7.sup.+) cell lines than CD28Ig. Other experiments
demonstrated that CTLA4 is a higher affinity receptor for B7
antigen than CD28 receptor. Additionally, CTLA4Ig was shown to bind
a single protein on lymphoblastoid cells which is similar in size
to the B7 antigen. CTLA4Ig inhibited T cell proliferation and
inhibited T.sub.h-induced IgM production.
[0090] In another preferred embodiment, hybrid fusion proteins
having amino acid sequences corresponding to fragments of different
receptor proteins were constructed. For example, amino acid
sequences corresponding to selected fragments of the extracellular
domains of CD28 and CTLA4 were linked to form soluble CD28/CTLA4
hybrid fusion proteins, e.g. a CD28/CTLA4Ig fusion protein. This
protein was obtained having a first amino acid sequence containing
amino acid residues corresponding to a fragment of the
extracellular domain of CD28 joined to a second amino acid sequence
corresponding to a fragment of the extracellular domain of CTLA4Ig
and to a third amino acid sequence corresponding to the hinge, CH2
and CH3 regions of human IgC.gamma.1.
[0091] One embodiment of the hybrid fusion proteins is a
CD28/CTLA4Ig fusion construct having a first amino acid sequence
containing amino acid residues from about position 1 to about
position 94 of the amino acid sequence corresponding to the
extracellular domain of CD28, joined to a second amino acid
sequence containing amino acid residues from about position 94 to
about position 125 of the amino acid sequence corresponding to the
extracellular domain of CTLA4, joined to a third amino acid
sequence corresponding to the hinge, CH2 and CH3 regions of human
IgC.gamma.1.
[0092] The techniques for cloning and expressing DNA sequences
encoding the amino acid sequences corresponding to the CTLA4
receptor protein, soluble fusion proteins and hybrid fusion
proteins, e.g synthesis of oligonucleotides, PCR, transforming
cells, constructing vectors, expression systems, and the like are
well-established in the art, and most practitioners are familiar
with the standard resource materials for specific conditions and
procedures. However, the following paragraphs are provided for
convenience and notation of modifications where necessary, and may
serve as a guideline.
Cloning and Expression of Coding Sequences for Receptors and Fusion
Proteins
[0093] Fusion protein constructs corresponding to CD28IgC.gamma.1
and B7IgC.gamma.1 for characterizing the CTLA4 Ig of the present
invention, and for preparing CD28/CTLA4 hybrid fusion proteins,
were prepared as described by Linsley et al., J. Exp. Med.
173:721-730 (1991), incorporated by reference herein.
Alternatively, cDNA clones may be prepared from RNA obtained from
cells expressing B7 antigen and CD28 receptor based on knowledge of
the published sequences for these proteins (Aruffo and Seed, and
Freeman, supra) using standard procedures.
[0094] CTLA4Ig fusions consisting of DNA encoding amino acid
sequences corresponding to the extracellular domain of CTLA4 and
the hinge, CH2 and CH3 regions of human IgC.gamma.1 were
constructed by ligation of PCR fragments. The cDNA encoding the
amino acid sequences is amplified using the polymerase chain
reaction ("PCR") technique (U.S. Pat. Nos. 4,683,195 and 4,683,202
to Mullis et al. and Mullis & Faloona, Methods Enzymol.
154:335-350 (1987)). CTLA4Ig fusion polypeptides were obtained
having DNA encoding amino acid sequences containing amino acid
residues from about position 1 to about position 125 of the amino
acid sequence corresponding to the extracellular domain of CTLA4
and DNA encoding amino acid sequences corresponding to the hinge,
CH2 and CH3 regions of Ig C.gamma.1.
[0095] Because the expression of CTLA4 receptor protein in human
lymphoid cells has not been previously reported, it was necessary
to locate a source of CTLA4 mRNA. PCR cDNA made from the total
cellular RNA of several human leukemia cell lines was screened,
using as primers, oligonucleotides from the published sequence of
the CTLA4 gene (Dariavach et al., supra). Of the cDNA tested, H38
cells (an HTLV II-associated leukemia line) provided the best yield
of PCR products having the expected size. Since a signal peptide
for CTLA4 was not identified in the CTLA4 gene, the N terminus of
the predicted sequence of CTLA4 was fused to the signal peptide of
oncostatin M (Malik et al., Molec. and Cell. Biol. 9:2847 (1989))
in two steps using oligonucleotides as described in the Examples,
infra. The product of the PCR reaction was ligated with cDNA
encoding the amino acid sequences corresponding to the hinge, CH2
and CH3 regions of Ig C.gamma.1 into an expression vector, such as
CDM8 or .pi.LN.
[0096] To obtain DNA encoding full length human CTLA4, a cDNA
encoding the transmembrane and cytoplasmic domains of CTLA4 was
obtained by PCR from H38 cells and joined with a fragment from
CTLA4Ig, obtained as described above, encoding the oncostatin M
signal peptide fused to the N terminus of CTLA4, using
oligonucleotide primers as described in the Examples, infra. PCR
fragments were ligated into the plasmid CDM8, resulting in an
expression plasmid encoding the full length CTLA4 gene, and
designated OMCTLA4.
[0097] For construction of DNA encoding the amino acid sequence
corresponding to hybrid fusion proteins, DNA encoding amino acids
corresponding to portions of the extracellular domain of one
receptor gene is joined to DNA encoding amino acids corresponding
to portions of the extracellular domain of another receptor gene,
and to DNA encoding the amino acid sequences corresponding to the
hinge, CH2 and CH3 regions of human IgC.gamma.1 using procedures as
described above for the B7Ig, CD28Ig and CTLA4Ig constructs. Thus,
for example, DNA encoding amino acid residues from about position 1
to about position 94 of the amino acid sequence corresponding to
the extracellular domain of the CD28 receptor is joined to DNA
encoding amino acid residues from about position 94 to about
position 125 of the amino acid sequence corresponding to the
extracellular domain of the CTLA4 receptor and to DNA encoding the
amino acid sequences corresponding to the hinge, CH2 and CH3
regions of human IgC.gamma.1.
[0098] To produce large quantities of cloned DNA, vectors
containing DNA encoding the fusion constructs of the invention are
transformed into suitable host cells, such as the bacterial cell
line E. coli strain MC1061/p3 (Invitrogen Corp., San Diego, Calif.)
using standard procedures, and colonies are screened for the
appropriate plasmids.
[0099] The clones containing DNA encoding fusion constructs
obtained as described above are then transfected into suitable host
cells for expression. Depending on the host cell used, transfection
is performed using standard techniques appropriate to such cells.
For example, transfection into mammalian cells is accomplished
using DEAE-Dextran.TM. mediated transfection, CaPO.sub.4
co-precipitation, lipofection, electroporation, or protoplast
fusion, and other methods known in the art including: lysozyme
fusion or erythrocyte fusion, scraping, direct uptake, osmotic or
sucrose shock, direct microinjection, indirect microinjection such
as via erythrocyte-mediated techniques, and/or by subjecting host
cells to electric currents. The above list of transfection
techniques is not considered to be exhaustive, as other procedures
for introducing genetic information into cells will no doubt be
developed.
[0100] Expression in eukaryotic host cell cultures derived from
multicellular organisms is preferred (Tissue Cultures, Academic
Press, Cruz and Patterson, Eds. (1973)). These systems have the
additional advantage of the ability to splice out introns and thus
can be used directly to express genomic fragments. Useful host cell
lines include Chinese hamster ovary (CHO), monkey kidney (COS),
VERO and HeLa cells. In the present invention, cell lines stably
expressing the fusion constructs are preferred.
[0101] Expression vectors for such cells ordinarily include
promoters and control sequences compatible with mammalian cells
such as, for example, CMV promoter (CDM8 vector) and avian sarcoma
virus (ASV) (.pi.LN vector). Other commonly used early and late
promoters include those from Simian Virus 40 (SV 40) (Fiers, et
al., Nature 273:113 (1973)), or other viral promoters such as those
derived from polyoma, Adenovirus 2, and bovine papilloma virus. The
controllable promoter, hMTII (Karin, et al., Nature 299:797-802
(1982)) may also be used. General aspects of mammalian cell host
system transformations have been described by Axel (U.S. Pat. No.
4,399,216 issued Aug. 16, 1983). It now appears, that "enhancer"
regions are important in optimizing expression; these are,
generally, sequences found upstream or downstream of the promoter
region in non-coding DNA regions. Origins of replication may be
obtained, if needed, from viral sources. However, integration into
the chromosome is a common mechanism for DNA replication in
eukaryotes.
[0102] Although preferred host cells for expression of the fusion
constructs include eukaryotic cells such as COS or CHO cells, other
eukaryotic microbes may be used as hosts. Laboratory strains of
Saccharomyces cerevisiae, Baker's yeast, are most used although
other strains such as Schizosaccharomyces pombe may be used.
Vectors employing, for example, the 2.mu. origin of replication of
Broach, Meth. Enz. 101:307 (1983), or other yeast compatible
origins of replications (for example, Stinchcomb et al., Nature
282:39 (1979)); Tschempe et al., Gene 10:157 (1980); and Clarke et
al., Meth. Enz. 101:300 (1983)) may be used. Control sequences for
yeast vectors include promoters for the synthesis of glycolytic
enzymes (Hess et al., J. Adv. Enzyme Reg. 7:149 (1968); Holland et
al., Biochemistry 17:4900 (1978)). Additional promoters known in
the art include the CMV promoter provided in the CDM8 vector
(Toyama and Okayama, FEBS 268:217-221 (1990); the promoter for
3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255:2073
(1980)), and those for other glycolytic enzymes. Other promoters,
which have the additional advantage of transcription controlled by
growth conditions are the promoter regions for alcohol
dehydrogenase 2, isocytochrome C, acid phosphatase, degradative
enzymes associated with nitrogen metabolism, and enzymes
responsible for maltose and galactose utilization. It is also
believed terminator sequences are desirable at the 3' end of the
coding sequences. Such terminators are found in the 3' untranslated
region following the coding sequences in yeast-derived genes.
[0103] Alternatively, prokaryotic cells may be used as hosts for
expression. Prokaryotes most frequently are represented by various
strains of E. coli; however, other microbial strains may also be
used. Commonly used prokaryotic control sequences which are defined
herein to include promoters for transcription initiation,
optionally with an operator, along with ribosome binding site
sequences, include such commonly used promoters as the
beta-lactamase (penicillinase) and lactose (lac) promoter systems
(Chang et al., Nature 198: 1056 (1977)), the tryptophan (trp)
promoter system (Goeddel et al., Nucleic Acids Res. 8:4057 (1980))
and the lambda derived P.sub.L promoter and N-gene ribosome binding
site (Shimatake et al., Nature 292:128 (1981)).
[0104] The nucleotide sequences encoding CD28Ig and CTLA4 Ig
proteins, and fusion hybrid proteins such as CD28/CTLA4Ig, may be
expressed in a variety of systems as set forth below. The cDNA may
be excised by suitable restriction enzymes and ligated into
suitable prokaryotic or eukaryotic expression vectors for such
expression. Because CD28 and CTLA4 receptor proteins occur in
nature as dimers, it is believed that successful expression of
these proteins requires an expression system which permits these
proteins to form as dimers. Truncated versions of these proteins
(i.e. formed by introduction of a stop codon into the sequence at a
position upstream of the transmembrane region of the protein)
appear not to be expressed. The expression of CD28 and CTLA4
receptors as fusion proteins permits dimer formation of these
proteins. Thus, expression of CTLA4 protein as a fusion product is
preferred in the present invention.
[0105] A stable CHO line of the invention, designated Chinese
Hamster Ovary Cell Line CTLA4Ig-24, is preferred for expression of
CTLA4Ig and has been deposited with the ATCC under the terms of the
Budapest Treaty on May 31, 1991, and accorded ATCC accession number
10762.
[0106] Expression of the CTLA4 receptor of the invention is
accomplished transfecting a cell line such as COS cells, and
detecting expression by binding of the CTLA4-transfected cells to a
ligand for the CTLA4 receptor, for example by testing for binding
of the cells to B7Ig fusion protein.
[0107] Sequences of the resulting constructs are confirmed by DNA
sequencing using known procedures, for example as described by
Sanger et al., Proc. Natl. Acad. Sci. USA 74:5463 (1977), as
further described by Messing et al., Nucleic Acids Res. 9:309
(1981), or by the method of Maxam et al. Methods Enzymol. 65:499
(1980).
Recovery of Protein Products
[0108] As noted above, CD28 and CTLA4 receptor genes are not
readily expressed as mature proteins using direct expression of DNA
encoding the truncated protein. To enable homodimer formation, DNA
encoding the amino acid sequence corresponding to the extracellular
domains of CD28 and CTLA4, and including the codons for a signal
sequence such as that of oncostatin M in cells capable of
appropriate processing, is fused with DNA encoding the amino acid
sequence corresponding to the Fc domain of a naturally dimeric
protein. Purification of these fusion protein products after
secretion from the cells is thus facilitated using antibodies
reactive with the anti-immunoglobulin portion of the fusion
proteins. When secreted into the medium, the fusion protein product
is recovered using standard protein purification techniques, for
example by application to protein A columns.
Use
[0109] CTLA4Ig fusion protein and/or fragments of the fusion
protein may be used to react with B7 positive cells, such as B
cells, to regulate immune responses mediated by T cell interactions
with the B7 antigen positive cells or in vitro for leukocyte typing
so as to define B cell maturational stages and/or B cell associated
diseases (Yokochi et al. J. Immuno. 128(2):823). Surface
immunostaining of leukocytes is accomplished by immunofluorescent
technology or immunoenzymatic methods but other means of detection
are possible.
[0110] Soluble CTLA4 proteins and CTLA4/CD28 hybrid fusion
proteins, and/or fragments and derivatives of these proteins, may
also be used to react with B7 positive cells, including B cells, to
regulate immune responses mediated by T cell dependent B cell
responses. The term "fragment" as used herein means a portion of
the amino acid sequence encoding the protein referred to as
"CTLA4". A fragment of the soluble CTLA4 protein that may be used
is a polypeptide having an amino acid sequence corresponding to
some portion of the amino acid sequence corresponding to the CTLA4
receptor used to obtain the soluble CTLA4 protein as described
herein.
[0111] The B7 antigen expressed on activated B cells and cells of
other lineages, and the CD28 receptor expressed on T cells, can
directly bind to each other, and this interaction can mediate
cell-cell interaction. Such interactions directly trigger the CD28
activation pathway in T cells, leading to cytokine production, T
cell proliferation, and B cell differentiation into immunoglobulin
producing cells. The activation of B cells that occurs, can cause
increased expression of B7 antigen and further CD28 stimulation,
leading to a state of chronic inflammation such as in autoimmune
diseases, allograft rejection, graft versus host disease or chronic
allergic reactions. Blocking or inhibiting this reaction may be
effective in preventing T cell cytokine production and thus
preventing or reversing inflammatory reactions.
[0112] Soluble CTLA4, e.g. CTLA4Ig, is shown herein to be a potent
inhibitor of in vitro lymphocyte functions requiring T and B cell
interaction. This indicates the importance of interactions between
the B7 antigen and its counter-receptors, CTLA4 and/or CD28. The
cytoplasmic domains of murine and human CTLA4 are similar
(Dariavach et al., supra, 1988), suggesting that this region has
important functional properties. The cytoplasmic domains of CD28
and CTLA4 also share homology.
[0113] CTLA4 is a more potent inhibitor in vitro of lymphocyte
responses than either anti-BB1, or anti-CD28 mAbs. CTLA4Ig does not
have direct stimulatory effects on T cell proliferation to
counteract its inhibitory effects. Therefore, the CTLA4Ig fusion
protein may perform as a better inhibitor in vivo than anti-CD28
monoclonal antibodies. The immunosuppressive effects of CTLA4Ig in
vitro suggests its use in therapy for treatment of autoimmune
disorders involving abnormal T cell activation or Ig
production.
[0114] The CTLA4Ig fusion protein is expected to exhibit inhibitory
properties in vivo. Thus, it is expected that CTLA4Ig will act to
inhibit T cells in a manner similar to the effects observed for the
anti-CD28 antibody, under similar conditions in vivo. Under
conditions where T cell/B cell interactions are occurring as a
result of contact between T cells and B cells, binding of
introduced CTLA4Ig to react with B7 antigen positive cells, for
example B cells, may interfere, i.e. inhibit, the T cell/B cell
interactions resulting in regulation of immune responses. Because
of this exclusively inhibitory effect, CTLA4 Ig is expected to be
useful in vivo as an inhibitor of T cell activity, over
non-specific inhibitors such as cyclosporine and glucosteroids.
[0115] In one embodiment, the CTLA4Ig fusion protein or
CTLA4/CD28Ig hybrid proteins, may be introduced in a suitable
pharmaceutical carrier in vivo, i.e. administered into a human
subject for treatment of pathological conditions such as immune
system diseases or cancer.
[0116] Introduction of the fusion protein in vivo is expected to
result in interference with T cell interactions with other cells,
such as B cells, as a result of binding of the ligand to B7
positive cells. The prevention of normal T cell interactions may
result in decreased T cell activity, for example, decreased T cell
proliferation. In addition, administration of the fusion protein in
vivo is expected to result in regulation of in vivo levels of
cytokines, including, but not limited to, interleukins, e.g.
interleukin ("IL")-2, IL-3, IL-4, IL-6, IL-8, growth factors
including tumor growth factor ("TGF"), colony stimulating factor
("CSF"), interferons ("IFNs"), and tumor necrosis factor ("TNF") to
promote desired effects in a subject. For example, when the fusion
protein is introduced in vivo, it may block production of
cytokines, which contribute to malignant growth, for example of
tumor cells. The fusion protein may also block proliferation of
viruses dependent on T cell activation, such as the virus that
causes AIDS, HTLV1.
[0117] Under some circumstances, as noted above, the effect of
administration of the CTLA4Ig fusion protein or its fragments in
vivo is inhibitory, resulting from blocking by the fusion protein
of the CTLA4 and CD28 triggering resulting from T cell/B cell
contact. For example, the CTLA4Ig protein may block T cell
proliferation. Introduction of the CTLA4Ig fusion protein in vivo
will thus produce effects on both T and B cell-mediated immune
responses. The fusion protein may also be administered to a subject
in combination with the introduction of cytokines or other
therapeutic reagents.
[0118] In an additional embodiment of the invention, other
reagents, including derivatives reactive with the CTLA4Ig fusion
protein or the CTLA4 receptor are used to regulate T cell
interactions. For example, antibodies, and/or antibody fragments
reactive with the CTLA4 receptor may be screened to identify those
capable of inhibiting the binding of the CTLA4 Ig fusion protein to
the B7 antigen. The antibodies or antibody fragments such as Fab or
F(ab').sub.2 fragments, may then be used to react with the T cells,
for example, to inhibit T cell proliferation.
[0119] Monoclonal antibodies reactive with CTLA4 receptor, may be
produced by hybridomas prepared using known procedures, such as
those introduced by Kohler and Milstein (Kohler and Milstein,
Nature, 256:495-97 (1975)), and modifications thereof, to regulate
cellular interactions.
[0120] These techniques involve the use of an animal which is
primed to produce a particular antibody. The animal can be primed
by injection of an immunogen (e.g. the B7Ig fusion protein, CTLA4Ig
fusion protein or CD28/CTLA4Ig hybrid fusion protein or other
functional, soluble forms thereof) to elicit the desired immune
response, i.e. production of antibodies from the primed animal. A
primed animal is also one which is expressing a disease.
Lymphocytes derived from the lymph nodes, spleens or peripheral
blood of primed, diseased animals can be used to search for a
particular antibody. The lymphocyte chromosomes encoding desired
immunoglobulins are immortalized by fusing the lymphocytes with
myeloma cells, generally in the presence of a fusing agent such as
polyethylene glycol (PEG). Any of a number of myeloma cell lines
may be used as a fusion partner according to standard techniques;
for example, the P3-NS1/1-Ag4-1, P3-x63-Ag8.653, Sp2/0-Ag14, or
HL1-653 myeloma lines. These myeloma lines are available from the
ATCC, Rockville, Md.
[0121] The resulting cells, which include the desired hybridomas,
are then grown in a selective medium such as HAT medium, in which
unfused parental myeloma or lymphocyte cells eventually die. Only
the hybridoma cells survive and can be grown under limiting
dilution conditions to obtain isolated clones. The supernatants of
the hybridomas are screened for the presence of the desired
specificity, e.g. by immunoassay techniques using the CTLA4Ig
protein that has been used for immunization. Positive clones can
then be subcloned under limiting dilution conditions, and the
monoclonal antibody produced can be isolated.
[0122] Various conventional methods can be used for isolation and
purification of the monoclonal antibodies so as to obtain them free
from other proteins and contaminants. Commonly used methods for
purifying monoclonal antibodies include ammonium sulfate
precipitation, ion exchange chromatography, and affinity
chromatography (Zola et al., in Monoclonal Hybridoma Antibodies:
Techniques and Applications, Hurell (ed.) pp. 51-52 (CRC Press,
1982)). Hybridomas produced according to these methods can be
propagated in vitro or in vivo (in ascites fluid) using techniques
known in the art (Fink et al., Prog. Clin. Pathol., 9:121-33
(1984), FIG. 6-1 at p. 123).
[0123] Generally, the individual cell line may be propagated in
vitro, for example, in laboratory culture vessels, and the culture
medium containing high concentrations of a single specific
monoclonal antibody can be harvested by decantation, filtration, or
centrifugation.
[0124] In addition, fragments of these antibodies containing the
active binding region reactive with the extracellular domain of
CTLA4 receptor, such as Fab, F(ab').sub.2 and Fv fragments may be
produced. Such fragments can be produced using techniques well
established in the art (e.g. Rousseaux et al., in Methods Enzymol.,
121:663-69, Academic Press (1986)).
[0125] Anti-B7 monoclonal antibodies prepared as described above
may be used to bind to B7 antigen to inhibit interactions of
CD28-positive or CTLA4-positive T cells with B7 positive cells.
Anti-CTLA4 monoclonal antibodies may be used to bind to CTLA4
receptor to inhibit the interaction of CTLA4-positive T cells with
other cells.
[0126] In another embodiment, the CTLA4Ig fusion protein may be
used to identify additional compounds capable of regulating the
interaction between CTLA4 and the B7 antigen. Such compounds may
include small naturally occurring molecules that can be used to
react with B cells and/or T cells. For example, fermentation broths
may be tested for the ability to inhibit CTLA4/B7 interactions. In
addition, derivatives of the CTLA4Ig fusion protein as described
above may be used to regulate T cell proliferation. For example,
the fragments or derivatives may be used to block T cell
proliferation in graft versus host (GVH) disease which accompanies
allogeneic bone marrow transplantation.
[0127] The CD28-mediated T cell proliferation pathway is
cyclosporine-resistant, in contrast to proliferation driven by the
CD3/Ti cell receptor complex (June et al., 1987, supra).
Cyclosporine is relatively ineffective as a treatment for GVH
disease (Storb, Blood 68:119-125 (1986)). GVH disease is thought to
be mediated by T lymphocytes which express CD28 antigen (Storb and
Thomas, Immunol. Rev. 88:215-238 (1985)). Thus, the CTLA4Ig fusion
protein may be useful alone, or in combination with
immunosuppressants such as cyclosporine, for blocking T cell
proliferation in GVH disease.
[0128] Regulation of CTLA4-positive T cell interactions with B7
positive cells, including B cells, by the methods of the invention
may thus be used to treat pathological conditions such as
autoimmunity, transplantation, infectious diseases and
neoplasia.
[0129] The B7-binding molecules and IL4-binding molecules described
herein may be in a variety of dosage forms which include, but are
not limited to, liquid solutions or suspensions, tablets, pills,
powders, suppositories, polymeric microcapsules or microvesicles,
liposomes, and injectable or infusible solutions. The preferred
form depends upon the mode of administration and the therapeutic
application.
[0130] The most effective mode of administration and dosage regimen
for the molecules of the present invention depends upon the
severity and course of the disease, the subject's health and
response to treatment and the judgment of the treating physician.
Accordingly, the dosages of the molecules should be titrated to the
individual subject.
[0131] The interrelationship of dosages for animals of various
sizes and species and humans based on mg/m.sup.2 of surface area is
described by Freireich, E. J., et al. (Quantitative Comparison of
Toxicity of Anticancer Agents in Mouse, Rat, Hamster, Dog, Monkey
and Man. Cancer Chemother, Rep., 50, No. 4, 219-244, May 1966).
[0132] Adjustments in the dosage regimen may be made to optimize
the growth inhibiting response. Doses may be divided and
administered on a daily basis or the dose may be reduced
proportionally depending upon the situation. For example, several
divided doses may be administered daily or the dose may be
proportionally reduced as indicated by the specific therapeutic
situation.
[0133] In accordance with the practice of the invention an
effective amount for treating a subject may be between about 0.1
and about 10 mg/kg body weight of subject. Also, the effective
amount may be an amount between about 1 and about 10 mg/kg body
weight of subject.
[0134] Advantages of the Invention: The subject invention overcomes
the problems associated with current therapies directed to
preventing the rejection of tissue or organ transplants. In
contrast to present therapies, the present invention affects only
immunological responses mediated by B7 interactions.
[0135] For example, the present invention affects the transplant
antigen-specific T cells, thus inducing donor-specific and
antigen-specific tolerance. The binding of CD28 by its ligand,
B7/BB1 (B7), during T cell receptor engagement is critical for
proper T cell signaling in some systems (M. K. Jenkins, P. S.
Taylor, S. D. Norton, K. B. Urdahl, J. Immunol. 147:2461 (1991); C.
H. June, J. A. Ledbetter, P. S. Linsley, C. B. Thompson, Immunol.
Today 11:211 (1990); H. Reiser, G. J. Freeman, Z. Razi-Wolf, C. D.
Gimmi, B. Benacerraf, L. M. Nadler, Proc. Natl. Acad. Sci. U.S.A.
89:271 (1992); N. K. Damle, K. Klussman, P. S. Linsley, A. Aruffo,
J. Immunol. 148:1985 (1992)).
[0136] When the interaction of CD28 with its ligand is blocked,
antigen-specific T cells are inappropriately induced into a state
of antigen-specific T cell anergy (M. K. Jenkins, P. S. Taylor, S.
D. Norton, K. B. Urdahl, J. Immunol. 147:2461 (1991); F. A.
Harding, J. G. McArthur, J. A. Gross, D. H. Raulet, J. P. Allison,
Nature 356:607 (1992)).
[0137] CTLA4Ig fusion protein binds to both human and murine B7
(with a 20-fold greater affinity than CD28), blocks the binding of
CD28 to B7, inhibits T cell activation, and induces T cell
unresponsiveness in vitro (F. A. Harding, J. G. McArthur, J. A.
Gross, D. H. Raulet, J. P. Allison, Nature 356:607 (1992); P. S.
Linsley et al., J. Exp. Med. 174:561 (1991)).
[0138] Moreover, the present invention would be useful to obtain
expression of a soluble protein product of the heretofore
unexpressed CTLA4 gene, and to identify a natural ligand for CTLA4
that is involved in functional responses of T cells. The soluble
protein product could then be used to regulate T cell responses in
vivo to treat pathological conditions.
[0139] The following examples are presented to illustrate the
present invention and to assist one of ordinary skill in making and
using the same. The examples are not intended in any way to
otherwise limit the scope of the invention.
Example 1
Preparation of B7Ig and CD28Ig Fusion Proteins
[0140] Receptor-immunoglobulin C gamma (IgC.gamma.) fusion proteins
B7Ig and CD28Ig were prepared as described by Linsley et al., in J.
Exp. Med. 173:721-730 (1991), incorporated by reference herein.
Briefly, DNA encoding amino acid sequences corresponding to the
respective receptor protein (e.g. B7) was joined to DNA encoding
amino acid sequences corresponding to the hinge, CH2 and CH3
regions of human IgC.gamma.1. This was accomplished as follows.
[0141] Polymerase Chain Reaction (PCR). For PCR, DNA fragments were
amplified using primer pairs as described below for each fusion
protein. PCR reactions (0.1 ml final volume) were run in Taq
polymerase buffer (Stratagene, La Jolla, Calif.), containing 20
.mu.moles each of dNTP; 50-100 pmoles of the indicated primers;
template (1 ng plasmid or cDNA synthesized from .ltoreq.1 .mu.g
total RNA using random hexamer primer, as described by Kawasaki in
PCR Protocols, Academic Press, pp. 21-27 (1990), incorporated by
reference herein); and Taq polymerase (Stratagene). Reactions were
run on a thermocycler (Perkin Elmer Corp., Norwalk, Conn.) for
16-30 cycles (a typical cycle consisted of steps of 1 min at
94.degree. C., 1-2 min at 50.degree. C. and 1-3 min at 72.degree.
C.). Plasmid Construction. Expression plasmids containing cDNA
encoding CD28, as described by Aruffo and Seed, Proc. Natl. Acad.
Sci. USA 84:8573 (1987), were provided by Drs. Aruffo and Seed
(Mass General Hospital, Boston, Mass.). Plasmids containing cDNA
encoding CD5, as described by Aruffo, Cell 61:1303 (1990), were
provided by Dr. Aruffo. Plasmids containing cDNA encoding B7, as
described by Freeman et al., J. Immunol. 143:2714 (1989), were
provided by Dr. Freeman (Dana Farber Cancer Institute, Boston,
Mass.). For initial attempts at expression of soluble forms of CD28
and B7, constructs were made (OMCD28 and OMB7) as described by
Linsley et al., J. Exp. Med., supra, in which stop codons were
introduced upstream of the transmembrane domains and the native
signal peptides were replaced with the signal peptide from
oncostatin M (Malik et al., Mol. Cell. Biol. 9:2847 (1989)). These
were made using synthetic oligonucleotides for reconstruction
(OMCD28) or as primers (OMB7) for PCR. OMCD28, is a CD28 cDNA
modified for more efficient expression by replacing the signal
peptide with the analogous region from oncostatin M. CD28Ig and
B7Ig fusion constructs were made in two parts. The 5' portions were
made using OMCD28 and OMB7 as templates and the oligonucleotide,
CTAGCCACTGAAGCTTCACCATGGGTGTACTGCTCACAC (SEQ ID NO:1), (encoding
the amino acid sequence corresponding to the oncostatin M signal
peptide) as a forward primer, and either
TGGCATGGGCTCCTGATCAGGCTTAGAAGGTCCGGGAAA (SEQ ID NO:2), or,
TTTGGGCTCCTGATCAGGAAAATGCTCTTGCTTGGTTGT (SEQ ID NO:3) as reverse
primers, respectively. Products of the PCR reactions were cleaved
with restriction endonucleases (Hind III and BclI) as sites
introduced in the PCR primers and gel purified.
[0142] The 3' portion of the fusion constructs corresponding to
human IgC.gamma.1 sequences was made by a coupled reverse
transcriptase (from Avian myeloblastosis virus; Life Sciences
Associates, Bayport, N.Y.)-PCR reaction using RNA from a myeloma
cell line producing human-mouse chimeric mAb L6 (provided by Dr. P.
Fell and M. Gayle, Bristol-Myers Squibb Company, Pharmaceutical
Research Institute, Seattle, Wash.) as template. The
oligonucleotide,
AAGCAAGAGCATTTTCCTGATCAGGAGCCCAAATCTTCTGACAAAACTCAC
ACATCCCCACCGTCCCCAGCACCTGAACTCCTG (SEQ ID NO:4), was used as
forward primer, and CTTCGACCAGTCTAGAAGCATCCTCGTGCGACCGCGAGAGC (SEQ
ID NO:5) as reverse primer. Reaction products were cleaved with
BclI and XbaI and gel purified. Final constructs were assembled by
ligating HindIII/BclI cleaved fragments containing CD28 or B7
sequences together with BclI/XbaI cleaved fragment containing
IgC.gamma.1 sequences into HindIII/XbaI cleaved CDM8. Ligation
products were transformed into MC1061/p3 E. coli cells and colonies
were screened for the appropriate plasmids. Sequences of the
resulting constructs were confirmed by DNA sequencing.
[0143] The construct encoding B7 contained DNA encoding amino acids
corresponding to amino acid residues from approximately position 1
to approximately position 215 of the extracellular domain of B7.
The construct encoding CD28 contained DNA encoding amino acids
corresponding to amino acid residues from approximately position 1
to approximately position 134 of the extracellular domain of
CD28.
[0144] CD5Ig was constructed in identical fashion, using
CATTGCACAGTCAAGCTTCCATGCCCATGGGTTCTCTGGCCACCTTG (SEQ ID NO:6), as
forward primer and ATCCACAGTGCAGTGATCATTTGGATCCTGGCATGTGAC (SEQ ID
NO:7) as reverse primer. The PCR product was restriction
endonuclease digested and ligated with the IgC.gamma.1 fragment as
described above. The resulting construct (CD5Ig) encoded a mature
protein having an amino acid sequence containing amino acid
residues from position 1 to position 347 of the sequence
corresponding to CD5, two amino acids introduced by the
construction procedure (amino acids DQ), followed by DNA encoding
amino acids corresponding to the IgC.gamma.1 hinge region.
[0145] Cell Culture and Transfections. COS (monkey kidney cells)
were transfected with expression plasmids expressing CD28 and B7
using a modification of the protocol of Seed and Aruffo (Proc.
Natl. Acad. Sci. 84:3365 (1987)), incorporated by reference herein.
Cells were seeded at 10.sup.6 per 10 cm diameter culture dish 18-24
h before transfection. Plasmid DNA was added (approximately 15
.mu.g/dish) in a volume of 5 mls of serum-free DMEM.TM. containing
0.1 mM chloroquine and 600 .mu.g/ml DEAE Dextran.TM., and cells
were incubated for 3-3.5 h at 37.degree. C. Transfected cells were
then briefly treated (approximately 2 min) with 10% dimethyl
sulfoxide in PBS and incubated at 37.degree. C. for 16-24 h in
DMEM.TM. containing 10% FCS. At 24 h after transfection, culture
medium was removed and replaced with serum-free DMEM.TM. (6
ml/dish). Incubation was continued for 3 days at 37.degree. C., at
which time the spent medium was collected and fresh serum-free
medium was added. After an additional 3 days at 37.degree. C., the
spent medium was again collected and cells were discarded.
[0146] CHO cells expressing CD28, CD5 or B7 were isolated as
described by Linsley et al., (1991) supra, as follows: Briefly,
stable transfectants expressing CD28, CD5, or B7, were isolated
following cotransfection of dihydrofolate reductase-deficient
Chinese hamster ovary (dhfr.sup.- CHO) cells with a mixture of the
appropriate expression plasmid and the selectable marker, pSV2dhfr
(Linsley et al., Proc. Natl. Acad. Sci. USA 87:5031 (1990)),
incorporated by reference herein. Transfectants were then grown in
increasing concentrations of methotrexate to a final level of 1
.mu.M and were maintained in DMEM.TM. supplemented with 10% fetal
bovine serum (FBS), 0.2 mM proline and 1 .mu.M methotrexate. CHO
lines expressing high levels of CD28 (CD28.sup.+CHO) or B7 (B7+CHO)
were isolated by multiple rounds of fluorescence-activated cell
sorting (FACS.sup.R) following indirect immunostaining with mAbs
9.3 or BB-1. Amplified CHO cells negative for surface expression of
CD28 or B7 (dhfr.sup.+ CHO) were also isolated by FACS.sup.R from
CD28-transfected populations.
[0147] Immunostaining and FACS.sup.R Analysis. Transfected CHO or
COS cells or activated T cells were analyzed by indirect
immunostaining. Before staining, CHO cells were removed from their
culture vessels by incubation in PBS containing 10 mM EDTA. Cells
were first incubated with murine mAbs 9.3 (Hansen et al.,
Immunogenetics 10:247 (1980)) or BB-1 (Yokochi et al., J. Immunol.
128:823 (1981)), or with Ig fusion proteins (all at 10 .mu.g/ml in
DMEM.TM. containing 10% FCS) for 1-2 h at 4.degree. C. Cells were
then washed, and incubated for an additional 0.5-2 h at 4.degree.
C. with a FITC-conjugated second step reagent (goat anti-mouse Ig
serum for murine mAbs, or goat anti-human Ig C.gamma. serum for
fusion proteins (Tago, Inc., Burlingame, Calif.)). Fluorescence was
analyzed on a FACS IV.sup.R cell sorter (Becton Dickinson and CO.,
Mountain View, Calif.) equipped with a four decade logarithmic
amplifier.
[0148] Purification of Ig Fusion Proteins. The first, second and
third collections of spent serum-free culture media from
transfected COS cells were used as sources for the purification of
Ig fusion proteins. After removal of cellular debris by low speed
centrifugation, medium was applied to a column (approximately
200-400 ml medium/ml packed bed volume) of immobilized protein A
(Repligen Corp., Cambridge, Mass.) equilibrated with 0.05 M sodium
citrate, pH 8.0. After application of the medium, the column was
washed with 1 M potassium phosphate, pH 8, and bound protein was
eluted with 0.05 M sodium citrate, pH 3. Fractions were collected
and immediately neutralized by addition of 1/10 volume of 2 M Tris,
pH 8. Fractions containing the peak of A.sub.280 absorbing material
were pooled and dialyzed against PBS before use. Extinction
coefficients of 2.4 and 2.8 ml/mg for CD28Ig and B7Ig,
respectively, were determined by amino acid analysis of solutions
of known absorbance. The recovery of purified CD28Ig and B7Ig
binding activities was nearly quantitative as judged by FACS.sup.R
analysis after indirect fluorescent staining of B7.sup.+ and
CD28.sup.+ CHO cells.
Example 2
Preparation of CTLA4Ig Fusion Protein
[0149] A soluble genetic fusion encoding CTLA4 Ig between the
extracellular domain of CTLA4 and an IgC.gamma.1 domain was
constructed in a manner similar to that described above for the
CD28Ig construct. The extracellular domain of the CTLA4 gene was
cloned by PCR using synthetic oligonucleotides corresponding to the
published sequence (Dariavach et al., Eur. Journ. Immunol.
18:1901-1905 (1988)).
[0150] Because a signal peptide for CTLA4 was not identified in the
CTLA4 gene, the N-terminus of the predicted sequence of CTLA4 was
fused to the signal peptide of oncostatin M (Malik et al., Mol. and
Cell. Biol. 9:2847 (1989)) in two steps using overlapping
oligonucleotides. For the first step, the oligonucleotide,
CTCAGTCTGGTCCTTGCACTCCTG TTTCCAAGCATGGCGAGCATGGCAATGCACGTGGCCCAGCC
(SEQ ID NO:8) (which encoded the C terminal 15 amino acids from the
oncostatin M signal peptide fused to the N terminal 7 amino acids
of CTLA4) was used as forward primer, and
TTTGGGCTCCTGATCAGAATCTGGGCACGGTTG (SEQ ID NO:9) (encoding amino
acid residues 119-125 of the amino acid sequence encoding CTLA4
receptor and containing a Bcl I restriction enzyme site) as reverse
primer. The template for this step was cDNA synthesized from 1
.mu.g of total RNA from H38 cells (an HTLV II infected T cell
leukemic cell line provided by Drs. Salahudin and Gallo, NCI,
Bethesda, Md.). A portion of the PCR product from the first step
was reamplified, using an overlapping forward primer, encoding the
N terminal portion of the oncostatin M signal peptide and
containing a Hind III restriction endonuclease site,
CTAGCCACTGAAGCTTCACCAATGGGTGTACTGCTCACACAGAGGACGCTG
CTCAGTCTGGTCCTTGCACTC (SEQ ID NO:10) and the same reverse primer.
The product of the PCR reaction was digested with Hind III and Bcl
I and ligated together with a Bcl 1/Xba I cleaved cDNA fragment
encoding the amino acid sequences corresponding to the hinge, CH2
and CH3 regions of IgC.gamma.1 into the Hind III/Xba I cleaved
expression vector, CDM8 or Hind III/Xba I cleaved expression vector
.pi.LN (provided by Dr. Aruffo).
[0151] A map of the resulting CTLA4Ig fusion construct is shown in
FIG. 1. Sequences displayed in this figure show the junctions
between CTLA4 (upper case letters, unshaded regions) and the signal
peptide, SP, of oncostatin M (dark shaded regions), and the hinge,
H, of IgC.gamma.1 (stippled regions). The amino acid in parentheses
was introduced during construction. Asterisks (*) indicate cysteine
to serine mutations introduced in the IgC.gamma. hinge region. The
immunoglobulin superfamily V-like domain present in CTLA4 is
indicated, as are the CH2 and CH3 domains of IgC.gamma.1.
[0152] Expression plasmids, CDM8, containing CTLA4Ig were then
transfected into COS cells using DEAE/Dextran.TM. transfection by
modification (Linsley et al., 1991, supra) of the protocol
described by Seed and Aruffo, 1987, supra.
[0153] Expression plasmid constructs (.pi.LN or CDM8) containing
cDNA encoding the amino acid sequence of CTLA4Ig, was transfected
by lipofection using standard procedures into dhfr.sup.- CHO lines
to obtain novel cell lines stably expressing CTLA4Ig.
[0154] DNA encoding the amino acid sequence corresponding to
CTLA4Ig has been deposited with the ATCC under the Budapest Treaty
on May 31, 1991, and has been accorded ATCC accession number
68629.
[0155] A preferred stable transfectant, expressing CTLA4Ig,
designated Chinese Hamster Ovary Cell Line, CTLA4Ig-24, was made by
screening B7 positive CHO cell lines for B7 binding activity in the
medium using immunostaining. Transfectants were maintained in
DMEM.TM. supplemented with 10% fetal bovine serum (FBS), 0.2 mM
proline and 1 .mu.M methotrexate.
[0156] The CTLA4Ig-24 CHO cell line has been deposited with the
ATCC under the Budapest Treaty on May 31, 1991 and has been
accorded accession number ATCC 10762.
[0157] CTLA4Ig was purified by protein A chromatography from
serum-free conditioned supernatants (FIG. 2). Concentrations of
CTLA4Ig were determined assuming an extinction coefficient at 280
nm of 1.6 (experimentally determined by amino acid analysis of a
solution of known absorbance). Molecular weight standards (lanes 1
and 3, FIG. 2) and samples (1 .mu.g) of CTLA4Ig (lanes 2 and 4)
were subjected to SDS-PAGE (4-12% acrylamide gradient) under
non-reducing conditions (-.beta.ME, lanes 1 and 2) or reducing
conditions (+.beta.ME, lanes 3 and 4) Proteins were visualized by
staining with Coomassie Brilliant Blue.
[0158] Under non-reducing conditions, CTLA4Ig migrated as a M.sub.r
approximately 100,000 species, and under reducing conditions, as a
M.sub.r approximately 50,000 species (FIG. 2). Because the
IgC.gamma. hinge disulfides were eliminated during construction,
CTLA4Ig, like CD28Ig, is a dimer presumably joined through a native
disulfide linkage.
Example 3
CTLA4 Receptor
[0159] To reconstruct DNA encoding the amino acid sequence
corresponding to the full length human CTLA4 gene, cDNA encoding
amino acids corresponding to a fragment of the transmembrane and
cytoplasmic domains of CTLA4 was cloned by PCR and then joined with
cDNA encoding amino acids corresponding to a fragment from CTLA4Ig
that corresponded to the oncostatin M signal peptide fused to the
N-terminus of CTLA4. Procedures for PCR, and cell culture and
transfections were as described above in Example 1 using COS cells
and DEAE-Dextran.TM. transfection.
[0160] Because the expression of CTLA4 receptor protein in human
lymphoid cells has not been previously reported, it was necessary
to locate a source of CTLA4 mRNA. PCR cDNA reverse transcribed from
the total cellular RNA of H38 cells, as noted above, was used for
cloning by PCR. For this purpose, the oligonucleotide,
GCAATGCACGTGGCCCAGCCTGCTGTGGTAGTG (SEQ ID NO: 11), (encoding the
first 11 amino acids in the predicted coding sequence) was used as
a forward primer, and TGATGTAACATGTCTAGATCAATTGATGGGAATAAAATAAGGCTG
(SEQ ID NO:12) (homologous to the last 8 amino acids in CTLA4 and
containing a Xba I site) as reverse primer. The template again was
a cDNA synthesized from 1 .mu.g RNA from H38 cells. Products of the
PCR reaction were cleaved with the restriction endonucleases Nco I
and Xba I and the resulting 316 bp product was gel purified. A 340
bp Hind III/Nco I fragment from the CTLAIg fusion described above
was also gel-purified, and both restriction fragments were ligated
into Hind III/Xba I cleaved CDM8 to form OMCTLA.
[0161] The resulting construct corresponded to full length CTLA4
(SEQ ID NOs: 13 and 14) and the oncostatin M signal peptide. The
construct is shown in FIG. 3 and was designated OMCTLA4. The
sequence for CTLA4 shown in FIG. 3 differs from the predicted human
CTLA4 DNA sequence (Dariavach et al., supra) by a base change such
that the previously reported alanine at amino acid position 111 of
the amino acid sequence shown, encodes a threonine. This threonine
is part of a newly identified N-linked glycosylation site that may
be important for successful expression of the fusion protein.
[0162] Ligation products were transformed into MC1061/p3 E. coli
cells and colonies were screened for the appropriate plasmids.
Sequences of the resulting constructs were confirmed by DNA
sequence analysis.
Example 4
Characterization of CTLA4Ig
[0163] To characterize the CTLA4Ig constructs, several isolates,
CD28Ig, B7Ig, and CD5Ig, were prepared as described above and were
transfected into COS cells as described in Examples 2 and 3, and
were tested by FACS.sup.R analysis for binding of B7Ig. In addition
to the above-mentioned constructs, CDM8 plasmids containing cDNAs
encoding CD7 as described by Aruffo and Seed, (EMBO Jour.
6:3313-3316 (1987)), incorporated by reference herein, were also
used.
[0164] mAbs. Murine monoclonal antibodies (mAbs) 9.3 (anti-CD28)
and G19-4 (anti-CD3), G3-7 (anti-CD7), BB-1 (anti-B7 antigen) and
rat mAb 187.1 (anti-mouse K chain) have been described previously
(Ledbetter et al., Proc. Natl. Acad. Sci. 84:1384-1388 (1987);
Ledbetter et al., Blood 75:1531 (1990); Yokochi et al., supra) and
were purified from ascites before use. The hybridoma producing mAb
OKT8 was obtained from the ATCC, Rockville, Md., and the mAb was
also purified from ascites before use. mAb 4G9 (anti-CD19) was
provided by Dr. E. Engleman, Stanford University, Palo Alto,
Calif.). Purified human-mouse chimeric mAb L6 (having human
C.gamma.1 Fc portion) was a gift of Dr. P. Fell and M. Gayle
(Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle,
Wash.).
[0165] Immunostaining and FACS.sup.R Analysis. Prior to staining,
COS or CHO cells were removed from their culture vessels by
incubation in PBS containing 10 mM EDTA. Cells were first incubated
with mAbs or Ig fusion proteins at 10 .mu.g/ml in DMEM.TM.
containing 10% FBS for 1-2 hr at 4.degree. C. Cells were then
washed, and incubated for an additional 0.5-2 hrs at 4.degree. C.
with FITC-conjugated goat anti-mouse immunoglobulin or with
FITC-conjugated goat anti-human Ig C.gamma. serum (both from Tago,
Burlingame, Calif.). When binding of both mAbs and Ig fusion
proteins were measured in the same experiment, FITC-conjugated
anti-mouse and anti-human second step reagents were mixed together
before use. Fluorescence on a total of 10,000 cells was then
analyzed by FACS.sup.R.
[0166] Peripheral Blood Lymphocyte Separation and Stimulation.
Peripheral blood lymphocytes (PBLs) were isolated by centrifugation
through Lymphocyte Separation Medium.TM. (Litton Bionetics,
Kensington, Md.). Alloreactive T cells were isolated by stimulation
of PBL in a primary mixed lymphocyte reaction (MLR). PBL were
cultured at 10.sup.6/ml irradiated (5000 rad) T51 LCL.
EBV-transformed lymphoblastoid cell lines (LCL), PM (Bristol-Myers
Squibb Co.) and T51 (Bristol-Myers Squibb Co.) were maintained in
RPMI.TM. supplemented with 10% FBS. After 6 days, alloreactive
"blasts" cells were cryopreserved. Secondary MLR were conducted by
culturing thawed alloreactive blasts together with fresh irradiated
T51 LCL in the presence and absence of mAbs and Ig fusion proteins.
Cells were cultured in 96 well flat bottom plates (4.times.10.sup.4
alloreactive blasts and 1.times.10.sup.4 irradiated T51 LCL
cells/well, in a volume of 0.2 ml) in RPMI.TM. containing 10% FBS.
Cellular proliferation of quadruplicate cultures was measured by
uptake of [.sup.3H]-thymidine during the last 6 hours of a 2-3 day
culture.
[0167] PHA-activated T cells were prepared by culturing PBLs with 1
.mu.g/ml PHA (Wellcome, Charlotte, N.C.) for five days, and one day
in medium lacking PHA. Viable cells were collected by sedimentation
through Lymphocyte Separation Medium.TM. before use. Cells were
stimulated with mAbs or transfected CHO cells for 4-6 hr at
37.degree. C., collected by centrifugation and used to prepare
RNA.
[0168] CD4+ T cells were isolated from PBLs by separating PBLs from
healthy donors into T and non-T cells using sheep erythrocyte
rosetting technique and further separating T cells by panning into
CD4+ cells as described by Damle et al., J. Immunol. 139:1501
(1987), incorporated by reference herein.
[0169] B cells were also purified from peripheral blood by panning
as described by Wysocki and Sato, Proc. Natl. Acad. Sci. USA
75:2844 (1978), incorporated by reference herein, using anti-CD19
mAb 4G9. To measure T.sub.h-induced Ig production, 10.sup.6 CD4+ T
cells were mixed with 10.sup.6 CD19+B cells in 1 ml of RPMI.TM.
containing 10% FBS. Following culture for 6 days at 37.degree. C.,
production of human IgM was measured in the culture supernatants
using solid phase ELISA as described by Volkman et al., Proc. Natl.
Acad. Sci. USA 78:2528 (1981), incorporated by reference
herein.
[0170] Briefly, 96-well flat bottom microtiter ELISA plates
(Corning, Corning, N.Y.) were coated with 200 .mu.l/well of sodium
carbonate buffer (pH 9.6) containing 10 .mu.g/ml of
affinity-purified goat anti-human IgG or IgM antibody (Tago,
Burlingame, Calif.), incubated overnight at 4.degree. C., and then
washed with PBS and wells were further blocked with 2% BSA in PBS
(BSA-PBS).
[0171] Samples to be assayed were added at appropriate dilution to
these wells and incubated with 200 .mu.l/well of 1:1000 dilution of
horseradish peroxidase (HRP)-conjugated F(ab').sub.2 fraction of
affinity-purified goat anti-human IgG or IgM antibody (Tago). The
plates were then washed, and 100:1/well of o-phenylenediamine
(Sigma Chemical Co., St. Louis, Mo.) solution (0.6 mg/ml in
citrate-phosphate buffer with pH 5.5 and 0.045% hydrogen peroxide).
Color development was stopped with 2 N sulfuric acid. Absorbance at
490 nm was measured with an automated ELISA plate reader.
[0172] Test and control samples were run in triplicate and the
values of absorbance were compared to those obtained with known IgG
or IgM standards run simultaneously with the supernatant samples to
generate the standard curve using which the concentrations of Ig in
the culture supernatant were quantitated. Data are expressed as
ng/ml of Ig.+-.SEM of either triplicate or quadruplicate
cultures.
[0173] Immunoprecipitation Analysis and SDS PAGE. Cells were
surface-labeled with .sup.125I and subjected to immunoprecipitation
analysis. Briefly, PHA-activated T cells were surface-labeled with
.sup.125I using lactoperoxidase and H.sub.2O.sub.2 as described by
Vitetta et al., J. Exp. Med. 134:242 (1971), incorporated by
reference herein. SDS-PAGE chromatography was performed on linear
acrylamide gradients gels with stacking gels of 5% acrylamide. Gels
were stained with Coomassie Blue, destained, and photographed or
dried and exposed to X ray film (Kodak.TM. XAR-5).
[0174] Binding Assays. B7Ig was labeled with .sup.125I to a
specific activity of approximately 2.times.10.sup.6 cpm/pmole.
Ninety-six well plastic dishes were coated for 16-24 hrs with a
solution containing CTLA4Ig (0.5 .mu.g in a volume of 0.05 ml of 10
mM Tris, pH 8). Wells were blocked with binding buffer (DMEM.TM.
containing 50 mM BES (Sigma Chemical Co.), pH 6.8, 0.1% BAS, and
10% FCS) before addition of a solution (0.09 ml) containing
.sup.125I B7Ig (approximately 5.times.10.sup.5 cpm) in the presence
or absence of competitor. Following incubation for 2-3 hrs at
23.degree. C., wells were washed once with binding buffer, and four
times with PBS. Bound radioactivity was then solubilized by
addition of 0.5N NaOH, and quantified by gamma counting.
[0175] Binding to B7Ig. The functional activity of the OMCTLA4
construct encoding the complete human CTLA4 DNA gene, is shown in
the experiment shown in FIG. 4. COS cells were transfected with
expression plasmids CD7, OMCD28 and OMCTLA4 as described above.
Forty-eight hours following transfection, cells were collected and
incubated with medium only (no addition) or with mAbs 9.3, B7Ig,
CD5Ig or G3-7. Cells were then washed and binding was detected by a
mixture of FITC-conjugated goat anti-mouse Ig and FITC-conjugated
goat anti-human Ig second step reagents. Transfected cells were
tested for expression of the appropriate cell surface markers by
indirect immunostaining and fluorescence was measured using
FACS.sup.R analysis as described above.
[0176] As shown in FIG. 4, mAb 9.3 bound to CD28-transfected COS
cells, but not to CTLA4-transfected cells. In contrast, the B7Ig
fusion protein (but not control CD5Ig fusion protein) bound to both
CD28- and CTLA4-transfected cells. CD7-transfected COS cells bound
neither mAb 9.3 nor either of the fusion proteins. This indicates
that CD28 and CTLA4 both bind the B cell activation antigen, B7.
Furthermore, mAb 9.3 did not detectably bind CTLA4.
[0177] Binding of CTLA4Ig on B7 Positive CHO cells. To further
characterize the binding of CTLA4Ig and B7, the binding activity of
purified CTLA4Ig on B7+CHO cells and on a lymphoblastoid cell line
(PM LCL) was measured in the experiment shown in FIG. 5. Amplified
transfected CHO cell lines and PM LCLs were incubated with medium
only (no addition) or an equivalent concentration of human
IgC.gamma.1-containing proteins (10 .mu.g/ml) of CD5Ig, CD28Ig or
CTLA4Ig. Binding was detected by FACS.sup.R following addition of
FITC-conjugated goat anti-human Ig second step reagents. A total of
10,000 stained cells were analyzed by FACS.sup.R.
[0178] As shown in FIG. 5, CD28Ig bound to B7+CHO cells but not to
PM LCL, a cell line which expresses relatively low levels of the B7
antigen (Linsley et al., supra, 1990). CTLA4Ig bound more strongly
to both cell lines than did CD28Ig, suggesting that it bound with
higher affinity. Neither CD28Ig nor CTLA4Ig bound to CD28.sup.+ CHO
cells.
[0179] Affinity of Binding of CTLA4Ig and B7Ig. The apparent
affinity of interaction between CTLA4Ig and B7Ig was then measured
using a solid phase competition binding assay. Ninety-six well
plastic dishes were coated with CTLA4Ig as described above. B7Ig
was radiolabeled with .sup.125I (5.times.10.sup.5 cpm,
2.times.10.sup.6 cpm/pmole), and added to a concentration of 4 nM
in the presence of the indicated concentrations (FIG. 6) of
unlabeled chimeric mAb L6, mAb 9.3, mAb BB-1 or B7Ig. Plate-bound
radioactivity was determined and expressed as a percentage of
radioactivity bound to wells treated without competitor (28,300
cpm). Each point represents the mean of duplicate determinations;
replicates generally varied from the mean by .ltoreq.20%.
Concentrations were calculated based on a M.sub.r of 75,000 per
binding site for mAbs and 51,000 per binding site for B7Ig.
[0180] As shown in FIG. 6, only mAb BB-1 and unlabeled B7Ig
competed significantly for .sup.125I-B7Ig binding (half maximal
effects at approximately 22 nM and approximately 175 nM,
respectively). Neither chimeric mAb L6, nor mAb 9.3 competed
effectively at the concentrations tested. In other experiments, the
concentrations of mAb 9.3 used were sufficient to inhibit binding
of .sup.125I-B7Ig to immobilized CD28Ig or to cell surface
expressed CD28 by .gtoreq.90%.
[0181] When the competition data from FIG. 6 were plotted in a
Scatchard representation, a dissociation constant, K.sub.d, of
approximately 12 nM was calculated for binding of .sup.125I-B7 to
immobilized CTLA4Ig (FIG. 7). This value is approximately 20 fold
lower than the previously determined K.sub.d of binding between
.sup.125I-B7Ig and CD28 (approximately 200 nM) (Linsley et al,
(1991), supra) indicating that CTLA4 is a higher affinity receptor
for the B7 antigen than CD28 receptor.
[0182] To identify the molecule(s) on lymphoblastoid cells which
bound CTLA4Ig (FIG. 7), .sup.125I-surface labeled cells were
subjected to immunoprecipitation analysis (FIG. 8). B7+CHO and PM
LCL cells were surface-labeled with .sup.125I, and extracted with a
non-ionic detergent solution as described above. Aliquots of
extracts containing approximately 1.5.times.10.sup.7 cpm in a
volume of 0.1 ml were subjected to immunoprecipitation analysis as
described above with no addition, or 2 .mu.g each of CD28Ig,
CTLA4Ig or CD5Ig. Washed immunoprecipitates were then analyzed by
SDS-PAGE (10-20% acrylamide gradient) under reducing conditions.
The gel was then dried and subjected to autoradiography. The left
panel of FIG. 8 shows an autoradiogram obtained after a 1 day
exposure. The right panel of FIG. 8 shows an autoradiogram of the
same gel after a 10 day exposure. The autoradiogram in the center
panel of FIG. 8 was also exposed for 10 days. Positions of
molecular weight standard are also indicated in this figure.
[0183] As shown by FIG. 8, a diffusely migrating (M.sub.r
approximately 50,000-75,000; center at approximately 60,000)
radiolabeled protein was immunoprecipitated by CTLA4Ig, but not by
CD28Ig or CD5 .mu.g. This molecule co-migrated with B7
immunoprecipitated from B7+CHO cells by CTLA4Ig, and much more
weakly, by CD28Ig. These findings indicate that CTLA4Ig binds a
single protein on lymphoblastoid cells which is similar in size to
the B7 antigen.
Inhibition of Immune Responses In Vitro by CTLA4Ig
[0184] Inhibition of Proliferation. Previous studies have shown
that the anti-CD28 mAb, mAb 9.3, and the anti-B7 mAb, mAb BB-1,
inhibit proliferation of alloantigen specific T.sub.h cells, as
well as immunoglobulin secretion by alloantigen-presenting B Cells
(Damle, et al., Proc. Natl. Acad. Sci. 78:5096 (1981); Lesslauer et
al., Eur. J. Immunol. 16:1289 (1986)). Because CTLA4 is a high
affinity receptor for the B7 antigen as demonstrated herein,
soluble CTLA4Ig was tested for its ability to inhibit these
responses. The effects of CTLA4Ig on T cell proliferation were
examined in the experiment shown in FIG. 9.
[0185] Primary mixed lymphocyte reaction (MLR) blasts were
stimulated with irradiated T51 lymphoblastoid cells (LC) in the
absence or presence of concentrations of murine mAb 9.3 Fab
fragments, or B7Ig, CD28Ig or CTLA4Ig immunoglobulin C.gamma.
fusion proteins. Cellular proliferation was measured by
[.sup.3H]-thymidine incorporation after 4 days and is expressed as
the percentage of incorporation by untreated cultures (21,000 cpm).
FIG. 9 shows the means of quadruplicate determinations
(SEM.ltoreq.10%).
[0186] As shown in FIG. 9, CTLA4Ig inhibited the MLR reaction in a
dose-dependant fashion by a maximum of >90% with a 1/2 maximal
response at approximately 30 ng/ml (approximately 0.8 nM). The Fab
fragment of mAb 9.3, which previously was shown to be a more potent
inhibitor of MLR than whole mAb 9.3 (Damle et al., J. Immunol.
140:1753-1761 (1988)), also inhibited the MLR, but at higher
concentrations (approximately 800 ng/ml or approximately 30 nM for
1/2 maximal response). B7Ig and CD28Ig did not significantly
inhibit the MLR even at higher concentrations. In another
experiment, addition of B7Ig together with CTLA4Ig partially
overcame the inhibition of MLR by CTLA4Ig, indicating that the
inhibition was specifically due to interactions with B7
antigen.
[0187] Inhibition of Immunoglobulin Secretion. The effects of
CTLA4Ig on helper T cell (T.sub.h)-induced immunoglobulin secretion
were also examined (FIG. 10). CD4.sup.+ T cells were mixed with
allogeneic CD19.sup.+ B cells in the presence or absence of the
indicated immunoglobulin molecules as described above. Murine mAbs
OKT8, 9.3 and BB-1 were added at 20 .mu.g/ml, and Ig fusion
proteins at 10 .mu.g/ml. After 6 days of culture, concentrations of
human IgM (SEM<5%) in culture supernatants were determined by
enzyme immunoassay (ELISA) as described above. IgM production by B
cells cultured in the absence of CD4.sup.+ T cells was 11
ng/ml.
[0188] As shown in FIG. 10, CD4.sup.+ T cells stimulated IgM
production by allogenic CD19.sup.+ B Cells (in the absence of
CD4.sup.+ T cells, IgM levels were reduced by 93%). mAbs 9.3 and
BB-1 significantly inhibited T.sub.h-induced IgM production (63%
and 65% inhibition, respectively). CTLA4Ig was even more effective
as an inhibitor (89% inhibition) than were these mAbs. Inhibition
by control Ig molecules, mAb OKT8 and CD5Ig, was much less
(.ltoreq.30% inhibition). None of these molecules significantly
inhibited Ig production measured in the presence of Staphylococcal
aureus enterotoxin B. Similar results were obtained with CD4.sup.+
T cells and B cells derived from other donors. These results
indicate that the inhibition by CTLA4Ig is specific.
[0189] The above data also demonstrate that the CTLA4 and CD28
receptors are functionally as well as structurally related. Like
CD28, CTLA4 is also a receptor for the B cell activation antigen,
B7. CTLA4Ig bound .sup.125I-B7 with an affinity constant, K.sub.d,
of approximately 12 nM, a value some 20 fold higher than the
affinity between CD28 and B7Ig (approximately 200 nM). Thus, CTLA4
and CD28 may be thought of as high and low affinity receptors,
respectively, for the same ligand, the B7 antigen.
[0190] The apparent affinity between CD28 and B7 is similar to the
affinity reported for binding of soluble alloantigen to the T cell
receptor of a murine T cell hybridoma (approximately 100 nM; Schnek
et al., Cell 56:47 (1989)), and is higher affinity than
interactions between CD2 and LFA3 (Recny et al., J. Biol. Chem.
265:8542 (1990)), or CD4 and MHC class II molecules (Clayton et
al., Nature 339:548 (1989)). The apparent affinity constant,
K.sub.d, between CTLA4 and B7 is even greater, and compares
favorably with higher affinity mAbs (K.sub.d 2-10,000 nM; Alzari et
al., Ann. Rev. Immuno. 6:555 (1988)). The K.sub.d between CTLA4 and
B7 is similar to or greater than K.sub.d values of integrin
receptors and their ligands (10-2000 nM; Hautanen et al., J. Biol.
Chem. 264:1437-1442 (1989); Di Minno et al., Blood 61:140-148
(1983); Thiagarajan and Kelley, J. Biol. Chem. 263:035-3038
(1988)). The affinity of interaction between CTLA4 and B7 is thus
among the highest yet reported for lymphoid adhesion systems.
[0191] These results demonstrate the first expression of a
functional protein product of CTLA4 transcripts. CTLA4Ig, a fusion
construct containing the extracellular domain of CTLA4 fused to an
IgC.gamma.1 domain, forms a disulfide-linked dimer of M.sub.r
approximately 50,000 subunits (FIG. 1). Because no interchain
disulfides would be predicted to form in the Ig portion of this
fusion, it seems likely that cysteines from CTLA4 are involved in
disulfide bond formation. The analogous CD28Ig fusion protein
(Linsley et al, supra, 1991) also contains interchain disulfide
linkage(s). These results suggest that CTLA4 receptor, like CD28
(Hansen et al., Immunogenetics 10:247-260 (1980)), exists on the T
cell surface as a disulfide linked homodimer. Although CD28 and
CTLA4 are highly homologous proteins, they are immunologically
distinct, because the anti-CD28 mAb, mAb 9.3, does not recognize
CTLA4 (FIGS. 4 and 5).
[0192] It is not known whether CTLA4 can activate T cells by a
signalling pathway analogous to CD28. The cytoplasmic domains of
murine and human CTLA4 are identical (Dariavach et al., supra
1988), suggesting that this region has important functional
properties. The cytoplasmic domains of CD28 and CTLA4 also share
homology, although it is unclear if this is sufficient to impart
similar signaling properties to the two molecules.
[0193] CTLA4Ig is a potent inhibitor of in vitro lymphocyte
functions requiring T cell and B cell collaboration (FIGS. 9 and
10). These findings, together with previous studies, indicate the
fundamental importance of interactions between B7 antigen and its
counter-receptors, CD28 and/or CTLA4, in regulating both T and B
lymphocyte responses. CTLA4Ig should be a useful reagent for future
investigations on the role of these interactions during immune
responses. CTLA4Ig is a more potent inhibitor of in vitro
lymphocyte responses than either mAb BB-1 or mAb 9.3 (FIGS. 9 and
10). The greater potency of CTLA4Ig over mAb BB-1 is most likely
due to the difference in affinities for B7 between these molecules
(FIG. 6). CTLA4Ig is also more potent than mAb 9.3, probably
because, unlike the mAb, it does not also have direct stimulatory
effects on T cell proliferation (June et al., Immunology Today
11:211 (1989)) to counteract its inhibitory effects. The
immunosuppressive effects of CTLA4Ig in vitro suggest that future
investigations are warranted into possible therapeutic effects of
this molecule for treatment of autoimmune disorders involving
aberrant T cell activation or Ig production.
Example 5
[0194] Female BALB/c (H-2.sup.d) and C57BL/6 (H-2.sup.d)mice, 6 to
8 wk. of age were obtained from The Jackson Laboratory (Bar Harbor,
Me.).
[0195] Human pancreatic islets cells were purified after
collagenase digestion as described (C. Ricordi et al.
Transplantation 52:519 (1991); A. G. Tzakis et al. Lancet 336:402
(1990); C. Ricordi, P. E. Lacy, E. H. Finke, B. J. Olack, D. W.
Scharp, Diabetes 37:413 (1988)).
[0196] B6 or B10 mice, treated with streptozocin (175 mg per
kilogram of body weight) 3 to 5 days before transplant and
exhibiting nonfasting plasma glucose levels of greater than 280
mg/dl (with the majority over 300 mg/ml), were used as
recipients.
[0197] Each animal received approximately 800 fresh human islets of
150 .mu.m in diameter beneath the left renal capsule (D. Faustman
and C. Coe, Science 252:1700 (1991); Y. J. Zeng et al.
Transplantation 53:277 (1992)). Treatment was started immediately
after transplantation.
[0198] Control animals were treated with PBS (solid lines) or L6
(dotted lines) at 50 .mu.g every other day for 14 days immediately
after transplantation (FIG. 11A). Islet transplants were considered
rejected when glucose levels were greater than 250 mg/dl for three
consecutive days. Animals treated with PBS (n=14) and L6 (n=8) had
mean graft survivals of 5.6 and 6.4 days, respectively.
[0199] Animals were treated with 10 .mu.g of CTLA4Ig for 14
consecutive days immediately after transplant (n=7) (FIG. 11B).
Three out of seven animals maintained their grafts for >80 days.
The remaining four animals had a mean graft survival of 12.75
days.
[0200] Animals were treated with 50 .mu.g of CTLA4Ig every other
day for 14 days immediately after human islet transplantation (FIG.
11C). All animals (n=12) treated with this dose maintained grafts
throughout the analysis (FIG. 11C). Selected mice were
nephrectomized on days 21 and 29 after the transplant to assess the
graft's function (FIG. 11C).
[0201] Histology was performed on kidneys transplanted with human
islet cells (FIGS. 12A, 12B, 12C, 12D). The slides were analyzed
blindly.
[0202] Hematoxylin and eosin staining of a control human islet
grafted mouse 29 days after transplantation showed a massive
lymphocyte infiltration (FIG. 12A). The same tissue, stained for
insulin, showed no detectable insulin production (FIG. 12B).
[0203] Histological examination of tissue from a CTLA4Ig-treated
mouse 21 days after transplant showed intact islets under the
kidney capsule with very few lymphocytes infiltrating the
transplanted tissue (FIG. 12C). The tissue was stained with
hematoxylin and eosin. The same tissue from the CTLA4Ig-treated
mouse, stained for insulin, showed the production of insulin by the
grafted islets (FIG. 12D). Similar results were observed in graft
tissue examined at later time points. The upper, middle, and lower
arrowheads identify the kidney capsule, islet transplant, and
kidney parenchyma, respectively.
[0204] In the histopathology assay all tissues were fixed in 10%
buffered formalin and processed, and 5-.mu.m sections were stained
either with hematoxylin and eosin or for insulin with the
avidin-biotin-peroxidase method (S. M. Hsu, L. Raine, H. Fanger, J.
Histochem, Cytochem, 29:577 (1981)). Magnification was
.times.122.
[0205] In FIG. 13 streptozotocin-treated animals were transplanted
as described hereinabove for FIG. 11. The mice were treated either
with PBS (dotted lines) or with MAb to human B7 (solid lines) at a
dose of 50 .mu.g every other day for 14 days (FIG. 13). Control
animals (treated with PBS) (n=3) had a mean graft survival of 3.5
days, whereas anti-B7-treated animals (n=5) maintained grafts from
9 to >50 days (FIG. 13).
[0206] In FIG. 14 normal glycemic, CTLA4Ig-treated, transplanted
mice (dotted lines) were nephrectomized on day 44 after transplant
and immediately retransplanted with either 1000 first party donor
islets (dotted lines, solid circles) or 1000 second party islets
(dotted lines, open circles) beneath the remaining kidney
capsule.
[0207] These islets, frozen at the time of the first transplant,
were thawed and cultured for 3 days before transplant to ensure
islet function. B10 mice that had been treated with streptozotocin
and exhibited nonfasting glucose levels of greater than 280 mg/dl
were used as controls (solid lines) (FIG. 14). No treatment was
given after transplantation.
[0208] Control animals rejected both the first party (solid lines,
closed circles) and the second party (solid lines, open circles)
islet grafts by day 4 after transplant (FIG. 14). The
CTLA4Ig-treated mice retransplanted with second party islets had a
mean graft survival of 4.5 days, whereas animals retransplanted
with first party donor islets maintained grafts for as long as
analyzed (>80 days) (FIG. 14).
CTLA4Ig Significantly Prolongs Human Islet Graft Survival in Mice
in a Donor-Specific Manner Thereby Providing an Approach to
Immunosuppression
[0209] C57BL/6 (B6) or C57BL/10 (B10) mice were treated with
streptozotocin to eliminate mouse pancreatic islet B cell function.
Diabetic animals were grafted under the kidney capsule, and
treatment was started immediately after surgery. Survival of the
islet grafts was monitored by the analysis of blood glucose
concentrations.
[0210] Transplanted control animals, treated with either
phosphate-buffered saline (PBS)(n=14) or L6 (a human IgG1 chimeric
MAb; n=8), had a mean graft survival of 5.6 and 6.4 days,
respectively (FIG. 11A).
[0211] In contrast, islet rejection was delayed in animals treated
with CTLA4Ig (10 .mu.g per day for 14 days), with four out of the
seven animals exhibiting moderately prolonged mean graft survival
(12.75 days), whereas the remaining three animals maintained normal
glucose levels for >80 days (FIG. 11B). This eventual increase
in glucose concentration may be a result of islet exhaustion
because no evidence of active cellular rejection was observed.
[0212] In the three mice that maintained long-term islet grafts,
the transient increase in glucose concentrations around day 21
after the transplant may have represented a self-limited rejection
episode consistent with the pharmacokinetics of CTLA4Ig clearance
after therapy (P. S. Linsley et al., Science 257:792 (1992)).
[0213] In subsequent experiments, the dose of CTLA4Ig was increased
to 50 .mu.g per animal every other day for about 14 days. This
treatment resulted in 100% of the animals maintaining normal islet
function throughout the experiment with no signs of a rejection
crisis (FIG. 11C).
[0214] In order to confirm that insulin production originated from
the transplanted islets and not from the native mouse pancreas, we
nephrectomized selected animals at days 21 and 29 to remove the
islet grafts (FIG. 11C). In these animals, glucose concentrations
increased to above 350 mg/dl within 24 hours, which indicated that
the islet xenograft was responsible for maintaining normal glucose
levels. It appears that the blocking of the CD28-B7 interaction
inhibits xenogenic islet graft rejection.
[0215] The effects of treatment with the soluble receptor, namely
CTLAIg fusion protein, were not a result of Fc binding (L6 did not
effect graft rejection) or general effects on T cell or B cell
function in vivo.
[0216] Historical analyses of islet xenograft from control (PBS
treated) and CTLA4Ig treated mice were done (FIGS. 12A, 12B, 12C,
12D). The islet tissue from the control animal demonstrated
evidence of immune rejection, with a marked lymphocytic infiltrate
into the graft and few remaining islets (FIG. 12A).
[0217] Immunohistochemical staining showed that insulin-positive
cells were present only rarely, and no somatostatin-positive cells
were present at all (FIG. 12B). In contrast, transplant tissue from
the CTLA4Ig-treated mice was devoid of any lymphocytic infiltrate
(FIG. 12C).
[0218] The grafts were intact, with many islets visible. In
addition, the B cells observed in the human islet tissue produced
human insulin (FIG. 12D) and somatostatin.
[0219] The human CTLA4Ig used in this study reacts with both murine
and human B7. One advantage of the xenogeneic transplant model is
the availability of a MAb to human B7 that does not react with
mouse B7 (T. Yokochi, R. D. Holly, E. A. Clark, J. Immunol. 128:823
(1982)). Thus, the role of human B7-bearing antigen-presenting
cells (APCs) could be directly examined.
[0220] The mice were transplanted as described and then treated
with 50 .mu.g of MAb to human B7 every other day for 14 days after
transplant. This treatment prolonged graft survival in treated mice
(9 to >50 days) in comparison to that for control mice (FIG.
13). The anti-B7 MAb is unable to block rejection as effectively as
CTLA4Ig.
[0221] The CTLA4 Ig therapy resulted in graft acceptance in the
majority of mice. However, the animals may not be tolerant.
Transient immunosuppression can lead to permanent islet graft
acceptance because of graft adaptation (the loss of immunogenicity
as a result of the loss of APC function) (L. Hao, Y. Wang, R. G.
Gill, K. J. Lafferty, J. Immunol. 139:4022 (1987); K. J. Lafferty,
S. J. Prowse, M. Simeonovic, Annu. Rev. Immunol. 1:143 (1983)).
[0222] In order to differentiate between these possibilities, we
nephrectomized selected xenografted, CTLA4Ig-treated mice (day 40)
and retransplanted them under the remaining kidney capsule with
either the original donor islets (first party) or unrelated second
party human islets (FIG. 14).
[0223] Streptozotocin-treated control animals, having never
received an islet graft, were also transplanted with either first
or second party islets. No treatment after the transplant was
given. Control animals rejected the first and second party islets
by day 4. The CTLA4Ig-treated animals that had received the second
party islets rejected these islets by day 5, whereas animals
receiving first party donor islets maintained the grafts for >80
days (FIG. 14).
[0224] These results suggest that the CTLA4Ig treatment resulted in
prolonged donor-specific unresponsiveness to the xenogeneic islets.
The ability of the murine immune response to distinguish
differences among the human islet donors also supports the direct
recognition of the polymorphic MHC products expressed on the human
islet cells.
Example 6
[0225] Female BALB/c (H-2.sup.d) and C57BL/6 (H-2.sup.d)mice, 6 to
8 wk. of age were obtained from The Jackson Laboratory (Bar Harbor,
Me.).
[0226] Monoclonal antibody 11B11 is a rat IgG1 anti-murine IL-4
(Ohara, J., and W. E. Paul, 1985, Production of a monoclonal
antibody to and molecular characterization of B-cell stimulatory
factor-1. Nature 315:333) (Verax (Lebanon, N.H.)).
[0227] BALB/c mice (five per group) were immunized intravenously
with 10.sup.8 SRBC alone or together with 200 .mu.g chimeric L6 mAb
or human CTLA4Ig fusion protein. The indicated groups were treated
2 hrs. prior to injection of SRBCs by intraperitoneal injection of
2 mls of either rat immunoglobulin or rat anti-murine IL-4 mAb
11B11 at 5 mg/ml. Treatment with chimeric L6 mAb or CTLA4Ig was
repeated daily for 4 additional days.
[0228] All animals were given intravenous injections of SRBCs (FIG.
15) or KLH (FIG. 16) on day 46. Specifically, in FIG. 15, the
closed circle represents mice who were administered with only SRBC
at day 0 and day 46. The open circle represents mice administered
with only SRBC at day 46. The remaining mice represented in FIG. 15
were further administered with SRBC at day 46. In contrast, in FIG.
16, the mice were administered with a different immunogen, KLH, at
day 46 only.
[0229] Serum concentrations of mice measured as having antibodies
directed against SRBCs or KLH were determined by ELISA as described
(Linsley et al., Science 1992).
[0230] Serum antibody titers were calculated as the dilution giving
an A.sub.450 of five times background. Serum antibody titer values
from FIG. 15 were determined from pooled sera from five mice per
group, while serum antibody titer values from FIG. 16 represents
mean titers of five individual sera. Arrows indicate an SRBC or KLH
injection at day 46.
[0231] FIGS. 15 and 16 show that the immunological response in mice
injected concurrently with both CTLA4Ig and anti-IL4 (open
triangle) is suppressed in an antigen-specific manner.
[0232] FIG. 15 shows that there is no rise in serum antibody titer
(i.e. no primary or secondary immunological response) in mice
injected concurrently with CTLA4Ig and anti-IL4 and injected with
SRBC at day 0 and day 46. The combination of CTLA4Ig and anti-IL4
suppresses a primary and secondary immune response and induces long
lasting immunological non-responsiveness to SRBC.
[0233] Additionally, FIG. 15 shows that there is no primary
immunological response in mice injected concurrently with CTLA4Ig
and the control rat Ig (Cappel, Organontecknika, Palo Alto,
Calif.). However, these mice exhibit a secondary immunological
response after injection with SRBC at day 46 (closed triangle, FIG.
15).
[0234] FIG. 16 shows that administration of CTLA4Ig and anti-IL4,
followed by a different immunogen, KLH, at day 46 in mice does not
suppress a primary immune response to KLH in mice. Instead, these
mice exhibited a primary immune response to KLH (open triangle,
FIG. 16). Thus, mice treated with CTLA4 Ig and anti-IL4 exhibited a
highly specific immune response depending on the antigen
administered therein.
Example 7
[0235] By site-specific and homolog mutagenesis, we have identified
regions in CTLA4Ig which are required for its high avidity binding
to B7-1. The following is a description of how to make soluble
CTLA4/CD28 hybrid fusion proteins which bind B7.
Materials and Methods
[0236] Monoclonal antibodies (mAbs). Murine mAb's specific for
CTLA4 were prepared and characterized as previously described
(Linsley et al. J. Ex. Med., (1992) 176:1595-1604). Antibody 9.3
(anti-CD28) has been described previously (Hansen et al.,
Immunogenetics 10:247-260 (1980)).
[0237] Cell Culture. The preparation of stably transfected B7-1
positive CHO cells has been previously described (Linsley et al.,
in J. Exp. Med. 173:721-730 (1991); P. S. Linsley et al., J. Exp.
Med. 174:561 (1991)).
[0238] Cells were maintained in DMEM.TM. supplemented with 10%
fetal bovine serum (FBS), 0.2 mM proline, and 1 .mu.M methotrexate.
COS cells were grown in DMEM.TM. supplemented with 10% FBS. CTLA4Ig
was prepared in CHO cells as previously described (Example 2).
[0239] CTLA4Ig and CD28Ig site-directed mutant expression plasmids.
Site-directed mutagenesis was performed on a vector encoding
soluble chimeric form of CTLA4 (CTLA4 Ig) in which the
extracellular domain of CTLA4 was genetically fused to the hinge
and constant regions of a human IgG heavy chain (Example 2).
CTLA4Ig site-directed mutants were prepared by encoding the desired
mutation in overlapping oligonucleotide primers and generating the
mutants by PCR (Ho et al., 1989, supra.) using the CTLA4Ig plasmid
construct as a template.
[0240] Six mutants were prepared which encoded substitutions to
alanine in the highly conserved hexapeptide 98MYPPPY103 (SEQ ID
NO:23) forming part of the putative CDR3-like domain (FIG. 17) (Ho
et al., 1989, supra.). These mutants are described in Table II.
[0241] In addition, two mutants encoding the residues P103A and
Y104A (MYPPAY (SEQ ID NO:31) and MYPPPA (SEQ ID NO:32),
respectively) from the CD28Ig 99MYPPPY104 (SEQ ID NO:23)
hexapeptide using CD28Ig as a template were also prepared by the
same method. These mutants are also described in Table II.
[0242] Primers required for PCR reactions but not for introducing
mutations included (1) a CDM8 forward (CDM8FP) primer encoding a
complementary sequence upstream of the HindIII restriction site at
the 5' end of the CDM8 stuffer region, and (2) a reverse primer
(CDM8RP) encoding a complementary sequence downstream of the XbaI
site at the 3' end of the CDM8 stuffer region.
[0243] These primers encoded the following sequences:
TABLE-US-00001 CDM8FP: 5'-AATACGACTCACTATAGG (SEQ ID NO: 15)
CDM8RP: 5'-CACCACACTGTATTAACC (SEQ ID NO: 16)
[0244] PCR conditions consisted of 6 min at 94.degree. C. followed
by 25 cycles of 1 min at 94.degree. C., 2 min at 55.degree. C. and
3 min at 72.degree. C. Taq polymerase and reaction conditions were
used as suggested by the vendor (Perkin Elmer Cetus, Emeryville,
Calif.). PCR products were digested with HindIII and XbaI and
ligated to HindIII/XbaI-cut CDM8 expression vector.
[0245] To confirm that the desired mutations had been inserted and
to verify the absence of secondary mutations, each CTLA4Ig mutant
fusion protein (an example of a soluble CTLA4 mutant fusion
protein) was sequenced by the dideoxy chain termination/extension
reaction with Sequenase reagents used according to the
manufacturers recommendations (United States Biochemical Corp.,
Cleveland, Ohio).
[0246] Plasmids were transfected into COS cells (Aruffo et al.,
Cell 61:1303 (1990)) and the conditioned media was used as a source
for the resulting Ig mutant fusion proteins.
[0247] CTLA4/CD28Ig hybrid expression plasmids. CTLA4/CD28Ig hybrid
scan plasmids encoding the constructs HS2, HS4, HS4-A, HS4-B, and
HS5 (FIG. 19 and Table I) were prepared by PCR using overlapping
oligonucleotide primers designed to introduce CTLA4 sequences into
CD28Ig while, at the same time, deleting the equivalent region from
CD28. The same CDM8 forward and reverse PCR primers described above
were also used.
[0248] The following is a list of the CTLA4/CD28 hybrid fusion
proteins which were made.
TABLE-US-00002 DESIGNATION FRAMEWORK MODIFICATIONS HS1 CTLA4 1-24
OF CD28 97-125 OF CD28 HS2 CD28 1-22 OF CTLA4 96-125 OF CTLA4 HS3
CTLA4 96-125 OF CTLA4 HS4 CD28 96-123 OF CTLA4 HS4A CD28 96-113 OF
CTLA4 HS4B CD28 114-123 OF CTLA4 HS5 CD28 25-32 OF CTLA4 HS6 CTLA4
25-32 OF CD28 HS7 CD28 96-123 OF CTLA4 25-32 OF CTLA4 HS8 CD28
25-32 OF CTLA4 96-113 OF CTLA4 HS9 CD28 25-32 OF CTLA4 114-123 OF
CTLA4 HS10 CD28 96-123 OF CTLA4 51-58 OF CTLA4 HS11 CD28 25-32 OF
CTLA4 51-58 OF CTLA4 96-123 OF CTLA4 HS12 CD28 51-58 OF CTLA4
96-113 OF CTLA4 HS13 CD28 25-32 OF CTLA4 51-58 OF CTLA4 96-113 OF
CTLA4 HS14 CD28 51-58 OF CTLA4
[0249] Each cDNA construct was genetically linked to cDNA encoding
the hinge and constant regions of a human IgG1 in order to make
soluble chimeras.
[0250] A HS6 hybrid was prepared in a similar manner to that
described above except that the CDR1-like region in CTLA4Ig was
replaced with the equivalent region from CD28Ig.
[0251] HS7, HS8, and HS9 constructs were prepared by replacing a
.about.350 base-pair HindIII/HpaI 5' fragment of HS4, HS4-A, and
HS4-B, respectively, with the equivalent cDNA fragment similarly
digested from HS5 thus introducing the CDR1-like loop of CTLA4 into
those hybrids already containing the CTLA4 CDR3-like region.
[0252] HS10-HS13 constructs are domain homolog mutants which were
prepared by introducing the CDR2-like loop of CTLA4Ig into
previously constructed homolog mutants. This was done by
overlapping PCR mutagenesis whereby primers were designed to
introduce CTLA4 CDR2-like sequences into homolog templates while at
the same time deleting the equivalent CD28 CDR2-like region from
the molecule.
[0253] Accordingly, HS4 served as a template to make HS10; HS7
served as a template to make HS11; HS4-A served as a template to
make HS12; and HS8 served as a template to make HS13 (FIG. 19 and
Table I). The CDM8 primers described above were also used in these
constructions.
[0254] The HS14 hybrid construct was prepared by replacing the
CDR2-like loop of CD28 with the equivalent loop from CTLA4Ig (FIG.
19 and Table I).
[0255] Oligonucleotide primers designed to introduce these changes
were used in overlapping PCR mutagenesis identical to that
described for other mutants.
[0256] PCR reactions and subcloning into CDM8 were performed as
described above. Again all mutants were sequenced by the dideoxy
chain termination/extension reaction.
[0257] Plasmids encoding each of the mutants were transfected into
COS cells and the resulting soluble Ig fusion proteins were
quantitated in culture media and visualized by Western blot as
described in following sections.
[0258] Quantitation of the resulting Ig fusion proteins in culture
media. Soluble mutant fusion proteins were quantitated in an enzyme
immunoassay by determining the amount of Ig present in serum-free
COS cell culture media.
[0259] Microtiter plates (Immulon2; Dynatech Labs., Chantilly, Va.)
were coated with 0.5 .mu.g/ml goat anti-human IgG (Jackson
Immunoresearch Labs., West Chester, Pa.) for 16-24 h at 4.degree.
C. Wells were blocked for 1 h with specimen diluent (Genetic
Systems, Seattle, Wash.), then washed with PBS containing 0.05%
Tween 20 (PBS-Tw).
[0260] COS cell culture media containing fusion proteins was added
at various dilutions and incubated for 1 h at 22.degree. C. Known
concentrations of CTLA4Ig were also added to separate wells on each
plate for a standard curve.
[0261] After washing, horseradish peroxidase (HRP)-conjugated goat
anti-human IgG (Tago, Burlingame, Calif.) diluted 1:12,000 was
added and incubated for 1 h at 22.degree. C. Wells were then washed
and incubated with 3,3',5,5' tetramethylbenzidine (TMB) substrate
(Genetic Systems) for 15 min before stopping the reaction by the
addition of 1N H.sub.2SO.sub.4. Optical density was measured at
dual wavelengths of 450 and 630 nm on a microtiter plate reader
(Genetic Systems).
[0262] Concentration of mutant Ig fusion protein was determined by
comparison with a standard curve of known concentrations of
CTLA4Ig.
[0263] Immunoprecipitation and Western blot analysis. CTLA4/CD28Ig
hybrid fusion proteins present in culture media were adsorbed to
protein A-Sepharose.TM. by overnight incubation at 4.degree. C. The
beads were washed with PBS containing 0.1% Nonidet-P40 (NP40) then
SDS PAGE sample buffer was added and the eluted protein was loaded
onto an SDS polyacrylamide gel.
[0264] Western blot transfer of protein onto nitrocellulose was
done by standard procedures. Nitrocellulose membranes were then
blocked with PBS containing 0.1% NP40 and 1% non-fat dry milk
powder.
[0265] After washing in PBS-Tw membranes were incubated with
alkaline phosphatase-conjugated goat anti-human IgG (Boehringer
Mannheim, Indianapolis, Ind.) diluted 1:1,000 and incubated for 1 h
at 22.degree. C. Blots were then washed and developed using
standard procedures.
[0266] B7 positive CHO cell enzyme immunoassay. The ability of
CTLA4Ig mutant fusion proteins, and CTLA4/CD28Ig hybrid fusion
proteins to bind B7-1 stably expressed on CHO cells was determined
by an enzyme immunoassay.
[0267] Round bottom tissue culture treated 96 well microtiter
plates (Corning, Corning, N.Y.) were seeded with B7-1 positive CHO
cells at 10.sup.3 cells/well. Two days later the confluent cells
were fixed in 95% ethanol for 15 min.
[0268] After washing with PBS-Tw, mutant Ig fusion proteins were
added at various concentrations and incubated for 1 h at 4.degree.
C. After washing, HRP-conjugated goat anti-human IgG (Tago) diluted
1:10,000 was added and incubated for 1 h at 22.degree. C.
[0269] Wells were then washed and TMB substrate added as above and
allowed to react for 30 min before stopping the reaction with 1N
H.sub.2SO.sub.4. Absorbance of the wells was measured at 450
nm.
[0270] CD28Ig site-directed mutant fusion protein binding assay.
Site-directed mutant fusion proteins of CD28Ig were assayed for
their ability to bind to B7-1 by an indirect enzyme
immunoassay.
[0271] Wells of ELISA plates were coated with a chimeric fusion
protein containing the extracellular domain of human B7-1 fused to
a mouse IgG1 Fc region, at 5 .mu.g/ml for 16 h at 4.degree. C.
Wells were blocked for 1 h with specimen diluent (Genetic Systems)
then washed with PBS-Tw. COS cell culture media containing known
concentrations of mutant fusion protein was added at various
concentrations and incubated for 1 h at 22.degree. C.
[0272] Known concentrations of CD28 .mu.g were also added to
separate wells on each plate. After washing, HRP-conjugated goat
anti-human IgG (Tago) diluted 1:10,000 was added and incubated for
1 h at 22.degree. C. TMB substrate was added and optical densities
read as described for quantitation of Ig fusion proteins in culture
media.
[0273] mAb binding to Ig fusion proteins. The ability of anti-CTLA4
mAb's and the anti-CD28 mAb 9.3 to bind CTLA4/CD28Ig hybrid fusion
proteins and CTLA4Ig mutant fusion proteins was assessed by an
enzyme immunoassay.
[0274] Wells of microtiter plates (Immulon 2) were coated with 0.5
.mu.g/ml of goat anti-human IgG (Jackson) for 16-24 h at 4.degree.
C. Plates were blocked for 1 h with specimen diluent (Genetic
Systems), washed with PBS-Tw, then incubated with the Ig fusion
proteins for 1 h at 22.degree. C. After washing, wells were
incubated with mAb at 1 .mu.g/ml for 1 h at 22.degree. C.
[0275] After further washing, HRP-conjugated goat anti-mouse Ig
(Tago) diluted 1:10,000 was added and incubated for 1 h at
22.degree. C. TMB substrate was added and optical density measured
as described above.
[0276] CTLA4 molecular model. An approximate three-dimensional
model of the CTLA4 extracellular domain was generated based on the
conservation of consensus residues of IGSF variable-like
domains.
[0277] Using such IGSF consensus residues as "anchor points" for
sequence alignments, CTLA4 residues were assigned to the A, B, C,
C', C'', D, E, F, G strands of an Ig variable fold
(Williams/Barclay, 1988, supra.) and the connecting loop regions
(FIG. 21).
[0278] The CTLA4 model was built (InsightII, Discover, Molecular
Modeling and Mechanics Programs, respectively, Biosym Technologies,
Inc., San Diego) using the variable heavy chain of HyHEL-5 (Sheriff
et al., 1987 PNAS 84:8075-8079) as template structure. Side-chain
replacements and loop conformations were approximated using
conformational searching (Bruccoleri et al., 1988 335:564-568).
[0279] Several versions of the model with modified assignments of
some residues to .beta.-strands or loops were tested using
3D-profile analysis (Luthy et al., 1992, Nature 336:83-85) in order
to improve the initial alignment of the CTLA4 extracellular region
sequence with an IGSF variable fold.
RESULTS
[0280] Construction and binding activity of CTLA4Ig and CD28Ig
mutant fusion proteins. A sequence alignment of various homologues
of CD28 and CTLA4 is demonstrated in FIG. 17. In FIG. 17, sequences
of human (H), mouse (M), rat (R), and chicken (Ch) CD28 are aligned
with human and mouse CTLA4. Residues are numbered from the mature
protein N-terminus with the signal peptides and transmembrane
domains underlined and the CDR-analogous regions noted. Dark shaded
areas highlight complete conservation of residues while light
shaded areas highlight conservative amino acid substitutions in all
family members.
[0281] Regions of sequence conservation are scattered throughout
the extracellular domains of these proteins with the most rigorous
conservation seen in the hexapeptide MYPPPY (SEQ ID NO:23) motif
located in the CDR3-like loop of both CTLA4 and CD28 (FIG. 17).
This suggests a probable role for this region in the interaction
with a B7 antigen, e.g., B7-1 and B7-2.
[0282] To test this possibility, site-directed alanine scanning
mutations were introduced into this region of CTLA4Ig using PCR
oligonucleotide primer-directed mutagenesis thereby resulting in
CTLA4Ig mutant fusion proteins. Similarly two alanine mutations
were introduced into the CD28Ig MYPPPY (SEQ ID NO:23) motif thereby
resulting in CD28Ig mutant fusion proteins.
[0283] All cDNA constructs were sequenced to confirm the desired
mutations before transfection into COS cells. The concentrations of
mutant Ig fusion proteins in serum-free COS cell culture media were
determined by an Ig quantitation assay.
[0284] The ability of each CTLA4Ig mutant fusion protein to bind to
B7-1 expressed on stably transfected CHO cells was then determined
by an indirect cell binding immunoassay. Binding of CD28Ig mutant
fusion proteins to B7-1 was assessed by an indirect enzyme
immunoassay. Each of these assays are described in Materials and
Methods.
[0285] Mutagenesis of each residue of the CTLA4Ig MYPPPY (SEQ ID
NO:23) motif to Ala had a profound effect on binding to B7-1 as
shown in FIG. 18. FIG. 18 shows that mutations in the MYPPPY (SEQ
ID NO:23) motif of CTLA4Ig and CD28Ig disrupt binding to B7-1.
Site-directed mutant Ig fusion proteins were produced in
transiently transfected COS cells, quantitated and tested for their
ability to bind to B7-1.
[0286] In FIG. 18 fusion protein quantitations were repeated at
least twice with replicate determinations. Specifically, FIG. 18
shows that CTLA4 Ig mutants bind to stably transfected,
ethanol-fixed B7-1+ CHO cells grown to confluency in ELISA tissue
culture plates. Binding data is expressed as the average of
duplicate wells and is representative of at least two
experiments.
[0287] Y99A and P101A mutants bound to B7-1 but with considerably
reduced ability relative to wild-type CTLA4Ig. In contrast, the
mutants M98A, P100A, P102A and Y103A showed an almost complete loss
of binding. Furthermore, the CD28Ig MYPPPY (SEQ ID NO:23) mutants
P103A and Y104A did not display detectable binding to B7-1
immobilized on wells of ELISA plates (FIG. 18).
[0288] B7-1 transfected CHO cells which were incubated with CTLA4Ig
mutant fusion protein, labeled with anti-human FITC, and assayed
using a FACSCAN showed equivalent results. These results clearly
demonstrate a critical role for the MYPPPY (SEQ ID NO:23) motif in
both CTLA4Ig and CD28Ig binding to B7-1.
[0289] Characterization of CTLA4/CD28Ig hybrid fusion proteins.
Since the MYPPPY (SEQ ID NO:23) motif is common to both CTLA4Ig and
CD28Ig, it alone cannot account for the observed differences in
binding to B7-1 seen with CTLA4Ig and CD28Ig. The contribution of
less well conserved residues to high avidity binding B7-1 was
assessed using a series of homolog mutants.
[0290] The three CDR-like regions of CD28 were replaced in various
combinations with the equivalent regions from the CTLA4
extracellular domain (FIG. 19 and Table I). FIG. 19 is a map of
CTLA4/CD28Ig mutant fusion proteins showing % binding activity to
B7-1+CHO cells relative to CTLA4-Ig. Conserved cysteine residues
(C) are shown at positions 22, 93 and 121 respectively (CTLA4
numbering). Also shown is the position of the MYPPPY (SEQ ID NO:23)
motif. Open areas represent CD28 sequence; filled areas represent
CTLA4 sequence; cross-hatched areas represent beginning of IgG Fc
(also refer to Table D. Percent binding activities were determined
by comparing binding curves (FIGS. 20a and 20b) relative to
CTLA4-Ig and finding the concentration of a mutant required to give
the same O.D. as that found for CTLA4-Ig. The ratio of mutant
protein to CTLA4-Ig concentration at a particular O.D. was then
expressed as % binding activity. At least two A450 readings were
taken from the linear part of the CTLA4-Ig binding curve and the
average % binding activity determined.
[0291] A total of 14 hybrid cDNA constructs were prepared,
sequenced, and transfected into COS cells. Concentrations of Ig
fusion proteins in serum-free culture media were determined and
their electrophoretic mobility compared by SDS-PAGE including
Western blotting analysis.
[0292] Under reducing conditions each chimeric protein migrated
with a relative molecular mass ranging between that of CTLA4Ig
(Mr-50 kDa) and CD28Ig (Mr-70 kDa) depending on the size of the
exchanged region.
[0293] Under non-reducing conditions the proteins migrated
primarily between 100-140 kDa indicating that these fusion proteins
existed as disulfide-linked dimers despite mutagenesis of the
cysteine residues in the hinge region of the Fc.
[0294] Since four of the five conserved cysteine residues in CTLA4
and CD28 are thought to be involved in intrachain disulfide bonds,
dimerization of the fusion proteins was therefore most likely
attributable to the fifth conserved cysteine residue at position
121 in CTLA4 (position 123 in CD28).
[0295] Binding of CTLA4/CD28Ig hybrid fusion proteins to B7-1. The
hybrid fusion proteins were tested for their ability to bind to
B7-1 by the same indirect cell binding immunoassay used to assay
the site-specific CTLA4Ig and CD28Ig mutant fusion proteins.
[0296] Under these conditions the binding between CD28 .mu.g and
B7-1 is barely detectable (FIGS. 20a/b). However, replacing
residues 97 to 125 (the CDR3-like extended region) of CD28 with the
corresponding residues of CTLA4 resulted in an approximately two
and a half orders of magnitude increase in binding of the CD28Ig
analog to B7-1 (FIG. 20a/b). FIG. 20a/b shows that CTLA4/CD28Ig
mutant fusion proteins demonstrate involvement of CDR-analogous
regions in high avidity binding to B7-1 CHO cells. Mutants were
assayed as described in FIG. 2. Data is expressed as the average of
duplicate wells and is representative of at least three
experiments. From these curves % binding activity relative to
CTLA4-Ig was determined as explained and shown in FIG. 19.
[0297] Binding to B7-1 by this construct, termed HS4 (FIG. 19), is
approximately five fold less than wild type CTLA4Ig. The HS2 hybrid
which includes additional N-terminal residues of CTLA4 (amino acids
1-22), did not improve the ability of the hybrid molecule to bind
to B7-1 relative to HS4.
[0298] The HS6 construct which represents the CTLA4Ig sequence
except that it contains the CDR1-like region of CD28 (residues
25-32), bound similarly. However, the additional inclusion of the
CTLA4 CDR1-like region (residues 25-32) into the HS4 construct
(termed HS7), showed further improved binding so that the binding
affinity is approximately 44% of CTLA4Ig (FIG. 19).
[0299] In contrast, inclusion of the CDR2-like region of CTLA4
(residues 51-58) into HS4 (construct HS10), did not further
increase binding (FIG. 19). A similar result was found for
construct HS11 which had all three CDR-like region sequences of
CTLA4 included into CD28Ig. The HS5 hybrid which contained only the
CDR1-like domain of CTLA4 bound at very low levels.
[0300] The CTLA4/CD28Ig hybrid HS4-A encoded CTLA4Ig residues
96-113 in the C-terminally extended CDR3-like region; nine CTLA4
derived residues fewer than HS4 (FIG. 19 and Table 1). HS4-A bound
B7-1 CHO cells less well than HS4 (FIGS. 19 and 20b). However,
addition of the CTLA4 CDR1-like loop (HS8 hybrid), increased B7-1
binding from about 2% to nearly 60% of wild type binding.
[0301] On the other hand, addition of the CTLA4 CDR2-like loop into
HS4-A (HS12) did not increase binding relative to HS4-A; neither
did addition of all three CTLA4 CDR-like regions (HS 13, FIG.
19).
[0302] Another hybrid called HS4-B, encoded the CD28 CDR3-like
region including the MYPPPY (SEQ ID NO:23) motif followed by CTLA4
residues 114-122 (Table I and FIG. 19).
[0303] HS4-B and HS4-A displayed similar binding to B7-1. Unlike
HS4-A, however, the inclusion of the CTLA4 CDR1-like loop into
HS4-B (HS9) did not improve binding (FIG. 19), suggesting that
residues immediately adjacent to the CTLA4Ig MYPPPY (SEQ ID NO:23)
motif were important determinants in high avidity binding.
[0304] Monoclonal antibody binding to CTLA4/CD28Ig hybrid fusion
proteins. The structural integrity of each hybrid fusion protein
was examined by assessing their ability to bind mAb's specific for
CTLA4 or CD28 in an enzyme immunoassay. The CTLA4 specific mAb's
7F8, 11D4 and 10A8 block ligand binding (Linsley et al. (1992)
supra.).
[0305] These antibodies bound to each of the CTLA4Ig mutant fusion
proteins except 11D4 which failed to bind to P100A and P102A (Table
II). Since 7F8 and 10A8 bound to these mutants, the lack of binding
by 11D4 can probably be attributed to mutagenesis perturbing the
epitope recognized by 11D4.
[0306] Conversely, each antibody failed to bind to any of the
homolog scan hybrid fusion proteins except 7F8 which bound to HS6,
and 11D4 which bound weakly to HS8. As many of these homolog hybrid
fusion proteins were, to some extent, able to bind to B7-1, it is
likely that lack of binding by the antibodies was due to disruption
of conformational epitopes formed by spatially adjacent but
non-linear sequences.
[0307] The CD28 specific mAb 9.3 (Linsley et al. (1992) supra.)
failed to bind to either of the CD28 site-directed mutant fusion
proteins but bound to the hybrid fusion proteins HS4, HS4-A, HS7
and HS8. With HS2, weaker binding was observed. No binding was seen
with the HS5 and HS6 constructs.
[0308] CTLA4 model. FIG. 21 shows a schematic representation of the
CTLA4 model. The assignment of CTLA4 residues to CDR-like regions
is shown in FIG. 17. The CTLA4 model suggests the presence of an
additional (non-Ig) disulfide bond between residues Cys49 and Cys67
which supports the similarity of CTLA4 and the Ig variable
fold.
[0309] The two possible N-linked glycosylation sites in CTLA4 map
to solvent exposed positions of the Ig .beta.-strand framework
regions. 3D-profile analysis indicated that the CTLA4 sequence is
overall compatible with an Ig V-fold, albeit more distantly
related.
[0310] Residue Val 115 represents the last residue of the
CTLA4Ig-like domain. The conformation of the region between Val 115
and the membrane-proximal Cys121 which is thought to form the CTLA4
homodimer is highly variable in the CD28 family. The picture that
emerges is that CD28 family members mainly utilize residues in two
of three CDR-like regions for binding to B7-1.
[0311] The MYPPPY (SEQ ID NO:23) motif represents a conserved
scaffold for binding which appears to be augmented by its
C-terminal extension and which is specifically modulated by the
highly variable CDR1-like region. CDR3 and CDR1-like regions are
spatially contiguous in Ig-variable folds. The CDR2 like region is
spatially distant and does not, in the case of the CD28 family,
significantly contribute to the binding to B7-1.
[0312] As will be apparent to those skilled in the art to which the
invention pertains, the present invention may be embodied in forms
other than those specifically disclosed above without departing
from the spirit or essential characteristics of the invention. The
particular embodiments of the invention described above, are,
therefore, to be considered as illustrative and not restrictive.
The scope of the present invention is as set forth in the appended
claims rather than being limited to the examples contained in the
foregoing description.
TABLE-US-00003 TABLE I CTLA4/CD28Ig homolog mutant junction
sequences. MUTANT HS1 -22CKYasp27- -93ckvEVM99- -123CPSDQE- HS2
-20fvcKYS25- -94CKIelm98- -121cpdDQE- HS3 -93ckvEVM99- -123CPSDQE-
HS4 -94CKIelm98- -121cpdDQE- HS5 -22CKYasp27- -30ateFRA35-
-123CPSDQE- HS6 -22ceySYN27- -30SREvrv35- -121cpdDQE- HS4-A
-94CKIelm98- -111tqiHVK118- -123CPSDQE- HS4-B -113TIIyvi116-
-121cpdDQE- HS7 -22CKYasp27- -30ateFRA35- -94CKIelm98- -121cpdDQE-
HS8 -22CKYasp27- -30ateFRA35- -94CKIelm98- -111tqiHVK118-
-123CPSDQE- HS9 -22CKYasp27- -30ateFRA35- -113TIIyvi116-
-121cpdDQE- HS10 -47VCVaty53- -56gneLQV60- -94CKIelm98- -121cpdDQE-
HS11 -22CKYasp27- -30ateFRA35- -47VCVaty53- -56gneLQV60-
-94CKIelm98- -121cpdDQE- HS12 -47VCVaty53- -56gneLQV60-
-94CKLelm98- -111tqiHVK118- -123CPSDQE- HS13 -22CKYasp27-
-30ateFRA35- -47VCVaty53- -56gneLQV60- -94CKIelm98- -111tqiHVK118-
-123CPSDQE- HS14 -47VCVaty53- -56gneLQV60- -123CPSDQE- Junction
sequences of the CTLA4/CD28-Ig hybrid fusion proteins. Amino acids
are denoted by their single letter code with those in upper case
being CD28 residues, those in lower case being CTLA4 residues and
those in bold upper case being human IgG1 residues. Numbering in
the table is from the N-terminal methionine of the respective
proteins and refers to the adjacent amino acids.
TABLE-US-00004 TABLE II Binding of CTLA4 and CD28 monoclonal
antibodies to CTLA4Ig and CD28Ig mutant fusion proteins and to
CTLA4/CD28Ig hybrid fusion proteins. anti-CTLA4 anti-CD28 mAbs mAb
7F8 11D4 10A8 9.3 CTLA4Ig MUTANT FUSION PROTEIN AYPPPY (SEQ ID NO:
24) +++ +++ +++ - MAPPPY (SEQ ID NO: 25) ++ + ++ - MYAPPY (SEQ ID
NO: 26) + - + - MYPAPY (SEQ ID NO: 27) +++ +++ +++ - MYPPAY (SEQ ID
NO: 28) +++ - + - MYPPPA (SEQ ID NO: 29) +++ ++ +++ - AAPPPY (SEQ
ID NO: 30) + ++ +++ - CD28Ig MUTANT FUSION PROTEIN MYPPAY (SEQ ID
NO: 31) - - - - MYPPPA (SEQ ID NO: 32) - - - + CTLA4/CD28Ig HYBRID
FUSION PROTEINS HS1 - - - - HS2 - - - + HS3 - - - - HS4 - - - +++
HS5 - - - - HS6 + - - - HS4-A - - - ++ HS4-B - - - ++ HS7 - - - +++
HS8 - + - +++ HS9 - + - - HS10 - - - - HS11 - - - + HS12 - - - -
HS13 - - - - HS14 - - - - CTLA4Ig +++ +++ +++ - CD28Ig - - - +++
Antibody binding was rated from that seen for wild type protein
(+++) to above background (+), and no detectable binding (-).
Sequence CWU 1
1
34139DNAArtificialOncostatin M signal peptide forward primer
1ctagccactg aagcttcacc atgggtgtac tgctcacac
39239DNAArtificialOncostatin M signal peptide reverse primer
2tggcatgggc tcctgatcag gcttagaagg tccgggaaa
39339DNAArtificialOncostatin M signal peptide reverse primer
3tttgggctcc tgatcaggaa aatgctcttg cttggttgt 39484DNAArtificialHuman
IgCgamma1 forward primer 4aagcaagagc attttcctga tcaggagccc
aaatcttctg acaaaactca cacatcccca 60ccgtccccag cacctgaact cctg
84541DNAArtificialHuman IgCgamma1 reverse primer 5cttcgaccag
tctagaagca tcctcgtgcg accgcgagag c 41647DNAArtificialCD5Ig forward
primer 6cattgcacag tcaagcttcc atgcccatgg gttctctggc caccttg
47739DNAArtificialCD5Ig reverse primer 7atccacagtg cagtgatcat
ttggatcctg gcatgtgac 39865DNAHomo sapiens 8ctcagtctgg tccttgcact
cctgtttcca agcatggcga gcatggcaat gcacgtggcc 60cagcc 65933DNAHomo
sapiens 9tttgggctcc tgatcagaat ctgggcacgg ttg 331072DNAHomo sapiens
10ctagccactg aagcttcacc aatgggtgta ctgctcacac agaggacgct gctcagtctg
60gtccttgcac tc 721133DNAHomo sapiens 11gcaatgcacg tggcccagcc
tgctgtggta gtg 331245DNAHomo sapiens 12tgatgtaaca tgtctagatc
aattgatggg aataaaataa ggctg 4513561DNAHomo sapiensCDS(1)..(561)
13gca atg cac gtg gcc cag cct gct gtg gta ctg gcc agc agc cga ggc
48Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly1
5 10 15atc gcc agc ttt gtg tgt gag tat gca tct cca ggc aaa gcc act
gag 96Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr
Glu 20 25 30gtc cgg gtg aca gtg ctt cgg cag gct gac agc cag gtg act
gaa gtc 144Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr
Glu Val 35 40 45tgt gcg gca acc tac atg atg ggg aat gag ttg acc ttc
cta gat gat 192Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe
Leu Asp Asp 50 55 60tcc atc tgc acg ggc acc tcc agt gga aat caa gtg
aac ctc act atc 240Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val
Asn Leu Thr Ile65 70 75 80caa gga ctg agg gcc atg gac acg gga ctc
tac atc tgc aag gtg gag 288Gln Gly Leu Arg Ala Met Asp Thr Gly Leu
Tyr Ile Cys Lys Val Glu 85 90 95ctc atg tac cca ccg cca tac tac ctg
ggc ata ggc aac gga acc cag 336Leu Met Tyr Pro Pro Pro Tyr Tyr Leu
Gly Ile Gly Asn Gly Thr Gln 100 105 110att tat gta att gat cca gaa
ccg tgc cca gat tct gac ttc ctc ctc 384Ile Tyr Val Ile Asp Pro Glu
Pro Cys Pro Asp Ser Asp Phe Leu Leu 115 120 125tgg atc ctt gca gca
gtt agt tcg ggg ttg ttt ttt tat agc ttt ctc 432Trp Ile Leu Ala Ala
Val Ser Ser Gly Leu Phe Phe Tyr Ser Phe Leu 130 135 140ctc aca gct
gtt tct ttg agc aaa atg cta aag aaa aga agc cct ctt 480Leu Thr Ala
Val Ser Leu Ser Lys Met Leu Lys Lys Arg Ser Pro Leu145 150 155
160aca aca ggg gtc tat gtg aaa atg ccc cca aca gag cca gaa tgt gaa
528Thr Thr Gly Val Tyr Val Lys Met Pro Pro Thr Glu Pro Glu Cys Glu
165 170 175aag caa ttt cag cct tat ttt att ccc atc aat 561Lys Gln
Phe Gln Pro Tyr Phe Ile Pro Ile Asn 180 18514187PRTHomo sapiens
14Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly1
5 10 15Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr
Glu 20 25 30Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr
Glu Val 35 40 45Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe
Leu Asp Asp 50 55 60Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val
Asn Leu Thr Ile65 70 75 80Gln Gly Leu Arg Ala Met Asp Thr Gly Leu
Tyr Ile Cys Lys Val Glu 85 90 95Leu Met Tyr Pro Pro Pro Tyr Tyr Leu
Gly Ile Gly Asn Gly Thr Gln 100 105 110Ile Tyr Val Ile Asp Pro Glu
Pro Cys Pro Asp Ser Asp Phe Leu Leu 115 120 125Trp Ile Leu Ala Ala
Val Ser Ser Gly Leu Phe Phe Tyr Ser Phe Leu 130 135 140Leu Thr Ala
Val Ser Leu Ser Lys Met Leu Lys Lys Arg Ser Pro Leu145 150 155
160Thr Thr Gly Val Tyr Val Lys Met Pro Pro Thr Glu Pro Glu Cys Glu
165 170 175Lys Gln Phe Gln Pro Tyr Phe Ile Pro Ile Asn 180
1851518DNAArtificialCDM8 forward primer 15aatacgactc actatagg
181618DNAArtificialCDM8 reverse primer 16caccacactg tattaacc
1817223PRTHomo sapiens 17Met Ala Cys Leu Gly Phe Gln Arg His Lys
Ala Gln Leu Asn Leu Ala1 5 10 15Ala Arg Thr Trp Pro Cys Thr Leu Leu
Phe Phe Leu Leu Phe Ile Pro 20 25 30Val Phe Cys Lys Ala Met His Val
Ala Gln Pro Ala Val Val Leu Ala 35 40 45Ser Ser Arg Gly Ile Ala Ser
Phe Val Cys Glu Tyr Ala Ser Pro Gly 50 55 60Lys Ala Thr Glu Val Arg
Val Thr Val Leu Arg Gln Ala Asp Ser Gln65 70 75 80Val Thr Glu Val
Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr 85 90 95Phe Leu Asp
Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val 100 105 110Asn
Leu Thr Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile 115 120
125Cys Lys Val Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly
130 135 140Asn Gly Thr Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro
Asp Ser145 150 155 160Asp Phe Leu Leu Trp Ile Leu Ala Ala Val Ser
Ser Gly Leu Phe Phe 165 170 175Tyr Ser Phe Leu Leu Thr Ala Val Ser
Leu Ser Lys Met Leu Lys Lys 180 185 190Arg Ser Pro Leu Thr Thr Gly
Val Tyr Val Lys Met Pro Pro Thr Glu 195 200 205Pro Glu Cys Glu Lys
Gln Phe Gln Pro Tyr Phe Ile Pro Ile Asn 210 215 22018223PRTMus
musculus 18Met Ala Cys Leu Gly Leu Arg Arg Tyr Lys Ala Gln Leu Gln
Leu Pro1 5 10 15Ser Arg Thr Trp Pro Phe Val Ala Leu Leu Thr Leu Leu
Phe Ile Pro 20 25 30Val Phe Ser Glu Ala Ile Gln Val Thr Gln Pro Ser
Val Tyr Leu Ala 35 40 45Ser Ser His Gly Tyr Ala Ser Phe Pro Cys Glu
Tyr Ser Pro Ser His 50 55 60Asn Thr Asp Glu Val Arg Val Thr Val Leu
Arg Gln Thr Asn Asp Gln65 70 75 80Met Thr Glu Val Cys Ala Thr Thr
Phe Thr Glu Lys Asn Thr Val Gly 85 90 95Phe Leu Asp Tyr Pro Phe Cys
Ser Gly Thr Phe Asn Glu Ser Arg Val 100 105 110Asn Leu Thr Ile Gln
Gly Leu Arg Ala Val Asp Thr Gly Leu Tyr Leu 115 120 125Cys Lys Val
Glu Leu Met Tyr Pro Pro Pro Tyr Phe Val Gly Met Gly 130 135 140Asn
Gly Thr Gln Ile Tyr Tyr Ile Asp Pro Glu Pro Cys Pro Asp Ser145 150
155 160Asp Phe Leu Leu Trp Ile Leu Tyr Ala Val Ser Leu Gly Leu Phe
Phe 165 170 175Tyr Ser Phe Leu Val Ser Ala Val Ser Leu Ser Lys Met
Leu Lys Lys 180 185 190Arg Ser Pro Leu Thr Thr Gly Val Tyr Val Lys
Met Pro Pro Thr Glu 195 200 205Pro Glu Cys Glu Lys Gln Phe Gln Pro
Tyr Phe Ile Pro Ile Asn 210 215 22019218PRTMus musculus 19Met Thr
Leu Arg Leu Leu Phe Leu Ala Leu Asn Phe Phe Ser Val Gln1 5 10 15Val
Thr Glu Asn Lys Ile Leu Val Lys Gln Ser Pro Leu Leu Tyr Val 20 25
30Asp Ser Asn Glu Val Ser Leu Ser Cys Arg Tyr Ser Tyr Asn Leu Leu
35 40 45Ala Lys Glu Phe Arg Ala Ser Leu Tyr Lys Gly Val Asn Ser Asp
Val 50 55 60Glu Val Cys Val Gly Asn Gly Asn Phe Thr Tyr Gln Pro Gln
Phe Arg65 70 75 80Ser Asn Ala Glu Phe Asn Cys Asp Gly Asp Phe Asp
Asn Glu Thr Val 85 90 95Thr Phe Arg Leu Trp Asn Leu His Val Asn His
Thr Asp Ile Tyr Phe 100 105 110Cys Lys Ile Glu Phe Met Tyr Pro Pro
Pro Tyr Leu Asp Asn Glu Arg 115 120 125Ser Asn Gly Thr Ile Ile His
Ile Lys Glu Lys His Leu Cys His Thr 130 135 140Gln Ser Ser Pro Lys
Leu Phe Trp Ala Leu Tyr Val Val Ala Gly Val145 150 155 160Leu Phe
Cys Tyr Gly Leu Leu Val Thr Val Ala Leu Cys Val Ile Trp 165 170
175Thr Asn Ser Arg Arg Asn Arg Leu Leu Gln Val Thr Tyr Met Asn Met
180 185 190Thr Pro Arg Arg Pro Gly Leu Thr Arg Lys Pro Tyr Gln Pro
Tyr Ala 195 200 205Pro Ala Arg Asp Phe Ala Ala Tyr Arg Pro 210
21520218PRTRattus norvegicus 20Met Thr Leu Arg Leu Leu Phe Leu Ala
Leu Ser Phe Phe Ser Val Gln1 5 10 15Val Thr Glu Asn Lys Ile Leu Val
Lys Gln Ser Pro Leu Leu Val Tyr 20 25 30Asp Asn Asn Glu Val Ser Leu
Ser Cys Arg Tyr Ser Tyr Asn Leu Leu 35 40 45Ala Lys Glu Phe Arg Ala
Ser Leu Tyr Lys Gly Val Asn Ser Asp Val 50 55 60Glu Val Cys Val Gly
Asn Gly Asn Phe Thr Tyr Gln Pro Gln Phe Arg65 70 75 80Pro Asn Val
Gly Phe Asn Cys Asp Gly Asn Phe Asp Asn Glu Thr Val 85 90 95Thr Phe
Arg Leu Trp Asn Leu Asp Val Asn His Thr Asp Ile Tyr Phe 100 105
110Cys Lys Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys
115 120 125Ser Asn Gly Thr Ile Ile His Ile Lys Glu Lys His Leu Cys
His Ala 130 135 140Gln Thr Ser Pro Lys Leu Phe Trp Pro Leu Val Val
Val Ala Gly Val145 150 155 160Leu Leu Cys Tyr Gly Leu Leu Tyr Thr
Val Thr Leu Cys Ile Ile Trp 165 170 175Thr Asn Ser Arg Arg Asn Arg
Leu Leu Gln Ser Asp Tyr Met Asn Met 180 185 190Thr Pro Arg Arg Leu
Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala 195 200 205Pro Ala Arg
Asp Phe Ala Ala Tyr Arg Pro 210 21521220PRTHomo sapiens 21Met Leu
Arg Leu Leu Leu Ala Leu Asn Leu Phe Pro Ser Ile Gln Val1 5 10 15Thr
Gly Asn Lys Ile Leu Val Lys Gln Ser Pro Met Leu Val Ala Tyr 20 25
30Asp Asn Ala Tyr Asn Leu Ser Cys Lys Tyr Ser Tyr Asn Leu Phe Ser
35 40 45Arg Glu Phe Arg Ala Ser Leu His Lys Gly Leu Asp Ser Ala Val
Glu 50 55 60Val Cys Val Val Tyr Gly Asn Tyr Ser Gln Gln Leu Gln Val
Tyr Ser65 70 75 80Lys Thr Gly Phe Asn Cys Asp Gly Lys Leu Gly Asn
Glu Ser Val Thr 85 90 95Phe Tyr Leu Gln Asn Leu Tyr Val Asn Gln Thr
Asp Ile Tyr Phe Cys 100 105 110Lys Ile Glu Val Met Tyr Pro Pro Pro
Tyr Leu Asp Asn Glu Lys Ser 115 120 125Asn Gly Thr Ile Ile His Val
Lys Gly Lys His Leu Cys Pro Ser Pro 130 135 140Leu Phe Pro Gly Pro
Ser Lys Pro Phe Trp Val Leu Val Val Val Gly145 150 155 160Gly Val
Leu Ala Cys Tyr Ser Leu Leu Tyr Thr Val Ala Phe Ile Ile 165 170
175Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met
180 185 190Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr
Gln Pro 195 200 205Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser
210 215 22022221PRTGallus gallus 22Met Leu Gly Ile Leu Val Val Leu
Cys Leu Ile Pro Ala Ala Asp Val1 5 10 15Thr Glu Asn Lys Ile Leu Val
Ala Gln Arg Pro Leu Leu Ile Val Ala 20 25 30Asn Arg Thr Ala Thr Leu
Val Cys Asn Tyr Thr Tyr Asn Gly Thr Gly 35 40 45Lys Glu Phe Arg Ala
Ser Leu His Lys Gly Thr Asp Ser Ala Val Glu 50 55 60Val Cys Phe Ile
Ser Trp Asn Met Thr Lys Ile Asn Ser Asn Ser Asn65 70 75 80Lys Glu
Phe Asn Cys Arg Gly Ile His Asp Lys Asp Lys Val Ile Phe 85 90 95Asn
Leu Trp Asn Met Ser Ala Ser Gln Thr Asp Ile Tyr Phe Cys Lys 100 105
110Ile Glu Ala Met Tyr Pro Pro Pro Tyr Val Tyr Asn Glu Lys Ser Asn
115 120 125Gly Thr Val Ile His Tyr Arg Glu Thr Pro Ile Gln Thr Gln
Glu Pro 130 135 140Glu Ser Ala Thr Ser Tyr Trp Val Met Tyr Ala Val
Thr Gly Leu Leu145 150 155 160Gly Phe Tyr Ser Met Leu Ile Thr Ala
Val Phe Ile Ile Tyr Arg Gln 165 170 175Lys Ser Lys Arg Asn Arg Tyr
Arg Gln Ser Asp Tyr Met Asn Met Thr 180 185 190Pro Arg His Pro Pro
His Gln Lys Asn Lys Gly Tyr Pro Ser Tyr Ala 195 200 205Pro Thr Arg
Asp Tyr Thr Ala Tyr Arg Ser Trp Gln Pro 210 215
220236PRTArtificialCTLA4/CD28 23Met Tyr Pro Pro Pro Tyr1
5246PRTArtificialCTLA4Ig mutant fusion protein 24Ala Tyr Pro Pro
Pro Tyr1 5256PRTArtificialCTLA4Ig mutant fusion protein 25Met Ala
Pro Pro Pro Tyr1 5266PRTArtificialCTLA4Ig mutant fusion protein
26Met Tyr Ala Pro Pro Tyr1 5276PRTArtificialCTLA4Ig mutant fusion
protein 27Met Tyr Pro Ala Pro Tyr1 5286PRTArtificialCTLA4Ig/CD28Ig
mutant fusion protein 28Met Tyr Pro Pro Ala Tyr1
5296PRTArtificialCTLA4Ig/CD28Ig mutant fusion protein 29Met Tyr Pro
Pro Pro Ala1 5306PRTArtificialCTLA4Ig mutant fusion protein 30Ala
Ala Pro Pro Pro Tyr1 5316PRTArtificialCTLA4Ig/CD28Ig mutant fusion
protein 31Met Tyr Pro Pro Ala Tyr1 5326PRTArtificialCTLA4Ig/CD28Ig
mutant fusion protein 32Met Tyr Pro Pro Pro Ala1
53310PRTArtificialCTLA4Ig 33Ser Met Ala Ser Met Ala Met His Val
Ala1 5 103422PRTArtificialCTLA4Ig 34Pro Cys Pro Asp Ser Asp Gln Glu
Pro Lys Ser Ser Asp Lys Thr His1 5 10 15Thr Ser Pro Pro Ser Pro
20
* * * * *