NZ619473B2 - Inhibitors of t-cell activation - Google Patents
Inhibitors of t-cell activation Download PDFInfo
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- NZ619473B2 NZ619473B2 NZ619473A NZ61947312A NZ619473B2 NZ 619473 B2 NZ619473 B2 NZ 619473B2 NZ 619473 A NZ619473 A NZ 619473A NZ 61947312 A NZ61947312 A NZ 61947312A NZ 619473 B2 NZ619473 B2 NZ 619473B2
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/70532—B7 molecules, e.g. CD80, CD86
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/70539—MHC-molecules, e.g. HLA-molecules
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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- C—CHEMISTRY; METALLURGY
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- C07K2319/00—Fusion polypeptide
Abstract
Disclosed is a bispecific biologic comprising a ligand specific for CTLA-4 and a ligand specific for a pMHC complex spaced apart by a linker. Also disclosed is the use of a bispecific biologic comprising a ligand specific for CTLA-4 and a ligand specific for a pMHC complex spaced apart by a linker, for the preparation of a medicament for the treatment of an autoimmune disease or transplant rejection. r, for the preparation of a medicament for the treatment of an autoimmune disease or transplant rejection.
Description
Inhibitors of T -cell activation
Cross -reference to Related Application
This application claims the benefit of the following U.S. Provisional Application No.:
61/503,282, filed June 30, 2011, the entire contents of which are incorporated herein
by reference .
Background of the Invention
Cell therapy using freshly isolated, ex vivo expanded or in vitro d Tregs in
models of autoimmune diseases or organ transplants have demonstrated that adoptive
transfer of Tregs can restore the balance of Tregs versus or T cells, thereby
controlling autoimmunity associated with these diseases (Allan et al., (2008)
Immunol. Rev. 223:391 -421 ; Jiang et al., (2006 ) Expert revie w of al immunology
2:387 -392; Riley et al., (2009 ) Immunity 30:656 -665 ; Tang et al., (2012 ) Journal of
molecular cell biology 4:11 -21) . However, the use of adoptive transfer as a
therapeutic strategy presents several nges to translation into the clinic. The
number of autologous Tregs that can be isolated from peripheral blood of a human
subject is limiting and extensive ex vivo expansion of the Tregs may alter their
functionality and/or purity. As the isolated Tregs are polyclonal, they can exert a pan -
immune suppressive function on non t effector T cells. antly, the
plasticity of Tregs poses a significant challenge (Bluestone e t al., (2009) Nat Rev
Immunol 9:811 -816 ; Zhou et al., (2009a) Curr Opin Immunol 21:281 -285 ), as
adoptively transferred Tregs can lose Foxp3 expression and erentiate into Th17
cells (Koenen et al., (2008) Blood 40 -2352. ) or pathogenic memory T c ells
(Zhou et al., (2009b) Nat l 10:1000 -1007 ) which raises the risk of aggravating
the autoimmunity or inflammation.
A therapeutic that induces the generation of Tregs in an antigen -specific manner in
situ would have advantages over adoptive Treg cell therapy. Cytotoxic T lymphocyte
associated antigen -4 (CTLA -4; CD 152) is a well -established negative regulator of the
T cell response, is important for the maintenance of T cell homeostasis and self -
nce. CTLA -4 is homologous to the co -stimulat ory molecule CD28 and shares
the same ligands, CD80 (B7.1) and CD86 (B7.2), which are expressed on the surface
of antigen presenting cells (APCs). However, ential binding of CD80/CD86 on
APCs to CD28 and CTLA -4 on or T cells leads to opposing outcomes, with
CD28 triggering T cell activation and CTLA -4 causing T cell inhibition.
Because CD28 is constitutively expressed on T cells and the expression of CTLA -4 is
only induced following T cell tion, peaking 2 -3 days later (Jago et al., (2004 )
Clinical & Experimental Immunology , 136: 463 -471), extensive T cell tion
would have occurred prior to CTLA -4 engagement. Hence, the main role of CTLA -4
is to act as a safeguard against an excessive T cell response rather than to inhibit T
cell act ivation. However, early engagement of CTLA -4 by its ligand and its
subsequent crosslinking to the T cell receptor (TCR) can prematurely dampen TCR
signaling, causing T cell inhibition and hyporesponsiveness, or anergy. This concept
has been validated exp erimentally using a y of methods, including the
following: (i) crosslinking T cell -activating antibodies (anti -CD3/anti CD28) using an
agonistic anti -CTLA -4 antibody by co -immobilization on a bead or via a secondary
antibody (Blair et al., (1998) J. Immunol. 160: 12 -15; Krummel and Allison, (1996) J
Exp Med 183:2533 -2540 ; s et al., (1996) J. Exp. Med. 183:2541 -2550); (ii)
molecularly engineering a surface -linked agonistic scFv against CTLA -4 on an APC
(Fife et al., (2006) J. Clin. Invest. 116(8) :2252 - 61; Griffin et al., (2001) J. Immunol.
Methods. 248(1 -2):77 -90; Griffin et al., (2000) J. Immunol. 164(9):4433 -42); and (iii)
chemically crosslinking antibodies that recognize specific antigens on an APC to an
agonistic anti -CTLA -4 antibody (Li et a l., . J. Immunol. :5191 -203; Rao
et al., (2001) Clin. Immunol. 101(2):136 -45; Vasu et al., (2004) J. l.
173(4):2866 - 76).
Restoring the balance of Tregs versus effector T cells is a promising means of treating
autoimmune disease. However , cell therapy involving transfer of Tregs has certain
limitations. Accordingly, therapeutics that can induce the generation of Tregs (e.g.,
CTLA -4) in an antigen -specific manner for the treatment of mune disease are
ly required.
Summary of the Invention
The present invention relates to ligands which crosslink ligand -engaged cytotoxic T
lymphocyte antigen -4 (CTLA -4) to the T cell receptor (TCR) during the early phase
of T cell activation and thereby attenuate TCR signaling, leading to T cell inhibition.
To p an agent that can inhibit T cell activation, a bispecific fusion protein
comprising moieties that selectively bind and activate CTLA -4 and co -ligate it to the
TCR was generated. In contrast to the approaches of the prior art, the bi ic fusion
protein was engineered to crosslink MHC to CTLA -4; both are then drawn to the
TCR, generating the CTLA -4/MHCII/TCR tri -molecular complex within the immune
synapses.
Crosslinking ligand -engaged cytotoxic T lymphocyte antigen -4 (CTLA -4) to th e TCR
with a bispecific fusion protein (BsB) comprising a mutant mouse CD80 and
lymphocyte activation antigen -3 in an allogenic MLR attenuated TCR signaling and
direct T cell differentiation towards Foxp3 + regulatory T cells (Tregs). As described
herein, a ntigen -specific Tregs can also be induced in an antigen fic setting.
Treatment of non -obese diabetic (NOD) mice with a short course of BsB moderately
delayed the onset of mune type 1 diabetes (T1D) with a transient increase of
Tregs in blood. Ho wever, a longer course of treatment of NOD s with BsB
significantly delayed the onset of disease as well as reduced the incidence of s
presenting with diabetes. Histopathological analysis of the pancreata of BsB -treated
mice that ed non -diabetic revealed the presence of Tregs that were intermixed
with other CD3 + T cells and non -T cell leukocytes around the islets. This peri -insulitis
was associated with l invasive insulitis and no notable destruction of the
insulin -producing β-cells. Thus, tional proteins capable of engaging CTLA -4
and MHCII and indirectly co -ligating it to the TCR may induce antigen fic Tregs
in vivo to t mice from T1D or other autoimmune diseases.
In ular, the invention describes bispecific f usion proteins which cross -link
CTLA -4 to the pMHCII complex. For example, there is described a bispecific fusion
n comprising a mutant mouse CD80 (CD80w88a) and lymphocyte activation
antigen -3 (LAG -3) which is engineered to concurrently engage CTLA -4 and crosslink
it to the TCR via pMHCII. In a first aspect, therefore, there is provided a bispecific
biologic comprising a ligand specific for CTLA -4 and a ligand specific for a pMHC
complex.
In one aspect, the invention provides a bispecific biologic c omprising a ligand
specific for CTLA -4 and a ligand ic for a pMHC complex spaced apart by a
linker. The bispecific biologic according to the invention is capable of cross -linking
CTLA -4, present on T -cells, with the peptide MHC (pMHC) complex on ant igen -
presenting cells (APC). The peptide MHC complex is bound by the cognate T -cell
receptor (TCR) on T -cells, meaning that the bispecific ic according to the
invention gives rise to a tripartite CTLA -4/MHC/TCR complex.
In s embodiments of the aspects delineated herein, the ligand specific for
CTLA -4 is ed from an dy specific for CTLA -4, and CD80 (B7 -1) or
CD86 (B7 -2). In a particular embodiment, the antibody specific for CTLA -4, and
CD80 (B7 -1) or CD86 (B7 -2) is an agonistic antibo dy. Antibodies specific for
CTLA -4 can be engineered, and both CD80 and CD86 are natural ligands for CTLA -
4. In one aspect, CD80 or a mutant thereof is used, since CD80 binds preferentially to
CTLA -4 over CD28, and thus promotes T -cell inactivation as op posed to tion.
In various embodiments of the aspects delineated herein, the ligand specific for the
pMHC complex can be selected from an anti -MHC antibody and LAG -3. The LAG -3
polypeptide is a natural ligand for the MHCII protein. In one embodiment , the MHC
is MHC -II (which interacts with CD4 + T-cells). In another embodiment the MHC is
MHC -I, which interacts with CD8 + T-cells.
In the bispecific biologic according to the invention, the ligand ic for CTLA -4
and the ligand specific for the pMHC complex are preferably spaced apart by a linker.
The linker can take the form of one or more of a polyamino acid sequence and an
antibody Fc domain. A suitable polyamino acid sequence is G9 (Gly -9).
In various embodiments of the aspects ated herein , the ligand specific for
CTLA -4 is CD80, or a mutant thereof which is mutated to se specificity for
CTLA -4 over CD28. In one embodiment, the mutated CD80 ses one or more
mutations selected from W88A, K75G. K75V, S112G, R126S, R126D, G127L,
S193 A, and S204A, using sequence numbering in mouse CD80 precursor, or their
human CD80 counterparts (W84A, K71G, K71V, S109G, R123S, R123D, G124L,
S190A, and S201A) and in addition R63A, M81A, N97A, E196A.
In one embodiment, the bispecific biologic comprises CD80, which comprises the
mutation W84A (human) or W88A (mouse).
In a particular embodiment, the ligand specific for the MHCII complex is LAG -3.
Advantageously, LAG -3 is mutated to increase specificity for pMHCII. For example,
LAG -3 comprises one or more m utations selected from R73E, R75A, R75E and R76E
(Huard et al., (1997) Proc. Natl. Acad. Sci. USA. 94(11): 5744 -5749. In one
embodiment, LAG -3 ses the mutation R75E.
Preferential binding of the bispecific fusion n to CTLA -4 over CD28 was
attai ned using mutant CD80 (CD80w88a), which contains alanine instead of
tryptophan at amino acid 88 (numbered in mouse CD80), as the ligand. CD80w88a
binds CTLA -4 but exhibits minimal affinity for CD28 (Wu et al., (1997), J. Exp. Med.
185:1327 -1335).
Lymphocyt e activation gene -3 (LAG -3), a natural ligand of MHCII, was selected as
the other binding component of the bispecific fusion protein (Baixeras et al., (1992) J.
Exp. Med. 176:327 - 337; Triebel et al., (1990) J. Exp. Med. 171:1393 -1405). We
show that a fusi on protein with such bi ionality effectively inhibits T cell
activation and stimulates anti -inflammatory cytokines IL -10 and TGF -β production.
More antly, this bispecific fusion protein also directed T cell differentiation
into highly suppressiv e Foxp3 + Tregs. This did not occur when the well -established
co latory inhibitor CTLA -4Ig was used instead (Bluestone et al., (2006)
ty 24:233 -238; y and Nadler (2009) Immunol. Rev. 229:307 -321).
Therefore, early engagement of CTLA -4 and c rosslinking of CTLA -4 to the TCR
during T cell activation could ly influence T cell differentiation. Such ific
fusion proteins might thus represent a novel class of biologics that could be used to
control excessive T cell responses in autoimmun e es.
In a second aspect of the invention, there is provided the use of a bispecific biologic
sing a ligand specific for CTLA -4 and a ligand specific for a pMHC complex
according to the first aspect of the invention, for the tolerisation of a T -cell by
contacting said T -cell with an antigen -presenting cell which is presenting a peptide
derived from said antigen complexed to a MHC molecule and said bispecific biologic.
In a third aspect, there is provided the use of a bispecific biologic comprisi ng a ligand
specific for CTLA -4 and a ligand specific for a pMHC complex according to the first
aspect of the invention, in the treatment of a disease selected from an autoimmune
disease and transplant rejection.
For e, the autoimmune disease is type 1 diabetes (T1D, Systemic Lupus
matosus (SLE), Rheumatoid Arthritis (RA) and inflammatory bowel disease
(IBD) (including ulcerative colitis (UC) and Crohn’s disease (CD)), multiple sis
(MS), scleroderma, and other diseases and disorders, such as PV (pemphigus
vulgaris), psoriasis, atopic dermatitis, celiac disease, Chronic Obstructive Lung
disease, Hashimoto’s thyroiditis, Graves’ e (thyroid), Sjogren’s syndrome,
Guillain -Barre syndrome, Goodpasture’s syndrome, Addison’s disease, Wegener’ s
granulomatosis, primary biliary sclerosis, sing cholangitis, autoimmune
hepatitis, polymyalgia tica. Raynaud’s phenomenon, temporal arteritis, giant
cell arteritis, autoimmune tic anemia, pernicious anemia, polyarteritis nodosa.
Behcet’ s disease, primary bilary cirrhosis, uveitis, myocarditis, rheumatic fever,
ankylosing spondylitis, glomerulenephritis, sarcoidosis, dermatomyositis, myasthenia
gravis, polymyositis, alopecia areata, and vitilgo.
In a fourth aspect, there is provided a met hod of tolerising a T -cell to an antigen,
comprising contacting said T -cell with an antigen -presenting cell which is presenting
a peptide derived from said antigen complexed to a MHC molecule and a bispecific
biologic according to the first aspect of the i nvention.
In a fifth aspect, there is provided a method for treating a subject suffering from a
ion selected from an autoimmune disease and transplant rejection, comprising
the steps of administering to a subject in need thereof a bispecific biologic sing
a ligand specific for CTLA -4 and a ligand specific for a pMHC complex according to
the first aspect of the ion.
For example, the autoimmune disease is Type 1 diabetes (T1D).
Description of the Figures
Figure 1. Designs of BsB and BsB Δ. (A) Schematic drawings of the BsB (bispecific
biologics) and BsB Δ fusion ns. (B) Schematic drawing of pMHCII, the TCR
and co -stimulatory molecules in the immune synapse, as well as the proposed scheme
for BsB -mediat ed crosslinking of CTLA -4 to the TCR via the CTLA -4/MHC 11/TCR
tri -molecular complex. The fusion protein s CTLA -4 and indirectly ligates the
TCR via binding to MHCII in the immune e. The two solid sides of the triangle
denote crosslinking of MH CI I and CTLA -4 as well as MHCI I and TCR; the dashed
side depicts ligation of CTLA -4 to TCR. The dotted line indicates inhibition of TCR
ing by BsB -engaged CTLA -4. C. Schematic drawing showing that the action of
BsB Δ is similar to that of BsB , it is unable to ligate the TCR.
Figure 2. Inhibition of allogenic T cell activation by BsB in a mixed lympho cyte
reaction. Naïve T cells from C57BL/6 mice and LPS -treated and irradiated BALB/c
APCs were mixed with the test constructs for 2 days. Culture media were then
harvested and assayed for IL -2. Only BsB and CTLA -4Ig inhibited T cell tion,
as indicate d by a decreased amount of IL -2 in the media. The figure is representative
of more than five independent but similar studies.
Figure 3. Induction of Foxp3 + Tregs and IL -10 and TGF -β production by BsB.
(A) Allogenic mixed lymphocyte reactions were set up as described in the legend to
Figure 2, using naïve CD4 +CD62L hi CD25 -GFP” cells that had been isolated from
Foxp3 -EGFP knock -in mice in the presence of the test ucts. Five days post -
activation, CD4 + T cells were analyzed for GFP sion by flow cytometry. Tregs
were gated as GFP + and CD25 + cells. Only BsB treatment led to GFP expression,
ting ion of Foxp3 + Tregs (middle left panel). Culture media were
ted fo r cytokine analysis (right panels), which ed elevated IL -10 and
TGF -β levels in the presence of BsB. The data are representative of numerous
independent but similar studies. (B) Requirement of autocrine TGF -β for Treg
induction is indicated by the complete blockade of Treg induction in the presence of a
blocking antibody t o TGF -β, wheraeas control Ab did not noticeably impact Treg
induction.
Figure 4. BsB -mediated induction of antigen -specific Tregs in vitro . (A) In vitro
induction of Ova 233 -339 -specific Tregs. Naïve OT -II T cells were mixed with LPS -
activated and irradiate d syngeneic APC in the presence of 0.5 µg/ml Ova 233 -239
e. Control mIgG2a, BsB, and BsB plus an anti -TGF -β antibody ( αTGF -β) were
then added and tested as ted (left panels). Cells were cultured for 5 days and
then labeled with anti -CD25 and ant i-Foxp3 antibodies before being analyzed by flow
cytometry. IL -2, IL -10 and TGF -β levels in the culture media were assayed by ELISA
(right panels). (B) Monitoring of induced Tregs proliferation. Studies were conducted
as in A except naïve OT -II T cells wer e pre -labeled with CFSE before being mixed
with APCs. Cells were gated on Foxp3 and CFSE fluorescent channels.
Figure 5. Suppressive function of BsB -induced Tregs. (A) BsB - or TGF -β-induced
Tregs were ed by flow cytometry and mixed with CFSE -labeled naïve responder
T cells prepared from C57BL/6 mice at the indicated ratios in transwells (filled
columns) or regular culture wells (hatched s). LPS -treated allogenic BALB/c
APCs were added to stimulate T cell activation. The results (mean + standard
deviation) indicate the percentage of proliferating responder T cells (Tresp), based on
a CFSE dilution without Tregs (Tresp + APC only) set to 100%. (B) Anti -IL -10 and
anti -TGF -β antibodies were added to cells in regular culture wells at a Tresp:Treg
ratio of 1:1 to determine the cytokines’ contribution to T cell proliferation. The anti -
TGF -β antibody partially inhibited the suppressive function of TGF -β-induced Tregs
(left panel ) but did not affect BsB -induced Tregs (right panel). The figure is
representative of more than three independent but r studies.
Figure 6. Down -regulation of AKT and mTOR phosphorylation by BsB. Naïve T
cells were cultured in round -bottom 96 -well pla tes co -coated with anti -CD3, anti -
CD28 and BsB, mouse IgG (mIgG) or mouse PD -L1 (mPD -L1) for 18 h. Cells
deemed not activated were cultured in wells coated with IgG only. The
phosphorylation status of AKT and mTOR was then monitored by flow cytometry
after staining with fluorescently labeled antibodies to phosphorylated AKT and
mTOR. MFI denotes mean scent intensity. This figure represents one of three
independent experiments.
Figure 7. Sustained Foxp3 expression in Tregs in se to continuous
sti mulation with BsB. Round -bottom 96 -well plates were co -coated with anti -CD3,
anti -CD28 and BsB or mouse IgG. Naïve T cells from Foxp3 -EGFP knock -in mice
were cultured for 5 days to induce Tregs (left panels), which were then purified from
the BsB -treated c ells (red square) and re -stimulated in another round of culture in co -
coated wells, as above, for 5 days, before is by flow cytometry for GFP + cells.
Re ring of ed Tregs with the mouse IgG control for 5 days resulted in a loss
of Foxp3 + ex pression in ~60% of cells (upper right quadrant of upper right panel),
whereas less than 7% of the Tregs re -cultured with BsB had lost Foxp3 + sion
(upper right quadrant of bottom right panel). This figure represents one of three
ndent experimen ts.
Figure 8. Pharmacokinetics of BsB in vivo and biochemical analysis. (A)
Pharmacokinetic profile of BsB in mice. Normal C57BL/6 mice (n=5) were dosed
intraperitoneally with 20 mg/kg of BsB. Blood samples were collected at the different
time points indic ated and the levels of BsB levels ined using an ELISA. (B)
Comparison of the binding of BsB and mouse IgG2a to FcRn. FcRn were
immobilized to a Biacore chip. BsB or control mouse IgG2a was loaded onto the chip
at various concentrations and the signal s then recorded.
Figure 9. Analysis of asparagine -linked glycosylation on BsB. The amino acid
sequence of BsB was ted to the yc 1.0 Server for prediction of Asn -
linked glycosylation sites. A total of 10 Asn d ylation sites (denoted N)
were predicted; other amino acids are presented as dots. Monosaccharide composition
of BsB was also performed to determine the composition of the glycans fucose (Fuc),
, N -acetylglucosamine (GlcNAc), galactose (Gal), , mannose (Man), sialic acid (N -
acet ylneuramic acid). A sialic acid:galactose ratio of 0.68 indicates that about a third
of the galactose residues are ble for binding to the asialoglycoprotein receptor.
Figure 10. Treatment of non -diabetic (NOD) mice with BsB delayed the onset of
type 1 diabetes (T1D) in a late prevention treatment paradigm. (A) Levels of
Foxp3 + Tregs in the blood of BsB -treated NOD (closed circles, n=15) and saline -
treated control NOD mice (closed triangles, n=14). There was a moderate but
significant increase in the n umber of Tregs in the BsB -treated animals over that noted
in the control animals. (B) Cumulative incidences of overt diabetes in NOD animals
treated with BsB (filled circles) or saline (filled triangles).
Figure 11. Treatment of NOD mice with BsB d the onset of T1D in an
early tion treatment paradigm. (A) Levels of Foxp3 + Tregs in the blood of
mice treated with BsB (closed circles, n=10), saline (closed triangles, n=10), CTLA -
4Ig (closed squares, n=10) and mouse IgG2a (open squares, n=10). No i ncrease in the
number of Foxp3 + Tregs was detected after two weeks of treatment with BsB when
compared to saline or mIgG2a -treated controls. However, treatment with CTLA -4Ig
resulted in a statistically significant decrease in the number of Foxp3 + Tregs in the
blood. (B) tive incidences of overt diabetes in animals treated with BsB or
controls. BsB treatment resulted in a significant delay in the onset of T1D when
compared to the saline or mouse IgG2a -treated control groups before 24 weeks of age
(p=0. 04). However, no significant difference between the groups was noted at the end
of the study. Data represent one of two separate studies with similar results, with total
of 26 NOD mice in each group.
Figure 12. Longer -term treatment of NOD mice with BsB si gnificantly delayed
the onset of T1D in NOD mice. (A) tive nces of overt diabetes in BsB -
treated (n=16) and untreated mice (n=16). BsB treatment significantly reduced the
incidence of T1D when compared to those treated with saline ( p<0.01). (B)
Histopathological analysis of pancreatic tissues from s treated with saline or
BsB. Panels a -c represent sections from saline -treated mice that remained non -
diabetic with H&E, an antibody to insulin, or anti -CD3 and forkhead box P3 (Foxp3),
respecti vely. Similar observations were noted in BsB -treated NOD mice that
remained disease -free. No evidence of infiltration or insulitis was noted in any of the
sections; a few Foxp3 + Tregs may be present (arrows in panel c). Panels d -f represent
pancreatic sect ions from diabetic NOD s. Invasive insulitis was clearly evident
and insulin -producing β-cells were completely destroyed (e). Some CD3 + T cell
infiltrations were also detected, along with few Tregs and many non -T cell leukocytes
with blue nuclei (f). Panels g -i shows islets of BsB treated animals that remained non -
diabetic exhibited characteristic peri itis. Leukocyte infiltrations were noted but
that were restricted to the periphery of the islets. Moreover, they were no notable
destruction of th e insulin -producing s. Most of the leukocytes at the periphery
were non -T cells (blue nuclei). Enlarged inset (panel j, represents red square in i)
indicated Foxp3 + Tregs (yellow arrow head) were intermixed with other CD3 + T cells
and non -T cell leukocytes (blue ) at the periphery of islets. Images were
acquired with a 40x ive; the inset was acquired with a 60x objective, which was
then further enlarged 3x digitally.
Detailed Description of the Invention
Unless ise stated, all technical and scientifi c terms used herein have the same
meanings as ly understood by one of ordinary skill in the art to which this
invention belongs. Any methods and materials with similar or equivalent function to
those described herein can be used in the practice or te sting of the present invention.
Methods, devices, and materials suitable for such uses are now described. All
publications cited herein are orated herein by nce in their entirety for the
purpose of bing and sing the methodologies, r eagents, and tools reported
in the publications that might be used in connection with the invention.
The methods and ques of the present application are generally performed
according to conventional methods well known to those of skill in the art and as
described in various general and more specific references that are cited and discussed
hout the present specification unless otherwise indicated. Such techniques are
ned fully in the literature. See, e.g., Gennaro, A. R., ed. (1990) Remingt on’s
Pharmaceutical Sciences, 18th ed., Mack Publishing Co.; Hardman, J. G., Limbird, L.
E., and Gilman, A. G., eds. (2001) The Pharmacological Basis of Therapeutics, 10th
ed., McGraw -Hill Co.; Colowick, S. et al., eds., Methods In Enzymology, ic
Pre ss, Inc.; Weir, D. M. , and Blackwell, C. C., eds. (1986) Handbook of
Experimental Immunology, Vols. I -IV, Blackwell Scientific Publications; Maniatis,
T. et al., eds. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Vols. I -
III, Cold Spring Har bor Laboratory Press; Ausubel, F. M. et al., eds. (1999) Short
Protocols in Molecular Biology, 4th edition, John Wiley & Sons; Ream et al., eds.
(1998) Molecular Biology Techniques: An Intensive Laboratory Course, Academic
Press; Newton, C. R., and Graham, A., eds. (1997) PCR (Introduction to
Biotechniques Series), 2nd ed., er -Verlag.
The term “antibody”, unless ted otherwise, is used to refer to entire antibodies
as well as antigen -binding fragments of such antibodies. For example, the term
enc ompasses four -chain IgG les, as well as antibody fragments.
As used herein, the term ody fragments” refers to portions of an intact full
length antibody - such as an antigen binding or variable region of the intact antibody.
Examples of antibod y nts include Fab, Fab’, F(ab ’)
2, and Fv fragments;
diabodies; linear antibodies; single -chain antibody les (e.g., scFv);
multispecific antibody fragments such as bispecific, trispecific, and multispecific
antibodies (e.g., ies, triabodie s, tetrabodies); binding -domain immunoglobulin
fusion proteins; camelized antibodies; minibodies; chelating recombinant dies;
tribodies or es; intrabodies; nanobodies; small modular
immunopharmaceuticals (SMIP), VHH containing antibodies; and a ny other
polypeptides formed from dy fragments, for example as further described
below.
Antibodies may be of any class, such as IgG, IgA, or IgM; and of any subclass, such
as IgG1 or IgG4. Different classes and subclasses of immunoglobulin have diffe rent
properties, which may be advantageous in different applications.
Specificity, in the context of the t invention, requires that the claimed antibody
be capable of selectively binding its defined cognate n, which is either CTLA -4
or the pMHC complex.
Naturally occurring immunoglobulins have a common core structure in which two
identical light chains (about 24 kD) and two identical heavy chains (about 55 or 70
kD) form a tetramer. The amino -terminal portion of each chain is known as the
variab le (V) region and can be distinguished from the more conserved nt (C)
s of the remainder of each chain. Within the variable region of the light chain
(also called the V L domain) is a C -terminal portion known as the J region. Within the
variable region of the heavy chain (also called the V H domain), there is a D region in
addition to the J region. Most of the amino acid sequence variation in
globulins is confined to three separate locations in the V s known as
hypervariable regions or complementarity determining regions (CDRs) which are
directly involved in antigen binding. Proceeding from the amino -terminus, these
regions are designated CDR1, CDR2 and CDR3, respectively. The CDRs are held in
place by more conserved framework regions ( FRs). Proceeding from the amino -
terminus, these regions are designated FR1, FR2, FR3 and FR4, respectively. The
locations of CDR and FR regions and a numbering system have been defined by
Kabat et al. (Kabat, E.A., et al., (1991) Sequences of Proteins of I mmunological
Interest, Fifth Edition, U.S. Department of Health and Human Services, U.S.
Government ng Office, and updates f which may be found online.
A humanized monoclonal antibody, as referred to herein, is an antibody which is
ed of a human antibody framework, into which have been grafted CDRs from
a non -human antibody. ures for the design and production of humanized
antibodies are well known in the art, and have been described, for example, in Cabilly
et al., U.S. Patent No. 4, 7; Cabilly et al., European Patent Application 0 125
023; Boss et al., U.S. Patent No. 4,816,397; Boss et al., European Patent Application
0 120 694; Neuberger, M.S. et al., WO 86/01533; Neuberger, M.S. et al., European
Patent Application 0 194 276 B l; Winter, U.S. Patent No. 5,225,539; Winter,
European Patent Application 0 239 400; Padlan, E.A. et al., European Patent
Application 0 519 596. Further details on antibodies, humanized antibodies, human
engineered antibodies, and methods for their prepara tion can be found in Kontermann,
R. and Dübel, S. eds. (2001, 2010) Antibody Engineering, 2nd ed., Springer -Verlag,
New York, NY, 2001.
Constant regions may be derived from any human antibody constant regions.
Typically, variable region genes are cloned in to expression s in frame with
constant region genes to s heavy and light immunoglobulin chains. Such
expression vectors can be transfected into antibody producing host cells for antibody
synthesis.
Required antibody variable and constant region s may be derived from sequence
databases. For example, immunoglobulin sequences are ble in the IMGT/LIGM
database (Giudicelli et al., (2006) Nucleic Acids Res. 34:(suppl. 1):D781 -D784) or
VBase (vbase.mrccpe.cam.ac.uk).
“Nucleic acids” as referred to herein typically include DNA molecules which encode
the antibodies of the invention. Preferred are expression vectors, which are suitable
for expressing the antibody genes in a host cell. sion vectors and host cells for
antibody gene expression are known in the art; see, for example, Morrow, K.J. (2008)
Genetic ering & Biotechnology News. (June 15, 2008) 28(12), and Backliwal,
G., et al. (2008) Nucleic Acids Res. 36(15): e96 -e96.
“CD80”, as used herein, refers to mammalian CD80 antigen as well as to mutants
thereof which have increased binding y or icity for CTLA -4. See Linsley
et al., (1994) Immunity 1:793 -801, and Wu et al., (1997) J. Exp. Med. 185(7):1327 -
1335, incorporated herein by reference. Mammalian CD80 can be selected from
rodent, such as mouse, or human CD80.
“CD86”, as used herein, refers to mammalian CD86 antigen as well as to mutants
thereof which have increased g avidity or specificity for CTLA -4. See Linsley
et al., (1994) Immunity 1:793 -801, incorporated herein by reference. Mammalian
CD86 can be selected from rodent, such as mouse, or human CD86.
“CTLA -4”, as used herein, refers to mammalian cytotoxic lymphocyte -associated
antigen -4 (CTLA -4). The sequence of human CTLA -4 can be found in GenBank,
ion num ber AAH74893.1, GI:49904741. ian CTLA -4 can be selected
from rodent, such as mouse, or human CTLA -4.
“LAG -3”, as used , refers to mammalian lymphocyte activation antigen 3
(LAG -3). The sequence for human LAG -3 can be found in Huard et al., (19 97) Proc.
Natl. Acad. Sci, USA 4 -5749, incorporated herein by reference. Mammalian
LAG -3 can be selected from rodent, such as mouse, or human LAG -3.
The “MHC” is the complex involved in the presentation of antigen derived es
by antigen -present ing cells, which is ised by the TCR. In a certain aspect, the
MHC is MHCII, which presents antigen to CD4 + helper T -cells. See, for example,
Wucherpfennig et al., CSH Perspect. Biol. 2(4): a005140, epub 2010 Mar 17.
A ific biologic, which may b e referred to as a bispecific ligand, is a ligand
which is capable of binding, or being bound by, two targets contemporaneously.
Bispecific antibodies are known in the art, and are further described below. In the
context of the present invention, the two t argets are the CTLA -4 le on a T -cell
and the MHC peptide complex on an APC. The bispecific biologic ing to the
invention can cross -link the two targets; by virtue of the pMHC binding to the TCR in
the immune synapse, it therefore cross -links th e CTLA -4 molecule to the TCR. A
“biologic”, in general, is a biological therapeutic or agent, which may be useful for,
inter alia, therapeutic, diagnostic and/or research purposes.
A linker is any amino acid sequence which joins and separates two polypepti de
domains in a protein. In the context of the bispecific ligand of the ion, the linker
is the ce which joins the CTLA -4 ligand to the MHC ligand. Exemplary linkers
are sequences of amino acid, such as polyglycine, for example Gly -9. An alterna tive
linker is an antibody Fc region. Such a linker spaces the two ligand domains by
approximately 120Å .
A ligand according to the invention may comprise antibody and non -antibody ligands
in any combination. For example, the CTLA -4 ligand may be an anti -CT LA -4
antibody, and the MHC ligand may be LAG -3. Alternatively, CD80 may be used as
the CTLA -4 ligand, in combination with LAG -3 or an anti -MHC antibody. Both
ligands may be antibodies, or both may be the natural ligands, CD80 and LAG -3.
Cytotoxic Lymphocyt e-associated Antigen -4 (CTLA -4)
Cytotoxic T lymphocyte associated antigen -4 (CTLA -4), also known as CD152, is a
negative regulator of the T cell se, which plays an important role in the
maintenance of T cell homeostasis and in the induction of self -tolerance (Karandikar
et al., (1996 ) J Exp Med 184:783 -788 ; Krummel and Allison, (1995 ) J Exp Med
182:459 -46 5; Linsley and Golstein, (1996 ) Curr Biol 6:398 -400 ; s and
Bluestone, (1998 ) J Immunol 160:3855 -3860 ; Walunas et al., (1994 ) J Immunol
160:3855 -3860 ). Mice deficient in CTLA -4 develop multi -organ autoimmune disease
and typically succumb to the ailment by 4 weeks of age (Tivol et al., (1995 ) ty
3:541 -547 ; ouse et al., (1995 ) Science 270:985 -988 ). The lar
mechanisms through which CTLA -4 modulate T cell activity are multifaceted an d are
thought to occur either intrinsically on tional T cells or extrinsically through
regulatory T cells (Tregs) (Ise et al., (2010 ) Nat Immunol 11:129 -135 ; Jain et al.,
(2010 ) Proc Natl Acad Sci U S A 107:1524 -1528 ; Paterson and Sharpe, (2010 ) Nat
Immunol 11:109 -111 ).
These mechanisms include competing w ith CD28 for ligand binding (Linsley et al.,
(1994 ) Immunity 1:793 -801 ), inducing the production of the tolerogenic enzyme
indoleamine 2,3 dioxygenase in APC (Grohmann et al., (2002 ) Nat Immunol 3:1097 -
1101 ; Onodera et al., (2009 ) J Immunol 183:5608 -5614 ), and displa cing CD28 from
the immunological synapse (Pentcheva -Hoang et al., (2004 ) Immunity 21:401 -413 ).
CTLA -4 is homologous to the co -stimulatory molecule CD28 and shares the same
ligands, CD80 (B7.1) and CD86 (B7.2), which are expressed on the surf ace of antigen
presenting cells (APCs). However, differential binding of CD80/CD86 on APCs to
CD28 and CTLA -4 on effector T cells leads to ng outcomes, with CD28
triggering T cell activation and CTLA -4 causing T cell tion. Engagement of
CTLA -4 by its ligands (CD80/86) on APC also stimulates the recruitment of the
phosphatases SHP -1 (Guntermann and Alexander, (2002 ) J l 168:4420 -4429 )
and PP2A (Baroja et al., (2002 ) J Immunol 168:5070 -5078 ; Chuang et al., (2000 )
Immunity 13:313 -322 ) to the vicinity of t he TCR of T cells undergoing activation.
Consequent dephosphorylation of key signaling molecules associated with the TCR
results in termination of T cell activation (Griffin et al., (2000 ) J Immunol 33 -
4442 ). Moreover, interventions that promote early engagement of CTLA -4 with its
ligands and crosslinking to the TCR result in premature dampening of key signaling
signatures and consequent inhibition of T cell activation, leading to T cell
hyporesponsiveness or anergy (Blair et al., (1998 ) Immunol 160:12 -15 ; Griffin et a l.,
(2000 ) J Immunol 33 -4442 ; Krummel and Allison, (1996 ) J Exp Med 182:459 -
465 ; Walunas et al., (1996 ) J Exp Med 41 -2550 ).
To promote crosslinking of CTLA -4 to the TCR during the early phase of T cell
activation a bispecific fusion protein (designated as “BsB”) sing a mutant
CD80 (CD80w88a) and lymphocyte activation gene -3 (LAG -3) was ted. BsB
was ed to concurrently en gage CTLA -4 and MHCII in the immune synapse and
thereby indirectly crosslink it to the TCR via the e pairing of MHCII with the
TCR (Karman et al., (2012 ) J Biol Chem epub 2012 Feb 15 ). In an allogenic MLR,
BsB was shown to be effective at in hibiting T cell tion. Surprisingly, BsB also
induced the production of IL -10 and TGF -β and promoted the differentiation of T
cells undergoing activation to Tregs. IL -10 can exert broad immune suppressive
properties through its ability to control the activation of hages and dendritic
cells (DCs), as well as self -regulate Th1 cells (Ohata et al., (2007 ) Arthritis Rheum
56:2947 -2956 ). TGF -β can act as an inhibitor of T cell differentiation (Kehrl et al.,
(1986 ) J Exp Med 37 -1050 ), macrophage activation (Tsunawaki et al., (1988 )
Nature 334:260 -262 ; Wahl et al., (1990 ) Ann N Y Acad Sci 593:188 -196 ) and
dendritic cell maturation (Ste inman et al., (2003 ) Annu Rev Immunol 21:685 -711 ). In
addition to their anti -inflammatory functions, IL -10 and TGF -β also purportedly can
influence Treg function. For example, IL -10 has been shown to induce IL -10
producing Tr1 cells rolo et al., (2006 ) Immunol Rev 212:28 -50 ) and to act on
Foxp3 + Tregs to maintain expression of Foxp3 and thereby propagate their
suppressive function (Murai et al., (2009 ) Nat Immunol 8 -1184 ). Similarly,
TGF -β has been reported to be necessary for the induction of Tregs (Chen et al.,
(2003 ) J Exp Med 198:1875 -1886 ; Zheng et al., (2002 ) J Immunol 169:4183 -4189 )
and in maintaining their suppressive function by promoting Foxp3 expression (Marie
et al., (2005 ) J Exp Med 201:1061 -1067 ).
Regulatory T cells (Tregs)
Tregs are a functionally distinct subpopulation of T cells capable of controlling the
immune responses to self and non -self antigens. A deficiency of Tregs s in a
heightened immune response and presentation of autoimmune diseases (Sakaguchi et
al., (1995 ) J Immunol 155:1151 -1164 ). Extensive research has established a role of
these specialized T cells in controlling all aspects of immune responses, in particular
in engendering self -tolerance. Without being bound to a particular theory, these
findings te that agents e of ng the in situ production of Tregs or the
adoptive transfer of Tregs may be deployed to treat autoimmune diseases. Indeed,
Treg cell -based therapies using freshly isolated or ex vivo expanded Tregs have been
shown to be effective in ng animal models of type 1 dia betes (T1D) (Tang et al.,
(2004 ) J Exp Med 55 -1465 ; Tarbell et al., (2007 ) J Exp Med 204:191 -201 ) and
gr aft -versus -host disease (Anderson et al., (2004 ) ; Taylor et al., (2002 ) Blood
99:3493 -3499 ; Zhao et al., (2008 ) Blood 112:2129 -2138 ). In lieu of isolating and
expanding Foxp3 +CD4 +CD25 + Tregs (often designated as natural Tregs or )
from peripheral blood or lymph nodes, Tregs can be induced from naïve CD4 +CD25 -
T cells in the context of TCR activation and in the itant presence of TGF -β.
These Tregs are often referred to as ve Tregs (aTregs) or induced Tregs
s). They are also Foxp3 + and purportedly exhibit equally potent suppressive
funct ions as nTregs (Chen et al., (2003 ) J Exp Med 198:1875 -1886 ; Yamagiwa et al.,
(2001 ) J Immunol 166:7282 -7289 ; Zheng et al., (2002 ) J Immunol 169:4183 -4189 ).
Adoptive ers of aTregs or iTregs have been shown to be effective in conferring
protection against autoimmune disease in an animal model of collagen -induced
arthritis (Gonzalez -Rey et al., (2006 ) Arthritis Rheum 54:864 -876 ). However, it is
becoming more evident that n -specific Tregs offer a signifi cantly higher
therapeutic quotient than polyclonal Tregs with a pan -TCR repertoire (Masteller et al.,
(2005 ) J Immunol 175:3053 -3059 ; Tang et al., (2004 ) J Exp Med 199:1455 -1465 ;
Tarbell et al., (2007 ) J Exp Med 204:191 -201 ), with less potential side effect on pan -
immune suppression. For this reason, we soug ht to evaluate the relative merits of
BsB at producing n -specific Tregs in an antigen fic T cell activation
setting in vitro . Moreover, we tested its potential in treating autoimmune diabetes in
the non -obese diabetic (NOD) mouse.
Type 1 Diabet es
Type 1 es (T1D) is an autoimmune disease caused by tissue ic destruction
of insulin -producing pancreatic β-cells with consequent development of
hyperglycemia. Non -obese diabetic (NOD) mice (female mice in particular)
spontaneously develop a ctive T cells towards islet -specific self -antigens (e.g.
insulin and ic acid decarboxylase 65). In concert with other lymphocytes,
these autoreactive T cells initiate the development of peri -insulitis between 3 and 4
weeks of age followed by i nvasive insulitis at 9 weeks and spontaneous overt diabetes
between 12 and 35 weeks (Anderson and Bluestone, (2005 ) Annu Rev I mmunol
23:447 -485 ). NOD mice share many similarities to the disease in human ts
such as the production of pancreas -specific autoantibodies and tion of
autoreactive CD4 + and CD8 + T cells. Susceptibility of these mice to autoimmunity, as
in hu mans, is influenced by genes for the major histocompatibility complex (MHC),
CTLA -4, and LAG -3. NOD mice harbor a unique major histocompatibility complex
(MHC) haplotype (H -2g7 ), which reportedly confers the highest risk for disease
susceptibility (McDevitt et al., (1996 ) Hormone and metabolic research 28:287 -288 ;
Wicker et al., (1995 ) Annu Rev Immunol 13:179 -200 ). CTLA -4 polymorphism has
also been noted in NOD mice (Ueda et al., (2003 ) Nature 423:506 -511 ) and in
humans (Qu et al., (2009 ) Genes and immunity 10 Suppl 1:S27 -32 ) and a deficiency
of LAG -3 on the NOD background rates T1D onset with 100% penetrance
(Bettini et al., (2011 ) J Immunol 187:3493 -3498 ). Because BsB engages all these
targets, the therapeutic merits of BsB were tested in this murine model of T1D.
Antibodies
The invention asses antigen -binding fragments of the antibodies set forth in
the claims. As used herein, the term “fragments” refers to portions of the intact full
length antibody - such as an antigen binding or variable region of the intact antibody.
Examples of antibody fragments are set forth above.
The term “fragments” as used herein refers to fragments capable of binding the targets
specified, the CTLA -4 molecule or the pMHC complex. These fragments may lack
the Fc fragment of an intact antibody, clear more y from the circulation, and can
have less non specific tissue bi nding than an intact antibody. These fragments can be
produced from intact dies using well known methods, for example by
proteolytic cleavage with enzymes such as papain (to produce Fab fragments) or
pepsin (to produce F(ab ’)
2 fragments), or expressi on of such fragments by
recombinant technology.
The antibodies and fragments also encompass single -chain antibody nts (scFv)
that bind to the CTLA -4 molecule or the pMHC complex. An scFv comprises an
dy heavy chain variable region (V H) operably linked to an antibody light chain
variable region (V L) wherein the heavy chain variable region and the light chain
variable region, together or individually, form a binding site that binds CTLA -4
molecule or the pMHC complex. An scFv may comprise a VH reg ion at the amino -
terminal end and a V L region at the carboxy -terminal end. Alternatively, scFv may
comprise a V L region at the amino -terminal end and a VH region at the carboxy -
terminal end. rmore, although the two domains of the Fv fragment, V L and VH,
are coded for by separate genes, they can be joined, using recombinant methods, by a
synthetic linker that enables them to be made as a single protein chain in which the V L
and V H regions pair to form lent les (known as single chain Fv (scF v)).
A scFv may ally further comprise a ptide linker between the heavy chain
variable region and the light chain variable region.
The antibodies and fragments also encompass domain antibody (dAb) fragments as
described in Ward, E.S. et al. (198 9) Nature 341:544 -546 which consist of a VH
domain.
The antibodies and fragments also encompass heavy chain antibodies (HCAb). These
antibodies can apparently form antigen -binding regions using only heavy chain
variable region, in that these functional ant ibodies are dimers of heavy chains only
(referred to as “heavy -chain antibodies” or “HCAbs”). ingly, antibodies and
fragments may be heavy chain antibodies (HCAb) that specifically bind to the CTLA -
4 or pMHC s.
The antibodies and fragments also encompass antibodies that are SMIPs or binding
domain immunoglobulin fusion ns ic for the CTLA -4 or pMHC targets.
These constructs are single -chain ptides comprising antigen -binding domains
fused to immunoglobulin domains necessary to ca rry out antibody effector functions
(see ).
The antibodies and fragments also encompass diabodies. These are bivalent
antibodies in which VH and V L domains are expressed on a single polypeptide chain,
but using a linker that is too short to a llow for pairing n the two domains on the
same chain. This forces the domains to pair with complementary domains of r
chain and thereby creates two antigen -binding sites (see, for example, WO 93/11161).
Diabodies can be bispecific or monospecif ic.
The antibody or antibody fragment thereof according to the invention does not cross -
react with any target other than the intended CTLA -4 or pMHC targets.
The dies and fragments thereof may themselves be bispecific. For example,
bispecific antibod ies may resemble single antibodies (or antibody nts) but have
two ent antigen binding sites (variable regions). Bispecific antibodies can be
produced by various methods – such as chemical techniques, “polydoma” techniques
or recombinant DNA tec hniques. Bispecific antibodies may have g specificities
for at least two different epitopes, for example one epitope on each of the CTLA -4
and pMHC targets.
Bispecific antibodies sing complementary pairs of VH and V L regions are
known in the ar t. These bispecific antibodies comprise two pairs of VH and V L, each
VHVL pair binding to a single antigen or epitope. Such ific antibodies include
hybrid hybridomas (Milstein, C. and Cuello, A.C., (1983) Nature 305 (5934): 537 -
40), minibodies (Hu et al., (1996) Cancer Res. 56:.3055 , diabodies (Holliger et
al., (1993) Proc. Natl. Acad. Sci. USA 90:6444 -6448; WO 94/13804), chelating
recombinant antibodies (CRAbs) (Neri et al., (1995) J. Mol. Biol. 246,367 - 373),
biscFv (e.g., Atwell et al., (1996 ) Mol. l. 33:1301 -1312), “knobs in holes”
stabilised antibodies (Carter et al., (1997) Protein Sci. 6:781 -788). In each case each
antibody s comprises two antigen -binding sites, each fashioned by a
complementary pair of VH and V L domains. Each antibody is thereby able to bind to
two different antigens or epitopes at the same time, with the g to each antigen
or epitope mediated by a VH and its complementary V L domain.
Natural autoantibodies have been described that are polyreactive (Casali and Notkins
(1989) Ann. Rev. Immunol . 7: 515 -531), reacting with at least two (usually more)
different antigens or epitopes that are not structurally related. It has also been shown
that selections of random peptide repertoires using phage display technolo gy on a
monoclonal antibody will identify a range of peptide sequences that fit the antigen
binding site. Some of the sequences are highly related, fitting a consensus sequence,
whereas others are very ent and have been termed pes (Lane and Ste phen
(1993) Current Opinion in Immunology 5:268 -271). It is therefore clear that the
binding site of an dy, comprising associated and complementary VH and V L
domains, has the potential to bind to many different antigens from a large universe of
known antigens.
WO 03/002609 (Domantis) bes the production of dual specific antibodies in
which each V HVL pair possesses a dual specificity, i.e., is able to bind two epitopes on
the same or different antigens. The mation can be open or closed; in a n open
mation, the two es may be bound simultaneously, but in the closed
conformation binding to the first epitope prevents or discourages binding to the
second.
Non -immunoglobulin proteins with multiple binding specificities are known in
natur e; for example, a number of transcription factors bind both DNA and other
protein molecules. r, methods for selecting binding es in the prior art
only select peptides with single, not dual or multiple specificities.
Different research teams hav e previously tethered polypeptides with cysteine residues
to a synthetic molecular structure (Kemp, D. S. and McNamara, P. E., (1985) J. Org.
Chem. man, P. et al., (2005) ChemBioChem. 6(5):821 -4). Meloen and co -
workers had used romomethyl)benze ne and related les for rapid and
quantitative cyclisation of multiple peptide loops onto synthetic scaffolds for
structural mimicry of protein surfaces (Timmerman, P. et al., (2005) ibid ). Methods
for the tion of candidate drug compounds wherei n said compounds are
generated by g cysteine containing polypeptides to a molecular scaffold as for
example tris(bromomethyl)benzene are disclosed in and WO
2006/078161. The selection of such molecules using y technology is descr ibed
in . Dual specific embodiments are further described in WO
2010/089117.
The ligand, such as an antibody or fragment thereof, may be modified in order to
increase its serum half -life, for example, by adding molecules - such as PEG or othe r
water soluble polymers, including polysaccharide polymers to se the half -life.
In one embodiment, an antibody Fc region may be added to the bispecific linker
according to the invention, to increase circulating half -life.
Antibody production
Antibod y production can be performed by any technique known in the art, ing
in transgenic organisms such as goats (see Pollock et al. (1999) J. lmmunol. Methods
231:147 - 157), chickens (see Morrow, KJJ (2000) Genet. Eng. News 20:1 -55), mice
(see Pollock et al. ibid) or plants (see Doran PM (2000) Curr. Opinion Biotechnol.
11:199 -204; Ma, JK -C (1998) Nat.Med. 4:601 -606; Baez, J. et al. (2000)
BioPharm. 13:50 -54; Stoger, E. et al. (2000) Plant Mol. Biol. 42:583 -590). Antibodies
may also be produced by al synthesis; however expression of genes encoding
the antibodies in host cells is preferred.
A polynucleotide encoding the antibody is isolated and inserted into a replicable
uct or vector such as a plasmid for further propagation or expression in a h ost
cell. Constructs or vectors (e.g., expression vectors) le for the sion of a
humanized immunoglobulin according to the invention are available in the art. A
variety of vectors are ble, including vectors which are maintained in single c opy
or multiple copies in a host cell, or which become integrated into the host cell’s
chromosome(s). The constructs or s can be introduced into a suitable host cell,
and cells which s a humanized immunoglobulin can be produced and
maintained i n e. A single vector or le vectors can be used for the
expression of a humanized immunoglobulin.
Polynucleotides encoding the antibody are readily isolated and sequenced using
conventional procedures (e.g., oligonucleotide probes). Vectors that may be used
include d, virus, phage, transposons, minichromsomes of which plasmids are a
typical ment. Generally such vectors further include a signal sequence, origin
of replication, one or more marker genes, an enhancer element, a promoter a nd
transcription termination sequences operably linked to the light and/or heavy chain
polynucleotide so as to facilitate expression. Polynucleotides encoding the light and
heavy chains may be inserted into te vectors and introduced (e.g., by
transfo rmation, transfection, electroporation or transduction) into the same host cell
concurrently or sequentially or, if desired both the heavy chain and light chain can be
inserted into the same vector prior to such introduction.
A promoter can be provided for expression in a suitable host cell. Promoters can be
constitutive or inducible. For example, a promoter can be operably linked to a nucleic
acid encoding a humanized immunoglobulin or immunoglobulin chain, such that it
directs expression of the encoded po lypeptide. A variety of suitable promoters for
prokaryotic and otic hosts are available. Prokaryotic promoters include lac, tac,
T3. T7 promoters for E. coil; 3- phosphoglycerate kinase or other glycolytic enzymes
e.g., enolase, glyceralderhyde 3 - ph osphate dehydrogenase, hexokinase, pyruvate
decarboxylase, phosphofructokinase, glucose 6 ate isomerase, 3 -
phosphoglycerate mutase and inase. Eukaryotic promoters include inducible
yeast promoters such as alcohol dehydrogenase 2, isocytochrome C, acid phosphatase,
metallothionein and enzymes responsible for nitrogen metabolism or
maltose/galactose utilization; RNA rase II promoters ing viral promoters
such as polyoma, fowlpox and adenoviruses (e.g., adenovirus 2), bovine papilloma
vi rus, avian sarcoma virus, cytomegalovirus (in particular the immediate early gene
er), retrovirus, hepatitis B virus, actin, rous sarcoma virus (RSV) promoter and
the early or late Simian virus 40 and non -viral promoters such as EF -1 alpha
(Mizushima and Nagata (1990) Nucleic Acids Re s. 18(17):5322). Those of skill in the
art will be able to select the appropriate promoter for expressing a humanized
antibody or portion thereof of the invention.
Where appropriate, e.g., for expression in cells of highe r eukaroytes, additional
enhancer elements can be included instead of or as well as those found located in the
promoters described above. le mammalian enhancer ces e
enhancer elements from globin, elastase, albumin, fetoprotein, metalloth ionine and
insulin. Alternatively, one may use an enhancer t from a eukaroytic cell virus
such as SV40 enhancer, cytomegalovirus early promoter enhancer, a er,
baculoviral enhancer or murine IgG2a locus (see WO 04/009823). Whilst such
enh ancers are typically located on the vector at a site upstream to the promoter, they
can also be located elsewhere e.g., within the untranslated region or downstream of
the polyadenylation signal. The choice and positioning of enhancer may be based
upon com patibility with the host cell used for expression.
In addition, the vectors (e.g., expression vectors) typically comprise a selectable
marker for selection of host cells carrying the vector and, in the case of a replicable
vector, an origin of replication. Genes encoding products which confer antibiotic or
drug resistance are common selectable markers and may be used in prokaryotic (e.g.,
β-lactamase gene (ampicillin resistance), Tet gene (tetracycline resistance) and
eukaryotic cells (e.g., neomycin (G418 or cin), gpt (mycophenolic acid),
ampicillin, or hygromycin resistance genes). Dihydrofolate reductase marker genes
permit selection with methotrexate in a variety of hosts. Genes encoding the gene
product of ophic markers of the host (e.g., LE U2, URA3, HIS3) are often used
as selectable markers in yeast. Use of viral (e.g., baculovirus) or phage vectors, and
vectors which are e of integrating into the genome of the host cell, such as
retroviral vectors, are also contemplated.
In eukaryoti c s, polyadenylation and termination signals are operably linked to
polynucleotide encoding the antibody of this invention. Such signals are lly
placed 3’ of the open reading frame. In mammalian systems, non -limiting examples of
polyadenylation /termination s include those derived from growth hormones,
elongation factor -1 alpha and viral (e.g., SV40) genes or retroviral long terminal
repeats. In yeast s, non -limiting examples of polydenylation/termination
s include those derived from the phosphoglycerate kinase (PGK) and the
alcohol dehydrogenase 1 (ADH) genes. In yotic systems polyadenylation
signals are typically not required and it is instead usual to employ shorter and more
defined terminator sequences. The choice of po lyadenylation/termination sequences
may be based upon compatibility with the host cell used for expression. In on to
the above, other features that can be employed to enhance yields include chromatin
remodeling elements, introns and host -cell specifi c codon modification. The codon
usage of the antibody of this invention thereof can be modified to accommodate
codon bias of the host cell such to t ript and/or product yield (e.g.,
Hoekema, A. et al. (1987) Mol Cell Biol. 7(8):2914 -24). The ch oice of codons may be
based upon compatibility with the host cell used for expression.
The invention thus relates to isolated nucleic acid molecules that encode the
humanized immunoglobulins, or heavy or light chains, thereof, of this invention. The
invent ion also relates to ed nucleic acid molecules that encode an antigen -
binding portion of the immunoglobulins and their chains.
The antibodies according to this invention can be produced, for example, by the
expression of one or more recombinant nuclei c acids encoding the antibody in a
suitable host cell. The host cell can be produced using any suitable method. For
e, the expression constructs (e.g., one or more s, e.g., a mammalian cell
expression vector) described herein can be introduced into a suitable host cell, and the
resulting cell can be ined (e.g., in culture, in an animal, in a plant) under
conditions suitable for expression of the construct(s) or vector(s). le host cells
can be prokaryotic, including ial cells su ch as E. coli (e.g., strain DH5a TM
(Invitrogen, Carlsbad, CA), PerC6 cells (Crucell, Leiden, NL), B. subtilis and/or other
suitable bacteria; eukaryotic cells, such as fungal or yeast cells (e.g., Pichia pastoris,
Aspergillus sp., Saccharomyces cerevisiae, Schizosaccharomyces pombe, Neurospora
crassa), or other lower eukaryotic cells, and cells of higher eukaryotes such as those
from insects (e.g., Drosophila Schnieder S2 cells, Sf9 insect cells (WO 94/126087
(O’Connor), TN5BI -4 (HIGH FIVE TM ) insect cells ( Invitrogen), s (e.g., COS
cells, such as COS -I (ATCC Accession No. CRL -1650) and COS -7 (ATCC
ion No. CRL -1651), CHO (e.g., ATCC Accession No. CRL -9096), CHO
DG44 (Urlaub, G. and Chasin, LA., (1980) Proc. Natl. Acac. Sci. USA, 77(7):4216 -
4220), 293 (ATCC Accession No. CRL - 1573), HeLa (ATCC Accession No. CCL -2),
CVI (ATCC Accession No. CCL -70), WOP y, L., et al., (1985) J. Virol.,
54:739 -749), 3T3, 293T (Pear, W. S., et al., (1993) Proc. Natl. Acad. Sci. USA.
90:8392 -8396), NSO cells, SP2/0 cells, HuT 78 cells and the like, or plants (e.g.,
tobacco, lemna (duckweed), and algae). See, for example, Ausubel, F.M. et al., eds.
Current Protocols in Molecular Biology, Greene Publishing ates and John
Wiley & Sons Inc. (1993). In some embodime nts, the host cell is not part of a
ellular organism (e.g., plant or animal), e.g., it is an isolated host cell or is part
of a cell culture.
Host cells may be cultured in spinner flasks, shake flasks, roller bottles, wave
bioreactors (e.g., System 1 000 from wavebiotech.com) or hollow fibre systems but it
is preferred for large scale production that stirred tank bioreactors or bag bioreactors
(e.g., Wave h, Somerset, New Jersey USA) are used particularly for suspension
cultures. Typically stirre d tank bioreactors are adapted for on using e.g.,
spargers, baffles or low shear impellers. For bubble columns and airlift ctors,
direct aeration with air or oxygen bubbles maybe used. Where the host cells are
cultured in a serum free culture m edium, the medium can be supplemented with a cell
protective agent such as pluronic F -68 to help prevent cell damage as a result of the
aeration s. Depending on the host cell characteristics, microcarriers maybe used
as growth substrates for anchorag e dependent cell lines, or the cells maybe adapted to
suspension culture. The culturing of host cells, particularly rate host cells, may
utilize a variety of operational modes such as batch, fed -batch, repeated batch
processing (see Drapeau et al (19 94) Cytotechnology 15: 103 -109), extended batch
process or perfusion culture. Although recombinantly transformed mammalian host
cells may be cultured in serum -containing media such media comprising fetal calf
serum (FCS), it is preferred that such host cel ls are ed in serum free media such
as disclosed in Keen et al (1995) Cytotechnology 17:153 -163, or commercially
available media such as ProCHO -CDM or HO TM (Cambrex NJ, USA),
supplemented where necessary with an energy source such as glucose and synthetic
growth factors such as recombinant insulin. The serum -free culturing of host cells
may require that those cells are adapted to grow in serum free conditions. One
adaptation ch is to culture such host cells in serum containing media and
rep eatedly exchange 80% of the culture medium for the serum -free media so that the
host cells learn to adapt in serum free ions (see e.g., enberg K .et al
(1995) in Animal Cell Technology Developments Towards the 21st Century (Beuvery
E.C. et al e ds), pp619 -623, Kluwer Academic publishers).
Antibodies according to the invention may be secreted into the medium and recovered
and purified therefrom using a variety of techniques to provide a degree of
purification le for the intended use. For exa mple, the use of therapeutic
antibodies of the invention for the treatment of human patients lly mandates at
least 95% purity as determined by reducing SDS -PAGE, more typically 98% or 99%
purity, when compared to the culture media comprising the ther apeutic antibodies. In
the first instance, cell debris from the culture media is typically removed using
centrifugation followed by a clarification step of the supernatant using e.g.,
microfiltration, ultrafiltration and/or depth filtration. atively, the antibody can
be harvested by iltration, ultrafiltration or depth filtration without prior
centrifugation. A variety of other techniques such as is and gel electrophoresis
and chromatographic techniques such as hydroxyapatite (HA), affinity
chromatography (optionally involving an affinity tagging system such as
polyhistidine) and/or hydrophobic interaction chromatography (HIC) (see US
5,429,746) are available. In one embodiment, the antibodies of the invention,
following various clarificatio n steps, are captured using Protein A or G affinity
chromatography followed by further chromatography steps such as ion exchange
and/or HA chromatography, anion or cation exchange, size exclusion chromatography
and ammonium sulphate precipitation. Typicall y, various virus removal steps are also
employed (e.g., nanofiltration using, e.g., a DV -20 filter). Following these various
steps, a purified preparation comprising at least 10mg/m1 or greater e.g., 100mg/m1
or greater of the antibody of the ion is provided and therefore forms an
embodiment of the invention. Concentration to 100mg/m1 or greater can be generated
by ultracentrifugation. Such preparations are ntially free of aggregated forms of
antibodies of the invention.
Bacterial systems are pa arly suited for the expression of antibody fragments.
Such fragments are localized ellularly or within the periplasm. ble
periplasmic ns can be extracted and refolded to form active proteins ing
to methods known to those skill ed in the art, see Sanchez et al. (1999) J. Biotechnol.
72:13 -20; Cupit, PM et al. (1999) Lett. Appl. Microbiol. 29:273 -277.
The t invention also relates to cells comprising a c acid, e.g., a vector, of
the invention (e.g., an expression vector ). For example, a c acid (i.e., one or
more nucleic acids) encoding the heavy and light chains of a humanized
immunoglobulin according to the invention, or a uct (i.e., one or more
constructs, e.g., one or more vectors) comprising such nucleic acid(s), can be
introduced into a suitable host cell by a method appropriate to the host cell ed
(e.g., transformation, transfection, electroporation, infection), with the nucleic acid(s)
being, or becoming, operably linked to one or more sion control elements (e.g.,
in a , in a construct created by processes in the cell, integrated into the host cell
genome). Host cells can be maintained under conditions suitable for expression (e.g.,
in the presence of inducer, suitable media supplemente d with appropriate salts,
growth factors, antibiotic, nutritional supplements, etc.), whereby the d
polypeptide(s) are produced. If desired, the encoded humanised antibody can be
isolated, for example, from the host cells, culture medium, or milk. Th is process
encompasses expression in a host cell (e.g., a mammary gland cell) of a transgenic
animal or plant (e.g., tobacco) (see e.g., WO 92/03918).
CD80 Ligands
The design and construction of CD80 ligands is intended to se the icity
of the l igand for CTLA -4 over CD28. The sequence of CD80 is known in the art,
cited example in Wu et al., 1997. CD80 comprises an extracellular Ig -V variable -like
domain, and an intracellular Ig C constant -like domain. In a preferred ment, the
extracellular domain of CD80 is used as a ligand. For example, see SEQ ID NO: 15,
especially residues 1 -241.
Mutations can be made in human CD80 to improve binding affinity, and to improve
selectivity for CTLA4 over CD28. See, for example, Wu et al., 1997.
Mutants other than W84A may be made, including K71G, K71V, S109G, R123S,
R123D, G124L, S190A, S201A, R63A, M81A, N97A, E196A. See Peach et al., JBC
1995. 270(6): 21181 -21187. Assessment of binding ty of mutants for both
CTLA -4 and CD28 can be effected by site -dir ected mutagenesis followed by
sion of the mutant polypeptides, and determination of Kd by surface plasmon
resonance using CTLA -4 and CD28 Biacore chips. See, for example. Guo et al.,
(1995) J. Exp. Med. 181:1345 -55.
s having advantageous bindin g and selectivity profiles can be selected, and
r assessed in cell based assays. For example, flow cytometry can be used to
assay the effect of wild -type or mutant CD80 transfected into the cells.
LAG -3 Ligands
LAG -3 has been described in the art, an d the binding site to the MHCII protein
characterised. See Huard et al., (1997) Proc. Natl. Acad. Sci. USA 94(11):5744 -9.
LAG -3 has four ellular lg -like domains, and mutations can be introduced into
these s to se binding to MHCII.
The eff ectiveness of mutations can be analysed as described above in respect of CD80
ligands.
In one aspect, only domains 1 and 2 (D1 and D2) of the four lg -like domains of LAG -
3 are used in a ligand according to the invention. It is believed that these domains a re
responsible for interaction with the MHCII protein.
Bispecific Ligand Constructs
The construction of a bispecific ligand follows the general formula “ligand -linker -
”. Bispecific antibodies are known in the art, and are described above.
Constructio n of bispecific ligands preferably ed construction and expression of
an appropriate gene encoding the desired polypeptide. Other methods of ucting
by mixing the two polypeptides under conditions that permit covalent, ionic, or
hydrophobic bondi ng. In preferred embodiments, it comprises covalently bonding the
polypeptides. Where a bispecific molecule comprising three components is
constructed, such as a CTLA -4 , a linker and an MHC ligand, two of the three
may be combined, bound together, a nd the third polypeptide subsequently added to
the fusion product, and bound to create a fusion product comprising all three
ptides.
Polypeptides in accordance with the present invention can be produced by any desired
technique, including chemical sy nthesis, isolation from biological samples and
expression of a nucleic acid encoding such a polypeptide. c acids, in their turn,
can be synthesised or isolated from biological sources, and ed by site -directed
mutagenesis if desired.
The inventi on thus relates to vectors encoding a bispecific ligand ing to the
invention, or a fragment thereof. The vector can be, for example, a phage, plasmid,
viral, or retroviral vector.
c acids according to the invention can be part of a vector conta ining a
selectable marker for propagation in a host. Generally, a d vector is introduced
in a precipitate, such as a calcium phosphate precipitate, or in a complex with a
d lipid.
If the vector is a virus, it can be ed in vtro using an ap propriate packaging cell
line and then transduced into host cells.
The nucleic acid insert is operatively linked to an appropriate promoter, such as the
phage lambda PL promoter, the E. coli lac, trp, phoA and tac promoters, the SV40
early and late promote rs and promoters of retroviral LTRs. Other suitable ers
are known to those skilled in the art. The expression constructs further contain sites
for ription initiation, termination, and, in the transcribed region, a ribosome
binding site for tran slation. The coding portion of the transcripts expressed by the
constructs preferably includes a translation initiating codon at the beginning and a
termination codon (UAA, UGA or UAG) appropriately positioned at the end of the
polypeptide to be translated .
As indicated, the expression s preferably include at least one selectable marker.
Such markers include dihydrofolate reductase, G418 or neomycin resistance for
eukaryotic cell
culture and tetracycline, kanamycin or ampicillin resistance genes for c ulturing in E.
coil and other bacteria. Representative es of appropriate hosts include, but are
not limited to, ial cells, such as E. coli , Streptomyces and Salmonella
typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces ce revisiae or
Pichia pastoris); insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal
cells such as CHO. COS, HEK293, and Bowes melanoma cells; and plant cells.
Appropriate culture media and conditions for the above -described host cells are
kno wn in the art and available commercially.
Among vectors red for use in bacteria include pQE70, pQE60 and pQE -9,
available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNH8A,
pNH16a, pNH18A, pNH46A, available from Stratagene Cloning Sy stems, Inc.; and
ptrc99a, pKK2233, pKK233 -3, pDR540, pRIT5 available from Pharmacia h,
Inc. Among preferred otic vectors are pWLNEO, pSV2CAT, p0G44, pXTI and
pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from
Pharmacia . Among vectors preferred for use in mammalian cell expression include
pSG5 Vector, pCMV •SPORT6, pcDNA, pCEP4, pREP4, pCI, pSI and pBICEP -
CMV. Preferred expression s for use in yeast systems include, but are not
limited to pYES2, pYDI, pTEFI/Zeo, pYE S2/GS, pPICZ, pGAPZ, pGAPZalph,
pPIC9, pPIC3.5, pHIL D2, pHIL -SI, pPIC3.5K, pPIC9K, and PA0815 (all ble
from Invitrogen, Carlsbad, CA).
Introduction of the construct into the host cell can be effected by calcium phosphate
transfection. DEAE -dextran m ediated transfection, cationic lipid -mediated
transfection, electroporation, transduction, infection, or other methods. Such methods
are described in many standard laboratory manuals, such as Sambrook et al., referred
to above. A polypeptide according to t he invention can be recovered and purified
from recombinant cell cultures by well -known methods including ammonium
sulphate or ethanol itation, acid tion, anion or cation exchange
chromatography, phosphocellulose chromatography, hydrophobic int eraction
chromatography, affinity chromatography, hydroxylapatite chromatography and lectin
chromatography. Most preferably, high performance liquid chromatography
(“HPLC”) is ed for purification.
Polypeptides according to the present invention can a lso be recovered from biological
s, including bodily , tissues and cells, especially cells derived from
tumour tissue or suspected tumour tissues from a subject.
In addition, polypeptides according to the invention can be ally synthesised
using techniques known in the art (for e, see Creighton, 1983, Proteins:
Structures and Molecular Principles, W. H. Freeman & Co. , N. Y. , and Hunkapiller
et al., (1984) Nature , 310:105 -111). For example, a polypeptide comprising all or part
of a b ispecific ligand ing to the invention can be synthesised by use of a
e synthesiser.
ific ligands in accordance with the invention are described in detail in the SEQ
IDs appended hereto. SEQ ID NOs: 1 and 2 provide the mouse surrogate DNA and
protein sequences of ific ligands in which the CTLA -4 ligand 8a is
paired with the MHC ligand LAG -3, separated by the IGg2a Fc region and a Gly -9
(G9) sequence. A terminal His tag (H6) sequence is provided at the C -terminus. SEQ
ID NOs: 3 a nd 4 provide mouse surrogate DNA and protein sequences for the same
constructs as SEQ ID NOs: 1 and 2, except that the IgG2a Fc region is placed C -
terminal to the LAG -3 ptide, such that the CD80 and LAG -3 peptides are
separated by G9 alone. The two a ments, with the Fc region between the ligands
or C nal thereto, are referred to as gene 1 and gene 2 constructs, respectively.
SEQ ID NOs: 5 and 6 provide human DNA and protein sequences in which wild -type
sequence has been preserved. No mutatio ns are made, either to CD80 or LAG -3.
In SEQ ID NOs: 7 and 8, a W84A mutation has been made to human CD80 (the
equivalent of W88A in mouse) and an R75E mutation has been made in LAG -3. The
remaining SEQ IDs (NOs: 7 - 14) describe other mutations in the CD80 and LAG -3
sequences.
Therapeutic Applications
Suppression of T cell activity is desirable in a number of situations in which
immunosuppression is warranted, and/or an autoimmune condition occurs.
Accordingly, targeting of the CTLA 4/MHC ction is ind icated in the ent of
diseases involving an inappropriate or undesired immune se, such as
inflammation, autoimmunity, and conditions ing such mechanisms. In one
embodiment, such disease or disorder is an autoimmune and/or inflammatory dise ase.
Examples of such autoimmune and/or inflammatory diseases are set forth above.
In one embodiment, such disease or disorder is Type 1 Diabetes (T1D).
In another embodiment, the ligands according to the invention are used to aid
transplantation by immuno suppressing the subject. Such use alleviates graft -versus -
host disease. For a description of existing treatments for graft -versus -host disease, see
Svennilson, (2005) Bone Marrow Transplantation 35:S65 –S67, and references cited
therein. Advantageously, the antibodies of the invention may be used in combination
with other ble therapies.
With regard to the treatment of autoimmune diseases, combination therapy may
include administration of a ligand of the present invention together with a
medicament, whi ch together with the ligand comprise an effective amount for
preventing or treating such autoimmune diseases. Where said mune e is
Type 1 diabetes, the combination therapy may encompass one or more of an agent
that promotes the growth of pancre atic beta -cells or enhances beta -cell
lantation, such as beta cell growth or al factors or immunomodulatory
antibodies. Where said autoimmune disease is rheumatoid arthritis, said combination
y may encompass one or more of methotrexate, a n anti -TNF -α antibody, a
TNF -α receptor -Ig fusion protein, an anti -IL -6, or anti -IL17, or anti -IL -15 or anti -IL -
21 antibody, a non -steroidal anti -inflammatory drug (NSAID), or a disease -
modifying anti - rheumatic drug (DMARD). For example, the additional agent may be
a biological agent such as an anti -TNF agent (e.g., Enbrel® , infliximab (Remicade®
and adalimumab (Humira® ) or rituximab (Rituxan® ). Where said autoimmune
disease is hematopoietic transplant rejection, hematopoietic growth factor(s) (such as
erythropoieti n, G -CSF, GM -CSF, IL -3, IL -11, thrombopoietin, etc.) or
antimicrobial(s) (such as antibiotic, antiviral, antifungal drugs) may be administered.
Where said autoimmune disease is psoriasis, the additional agent may be one or more
of tar and derivatives there of, phototherapy, corticosteroids, porine A, vitamin
D analogs, rexate, p38 mitogen -activated protein kinase (MAPK) inhibitors, as
well as biologic agents such as anti -TNF -α agents and Rituxan® . Where said
autoimmune disease is an inflammatory bowel e (IBD) such as, for example,
Crohn’s Disease or ulcerative s, the additional agent may be one or more of
aminosalicylates, corticosteroids, immunomodulators, antibiotics, or biologic agents
such as Remicade® and Humira® .
The combination treatment may be carried out in any way as deemed necessary or
convenient by the person skilled in the art and for the purpose of this specification, no
limitations with regard to the order, amount, repetition or relative amount of the
compounds to be used in combination is contemplated. Accordingly, the dies
ing to the present ion for use in therapy may be ated into
pharmaceutical compositions. The present invention is also related to pharmaceutical
compositions comprising peptides according to the present invention.
Pharmaceutical Compositions
In a preferred ment, there is provided a ceutical composition
comprising a bispecific ligand according to the invention, or a ligand or ligands
identifiable by an assay method as d efined in the previous aspect of the invention.
Ligands may be immunoglobulins, peptides, nucleic acids or small les, as
discussed herein. They are referred to, in the following discussion, as “compounds”.
A pharmaceutical composition according to th e invention is a ition of matter
comprising a compound or compounds e of modulating T -cell activity as an
active ingredient. Typically, the compound is in the form of any pharmaceutically
acceptable salt, or e.g., where riate, an , free base form, er,
enantiomer racemate, or combination thereof. The active ingredients of a
ceutical composition comprising the active ingredient according to the
invention are contemplated to exhibit excellent therapeutic activity, for exampl e, in
the treatment of graft -versus -host disease, when administered in amount which
depends on the particular case.
In another embodiment, one or more compounds of the invention may be used in
combination with any art recognized compound known to be suitab le for treating the
ular indication in treating any of the aforementioned conditions. Accordingly,
one or more compounds of the invention may be combined with one or more art
recognized compounds known to be suitable for treating the foregoing indica tions
such that a ient, single composition can be administered to the subject. Dosage
regimen may be adjusted to provide the optimum therapeutic response.
For example, several divided doses may be administered daily or the dose may be
proportionally d as indicated by the exigencies of the therapeutic situation.
The active ient may be administered in a convenient manner such as by the
oral, intravenous (where water soluble), intramuscular, subcutaneous, intranasal,
intradermal or suppositor y routes or implanting (e.g., using slow release molecules).
Depending on the route of administration, the active ient may be required to be
coated in a al to protect said ingredients from the action of enzymes, acids and
other natural conditio ns which may inactivate said ingredient.
In order to administer the active ient by other than parenteral administration, it
will be coated by, or administered with, a material to prevent its inactivation. For
example, the active ingredient may be adm inistered in an adjuvant, co administered
with enzyme inhibitors or in liposomes. Adjuvant is used in its broadest sense and
includes any immune stimulating compound such as interferon. Adjuvants
contemplated herein include resorcinols, non -ionic surfactan ts such as
polyoxyethylene oleyl ether and n -hexadecyl polyethylene ether. Enzyme tors
include pancreatic trypsin.
Liposomes include water -in -oil -in -water CGF emulsions as well as conventional
liposomes.
The active ingredient may also be administered parenterally or intraperitoneally.
Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and
mixtures thereof and in oils. Under ry conditions of storage and use, these
preparations contain a preservative to prevent the growth o f microorganisms.
The pharmaceutical forms le for injectable use include sterile aqueous solutions
(where water soluble) or sions and sterile powders for the extemporaneous
preparation of sterile injectable solutions or dispersion. In all cases the form must be
sterile and must be fluid to the extent that easy syringability exists. It must be stable
under the conditions of manufacture and storage and must be preserved against the
contaminating action of microorganisms such as ia and fungi. The carrier can be
a solvent or dispersion medium containing, for example, water, l, polyol (for
e, glycerol, propylene glycol, and liquid polyethylene glycol, and the like),
suitable mixtures thereof, and vegetable oils. The proper fluidity c an be ined,
for example, by the use of a coating such as in, by the maintenance of the
required particle size in the case of dispersion and by the use of surfactants.
The prevention of the action of microorganisms can be brought about by variou s
antibacterial and ngal agents, for example, parabens, chlorobutanol, phenol,
sorbic acid, thirmerosal, and the like. In certain cases, it may be preferable to include
ic agents, for e, sugars or sodium chloride. Prolonged absorption of the
injectable compositions can be brought about by the use in the itions of agents
delaying absorption, for example, aluminium monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active ingredient in the
required amount in the appropriate solvent with various of the other ingredients
enumerated above, as required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the sterilized active ingredient into a sterile
vehicle which c ontains the basic dispersion medium and the required other ingredients
from those enumerated above. In the case of sterile powders for the preparation of
sterile injectable solutions, the preferred methods of preparation are vacuum drying
and the freeze -dr ying que which yield a powder of the active ingredient plus
any additional desired ingredient from previously sterile -filtered solution thereof.
As used herein “pharmaceutically acceptable r and/or diluent” es any and
all solvents, disper sion media, coatings, antibacterial and antifungal agents, isotonic
and absorption delaying agents and the like. The use of such media and agents for
pharmaceutical active substances is well known in the art. Except insofar as any
conventional media or age nt is incompatible with the active ingredient, use thereof in
the therapeutic compositions is contemplated. Supplementary active ingredients can
also be incorporated into the compositions.
It is especially advantageous to formulate parenteral compositions in dosage unit form
for ease of stration and uniformity of dosage. Dosage unit form as used herein
refers to physically discrete units suited as unitary dosages for the mammalian
subjects to be treated; each unit containing a predetermined quantity o f active material
calculated to produce the desired therapeutic effect in association with the required
pharmaceutical r. The specification for the novel dosage unit forms of the
invention are dictated by and directly dependent on (a) the unique char acteristics of
the active material and the particular therapeutic effect to be ed, and (b) the
limitations inherent in the art of compounding such as active al for the
treatment of disease in living subjects having a diseased condition in which bodily
health is impaired.
The principal active ingredients are compounded for convenient and effective
administration in effective s with a suitable pharmaceutically acceptable
carrier in dosage unit form. In the case of compositions containing sup tary
active ingredients, the dosages are determined by reference to the usual dose and
manner of administration of the said ingredients.
In order to facilitate delivery of peptide compounds, including antibodies, to cells,
peptides may be modified in order to improve their y to cross a cell ne.
For example, US 5,149,782 discloses the use of fusogenic peptides, ion -channel
forming peptides, membrane peptides, long -chain fatty acids and other ne
blending agents to increase protein tran sport across the cell membrane. These and
other methods are also described in WO 97/37016 and US 5,108,921, incorporated
herein by reference.
In a further aspect there is ed the active ingredient of the invention as
hereinbefore defined for use in th e treatment of disease either alone or in combination
with art recognized compounds known to be suitable for treating the particular
indication. Consequently there is provided the use of an active ingredient of the
invention for the manufacture of a medica ment for the treatment of disease associated
with an aberrant immune response.
er, there is provided a method for treating a condition associated with an
aberrant immune se, comprising administering to a subject a therapeutically
effective amou nt of a ligand identifiable using an assay method as described above.
The invention is further described, for the purposes of illustration only, in the
following examples.
Examples
Design of a bispecific fusion protein that engages CTLA -4 and cro sslinks it to the
TCR via MHC 11.
To generate a bispecific fusion protein that selectively and tically s
CTLA -4 and simultaneously ligates it to the TCR, mutant CD80 (CD80w88a, referred
to hereafter as CD80wa) that binds CTLA -4 but has minimal affinity for CD28 (Wu et
al., 1997) was fused to LAG -3, a l ligand of MHCII (Baixeras et al., 1992;
Triebel et al., 1990). CD80wa was joined to LAG -3 using a linker composed of nine
glycines, which in turn was attached to the Fc n of mouse IgG2 a to purportedly
increase its circulating half -life (Fig. 1A). In response to a ligand of this
configuration, CTLA -4 engagement and ligation to the TCR were expected to occur
indirectly, via formation of the tri -molecular complex (CTLA -4/MHCII/TTCR) in th e
immune synapses during early T cell tion (Fig. 1B). Conceptually, outside of
the context of the immune synapse, g of the bispecific fusion protein to either
CTLA -4 or MHCII alone or to both CTLA -4 and MHCII should not lead to inhibition
of T cell activity. The engagement of CTLA -4 by CD80wa was designed to trigger
CTLA -4 signaling via the recruitment of phosphatases to the cytoplasmic tail of
CTLA -4. Meanwhile, binding of LAG -3 to MHCII was intended to bring CTLA -4
into the proximity of the cognate TCR, which binds the pMHCII complex in the
immune synapse (Fig. 1B). The combination of these two binding events was
expected to deliver an inhibitory signal to the TCR. A control fusion protein
sing CD80wa and IgG2a Fc was also constructed (Fig. 1A), which should not
be capable of crosslinking CTLA -4 to the TCR (Fig. 1C) as it lacks LAG -3.
The test and control fusion proteins were expressed in Chinese hamster ovary cells
and purified with affinity tography on a protein G column. Aggr egates were
removed using size exclusion chromatography. The test bispecific fusion protein
(CD80wa -LAG Fc) is referred to as BsB (nucleotide sequence: SEQ ID NO. 3;
amino acid sequence: SEQ ID NO: 4), and the control construct (CD80wa -Fc) is
known as BsB Δ (nucleotide sequence: SEQ ID NO. 16; amino acid sequence: SEQ
ID NO: 17). As expected, both fusion proteins appeared as dimers on non -reducing
SDS -PAGE gels (BsB, 200 kDa; BsB Δ 140 kDa) and as monomers (BsB, 100 kDa;
BsB Δ 70 kDa) on reducing SDS -PAGE gels. Their identities were further med
by Western blotting, using antibodies against LAG -3 and CD80.
Example 2
BsB inhibits T cell tion in an allogenic mixed lymphocyte reaction.
The relative ability of BsB and BsB Δ to inhibit T cell acti vation was assessed in an
nic mixed lymphocyte reaction by measuring the production of IL -2. Naïve
CD4 +CD25 - CD62L high CD44 1ow T cells that had been purified from BALB/c mice
were mixed with APCs isolated from C57BL/6 mice in the ce or absence of the
BsB or BsB Δ. Murine IgG2a and CTLA -4Ig, a co lation inhibitor that binds to
CD80/86 and blocks their binding to CD28, were included as negative and ve
controls, respectively. Inclusion of BsB but not BsB Δ in the mixed lymphocyte
reaction inhibited IL -2 production albeit not to the same extent as that achieved by
CTLA -4Ig (Fig. 2). This difference was likely the result of BsB -mediated T cell
inhibition occurring later than CTLA -4Ig -mediated inhibition. More specifically, for
BsB, inhibition only occurre d after CTLA -4 was upregulated following T cell
activation. The inability of BsB Δ to reduce IL -2 production strongly suggests that
engagement of CTLA -4 alone is icient to prevent T cell activation because
concurrent crosslinking to the TCR is required. To exclude the possibility that the
LAG -3 portion of BsB plays a role in T ce ll inhibition, LAG -3Ig was tested in this
assay and verified not to inhibit T cell activation.
Example 3
BsB s T cell differentiation into Tregs.
Early termination of TCR signaling by withdrawal of antigen stimulation, inhibition
of mTOR signaling, s uboptimal TCR stimulation due to a low affinity antigen, or
weak co -stimulation during T cell activation have been shown to induce Foxp3 +
expression and skew T cell differentiation toward a Treg phenotype (Delgoffe et al.,
(2009) Immunity 30:832 -844; Haxhi nasto et al., (2008) J. Exp. Med. 205:565 -574;
Sauer et al., (2008) Proc. Natl. Acad. Sci. USA 105:7797 -7802). As BsB forces early
engagement of the TCR by activation -induced CTLA -4 with consequent attenuation
of TCR signaling, its ability to generate Fo xp3 + Tregs was also evaluated. Naïve
CD4 +CD62L high GFP” T cells prepared from Foxp3 -EGFP knock -in mice (Haribhai et
al., (2007) J. Immunol 178:2961 -2972) were mixed with LPS -treated allogenic APCs
in the presence of BsB or BsB Δ. Flow cytometry analysis of the cells after five days
of culture revealed a large number of CD4 +CD25 +GFP + T cells among the BsB -
treated cells (Fig. 3A, middle left panel) but not among cells treated with mouse
IgG2a (Fig. 3A, top left panel) or the B sB Δ control (Fig. 3A, bottom left panel),
suggesting that these CD4 +CD25 +GFP + T cells were Foxp3 + Tregs. To confirm this
g, cell culture media were collected and d for the signature Treg
cytokines, IL -10 and TGF -β (Cools et al., (2008) J. Cell Mo/. Med. 12:690 -700).
Large amounts of IL -10 and TFG -β were detected in the media of BsB -treated cells
(Fig. 3A, left panels) but not in media of cells treated with BsB Δ or mIgG2a.
Surprisingly, CTLA -4Ig did not induce generation of GFP + Tregs or IL -10 and TGF -β
production. Without being bound to a particular , the ism by which
CTLA -4lg curtails the T cell response is different from that of BsB. LAG -3Ig alone
or in combination with BsB Δ also failed to induce generation of GFP + Tregs,
sugges ting that BsB -mediated crosslinking of CTLA -4 with the TCR was required for
Treg induction.
Example 4
Induction of Tregs by BsB requires self -stimulated TGF -β
The rent detection of elevated levels of IL -10 and TGF -β following treatment
with BsB rais ed the possibility that the cytokines, TGF -β in particular, played a role in
facilitating the generation of Tregs A). To s this, culture media were
collected over a period of five days and analyzed for ne and Foxp3 + Treg
t. Elevat ed IL -10 and TGF -β levels were detected as early as day 2 post -
treatment, and Foxp3 + Tregs were detected after day 3. Without being bound to a
particular theory, the endogenous production of TGF -β presumably stimulated by
BsB, is involved in Treg differen tiation. Addition of an anti -TGF -β antibody (clone
1D11), but not an isotype control IgG (clone 13C4), to the Treg induction assay
completely blocked the appearance of Foxp3 + Tregs (Fig. 3B). Without being bound
to a ular theory, the early engageme nt of CTLA -4 and its uent
inking to the TCR by BsB stimulated endogenous TGF -β production, which in
turn encouraged Treg differentiation. Crosslinking of CTLA -4 and the TCR has been
previously ed to induce TGF -β production (Chen et al., (1998) J. Exp. Med.
188:1849 -1857), although Treg differentiation was not assessed in this study.
Tregs have shown considerable therapeutic potential in modulating the disease
manifestations in several animal models of autoimmune diseases. However, the
im portance of the specificity of the induced Tregs against the relevant antigens has
been highlighted. Non -antigen specific Tregs that will not be activated against
ular autoantigens in the context of autoantigen -specific reactive T cells are
presumab ly not functionally immunosuppressive. Hence, approaches that facilitate
the generation of large s of antigen -specific Tregs are highly desirable for
treating these ailments. Moreover, strategies that facilitate the de novo induction of
antigen -spec ific Tregs in situ (e.g. in islets of pancreas for T1D or in the lamina
propria for ulcerative s or Crohn’s disease) are preferred over the use of adoptive
transfer of in vitro differentiated or expanded Tregs.
Example 5
BsB -induced Tregs are functi onally suppressive in a cell -cell t -dependent
manner.
To assess whether the BsB -induced Tregs were functionally suppressive, BsB -
induced Tregs and TGF -β-induced Tregs, which served as a l, were purified
using fluorescence -activated cell g (FACS) and mixed with CFSE ed
syngeneic responder T cells at different ratios and allogenic APCs. Cells were co -
cultured for three days in either transwells or regular culture wells, after which the
proliferation of der T cells was analyzed us ing flow cytometry. As
ized in Fig. 5 A, both BsB - and TGF -β-induced Tregs cultured in regular
culture wells almost completely inhibited the proliferation of the responder T cells.
The potency of the suppressive activity of the BsB -induced Tregs was comparable to
that of TGF -β-induced Tregs. In contrast, Tregs generated by either BsB or TGF -β did
not icantly t the proliferation of responder T cells when the T cells were
separated from the Tregs in a transwell. Without being bound to a particular theory,
Treg suppressive activity d d on cell -cell contact and was not mediated by
secreted cytokines or other factors. Supporting this notion, inclusion of an antibody
to IL -10 (clone JES5 -2A5) in the regular culture well did not affect the suppressive
activity of either the BsB - or the TGF -β-induced Tregs (Fig. 5B). The addition of an
antibody to TGF -β1D11 also did not affect the suppressive activity of BsB ed
Tregs, although it partially reduced suppression by TGF -β-induced Tregs (Fig. 5B).
Example 6
BsB directs differentiation of OT -II T cells into antigen -specific Tregs.
As it was found that a bifunctional fusion protein comprising CD80wa and LAG3
(BsB) that crosslinks CTLA -4 to the TCR (via MHCII) can induce the production of
Foxp3 + Tregs in an allogenic MLR, the potential of BsB at eliciting the production of
antigen -specific Tregs was examined. To investigate this prospect, naïve OT -II T
cells were purified from transgenic mice ing enes encoding the TCR ( αand
β- subunits) specific for a n ovalbumin peptide (323 -339) (Barnden et al.,
1998 ) and mixed with syngeneic APCs in the presence of Ova323 -339. After 5 days
of culture, significantly greater amounts of Foxp3 + Tregs were detected in OT -II T
cells that had been treated with BsB (Fig. 4A, middle left panel) than by the mIgG
control (Fig. 4A, upper left panel) or by CTLA -4Ig (data not shown). This induction
of Tregs was inhibited by the inclusion of anti -TGF -β antibody in the cultures (Fig.
4A, bottom left . Without being bound to a particular theory, entiation
was mediated by endogenously produced TGF -β in an autocrine or paracrine manner.
Levels of IL -2 were decreased while those of IL -10 and TGF -β were sed in the
media of BsB -treated cells (Fig. 4A, right panels).
To monitor the proliferative activity of the induced Tregs, OT -II cells were preloaded
with the fluorescent tracer, CFSE. As shown in Fig. 4B, BsB -induced Foxp3 + Tregs
were determined to be erative as ted by a dilution of the CFSE signal. As
ex pected, the addition of CTLA -4Ig, a co -stimulatory blocker, reduced T cell
proliferation. Hence, BsB was able to inhibit T cell activation and induce the
production of Tregs in both an allogenic MLR and antigen -specific setting.
Example 7
Induction of Tr egs by BsB may involve attenuation of the AKT/mTOR ing
pathway.
Recent reports have indicated that the AKT and mTOR signaling pathways play
important roles in determining T cell fate. The presence of constitutively active AKT
in T cells diminishes T reg differentiation in a rapamycin -sensitive manner
(Haxhinasto et al., 2008), suggesting that the AKT and mTOR ing pathways
intersect to influence Treg fate. er, T cells deficient in mTOR differentiate to
Tregs more readily than normal control T cells (Delgoffe et al., (2009) Immunity
30:832 -844). An obligatory role for the co -inhibitory molecules PD -1/PD -L1 in
controlling adaptive Treg development by antagonizing AKT/mTOR has also been
reported isco et al., (2009) J. Exp. Med. 206:3015 -3029). To determine if these
pathways are also involved in BsB ted induction of Tregs, anti -CD3 and anti -
CD28 antibodies were co -immobilized with BsB, mIgG, or PD -L1 on 96 -well plates,
onto which naïve T cells were seeded. Eighteen hours post -activati on, the cells were
stained with fluorescently -labeled antibodies against phosphorylated AKT and mTOR
and analyzed by flow cytometry. Phosphorylation of both AKT and mTOR was
ated by BsB and PD -L1 co -immobilization (Fig. 6). Without being bound to a
particular theory, signaling events mediated by CTLA -4 and PD -L1 inhibitory
molecules may converge at some point along the AKT/mTOR signaling pathway
during T cell activation to regulate Treg differentiation.
Example 8
Exposure to BsB sustains Foxp3 + expre ssion in d Tregs.
In vitro -induced Tregs, unlike fully ted natural Tregs, are reportedly less
stable and can lose Foxp3 + expression upon extended culture in the absence of the
initial inducer (e.g., TGF -β or retinoic acid) raj and Geiger, (2007) J. lmmunol.
178:7667 -7677). In the current study, BsB -induced Tregs showed similar ility,
with some cells losing Foxp3 expression following repeated culture (Fig. 7). To test
whether re -stimulation by BsB could prolong Foxp3 expression, Tre gs were first
induced by coating 96 -well plates with both anti -CD3/anti -CD28 antibodies and BsB.
Purified Tregs were then subjected to an additional round of culture in the presence or
absence of BsB. Re lation of the purified Tregs with BsB allowed for
nance of a large population (~93% of total Tregs) of Foxp3 + Tregs (Fig. 7,
bottom right panel), compared to ~40% Foxp3 expression in response to the IgG
control (Fig. 7, upper right panel).
Example 9
Pharmacokinetics of BsB in mice.
Prior to te sting the therapeutic utility of BsB in animal models of autoimmune
diseases, its pharmacokinetic profile was determined to help design a dosing n
in vivo . eritoneal injection of BsB into 6 mice resulted in a measurable
rise in circulati ng levels followed by rapid clearance with an estimated plasma half -
life (t 1/2 ) of ~12 hr (Fig. 8A). This profile was unexpected since the pharmacokinetics
of Fc -containing fusion proteins or antibodies is typically more prolonged. As
binding of antibodi es to the neonatal Fc receptor (FcRn) is primarily responsible for
their prolonged half -lives (Roopenian and Akilesh, 2007 ), the relative abilities of BsB
and a control mouse IgG2a to bind FcRn we re compared. Figure 8B shows that the
binding characteristics of both proteins to the FcRn were very r indicating that a
defect in the binding of BsB to FcRn was unlikely to be the cause of its rapid
clearance from the circulation.
Another potentia l explanation for the rapid clearance of BsB could be due to its
uptake by carbohydrate receptors on non -target cells. Examples of such receptors
e the glycoprotein receptor (ASGPR) on hepatocytes (Weigel, 1994 ) and
the e or on hages and endothelial cells of the reticuloendothelial
system (Pontow et al., 1992 ). Analysis of BsB using the NetNGlyc server suggested it
has the potential to harbor up to 10 asparagine -linked o ligosaccharide side chains per
monomer (Fig. 9). A monosaccharide composition analysis indicated that BsB
contained approximately 37 mannose residues, and all the predicted asparagine -linked
glycosylation sites may have been used e each of these asp aragine d
oligosaccharide glycans contains the core -mannose structure with three mannose
residues (a total of 30 mannose residues). In addition, a small amount of high -
mannose type oligosaccharides may also exist to account for the extra mannose
resid ues. Indeed, significant amounts of under -sialylated tri - and tetra -antennary
asparagine d, as well as some high -mannose type oligosaccharides were
identified by mass spectrometry of permethylated glycans released from the protein.
This projection is also consistent with BsB’s molecular weight of 100 kDa as
indicated by an SDS -PAGE analysis, as opposed to BsB’s calculated weight of 80
kDa. The added presence of oligosaccharides contributed to the difference (20 kDa)
in molecular weight. Moreover, Bs B exhibited a ratio of sialic acids to galactose of
0.68 (Fig. 9), indicating that the glycans were incompletely sialylated. Without being
bound to a particular , the carbohydrate -mediated clearance of BsB by the
ASGPR contributed to its rapid clear ance from circulation.
Example 10
A short course of treatment with BsB delayed the onset of autoimmune diabetes
in NOD mice.
As the EC 50 of BsB for inducing Tregs in vitro was estimated to be about 100 nM and
its circulating half -life was short (t 1/2 at ~12h), BsB was tested in NOD mice in a late
prevention paradigm. NOD mice were administered BsB over a short interval (every
other day for 4 weeks) when they were between 9 and 12 weeks of age. At this age,
autoreactive T cells and insulitis are already t but the mice have yet to develop
overt es. As shown in Figure 10A, NOD mice treated for 2 weeks with BsB
showed a modest but statistically significantly increase (25%) in the number of
Foxp3 + Treg in the blood when compared to saline -treated controls. However, this
increase in Tregs was transient as a difference in the number of Tregs after 4 weeks of
ent or at later times points was unable to be detected. A similar transient
increase in Tregs in lymphoid organs was noted previously fo llowing treatment of
NOD mice with an anti -CD3 antibody (Nishio et al., 2010 ). Without being bound to a
particular theory, the BsB -induced Tregs may have reverte d to Foxp3 - T cells after
cessation of treatment. They may also have been recruited by specific target tissues
(e.g. pancreas) to execute their function. Regardless, this short course of ent
with BsB in a late prevention treatment paradigm appears t o ly delay the onset
of disease and decrease the number of mice presenting with overt T1D (Fig. 10B).
The modest response noted may have been due to the ce of active insulitis in
the 9 week -old NOD mice prior to cement of therapy. An inf lammatory
milieu has been shown to favor the conversion of activating T cells to Th17 cells and
suppress their conversion to Tregs. matory nes such as IL -6 or IL -4 have
also been shown to inhibit Treg conversion and promote the loss of Foxp3 + expression
in Tregs (Caretto et al., 2010 ; Kastner et al., 2010 ; Koenen et al., 2008 ). To
vent these challenges, NOD mice were d starting at an earlier age (4
week -old) prior to overt induction of auto -reactive T cells and tis. CTLA -4Ig
was also ed as a positive control in this study as one and gues
(Lenschow et al., 1995 ) had demonstrated a benefit using this agent in this model;
mIgG2a was used as an additional negative l t o saline. In contrast to the results
in older mice (Fig. 10A), the number of Foxp3 + Tregs in the peripheral blood of
younger NOD mice treated for 2 weeks with BsB was not increased over those
administered saline or mIgG (Fig. 11A). Without being bound to a particular theory,
this might be because the number of auto -reactive T cells in 4 week -old NOD mice
(in contrast to 9 -12 week old mice used in the earlier study) was very low. The
number of induced antigen -specific Tregs was likely too small to registe r beyond the
basal levels present in the animals. A significantly lower incidence of T1D was noted
in NOD mice administered BsB when compared to the saline -treated controls prior to
24 weeks of age (Fig. 11B). However, this benefit was reduced at the lat er time
points.
Consistent with the report of NOD mice administered CTLA -4Ig (Salomon et al.,
2000 ), the levels of Tregs in the blood (Fig. 11A) were significan tly depressed
presumably because of CTLA -4Ig’s effects on CD28/B7 signaling (Tang et al., 2003 ).
Treatment with CTLA -4Ig also aggravated the disease with mice exhi biting an earlier
onset of disease (Fig. 11B) and higher penetrance of disease when compared to the
saline - and mIgG -treated controls (Fig. 11B). The reason for the discrepancy between
these findings and those reported by Bluestone and colleagues (Lenschow et al., 1995 )
is unclear but may be due to the differences in the CTLA -4Ig used or the dosing
n employed. In the present studies, a dose of 10 mg/kg of human CTLA -4Ig
(Orencia) was used instead of 2.5 mg/kg of mouse CTLA -4Ig by Bluestone and
colleagues. er, BsB ent was not extended beyond 7 weeks. Without
being bound to a particular theory, the use of a higher dose of CTLA -4Ig afforded a
mo re complete blockade of the co -stimulatory signal required for Treg homeostasis.
Example 11
A longer course of treatment with BsB significantly delayed the onset and
reduced the incidence of autoimmune diabetes in NOD mice.
Potential reasons for the obser ved modest benefits of BsB at addressing the e in
NOD mice in the r studies include the deployment of a relatively short e of
treatment, the moderate y of BsB at inducing the production of Tregs (EC 50 at
> 100 nM), and the short circ ulating half -life of BsB that may have limited its
exposure. As the potency and circulating half -life of BsB are intrinsic to the molecule
and therefore not amenable to facile change, a longer course of treatment was tested.
To this end, NOD mice were tre ated with BsB for 10 weeks instead of 4 weeks
starting when the mice were at 4 weeks of age. As shown in Figure 12A, NOD mice
treated for 10 weeks with BsB exhibited a significant delay in the onset of T1D.
Importantly, by 35 weeks of age, only ~13% of Bs B-treated NOD mice ped
T1D as compared to over 70% in the saline -treated controls. Thus, extended
ent of NOD mice with BsB appeared to have protected the animals from
developing autoimmune diabetes.
At the sion of the study (when mice w ere 35 week -old), the animals were
sacrificed and their pancreata were collected for histopathological analysis. Adjacent
serial sections were stained with H&E for a general assessment of the islets, probed
with an anti in antibody to detect the pres ence of insulin in the β-cells, and
double stained with anti -CD3 and anti -Foxp3 antibodies to locate T cells and Tregs.
Due to the genetic heterogeneity of the NOD mice, a small number of the untreated
animals did not develop disease at 35 weeks of age. A nalysis of the islets of these
non -diabetic animals (from the saline -treated cohort) showed the β-cells were intact
with no obvious evidence of lymphocytic infiltration or insulitis, (Fig. 12B, panels a -
c). A few Foxp3 + Treg cells were present in the isle ts of these mice (arrows in panel
c). In contrast, islets from diabetic NOD mice (from the saline -treated cohort)
ed the presence of invasive insulitis (Fig. 12B, panel d) and complete
destruction of the β-cells (panel e). In on to CD3 + T cel ls and Foxp3 + Tregs,
large numbers of non -T cell lymphocytes were also evident (Fig. 12B, panel f).
Similar histopathological findings were noted in the corresponding BsB -treated mice
that remained disease -free at the end of the study or that developed T1D during the
study. Interestingly, in ~50% of the islets of BsB -treated NOD mice that remained
non -diabetic, evidence of peri -insulitis were noted (Fig. 12B, panel g); however, the
β-cells were well preserved (Fig. 12B, panel h). Staining with dies indicated
that the cells at the periphery of the islets comprise primarily CD3 + T cells and Tregs.
(Fig. 12B, panel i). An enlargement of a section of the image (red square in Fig. 12B,
panel i) clearly revealed the presence of numerous Foxp3 + Tregs (yellow arrows in
Fig. 12B, panel j) that were interspersed with non -Foxp3 + but CD3 + T cells (black
arrow heads in Fig. 12B, panel j) as well as non -T cell mononucleocytes (blue nuclei).
The development of peri -insulitis has been noted in young (4 -10 week -old) NOD
mice (Anderson and Bluestone, 2005 ) and in ol der mice treated with other cious
therapeutic agents that delayed or reversed new onset T1D in NOD mice (Chatenoud
et al., 1994 ; Daniel et al., 2011 ; Simon et al., 2008 ; Vergani et al., 2010 ). Hence, a
longer course of treatment of NOD mice with BsB protected the animals from
developing ve insulitis and overt T1D. Without being bound to a particular
theory, this was ed, at least in part, by the de novo and possibly in situ
induction of islet antigen -specific Tregs.
Crosslinking CTLA -4 a nd TCR via MHCII using a novel bispecific fusion n
(BsB) efficiently induced the production of antigen -specific Tregs as well as the anti -
inflammatory cytokines, IL -10 and TGF -β . Previous studies showed that Tregs are
critical for conferring immune tolerance and that antigen -specific Tregs are more
efficacious in animal models of autoimmune diseases. BsB was further evaluated in
animal models of autoimmune diseases, such as T1D. Without being bound to a
particular theory, it was hypothesized that i f BsB promoted the induction of antigen -
specific Tregs during the early phase of activation of autoreactive T cells in NOD
mice it can delay the onset or halt the progression of disease by converting the
autoreactive T cells that are undergoing activation to Tregs.
Despite BsB exhibiting a modest potency (due to its moderate affinity for the MHC -II
and TCR) and a short circulating half -life (which limited its exposure), a short course
of treatment reproducibly delayed the onset of T1D in NOD mice treated at an early
age en 4 -6 weeks of age) and when they were older (between 9 -12 weeks of
age). However, the observed benefits were modest and ained. A longer course
of treatment (10 weeks) of NOD mice (between 4 and 13 weeks of age) with BsB
signif icantly delayed the onset of disease and the nce of animals developing
T1D. Without being bound to a particular theory, this benefit was imparted by the de
novo generation of induced Tregs that were either produced locally (e.g. in the
pancreas or atic draining lymph nodes) or distally that were then recruited to
the pancreas to protect the islets from ction by autoreactive T cells and other
non -T cell leukocytes. Immunohistochemical staining of sections of pancreatic tissues
of 35 week -old BsB -treated mice that remained non -diabetic clearly ted an
increase in the number of Foxp3 + Tregs at the periphery of the islets. Visually, they
appeared to be preventing CD3 + T cells and non -T cell lymphocytes from ng the
islets. This phe nomenon was observed in ~50% of the islets of BsB -treated NOD
mice that remained non tic at the end of the study but in none of the islets of
diabetic animals in the control group. The islets of a few non -diabetic mice in the
control group remained d evoid of lymphocytic infiltrations and were insulitis -free. It
is known that because of the genetic heterogeneity of NOD mice, a few animals in a
cohort of this size never develop diabetes within this timeframe. In the remaining
~50% of the non -diabetic animals in the BsB -treated group, the islets were also
devoid of lymphocytic infiltrations and insulitis -free. Possibilities for the disease -free
status of these mice include BsB treatment and the genetic background.
Consistent with the histopathological gs, a small but statistically significant
increase in the number of Foxp3 + Tregs was detected in the blood of BsB -treated
animals (treated from 9 -12 weeks of age) when compared to ted controls. This
increase was not evident in mice that starte d treatment at a younger age (4 week -old).
Without being bound to a particular theory, this may be because more active T
cells were undergoing tion in the 9 week -old than in the 4 week -old mice. The
low levels of autoreactive T cells in the 4 week -old mice might have precluded
ion of induced Tregs beyond that in the existing milieu of Tregs. The increase
in Tregs was also transient in nature. As a similar observation was noted in animals
subjected to anti -CD3 therapy (Nishio et al., 2010 ), it is possible that the induced
Tregs were unstable and lost expression of Foxp3. It is more conceivable that the
Tregs were recruited from circulation to a d target tissues. In contrast, NOD
mice treated with CTLA -4Ig exhibited a significant decrease in the number of
circulating Tregs. Treatment also aggravated the disease as evidenced by an
expedited onset of e and a higher incidence of animals displaying overt disease.
This is consistent with previous reports showing that the co latory pathway is
involved in Treg homeostasis and that a lack of co -stimulation reduces the production
of Tregs. Blocking or knocking -out CD80 or CD86 in NOD mi ce also results in an
earlier onset of T1D (Salomon et al., 2000 ; Tang et al., 2003 ).
The appearance of peri -insulitis is typically observed in the pancreas of NOD mice
between 4 and 9 weeks of age. If uncontrolled, invasive insulitis ensues leading to the
complete destruction of β-cells and the development of overt diabetes between 12 and
weeks of age. The pancreata of non tic NOD mice that had been treated for
10 weeks with BsB and analyzed at 35 weeks of age ted evidence of peri -
insulitis that appeared to be arres ted in their progression. No indication of invasive
insulitis or excessive destruction of insulin cing β-cells was noted. There are
other reports of different therapeutic entions similarly delaying or preventing
disease in NOD mice (Shoda et al., 2005 ). The s here are most ak in to those
reported by Lee et al. (2010), who showed that er of diabetogenic CD4 +CD25 -
BDC2.5 T cells depleted of CD4 +CD25 + Tregs into female NOD/SCID mice
expedited the development of invasive insulitis when compared to mice administered
total CD4 + T cells containing CD4 +CD25 + Tregs. Invasive insulitis was largely
dominated by infiltration of tic cells (DC) rather than by BDC2.5 T cells per se.
The authors surmised from their study that Tregs regulated the invasiveness of DCs
into the islets by modulating, at least in part, the chemotaxis of DCs in response to the
chemokines CCL19 and CCL21 secreted by the . The immunohistochemical
staining patterns for Foxp3 + Tregs, CD3 + T cells and non -T cell leukocytes noted in
the atic section s of BsB -treated, non -diabetic NOD mice are consistent with
their findings (Fig. 12B). Without being bound to a particular , Tregs produced
in NOD mice in response to BsB likely acted to halt the migration of autoreactive T
cells and non -T cell lymp hocytes into the islets. A longer course of treatment with
BsB was more effective because this generated a more robust and sustained induction
of Tregs. That continuous stimulation of induced Tregs with BsB in cell cultures
extended the expression of Fox p3 + in Tregs is tive of this notion (Karman et
al., 2012 ).
Cell therapy using freshly isolated, ex vivo expanded or in vitro induced Tregs in
animal models of autoimmune diseases or organ transplants have demonstrated that
adoptive transfer o f Tregs can restore the balance of Tregs versus effector T cells,
thereby controlling the exuberant autoimmunity associated with these diseases (Allan
et al., 2008 ; Jiang et al., 2006 ; Riley et al., 2009 ; Tang et al., 2012 ). However, the use
of adoptive transfer a s a therapeutic strategy presents several challenges to translation
into the clinic. Firstly, the number of gous Tregs that can be isolated from
peripheral blood of a human subject is limiting. Hence extensive ex vivo expansion of
the Tregs is often necessary, which may alter their functionality and purity. Secondly,
as the isolated Tregs are polyclonal, they can exert a pan -immune suppressive
function on non -target effector T cells. Thirdly, and most importantly, the plasticity
of Tregs poses a sig nificant challenge (Bluestone et al., 2009 ; Zhou et al., 2009a ). It
has been shown that adoptively err ed Tregs can lose Foxp3 expression and
redifferentiate into Th17 cells (Koenen et al., 2008 ) or pathogenic memory T cells
(Zhou et al., 2009b ) which raises the risk of aggravating the autoimmunity or
inflammation. Conse quently, a eutic that induces the generation of Tregs in an
n -specific manner in situ is more advantageous over adoptive Treg cell y.
The s presented herein demonstrate the utility and effectiveness of such an agent
(BsB) that cros slinks CTLA -4 to MHCII in the context of a mouse model of T1D.
The combined demonstration of production of IL -10, TGF -β and Tregs in response to
treatment with BsB as well as efficacy in the NOD mouse model of T1D has the
potential to provide a novel therapeutic concept. BsB also offers additional
advantages over other immune modulators in that it does not affect resting T cells or
other lymphocytes. The numbers and tages of CD4 + T cells and CD19 + B cells
in the periphery remained the same in all our NOD studies. Without being bound to a
particular theory, this approach is effective in delaying or halting disease progression.
The development of BsB variants that are more potent and that harbor a more
favorable pharmacokinetic e should confirm these studies. Thus, this concept
may also be applied towards the ment of other immune ted es.
Res ults reported herein were ed using the following methods and materials
unless indicated otherwise.
Animals. Female wild -type C57BL/6 (H -2b), BALB/c(H -2d), transgenic OT -II mice
expressing the mouse α-chain and β-chain T cell receptor specific for ch icken
ovalbumin 323 -339(Ova 323 -339 ) in C57BL/6 genetic background, and female non -
obese diabetic (NOD/LtJ) mice were purchased from The n Laboratory.
Animals were maintained in a pathogen -free facility and studies were conducted in
accordance with t he guidelines issued by the U.S. Department of Health and Human
Services (NIH Publication No 86 -23) and by Genzyme’s Institutional Animal Care
and Use committee.
Antibodies and ts. Functional grade or fluorescently -labeled anti -mouse CD3
(clone 145 -2C11), CD25, insulin and Foxp3 + dies were purchased from
eBioscience or BD ences. Murine CTLA Fc and human CTLA -4Ig (Orencia)
were purchased from R&D Systems, Inc. and Bristol -Myers Squibb , respectively.
Mouse IgG2a isotype control was obtai ned from BioXCell Inc. CFSE, ultralow Ig
fetal bovine serum (FBS), and other cell culture media were from Invitrogen .
n Ova 323 -339 peptide was obtained from New England Peptide.
Construction and production of the bispecific fusion protein BsB. Con struction and
expression of the bispecific fusion protein (BsB) comprising the extracellular domains
of CD80w88a and LAG -3 as well as the Fc of mouse IgG2a (CD80wa -LAG Fc,
BsB) were described previously (Karman et al., 2012 ).
e assays and monosaccharide ition analysis. Biacore was used to
compare the binding of BsB and mIgG2a to the mouse neonatal Fc receptor (FcRn).
Briefly, a CM5 chip was immobilized with ~1430 RU of mouse FcRn -HPC4 using
amine chemistry. Each sample was ly diluted 1:2 to final trations of
between 200 and 6.25 nM in PBSP (PBS with 0.005% Surfactant P -20), pH 6.0 and
injected for 3 min in duplicate, followed by 3 min washwith dissociation buffer. The
surface was regenerated with 10mM sodium borate and 1 M NaCl, pH 8.5. The
carbohydrate monosaccharide composition of BsB was ed according to the
protocol described by Zhou et al. (Zhou et al., 2011 ).
Isolation o f naïve T cells. Naive T cells from the spleens and lymph nodes of 8 -12
week old female BALB/c or OT -II mice were purified by magnetic separation
followed by fluorescence -activated cell sorting. Cells were first negatively selected
by magnetic cell separ ation (Miltenyi Biotech) and then sorted as CD4 +CD25 -
CD62L hi CD44 low cells to a purity of greater than 98%.
n -specific Treg induction assay. Assays in an allogenic MLR setting was
performed as previously reported (Karman et al., 2012 ). For a ntigen -specific T cell
activation, 10 5 naïve OT -II T cells were mixed in round -bottom 96 -well plates with
5 irradiated syngeneic APCs in the presence of Ova
323 -329 at 0.5 µg/ml and 1 µg/ml
e anti -CD28 (clone 37.51, eBioscience). The test constructs , mouse IgG2a, or
mouse CTLA -4Ig were added to the cultured cells at a saturating concentration of 100
µg/ml. The cells were cultured for 5 days to induce production of Tregs and analyzed
by flow cytometry. Media were collected for analysis of IL -2, IL -10 and TGF -β using
ELISA kits per the manufacturer’s instructions. To assess T cell proliferation,
ed naïve OT -II T cells were labeled with 5 µM CFSE for 5 min at 37 oC. They
were then washed to remove unbound CFSE and used in Treg induction assays as
described above. Cells were cultured for 5 days to allow them to divide before being
analyzed by flow cytometry. To detect Foxp3 + in T cells, cells were stained for
surface markers as described above followed by permeabilizing with Fix/Perm buffer
(eBiosc ience) and staining with PE -Cy7 conjugated anti -Foxp3 antibody (clone FJK -
16s, eBioscience).
Pharmacokinetics measurements of BsB in mice. The pharmacokinetics of BsB was
determined in 8 week -old C57BL/6 mice. 20 mg/kg of BsB was administered into
mice b y intraperitoneal injection. Blood was collected by saphenous vein bleeding at
1 hr, 5 hr, 24 hr, 48 hr, and 72 hr after administration. The levels of BsB at each time
point were measured using an ELISA assay. Briefly, 100 µl (1 µg/ml) of an anti -
mouse CD8 0 dy in PBS were coated onto 96 -well plates and ted
overnight at 4 oC. Plates were blocked with 5% fetal bovine serum for 1 h, after which
they were washed 4 times with PBS. 100 µl of blood samples at various dilutions
were then added into the wells. The plates were incubated for 2 hr with gentle
shaking at room temperature and washed 4 times with PBS. ylated anti -mouse
LAG -3 antibody (1 µg/ml) was added and incubated for 2hr. The plates were washed
4 times with PBS after which av idine -HRP was added. After 30 min, the plates
were washed 6 times with PBS and developed for colorimetric measuring. Purified
BsB diluted in assay diluent at various concentrations were used as standards.
Treatment of NOD mice with BsB . In the short cour se treatment, 4 week -old female
NOD mice were treated with , 20 mg/kg BsB, 20 mg/kg mouse IgG2a, or 10
mg/kg human CTLA -4Ig (Orencia) three times a week by intraperitoneal injection
over a period of 2.5 weeks. For the late prevention model, 9 -12 wee k-old NOD mice
were treated with saline or 20 mg/kg BsB as above for 4 weeks. For longer course
treatment, NOD mice were treated with BsB or saline as above for 10 weeks from age
of 4 weeks to 13 weeks. Non ng blood glucose levels were monitored week ly
starting at 8 weeks of age. Mice were deemed diabetic when their glucose readings
were greater than 300 mg/dL for three consecutive readings. Foxp3 + Tregs in
peripheral blood was ed after two weeks of treatment by flow cytometry.
Briefly, 50 µl o f whole blood was blocked with unlabeled anti -Fc γRIIb and FcgRIII
(clone 93, eBioscience) for 20 min. Cells were uently stained with
fluorescently -labeled anti -CD4 antibody for 30 min and then washed. Red blood cells
were lysed using FACS Lysing solution (BD Biosciences) for 5 min. After washin g,
cells were fixed, permeabilized and d with a FITC ed anti -Foxp3 antibody
for 30 min as bed above. Pancreata were dissected in half with one half fixed in
neutral buffer formalin and the other placed into OCT compound and then frozen on
dry ice.
Statistical analysis. Cumulative incidences of NOD mice ting with T1D and
hyperglycemia following treatment with BsB or controls were compared using the
log -rank (Cox -Mantel) test in Prism 5 (Graphpad, city and state). A value of p<0.05
was considered statistically significant.
Histopathological analysis. Neutral buffer formalin -fixed pancreata was stained for
CD3, Foxp3 + cells using an automated ser. Tissue sections were dewaxed
using xylene -ethanol, the antigens retrieved by incu bating for 25 min in citrate buffer
and then blocked with serum. Slides were incubated with an anti -CD3 antibody for 45
min, followed by a goat anti -rabbit horse radish peroxidase polymer for 20 min.
Chromogen visualization of CD3 was obtained by incubati ng with 3,3’ -
diaminobenzidine tetrahydrochloride for 2 -4 min. To detect Foxp3 +, sections were re -
blocked with serum, followed by exposure to an anti -Foxp3 antibody for 45 min.
Slides were then incubated with a rabbit anti -rat IgG antibody for 30 min, fol lowed by
a goat anti -rabbit alkaline phosphatase polymer. Chromogen visualization was
achieved using Fast Red for 10 min. Tissue sections were counterstained using
hematoxylin for 2 min and washed 3 times with 0.05% Tween -20/Tris buffered saline
n steps. Adjacent serial sections were stained using an anti -insulin dy as
described above. Pictures were taken using a Nikon Eclipse E800 fluorescent
microscope with an ed digital camera from Diagnostic Inc. and images acquired
using the Spot Advanced re.
Sequences
Legend
CD80w88a = CTLA -4 ligand
IgG2a = IgG2 Fc region
G9 = Gly 9
Lag -3 = MHC ligand
H6 = His 6
SEQ ID NO. 1:
CTLA -4 BsB (Gene1) = mouse CD80w88a(aa1 -235) -IgG2a(aa241 -474) -G9 -Lag -
3(aa25 -260) - H6
Nucleotide sequence of mouse surrogate construct (Gene1):
ATGGCTTGCAATTGTCAGTTGATGCAGGATACACCACTCCTCAAGTTTCCATGTC
CAAGGCTCATTCTTCTCTTTGTGCTGCTGATTCGTCTTTCACAAGTGTCTTCAGA
TGAACAACTGTCCAAGTCAGTGAAAGATAAGGTATTGCTGCCTTGCCG
TTACAACTCTCCTCATGAAGATGAGTCTGAAGACCGAATCTACTGGCAAAAACAT
GACAAAG TGGTGCTGTCTGTCATTGCTGGGAAACTAAAAGTGGCGCCCGAGTAT
AAGAACCGGACTTTATATGACAACACTACCTACTCTCTTATCATCCTGGGCCTGG
TCCTTTCAGACCGGGGCACATACAGCTGTGTCGTFCAAAAGAAGGAAAGAGGAA
CGTATGAAGTTAAACACTTGGCTTTAGTAAAGTTGTCCATCAAAGCTGACTTCTC
CAACATAACTGAGTCTGGAAACCCATCTGCAGACACTAA AAGGATT AC
TGCTFCCGGGGGITTCCCAAAGCCTCGCTTCTCTTGGTTGGAAAATGG
AAGAGAATTACCTGGCATCAATACGACAATTTCCCAGGATCCTGAATCTGAATTG
TACACCATTAGT AGCCAACTAGATTTCAATACGACTCGCAACCACACCATTAAGT
GTCTCATFAAATATGGAGATGCTCACGTGTCAGAGGACTTCACCTGGGAGCCCA
GAGGGCCCACAATCAAGCCCTGTCCTCCA TGCAAATGCCCAGCACCTAACCTCT
TGGGTGGACCATCCGTCTTCATCTTCCCTCCAAAGATCAAGGATGTACTCATGA
TCTCCCTGAGCCCCATGGTCACATGTGTGGTGGTGGATGTGAGCGAGGATGAC
CCAGATGTCCAGATCAGCTGGTTCGTGAACAACGTGGAAGTACTCA CAGCTCAG
ACACAAACCCATAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTC
CCCATCCAGCACCAGG ACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAA
CAACAAAGCCCTCCCAGCGCCCATCGAGAGAACCATCTCAAAACCCAAAGGGT
CAGTAAGAGCTCCACAGGTATATGTCTTGCCTCCACCAGAAGAAGAGATGACTA
AGAAACAGGTCACTCTGACCTGCATGGTCACAGACTTCATGCCTGAAGACATTT
ACGTGGAGTGGACCAACAACGGGAAAACAGAGCTAAACTACAAGAACACTGAA
CCAGT CTCTGATGGTTCTTACTTCATGTACAGCAAGCTGAGAGTGGAA
AAGAAGAACTGGGTGGAAAGAAATAGCTACTCCTGCTCAGTGGTCCACGAGGG
TCTGCACAATCACCACACGACTAAGAGCTTCTCCCGGACTCCGGGTAAAGGCG
GTGGCGGCGGAGGCGGTGGCGGTGGGCCTGGGAAAGAGCTCCCCGTGGTGT
GGGCCCAGGAGGGAGCTCCCGTCCATCTTCCCTGCAGCCTCAAATCCCCC AAC
CTGGATCCTAACTTTCTACGAAGAGGAGGGGTTATCTGGCAACATCAACCAGAC
AGTGGCCAACCCACTCCCATCCCGGCCCTTGACCTTCACCAGGGGATGCCCTC
GCCTAGACAACCCGCACCCGGTCGCTACACGGTGCTGAGCGTGGCTCCAGGA
GGCCTGCGCAGCGGGAGGCAGCCCCTGCATCCCCACGTGCAGCTGGAGGAGC
GCGGCCTCCAGCGCGGGGACTTCTCTCTGTGGTTGCGCCCAG CTCTGCGCAC
CGATGCGGGCGAGTACCACGCCACCGTGCGCCTCCCGAACCGCGCCCTCTCC
TGCAGTCTCCGCCTGCGCGTCGGCCAGGCCTCGATGATTGCTAGTCCCTCAGG
AGTCCTCAAGCTGTCTGATTGGGTCCTTTTGAACTGCTCCTTCAGCCGTCCTGA
CCGCCCAGTCTCTGTGCACTGGTTCCAGGGCCAGAACCGAGTGCCTGTCTACA
ACTCACCGCGTCATTTTTTAGCTGAAACTTTCCT GTTACTGCCCCAAGTCAGCCC
CCTGGACTCTGGGACCTGGGGCTGTGTCCTCACCTACAGAGATGGCTTCAATG
TCTCCATCACGTACAACCTCAAGGTTCTGGGTCTGGAGCCCGTAGCCCACCATC
ACCATCATCACTGA
SEQ ID NO. 2:
CTLA -4 BsB (Gene1) = mouse CD80w88a(aa1 -235) -IgG2a(aa241 -474) -G9 -Lag -
3(aa25 -260) - H6
ated pro tein sequence of mouse ate construct (Gene1):
MACNCQLMQDTPLLKFPCPRLILLFVLLIRLSQVSSDVDEQLSKSVKDKVLLPCRYN
SPHEDESEDRIYWQKHDKVVLSVIAGKLKVAPEYKNRTLYDNTTYSLIILGLVLSDRG
TYSCVVQKKERGTYEVKHLALVKLSIKADFSTPNITESGNPSADTKRITCFASGGFPK
PRFSWLENGRELPGINTTISQDPESELYTIS SQLDFNTTRNHTIKCLIKYGDAHVSED
FTWEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPMVTCVVVDVSE
DDPDVQISWFVNNVEVLTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKV
NNKALPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVE
WTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHN
HHTTKS KGGGGGGGGGGPGKELPVVWAQEGAPVHLPCSLKSPNLDPNF
LRRGGVIWQHQPDSGQPTPIPALDLHQGMPSPRQPAPGRYTVLSVAPGGLRSGR
QPLHPHVQLEERGLQRGDFSLWLRPALRTDAGEYHATVRLPNRALSCSLRLRVGQ
ASMIASPSGVLKLSDWVLLNCSFSRPDRPVSVHWFQGQNRVPVYNSPRHFLAETF
LLLPQVSPLDSGTWGCVLTYRDGFNVSITYNLKVLGLEPVAHHH HHH
SEQ ID NO. 3:
CTLA -4 BsB ) = mouse CD80w88a(aa1 -235) -G9 -Lag -3(aa25 -260) -
IgG2a(aa241 -474)
Nucleotide sequence of mouse surrogate construct (Gene 2):
ATGGCTTGCAATTGTCAGTTGATGCAGGATACACCACTCCTCAAGTTTCCATGTC
CAAGGCTCATTCTTCTCTTTGTGCTGCTGATTCGTCTTTCACA AGTGTCTTCAGA
TGTTGATGAACAACTGTCCAAGTCAGTGAAAGATAAGGTATTGCTGCCTTGCCG
TTACAACTCTCCTCATGAAGATGAGTCTGAAGACCGAATCTACTGGCAAAAACAT
GACAAAGTGGTGCTGTCTGTCATTGCTGGGAAACTAAAAGTGGCGCCCGAGTAT
AAGAACCGGACTTTATATGACAACACTACCTACTCTCTTATCATCCTGGGCCTGG
TCCTTTCAGACCGGGGCACATACAGC TGTGTCGTTCAAAAGAAGGAAAGAGGAA
CGTATGAAGTTAAACACTTGGCTTTAGTAAAGTTGTCCATCAAAGCTGACTTCTC
TACCCCCAACATAACTGAGTCTGGAAACCCATCTGCAGACACTAAAAGGATTAC
CTGCTTTGCTTCCGGGGGTTTCCCAAAGCCTCGCTTCTCTTGGTTGGAAAATGG
AAGAGAATTACCTGGCATCAATACGACAATTTCCCAGGATCCTGAATCTGAATTG
TACACCATTA GTAGCCAACTAGATTTCAATACGACTCGCAACCACACCATTAAGT
TTAAATATGGAGATGCTCACGTGTCAGAGGACTTCACCTGGGGCGGTG
GCGGCGGAGGCGGTGGCGGTGGGCCTGGGAAAGAGCTCCCCGTGGTGTGGG
CCCAGGAGGGAGCTCCCGTCCATCTTCCCTGCAGCCTCAAATCCCCCAACCTG
GATCCTAACTTTCTACGAAGAGGAGGGGTTATCTGGCAACATCAACCAGACAG T
GGCCAACCCACTCCCATCCCGGCCCTTGACCTTCACCAGGGGATGCCCTCGCC
TAGACAACCCGCACCCGGTCGCTACACGGTGCTGAGCGTGGCTCCAGGAGGC
CTGCGCAGCGGGAGGCAGCCCCTGCATCCCCACGTGCAGCTGGAGGAGCGCG
GCCTCCAGCGCGGGGACTTCTCTCTGTGGTTGCGCCCAGCTCTGCGCACCGAT
GCGGGCGAGTACCACGCCACCGTGCGCCTCCCGAACCGCGCCCTC TCCTGCA
GTCTCCGCCTGCGCGTCGGCCAGGCCTCGATGATTGCTAGTCCCTCAGGAGTC
CTCAAGCTGTCTGATTGGGTCCTTTTGAACTGCTCCTTCAGCCGTCCTGACCGC
TCTGTGCACTGGTTCCAGGGCCAGAACCGAGTGCCTGTCTACAACTC
ACCGCGTCATTTTTTAGCTGAAACTTTCCTGTTACTGCCCCAAGTCAGCCCCCT
GGACTCTGGGACCTGGGGCTGTGTCCTCACCTACA GAGATGGCTTCAATGTCT
CCATCACGTACAACCTCAAGGTTCTGGGTCTGGAGCCCGTAGCCCCCAGAGGG
CCCACAATCAAGCCCTGTCCTCCATGCAAATGCCCAGCACCTAACCTCTTGGGT
GGACCATCCGTCTTCATCTTCCCTCCAAAGATCAAGGATGTACTCATGATCTCC
CTGAGCCCCATGGTCACATGTGTGGTGGTGGATGTGAGCGAGGATGACCCAGA
TGTCCAGATCAGCTGGTTCGTGAA CAACGTGGAAGTACTCACAGCTCAGACACA
TAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCCCCAT
CCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACA
AAGCCCTCCCAGCGCCCATCGAGAGAACCATCTCAAAACCCAAAGGGTCAGTA
AGAGCTCCACAGGTATATGTCTTGCCTCCACCAGAAGAAGAGATGACTAAGAAA
CAGGTCACTCTG ACCTGCATGGTCACAGACTTCATGCCTGAAGACATTTACGTG
GAGTGGACCAACAACGGGAAAACAGAGCTAAACTACAAGAACACTGAACCAGT
CCTGGACTCTGATGGTTCTTACTTCATGTACAGCAAGCTGAGAGTGGAAAAGAA
GAACTGGGTGGAAAGAAATAGCTACTCCTGCTCAGTGGTCCACGAGGGTCTGC
ACAATCACCACACGACTAAGAGCTTCTCCCGGACTCCGGGTAAATGA
SEQ ID NO. 4:
CTLA -4 BsB ) = mouse CD80w88a(aa1 -235) -G9 -Lag 5 -260) -
IgG2a(aa241 -474)
Translated protein sequence of mouse surrogate construct (Gene 2):
MACNCQLMQDTPLLKFPCPRLILLFVLLIRLSQVSSDVDEQLSKSVKDKVLLPCRYN
SPHEDESEDRIYWQKHDKVVLSVIAGKLKVAPEYKNRTLYDNT TYSLIILGLVLSDRG
TYSCVVQKKERGTYEVKHLALVKLSIKADFSTPNITESGNPSADTKRITCFASGGFPK
PRFSWLENGRELPGINTTISQDPESELYTISSQLDFNTTRNHTIKCLIKYGDAHVSED
FTWGGGGGGGGGGPGKELPVVWAQEGAPVHLPCSLKSPNLDPNFLRRGGVIWQ
HQPDSGQPTPIPALDLHQGMPSPRQPAPGRYTVLSVAPGGLRSGRQPLHPHVQLE
ERGLQRGDFSLWLRPAL RTDAGEYHATVRLPNRALSCSLRLRVGQASMIASPSGVL
KLSDWVLLNCSFSRPDRPVSVHWFQGQNRVPVYNSPRHFLAETFLLLPQVSPLDS
GTWGCVLTYRDGFNVSITYNLKVLGLEPVAPRGPTIKPCPPCKCPAPNLLGGPSVFI
DVLMISLSPMVTCVVVDVSEDDPDVQISWFVNNVEVLTAQTQTHREDYNST
LRVVSALPIQHQDWMSGKEFKCKVNNKALPAPIERTISKPKGSVRAPQ VYVLPPPEE
EMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLR
VEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK
SEQ ID NO. 5:
CTLA -4 BsB human construct wildtype nucleotide sequence = (human
CD80(aa1 -234) - G9 -Lag -3(aa27 -262 -IgGla(aa240 -471 )
ATGGGCCACACACGGAGGCAGGGAA CATCACCATCCAAGTGTCCATACCTCAA
TTTCTTTCAGCTCTTGGTGCTGGCTGGTCTTTCTCACTTCTGTTCAGGTGTTATC
CACGTGACCAAGGAAGTGAAAGAAGTGGCAACGCTGTCCTGTGGTCACAATGT
TTCTGTTGAAGAGCTGGCACAAACTCGCATCTACTGGCAAAAGGAGAAGAAAAT
GGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACC
GGACCATCTTTGA TATCACTAATAACCTCTCCATTGTGATCCTGGCTCTGCGCCC
ATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGACGCTTT
CAAGCGGGAACACCTGGCTGAAGTGACGTTATCAGTCAAAGCTGACTTCCCTAC
ACCTAGTATATCTGACTTTGAAATTCCAACTTCTAATATTAGAAGGATAATTTGCT
CAACCTCTGGAGGTTTTCCAGAGCCTCACCTCTCCTGGTTGGAAAATGGA GAAG
AATTAAATGCCATCAACACAACAGTTTCCCAAGATCCTGAAACTGAGCTCTATGC
CAGCAAACTGGATTTCAATATGACAACCAACCACAGCTTCATGTGTCTC
ATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACAACCGGC
GGTGGCGGCGGAGGCGGTGGCGGTTCCGGAGCTGAGGTCCCGGTGGTGTGG
GCCCAGGAGGGGGCTCCTGCCCAGCTCCCCTGCAGCC CCACAATCCCCCTCC
AGGATCTCAGCCTTCTGCGAAGAGCAGGGGTCACTTGGCAGCATCAGCCAGAC
AGTGGCCCGCCCGCTGCCGCCCCCGGCCATCCCCTGGCCCCCGGCCCTCACC
CGGCGGCGCCCTCCTCCTGGGGGCCCAGGCCCCGCCGCTACACGGTGCTGAG
TCCCGGAGGCCTGCGCAGCGGGAGGCTGCCCCTGCAGCCCCGCGT
CCAGCTGGATGAGCGCGGCCGGCAGCGCGGGGA CTTCTCGCTATGGCTGCGC
CCAGCCCGGCGCGCGGACGCCGGCGAGTACCGCGCCGCGGTGCACCTCAGG
GACCGCGCCCTCTCCTGCCGCCTCCGTCTGCGCCTGGGCCAGGCCTCGATGA
CTGCCAGCCCCCCAGGATCTCTCAGAGCCTCCGACTGGGTCATTTTGAACTGCT
CCTTCAGCCGCCCTGACCGCCCAGCCTCTGTGCATTGGTTTCGGAACCGGGGC
CAGGGCCGAGTCCCTGTCCGGGAGTCC CCCCATCACCACTTAGCGGAAAGCTT
CCTCTTCCTGCCCCAAGTCAGCCCCATGGACTCTGGGCCCTGGGGCTGCATCC
TCACCTACAGAGATGGCTTCAACGTCTCCATCATGTATAACCTCACTGTTCTGG
TGGTGCCCCGGGGCTCCGAGCCCAAATCTTGTGACAAAACTCACACA
TGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTT
CCCCCCAAAACCCAAGG ACACCCTCATGATCTCCCGGACCCCTGAGGTCACAT
GCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTAC
GTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGT
ACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGG
CTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCC
CATCGAGAA CTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGT
ACACCCTGCCCCCATCTCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACC
TGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAA
TGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACG
GCTCCTTCTTCCTATACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG
GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACG
CAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
SEQ ID NO. 6:
CTLA -4 BsB human construct wildtype translated protein sequence = (human
CD80(aa1 - 234) -G9 -Lag -3(aa27 -262 -IgGla(aa240 -471 )
MGHTRRQGTSPSKCPYLNFFQLLVLAGLSH FCSGVIHVTKEVKEVATLSCGHNVSV
EELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTY
ECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHL
SWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTF
NWNTTGGGGGGGGGSGAEVPWWAQEGAPAQLPCSPTIPLQDLSLLRRAGVTWQ
HQPD SGPPAAAPGHPLAPGPHPAAPSSWGPRPRRYTVLSVGPGGLRSGRLPLQP
RVQLDERGRQRGDFSLWLRPARRADAGEYRAAVHLRDRALSCRLRLRLGQASMT
ASPPGSLRASDVVVILNCSFSRPDRPASVHWFRNRGQGRVPVRESPHHHLAESFL
FLPQVSPMDSGPWGCILTYRDGFNVSIMYNLTVLGLLVPRGSEPKSCDKTHTCPPC
PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVH
NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO. 7:
CTLA -4 BsB human construct t nucleotide sequence 1 = (huma n
CD80W84A/S190A(aa1 -234) -G9 -Lag -3R316/75E(aa27 -262 -
IgGlaN596/297Q(aa240 -471)
ATGGGCCACACACGGAGGCAGGGAACATCACCATCCAAGTGTCCATACCTCAA
TTTCTTTCAGCTCTTGGTGCTGGCTGGTCTTTCTCACTTCTGTTCAGGTGTTATC
ACCAAGGAAGTGAAAGAAGTGGCAACGCTGTCCTGTGGTCACAATGT
TGAAGAGCTGG CACAAACTCGCATCTACTGGCAAAAGGAGAAGAAAAT
GGTGCTGACTATGATGTCTGGGGACATGAATATAGCCCCCGAGTACAAGAACC
GGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATCCTGGCTCTGCGCCC
ATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGACGCTTT
CAAGCGGGAACACCTGGCTGAAGTGACGTTATCAGTCAAAGCTGACTTCCCTAC
ACC TAGTATATCTGACTTTGAAATTCCAACTTCTAATATTAGAAGGATAATTTGCT
CAACCTCTGGAGGTTTTCCAGAGCCTCACCTCTCCTGGTTGGAAAATGGAGAAG
ATGCCATCAACACAACAGTTGCCCAAGATCCTGAAACTGAGCTCTATGC
TGTTAGCAGCAAACTGGATTTCAATATGACAACCAACCACAGCTTCATGTGTCTC
ATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAAC TGGAATACAACCGGC
GGTGGCGGCGGAGGCGGTGGCGGTTCCGGAGCTGAGGTCCCGGTGGTGTGG
GCCCAGGAGGGGGCTCCTGCCCAGCTCCCCTGCAGCCCCACAATCCCCCTCC
AGGATCTCAGCCTTCTGCGAAGAGCAGGGGTCACTTGGCAGCATCAGCCAGAC
AGTGGCCCGCCCGCTGCCGCCCCCGGCCATCCCCTGGCCCCCGGCCCTCACC
CGGCGGCGCCCTCCTCCTGGGGGCCCAGGCCCG ACACGGTGCTGAG
CGTGGGTCCCGGAGGCCTGCGCAGCGGGAGGCTGCCCCTGCAGCCCCGCGT
CCAGCTGGATGAGCGCGGCCGGCAGCGCGGGGACTTCTCGCTATGGCTGCGC
CGGCGCGCGGACGCCGGCGAGTACCGCGCCGCGGTGCACCTCAGG
GACCGCGCCCTCTCCTGCCGCCTCCGTCTGCGCCTGGGCCAGGCCTCGATGA
CTGCCAGCCCCCCAGGATCTCTCAGAGCCTC CGACTGGGTCATTTTGAACTGCT
CCTTCAGCCGCCCTGACCGCCCAGCCTCTGTGCATTGGTTTCGGAACCGGGGC
CAGGGCCGAGTCCCTGTCCGGGAGTCCCCCCATCACCACTTAGCGGAAAGCTT
CCTCTTCCTGCCCCAAGTCAGCCCCATGGACTCTGGGCCCTGGGGCTGCATCC
TCACCTACAGAGATGGCTTCAACGTCTCCATCATGTATAACCTCACTGTTCTGG
GTCTGCTGGTGCCCCGGGGC CCCAAATCTTGTGACAAAACTCACACA
AGCCCACCGAGCCCAGCACCTGAACTCCTGGGGGGATCCTCAGTCTTCCTCTT
CCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACAT
GCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTAC
GTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGT
ACCAGAGCACGT ACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGG
CTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCC
CATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGT
ACACCCTGCCCCCATCTCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACC
TGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAA
TGG GGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACG
GCTCCTTCTTCCTATACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG
GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACG
CAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
SEQ ID NO. 8:
CTLA -4 BsB human construct variant translated prote in sequence 1 = (human
CD80W84A/S190A(aa1 -234) -G9 -Lag -3R316/75E(aa27 -262 -
IgGlaN596/297Q(aa240 -471)
MGHTRRQGTSPSKCPYLNFFQLLVLAGLSHFCSGVIHVTKEVKEVATLSCGHNVSV
EELAQTRIYWQKEKKMVLTMMSGDMNIAPEYKNRTIFDITNNLSIVILALRPSDEGTY
ECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNI RRIICSTSGGFPEPHL
SWLENGEELNAINTTVAQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTF
NWNTTGGGGGGGGGSGAEVPVVWAQEGAPAQLPCSPTIPLQDLSLLRRAGVTW
GPPAAAPGHPLAPGPHPAAPSSWGPRPERYTVLSVGPGGLRSGRLPLQ
PRVQLDERGRQRGDFSLWLRPARRADAGEYRAAVHLRDRALSCRLRLRLGQASM
TASPPGSLRASDWVILNCSFSRP DRPASVHWFRNRGQGRVPVRESPHHHLAESFL
FLPQVSPMDSGPWGCILTYRDGFNVSIMYNLTVLGLLVPRGSEPKSCDKTHTSPPS
PAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH
NAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO. 9:
CTLA -4 BsB human construct variant nucleotide ce 2 = (human
CD80W84A/S190AS201A(aal -234) -G9 -Lag /75E(aa27 -262 -
IgGlaN596/297Q(aa240 - 471)
ATGGGCCACACACGGAGGCAGGGAACATCACCATCCAAGTGT CCATACCTCAA
TTTCTTTCAGCTCTTGGTGCTGGCTGGTCTTTCTCACTTCTGTTCAGGTGTTATC
CACGTGACCAAGGAAGTGAAAGAAGTGGCAACGCTGTCCTGTGGTCACAATGT
TTCTGTTGAAGAGCTGGCACAAACTCGCATCTACTGGCAAAAGGAGAAGAAAAT
GGTGCTGACTATGATGTCTGGGGACATGAATATAGCCCCCGAGTACAAGAACC
GGACCATCTTTGATATCACTAATAACCTCT CCATTGTGATCCTGGCTCTGCGCCC
ATCTGACGAGGGCACATACGAGTGTGTTGTICTGAAGTATGAAAAAGACGCTTTC
AAGCGGGAACACCTGGCTGAAGTGACGTTATCAGTCAAAGCTGACTTCCCTACA
CCTAGTATATCTGACTTTGAAATTCCAACTTCTAATATTAGAAGGATAATTTGCTC
AACCTCTGGAGGTTTTCCAGAGCCTCACCTCTCCTGGTTGGAAAATGGAGAAGA
ATTAAATGCCAT CAACACAACAGTTGCCCAAGATCCTGAAACTGAGCTCTATGCT
GTTGCCAGCAAACTGGATTTCAATATGACAACCAACCACAGCTTCATGTGTCTCA
TCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACAACCGGCG
GTGGCGGCGGAGGCGGTGGCGGTTCCGGAGCTGAGGTCCCGGTGGTGTGGG
CCCAGGAGGGGGCTCCTGCCCAGCTCCCCTGCAGCCCCACAATCCCCCTCCA
CAGCCTTCTGCGAAGAGCAGGGGTCACTTGGCAGCATCAGCCAGACA
GTGGCCCGCCCGCTGCCGCCCCCGGCCATCCCCTGGCCCCCGGCCCTCACCC
GGCGGCGCCCTCCTCCTGGGGGCCCAGGCCCGAGCGCTACACGGTGCTGAGC
GTGGGTCCCGGAGGCCTGCGCAGCGGGAGGCTGCCCCTGCAGCCCCGCGTC
CAGCTGGATGAGCGCGGCCGGCAGCGCGGGGACTTCTCGCTATGGCTGC GCC
CAGCCCGGCGCGCGGACGCCGGCGAGTACCGCGCCGCGGTGCACCTCAGGG
ACCGCGCCCTCTCCTGCCGCCTCCGTCTGCGCCTGGGCCAGGCCTCGATGACT
GCCAGCCCCCCAGGATCTCTCAGAGCCTCCGACTGGGTCATTTTGAACTGCTC
CTTCAGCCGCCCTGACCGCCCAGCCTCTGTGCATTGGTTTCGGAACCGGGGCC
AGGGCCGAGTCCCTGTCCGGGAGTCCCCCCATCACCACTTAGC CTTC
CTCTTCCTGCCCCAAGTCAGCCCCATGGACTCTGGGCCCTGGGGCTGCATCCT
CACCTACAGAGATGGCTTCAACGTCTCCATCATGTATAACCTCACTGTTCTGGG
TCTGCTGGTGCCCCGGGGCTCCGAGCCCAAATCTTGTGACAAAACTCACACAA
GCCCACCGAGCCCAGCACCTGAACTCCTGGGGGGATCCTCAGTCTTCCTCTTC
CCCCCAAAACCCAAGGACACCCTCATGATCTCC CGGACCCCTGAGGTCACATG
CGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACG
TGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTA
CCAGAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGC
TGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCC
AAAACCATCTCCAAAGCCA AAGGGCAGCCCCGAGAACCACAGGTGTA
CACCCTGCCCCCATCTCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCT
GCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAAT
GGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACG
GCTCCTTCTTCCTATACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG
GGGAACGTCTTCTCATG CTCCGTGATGCATGAGGCTCTGCACAACCACTACACG
CAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
SEQ ID NO. 10:
CTLA -4 BsB human construct t translated protein sequence 2 = (human
CD80W84A/S190AS201A(aa1 -234) -G9 -Lag -3R316/75E(aa27 -262 -
IgG1aN596/297Q(aa240 - 471)
MGHTRRQGTSPSKC PYLNFFQLLVLAGLSHFCSGVIHVTKEVKEVATLSCGHNVSV
EELAQTRIYWQKEKKMVLTMMSGDMNIAPEYKNRTIFDITNNLSIVILALRPSDEGTY
ECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHL
SWLENGEELNAINTTVAQDPETELYAVASKLDFNMTTNHSFMCLIKYGHLRVNQTF
NWNTTGGGGGGGGGSGAEVPVVWAQEGAPAQLPCSPTIPLQ DLSLLRRAGVTW
QHQPDSGPPAAAPGHPLAPGPHPAAPSSWGPRPERYTVLSVGPGGLRSGRLPLQ
ERGRQRGDFSLWLRPARRADAGEYRAAVHLRDRALSCRLRLRLGQASM
TASPPGSLRASDWVILNCSFSRPDRPASVHWFRNRGQGRVPVRESPHHHLAESFL
FLPQVSPMDSGPWGCILTYRDGFNVSIMYNLTVLGLLVPRGSEPKSCDKTHTSPPS
PAPELLGGSSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH
NAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO. 11:
CTLA -4 BsB human construct t nucleotide s equence 3 = (human
CD80E196A/5190A(aa1 -234) -G9 -Lag -3R316/75E(aa27 -262 -
IgG1aN596/297Q(aa240 -471)
ATGGGCCACACACGGAGGCAGGGAACATCACCATCCAAGTGTCCATACCTCAA
TCAGCTCTTGGTGCTGGCTGGTCTTTCTCACTTCTGTTCAGGTGTTATC
CACGTGACCAAGGAAGTGAAAGAAGTGGCAACGCTGTCCTGTGGTCACAATG T
TTCTGTTGAAGAGCTGGCACAAACTCGCATCTACTGGCAAAAGGAGAAGAAAAT
GGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACC
GGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATCCTGGCTCTGCGCCC
ATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGACGCTTT
CAAGCGGGAACACCTGGCTGAAGTGACGTTATCAGTCAA AGCTGACTTCCCTAC
ACCTAGTATATCTGACTTTGAAATTCCAACTTCTAATATTAGAAGGATAATTTGCT
CAACCTCTGGAGGTTTTCCAGAGCCTCACCTCTCCTGGTTGGAAAATGGAGAAG
AATTAAATGCCATCAACACAACAGTTGCCCAAGATCCTGAAACTGCCCTCTATGC
TGTTAGCAGCAAACTGGATTTCAATATGACAACCAACCACAGCTTCATGTGTCTC
ATCAAGTATGGACATTTAAGA CAGACCTTCAACTGGAATACAACCGGC
GGTGGCGGCGGAGGCGGTGGCGGTTCCGGAGCTGAGGTCCCGGTGGTGTGG
GCCCAGGAGGGGGCTCCTGCCCAGCTCCCCTGCAGCCCCACAATCCCCCTCC
AGGATCTCAGCCTTCTGCGAAGAGCAGGGGTCACTTGGCAGCATCAGCCAGAC
AGTGGCCCGCCCGCTGCCGCCCCCGGCCATCCCCTGGCCCCCGGCCCTCACC
CGGCGGCGCCCTCCT CCTGGGGGCCCAGGCCCGAGCGCTACACGGTGCTGAG
CGTGGGTCCCGGAGGCCTGCGCAGCGGGAGGCTGCCCCTGCAGCCCCGCGT
CCAGCTGGATGAGCGCGGCCGGCAGCGCGGGGACTTCTCGCTATGGCTGCGC
CCAGCCCGGCGCGCGGACGCCGGCGAGTACCGCGCCGCGGTGCACCTCAGG
GCCCTCTCCTGCCGCCTCCGTCTGCGCCTGGGCCAGGCCTCGATGA
CTGCCAGCCCCCC AGGATCTCTCAGAGCCTCCGACTGGGTCATTTTGAACTGCT
CCTTCAGCCGCCCTGACCGCCCAGCCTCTGTGCATTGGTTTCGGAACCGGGGC
CAGGGCCGAGTCCCTGTCCGGGAGTCCCCCCATCACCACTTAGCGGAAAGCTT
CCTCTTCCTGCCCCAAGTCAGCCCCATGGACTCTGGGCCCTGGGGCTGCATCC
TCACCTACAGAGATGGCTTCAACGTCTCCATCATGTATAACCTCACTGTTCTGG
GT CTGCTGGTGCCCCGGGGCTCCGAGCCCAAATCTTGTGACAAAACTCACACA
AGCCCACCGAGCCCAGCACCTGAACTCCTGGGGGGATCCTCAGTCTTCCTCTT
CCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACAT
GCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTAC
GTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGG AGCAGT
ACCAGAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGG
CTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCC
CATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGT
ACACCCTGCCCCCATCTCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACC
TGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGT GGAGTGGGAGAGCAA
TGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACG
GCTCCTTCTTCCTATACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG
GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACG
CAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
SEQ ID NO. 12:
CTLA -4 BsB human construct varia nt translated protein sequence 3 = (human
CD80E196A/S190A(aa1 -234) -G9 -Lag /75E(aa27 -262 -IgG1
aN596/297Q(aa240 -471)
MGHTRRQGTSPSKCPYLNFFQLLVLAGLSHFCSGVIHVTKEVKEVATLSCGHNVSV
EELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTY
ECVVLKYEKDAFKREHLAEVTL PTPSISDFEIPTSNIRRIICSTSGGFPEPHL
SWLENGEELNAINTTVAQDPETALYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTF
NWNTTGGGGGGGGGSGAEVPVVWAQEGAPAQLPCSPTIPLQDLSLLRRAGVTW
QHQPDSGPPAAAPGHPLAPGPHPAAPSSWGPRPERYTVLSVGPGGLRSGRLPLQ
PRVQLDERGRQRGDFSLWLRPARRADAGEYRAAVHLRDRALSCRLRLRLGQASM
TA SPPGSLRASDVVVILNCSFSRPDRPASVHWFRNRGQGRVPVRESPHHHLAESF
LFLPQVSPMDSGPWGCILTYRDGFNVSIMYNLTVLGLLVPRGSEPKSCDKTHTSPP
SPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV
HNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPP
SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO. 13:
CTLA -4 BsB human construct variant nucleotide ce 4 = (human
CD80E196A/S190AS201A(aa1 -234) -G9 -Lag -3R316/75E(aa27 -262 -IgG1a
N596/297Q(aa240 - 471)
ATGGGCCACACACGGAGG CAGGGAACATCACCATCCAAGTGTCCATACCTCAA
TCAGCTCTTGGTGCTGGCTGGTCTTTCTCACTTCTGTTCAGGTGTTATC
ACCAAGGAAGTGAAAGAAGTGGCAACGCTGTCCTGTGGTCACAATGT
TTCTGYFGAAGAGCTGGCACAAACTCGCATCTACTGGCAAAAGGAGAAGAAAAT
GGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACC
GGACCA TCTTTGATATCACTAATAACCTCTCCATTGTGATCCTGGCTCTGCGCCC
ATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGACGCTTT
CAAGCGGGAACACCTGGCTGAAGTGACGTTATCAGTCAAAGCTGACTTCCCTAC
ACCTAGTATATCTGACTTTGAAATTCCAACTTCTAATATTAGAAGGATAATTTGCT
CAACCTCTGGAGGTTTTCCAGAGCCTCACCTCTCCTGGTTGGA AAATGGAGAAG
AATTAAATGCCATCAACACAACAGTTGCCCAAGATCCTGAAACTGCCCTCTATGC
TGTTGCCAGCAAACTGGATTTCAATATGACAACCAACCACAGCTTCATGTGTCTC
ATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACAACCGGC
GGTGGCGGCGGAGGCGGTGGCGGTTCCGGAGCTGAGGTCCCGGTGGTGTGG
GCCCAGGAGGGGGCTCCTGCCCAGCTCCCC TGCAGCCCCACAATCCCCCTCC
AGGATCTCAGCCTTCTGCGAAGAGCAGGGGTCACTTGGCAGCATCAGCCAGAC
AGTGGCCCGCCCGCTGCCGCCCCCGGCCATCCCCTGGCCCCCGGCCCTCACC
CGGCGGCGCCCTCCTCCTGGGGGCCCAGGCCCGAGCGCTACACGGTGCTGAG
CGTGGGTCCCGGAGGCCTGCGCAGCGGGAGGCTGCCCCTGCAGCCCCGCGT
CCAGCTGGATGAGCGCGGCCGGCAGC GCGGGGACTTCTCGCTATGGCTGCGC
CGGCGCGCGGACGCCGGCGAGTACCGCGCCGCGGTGCACCTCAGG
GACCGCGCCCTCTCCTGCCGCCTCCGTCTGCGCCTGGGCCAGGCCTCGATGA
CTGCCAGCCCCCCAGGATCTCTCAGAGCCTCCGACTGGGTCATTTTGAACTGCT
CCTTCAGCCGCCCTGACCGCCCAGCCTCTGTGCATTGGTTTCGGAACCGGGGC
CAGGGCCGAGTCCCTGTCCG GGAGTCCCCCCATCACCACTTAGCGGAAAGCTT
CCTCTTCCTGCCCCAAGTCAGCCCCATGGACTCTGGGCCCTGGGGCTGCATCC
TCACCTACAGAGATGGCTTCAACGTCTCCATCATGTATAACCTCACTGTTCTGG
GTCTGCTGGTGCCCCGGGGCTCCGAGCCCAAATCTTGTGACAAAACTCACACA
AGCCCACCGAGCCCAGCACCTGAACTCCTGGGGGGATCCTCAGTCTTCCTCTT
CCCCCCAAAA CCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACAT
GCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTAC
GTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGT
ACCAGAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGG
CTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCC
CA AAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGT
ACACCCTGCCCCCATCTCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACC
TGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAA
TGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACG
TCTTCCTATACAGCAAGCTCACCGTGGACAAGAGCAGGTG GCAGCAG
GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACG
CAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
SEQ ID NO. 14:
CTLA -4 BsB human construct variant translated protein sequence 4 = (human
CD80E196A/S190AS201A(aa1 -234) -G9 -Lag -3R316/75E(aa27 -262 -
IgG1aN596/297Q(a a240 - 471)
MGHTRRQGTSPSKCPYLNFFQLLVLAGLSHFCSGVIHVTKEVKEVATLSCGHNVSV
EELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTY
ECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHL
SWLENGEELNAINTTVAQDPETALYAVASKLDFNMTTNHSFMCLIKYGHLRVNQTF
NWNTTGGGGGGGGGSG AEVPVVWAQEGAPAQLPCSPTIPLQDLSLLRRAGVTIN
QHQPDSGPPAAAPGHPLAPGPHPAAPSSWGPRPERYTVLSVGPGGLRSGRLPLQ
PRVQLDERGRQRGDFSLWLRPARRADAGEYRAAVHLRDRALSCRLRLRLGQASM
TASPPGSLRASDWVILNCSFSRPDRPASVHWFRNRGQGRVPVRESPHHHLAESFL
FLPQVSPMDSGPWGCILTYRDGFNVSIMYNLTVLGLLVPRGSEPKSCDKTHTSPP S
PAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH
NAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO. 15
Human CD80
MGHTRRQGTS PSKCPYLNFF QLLVLAGLSH FCSGVIHVTK EVKEVATLSC
GHNVSVEELA QTRIYWQKEK MSGD MNIWPEYKNR TIFDITNNLS
IVILALRPSD EGTYECVVLK KREH LAEVTLSVKA DFPTPSISDF
EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVS QDPETELYAV
SSKLDFNMTT NHSFMCLIKY GHLRVNQTFN WNTTKQEHFP DN LLPSWAIT
IFVI FAPR NERL RRESVRPV
SEQ ID NO. 16
BsB Δ (CD80wa -Fc) DNA = mouse CD80w88a(aa1 -235) -IgG2a(aa241 -474)
Nucleotide sequence of mouse surrogate construct (BsB Δ; CD80wa -Fc) :
ATGGCTTGCAATTGTCAGTT GATGCAGGATACACCACTCC TCAAGTTTCCATGTC
CAA GGCTCATTCTTCTCTTTGTG CTGCTGATTCGTCTTTCACA AGTGTCTTCAGA
TGTTGATGAACAACTGTCCA AGTCAGTGAAAGATAAGGTA CCTTGCCG
CTCTCCTCATGAAG ATGAGTCTGAAGACCGAATC TACTGGCAAAAACAT
GACAAAGTGGTGCTGTCTGT TGGGAAACTAAAAG TGGCGCCCGAGTAT
AAGAACCGGACTTTATATGA CAACACTACCTACTCTCTTA TCATCCTGGGCCTGG
TCCTTTCAGACCGGGGCACA TACAGCTGTGTCGTTCAAAA GAAGGAAAGAGGAA
CGTATGAAGTTAAACACTTG GCTTTAGTAAAGTTGTCCAT CAAAGCTGACTTCTC
CAACATAACTGAGT CTGGAAACCCATCTGCAGAC ACTAAAAGGATTAC
CTGCTTTGCTTCCGGGGGTT TCCCAAAGCCTCGCTTCTCT TGGTTGGAAAATGG
AAGAGAATTACCTGGCATCA ATACG ACAATTTCCCAGGATCCTGA ATCTGAATTG
TACACCATTAGTAGCCAACT AGATTTCAATACGACTCGCA ACCACACCATTAAGT
GTCTCATTAAATATGGAGAT GCTCACGTGTCAGAGGACTT CACCTGGGAGCCCA
GAGGGCCCACAATCAAGCCC TGTCCTCCATGCAAATGCCC AGCACCTAACCTCT
TGGGTGGACCATCCGTCTTC ATCTTCCCTCCAAAGATCAA GGATGTACTCATGA
TCTCCCTGA GCCCCATGGTCACATGTGTG GTGGTGGATGTGAGCGAGGA TGAC
CCAGATGTCCAGATCAGCTG GTTCGTGAACAACGTGGAAG TACTCACAGCTCAG
ACACAAACCCATAGAGAGGA TTACAACAGTACTCTCCGGG GTGCCCTC
CCCATCCAGCACCAGGACTG GATGAGTGGCAAGGAGTTCA AATGCAAGGTCAA
CAACAAAGCCCTCCCAGCGC CCATCGAGAGAACCATCTCA AAACCCAAAGG GT
GAGCTCCACAGGTA TATGTCTTGCCTCCACCAGA AGAAGAGATGACTA
AGAAACAGGTCACTCTGACC GTCACAGACTTCAT GCCTGAAGACATTT
ACGTGGAGTGGACCAACAAC GGGAAAACAGAGCTAAACTA CAAGAACACTGAA
CCAGTCCTGGACTCTGATGG TTCTTACTTCATGTACAGCA AGCTGAGAGTGGAA
AAGAAGAACTGGGTGGAAAG AAATAGCTACTCCTGCTCA GTGGTCCACGAGGG
TCTGCACAATCACCACACGA CTAAGAGCTTCTCCCGGACT CCGGGTAAAGGCG
GTGGCGGCGGAGGCGGTGGC GGTGGGCCTGGGAAAGAGCT GGGTCTGGAGC
CCGTAGCCCACCATCACCAT CATCACTGA
SEQ ID NO. 17
BsB Δ (CD80wa -Fc) Protein = mouse CD80w88a(aa1 -235) -IgG2a(aa241 -474)
Translated protein sequence of mouse ate construct ( BsB Δ; CD80wa -Fc ):
MACNCQLMQDTPLLKFPCPR LILLFVLLIRLSQVSSDVDE QLSKSVKDKVLLPCRYN
SPHEDESEDRIYWQKHDKVV LSVIAGKLKVAPEYKNRTLY DNTTYSLIILGLVLSDRG
TYSCVVQKKERGTYEVKHLA KADFSTPNITESGN RITCFASGGFPK
PRFSWLENGRELPGINTTIS QDPESELYTISSQLDFNTTR NHTIKCLIKYGDAHVSED
FTWEPRGPTIKPCPPCKCPA PNLLGGPSVFIFPPKIKDVL MISLSPMVTCVVVDVSE
DDPDVQISWFVNNVEVLTAQ TQTHREDYNSTLRVVSALPI QHQDWMSGKEFKCKV
NNKALPAPIERTISKPKGSV RAPQVYVL PPPEEEMTKKQVTLTCMVTD FMPEDIYVE
TELNYKNTEPVLDS DGSYFMYSKLRVEKKNWVER NSYSCSVVHEGLHN
HHTTKSFSRTPGKGGGGGGG GGGPGKELGLEPVAHHHHHH
Other Embodiments
From the foregoing description, it will be nt that variations and modifications
may be made to the inventi on described herein to adopt it to various usages and
conditions. Such embodiments are also within the scope of the following claims.
The tion of a listing of elements in any definition of a variable herein includes
definitions of that variable as a ny single element or combination (or subcombination)
of listed elements. The recitation of an embodiment herein includes that embodiment
as any single embodiment or in combination with any other ments or portions
thereof.
All patents and publicatio ns mentioned in this specification are herein orated by
reference to the same extent as if each independent patent and publication was
specifically and individually indicated to be incorporated by reference.
References
The following documents are ci ted herein.
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Claims (17)
1. A bispecific biologic compr ising a ligand specific for CTLA -4 and a ligand 5 specific for a pMHC complex spaced apart by a linker.
2. The bispecific biologic according to claim 1, where in the ligand specific for CTLA -4 is selected from an antibody specific for CTLA -4, and CD80 (B7 1) or CD86 (B7 2).
3. The bispecific biologic according to claim 2, wherein the ligand specific for the 10 pMHC complex is selected from an anti MHC antibody and LAG 3.
4. The bispecific biologic according to claim 1, n the linker is one or more of a polyamino acid ce and an antibody Fc domain.
5. The bispecific biologic according to claim 4, wherein the polyamino acid sequence is G9 (Gly 9). 15
6. The bispecific biologic according to claim 2, where in the ligand specific for CTLA -4 is CD80.
7. The bispecific biologic acco rding to claim 6, wherein CD80 is mutated t o increase specificity for CTLA -4.
8. The ific biologic according to claim 7, wherein CD80 is human CD80 20 comprising at least one of mutations W84A, K71G, K71V, S109G, R123S, R123D, G124L, S190A, S201A, R63A, M8 1A, N97A and E196A .
9. The bispecific biologic according to claim 8, wherein CD80 comprises the mutation W84A or E196A of human CD80.
10. The bispecific biologic according to claim 3, n the ligand specific for the 25 MHC x is LAG 3.
11. The bispecific biolo gic according to claim 10, wherein LAG 3 is mutated to se specificity for pMHCII.
12. The bispecific biologic according to claim 11, wherein LAG 3 is human LAG 3 sing at least one of mutations R73E, R75A, R75E and R76E .
13. The bispecific biologic acc ording to claim 12, wherein LAG 3 comprises the mutation R75A or R75E.
14. The use of a T-cell contacting with an n nting cell , which is presenting a peptide derived from an n complexed to a MHC molecule and 5 a bispecific biologic, according to any one of claims 1 -13 for the manufacture of a medicament of tolerising said T -cell to said antigen.
15. The use of a bispecific biologic comprising a ligand specific for CTLA -4 and a ligand specific for a pMHC complex according to any one of claims 1 -13, fo r the preparation of a medicament for the treatment of an autoimmune disease or 10 lant rejection.
16. The use according to claim 15, wherein the bispecific ic is in combination with a further immune ssant or modulator.
17. The use according to clai m 15 or 16, wherein the autoimmune disease is selected from type 1 diabetes (T1D), Systemic Lupus Erythematosus ( SLE), Rheumatoid 15 Arthritis (RA) , inflammatory bowel disease (IBD), ulcerative colitis (UC), s disease (CD), multiple sclerosis (MS), scle roderma, pemphigus vulgaris (PV ), psoriasis, atopic itis, celiac disease, Chronic Obstructive Lung disease, Hashimoto’s thyroiditis, Graves’ disease (thyroid). Sjogren’s syndrome, Guillain -Barre syndrome, Goodpasture’s syndrome, Addison’s disease, 20 r’s granulomatosis, primary biliary sclerosis, sclerosing gitis, autoimmune hepatitis, polymyalgia rheumatica, Raynaud’s phenomenon, temporal arteritis, giant cell arteritis, autoimmune hemolytic anemia, pernicious anemia, polyarteritis nodosa. Behcet’s disease, primary bilary sis, uveitis, myocarditis, rheumatic fever, ankylosing spondylitis, glomerulenephritis, 25 sarcoidosis, dermatomyositis, myasthenia gravis, polymyositis, alopecia areata, and vitiligo. B5B BSBA Inn-- u unu- pMHC Ifigure I
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