NZ619473B2 - Inhibitors of t-cell activation - Google Patents

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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Anderson, B.E., J.M. McNiff, C. Matte, I. At hanasiadis, W.D. Shlomchik, and M.J.

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Claims (17)

Claims 1.

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