NZ717726B2 - Anti-??tcr antibody - Google Patents

Abstract

Disclosed is a humanised multispecific antibody comprising a first binding domain specific for the human ??TCR/CD3 complex and a second binding domain specific for a tumor-specific antigen, wherein the first binding domain comprises a heavy chain variable region having an amino acid sequence selected from the group consisting of the sequences set forth in SEQ ID NOs: 7, 12, 13, 15 and 16, and a light chain variable region comprising the amino acid sequence set forth as SEQ ID NO: 14. d from the group consisting of the sequences set forth in SEQ ID NOs: 7, 12, 13, 15 and 16, and a light chain variable region comprising the amino acid sequence set forth as SEQ ID NO: 14.

Description

Anti -αβαβαβαβ TCR antibody The present invention relates to an antibody specific for the alpha beta T cell receptor (αβ TCR). In particular, the invention relates to a humanized anti -αβ TCR antibody, which is derived from the murine monoclonal antibody BMA031, and the use of said zed antibody in immunosuppressive therapy.

Introduction The use of immunosuppressive agents in autoimmune diseases and organ transplant therapy is well documented; however the s is far from optimal. Toxicity, opportunistic inf ections, cytokine storm and increased risk of cancer are prevalent in patients treated with these agents. The use of biologics in this arena has improved patient outcome to some degree yet these side effects remain evident.

The use of polyclonal antisera a gainst lymphocytes is well known for the purpose of immunosuppression. However, antisera are labor -intensive to produce, show ties which vary n batches, and the specificity which can be ed using polyclonal antisera is d.

Monoclonal antibody production by hybridoma technology was first described by Köhler and Milstein ( Nature 256:495 -497 (1975)). As compared to polyclonal antisera, monoclonal antibodies (mAbs) are more specific, and have more consistent properties. mAbs have been mo st frequently and successfully used for immunosuppressive therapy in clinical organ transplantation. r, most mAbs used as suppressive agents for ng mune diseases and in transplant patients have a broad suppressive capacity, th us undesirably influencing functions of a wide um of immune cells, presumably not all involved in graft rejection.

Mouse monoclonal antibodies against T cell surface receptor antigens were first produced in 1979 using hybridoma technology (Kung et a l. (1979) Science 206:347 -349). Of the three monoclonal antibodies discovered by Kung et al ., one antibody designated muromonab -CD3 (OKT3) had defined specificity to the CD3 receptor of the T cell, reacting with more that 95% of peripheral mature T cells w ithout affecting immature thymocytes.

Binding of OKT3 to the CD3 complex causes internalization of the CD3 receptor and loss of CD3 positive cells from the periphery. Successful OKT3 treatment is associated with a prompt decline in CD3 positive T cells fr om approximately 60% to less than 5%.

OKT3 has been extensively used for the treatment of ts undergoing acute allograft rejection after kidney lantation (Russell, P.S., Colvin, R.B., Cosimi, A.B. (1984) Annu. Rev. Med . 35:63 and , A.B., Burton, R.C., Colvin, R.B. et al . (1981) Transplantation 32:535). Moreover, OKT3 and rabbit complement were used for purging mature T cells from donor marrow to prevent acute graft versus host disease (GVHD) in allogeneic bone marrow transplantation (Pre ntice, H.G., Blacklock, H.A., Janossy, G. et al . (1982) Lancet 1:700 and ock, H.A., Prentice, H.G., Gilmore, M.J. et al . (1983) Exp.

Hematol . . Whereas OKT3 treatment seems to be ive in the prevention of GVHD in allogeneic bone marrow tra tation for acute leukemia, a combined in vitro /in vivo treatment with OKT3 failed to prevent GVHD during therapy for severe combined immunodeficiency (Hayward, A.R. et al . (1982) Clin. Lab. Observ . 100:665).

Treatment of T cells with OKT3 elicits sev eral responses inconsistent with immune suppression including T cell activation, production of immune mediators and T3 - modulation. The T3 en complex recognized by CD3 -mAbs ( e.g ., OKT3) is postulated to play a crucial role during T cell activation. Alp ha/beta T cytes recognize peptide – MHC ligands by means of a multimeric protein ensemble termed the αβ T cell antigen receptor (TCR)·CD3 complex. This structure is composed of a variable αβ TCR dimer which binds antigens and three invariant dimers (CD3 γε , δε and ζζ ) which are involved in TCR·CD3 surface transport, stabilization and signal transduction. The alpha beta T cell receptor ( αβ TCR) is expressed on the majority (approx. 95%) of T cells and has a critical role in T cell activation via engagement of antigen displayed on MHC. The remaining 5% of ce lls are gamma delta T cell receptor ( γδ TCR) ve. The γδ TCR positive cell population plays an important role in the innate immune response in defense against opportunistic infections of bacterial, viral and fungal origin. Gamma delta T cells do not play a role in graft rejection in transplantation. Therefore, targeting the αβ TCR positive cell population and sparing the γδ TCR positive population should allow for significant therapeutic efficacy whilst maintaining a baseline immune protection against o pportunistic infections.

The mouse IgG2b monoclonal antibody BMA031 (Borst et al . (Nov. 1990) Hum. Immunol. 175 -88; EP0403156) is specific for the common determinant on the TCR beta/CD3 complex, and does not bind to the gamma -delta TCR. BMA03 1 is highly immunosuppressive and is capable of inducing apoptosis of activated T cells via a mechanism of activation -induced cell death (AICD) (Wesselborg et al. (May 1993) J.

Immunol. ): 4338 -4345). In vitro it ts a mixed lymphocyte reaction and it has shown preliminary clinical efficacy in prevention of graft rejection in a number of solid organ lant ios as well as the treatment of acute graft versus host disease ) (Kurrle et al. (Feb 1989) Transplant Proc. 21(1): 1017 -1019). BMA031 does not engage human Fc gamma receptors (Fc γR) in the ty of the human population (ap proximately 10% of human possess Fc γRs which do bind to mouse IgG2b isotype). As such the antibody does not cause T cell activation via cross -linking of the T cell receptor and, therefore, it does not induce T cell activation or the associated cytokine rel ease. In this regard its profile is highly preferable over that of OKT3. However, BMA031 is a murine antibody and, as such, is not suitable for repeat dosing in human subjects in view of the human anti -mouse antibody (HAMA) response elicited therein .

Seve ral humanized versions of BMA031 have been described ( see, EP 6; also an et al ., (1991) J. Immunol . 147:4366 -4373). As noted in EP0403156, mere CDR grafting was not sful in retaining antigen binding. One clone with significant framework cations, EUCIV3, successfully bound to T cells; however, as noted in EP0403156, binding to the αβ TCR is not as effective as the parent BMA031 antibody as determined by flow cytometry competition assays. We have also shown that the ability of EuCIV3 to inhibit an in vitro immune response is significantly reduced as compared to BMA031 ( see, Fig. 2). In addition EuCIV3 was ally generated on a wild -type human IgG1 or IgG4 backbone which still retains Fc γR binding. These humanized antibodies there fore allowed for T cell activation, proliferation and the concomitant cytokine release and as such were significantly different to the original properties of BMA031.

The modification of antibody glycosylation is known in the art. For example, it is known that aglycosylated antibodies can have extensively modified functionality; see, Boyd et al . (1996) Mol. Immunol . 32:1311 -1318. However, aglycosylated forms of humanized BMA031, or derivatives with modified glycosylation patterns, have previously not been described.

There is a need in the art, therefore, for an anti -αβ TCR zed dy which improves on the binding properties of EUCIV3 and advantageously retains the immunosuppressive and non -T cell -activatory properties of BMA031.

Summary of the Invention In one aspect of the invention, there is provided a humani zed multispecific antibody comprising a first binding domain specific for the human αβ TCR/CD3 complex and a second binding domain ic for a tumor -specific antigen, wherein the first binding domain comprises a heavy chain variable region having an amin o acid ce selected from the group consisting of the sequences set forth in SEQ ID NOs: 7, 12, 13, 15 and 16, and a light chain variable region comprising the amino acid sequence set forth as SEQ ID NO: 14 . es of antibodies according to the firs t aspect include antibodies which comprise a heavy chain variable region selected from the heavy chains comprising the sequences set forth in SEQ ID NO: 7, SEQ ID NO: 12 and SEQ ID NO: 13 . es of humanized antibodies according to the first aspect incl ude antibodies which comprise a heavy chain variable region selected from the heavy chains comprising the sequences set forth in SEQ ID NO: 15 and SEQ ID NO: 16 .

In the sequence listing, CDRs are indicated by means of annotation or underlining.

Frameworks are all sequences outside of the CDRs, which are defined according to the "Kabat" numbering system and extended, where able, by use of “IMGT” CDR definition. If a framework residue is not indicated to be changed to match a donor sequence, it will o rdinarily be understood to be an acceptor residue.

The humanized antibodies may se a constant region. In one embodiment, the constant region is of human origin.

The humanized antibodies of the ion may be further modified by Fc engineering.

Imm unoglobulins are liable to cross -link Fc γ receptors, which can lead to constitutive T cell activation for anti -T cell antibodies. In order to avoid Fc γ cross -linking, antibodies can be modified to remove the Fc , such as by the generation of Fab or Fv fragments; however, truncated immunog lobulins lack beneficial effector functions and exhibit a lower serum half -life. ore, the Fc region of the humanized antibody can be modified to prevent Fc γ cross -linking. Exemplary techniques include generation of aglycosylated immunoglobulins, fo r instance by modification of the Fc region by an N297Q on.

Immunoglobulins which fail to bind Fc γ are also described by Armour et al ., (1999) Eur. J.

Immunol . 29:2613 -2624. The modification effected to IgG1 is known as the ∆ab modification, and con sists in a combination of the ∆a mutation, in which IgG residues are substituted at ons 327, 330 and 331, and IgG2 residues substituted at positions 233 -236, and the ∆b on, in which residue 236 is deleted. In another embodiment, the ylati on pattern of antibodies according to the invention can be modified.

In r aspect of the invention, there is provided a nucleic acid encoding at least a heavy chain variable region of a humanized monoclonal antibody according to the ing aspects of the described embodiments. The nucleic acid may encode variable and constant regions of the humanized antibody. Heavy and light chains may be encoded on separate nucleic acids or on the same nucleic acid molecule.

According to yet another aspect of th e invention , there is provided a cell which expresses a nucleic acid according to the preceding aspect. The cell is, for example, a cell adapted to express antibody molecules in e. The nucleic acid may include signal sequences and/or other ce s or modifications which are required for, or which modulate, expression of the dy le in the cell, and/or secretion of the antibody molecule from the cell.

In one embodiment, a humanized antibody is provided as described in the foregoing aspec ts, for use in suppressing a T cell mediated se in a subject.

For example, the T cell mediated response can be involved in a condition selected from tissue transplantation, including solid organ transplant and composite tissue transplant, tissue graf ting, multiple sclerosis and type 1 diabetes.

Moreover, another embodiment es a method for treating a subject suffering from a condition ing an aberrant T cell mediated response comprising stering to a subject in need thereof a pharmaceut ically effective dose of an antibody according to the described embodiments.

Humanized non -activatory anti -αβ TCR antibodies which do not induce cytokine release have thus been generated which are capable of selective modulation of the αβ TCR and of inducin g apoptosis of activated αβ TCR positive T cells. These antibodies have been generated for use as immunosuppressive agents in T cell ed diseases. These antibodies have been generated h humanization of a mouse anti -αβ TCR antibody BMA031 and by F c-engineering of the humanized antibodies to prevent ment of Fc gamma receptors. The antibodies according to the described embodiments retain the binding affinity of BMA031, unlike the humanized versions of BMA031 available in the art.

Further, as sh own in in vitro ion assays, the immunosuppressive properties of antibodies according to the described embodiments are or to those of BMA031. er, unlike the zed BMA031 antibodies of the prior art, the antibodies according to the desc ribed embodiments do not induce cytokine release in normal PBMC.

The multispecific antibody can comprise many different conformations; in one embodiment, it comprises an anti -TCR/CD3 scFv and an anti -tumor scFv.

In one embodiment, the multispecific antibod y is bispecific.

Brief Description of the Figures Fig. 1. BMA031 binds more strongly to αβαβαβαβ TCR compared to EuCIV3.

Competition of binding of PE -labeled BMA031 antibody by BMA031 MoIgG2b, BMA031 HuIgG1 and EuCIV3 HuIgG1 antibodies. EuCIV3 has a decreased potency compared to BMA031.

Fig. 2. EuCIV3 is less potent than BMA031 in an in vitro educ ation (IVE) assay.

Plot showing loss of performance of EuCIV3 humanized dy in ical assay when compared to parent BMA031 antibody. CD8+ T cells were treated with anti -αβ TCR antibodies at various concentrations (x -axis) and co -cultured with auto logous dendritic cells pulsed with the CMV peptide 495 -503 (pp65) for seven days.

Fig. 3. HEBE1 binds αβαβαβαβ TCR comparably to BMA031 in a competition assay.

Competition of binding of PE -labeled BMA031 antibody by BMA031 HuIgG1, HEBE1 HuIgG1 and EuCIV3 HuIgG1 dies. EuCIV3 has a decreased potency ed to BMA031 and HEBE1.

Fig. 4. HEBE1 has similar potency to EuCIV3 in an in vitro education (IVE) assay.

The IVE assay was performed as described in respect of Fig. 2.

[THE REMAINING LINES ON PAGE 6 HAVE BEEN LEFT BLANK INTENTIONALLY].

[PAGE 7 HAS BEEN LEFT BLANK IONALLY].

[THE NEXT SEQUENTIAL PAGE NUMBER IN THE SPECIFICATION IS PAGE 8].

Fig. 5. Schematic showing iterative rounds of mutagenesis of anti -αβαβαβαβ TCR variable domains.

Framework in shaded box depicts the FR region where certain mouse residues lie.

Shaded residues in first row of ons are the mouse amino acids that are useful to maintain off -rate. Shaded residues in second row of mutations are the mouse residues surrounding the CDR regions which were retained during the final ning process.

Fig. 6. Optimized humanized antibody has improved off -rate compared to .

The kinetics of antibody dissociation from αβ TCR on T cells was measured by flow cytometry. BMA031 had a better off -rate compared to EuCIV3 and HEBE1. By optimizing the binding domain of HEBE1 we were able to improve the off -rate of HEBE1.H10 compared to BMA031.

Fig. 7. zed humanized antibody has improved off -rate compared to BMA031.

The kinetics of antibody dissociation from αβ TCR on T cells was measured by flow cytometry. By optimizing the binding domain of HEBE1 we were able to e the off - rate of HEBE1.H66 compared to BMA031.

Fig. 8. Optimized humanized antibody has improved off -rate compared to BMA031 in both ∆∆∆∆ab and aglycosylated formats.

The kinetics of dy dissociation from αβ TCR on T cells was measured by flow cytometry.

Fig. 9. Optimization of HEBE1 leads to equivalent functionality as BMA031.

IVE assay as described in Fig. 4. BMA031 inhibited education of CD8+ T cells, as they were unable to lyse specific targets in a dose ent manner. The parental humanized antibody, HEBE1, was not as potent as BMA031 and was able to only inhibit education at the t dose ar results observed with a secon d non -improved humanized Ab, HEBE1 H13). Further improvements were made to the humanized antibody, HEBE1 H10, which had equivalent potency to BMA031 in this assay.

Fig. 10. IVE data with anti -αβ TCR antibodies.

Both HEBE1 and GL1 BM series antibodies showed improvements in IVE results in comparison with BMA031.

Fig. 11. Antigen positive cells from IVE assay as determined by antigen -specific tetramer binding.

Cells which are antigen -positive ( i.e ., have been educated within IVE assay) are able to bind to an MHC -tetramer molecule. When the IVE assay was conducted in the presence of antibody which has been able to prevent the education of T cells to antigen, there wer e fewer cells able to bind to the MHC -tetramer at the end of the assay.

Fig. 12. Proliferation of PBMCs in presence of anti -αβαβαβαβ TCR dies, OKT3 and atory beads.

The stimulatory activity of OKT3 was not seen in anti -αβ TCR antibodies in this comparison.

Fig. 13. Cytokine release from PBMCs in presence of anti βαβ TCR antibodies.

Cytokine release profile of anti -αβ TCR anti bodies was similar to the profile demonstrated by BMA031.

Fig. 14. IFN -gamma e from T cells in IVE assay.

CD8+ T cells were treated with anti -αβ TCR antibodies at various concentrations ( see Fig. 2, x -axis) and co -cultured with autologous dendritic c ells pulsed with the CMV peptide 495 -503 (pp65) for seven days in an in vitro education (IVE) assay. IFN -gamma release was measured in this assay.

Fig. 15. Activation -induced apoptosis by anti -αβαβαβαβ TCR antibodies.

Antigen stimulated CD8+ T cells were induc ed to apoptosis by binding of anti -αβ TCR antibodies BMA031 and HEBE1 H66. The ability of HEBE1 H66 to induce apoptosis was increased compared to BMA031.

Fig. 16. ion of glycosylation mutants and aglycosylated dies Coomassie -blue stained gel showing expression and purification of glycosylation mutants Fig. 17. g of αβ TCR antibody mutants to human Fc γRIIIa using Biacore. e was used to assess binding to recombinant human Fc γRIIIa (V158 & F158).

Fig 18. Binding of αβ TCR antibody mut ants to human Fc γRI using Biacore. e was used to assess binding to recombinant human and Fc γRI.

Fig. 19 . Cytokine release from PBMCs in presence of glycosylation mutant anti - αβαβ TCR antibodies (day 2) .

Cytokine release profile for TNFa, GM -CSF, IFNy a nd IL10 of anti -αβ TCR antibodies was similar to the profile demonstrated by BMA031 and H66 delta AB .

Fig. 20 . Cytokine release from PBMCs in presence of glycosylation mutant anti - αβαβ TCR antibodies (day 2) .

Cytokine release profile for IL6, IL4 and IL2 of a nti -αβ TCR antibodies was similar to the profile demonstrated by BMA031 and H66 delta AB .

Fig. 21 . Cytokine release from PBMCs in presence of glycosylation mutant anti - αβαβ TCR antibodies (day 4) .

Cytokine release profile for TNFa, GM -CSF, IFNy and IL10 of an ti -αβ TCR antibodies was similar to the profile demonstrated by BMA031 and H66 delta AB .

Fig. 22 . ne release from PBMCs in presence of glycosylation mutant anti - αβαβ TCR antibodies (day 4) .

Cytokine release profile for IL6, IL4 and IL2 of anti -αβ TCR ant ibodies was similar to the profile demonstrated by BMA031 and H66 delta AB .

Fig. 23: Binding profiles of TRACERS .

Binding profiles of bi -specific antibodies to both tumor target cells and human T cells assessed by flow cytometery.

Fig. 24: Cytotoxic activ ity of ent T cell recruitment arms .

A panel of humanised BMA031 antibodies have been d and from this panel a number of antibodies have been selected which y cytotoxic activity against tumor antigen expressing cell lines Fig. 25 : Cytokine release profile of ent T cell tment arms.

A panel of TRACERs with different T cell recruitment arms show similar cytokine release profiles. Large amounts of nes are detected following activation of T cells in the presence of target cell s whereas in the presence of only unstimulated human PBMC observed cytokine levels are significantly lower.

Fig. 26: Binding of CD52 antibody mutants to human Fc γRIIIa using Biacore.

Biacore was used to assess binding of ed anti -CD52 to recombinant human Fc γRIIIa (V158). Anti -CD52 comprising S298N/Y300S ons in the Fc domain were used to assess the effector function of the modified molecule. A: binding to CD52 peptide.

B: g to Fc γRIIIa (V158). C: control binding to mouse FcRn.

Detailed Description of the Invention Unless otherwise stated, all technical and scientific terms used herein have the same meanings as commonl y understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein can be used in the methods or techniques of the present invention. All publications cited herein ar e incorporated herein by reference in their entirety for the purpose of describing and disclosing the methodologies, reagents, and tools reported in the publications that might be used in connection with the invention.

The methods and techniques of the p resent application are generally performed according to conventional methods well known in the art and as described in various general and more ic references that are cited and discussed hout the present specification unless otherwise indicated . See , e.g ., Gennaro, A.R., ed. (1990) Remington's ceutical 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 Press, 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 g: A Laboratory Manual , 2 nd n, Vols. I -III, Cold Spring Harbor Laboratory Press; Ausubel, F.M. et al ., eds. (1999) Short Protocols in lar Biology , 4th edition, John Wiley & Sons; Ream et al. , eds. (1998) Molecular Biology Techniques: An Intensive tory Course , Aca demic Press; Newton, C.R. and Graham, A., eds. (1997) PCR (Introduction to Biotechniques Series) , 2nd ed., Springer -Verlag.

A humanized monoclonal antibody, as referred to , is an antibody which is composed of a human antibody framework, into which have been grafted complementarity determining s (CDRs) from a non -human antibody. Changes in the human acceptor framework may also be made. Procedures for the design and production of humanized antibodies are well known in the art, and have been des cribed, for example, in Cabilly et al., U.S. Patent No. 4,816,567; Cabilly et aI., 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; Neube rger, M.S. et al., European Patent Application 0 194 276 B1; 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 dies, humanized antibodies, human engineered dies, and methods for their ation can be found in Kontermann, R. and Dübel, S. eds. (2001, 2010) Antibody Engineering , 2nd ed., Springer -Verlag, New York, NY.

The term "antibody", unless indicated otherwise, is used to refer t o entire antibodies as well as antigen -binding fragments of such antibodies. For example, the term encompasses four -chain IgG molecules, as well as antibody fragments.

As used herein, the term “antibody fragments” refers to portions of an intact full -leng th antibody, 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. ent classes and sses of immunoglobulin have different ties, which may be advantage ous in different applications. For example, IgG4 antibodies have reduced binding to Fc receptors.

Specificity, in the context of the antibodies described herein, means that the claimed antibody be capable of selectively binding its d cognate antigen , which is the αβ TCR .CD3 complex. The antibodies of the ion bind the αβ TCR.CD3 complex expressed on cells .

The human αβ TCR/CD3 complex is the T cell receptor complex presented on the surface of T cells. See, Kuhns et al ., (2006) ty 24:133 –13 9. This complex is ed by the murine monoclonal antibody BMA031 ( see, European patent application EP 0 403 156; SEQ ID NOs: 1 and 2).

Naturally occurring immunoglobulins have a common core structure in which two cal light chains (about 24 kD) a nd two identical heavy chains (about 55 or 70 kD) form a tetramer. The amino -terminal portion of each chain is known as the variable (V) region and can be distinguished from the more conserved constant (C) regions 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 aci d sequence variation in globulins is confined to three separate locations in the V regions known as hypervariable regions or complementarity determining regions (CDRs) which are directly involved in antigen binding. ding from the amino - terminus , these regions are ated CDR1, CDR2 and CDR3, respectively. The CDRs are held in place by more conserved ork regions (FRs). Proceeding from the amino -terminus, these regions are ated FR1, FR2, FR3 and FR4, respectively. The locations of C DR and FR regions and a numbering system have been defined by Kabat et al. (Kabat, E.A. et al. , Sequences of Proteins of Immunological Interest , Fifth Edition, U.S.

Department of Health and Human Services, U.S. Government Printing Office (1991), and update s thereof which may be found online). In addition, CDR region boundaries have been further defined by IMGT nomenclature. le regions of antibodies according to the described embodiments may be found in SEQ ID NOs: 5 -7 and 12 -16, and may be ed b y humanizing BMA031, that is, by transferring the CDRs of BMA031 to a human framework. Two series of humanized antibodies are bed; the HEBE1 series, comprising SEQ ID NOs: 5 -7, 12 and 13, and the GL1BM series, comprising heavy chain variable regions as shown in SEQ ID NOs: 8, and 16. In both cases, the light chain variable region used is as shown in SEQ ID NO: 14 (GL1BM VK43).

The human frameworks used are IGH3 -23 in the case of HEBE1, and IGHV1 -3*01 and IGKV3 -11*01 in the case of GL1BM.

Consta nt s may be derived from any human antibody constant regions. Variable region genes may be cloned into expression vectors in frame with constant region genes to express heavy and light immunoglobulin chains. Such sion vectors can be transfecte d into antibody producing host cells for antibody synthesis.

Human antibody variable and constant regions may be derived from sequence databases.

For e, immunoglobulin sequences are available in the IMGT/LIGM database (Giudicelli et al ., (2006) Nu cleic Acids Res. 34 l. 1):D781 -D784) or VBase (vbase.mrc -cpe.cam.ac.uk).

Aglycosylated dies can have extensively modified functionality; see, Boyd et al . (1996) Mol. Immunol . 32:1311 -1318. A "delta ab" or ∆ab modification, referred to herein, is an Fc modification as described in Armour et al ., (1999) Eur. J. Immunol . 29:2613 -2624.

Techniques for modifying glycosylation of antibody Fc regions are known in the art, and e chemical, enzymatic and mutatio nal means, for example, mutation of the N297 on in the CH 2 domain. Techniques for mutating antibody genes for producing aglycosylated IgG molecules are described in Tao and Morrison (1989) J. l . 143:2595 -2601.

"Nucleic acids" as referred to he rein include DNA molecules which encode the antibodies of the invention. Preferred are expression vectors, which are le for expressing the antibody genes in a host cell. Expression s and host cells for antibody gene expression are known in th e art; see , for example , Morrow, K.J. Genetic Engineering & Biotechnology News (June 15, 2008) 28(12), and Backliwal, G. et al . (2008) Nucleic Acids Res. :e96 -e96. 1. Antibodies The invention encompasses antigen -binding fragments of the humanized a nti -αβ TCR antibodies. Fragments of the antibodies are capable of g the αβ TCR .CD3 complex.

They encompass Fab, Fab’, F(ab’) 2, and F(v) fragments, or the individual light or heavy chain variable regions or portion f. Fragments include, for example, Fab, Fab', F(ab') 2, Fv and scFv. These fragments lack the Fc portion of an intact antibody, clear more rapidly from the circulation, and can have less non -specific tissue binding than an intact antibody. These fragments can be produced from intact antibod ies 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).

The antibodies and fragments also encompass single -chain antibody fragments (scFv) that bin d to the αβ TCR .CD3 x. An scFv comprises an antibody heavy chain variable region (V H) operably linked to an antibody light chain le region (V L) wherein the heavy chain variable region and the light chain variable , together or individuall y, form a binding site that binds αβ TCR . An scFv may comprise a V H region at the amino - terminal end and a V L region at the y -terminal end. Alternatively, scFv may comprise a V L region at the amino -terminal end and a V H region at the carboxy -termina l end. Furthermore, although the two domains of the Fv fragment, V L and V H, 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 monovalent molecules (known as single chain Fv (scFv)). An scFv may optionally further comprise a polypeptide linker between the heavy chain variable region and the light chain variable region.

The antibodies and fragments also enc ompass domain antibody (dAb) fragments as described in Ward, E.S. et al . (1989) Nature 341:544 -546 which consist of a V H domain.

The antibodies and fragments also encompass heavy chain antibodies (HCAb). These antibodies are reported to form antigen -bi nding regions using only heavy chain variable , in that these functional antibodies are dimers of heavy chains only (referred to as "heavy -chain antibodies" or "HCAbs"). Accordingly, antibodies and fragments may be heavy chain antibodies (HCAb) that specifically bind to the αβ TCR .CD3 complex.

The antibodies and fragments also ass antibodies that are SMIPs or g domain immunoglobulin fusion proteins specific for αβ TCR .CD3 complex. These constructs are single -chain polypeptides comprisin g antigen -binding domains fused to immunoglobulin domains necessary to carry out antibody effector ons ( see , WO 17148).

The antibodies and fragments also ass diabodies. These are bivalent antibodies in which V H and V L s are expre ssed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain.

This forces the domains to pair with complementary domains of another chain and thereby creates two n -binding si tes ( see, for example , WO 93/11161). Diabodies can be bispecific or monospecific.

The antibody or antibody fragment thereof does not cross -react with any target other than the αβ TCR .CD3 complex.

The antibody or fragment thereof may be modified in orde r to increase its serum half -life, for example, by adding molecules - such as PEG or other water soluble rs, including polysaccharide polymers to increase the half -life.

The antibodies and fragments thereof may be bispecific. For example, bispec ific antibodies may resemble single antibodies (or antibody fragments) but have two different antigen g sites ble regions). Bispecific antibodies can be produced by various methods – such as chemical techniques, "polydoma" techniques or recombi nant DNA techniques. Bispecific dies may have binding specificities for at least two ent epitopes, at least one of which is the αβ TCR .CD3 complex. The other specificity may be selected from any useful or desired specificities including, for e xample, specificity for human serum albumin for the ion of half -life in vivo .

The use of bi -specifi c antibodies in the clinic for o ncology applications is now becoming reality with the tri ional Catumaxomab (Removmab ® ) approved for use in cases of malignant ascites and the bi -specific antibody Blinatumomab now in phase II trials in hematological malignancies. These molecules have in common a binding arm which binds to T cells and a second arm which binds to the tumor target cell which results in T cell mediated lysis of the tumor target. Also in common, these molecules recruit T cells via the CD3 protein located on the cell surface. An alternative to recruitment via CD3 is to make use of the αβ T cell receptor ( αβ TCR) which is also expressed on the e of the cell.

Accordingly, antibodies according to the present invention can be used to develop anti - tumor antibodies by combining a specificity for a tumor associated antigen with a specificity for the αβ T cell receptor ( αβ TCR) . 2. Antibody production The amino acid sequences of the variable domains of the dies described herein are set forth in SEQ ID NOs: 5 -7 and 12 -16. Antibody production can be performed by any technique known in the art, including in transgenic organisms such as g oats ( see, Pollock et al . (1999) J. lmmunol. Methods 7 -157), chickens ( see, Morrow, K.J.J. (2000) Genet. Eng. News 20:1 -55), mice ( see Pollock et al ., supra ) or plants ( see, Doran, P.M. (2000) Curr. Opinion hnol . 11:199 -204, Ma. J.K -C. (1998) Nat. Med . 4:601 -606, Baez, J. et al . (2000) BioPharm . 13:50 -54, , E. et al . (2000) Plant Mol. Biol . 42:583 - 590). Antibodies may also be produced by chemical synthesis or by expression of genes encoding the antibodies in host cells.

A polynucleotide en coding the antibody is isolated and ed into a replicable construct or vector such as a plasmid for further propagation or expression in a host cell. Constructs or vectors (e.g., expression s) suitable for the expression of a humanized glo bulin according to the described embodiments are available in the art. A variety of vectors are available, including vectors which are maintained in single copy or multiple copies in a host cell, or which become integrated into the host cell's chromosome( s). The constructs or vectors can be uced into a suitable host cell, and cells which express a humanized immunoglobulin can be produced and maintained in culture. A single vector or multiple vectors can be used for the expression of a zed immu noglobulin. cleotides encoding the antibody are readily isolated and sequenced using conventional ures ( e.g ., oligonucleotide probes). Vectors that may be used include plasmid, virus, phage, transposons, minichromosomes of which plasmids are a typical embodiment. Generally such vectors further include a signal sequence, origin of replication, one or more marker genes, an enhancer element, a promoter and transcription termination sequences ly linked to the light and/or heavy chain polynucl eotide so as to tate expression. Polynucleotides encoding the light and heavy chains may be ed into separate vectors and introduced ( e.g ., by transformation, 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 ed for expression in a suitable host cell. Promoters can be constitutive or inducible. For e, a promoter can be operably linked to a nucleic acid ng a humanized immunoglobulin or immunoglobulin chain, such that it directs expression of the encoded polypeptide. A variety of suitable promoters for prokaryotic and eukaryotic hosts are available. Prokaryotic promoters include lac, tac, T3, T7 ers for E. coli ; 3-phosphoglycerate kinase or other glycolytic s e.g ., enolase, glyceralderhyde 3 -phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose 6 phosp hate isomerase, 3 -phosphoglycerate mutase and glucokinase . E ukaryotic promoters include i nducible yeast promoters such as alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, othionein and enzymes responsible for nitrogen metabolism or maltos e/galactose ation ; RNA polymerase ll ers including viral promoters such as polyoma, fowlpox and adenoviruses ( e.g ., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus (in particular, the immediate early gene promoter), irus, 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 Res . 18(17):5322 ). Those of skill in the art will be able to select the riate promoter for expressing a humanized antibody or portion thereof.

Where appropriate, e.g ., for expression in cells of higher eukaroytes, additional enhancer elements can be included instead of or as well as those found located in the promoters described above. Suitable mammalian enhancer sequences include enhancer elements from globin, elastase, albumin, fetoprotein, metallothionine and insulin. atively, one may use an enhancer t from a eukaryotic cell virus such as SV40 e nhancer, cytomegalovirus early er enhancer, polyoma enhancer, baculoviral enhancer or murine lgG2a locus ( see, WO 04/009823). Whilst such enhancers are often located on the vector at a site am to the promoter, they can also be located elsewhere e.g ., within the slated region or downstream of the polyadenylation signal. The choice and positioning of enhancer may be based upon compatibility with the host cell used for expression.

In addition, the vectors (e.g., expression vectors) may compr ise a selectable marker for selection of host cells carrying the vector, and, in the case of a replicable vector, an origin of replication. Genes ng products which confer antibiotic or drug resistance are common selectable markers and may be used in prokaryotic (e.g., f3 mase 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 auxotrophic markers of the host (e.g., LEU2, URA3, HIS3 ) are often used as selectable markers in yeast. Use of viral (e.g., baculovirus) or phage vectors, an d vectors which are capable of integrating into the genome of the host cell, such as retroviral vectors, are also contemplated.

In eukaryotic systems, polyadenylation and termination signals are operably linked to cleotide encoding the antibody of th e invention. Such signals are lly placed 3' of the open reading frame. In mammalian systems, non -limiting examples of polyadenylation/termination s include those d from growth hormones, elongation factor -1 alpha and viral ( e.g ., SV40) gen es or iral long terminal repeats.

In yeast systems, non -limiting examples of polydenylation/termination signals e those derived from the phosphoglycerate kinase (PGK) and the alcohol dehydrogenase 1 (ADH) genes. In prokaryotic systems polyadeny lation signals are typically not required and it is instead usual to employ shorter and more defined terminator sequences. The choice of polyadenylation/termination sequences may be based upon compatibility with the host cell used for expression. In addit ion to the above, other features that can be employed to enhance yields include chromatin remodeling elements, introns and host cell specific codon modification. The codon usage of the dies of the invention can be modified to accommodate codon bias o f the host cell such to augment transcript and/or product yield ( e.g ., Hoekema, A. et al . (1987) Mol. Cell Biol . 7(8):2914 -24). The choice of codons may be based upon compatibility with the host cell used for expression.

The invention thus relates to isol ated nucleic acid molecules that encode the humanized immunoglobulins, or heavy or light chains, thereof. The invention also relates to ed c acid molecules that encode an antigen -binding portion of the immunoglobulins and their chains.

The anti bodies can be produced, for example, by the expression of one or more recombinant c acids encoding the antibody in a suitable host cell. The host cell can be produced using any suitable . For example, the expression constructs (e.g., one or mo re vectors, e.g., a mammalian cell expression vector) described herein can be introduced into a le host cell, and the resulting cell can be maintained (e.g., in culture, in an animal, in a plant) under conditions suitable for expression of the constr uct(s) or vector(s). Host cells can be prokaryotic, including bacterial cells such as E. coli (e.g., strain DH5a™) (Invitrogen, Carlsbad, CA), PerC6 (Crucell, Leiden, NL), B. subtilis and/or other suitable bacteria; eukaryotic cells, such as fungal or yeas t 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 i nsect cells) (WO 94/126087 (O'Connor)), BTI -TN -5B1 -4 (High Five™) insect cells (Invitrogen), mammals (e.g., COS cells, such as COS -1 (ATCC Accession No. CRL -1650) and COS -7 (ATCC ion No. CRL -1651), CHO (e.g., ATCC Accession No. CRL -9096), CHO DG44 (U rlaub, G. and Chasin, L.A. (1980) Proc. Natl. Acad. Sci. USA, 77(7):4216 -4220), 293 (ATCC Accession No. CRL -1573), HeLa (ATCC Accession No. CCL -2), CVl (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. U.S.A ., 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 e , Ausubel, F.M. et al., eds. Current Protocols in Molecular Biology, Greene hing ates and John Wiley & Sons Inc. (1993). In some embodiments, the host cell is not part of a multicellular organism (e.g., plant or animal), e.g., it is an ed host cell or is part of a cell culture.

Host cells may be cultured in r , shake flasks, roller bottles, wave reactors (e.g ., System 1000 from wavebiotech.com) or hollow fibre systems but it is preferred for large scale tion that d tank reactors or bag reactors ( e.g ., Wave Biotech, Somerset, New Jer sey USA) are used particularly for suspension cultures. Stirred tank reactors can be adapted for aeration using e.g. , spargers, baffles or low shear impellers.

For bubble columns and airlift rs, direct aeration with air or oxygen s maybe used. Where the host cells are cultured in a serum -free culture medium, 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 process. Depending on the host cell characteristics, microcarriers maybe used as growth substrates for anchorage dependent cell lines, or the cells maybe adapted to suspension culture. The culturing of host cells, particularly vertebrate host cells, may utilize a variety of operational modes such as batch, fed -batch, ed batch processing ( see, Drapeau et al . (1994) Cytotechnology 15:103 -109), extended batch process or perfusion culture. Although recombinantly ormed mammalian host cells may be cultured in serum -containing media such media comprisin g fetal calf serum (FCS), it is preferred that such host cells are cultured 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 UltraCHO™ (Cambrex NJ, USA), ment ed where necessary with an energy source such as glucose and tic 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 approach is to culture such host cells in serum containing media and repeatedly exchange 80% of the culture medium for the serum -free media so that the host cells learn to adapt in serum -free conditions ( see, e.g ., Scharfenberg, K. et al . (1995) Animal Cell Technology : Developments Towards the 21st Century (Beuvery, E.C. et al ., eds), pp.619 -623, Kluwer Academic publishers).

Antibodies according to the described embodiments may be secreted into the medium and recovered and purified therefrom using a variety of techniqu es to provide a degree of purification suitable for the intended use. For example, the use of therapeutic antibodies for the ent of human patients lly mandates at least 95% purity as determined by reducing SDS -PAGE, more typically 98% or 99% pu rity, when compared to the culture media comprising the therapeutic antibodies. In the first instance, cell debris from the culture media can be removed using fugation ed by a clarification step of the atant using e.g. , microfiltration, u ltrafiltration and/or depth filtration. Alternatively, the antibody can be harvested by iltration, ultrafiltration or depth filtration without prior centrifugation. A variety of other techniques such as dialysis and gel electrophoresis and chromatog raphic techniques such as hydroxyapatite (HA), affinity chromatography (optionally involving an affinity tagging system such as stidine) and/or hydrophobic interaction chromatography (HIC) ( see, US 5,429,746) are available. In one embodiment, the ant s, following various ication steps, are captured using Protein A or G ty chromatography followed by further chromatography steps such as ion exchange and/or HA chromatography, anion or cation exchange, size ion chromatography and am monium sulphate precipitation. Various virus removal steps may also be employed (e.g ., nanofiltration using, e.g ., a DV -20 filter). Following these various steps, a purified preparation comprising at least 10 mg/ml or greater, e.g ., 100 mg/ml or greater of the antibody of the invention is provided and, therefore, forms another embodiment of the invention. Concentration to 100 mg/ml or greater can be generated by ultracentrifugation.

Such preparations are substantially free of aggregated forms of antibodies of the invention.

Bacterial systems are particularly suited for the expression of antibody fragments. Such fragments are localized ellularly or within the periplasm. Insoluble periplasmic proteins can be extracted and refolded to form active protein s according to methods known to those skilled in the art, see, Sanchez et al . (1999) J. Biotechnol . 72:13 -20; Cupit, P.M. et al . (1999) Lett. Appl. Microbiol . 29:273 -277.

The present invention also s to cells sing a nucleic acid, e.g., a vector , of the invention (e.g., an sion vector). For e, a nucleic acid (i.e., one or more nucleic acids) encoding the heavy and light chains of a humanized globulin according to the described embodiments, or a construct (i.e., one or more const ructs, e.g., one or more vectors) comprising such nucleic ), can be introduced into a le host cell by a method appropriate to the host cell selected (e.g., transformation, transfection, electroporation, infection), with the nucleic acid(s) bein g, or becoming, operably linked to one or more expression control elements (e.g., in a vector, in a construct created by processes in the cell, integrated into the host cell genome). Host cells can be ined under conditions suitable for expression (e. g., in the presence of inducer, suitable media supplemented with appropriate salts, growth factors, antibiotic, nutritional supplements, etc. ), y the d polypeptide(s) are produced. If desired, the encoded humanized antibody can be isolated, for example, from the host cells, culture , or milk. This 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). 3. 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 αβ TCR.CD3 complex is indicated in the treatment of diseases involving an inappropriate or un desired immune response, such as inflammation, autoimmunity, and other conditions involving such mechanisms. In one embodiment, such disease or disorder is an autoimmune and/or inflammatory e. Examples of such autoimmune and/or matory diseases are ic Lupus Erythematosus (SLE), Rheumatoid Arthritis (RA) and inflammatory bowel disease (IBD) (including ulcerative colitis (UC) and Crohn's disease (CD)), multiple sclerosis (MS), scleroderma and type 1 diabetes (T1D), and other diseases and diso rders, such as PV (pemphigus vulgaris), psoriasis, atopic dermatitis, celiac disease, Chronic Obstructive Lung disease, Hashimoto's thyroiditis, Graves' disease (thyroid), Sjogren's me, Guillain -barré syndrome, Goodpasture's syndrome, Addison's disea se, Wegener's granulomatosis, primary biliary sclerosis, sclerosing cholangitis, autoimmune hepatitis, algia rheumatica, Raynaud's phenomenon, temporal arteritis, giant cell arteritis, autoimmune hemolytic anemia, pernicious anemia, polyarteritis nod osa, behcet's disease, primary bilary cirrhosis, uveitis, ditis, rheumatic fever, ankylosing spondylitis, glomerulenephritis, sarcoidosis, dermatomyositis, myasthenia gravis, polymyositis, alopecia areata, and vitilgo.

In one embodiment, such diseas e or disorder is SLE, RA or IBD. In one embodiment, such disease or disorder is MS.

In another embodiment, the antibodies according to the described embodiments are used to aid transplantation by immunosuppressing the t. Such use alleviates graft - ve rsus -host disease. For a description of existing treatments for graft -versus -host disease, see , e.g ., Svennilson, Bone Marrow Transplantation (2005) 35 :S65 –S67, and references cited therein. Advantageously, the antibodies of the ion may be used in combination with other available therapies.

With regard to the treatment of autoimmune diseases, combination therapy may e stration of an antibody of the present invention together with a ment, which together with the antibody comprises an effective amount for ting or treating such autoimmune diseases. Where said mune disease is Type 1 diabetes, the combination therapy may encompass one or more of an agent that promotes the growth of pancreatic beta -cells or enhances beta -cell transplantation, such as beta cell growth or survival factors or immunomodulatory antibodies. Where said autoimmune disease is rheumatoid arthritis, said combination therapy may encompass one or more of methotrexate, an anti -TNF -β antibody, a TNF -β receptor -lg fusion protein, an anti -IL -15 or anti -IL -21 antibody, a non -steroidal anti -inflammatory drug (NSAID), or a e - modifying anti -rheumatic drug ). For example, the additional agent may be a biological agent such as a n anti -TNF agent ( e.g. , Enbrel® , infliximab ade® ) and adalimumab (Humira® ) or rituximab (Rituxan® ). Where said autoimmune disease is hematopoietic lant rejection, hematopoietic growth factor(s) (such as erythropoietin, 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 thereof, phototherapy, corticoste roids, Cyclosporine A, vitamin D s, methotrexate, p38 mitogen -activated n kinase (MAPK) inhibitors, as well as biologic agents such as anti -TNF - agents and n® . Where said autoimmune disease is an inflammatory bowel disease (IBD) such as, for example, Crohn's e or ulcerative colitis, 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 ou t 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 contempl ated. Accordingly, the antibodies according to the described embodiments may be formulated into pharmaceutical itions for use in therapy. 4. ceutical Compositions In a preferred embodiment, there is ed a pharmaceutical composition compri sing an antibody according to the invention, or a ligand or ligands fiable by an assay method as defined in the previous aspect of the ion. Ligands may be immunoglobulins, peptides, c acids or small molecules, as discussed herein. They are referred to, in the following discussion, as “compounds”.

A ceutical composition according to the invention is a composition of matter comprising a compound or compounds capable of modulating T cell activity as an active ingredient. The compoun d is in the form of any pharmaceutically acceptable salt, or e.g ., where appropriate, an analog, free base form, tautomer, enantiomer racemate, or combination f. The active ingredients of a pharmaceutical composition comprising the active ingredient according to the invention are plated to exhibit therapeutic activity, for example, in the treatment of graft -versus -host e, 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 suitable for treating the particular 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 le for treating the ing indications such that a convenient, single composition can be stered to the subject. Dosage regima may be adjusted to provide the optimum therapeutic response.

For exa mple, several divided doses may be stered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

The active ingredient may be administered in a convenient manner such as by the oral, intravenous ( where water soluble), intramuscular, subcutaneous, intranasal, intradermal or suppository routes or implanting ( e.g ., using slow release molecules).

Depending on the route of stration, the active ient may be required to be coated in a material to protect said ingredients from the action of enzymes, acids and other l conditions which may inactivate said ingredient.

In order to administer the active ingredient by means other than parenteral administration, it will be coated by, or administer ed with, a material to prevent its inactivation. For example, the active ingredient may be administered in an adjuvant, co istered with enzyme inhibitors or in liposomes. Adjuvant is used in its broadest sense and includes any immune ating compo und such as interferon. Adjuvants contemplated herein include resorcinols, non -ionic surfactants such as polyoxyethylene oleyl ether and n -hexadecyl polyethylene ether. Enzyme inhibitors 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 hylene glycols, and mixtures thereof and in oils. Under ry c onditions of storage and use, these preparations n a preservative to t the growth of rganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions 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 m ust be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol ( for e , glycerol, propylene glycol, and liquid polyethyl ene , and the like ), suitable mixtures thereof, and vegetable oils. The proper ty can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the us e of superfactants.

The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal, and the like. In certain cases, it may be prefer able to include isotonic agents, for example, sugars or sodium chloride. Prolonged tion of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.

Sterile injectable solutions are prepared by orating the active ingredient in the required amount in the appropriate solvent with l of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersi ons are prepared by incorporating the sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile s for the preparation of steri le injectable solutions, the preferred methods of preparation are vacuum drying and the freeze -drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile -filtered solution thereof.

Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. Of course, any al used in preparing any dosage unit form should be ceutically pure and substantially non -toxic in the amounts employed. In addition, the active ingredient may be incorporated into sustained -release preparations and formulations.

As used herein "pharmaceutically acceptable carrier and/or t" includes any and all solvents, dispersion media, coatings, antibacterial and antifu ngal 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 agent is incompatible with the active ient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the itions.

It is especially ageous to formulate parenteral itions in dosage unit form for ease of administration and uniformity of . Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the ian subjects to be treated; each unit containing a predetermined quantity of active al calculated to produce the de sired eutic effect in association with the required pharmaceutical carrier. The specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the par ticular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such as active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired.

The principal acti ve ingredients are compounded for convenient and effective administration in effective amounts with a le pharmaceutically acceptable carrier in dosage unit form. In the case of compositions containing supplementary 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 dies, to cells, peptides may be modified in order to improve their ability to cross a cel l membrane. For example, US 5,149,782 discloses the use of nic peptides, ion -channel forming peptides, membrane peptides, long -chain fatty acids and other membrane blending agents to increase protein transport across the cell ne. These and othe r methods are also described in WO 97/37016 and US 5,108,921, incorporated herein by reference.

In a further aspect there is provided the active ingredient of the invention as hereinbefore defined for use in the treatment of disease either alone or in comb ination 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 medicament for the treatment of disease associated w ith an nt immune response.

Moreover, there is provided a method for treating a condition ated with an aberrant immune response, comprising administering to a subject a therapeutically effective amount of a ligand identifiable using an assay met hod as described above.

The invention is further described, for the es of illustration only, in the ing examples.

Comparative Example 1 Binding and biological activity of EuCIV3 is sed compared with BMA031 Using flow try, we have shown that EuCIV3 is inferior to BMA031 in T cell binding (Fig. 1). In this competition assay, T cells were incubated on ice in the presence of a fixed concentration of directly Phycoerythrin -labeled MoIgG2b -BMA031 (murine competitor) and an increasing co ncentration of anti -αβ TCR antibodies. After 20 s incubation, the cells were washed and surface bound directly Phycoerythrin -labeled MoIgG2b - BMA031 was detected by flow cytometry. The BMA031 HuIgG1 chimeric antibody es much more effectively tha n EuCIV3.

In order to assess its ability to inhibit T cell activity in vivo , CD8+ T cells were treated with anti -αβ TCR antibodies at various trations ( see, Fig. 2, x -axis) and co -cultured with autologous tic cells (DCs) pulsed with the CMV pept ide 495 -503 (pp65) for seven days in an in vitro education (IVE) assay.

Normal donor aphaeresis products from HLA -A2 + individuals were obtained from HemaCare Corp., Van Nuys, CA). PBMC were isolated by centrifugation over Ficoll (GE care, Piscataway , NJ). CD8+ T cells were isolated using magnetic beads (Invitrogen, Carlsbad, California) according to the manufacturer’s instructions. To generate autologous immature dendritic cells, PBMC were ended in RPMI 1640/5% human AB serum ), plated in triple flasks (Corning) and incubated for more than 2 hours at 37 oC/5%CO 2. The adherent monocytes were then rinsed with PBS and cultured for 6 days in RPMI 1640/5% human AB serum supplemented with GM -CSF (Immunex, Seattle, WA) and IL -4 (PeproTech, Rock y Hill, NJ). Prior to establishing the T C co - cultures, the DCs were pulsed with peptides ml) for 4 hours and then matured.

Mature dendritic cells were generated by the addition of l TNF -alpha, 25ng/ml IL - 1β, l IL -6, 500ng/ml PGE -2 (PeproTech, Rocky Hill, NJ) and culturing the dendritic cells for an additional 24 hours. The peptide -pulsed DCs were then added to the previously isolated CD8+ T cells at a T:DC ratio of 10:1. Cultures were immediately fed with IL -2 (100 IU/ml) added to the cultures. The es were supplemented with IL -2 (100 IU/ml) on day 4. The bulk cultures were assayed for peptide reactivity in a chromium release assay on day 7.

The graph in Fig. 2 shows lysis data from the chromiu m release assay, where untreated T cells were successfully educated against pp65 peptide and able to lyse ic targets at >50%. BMA031 inhibited education of these T cells, as they were unable to lyse ic targets in a dose -dependent manner. Humani zed antibody EuCIV3 was less potent than BMA031 and was only able to inhibit education at the highest dose.

Example 2 Fc Engineering of BMA031 chimeric antibodies In vitro profile We have assessed the in vitro profile of BMA031 in a panel of assays . Table 1 shows the in vitro profile of BMA031. BMA031 is compared to OKT3 in these assays.

In the PBMC proliferation assay, human PBMC were cultured with increasing concentrations of therapeutic antibody for 72 hours, 3H-thymidine was added and cells were ha rvested 18 hours later.

For the T cell depletion/Cytokine Release assay, human PBMC were cultured with increasing concentrations of therapeutic antibody and were analyzed daily for cell counts and viability (Vi -Cell, Beckman Coulter) out to day 7. Cell supernatants were also harvested, stored at -20 oC and analyzed on an 8 -plex cytokine panel (Bio -Rad).

BMA031 does not : (i) PBMC proliferation; (ii) T cell ion; (iii) CD25 expression; or (iv) cytokine release. In st, OKT3 does induce all of the aforementioned effects.

BMA031 and OKT3 are capable of blocking the education of CD8+ cells to peptide in an in vitro education (IVE) assay and are also e of blocking a mixed lymphocyte reaction (MLR). BMA031 also s apoptosis of activat ed T cells (activation -induced cell death; AICD).

Unlike BMA031, a chimeric version of BMA031 (HuIgG1) , with wild -type human IgG1 constant region , had an in vitro profile comparable to OKT3 (Table 1). We postulated that Fc γR involvement was critical for t his change of in vitro profile for HuIgG1 BMA031 compared to BMA031 MoIgG2b. ore we made F(ab’) 2 fragments of BMA031 HuIgG1 and found these to recover the profile of BMA031 MoIgG2b. By Fc engineering we incorporated modifications that removed Fc γR binding in mutations known as “delta ab” (Armour et al . (1999) Eur. J. Immunol ., 29:2613 -2624) and by generating an aglycosylated form of HuIgG4 (N297Q). HuIgG1 delta ab and HuIgG4 agly anti -αβ TCR antibodies had the same in vitro profile as BMA031 MoIgG2b (Table 1).

Normal PBMC Antigen Activated T -cells γγδδ TCR αβαβ TCR Fc γR PBMC CD25 Cytokine MLR Apoptosis / IVE Depletion binding binding binding Proliferation Expression Release Inhibition AICD Inhibition OKT3 + + + + + + + + ND + BMA031 MoIgG2b - + - - - - - + + + BMA031 HuIgG1 - + + + + + + ND ND + BMA031 F(ab)2 - + - - - - - ND ND + BMA031 ∆∆∆∆ab HuIgG1 - + - - - - - + + + HEBE1 ∆∆∆∆ab HuIgG1 - + - - - - - + + + HEBE1 IgG4 agly - + - - - - - + + + Table 1 Construction of humanized antibodies with improved binding We have generated two series of humanized versions of BMA031 called HEBE1 series (IGH3 -23) and GL1BM series (IGHV1 -3*01 & IGKV3 -11*01; see, VBase, vbase.mrc - m.ac.uk). Initial ng of BMA031 heavy chain CDR regions onto IGH3 -23 framework regions ( see , SEQ ID NOs: 5 and 6) improved the binding of the antibody to the αβ TCR as shown by a competition assay (Fig. 3); see, Example 2. However, this improvement did not ate into a functional improvement in the antibody as shown by an IVE assay (Fig. 4).

Example 4 Optimization of humanized antibodies The strategy for op timization of the humanized antibodies was based upon mutagenesis and onal screening. zation was started with block changes of amino acid residues in one of each of the four framework regions of the variable domains, from mouse to human. Key framework regions were identified in each of the GL1BM HC, GL1BM LC and HEBE1 HC series. Following this fication, dual residues within those framework regions were mutated to human germline residues from the original mouse sequence. Framework residues for which identity with the mouse sequence was found to be important to retaining the binding properties of the antibody were retained as mouse residues . Otherwise, framework residues were changed to match the human germline amino acid sequence . This was continued across the sequence until the minimal number of mouse residues, to retain the original binding properties of the dy, were identified. See Fig. 5. We have demonstrated that several of the antibodies from these series have an imp roved binding compared to BMA031 as determined by dy off -rate from T cells (Figs. 6, 7 and 8).

For off -rate assays, 10 5 human T cells were incubated for 30 -60 s at room temperature in 100uL full growth media containing 2ug/mL of the antibodi es expressed as HuIgG1 -Δab. The cells were then washed, resuspended in 50uL full growth media and 20ug/mL of HEBE1 F(ab’) 2 was added in order to prevent the rebinding of the dissociated candidate antibodies. At the end of this time course assay, the cells we re fixed and the level of remaining HuIgG1 -Δab antibody bound to the cell e was measured by flow cytometry via a PE labeled goat anti -HuIgG secondary antibody.

We have also demonstrated that the antibodies are active in preventing the immune se in an IVE (Figs. 9, 10 and 11) and a MLR assay. In the IVE assay, er binding was used as a quantitative measurement for the IVE. The percentage of cells which were antigen specific was determined by staining the T cells with a directly labeled tetra mer that is specific for the educating peptide. Briefly, day 7 CD8+ T cells from the IVE were stained with er by standard flow cytometry staining protocols and analyzed on BD FACSCalibur. In addition, the humanized dies demonstrated comparable levels of proliferative potential on PBMCs and cytokine release as compared to BMA031 (Figs. 12 and 13).

The antibodies also showed an ability to t the release of IFN γ from T cells in an IVE assay (Fig. 14). In addition we have shown that a number of these antibodies have an improved ability to elicit activation -induced cell death (AICD) of activated αβ TCR -positive T cells compared to BMA031 (Fig. 15) . In the AICD assay, antigen -specific CD8+ T cells were cultured with therapeutic antibody. At 24 hours, 48 hours and 72 hours cells were stained for apoptosis markers Annexin -V and 7 -AAD. Cells were also stained with tetramer to track sis with effects on n fic T cells.

In conclusion, we have made significant improvement over previou s attempts to humanize BMA031. The ery of antibodies with an improved off -rate compared to BMA031 is an cted finding via this process. This improvement in binding correlates with an improvement in y to suppress an immune response as dem onstrated in the IVE assay (Figs. 10 and 11). The specificity of the antibodies for αβ TCR, the decreased immunogenicity by humanization, the specific apoptosis of activated T cells and the lack of T cell activation upon antibody binding make these antibod ies excellent candidates for therapeutic purposes.

Example 5 Generation of Fc mutants for reduced effector function.

Engineered Fc variants was designed and generated where a glycosylation site is introduced at amino acid Ser 298 position, next to the n aturally -occurring Asn297 site. The glycosylation at Asn297 was either kept or knocked out by mutations. Mutations and glycosylation results are set forth in Table 2. # on Predicted result Expected Benefit 1 N297Q No glycosylation Agly Control No glycosylation Agly Control, 2 T299A n effector function No glycosylation at 297 but Reduced effector N297Q/S298N/Y300S 3 engineered glycosylation site function (NSY) at 298 No glycosylation at 297 but Reduced effector S298N/T299A/Y300S 4 engineered glycosyla tion site function (STY) at 298 Two potential glycosylation Positive control for sites at 297 & 298; Double reduced effector S298N/Y300S (SY) glycosylation? Mixed function glycosylation? Table 2 Mutations were made on the heavy chain of αβ T -cell receptor dy clone #66 by ange using a pENTR_LIC_IgG1 template. The VH domain of HEBE1 Δab IgG1 #66 was amplified with LIC primers, and cloned into mutated or wild type pENTR_LIC_IgG1 by LIC to create a full -length Ab mutants or wild t ype. The subcloning was verified with DraIII/XhoI double digest, ing ~1250 bp insert in the successful clones. Those full -length mutants were then cloned into an expression vector, pCEP4( -E+I)Dest, via Gateway cloning . The mutations were then conf irmed by DNA sequencing.

Two ucts, HEBE1 Agly IgG4 and HEBE1 Δab IgG1 in pCEP4, were used as controls in HEK293 transfection.

The mutants, wt and controls (Agly and Δab) were transfected into HEK293 -EBNA cells in triple -flask for expression. Proteins were purified from 160 ml of conditioned media (CM) with 1 ml HiTrap n A columns (GE) on multichannel peristaltic pump. Five micrograms of each supernatant were ed on 4 -20% Tris -Glycine reducing and non - reducing SDS -PAGE (see Figure 16) . The heavy chain of the agly cosylated mutants (N297Q, T299A, and Agly control, is lower (arrow in black), consistent with the loss of the glycans in these dy. The heavy chain s of the ered glycosylated antibodies (NSY, STY, SY, Δab, and wt control, arrows in red), however, migrate the same way as the wild -type control. This result is consistent with the ed e of engineered glycosylation site at 298 positions. SEC -HPLC analysis indicated that all s are expressed as monomers.

Glycosylation analysis by LC -MS.

The ered H66 IgG1 Fc variants were partially d with 20mM DTT at 37°C for min. The samples were analyzed by capillary LC/MS on an Agilent 1100 capillary HPLC system coupling with a QSTAR qq TOF hybrid system (Applied Biosystem).

Bayesian protein reconstruct with baseline correction and computer modeling in Analyst QS 1.1 (Applied Bisoystem) was used for data analysis. For mutant S298N/T299A/Y300S H66 antibody lead, one glycosylation site was obs erved at amino acid 298 with bi - antennary and tri - antennary complex -type glycans detected as the major species, as well as G0F, G1F and G2F.

Binding of ?? TCR antibody mutants to human Fc ?RIIIa and Fc ?RI using Biacore.

Biacore was used to assess binding to recombinant human Fc γRIIIa (V158 & F158) and Fc γRI. All 4 flowcells of a CM5 chip were immobilized with anti -HPC4 antibody via the standard amine coupling procedure provided by Biacore. The anti -HPC4 antibody was diluted to L in 10mM sodium acetate pH 5.0 for the coupling reaction and injected for 25 min at 5µL/min. Approximately 12,000 RU of antibody was immobilized to the chip e. Recombinant human Fc γRIIIa -V158 and Fc γRIIIa -F158 were diluted to 0.6µg/mL in binding buffer, HBS -P with 1mM Ca Cl 2, and injected to flowcells 2 and 4, respectively, for 3 min at 5µL/min to capture 300 – 400 RU receptor to the anti -HPC4 chip. In order to distinguish n the low binders, three times more rhFc γRIIIa was captured to the anti -HPC4 surface than usua lly used in this assay. Flowcells 1 and 3 were used as reference controls. Each antibody was diluted to 200nM in binding buffer and injected over all 4 flowcells for 4 min, followed by 5 min dissociation in . The surfaces were regenerated with 10m M EDTA in HBS -EP buffer for 3 min at 20µL/min.

The results are shown in Figure 17.

Biacore was also used to compare the Fc γRI binding. Anti -tetra His antibody was buffer exchanged into 10mM sodium acetate pH 4.0 using a Zeba Desalting column and d to 25µg/mL in the acetate buffer for amine coupling. Two flowcells of a CM5 chip we re lized with ~9000 RU of the anti -Tetra -His dy after 20 min injection at 5µL/min. Similar to the previous experiment, ten times more Fc γRI was ed to the anti -tetra -His surface in order to compare weak binders. Recombinant human Fc γRI w as diluted 10µg/mL in HBS -EP binding buffer and injected to flowcell 2 for 1 min at 5µL/min to capture ~1000 RU receptor to the anti -tetra -His chip. A single concentration of antibody, 100nM, was injected for 3 min at 30µL/min over the ed receptor a nd control surface. Dissociation was monitored for 3 min. The surface was regeneration with two 30 sec injections of 10mM glycine pH 2.5 at 20µL/min.

The results are shown in Figure 18.

The result suggests very little binding of the glycoengineered mut ants to Fc γRIIIa or Fc γRI. H66 S298N/T299A/Y300S in particular has almost completely abolished binding to both receptors. This mutant was chosen as the lead for detailed terization.

Stability characterization using Circular ism (CD).

The sta bility of the S298N/T299A/Y300S antibody mutant was monitored by a Far -UV CD thermo melting experiment where the CD signal at 216nm and 222nm was monitored as temperature increases that eventually leads to the unfolding of the antibody. The CD a were collected on a Jasco 815 spectrophotometer at a protein concentration of approximately 0.5 mg/mL in PBS buffer in a quartz cuvette a, Inc) with a path length of 10 mm. Temperature was controlled by a thermoelectric peltier (Jasco model AWC100) and was ramped at a rate of 1 ˚ C/min from 25 -89 ˚ C. CD signal and HT voltage were both collected. Data was obtained from 210 -260 nm with data intervals of 0.5 nm and at temperature intervals of 1 ˚ C. The scanning speed was 50 nm/min and a data pitch of 0.5 n m. A bandwidth of 2.5 nm was used with a sensitivity setting of medium. 4 replicate scans were performed for each sample. The result suggest that both delta AB H66 and the S298N/T299A/Y300S H66 mutant show similar thermal behavior and have the same onse t temperature for degradation around 63C. In other word, the mutant is as stable as the delta AB format.

See Figure 18.

Example 6 Functional analysis of Fc -engineered mutants PBMC eration and cytokine release assays were conducted as set forth in Exa mple 2. Normal donor PBMC were thawed and treated under the following ions (all in media containing complement): • Untreated • BMA031, moIgG2b 10ug/ml • OKT3, moIgG2a 10ug/ml • H66, huIgG1 deltaAB 10ug/ml, 1ug/ml and 0.1ug/ml • H66, huIgG1 S298N/T299A/Y300 S 10ug/ml, 1ug/ml and 0.1ug/ml Cytokines were harvested at day 2 ( D2 ) and day 4 (D4 ) for Bioplex Analysis (IL2, IL4, IL6, IL8, IL10, GM -CSF, IFNg, TNFa) . Cells were stained at D4 for CD4, CD8, CD25 and abTCR expression .

The results, shown in Figures 19 -22 , demonstrate that H66 S298N/T299A/Y300S d rly to the H66 deltaAB in all cell based assays , showing m inimal T -cell activation by CD25 expression ; binding to abTCR, gh with slightly different kinetics to deltaAB ; m inimal cytokine release at both D2 and D4 time points ; the mutant was in fact superior to deltaAB at D4 in respect of several of the cytokines .

The S298N/T299A/Y300S mutant thus eliminated effector function as effectively as the deltaAB mutation .

Example 7 Bispecific antibodies Bi-specific les were constructed comprised of two single chain dies (scFv) linked together via a short amino acid linker whereby one arm is capable of binding a tumor target and the other capable of binding T cells via the αβ TCR. The bis pecific molecule is referred to herein as a TRACER (T cell Receptor Activated Cytotoxic EnableR).

The following zed anti -αβ TCR scFv constructs were made : GL1BM ΔSxVK1 GL1BM ΔSxVK27 GL1BM ΔSVH11xVK1 GL1BM ΔSVH15xVK1 GL1BM ΔSVH28xVK43 GL1BM ΔSVH31xVK43 The sequence s of the heavy and light chains are set forth in SEQ ID nos 14 -16 and 20 -24 Characterization of these molecule s comprised an assessment of binding to tumor target and T cells, in vitro cytotoxic activity and cytokine release profile in the presence and absence of tumor target cells.

The binding profile assessed by flow tery shows that anti - αβ TCR bi -specific antibodies are able to bind both the tumor target cell line and T cells. See Figure 23.

In vitro cytotoxic activity measured by flow tery shows that T cells recruite d via anti - αβ TCR bi -specific antibody are capable of inducing T cell mediated lysis. See Figure 24. is of the cytokine release profile has shown that upon binding of both arms of the bi -specific antibody there is a high level of TH1/TH2 cytokine r elease from the T cells which is not seen in the absence of target cells. Taken together this mechanism of action shows a similar e to that of the CD3 based bispecifics described in the literature.

Example 8: Preparation and characterization of an en gineered Fc variant in anti - CD52 antibody.

In order to test the lity of the applicability of the Fc mutations described herein, ylation mutant S298N/Y300S was also prepared in an anti -CD52 antibody (clone 2C3) to see whether the effector functi on modulation with the loss of Fc γRIII binding applies to a different antibody backbone. S298N/Y300S 2C3 variant DNA was prepared by quick change mutagenesis. The protein was purified from ioned media after HEK293 transient transfection. Anti -CD52 2C 3 wild -type dy was produced in parallel as a control. Biacore was used to characterize the antigen -binding, Fc γRIII, and binding properties of the purified antibodies (see Figure 26 ).

The S298N/Y300S 2C3 variant bind s to CD52 peptide tightly and th e binding sensorgram is undistinguishable with the wild -type control, suggesting that this mutation on the Fc domain does not affect i ts antigen binding (Figure 26 A).

To assay Fc effector function, Fc γRIII receptor (Val158) was used in binding s. Th e mutant and wild -type control dy were diluted to 200nM and injected to HPC4 -tag captured Fc . Fc γRIII binding is almost undetectable for the S298N/Y300S mutant, which indicates loss of effector function with this variant (Figure 26 B). The mut ant also binds to FcRn receptor with the same affinity as the wild -type dy control so we expect no change in its circulation half -life or other pharmacokinetic properties. (see Figure 26 C) . We conclude that the S298N /Y300S mutation is applicable to antibodies in general, to reduce or eliminate undesired Fc effector on, for e through engagement of human Fc γ receptors.

Claims (17)

Claims

1. A humanized multispecific dy sing a first binding domain specific for the human ??TCR/CD3 x and a second binding domain specific for a tumor - specific antigen, wherein the first binding d omain comprises a heavy chain variable region having an amino acid sequence selected from the group consisting of the sequences set forth in SEQ ID NOs: 7, 12, 13, 15 and 16, and a light chain variable region comprising the amino acid sequence set forth as SEQ ID NO: 14.

2. The humanized multispecific antibody according to claim 1, wherein the heavy chain variable region is selected from the group ting of the amino acid sequences set forth as SEQ ID NO: 7, SEQ ID NO: 12 and SEQ ID NO: 13.

3. The humanized pecific antibody according to claim 1, wherein the heavy chain variable region is selected from the group consisting of the amino acid sequences set forth as SEQ ID NO: 15 and SEQ ID NO: 16.

4. The humanized multispecific antibody according to claim 1, wherein the antibody comprises an anti -?? TCR/CD3 scFv and an anti -tumor scFv.

5. The humanized multispecific antibody according to claim 1, n the antibody is bispecific.

6. The humanized multispecific anti body ing to any one of the preceding claims, further comprising a constant region of human origin.

7. The humanized multispecific antibody according to claim 6, further comprising an Fc modification which reduces Fc γ receptor binding.

8. The humanized pecific antibody ing to claim 7, which comprises a modified glycosylation pattern.

9. The humanized multispecific antibody according to claim 7, which comprises an aglycosylated Fc region or a delta ab modification.

10 . A n ucleic acid encoding the humanized multispecific antibody according to any one of claims 1 to 9.

11. An isolated cell which expresses the nucleic acid ing to claim 10, wherein transformed cells within a human host and cells capable of producing a human are ex cluded.

12. Use of the humanized multispecific antibody according to any one of claims 1 to 9 in the manufacture of a medicament for the treatment of a disease or disorder in a subject.

13. The use according to claim 12, wherein the disease or disorder is cancer.

14. The use according to claim 12, wherein the treatment results in the recruitment of a T cell to a tumor cell target.

15. The use according to claim 14, wherein the recruitment of the T cell to the tumor cell target results in a e o f one or more 2 cytokine from the T cell.

16. The use according to claim 15, wherein the one or more TH1/TH2 cytokine is selected from the group consisting of TNF -alpha and IFN -gamma.

17. The use according to claim 14, wherein the tment of the T cell to the tumor cell target results in T -cell -mediated lysis of the tumor cell target. GENZYME ATION WATERMARK INTELLECTUAL PROPERTY PTY LTD P38649NZ01 :03: Qmo_>_ 5:: 6:80 _m_ Fmo<_>_m_ m>_03m