NZ721138A - Bispecific t cell activating antigen binding molecules - Google Patents

Abstract

Discloses CD3 binding bispecific T cell activating antigen binding molecules comprising a crossover Fab molecule wherein either the variable or the constant regions of the Fab light chain and the Fab heavy chain are exchanged. Either the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety and the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain, or (ii) the first antigen binding moiety is fused at the C- terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding moiety and the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or the second subunit of the Fc domain. The T cell activating bispecific antigen binding molecule comprises not more than one antigen binding moiety capable of specific binding to CD3.

Description

Bispecific T cell activating antigen binding molecules Field of the Invention The present ion generally relates to bispecific antigen binding molecules for activating T cells. In addition, the present invention relates to polynucleotides encoding such bispecific antigen g molecules, and vectors and host cells comprising such polynucleotides. The invention further relates to methods for producing the bispecific antigen g molecules of the invention, and to methods of using these bispecific antigen binding molecules in the treatment of disease.

Background The selective destruction of an dual cell or a specific cell type is often desirable in a y of clinical settings. For example, it is a primary goal of cancer therapy to specifically destroy tumor cells, while leaving healthy cells and tissues intact and undamaged.

An attractive way of achieving this is by inducing an immune response against the tumor, to make immune effector cells such as natural killer (NK) cells or cytotoxic T lymphocytes (CTLs) attack and destroy tumor cells. CTLs constitute the most potent effector cells of the immune , however they cannot be activated by the effector mechanism mediated by the Fc domain of conventional eutic antibodies.

In this regard, ific dies designed to bind with one “arm” to a surface antigen on target cells, and with the second “arm” to an activating, invariant component of the T cell receptor (TCR) x, have become of interest in recent years. The simultaneous binding of such an antibody to both of its targets will force a temporary interaction between target cell and T cell, causing activation of any cytotoxic T cell and subsequent lysis of the target cell. Hence, the immune response is re-directed to the target cells and is independent of peptide antigen presentation by the target cell or the specificity of the T cell as would be relevant for normal MHC-restricted tion of CTLs. In this context it is crucial that CTLs are only activated when a target cell is ting the bispecific antibody to them, i.e. the immunological synapse is mimicked. Particularly desirable are ific antibodies that do not require lymphocyte preconditioning or co-stimulation in order to elicit efficient lysis of target cells.

Several bispecific antibody formats have been developed and their suitability for T cell mediated immunotherapy igated. Out of these, the so-called BiTE (bispecific T cell engager) molecules have been very well characterized and already shown some promise in the clinic (reviewed in Nagorsen and Bäuerle, Exp Cell Res 317, 1255-1260 (2011)). BiTEs are tandem scFv molecules n two scFv molecules are fused by a le linker. Further bispecific formats being evaluated for T cell engagement include diabodies (Holliger et al., Prot Eng 9, 299-305 (1996)) and derivatives f, such as tandem diabodies (Kipriyanov et al., J Mol Biol 293, 41-66 (1999)). A more recent development are the so-called DART (dual affinity retargeting) molecules, which are based on the y format but feature a C-terminal disulfide bridge for additional stabilization (Moore et al., Blood 117, 4542-51 (2011)). The so-called triomabs, which are whole hybrid mouse/rat IgG molecules and also currently being evaluated in clinical , represent a larger sized format (reviewed in z et al., Cancer Treat Rev 36, 458-467 (2010)).

The variety of formats that are being developed shows the great ial attributed to T cell re- direction and activation in immunotherapy. The task of generating bispecific antibodies suitable therefor is, r, by no means trivial, but involves a number of challenges that have to be met d to cy, ty, applicability and produceability of the antibodies.

Small constructs such as, for example, BiTE molecules – while being able to efficiently crosslink effector and target cells – have a very short serum half life requiring them to be administered to patients by continuous infusion. IgG-like formats on the other hand – while having the great t of a long half life – suffer from toxicity associated with the native effector functions inherent to IgG molecules. Their immunogenic potential constitutes another rable feature of IgG-like bispecific antibodies, especially non-human formats, for sful therapeutic development. Finally, a major challenge in the general development of bispecific antibodies has been the production of bispecific antibody constructs at a clinically sufficient quantity and purity, due to the mispairing of antibody heavy and light chains of different specificities upon co-expression, which decreases the yield of the correctly assembled construct and results in a number of non-functional side ts from which the desired bispecific antibody may be difficult to separate.

Given the difficulties and disadvantages associated with currently available bispecific antibodies for T cell mediated immunotherapy, there remains a need for novel, improved formats of such molecules. The present invention provides bispecific antigen binding molecules designed for T cell tion and re-direction that combine good efficacy and eability with low toxicity and favorable pharmacokinetic properties; and/or which at least provides the public with a useful choice.

In this specification where reference has been made to patent specifications, other external nts, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such al documents is not to be construed as an admission that such documents, or such sources of information, in any iction, are prior art, or form part of the common general knowledge in the art.

In the description in this specification reference may be made to subject matter that is not within the scope of the claims of the current application. That subject matter should be readily identifiable by a person skilled in the art and may assist in putting into ce the invention as defined in the claims of this application.

Summary of the Invention In a first aspect, the invention provides a T cell activating ific antigen binding molecule sing a first and a second antigen binding moiety, one of which is a Fab molecule capable of specific binding to CD3 and the other one of which is a Fab molecule capable of specific binding to a target cell antigen, and an Fc domain composed of a first and a second t e of stable association; wherein the first n binding moiety is a crossover Fab le wherein either the variable or the constant regions of the Fab light chain and the Fab heavy chain are exchanged; wherein (i) the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety and the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain, or (ii) the first antigen binding moiety is fused at the C- terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding moiety and the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or the second subunit of the Fc domain; and wherein the T cell activating bispecific antigen binding molecule comprises not more than one n binding moiety capable of specific binding to CD3.

In a second aspect, the invention provides an isolated polynucleotide encoding the T cell activating bispecific antigen binding molecule of the first aspect.

In a third aspect, the invention provides a vector, comprising the isolated polynucleotide of thesecond aspect.

In a fourth aspect, the invention provides a host cell comprising the ed polynucleotide of the second aspect or the vector of the third aspect, wherein the host cell is not a human cell within a human.

In a fifth aspect, the invention provides a method of producing the T cell activating bispecific antigen binding molecule of the first aspect, comprising the steps of a) culturing the host cell of the fourth aspect under conditions suitable for the expression of the T cell activating bispecific antigen binding molecule and b) recovering the T cell activating bispecific antigen g In a sixth aspect, the invention provides a pharmaceutical composition comprising the T cell activating bispecific antigen binding le of the first aspect and a pharmaceutically acceptable carrier.

In a seventh aspect, the invention provides use of the T cell activating bispecific antigen binding molecule of the first , for the manufacture of a medicament for the treatment of a disease in an individual in need thereof.

In an eighth aspect, the invention provides an in vitro method for inducing lysis of a target cell, comprising contacting a target cell with the T cell activating bispecific antigen binding molecule of the first aspect, in the ce of a T cell.

Brief Decription Broadly described is a T cell activating bispecific antigen binding le comprising a first and a second antigen binding , one of which is a Fab molecule capable of ic binding to an activating T cell n and the other one of which is a Fab le capable of specific binding to a target cell antigen, and an Fc domain ed of a first and a second subunit capable of stable association; wherein the first antigen binding moiety is (a) a single chain Fab molecule wherein the Fab light chain and the Fab heavy chain are connected by a peptide linker, or (b) a crossover Fab molecule wherein either the variable or the nt regions of the Fab light chain and the Fab heavy chain are exchanged.

In a particular embodiment, not more than one antigen binding moiety capable of specific binding to an activating T cell antigen is present in the T cell activating bispecific antigen g molecule (i.e. the T cell activating bispecific antigen binding molecule provides monovalent binding to the activating T cell n). In ular embodiments, the first antigen binding moiety is a ver Fab molecule. In even more particular ments, the first antigen binding moiety is a crossover Fab le wherein the constant regions of the Fab light chain and the Fab heavy chain are exchanged.

In some embodiments, the first and the second antigen binding moiety of the T cell activating bispecific antigen g molecule are fused to each other, optionally via a peptide linker. In one such embodiment, the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety. In another such embodiment, the first n binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding moiety. In yet another such embodiment, the second antigen binding moiety is fused at the inus of the Fab light chain to the N-terminus of the Fab light chain of the first antigen binding . In embodiments wherein the first antigen binding moiety is a crossover Fab molecule and wherein either (i) the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety or (ii) the first antigen g moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding moiety, additionally the Fab light chain of the first antigen binding moiety and the Fab light chain of the second antigen binding moiety may be fused to each other, optionally via a peptide linker.

In one embodiment, the second antigen binding moiety of the T cell activating bispecific antigen binding le is fused at the C-terminus of the Fab heavy chain to the inus of the first or the second subunit of the Fc domain. In another embodiment, the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain.

In one embodiment, the first and the second antigen binding moiety of the T cell ting bispecific antigen binding molecule are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain.

In certain embodiments, the T cell activating bispecific antigen binding molecule comprises a third antigen binding moiety which is a Fab molecule capable of ic binding to a target cell antigen. In one such embodiment, the third antigen g moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. In a particular embodiment, the second and the third antigen binding moiety of the T cell activating antigen g molecule are each fused at the C-terminus of the Fab heavy chain to the N- terminus of one of the subunits of the Fc domain, and the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding moiety. In another particular embodiment, the first and the third antigen binding moiety of the T cell ting antigen binding molecule are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain, and the second antigen binding moiety is fused at the inus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety. The components of the T cell activating bispecific antigen binding molecule may be fused directly or through suitable peptide linkers. In one embodiment the second and the third n binding moiety and the Fc domain are part of an immunoglobulin molecule. In a particular embodiment the immunoglobulin molecule is an IgG class immunoglobulin. In an even more ular embodiment the immunoglobulin is an IgG1 ss immunoglobulin. In another ment, the immunoglobulin is an IgG4 subclass immunoglobulin.

In a particular embodiment, the Fc domain is an IgG Fc domain. In a specific embodiment, the Fc domain is an IgG1 Fc domain. In r specific embodiment, the Fc domain is an IgG4 Fc domain. In an even more specific embodiment, the Fc domain is an IgG4 Fc domain comprising the amino acid substitution S228P (EU ing). In particular embodiments the Fc domain is a human Fc domain.

In particular embodiments the Fc domain ses a cation promoting the association of the first and the second Fc domain subunit. In a specific such ment, an amino acid residue in the CH3 domain of the first subunit of the Fc domain is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain of the first subunit which is positionable in a cavity within the CH3 domain of the second subunit, and an amino acid residue in the CH3 domain of the second subunit of the Fc domain is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second subunit within which the erance within the CH3 domain of the first subunit is positionable.

In a particular embodiment the Fc domain exhibits reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a native IgG1 Fc domain. In certain embodiments the Fc domain is engineered to have reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a non-engineered Fc domain. In one embodiment, the Fc domain comprises one or more amino acid substitution that s binding to an Fc receptor and/or or function. In one embodiment, the one or more amino acid substitution in the Fc domain that reduces binding to an Fc receptor and/or effector function is at one or more position selected from the group of L234, L235, and P329 (EU numbering). In ular embodiments, each subunit of the Fc domain comprises three amino acid substitutions that reduce binding to an Fc receptor and/or effector function wherein said amino acid substitutions are L234A, L235A and P329G. In one such embodiment, the Fc domain is an IgG1 Fc , particularly a human IgG1 Fc domain. In other embodiments, each subunit of the Fc domain comprises two amino acid substitutions that reduce binding to an Fc receptor and/or effector function n said amino acid substitutions are L235E and P329G. In one such embodiment, the Fc domain is an IgG4 Fc domain, particularly a human IgG4 Fc domain.

In one embodiment the Fc receptor is an Fcγ receptor. In one embodiment the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an ting Fc receptor. In a specific embodiment, the Fc receptor is human FcγRIIa, FcγRI, and/or FcγRIIIa. In one embodiment, the effector function is antibody-dependent cell-mediated xicity (ADCC).

In a particular embodiment, the activating T cell antigen that the bispecific antigen binding molecule is capable of binding is CD3. In other embodiments, the target cell antigen that the bispecific antigen binding molecule is capable of binding is a tumor cell antigen. In one embodiment, the target cell n is selected from the group consisting of: maassociated Chondroitin Sulfate Proteoglycan (MCSP), Epidermal Growth Factor Receptor , Carcinoembryonic Antigen (CEA), Fibroblast Activation Protein (FAP), CD19, CD20 and CD33.

Also described is an ed polynucleotide encoding a T cell activating bispecific antigen binding molecule described or a nt thereof. Also described are polypeptides encoded by the polynucleotidesdescribed. r bed is an expression vector comprising the isolated polynucleotide i described, and a host cell comprising the isolated polynucleotide or the sion vector described. In some embodiments the host cell is a eukaryotic cell, particularly a mammalian cell.

Also described is a method of producing the T cell activating bispecific antigen binding molecule described, comprising the steps of a) culturing the host cell described under conditions suitable for the expression of the T cell ting bispecific antigen binding molecule and b) recovering the T cell activating bispecific antigen binding molecule. Also described is a T cell activating bispecific antigen binding molecule produced by the method described.

Also described is a pharmaceutical composition comprising the T cell activating bispecific antigen binding molecule described and a pharmaceutically acceptable carrier.

Also described are methods of using the T cell activating bispecific antigen binding molecule and pharmaceutical composition described. bed is a T cell activating bispecific antigen binding molecule or a pharmaceutical composition described for use as a medicament. Described is a T cell activating bispecific antigen binding molecule or a pharmaceutical composition described for use in the treatment of a disease in an individual in need thereof. In a specific embodiment the e is cancer.

Also described is the use of a T cell activating bispecific antigen binding molecule described for the cture of a medicament for the treatment of a disease in an individual in need thereof; as well as a method of treating a disease in an individual, comprising stering to said individual a therapeutically effective amount of a composition comprising the T cell activating ific antigen binding molecule bed in a pharmaceutically acceptable form. In a specific embodiment the disease is cancer. In any of the above embodiments the individual preferably is a mammal, ularly a human.

Also described is a method for inducing lysis of a target cell, particularly a tumor cell, comprising contacting a target cell with a T cell activating bispecific antigen g molecule described in the presence of a T cell, particularly a cytotoxic T cell.

Brief Description of the gs FIGURE 1. ary configurations of the T cell activating bispecific antigen binding molecules described. Illustration of (A) the “1+1 IgG scFab, one armed”, and (B) the “1+1 IgG scFab, one armed inverted” molecule. In the “1+1 IgG scFab, one armed” molecule the light chain of the T cell targeting Fab is fused to the heavy chain by a linker, while the “1+1 IgG scFab, one armed inverted” molecule has the linker in the tumor targeting Fab. (C) Illustration of the “2+1 IgG scFab” molecule. (D) Illustration of the “1+1 IgG scFab” molecule. (E) Illustration of the “1+1 IgG ab” molecule. (F) ration of the “2+1 IgG Crossfab” molecule. (G) Illustration of the “2+1 IgG Crossfab” le with ative order of Crossfab and Fab components rted”). (H) Illustration of the “1+1 IgG Crossfab light chain (LC) fusion” molecule. (I) Illustration of the “1+1 CrossMab” molecule. (J) Illustration of the “2+1 IgG Crossfab, linked light chain” molecule. (K) Illustration of the “1+1 IgG ab, linked light chain” molecule. (L) Illustration of the “2+1 IgG Crossfab, inverted, linked light chain” molecule. (M) Illustration of the “1+1 IgG Crossfab, inverted, linked light chain” molecule.

Black dot: optional modification in the Fc domain promoting heterodimerization.

FIGURE 2. SDS PAGE (4-12% Bis/Tris, NuPage Invitrogen, Coomassie-stained) of “1+1 IgG scFab, one armed” (anti-MCSP/anti-huCD3) (see SEQ ID NOs 1, 3, 5), non reduced (A) and reduced (B), and of “1+1 IgG scFab, one armed inverted” MCSP/anti-huCD3) (see SEQ ID NOs 7, 9, 11), non reduced (C) and reduced (D).

FIGURE 3. Analytical size exclusion chromatography (Superdex 200 10/300 GL GE Healthcare; 2 mM MOPS pH 7.3, 150 mM NaCl, 0.02% (w/v) NaCl; 50 μg sample injected) of “1+1 IgG scFab, one armed” (anti-MCSP/anti-huCD3) (see SEQ ID NOs 1, 3, 5) (A) and “1+1 IgG scFab, one armed inverted” (anti-MCSP/anti-huCD3) (see SEQ ID NOs 7, 9, 11) (B).

FIGURE 4. SDS PAGE (4-12% Bis/Tris, NuPage Invitrogen, Coomassie-stained) of “1+1 IgG scFab, one armed” (anti-EGFR/anti-huCD3) (see SEQ ID NOs 43, 45, 57), non reduced (A) and reduced (B), and of “1+1 IgG scFab, one armed inverted” (anti-EGFR/anti-huCD3) (see SEQ ID NOs 11, 49, 51), non reduced (C) and reduced (D).

FIGURE 5. Analytical size exclusion chromatography (Superdex 200 10/300 GL GE Healthcare; 2 mM MOPS pH 7.3, 150 mM NaCl, 0.02% (w/v) NaCl; 50 μg sample injected) of “1+1 IgG scFab, one armed” (anti-EGFR/anti-huCD3) (see SEQ ID NOs 43, 45, 47) (A) and “1+1 IgG scFab, one armed inverted” EGFR/anti-huCD3) (see SEQ ID NOs 11, 49, 51) (B).

FIGURE 6. (A, B) SDS PAGE (4-12% Bis/Tris, NuPage Invitrogen, sie-stained) of “1+1 IgG scFab, one armed inverted” (anti-FAP/anti-huCD3) (see SEQ ID NOs 11, 51, 55), non reduced (A) and reduced (B). (C) Analytical size exclusion chromatography (Superdex 200 10/300 GL GE Healthcare; 2 mM MOPS pH 7.3, 150 mM NaCl, 0.02% (w/v) NaCl; 50 μg sample injected) of “1+1 IgG scFab, one armed inverted” (anti-FAP/anti-huCD3).

FIGURE 7. SDS PAGE (4-12% is, NuPage Invitrogen, Coomassie-stained) of (A) “2+1 IgG scFab, P329G LALA” (anti-MCSP/anti-huCD3) (see SEQ ID NOs 5, 21, 23), non reduced (lane 2) and d (lane 3); of (B) “2+1 IgG scFab, LALA” (anti-MCSP/anti-huCD3) (see SEQ ID NOs 5, 17, 19), non reduced (lane 2) and reduced (lane 3); of (C) “2+1 IgG scFab, wt” (anti-MCSP/anti-huCD3) (see SEQ ID NOs 5, 13, 15), non reduced (lane 2) and reduced (lane 3); and of (D) “2+1 IgG scFab, P329G LALA N297D” (anti-MCSP/anti-huCD3) (see SEQ ID NOs , 25, 27), non reduced (lane 2) and reduced (lane 3).

FIGURE 8. Analytical size exclusion chromatography dex 200 10/300 GL GE Healthcare; 2 mM MOPS pH 7.3, 150 mM NaCl, 0.02% (w/v) NaCl; 50 μg sample injected) of (A) “2+1 IgG scFab, P329G LALA” (anti-MCSP/anti-huCD3) (see SEQ ID NOs 5, 21, 23); of (B) “2+1 IgG scFab, LALA” (anti-MCSP/anti-huCD3) (see SEQ ID NOs 5, 17, 19); of (C) “2+1 IgG scFab, wt” (anti-MCSP/anti-huCD3) (see SEQ ID NOs 5, 13, 15); and of (D) “2+1 IgG scFab, P329G LALA N297D” (anti-MCSP/anti-huCD3) (see SEQ ID NOs 5, 25, 27).

FIGURE 9. (A, B) SDS PAGE (4-12% Bis/Tris, NuPage Invitrogen, Coomassie-stained) of “2+1 IgG scFab, P329G LALA” (anti-EGFR/anti-huCD3) (see SEQ ID NOs 45, 47, 53), non reduced (A) and reduced (B). (C) Analytical size exclusion chromatography (Superdex 200 10/300 GL GE Healthcare; 2 mM MOPS pH 7.3, 150 mM NaCl, 0.02% (w/v) NaCl; 50 μg sample ed) of “2+1 IgG scFab, P329G LALA” EGFR/anti-huCD3).

FIGURE 10. (A, B) SDS PAGE (4-12% Bis/Tris, NuPage Invitrogen, sie-stained) of “2+1 IgG scFab, P329G LALA” (anti-FAP/anti-huCD3) (see SEQ ID NOs 57, 59, 61), non d (A) and reduced (B). (C) Analytical size exclusion chromatography (Superdex 200 /300 GL GE Healthcare; 2 mM MOPS pH 7.3, 150 mM NaCl, 0.02% (w/v) NaCl; 50 μg sample injected) of “2+1 IgG scFab, P329G LALA” FAP/anti-huCD3).

FIGURE 11. (A, B) SDS PAGE (4-12% Tris-Acetate (A) or 4-12% Bis/Tris (B), NuPage Invitrogen, Coomassie-stained) of “1+1 IgG Crossfab, Fc(hole) P329G LALA / Fc(knob) wt” (anti-MCSP/anti-huCD3) (see SEQ ID NOs 5, 29, 31, 33), non reduced (A) and reduced (B). (C) Analytical size exclusion chromatography (Superdex 200 10/300 GL GE Healthcare; 2 mM MOPS pH 7.3, 150 mM NaCl, 0.02% (w/v) NaCl; 50 μg sample injected) of “1+1 IgG Crossfab, Fc(hole) P329G LALA / Fc(knob) wt” (anti-MCSP/anti-huCD3).

FIGURE 12. (A, B) SDS PAGE (4-12% Bis/Tris, NuPage Invitrogen, Coomassie-stained) of “2+1 IgG Crossfab” (anti-MCSP/anti-huCD3) (see SEQ ID NOs 3, 5, 29, 33), non reduced (A) and reduced (B). (C) Analytical size exclusion tography (Superdex 200 10/300 GL GE Healthcare; 2 mM MOPS pH 7.3, 150 mM NaCl, 0.02% (w/v) NaCl; 50 μg sample injected) of “2+1 IgG Crossfab” (anti-MCSP/anti-huCD3).

FIGURE 13. (A, B) SDS PAGE (4-12% Bis/Tris, NuPage Invitrogen, Coomassie-stained) of “2+1 IgG ab” (anti-MCSP/anti-cyCD3) (see SEQ ID NOs 3, 5, 35, 37), non reduced (A) and reduced (B). (C) Analytical size exclusion chromatography (Superdex 200 10/300 GL GE Healthcare; 2 mM MOPS pH 7.3, 150 mM NaCl, 0.02% (w/v) NaCl; 50 μg sample injected) of “2+1 IgG Crossfab” (anti-MCSP/anti-cyCD3).

FIGURE 14. (A, B) SDS PAGE (4-12% Bis/Tris, NuPage Invitrogen, Coomassie-stained) of “2+1 IgG Crossfab, inverted” (anti-CEA/anti-huCD3) (see SEQ ID NOs 33, 63, 65, 67), non reduced (A) and reduced (B). (C) Analytical size exclusion tography (Superdex 200 10/300 GL GE Healthcare; 2 mM MOPS pH 7.3, 150 mM NaCl, 0.02% (w/v) NaCl; 50 μg sample injected) of “2+1 IgG Crossfab, inverted” (anti-CEA/anti-huCD3).

FIGURE 15. (A) Thermal stability of )2-Fc” and “(dsscFv)2-Fc” (anti-MCSP (LC007)/anti-huCD3 (V9)). Dynamic Light Scattering, measured in a temperature ramp from 25- 75°C at 0.05°C/min. Black curve: )2-Fc”; grey curve: “(dsscFv)2-Fc”. (B) Thermal stability of “2+1 IgG scFab” (see SEQ ID NOs 5, 21, 23) and “2+1 IgG Crossfab” (anti- nti-huCD3) (see SEQ ID NOs 3, 5, 29, 33). Dynamic Light Scattering, measured in a temperature ramp from 25-75°C at 0.05°C/min. Black curve: “2+1 IgG scFab”; grey curve: “2+1 IgG Crossfab”.

FIGURE 16. Biacore assay setup for (A) determination of ction of various Fc-mutants with human FcγRIIIa, and for (B) simultaneous binding of T cell bespecific constructs with tumor target and human CD3γ(G4S)5CD3ε–AcTev–Fc(knob)–Avi/Fc(hole).

FIGURE 17. aneous binding of T-cell bispecific constructs to the D3 domain of human MCSP and human CD3γ(G4S)5CD3ε–AcTev–Fc(knob)–Avi/Fc(hole). (A) “2+1 IgG Crossfab” (see SEQ ID NOs 3, 5, 29, 33), (B) “2+1 IgG scFab” (see SEQ ID NOs 5, 21, 23).

FIGURE 18. Simultaneous binding of T-cell bispecific constructs to human EGFR and human CD3γ(G4S)5CD3ε–AcTev–Fc(knob)–Avi/Fc(hole). (A) “2+1 IgG scFab” (see SEQ ID NOs 45, 47, 53), (B) “1+1 IgG scFab, one armed” (see SEQ ID NOs 43, 45, 47), (C) “1+1 IgG scFab, one armed ed” (see SEQ ID NOs 11, 49, 51), and (D) “1+1 IgG scFab” (see SEQ ID NOs 47, 53, 213).

FIGURE 19. Binding of the “(scFv)2” molecule (50 nM) to CD3 expressed on Jurkat cells (A), or to MCSP on Colo-38 cells (B) measured by FACS. Mean fluorescence intensity compared to untreated cells and cells stained with the ary antibody only is depicted.

FIGURE 20. Binding of the “2+1 IgG scFab, LALA” (see SEQ ID NOs 5, 17, 19) construct (50 nM) to CD3 expressed on Jurkat cells (A), or to MCSP on Colo-38 cells (B) measured by FACS.

Mean fluorescence intensity compared to cells d with the nce anti-CD3 IgG (as ted), untreated cells, and cells stained with the secondary antibody only is depicted.

FIGURE 21. Binding of the “1+1 IgG scFab, one armed” (see SEQ ID NOs 1, 3, 5) and “1+1 IgG scFab, one armed inverted” (see SEQ ID NOs 7, 9, 11) constructs (50 nM) to CD3 expressed on Jurkat cells (A), or to MCSP on Colo-38 cells (B) measured by FACS. Mean fluorescence intensity compared to cells treated with the reference D3 or anti-MCSP IgG (as indicated), untreated cells, and cells stained with the secondary antibody only is depicted.

FIGURE 22. Dose dependent binding of the “2+1 IgG scFab, LALA” (see SEQ ID NOs 5, 17, 19) bispecific construct and the corresponding anti-MCSP IgG to MCSP on Colo-38 cells as measured by FACS.

FIGURE 23. Surface expression level of different activation markers on human T cells after incubation with 1 nM of “2+1 IgG scFab, LALA” (see SEQ ID NOs 5, 17, 19) or “(scFv)2” CD3-MCSP bispecific constructs in the presence or absence of Colo-38 tumor target cells, as ted (E:T ratio of PBMCs to tumor cells = 10:1). Depicted is the expression level of the early activation marker CD69 (A), or the late activation marker CD25 (B) on CD8+ T cells after 15 or 24 hours tion, respectively.

FIGURE 24. Surface expression level of the late activation marker CD25 on human T cells after tion with 1 nM of “2+1 IgG scFab, LALA” (see SEQ ID NOs 5, 17, 19) or “(scFv)2” CD3-MCSP bispecific constructs in the presence or absence of Colo-38 tumor target cells, as indicated (E:T ratio = 5:1). Depicted is the sion level of the late activation marker CD25 on CD8+ T cells (A) or on CD4+ T cells (B) after 5 days incubation.

FIGURE 25. Surface expression level of the late activation marker CD25 on cynomolgus CD8+ T cells from two different animals (cyno Nestor, cyno Nobu) after 43 hours incubation with the indicated trations of the “2+1 IgG Crossfab” bispecific construct (targeting cynomolgus CD3 and human MCSP; see SEQ ID NOs 3, 5, 35, 37), in the presence or absence of human MCSP-expressing MV-3 tumor target cells (E:T ratio = 3:1). As controls, the reference IgGs (anti-cynomolgus CD3 IgG, anti-human MCSP IgG) or the unphysiologic stimulus PHA-M were used.

FIGURE 26. IFN-γ levels, secreted by human pan T cells that were activated for 18.5 hours by the “2+1 IgG scFab, LALA” CD3-MCSP bispecific construct (see SEQ ID NOs 5, 17, 19) in the ce of U87MG tumor cells (E:T ratio = 5:1). As ls, the corresponding anti-CD3 and anti-MCSP IgGs were administered.

FIGURE 27. Killing (as measured by LDH release) of MDA-MB-435 tumor cells upon coculture with human pan T cells (E:T ratio = 5:1) and activation for 20 hours by different concentrations of the “2+1 IgG scFab” (see SEQ ID NOs 5, 21, 23), “2+1 IgG Crossfab” (see SEQ ID NOs 3, 5, 29, 33) and )2” bispecific molecules and corresponding IgGs.

FIGURE 28. Killing (as measured by LDH release) of MDA-MB-435 tumor cells upon coculture with human pan T cells (E:T ratio = 5:1), and activation for 20 hours by different concentrations of the bispecific ucts and corresponding IgGs. “2+1 IgG scFab” ucts differing in their Fc-domain (having either a wild-type Fc domain (see SEQ ID NOs 5, 13, 15), or a Fc-domain mutated to abolish (NK) effector cell function: P329G LALA (see SEQ ID NOs , 21, 23), P329G LALA N297D (see SEQ ID NOs 5, 25, 27)) and the “2+1 IgG Crossfab” (see SEQ ID NOs 3, 5, 29, 33) construct were compared.

FIGURE 29. Killing (as measured by LDH release) of Colo-38 tumor cells upon co-culture with human pan T cells (E:T ratio = 5:1), treated with CD3-MCSP bispecific “2+1 IgG scFab, LALA” (see SEQ ID NOs 5, 17, 19) construct, “(scFv)2” le or corresponding IgGs for 18.5 hours.

FIGURE 30. Killing (as measured by LDH release) of Colo-38 tumor cells upon co-culture with human pan T cells (E:T ratio = 5:1), treated with SP bispecific “2+1 IgG scFab, LALA” (see SEQ ID NOs 5, 17, 19) construct, the “(scFv)2” molecule or corresponding IgGs for 18 hours.

FIGURE 31. Killing (as measured by LDH release) of MDA-MB-435 tumor cells upon coculture with human pan T cells (E:T ratio = 5:1), and activation for 23.5 hours by different trations of the CD3-MCSP bispecific “2+1 IgG scFab, LALA” (see SEQ ID NOs 5, 17, 19) construct, “(scFv)2” molecule or corresponding IgGs.

FIGURE 32. Killing (as measured by LDH release) of Colo-38 tumor cells upon co-culture with human pan T cells (E:T ratio = 5:1) and activation for 19 hours by different concentrations of the CD3-MCSP bispecific “1+1 IgG scFab, one armed” (see SEQ ID NOs 1, 3, 5), “1+1 IgG scFab, one armed inverted” (see SEQ ID NOs 7, 9, 11) or “(scFv)2” constructs, or corresponding IgGs.

FIGURE 33. g (as measured by LDH e) of Colo-38 tumor cells upon co-culture with human pan T cells (E:T ratio = 5:1), d with “1+1 IgG scFab” CD3-MCSP ific construct (see SEQ ID NOs 5, 21, 213) or “(scFv)2” molecule for 20 hours.

FIGURE 34. Killing (as ed by LDH release) of MDA-MB-435 tumor cells upon coculture with human pan T cells (E:T ratio = 5:1), and activation for 21 hours by different trations of the bispecific constructs and corresponding IgGs. The CD3-MCSP bispecific “2+1 IgG Crossfab” (see SEQ ID NOs 3, 5, 29, 33) and “1+1 IgG Crossfab” (see SEQ ID NOs 5, 29, 31, 33) constructs, the “(scFv)2” molecule and corresponding IgGs were compared.

FIGURE 35. Killing (as measured by LDH release) of different target cells (MCSP-positive Colo-38 tumor target cells, mesenchymal stem cells derived from bone marrow or adipose , or pericytes from placenta; as ted) induced by the activation of human T cells by 135 ng/ml or 1.35 ng/ml of the “2+1 IgG Crossfab” CD3-MCSP bispecific construct (see SEQ ID NOs 3, 5, 29, 33) (E:T ratio = 25:1).

FIGURE 36. Killing (as measured by LDH release) of Colo-38 tumor target cells, measured after an overnight incubation of 21h, upon co-culture with human PBMCs and ent CD3- MCSP bispecific constructs (“2+1 IgG scFab, LALA” (see SEQ ID NOs 5, 17, 19) and )2”) or a glycoengineered anti-MCSP IgG (GlycoMab). The effector to target cell ratio was fixed at 25:1 (A), or varied as depicted (B). PBMCs were isolated from fresh blood (A) or from a Buffy Coat (B).

FIGURE 37. Time-dependent cytotoxic effect of the “2+1 IgG Crossfab” construct, targeting cynomolgus CD3 and human MCSP (see SEQ ID NOs 3, 5, 35, 37). Depicted is the LDH release from human MCSP-expressing MV-3 cells upon co-culture with primary cynomolgus PBMCs (E:T ratio = 3:1) for 24 h or 43 h. As controls, the reference IgGs (anti-cyno CD3 IgG and antihuman MCSP IgG) were used at the same molarity. PHA-M served as a control for (unphysiologic) T cell activation.

FIGURE 38. Killing (as measured by LDH release) of -positive MV-3 melanoma cells upon co-culture with human PBMCs (E:T ratio = 10:1), treated with different CD3-MCSP bispecific constructs (“2+1 IgG Crossfab” (see SEQ ID NOs 3, 5, 29, 33) and “(scFv)2”) for ~26 hours.

FIGURE 39. Killing (as measured by LDH release) of EGFR-positive LS-174T tumor cells upon co-culture with human pan T cells (E:T ratio = 5:1), treated with different CD3-EGFR bispecific ucts (“2+1 IgG scFab” (see SEQ ID NOs 45, 47, 53), “1+1 IgG scFab” (see SEQ ID NOs 47, 53, 213) and “(scFv)2”) or reference IgGs for 18 hours.

FIGURE 40. g (as measured by LDH release) of EGFR-positive LS-174T tumor cells upon co-culture with human pan T cells (E:T ratio = 5:1), treated with ent CD3-EGFR bispecific constructs (“1+1 IgG scFab, one armed” (see SEQ ID NOs 43, 45, 47), “1+1 IgG scFab, one armed ed” (see SEQ ID NOs 11, 49, 51), “1+1 IgG scFab” (see SEQ ID NOs 47, 53, 213) and “(scFv)2”) or nce IgGs for 21 hours.

FIGURE 41. Killing (as measured by LDH release) of EGFR-positive LS-174T tumor cells upon co-culture with either human pan T cells (A) or human naive T cells (B), treated with ent CD3-EGFR bispecific constructs (“1+1 IgG scFab, one armed” (see SEQ ID NOs 43, 45, 47), “1+1 IgG scFab, one armed inverted” (see SEQ ID NOs 11, 49, 51) and “(scFv)2”) or reference IgGs for 16 hours. The effector to target cell ratio was 5:1.

FIGURE 42. Killing (as measured by LDH release) of FAP-positive GM05389 fibroblasts upon co-culture with human pan T cells (E:T ratio = 5:1), treated with different P bispecific constructs (“1+1 IgG scFab, one armed inverted” (see SEQ ID NOs 11, 51, 55), “1+1 IgG scFab” (see SEQ ID NOs 57, 61, 213), “2+1 IgG scFab” (see SEQ ID NOs 57, 59, 61) and “(scFv)2”) for ~18 hours.

FIGURE 43. Flow cytrometric analysis of expression levels of CD107a/b, as well as in levels in CD8+ T cells that have been treated with different CD3-MCSP bispecific constructs (“2+1 IgG scFab, LALA” (see SEQ ID NOs 5, 17, 19) and “(scFv)2”) or corresponding control IgGs in the ce (A) or absence (B) of target cells for 6h. Human pan T cells were incubated with 9.43 nM of the different molecules in the presence or absence of Colo-38 tumor target cells at an effector to target ratio of 5:1. Monensin was added after the first hour of incubation to increase intracellular protein levels by preventing protein ort. Gates were set either on all CD107a/b positive, perforin-positive or double-positive cells, as depicted.

FIGURE 44. Relative proliferation of either CD8+ (A) or CD4+ (B) human T cells upon incubation with 1 nM of different SP bispecific constructs (“2+1 IgG scFab, LALA” (see SEQ ID NOs 5, 17, 19) or “(scFv)2”) or corresponding control IgGs in the presence or absence of Colo-38 tumor target cells at an effector to target cell ratio of 5:1. CFSE-labeled human pan T cells were characterized by FACS. The relative proliferation level was determined by setting a gate around the non-proliferating cells and using the cell number of this gate relative to the overall measured cell number as the reference.

FIGURE 45. Levels of different nes measured in the supernatant of human PBMCs after treatment with 1 nM of different CD3-MCSP bispecific constructs (“2+1 IgG scFab, LALA” (see SEQ ID NOs 5, 17, 19) or “(scFv)2”) or corresponding control IgGs in the presence (A) or absence (B) of Colo-38 tumor cells for 24 hours. The effector to target cell ratio was 10:1.

FIGURE 46. Levels of ent cytokines measured in the supernatant of whole blood after treatment with 1 nM of different CD3-MCSP bispecific constructs (“2+1 IgG scFab”, “2+1 IgG Crossfab” (see SEQ ID NOs 3, 5, 29, 33) or “(scFv)2”) or corresponding control IgGs in the presence (A, B) or absence (C, D) of Colo-38 tumor cells for 24 hours. Among the bispecific constructs were different “2+1 IgG scFab” constructs having either a wild-type Fc domain (see SEQ ID NOs 5, 13, 15), or an Fc domain mutated to abolish (NK) effector cell function (LALA (see SEQ ID NOs 5, 17, 19), P329G LALA (see SEQ ID NOs 5, 2, 23) and P329G LALA N297D (see SEQ ID NOs 5, 25, 27)).

FIGURE 47. CE-SDS es. Electropherogram shown as SDS PAGE of 2+1 IgG Crossfab, linked light chain (see SEQ ID NOs 3, 5, 29, 179). (lane 1: reduced, lane 2: non-reduced).

FIGURE 48. Analytical size exclusion chromatography of 2+1 IgG Crossfab, linked light chain (see SEQ ID NOs 3, 5, 29, 179) (final product). 20 µg sample were injected.

FIGURE 49. Killing (as ed by LDH release) of MCSP-positive MV-3 tumor cells upon co-culture by human PBMCs (E:T ratio = 10:1), treated with different CD3-MCSP bispecific constructs for ~ 44 hours (“2+1 IgG Crossfab” (see SEQ ID NOs 3, 5, 29, 33) and “2+1 IgG ab, linked LC” (see SEQ ID NOs 3, 5, 29, 179)). Human PBMCs were isolated from fresh blood of healthy volunteers.

FIGURE 50. Killing (as measured by LDH release) of MCSP-positive Colo-38 tumor cells upon co-culture by human PBMCs (E:T ratio = 10:1), treated with different CD3-MCSP bispecific constructs for ~22 hours (“2+1 IgG Crossfab” (see SEQ ID NOs 3, 5, 29, 33) and “2+1 IgG ab, linked LC” (see SEQ ID NOs 3, 5, 29, 179)). Human PBMCs were isolated from fresh blood of healthy eers.

FIGURE 51. Killing (as ed by LDH release) of MCSP-positive Colo-38 tumor cells upon co-culture by human PBMCs (E:T ratio = 10:1), treated with ent SP ific constructs for ~22 hours (“2+1 IgG Crossfab” (see SEQ ID NOs 3, 5, 29, 33) and “2+1 IgG Crossfab, linked LC” (see SEQ ID NOs 3, 5, 29, 179)). Human PBMCs were isolated from fresh blood of healthy volunteers.

FIGURE 52. Killing (as measured by LDH release) of MCSP-positive WM266-4 cells upon co- culture by human PBMCs (E:T ratio = 10:1), treated with different CD3-MCSP bispecific constructs for ~22 hours (“2+1 IgG Crossfab” (see SEQ ID NOs 3, 5, 29, 33) and “2+1 IgG Crossfab, linked LC” (see SEQ ID NOs 3, 5, 29, 179)). Human PBMCs were isolated from fresh blood of y volunteers.

FIGURE 53. Surface expression level of the early activation marker CD69 (A) and the late activation marker CD25 (B) on human CD8+ T cells after 22 hours tion with 10 nM, 80 pM or 3 pM of different CD3-MCSP bispecific constructs (“2+1 IgG Crossfab” (see SEQ ID NOs 3, 5, 29, 33) and “2+1 IgG Crossfab, linked LC” (see SEQ ID NOs 3, 5, 29, 179)) in the presence or absence of human MCSP-expressing Colo-38 tumor target cells (E:T ratio = 10:1).

FIGURE 54. CE-SDS analyses. (A) opherogram shown as SDS-PAGE of 1+1 IgG Crossfab; VL/VH exchange (LC007/V9) (see SEQ ID NOs 5, 29, 33, 181): a) non-reduced, b) reduced. (B) Electropherogram shown as SDS-PAGE of 1+1 CrossMab; CL/CH1 ge (LC007/V9) (see SEQ ID NOs 5, 23, 183, 185): a) reduced, b) non-reduced. (C) Electropherogram shown as SDS-PAGE of 2+1 IgG Crossfab, inverted; CL/CH1 exchange (LC007/V9) (see SEQ ID NOs 5, 23, 183, 187): a) reduced, b) non-reduced. (D) Electropherogram shown as SDS-PAGE of 2+1 IgG Crossfab; VL/VH exchange (M4-3 ML2/V9) (see SEQ ID NOs 33, 189, 191, 193): a) reduced, b) non-reduced. (E) Electropherogram shown as SDS-PAGE of 2+1 IgG Crossfab; CL/CH1 exchange (M4-3 ML2/V9) (see SEQ ID NOs 183, 189, 193, 195): a) reduced, b) duced. (F) Electropherogram shown as SDS-PAGE of 2+1 IgG Crossfab, inverted; CL/CH1 exchange (CH1A1A/V9) (see SEQ ID NOs 65, 67, 183, 197): a) reduced, b) non-reduced. (G) Electropherogram shown as SDS-PAGE of 2+1 IgG Crossfab; CL/CH1 ge (M4-3 ML2/H2C) (see SEQ ID NOs 189, 193, 199, 201): a) reduced, b) nonreduced.

(H) Electropherogram shown as SDS-PAGE of 2+1 IgG Crossfab, inverted; CL/CH1 exchange (431/26/V9) (see SEQ ID NOs 183, 203, 205, 207): a) d, b) non-reduced. (I) Electropherogram shown as SDS-PAGE of “2+1 IgG Crossfab light chain fusion” (CH1A1A/V9) (see SEQ ID NOs 183, 209, 211, 213): a) reduced, b) non-reduced. (J) SDS PAGE (4-12% Bis/Tris, NuPage Invitrogen, Coomassie-stained) of “2+1 IgG Crossfab” (anti-MCSP/antihuCD3 ) (see SEQ ID NOs 5, 23, 215, 217), non-reduced (left) and reduced (right). (K) Electropherogram shown as SDS-PAGE of “2+1 IgG Crossfab, inverted” (anti-MCSP/antihuCD3 ) (see SEQ ID NOs 5, 23, 215, 219): a) reduced, b) non-reduced. (L) SDS PAGE (4-12% is, NuPage Invitrogen, Coomassie-stained) of “1+1 IgG Crossfab” CD33/anti-huCD3) (see SEQ ID NOs 33, 213, 221, 223), reduced (left) and non-reduced (right). (M) SDS PAGE (4- 12% Bis/Tris, NuPage Invitrogen, Coomassie-stained) of “2+1 IgG Crossfab” (anti-CD33/antihuCD3 ) (see SEQ ID NOs 33, 221, 223, 225), reduced (left) and non-reduced (right). (N) SDS PAGE (4-12% Bis/Tris, NuPage ogen, Coomassie-stained) of “2+1 IgG Crossfab” (anti- CD20/anti-huCD3) (see SEQ ID NOs 33, 227, 229, 231), non-reduced.

FIGURE 55. Binding of bispecific constructs (CEA/CD3 “2+1 IgG Crossfab, inverted (VL/VH)” (see SEQ ID NOs 33, 63, 65, 67) and “2+1 IgG Crossfab, inverted (CL/CH1) 2 (see SEQ ID NOs 65, 67, 183, 197)) to human CD3, expressed by Jurkat cells (A), or to human CEA, expressed by LS-174T cells (B) as determined by FACS. As a control, the equivalent maximum concentration of the reference IgGs and the background staining due to the labeled 2ndary antibody (goat anti-human FITC-conjugated AffiniPure F(ab’)2 Fragment, Fcγ Fragment-specific, n Immuno Research Lab # 109098) were assessed as well.

FIGURE 56. Binding of ific constructs constructs (MCSP/CD3 “2+1 IgG Crossfab” (see SEQ ID NOs 3, 5, 29, 33) and “2+1 IgG Crossfab, inverted” (see SEQ ID NOs 5, 23, 183, 187)) to human CD3, expressed by Jurkat cells (A), or to human MCSP, sed by WM266-4 tumor cells (B) as determined by FACS.

FIGURE 57. Binding of the “1+1 IgG Crossfab light chain fusion” (see SEQ ID NOs 183, 209, 211, 213) to human CD3, expressed by Jurkat cells (A), or to human CEA, expressed by LS- 174T cells (B) as determined by FACS.

FIGURE 58. Binding of the “2+1 IgG Crossfab” (see SEQ ID NOs 5, 23, 215, 217) and the “2+1 IgG Crossfab, inverted” (see SEQ ID NOs 5, 23, 215, 219) constructs to human CD3, expressed by Jurkat cells (A), or human MCSP, expressed by WM266-4 tumor cells (B) as determined by FACS.

FIGURE 59. Surface expression level of the early activation marker CD69 (A) or the late activation marker CD25 (B) on human CD4+ or CD8+ T cells after 24 hours incubation with the indicated concentrations of the CD3/MCSP “1+1 CrossMab” (see SEQ ID NOs 5, 23, 183, 185), “1+1 IgG Crossfab” (see SEQ ID NOs 5, 29, 33, 181) and “2+1 IgG ab” (see SEQ ID NOs 3, 5, 29, 33) constructs. The assay was med in the presence or e of MV-3 target cells, as indicated.

FIGURE 60. Surface expression level of the early activation marker CD25 on CD4+ or CD8+ T cells from two different cynomolgus monkeys (A and B) in the ce or absence of huMCSP- positive MV-3 tumor cells upon co-culture with cynomolgus PBMCs (E:T ratio = 3:1, normalized to CD3+ numbers), treated with the “2+1 IgG Crossfab” (see SEQ ID NOs 5, 23, 215, 217) and the “2+1 IgG Crossfab, inverted” (see SEQ ID NOs 5, 23, 215, 219) for ~41 hours.

FIGURE 61. g (as ed by LDH release) of MKN-45 (A) or LS-174T (B) tumor cells upon co-culture with human PBMCs (E:T ratio = 10:1) and activation for 28 hours by different concentrations of the “2+1 IgG Crossfab, inverted (VL/VH)” (see SEQ ID NOs 33, 63, 65, 67) versus the “2+1 IgG Crossfab, inverted (CL/CH1)” (see SEQ ID NOs 65, 67, 183, 197) construct.

FIGURE 62. g (as measured by LDH release) of WM266-4 tumor cells upon co-culture with human PBMCs (E:T ratio = 10:1) and activation for 26 hours by different concentrations of the “2+1 IgG Crossfab (VL/VH)” (see SEQ ID NOs 33, 189, 191, 193) versus the “2+1 IgG Crossfab (CL/CH1)” (see SEQ ID NOs 183, 189, 193, 195) construct.

FIGURE 63. Killing (as measured by LDH release) of MV-3 tumor cells upon co-culture with human PBMCs (E:T ratio = 10:1) and activation for 27 hours by different concentrations of the “2+1 IgG Crossfab (VH/VL)” (see SEQ ID NOs 33, 189, 191, 193) versus the “2+1 IgG Crossfab (CL/CH1)” (see SEQ ID NOs 183, 189, 193, 195) constructs.

FIGURE 64. Killing (as measured by LDH release) of human MCSP-positive WM266-4 (A) or MV-3 (B) tumor cells upon co-culture with human PBMCs (E:T ratio = 10:1) and activation for 21 hours by ent concentrations of the “2+1 IgG Crossfab” (see SEQ ID NOs 3, 5, 29, 33), the “1+1 CrossMab” (see SEQ ID NOs 5, 23, 183, 185), and the “1+1 IgG ab” (see SEQ ID NOs 5, 29, 33, 181), as indicated.

FIGURE 65. g (as measured by LDH release) of MKN-45 (A) or LS-174T (B) tumor cells upon co-culture with human PBMCs (E:T ratio = 10:1) and activation for 28 hours by different concentrations of the “1+1 IgG Crossfab LC fusion” (see SEQ ID NOs 183, 209, 211, 213).

FIGURE 66. Killing (as measured by LDH release) of MC38-huCEA tumor cells upon coculture with human PBMCs (E:T ratio = 10:1) and tion for 24 hours by different concentrations of the “1+1 IgG ab LC fusion” (see SEQ ID NOs 183, 209, 211, 213) versus an untargeted “2+1 IgG Crossfab” reference.

FIGURE 67. Killing (as measured by LDH release) of human MCSP-positive MV-3 (A) or WM266-4 (B) tumor cells upon co-culture with human PBMCs (E:T ratio = 10:1), treated with the “2+1 IgG Crossfab (V9)” (see SEQ ID NOs 3, 5, 29, 33) and the “2+1 IgG Crossfab, inverted (V9)” (see SEQ ID NOs 5, 23, 183, 187), the “2+1 IgG Crossfab (anti-CD3)” (see SEQ ID NOs 5, 23, 215, 217) and the “2+1 IgG Crossfab, inverted (anti-CD3)” (see SEQ ID NOs 5, 23, 215, 219) constructs. ed ption of the Invention Definitions Terms are used herein as generally used in the art, unless otherwise defined in the following.

As used herein, the term "antigen binding molecule" refers in its st sense to a molecule that specifically binds an antigenic determinant. Examples of antigen binding molecules are immunoglobulins and derivatives, e.g. fragments, thereof.

The term “bispecific” means that the antigen binding molecule is able to specifically bind to at least two distinct antigenic determinants. Typically, a bispecific antigen binding molecule comprises two antigen binding sites, each of which is specific for a different antigenic determinant. In certain embodiments the bispecific antigen binding molecule is e of simultaneously g two antigenic determinants, particularly two antigenic determinants expressed on two distinct cells.

The term “valent” as used herein s the presence of a specified number of n binding sites in an antigen binding molecule. As such, the term “monovalent binding to an antigen” denotes the presence of one (and not more than one) antigen binding site specific for the antigen in the n binding le.

An “antigen binding site” refers to the site, i.e. one or more amino acid residues, of an antigen binding molecule which provides interaction with the n. For example, the antigen binding site of an antibody comprises amino acid residues from the complementarity determining regions (CDRs). A native immunoglobulin molecule typically has two antigen binding sites, a Fab molecule typically has a single n binding site.

As used herein, the term "antigen binding moiety" refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one embodiment, an antigen binding moiety is able to direct the entity to which it is attached (e.g. a second antigen binding moiety) to a target site, for example to a specific type of tumor cell or tumor stroma bearing the nic determinant. In another embodiment an antigen binding moiety is able to activate ing through its target antigen, for example a T cell receptor complex antigen. Antigen binding moieties include antibodies and fragments thereof as further defined herein. Particular antigen binding moieties include an antigen binding domain of an antibody, comprising an antibody heavy chain le region and an antibody light chain variable region. In certain embodiments, the antigen binding es may comprise antibody constant regions as r d herein and known in the art. Useful heavy chain constant regions include any of the five isotypes: α, δ, ε, γ, or μ. Useful light chain constant regions include any of the two isotypes: κ and λ.

As used herein, the term "antigenic inant" is synonymous with "antigen" and "epitope," and refers to a site (e.g. a contiguous stretch of amino acids or a conformational configuration made up of ent regions of non-contiguous amino acids) on a ptide macromolecule to which an antigen g moiety binds, forming an antigen g moiety-antigen complex.

Useful antigenic determinants can be found, for example, on the surfaces of tumor cells, on the surfaces of virus-infected cells, on the surfaces of other diseased cells, on the surface of immune cells, free in blood serum, and/or in the extracellular matrix (ECM). The proteins ed to as antigens herein (e.g. MCSP, FAP, CEA, EGFR, CD33, CD3) can be any native form the proteins from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g. mice and rats), unless otherwise indicated. In a ular embodiment the antigen is a human n. Where reference is made to a specific protein herein, the term encompasses the “fulllength” , unprocessed protein as well as any form of the protein that results from processing in the cell. The term also encompasses naturally occurring variants of the protein, e.g. splice ts or allelic variants. Exemplary human ns useful as antigens include, but are not limited to: Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP), also known as oitin Sulfate Proteoglycan 4 (UniProt no. Q6UVK1 (version 70), NCBI RefSeq no. NP_001888.2); Fibroblast Activation Protein (FAP), also known as Seprase (Uni Prot nos. Q12884, Q86Z29, Q99998, NCBI Accession no. NP_004451); Carcinoembroynic antigen (CEA), also known as Carcinoembryonic antigen-related cell adhesion molecule 5 (UniProt no. P06731 (version 119), NCBI RefSeq no. 354.2); CD33, also known as gp67 or Siglec-3 (UniProt no. P20138, NCBI Accession nos. NP_001076087, 171079); Epidermal Growth Factor Receptor (EGFR), also known as ErbB-1 or Her1 (UniProt no. P0053, NCBI Accession nos. NP_958439, NP_958440), and CD3, particularly the epsilon subunit of CD3 (see UniProt no. P07766 (version 130), NCBI RefSeq no. NP_000724.1, SEQ ID NO: 265 for the human sequence; or UniProt no.

Q95LI5 (version 49), NCBI GenBank no. BAB71849.1, SEQ ID NO: 266 for the cynomolgus [Macaca fascicularis] sequence). In certain embodiments the T cell activating bispecific antigen binding molecule described binds to an epitope of an ting T cell antigen or a target cell n that is conserved among the ting T cell antigen or target antigen from different species.

By "specific binding" is meant that the binding is selective for the antigen and can be discriminated from unwanted or non-specific ctions. The ability of an antigen binding moiety to bind to a specific antigenic determinant can be measured either through an enzymelinked immunosorbent assay (ELISA) or other techniques familiar to one of skill in the art, e.g. surface plasmon nce (SPR) technique (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)), and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the extent of binding of an antigen binding moiety to an unrelated protein is less than about 10% of the binding of the antigen g moiety to the antigen as ed, e.g., by SPR. In certain embodiments, an antigen binding moiety that binds to the antigen, or an antigen binding molecule sing that antigen binding moiety, has a dissociation constant (KD) of ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM (e.g. 10-8 M or less, e.g. from 10-8 M to 10-13 M, e.g., from 10-9 M to 10-13 M). ity” refers to the strength of the sum total of non-covalent interactions n a single binding site of a molecule (e.g., a or) and its binding partner (e.g., a ). Unless indicated otherwise, as used , “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction n members of a binding pair (e.g., an antigen binding moiety and an antigen, or a receptor and its ligand). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD), which is the ratio of dissociation and association rate constants (koff and kon, respectively). Thus, equivalent affinities may comprise different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured by well established s known in the art, including those described herein. A particular method for measuring affinity is Surface Plasmon Resonance (SPR).

“Reduced binding”, for example reduced binding to an Fc receptor, refers to a decrease in affinity for the respective interaction, as measured for example by SPR. For y the term includes also reduction of the ty to zero (or below the detection limit of the analytic ), i.e. complete abolishment of the interaction. Conversely, “increased binding” refers to an increase in binding affinity for the respective interaction.

An “activating T cell antigen” as used herein refers to an antigenic determinant sed on the surface of a T lymphocyte, particularly a cytotoxic T lymphocyte, which is capable of inducing T cell activation upon interaction with an antigen binding molecule. Specifically, interaction of an antigen binding molecule with an activating T cell antigen may induce T cell activation by triggering the signaling cascade of the T cell or complex. In a particular embodiment the activating T cell antigen is CD3.

“T cell activation” as used herein refers to one or more cellular response of a T lymphocyte, ularly a cytotoxic T lymphocyte, ed from: proliferation, differentiation, cytokine secretion, xic effector molecule release, cytotoxic activity, and expression of activation markers. The T cell activating bispecific antigen binding molecules described are capable of inducing T cell activation. Suitable assays to measure T cell activation are known in the art described herein.

A “target cell antigen” as used herein refers to an antigenic determinant presented on the surface of a target cell, for example a cell in a tumor such as a cancer cell or a cell of the tumor stroma.

As used herein, the terms “first” and “second” with respect to antigen binding moieties etc., are used for convenience of distinguishing when there is more than one of each type of moiety. Use of these terms is not ed to confer a specific order or orientation of the T cell activating bispecific antigen binding molecule unless explicitly so stated.

A “Fab le” refers to a protein consisting of the VH and CH1 domain of the heavy chain (the “Fab heavy chain”) and the VL and CL domain of the light chain (the “Fab light chain”) of an immunoglobulin.

By ” is meant that the components (e.g. a Fab molecule and an Fc domain subunit) are linked by peptide bonds, either directly or via one or more peptide linkers.

As used herein, the term "single-chain" refers to a molecule comprising amino acid monomers linearly linked by peptide bonds. In certain embodiments, one of the n binding moieties is a -chain Fab le, i.e. a Fab molecule wherein the Fab light chain and the Fab heavy chain are connected by a peptide linker to form a single peptide chain. In a particular such embodiment, the C-terminus of the Fab light chain is connected to the N-terminus of the Fab heavy chain in the single-chain Fab molecule.

By a “crossover” Fab molecule (also termed “Crossfab”) is meant a Fab molecule wherein either the variable regions or the constant regions of the Fab heavy and light chain are exchanged, i.e. the crossover Fab molecule comprises a peptide chain composed of the light chain variable region and the heavy chain constant region, and a peptide chain composed of the heavy chain variable region and the light chain constant region. For clarity, in a crossover Fab molecule wherein the variable regions of the Fab light chain and the Fab heavy chain are exchanged, the peptide chain comprising the heavy chain constant region is ed to herein as the “heavy chain” of the crossover Fab molecule. Conversely, in a ver Fab molecule wherein the constant regions of the Fab light chain and the Fab heavy chain are exchanged, the e chain comprising the heavy chain variable region is referred to herein as the “heavy chain” of the crossover Fab molecule.

The term “immunoglobulin molecule” refers to a protein having the structure of a naturally occurring antibody. For example, immunoglobulins of the IgG class are heterotetrameric glycoproteins of about 0 daltons, composed of two light chains and two heavy chains that are disulfide-bonded. From N- to inus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), also called a heavy chain constant region. Similarly, from N- to C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a nt light (CL) domain, also called a light chain constant region. The heavy chain of an immunoglobulin may be assigned to one of five types, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which may be further divided into subtypes, e.g. γ1 (IgG1), γ2 (IgG2), γ3 , γ4 (IgG4), α1 (IgA1) and α2 (IgA2). The light chain of an immunoglobulin may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain. An globulin essentially consists of two Fab molecules and an Fc domain, linked via the immunoglobulin hinge region.

The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not d to monoclonal antibodies, polyclonal antibodies, and antibody fragments so long as they exhibit the desired antigen-binding activity.

An "antibody nt" refers to a molecule other than an intact dy that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab’-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g. scFv), and single-domain antibodies. For a review of certain antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For a review of scFv fragments, see e.g. Plückthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); see also WO 93/16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For sion of Fab and F(ab')2 fragments comprising salvage receptor binding epitope residues and having sed in vivo half-life, see U.S. Patent No. 5,869,046. Diabodies are antibody fragments with two antigenbinding sites that may be nt or bispecific. See, for example, EP 404,097; ; Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat Med 9, 129-134 (2003). Single-domain antibodies are antibody fragments sing all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see e.g. U.S. Patent No. 6,248,516 B1). dy fragments can be made by various techniques, ing but not limited to proteolytic ion of an intact antibody as well as production by recombinant host cells (e.g. E. coli or phage), as described herein.

The term "antigen binding domain" refers to the part of an antibody that comprises the area which specifically binds to and is complementary to part or all of an antigen. An n g domain may be provided by, for example, one or more antibody variable domains (also called antibody variable regions). ularly, an antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

The term “variable region” or “variable ” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, tively) of a native dy generally have similar structures, with each domain comprising four ved framework regions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 . A single VH or VL domain may be sufficient to confer antigen-binding specificity.

The term “hypervariable region” or “HVR”, as used herein, refers to each of the s of an antibody variable domain which are ariable in sequence and/or form structurally defined loops (“hypervariable loops”). Generally, native four-chain antibodies comprise six HVRs; three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generally comprise amino acid residues from the hypervariable loops and/or from the complementarity determining regions (CDRs), the latter being of highest sequence variability and/or ed in antigen recognition.

With the exception of CDR1 in VH, CDRs generally comprise the amino acid residues that form the hypervariable loops. Hypervariable regions (HVRs) are also referred to as “complementarity determining regions” (CDRs), and these terms are used herein interchangeably in reference to ns of the variable region that form the antigen binding regions. This particular region has been described by Kabat et al., U.S. Dept. of Health and Human Services, ces of Proteins of Immunological st (1983) and by Chothia et al., J Mol Biol 196:901-917 (1987), where the definitions include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The appropriate amino acid residues which ass the CDRs as defined by each of the above cited references are set forth below in Table 1 as a comparison. The exact residue numbers which encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the le region amino acid sequence of the antibody.

TABLE 1. CDR Definitions1 CDR Kabat Chothia AbM2 VH CDR1 31-35 26-32 26-35 VH CDR2 50-65 52-58 50-58 VH CDR3 95-102 95-102 95-102 VL CDR1 24-34 26-32 24-34 VL CDR2 50-56 50-52 50-56 VL CDR3 89-97 91-96 89-97 1 Numbering of all CDR definitions in Table 1 is according to the numbering conventions set forth by Kabat et al. (see below). 2 "AbM" with a lowercase “b” as used in Table 1 refers to the CDRs as defined by Oxford Molecular's "AbM" antibody modeling software.

Kabat et al. also defined a numbering system for variable region sequences that is applicable to any antibody. One of ordinary skill in the art can unambiguously assign this system of "Kabat numbering" to any variable region sequence, without reliance on any experimental data beyond the sequence itself. As used herein, "Kabat numbering" refers to the numbering system set forth by Kabat et al., U.S. Dept. of Health and Human Services, nce of Proteins of logical Interest" (1983). Unless otherwise specified, references to the numbering of specific amino acid residue ons in an antibody variable region are according to the Kabat numbering system.

The polypeptide sequences of the sequence listing (i.e., SEQ ID NOs 1, 3, 5, 7, 9, 11, 13, 15 etc.) are not numbered according to the Kabat ing system. However, it is well within the ordinary skill of one in the art to convert the ing of the sequences of the Sequence Listing to Kabat numbering.

"Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally ts of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The ” of an antibody or immunoglobulin refers to the type of constant domain or constant region possessed by its heavy chain. There are five major s of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, tively.

The term “Fc domain” or “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term es native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an IgG heavy chain might vary slightly, the human IgG heavy chain Fc region is usually defined to extend from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be t.

Unless otherwise specified herein, ing of amino acid residues in the Fc region or nt region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, al Institutes of Health, Bethesda, MD, 1991. A “subunit” of an Fc domain as used herein refers to one of the two polypeptides g the dimeric Fc domain, i.e. a polypeptide comprising C- terminal constant regions of an immunoglobulin heavy chain, capable of stable self-association.

For example, a subunit of an IgG Fc domain comprises an IgG CH2 and an IgG CH3 constant domain.

A “modification promoting the association of the first and the second subunit of the Fc domain” is a manipulation of the peptide backbone or the post-translational modifications of an Fc domain subunit that reduces or prevents the association of a polypeptide comprising the Fc domain subunit with an identical polypeptide to form a homodimer. A modification promoting association as used herein particularly includes separate modifications made to each of the two Fc domain subunits desired to associate (i.e. the first and the second subunit of the Fc domain), wherein the modifications are complementary to each other so as to promote association of the two Fc domain subunits. For e, a modification promoting association may alter the structure or charge of one or both of the Fc domain subunits so as to make their association sterically or electrostatically favorable, respectively. Thus, (hetero)dimerization occurs between a ptide comprising the first Fc domain subunit and a ptide comprising the second Fc domain subunit, which might be non-identical in the sense that r components fused to each of the subunits (e.g. antigen binding moieties) are not the same. In some embodiments the modification promoting association comprises an amino acid mutation in the Fc domain, specifically an amino acid substitution. In a particular embodiment, the modification promoting ation comprises a separate amino acid mutation, ically an amino acid substitution, in each of the two subunits of the Fc .

The term “effector functions” refers to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement ent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine ion, immune complex-mediated antigen uptake by n presenting cells, down regulation of cell surface receptors (e.g. B cell or), and B cell activation.

As used herein, the terms “engineer, engineered, engineering”, are ered to include any manipulation of the peptide backbone or the post-translational modifications of a naturally occurring or inant polypeptide or fragment thereof. Engineering es modifications of the amino acid sequence, of the glycosylation pattern, or of the side chain group of individual amino acids, as well as combinations of these approaches.

The term “amino acid mutation” as used herein is meant to encompass amino acid substitutions, deletions, insertions, and modifications. Any combination of substitution, deletion, insertion, and modification can be made to arrive at the final construct, provided that the final uct possesses the desired characteristics, e.g., reduced binding to an Fc receptor, or increased association with another peptide. Amino acid sequence deletions and insertions e amino- and/or carboxy-terminal deletions and insertions of amino acids. Particular amino acid mutations are amino acid substitutions. For the purpose of altering e.g. the binding characteristics of an Fc region, non-conservative amino acid substitutions, i.e. replacing one amino acid with another amino acid having different structural and/or chemical properties, are ularly preferred.

Amino acid tutions include replacement by non-naturally occurring amino acids or by naturally occurring amino acid derivatives of the twenty standard amino acids (e.g. 4- hydroxyproline, ylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene sis and the like. It is contemplated that methods of altering the side chain group of an amino acid by s other than genetic engineering, such as al modification, may also be useful. Various designations may be used herein to te the same amino acid mutation. For example, a substitution from proline at position 329 of the Fc domain to glycine can be indicated as 329G, G329, G329, P329G, or Pro329Gly.

As used herein, term "polypeptide" refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain of two or more amino acids, and does not refer to a specific length of the product.

Thus, peptides, dipeptides, tripeptides, oligopeptides, "protein," "amino acid chain," or any other term used to refer to a chain of two or more amino acids, are included within the definition of "polypeptide," and the term "polypeptide" may be used instead of, or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to the ts of post-expression modifications of the ptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide may be derived from a natural biological source or produced by inant technology, but is not necessarily translated from a designated nucleic acid sequence. It may be generated in any , including by al synthesis. A polypeptide bed may be of a size of about 3 or more, 5 or more, or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides may have a defined threedimensional structure, although they do not necessarily have such structure. Polypeptides with a defined three-dimensional structure are referred to as folded, and polypeptides which do not possess a defined three-dimensional structure, but rather can adopt a large number of different conformations, and are referred to as unfolded.

By an ted" polypeptide or a variant, or derivative thereof is intended a polypeptide that is not in its natural milieu. No particular level of purification is required. For example, an ed polypeptide can be removed from its native or natural environment. inantly produced polypeptides and proteins expressed in host cells are considered isolated for the purpose of thedescription, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.

“Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a ate ce that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid ce ty can be ed in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, 2, ALIGN or Megalign AR) software. Those skilled in the art can determine appropriate ters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison er program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The 2 program is publicly ble from Genentech, Inc., South San Francisco, California, or may be compiled from the source code.

The ALIGN-2 program should be ed for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence ty of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or ses a certain % amino acid ce identity to, with, or against a given amino acid ce B) is calculated as follows: 100 times the fraction X/Y where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program’s alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

The term "polynucleotide" refers to an isolated c acid molecule or construct, e.g. messenger RNA (mRNA), virally-derived RNA, or plasmid DNA (pDNA). A polynucleotide may comprise a conventional odiester bond or a non-conventional bond (e.g. an amide bond, such as found in peptide nucleic acids (PNA). The term "nucleic acid molecule" refers to any one or more nucleic acid segments, e.g. DNA or RNA fragments, present in a polynucleotide.

By "isolated" nucleic acid molecule or polynucleotide is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment. For example, a inant polynucleotide encoding a polypeptide contained in a vector is considered isolated for the es of the presentdescription. Further examples of an isolated polynucleotide include inant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution. An isolated cleotide includes a polynucleotide molecule contained in cells that ordinarily contain the cleotide molecule, but the polynucleotide molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location. Isolated RNA molecules include in vivo or in vitro RNA transcripts, as well as positive and negative strand forms, and -stranded forms.

Isolated polynucleotides or nucleic acids according to the present description further include such molecules produced synthetically. In addition, a polynucleotide or a nucleic acid may be or may include a regulatory element such as a promoter, ribosome binding site, or a transcription terminator.

By a nucleic acid or polynucleotide having a nucleotide sequence at least, for e, 95% "identical" to a reference tide ce described, it is intended that the tide ce of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the 5’ or 3’ terminal positions of the reference tide sequence or re between those terminal positions, interspersed either dually among residues in the reference sequence or in one or more contiguous groups within the reference sequence. As a practical matter, r any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence described can be determined conventionally using known er programs, such as the ones discussed above for polypeptides (e.g. ALIGN-2).

The term "expression te" refers to a polynucleotide generated inantly or synthetically, with a series of specified c acid elements that permit transcription of a particular c acid in a target cell. The recombinant expression cassette can be incorporated into a plasmid, chromosome, ondrial DNA, plastid DNA, virus, or nucleic acid fragment.

Typically, the inant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter. In certain embodiments, the expression cassette comprises polynucleotide sequences that encode bispecific antigen binding molecules described or fragments thereof.

The term “vector” or "expression vector" is synonymous with "expression construct" and refers to a DNA molecule that is used to introduce and direct the expression of a specific gene to which it is operably associated in a target cell. The term includes the vector as a self-replicating nucleic acid structure as well as the vector orated into the genome of a host cell into which it has been introduced. The expression vector described comprises an expression cassette. sion s allow transcription of large amounts of stable mRNA. Once the expression vector is inside the target cell, the ribonucleic acid molecule or protein that is encoded by the gene is produced by the cellular transcription and/or translation machinery. In one embodiment, the expression vector described comprises an expression cassette that comprises polynucleotide sequences that encode bispecific n binding molecules described or fragments thereof.

The terms "host cell", "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, ing the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom t regard to the number of passages.

Progeny may not be tely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as ed or selected for in the originally transformed cell are included herein. A host cell is any type of cellular system that can be used to generate the bispecific antigen binding molecules described.

Host cells include ed cells, e.g. mammalian cultured cells, such as CHO cells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, insect cells, and plant cells, to name only a few, but also cells comprised within a transgenic animal, transgenic plant or cultured plant or animal tissue.

An “activating Fc receptor” is an Fc receptor that following ment by an Fc domain of an antibody elicits signaling events that stimulate the receptor-bearing cell to perform effector functions. Human activating Fc receptors e FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89).

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism g to the lysis of antibody-coated target cells by immune or cells. The target cells are cells to which antibodies or derivatives thereof comprising an Fc region specifically bind, generally via the protein part that is inal to the Fc region. As used herein, the term ed ADCC” is defined as either a reduction in the number of target cells that are lysed in a given time, at a given concentration of antibody in the medium nding the target cells, by the mechanism of ADCC d above, and/or an se in the concentration of antibody in the medium surrounding the target cells, required to achieve the lysis of a given number of target cells in a given time, by the mechanism of ADCC. The ion in ADCC is ve to the ADCC mediated by the same antibody produced by the same type of host cells, using the same standard production, purification, formulation and storage s (which are known to those skilled in the art), but that has not been engineered. For example the reduction in ADCC ed by an antibody sing in its Fc domain an amino acid substitution that reduces ADCC, is relative to the ADCC mediated by the same antibody without this amino acid substitution in the Fc . Suitable assays to measure ADCC are well known in the art (see e.g. PCT publication no. or PCT patent application no. ).

An "effective amount" of an agent refers to the amount that is necessary to result in a physiological change in the cell or tissue to which it is administered.

A "therapeutically effective amount" of an agent, e.g. a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic . A therapeutically effective amount of an agent for example eliminates, decreases, delays, minimizes or prevents adverse effects of a disease.

An “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g. cows, sheep, cats, dogs, and horses), primates (e.g. humans and non- human primates such as monkeys), rabbits, and rodents (e.g. mice and rats). Particularly, the individual or subject is a human.

The term "pharmaceutical composition" refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are ptably toxic to a subject to which the formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical composition, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to clinical intervention in an attempt to alter the natural course of a disease in the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of ent include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of ms, diminishment of any direct or indirect pathological consequences of the e, ting metastasis, decreasing the rate of disease ssion, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, T cell activating bispecific antigen binding molecules described are used to delay development of a disease or to slow the progression of a disease.

The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic ts.

The term “comprising” as used in this specification means “consisting at least in part of”. When interpreting each statement in this specification that includes the term ising”, features other than that or those ed by the term may also be present. Related terms such as “comprise” and “comprises” are to be interpreted in the same manner.

Detailed Description of the Embodiments Described is a T cell activating bispecific antigen binding molecule comprising a first and a second antigen binding moiety, one of which is a Fab molecule capable of specific binding to an activating T cell antigen and the other one of which is a Fab molecule e of specific binding to a target cell antigen, and an Fc domain composed of a first and a second subunit capable of stable association; wherein the first antigen binding moiety is (a) a single chain Fab molecule n the Fab light chain and the Fab heavy chain are connected by a peptide linker, or (b) a crossover Fab molecule wherein either the variable or the constant regions of the Fab light chain and the Fab heavy chain are exchanged.

T cell ting bispecific antigen binding molecule formats The components of the T cell activating bispecific n binding molecule can be fused to each other in a variety of configurations. Exemplary urations are depicted in Figure 1.

In some ments, the second antigen g moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or the second subunit of the Fc domain.

In a particular such embodiment, the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding . In a specific such embodiment, the T cell activating bispecific n binding molecule essentially consists of a first and a second antigen binding moiety, an Fc domain composed of a first and a second subunit, and optionally one or more peptide linkers, wherein the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the inus of the Fab heavy chain of the second antigen binding moiety, and the second n binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or the second subunit of the Fc domain. In an even more specific embodiment, the first antigen binding moiety is a single chain Fab molecule. Alternatively, in a particular embodiment, the first antigen binding moiety is a crossover Fab molecule. Optionally, if the first antigen binding moiety is a crossover Fab molecule, the Fab light chain of the first antigen binding moiety and the Fab light chain of the second antigen binding moiety may additionally be fused to each other.

In an alternative such embodiment, the first n binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. In a specific such embodiment, the T cell activating bispecific antigen binding molecule essentially consists of a first and a second antigen binding moiety, an Fc domain composed of a first and a second subunit, and optionally one or more peptide linkers, wherein the first and the second antigen binding moiety are each fused at the C-terminus of the Fab heavy chain to the N- terminus of one of the subunits of the Fc domain. In an even more specific embodiment, the first antigen binding moiety is a single chain Fab molecule. Alternatively, in a particular embodiment, the first antigen binding moiety is a crossover Fab molecule.

In yet another such embodiment, the second antigen binding moiety is fused at the C-terminus of the Fab light chain to the N-terminus of the Fab light chain of the first antigen g moiety. In a specific such embodiment, the T cell activating ific n binding molecule essentially consists of a first and a second n binding moiety, an Fc domain composed of a first and a second subunit, and optionally one or more peptide linkers, wherein the first antigen binding moiety is fused at the N-terminus of the Fab light chain to the C-terminus of the Fab light chain of the second antigen binding moiety, and the second n binding moiety is fused at the C- us of the Fab heavy chain to the N-terminus of the first or the second subunit of the Fc domain. In an even more specific embodiment, the first antigen binding moiety is a crossover Fab molecule.

In other embodiments, the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain.

In a particular such embodiment, the second antigen g moiety is fused at the inus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first n binding moiety.

In a specific such embodiment, the T cell activating ific antigen binding molecule ially consists of a first and a second antigen binding moiety, an Fc domain ed of a first and a second subunit, and optionally one or more e linkers, wherein the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety, and the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or the second subunit of the Fc domain. In an even more specific embodiment, the first antigen g moiety is a crossover Fab molecule. Optionally, the Fab light chain of the first antigen binding moiety and the Fab light chain of the second antigen binding moiety may additionally be fused to each other.

In ular of these ments, the first antigen binding moiety is capable of specific binding to an ting T cell antigen. In other embodiments, the first antigen binding moiety is capable of specific binding to a target cell antigen.

The antigen binding moieties may be fused to the Fc domain or to each other directly or through a peptide linker, comprising one or more amino acids, typically about 2-20 amino acids. Peptide linkers are known in the art and are described herein. Suitable, non-immunogenic peptide linkers include, for e, (G4S)n, (SG4)n, (G4S)n or G4(SG4)n peptide linkers. “n” is generally a number between 1 and 10, typically between 2 and 4. A particularly suitable peptide linker for fusing the Fab light chains of the first and the second antigen binding moiety to each other is (G4S)2. An exemplary peptide linker suitable for connecting the Fab heavy chains of the first and the second antigen binding moiety is EPKSC(D)-(G4S)2 (SEQ ID NOs 150 and 151).

Additionally, linkers may comprise (a portion of) an immunoglobulin hinge region. Particularly where an antigen binding moiety is fused to the N-terminus of an Fc domain subunit, it may be fused via an immunoglobulin hinge region or a portion thereof, with or without an onal peptide linker.

A T cell activating bispecific antigen binding molecule with a single antigen binding moiety capable of specific binding to a target cell antigen (for example as shown in Figure 1A, 1B, 1D, 1E, 1H, 1I, 1K or 1M) is useful, particularly in cases where internalization of the target cell n is to be expected following binding of a high affinity antigen binding moiety. In such cases, the presence of more than one antigen binding moiety specific for the target cell n may e internalization of the target cell n, thereby reducing its availablity.

In many other cases, however, it will be advantageous to have a T cell activating bispecific antigen binding molecule comprising two or more antigen binding moieties specific for a target cell antigen (see examples in shown in Figure 1C, 1F, 1G, 1J or 1L), for example to optimize targeting to the target site or to allow crosslinking of target cell antigens.

Accordingly, in certain embodiments, the T cell activating bispecific antigen g molecule described further comprises a third antigen binding moiety which is a Fab molecule capable of ic binding to a target cell antigen. In one embodiment, the third antigen g moiety is capable of ic g to the same target cell antigen as the first or second antigen binding moiety. In a particular embodiment, the first n binding moiety is capable of specific binding to an ting T cell antigen, and the second and third antigen binding moieties are capable of ic binding to a target cell antigen.

In one embodiment, the third antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. In a particular embodiment, the second and the third antigen binding moiety are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain, and the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding moiety. In one such embodiment the first antigen binding moiety is a single chain Fab molecule. In a particular such embodiment the first antigen g moiety is a crossover Fab molecule. Optionally, if the first antigen binding moiety is a crossover Fab molecule, the Fab light chain of the first antigen binding moiety and the Fab light chain of the second antigen binding moiety may additionally be fused to each other.

The second and the third antigen g moiety may be fused to the Fc domain directly or through a peptide linker. In a particular ment the second and the third antigen binding moiety are each fused to the Fc domain through an immunoglobulin hinge region. In a specific ment, the immunoglobulin hinge region is a human IgG1 hinge region. In one embodiment the second and the third antigen binding moiety and the Fc domain are part of an immunoglobulin molecule. In a particular embodiment the immunoglobulin le is an IgG class immunoglobulin. In an even more particular embodiment the immunoglobulin is an IgG1 subclass immunoglobulin. In another embodiment the immunoglobulin is an IgG4 subclass immunoglobulin. In a further particular embodiment the immunoglobulin is a human immunoglobulin. In other embodiments the immunoglobulin is a chimeric immunoglobulin or a humanized immunoglobulin. In one embodiment, the T cell activating bispecific antigen binding molecule essentially consists of an immunoglobulin molecule capable of specific binding to a target cell antigen, and an antigen binding moiety capable of ic binding to an activating T cell antigen wherein the antigen binding moiety is a single chain Fab molecule or a crossover Fab molecule, particularly a crossover Fab le, fused to the N-terminus of one of the immunoglobulin heavy chains, optionally via a peptide linker.

In an alternative embodiment, the first and the third antigen binding moiety are each fused at the C-terminus of the Fab heavy chain to the inus of one of the subunits of the Fc domain, and the second n binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety. In a specific such embodiment, the T cell activating bispecific antigen binding molecule essentially ts of a first, a second and a third antigen binding , an Fc domain composed of a first and a second subunit, and ally one or more peptide linkers, wherein the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety, and the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and wherein the third antigen binding moiety is fused at the inus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain. In a particular such embodiment the first antigen binding moiety is a ver Fab molecule. Optionally, the Fab light chain of the first antigen g moiety and the Fab light chain of the second antigen binding moiety may additionally be fused to each other.

In some of the T cell activating bispecific antigen binding molecule described, the Fab light chain of the first antigen binding moiety and the Fab light chain of the second antigen binding moiety are fused to each other, optionally via a linker peptide. Depending on the configuration of the first and the second antigen binding moiety, the Fab light chain of the first antigen binding moiety may be fused at its C-terminus to the N-terminus of the Fab light chain of the second antigen binding , or the Fab light chain of the second antigen binding moiety may be fused at its C-terminus to the N-terminus of the Fab light chain of the first n g moiety. Fusion of the Fab light chains of the first and the second antigen binding moiety further reduces mispairing of unmatched Fab heavy and light chains, and also reduces the number of plasmids needed for expression of some of the T cell activating bispecific antigen binding molecules described.

In n ments the T cell activating bispecific antigen binding molecule comprises a polypeptide wherein a first Fab light chain shares a carboxy-terminal peptide bond with a peptide linker, which in turn shares a carboxy-terminal peptide bond with a first Fab heavy chain, which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VL-CL-linker-VHCH1-CH2-CH2 (-CH4)), and a polypeptide wherein a second Fab heavy chain shares a carboxyterminal peptide bond with an Fc domain subunit (VH-CH1-CH2-CH3(-CH4)). In some embodiments the T cell activating bispecific antigen binding molecule further comprises a second Fab light chain polypeptide (VL-CL). In certain embodiments the polypeptides are covalently linked, e.g., by a disulfide bond.

In some embodiments, the T cell ting ific antigen binding molecule comprises a polypeptide wherein a first Fab light chain shares a carboxy-terminal peptide bond with a peptide linker, which in turn shares a carboxy-terminal peptide bond with a first Fab heavy chain, which in turn shares a carboxy-terminal peptide bond with a second Fab heavy chain, which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VL-CL-linker-VH-CH1- VH-CH1-CH2-CH3(-CH4)). In one of these embodiments that T cell activating bispecific antigen g molecule further comprises a second Fab light chain polypeptide (VL-CL). The T cell ting bispecific antigen binding molecule according to these embodiments may further se (i) an Fc domain subunit polypeptide (CH2-CH3(-CH4)), or (ii) a polypeptide wherein a third Fab heavy chain shares a carboxy-terminal e bond with an Fc domain subunit (VH-CH1-CH2-CH3(-CH4)) and a third Fab light chain polypeptide ). In n embodiments the polypeptides are covalently linked, e.g., by a disulfide bond.

In certain embodiments the T cell activating bispecific antigen binding molecule comprises a polypeptide wherein a first Fab light chain variable region shares a y-terminal peptide bond with a first Fab heavy chain constant region (i.e. a ver Fab heavy chain, wherein the heavy chain variable region is replaced by a light chain variable region), which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VL-CH1-CH2-CH2(-CH4)), and a polypeptide wherein a second Fab heavy chain shares a carboxy-terminal e bond with an Fc domain subunit (VH-CH1-CH2-CH3(-CH4)). In some ments the T cell activating bispecific antigen binding molecule further comprises a ptide wherein a Fab heavy chain variable region shares a carboxy-terminal e bond with a Fab light chain constant region (VH-CL) and a Fab light chain polypeptide (VL-CL). In certain embodiments the polypeptides are covalently linked, e.g., by a disulfide bond.

In alternative ments the T cell activating bispecific antigen binding molecule comprises a polypeptide wherein a first Fab heavy chain variable region shares a carboxy-terminal peptide bond with a first Fab light chain constant region (i.e. a crossover Fab heavy chain, wherein the heavy chain constant region is replaced by a light chain constant ), which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VH-CL-CH2-CH2(-CH4)), and a polypeptide wherein a second Fab heavy chain shares a carboxy-terminal peptide bond with an Fc domain subunit (VH-CH1-CH2-CH3(-CH4)). In some embodiments the T cell activating bispecific antigen g molecule further ses a polypeptide wherein a Fab light chain variable region shares a carboxy-terminal peptide bond with a Fab heavy chain constant region (VL-CH1) and a Fab light chain polypeptide (VL-CL). In certain embodiments the polypeptides are covalently linked, e.g., by a ide bond.

In some embodiments, the T cell activating bispecific antigen binding molecule comprises a polypeptide wherein a first Fab light chain variable region shares a carboxy-terminal peptide bond with a first Fab heavy chain constant region (i.e. a crossover Fab heavy chain, wherein the heavy chain le region is replaced by a light chain variable region), which in turn shares a carboxy-terminal peptide bond with a second Fab heavy chain, which in turn shares a carboxyterminal e bond with an Fc domain subunit (VL-CH1-VH-CH1-CH2-CH3(-CH4)). In other embodiments, the T cell activating bispecific antigen binding le comprises a polypeptide n a first Fab heavy chain variable region shares a carboxy-terminal peptide bond with a first Fab light chain constant region (i.e. a crossover Fab heavy chain, wherein the heavy chain constant region is replaced by a light chain nt ), which in turn shares a carboxy-terminal peptide bond with a second Fab heavy chain, which in turn shares a carboxy- terminal peptide bond with an Fc domain t (VH-CL-VH-CH1-CH2-CH3(-CH4)). In still other embodiments, the T cell activating bispecific antigen binding molecule comprises a polypeptide wherein a second Fab heavy chain shares a carboxy-terminal peptide bond with a first Fab light chain variable region which in turn shares a carboxy-terminal peptide bond with a first Fab heavy chain constant region (i.e. a ver Fab heavy chain, wherein the heavy chain variable region is replaced by a light chain variable region), which in turn shares a carboxyterminal peptide bond with an Fc domain t (VH-CH1-VL-CH1-CH2-CH3(-CH4)). In other embodiments, the T cell activating bispecific antigen binding molecule comprises a polypeptide wherein a second Fab heavy chain shares a carboxy-terminal peptide bond with a first Fab heavy chain le region which in turn shares a carboxy-terminal peptide bond with a first Fab light chain constant region (i.e. a crossover Fab heavy chain, wherein the heavy chain constant region is replaced by a light chain constant region), which in turn shares a carboxyterminal peptide bond with an Fc domain subunit (VH-CH1-VH-CL-CH2-CH3(-CH4)).

In some of these embodiments the T cell activating bispecific antigen binding molecule further comprises a crossover Fab light chain polypeptide, wherein a Fab heavy chain variable region shares a carboxy-terminal peptide bond with a Fab light chain constant region (VH-CL), and a Fab light chain ptide (VL-CL). In others of these embodiments the T cell activating bispecific antigen binding molecule further comprises a crossover Fab light chain polypeptide, wherein a Fab light chain variable region shares a carboxy-terminal peptide bond with a Fab heavy chain constant region (VL-CH1), and a Fab light chain polypeptide (VL-CL). In still others of these embodiments the T cell activating bispecific antigen binding molecule further ses a polypeptide wherein a Fab light chain variable region shares a carboxy-terminal peptide bond with a Fab heavy chain constant region which in turn shares a carboxy-terminal peptide bond with a Fab light chain polypeptide (VL-CH1-VL-CL), a ptide wherein a Fab heavy chain variable region shares a carboxy-terminal peptide bond with a Fab light chain constant region which in turn shares a carboxy-terminal e bond with a Fab light chain polypeptide (VH-CL-VL-CL), a polypeptide wherein a Fab light chain polypeptide shares a carboxy-terminal peptide bond with a Fab light chain le region which in turn shares a carboxy-terminal peptide bond with a Fab heavy chain constant region (VL-CL-VL-CH1), or a polypeptide wherein a Fab light chain polypeptide shares a carboxy-terminal peptide bond with a Fab heavy chain variable region which in turn shares a carboxy-terminal peptide bond with a Fab light chain constant region (VL-CL-VH-CL).

The T cell activating bispecific n binding molecule according to these embodiments may further comprise (i) an Fc domain subunit ptide (CH2-CH3(-CH4)), or (ii) a ptide wherein a third Fab heavy chain shares a carboxy-terminal e bond with an Fc domain subunit (VH-CH1-CH2-CH3(-CH4)) and a third Fab light chain polypeptide ). In certain embodiments the polypeptides are ntly linked, e.g., by a disulfide bond.

In one embodiment, the T cell ting bispecific antigen binding molecule comprises a polypeptide wherein a second Fab light chain shares a carboxy-terminal peptide bond with a first Fab light chain variable region which in turn shares a carboxy-terminal peptide bond with a first Fab heavy chain constant region (i.e. a crossover Fab light chain, wherein the light chain constant region is replaced by a heavy chain constant region) -VL-CH1), a polypeptide n a second Fab heavy chain shares a carboxy-terminal peptide bond with an Fc domain subunit (VH-CH1-CH2-CH3(-CH4)), and a polypeptide wherein a first Fab heavy chain variable region shares a carboxy-terminal peptide bond with a first Fab light chain constant region (VHCL ). In another embodiment, the T cell activating bispecific antigen binding molecule comprises a polypeptide wherein a second Fab light chain shares a carboxy-terminal peptide bond with a first Fab heavy chain variable region which in turn shares a carboxy-terminal peptide bond with a first Fab light chain constant region (i.e. a crossover Fab light chain, wherein the light chain variable region is replaced by a heavy chain variable region) -VH-CL), a polypeptide wherein a second Fab heavy chain shares a carboxy-terminal peptide bond with an Fc domain subunit (VH-CH1-CH2-CH3(-CH4)), and a polypeptide wherein a first Fab light chain variable region shares a carboxy-terminal peptide bond with a first Fab heavy chain constant region (VL- CH1). The T cell activating bispecific antigen binding molecule according to these embodiments may further comprise (i) an Fc domain subunit polypeptide (CH2-CH3(-CH4)), or (ii) a polypeptide wherein a third Fab heavy chain shares a carboxy-terminal e bond with an Fc domain subunit (VH-CH1-CH2-CH3(-CH4)) and a third Fab light chain polypeptide (VL-CL).

In certain ments the polypeptides are covalently linked, e.g., by a disulfide bond.

According to any of the above embodiments, ents of the T cell activating bispecific antigen binding molecule (e.g. antigen binding moiety, Fc domain) may be fused directly or through s linkers, ularly peptide linkers comprising one or more amino acids, typically about 2-20 amino acids, that are described herein or are known in the art. Suitable, nonimmunogenic peptide linkers include, for example, , , (G4S)n or G4(SG4)n peptide linkers, wherein n is generally a number between 1 and 10, typically n 2 and 4.

Fc domain The Fc domain of the T cell activating bispecific antigen binding molecule consists of a pair of polypeptide chains comprising heavy chain domains of an immunoglobulin le. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, each subunit of which comprises the CH2 and CH3 IgG heavy chain constant domains. The two subunits of the Fc domain are capable of stable association with each other. In one embodiment the T cell activating bispecific antigen binding molecule described comprises not more than one Fc domain.

In one embodiment the Fc domain of the T cell activating bispecific n binding molecule is an IgG Fc domain. In a particular ment the Fc domain is an IgG1 Fc domain. In another embodiment the Fc domain is an IgG4 Fc domain. In a more specific embodiment, the Fc domain is an IgG4 Fc domain comprising an amino acid substitution at position S228 (EU numbering), particularly the amino acid substitution S228P. This amino acid tution reduces in vivo Fab arm exchange of IgG4 antibodies (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). In a further particular embodiment the Fc domain is human. An exemplary sequence of a human IgG1 Fc region is given in SEQ ID NO: 149.

Fc domain modifications promoting heterodimerization T cell activating bispecific antigen binding molecules described comprise different antigen binding moieties, fused to one or the other of the two subunits of the Fc domain, thus the two subunits of the Fc domain are typically comprised in two non-identical polypeptide chains.

Recombinant co-expression of these polypeptides and subsequent dimerization leads to l possible combinations of the two polypeptides. To improve the yield and purity of T cell activating bispecific antigen binding molecules in recombinant production, it will thus be advantageous to introduce in the Fc domain of the T cell activating bispecific antigen binding molecule a modification promoting the association of the desired polypeptides.

Accordingly, in particular embodiments the Fc domain of the T cell activating bispecific antigen binding molecule described comprises a modification promoting the association of the first and the second subunit of the Fc domain. The site of most ive protein-protein interaction between the two ts of a human IgG Fc domain is in the CH3 domain of the Fc domain.

Thus, in one embodiment said cation is in the CH3 domain of the Fc domain.

In a specific embodiment said modification is a so-called “knob-into-hole” modification, comprising a “knob” modification in one of the two subunits of the Fc domain and a “hole” cation in the other one of the two ts of the Fc .

The knob-into-hole technology is described e.g. in US 5,731,168; US 936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J l Meth 248, 7-15 (2001). Generally, the method es introducing a protuberance (“knob”) at the interface of a first polypeptide and a corresponding cavity (“hole”) in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote dimer formation and hinder homodimer formation. erances are ucted by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g. tyrosine or tryptophan).

Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). ingly, in a particular embodiment, in the CH3 domain of the first subunit of the Fc domain of the T cell activating bispecific antigen binding molecule an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain of the first subunit which is onable in a cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain an amino acid residue is replaced with an amino acid residue having a smaller side chain , thereby generating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit is positionable.

The protuberance and cavity can be made by altering the nucleic acid encoding the polypeptides, e.g. by site-specific mutagenesis, or by peptide synthesis.

In a specific embodiment, in the CH3 domain of the first subunit of the Fc domain the threonine residue at position 366 is replaced with a phan residue (T366W), and in the CH3 domain of the second subunit of the Fc domain the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In one embodiment, in the second subunit of the Fc domain additionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A).

In yet a further embodiment, in the first subunit of the Fc domain additionally the serine residue at on 354 is replaced with a cysteine e (S354C), and in the second subunit of the Fc domain additionally the tyrosine residue at position 349 is ed by a cysteine e (Y349C). Introduction of these two cysteine es results in formation of a ide bridge between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

In a particular embodiment the antigen binding moiety e of binding to an activating T cell antigen is fused (optionally via the antigen binding moiety capable of binding to a target cell antigen) to the first subunit of the Fc domain (comprising the “knob” modification). Without wishing to be bound by theory, fusion of the n binding moiety capable of binding to an activating T cell n to the knob-containing subunit of the Fc domain will (further) minimize the generation of antigen binding molecules comprising two antigen binding moieties capable of binding to an activating T cell antigen (steric clash of two knob-containing ptides).

In an alternative embodiment a cation promoting association of the first and the second t of the Fc domain comprises a modification mediating electrostatic steering effects, e.g. as described in PCT publication . Generally, this method involves replacement of one or more amino acid residues at the interface of the two Fc domain subunits by charged amino acid residues so that homodimer formation becomes electrostatically unfavorable but heterodimerization electrostatically favorable.

Fc domain modifications reducing Fc receptor binding and/or effector on The Fc domain s to the T cell activating bispecific antigen binding molecule favorable pharmacokinetic properties, including a long serum half-life which butes to good accumulation in the target tissue and a favorable tissue-blood distribution ratio. At the same time it may, however, lead to undesirable targeting of the T cell ting bispecific antigen binding molecule to cells expressing Fc ors rather than to the preferred antigen-bearing cells.

Moreover, the co-activation of Fc receptor signaling pathways may lead to cytokine release which, in combination with the T cell activating properties and the long half-life of the antigen binding molecule, results in excessive activation of cytokine receptors and severe side effects upon ic administration. Activation of (Fc receptor-bearing) immune cells other than T cells may even reduce cy of the T cell activating bispecific antigen g molecule due to the potential destruction of T cells e.g. by NK cells.

Accordingly, in particular embodiments the Fc domain of the T cell ting bispecific antigen binding molecules described exhibits reduced binding affinity to an Fc or and/or reduced effector function, as compared to a native IgG1 Fc domain. In one such embodiment the Fc domain (or the T cell activating bispecific n binding molecule comprising said Fc domain) exhibits less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% of the binding affinity to an Fc receptor, as compared to a native IgG1 Fc domain (or a T cell activating bispecific antigen binding molecule comprising a native IgG1 Fc domain), and/or less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% of the effector function, as compared to a native IgG1 Fc domain domain (or a T cell activating bispecific antigen binding le comprising a native IgG1 Fc domain). In one embodiment, the Fc domain domain (or the T cell activating bispecific antigen binding molecule comprising said Fc domain) does not ntially bind to an Fc receptor and/or induce effector function. In a particular embodiment the Fc receptor is an Fcγ receptor. In one embodiment the Fc receptor is a human Fc receptor. In one embodiment the Fc receptor is an activating Fc receptor. In a specific embodiment the Fc receptor is an activating human Fcγ or, more specifically human FcγRIIIa, FcγRI or FcγRIIa, most ically human FcγRIIIa. In one embodiment the effector function is one or more selected from the group of CDC, ADCC, ADCP, and cytokine secretion. In a particular embodiment the effector function is ADCC. In one embodiment the Fc domain domain exhibits substantially similar binding affinity to neonatal Fc receptor , as compared to a native IgG1 Fc domain domain. Substantially similar binding to FcRn is achieved when the Fc domain (or the T cell activating bispecific n binding molecule comprising said Fc domain) ts greater than about 70%, particularly greater than about 80%, more particularly greater than about 90% of the binding affinity of a native IgG1 Fc domain (or the T cell activating ific antigen binding molecule sing a native IgG1 Fc domain) to FcRn.

In certain embodiments the Fc domain is engineered to have reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a non-engineered Fc domain. In ular embodiments, the Fc domain of the T cell activating bispecific antigen binding molecule comprises one or more amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor and/or effector on. Typically, the same one or more amino acid mutation is present in each of the two subunits of the Fc domain. In one embodiment the amino acid on reduces the binding affinity of the Fc domain to an Fc receptor. In one embodiment the amino acid on reduces the binding affinity of the Fc domain to an Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold. In embodiments where there is more than one amino acid mutation that reduces the binding affinity of the Fc domain to the Fc receptor, the combination of these amino acid mutations may reduce the binding ty of the Fc domain to an Fc receptor by at least 10-fold, at least 20-fold, or even at least 50-fold. In one embodiment the T cell activating bispecific antigen binding molecule comprising an engineered Fc domain exhibits less than 20%, particularly less than 10%, more particularly less than 5% of the binding affinity to an Fc receptor as compared to a T cell activating bispecific n binding molecule comprising a non-engineered Fc domain. In a particular embodiment the Fc receptor is an Fcγ receptor. In some embodiments the Fc or is a human Fc receptor. In some embodiments the Fc receptor is an activating Fc receptor. In a specific embodiment the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, most specifically human FcγRIIIa. Preferably, binding to each of these receptors is d. In some embodiments binding affinity to a complement component, specifically binding affinity to C1q, is also reduced. In one embodiment g affinity to neonatal Fc receptor (FcRn) is not reduced. Substantially similar g to FcRn, i.e. preservation of the g ty of the Fc domain to said receptor, is achieved when the Fc domain (or the T cell ting bispecific antigen binding molecule sing said Fc ) exhibits greater than about 70% of the binding affinity of a non-engineered form of the Fc domain (or the T cell activating bispecific antigen binding molecule comprising said non-engineered form of the Fc domain) to FcRn. The Fc domain, or T cell activating bispecific n g molecules described comprising said Fc domain, may exhibit greater than about 80% and even greater than about 90% of such affinity. In certain embodiments the Fc domain of the T cell ting bispecific antigen binding molecule is engineered to have d effector function, as ed to a non-engineered Fc domain. The reduced effector function can include, but is not limited to, one or more of the following: reduced complement dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune x-mediated antigen uptake by antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, d binding to polymorphonuclear cells, reduced direct signaling inducing apoptosis, reduced crosslinking of target-bound antibodies, reduced dendritic cell maturation, or reduced T cell priming. In one embodiment the reduced effector function is one or more selected from the group of reduced CDC, reduced ADCC, reduced ADCP, and reduced cytokine secretion. In a particular embodiment the reduced effector function is reduced ADCC. In one embodiment the reduced ADCC is less than 20% of the ADCC induced by a non-engineered Fc domain (or a T cell activating bispecific antigen binding le comprising a non-engineered Fc domain).

In one embodiment the amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor and/or effector function is an amino acid substitution. In one embodiment the Fc domain comprises an amino acid substitution at a position selected from the group of E233, L234, L235, N297, P331 and P329. In a more specific embodiment the Fc domain comprises an amino acid substitution at a position selected from the group of L234, L235 and P329. In some embodiments the Fc domain comprises the amino acid substitutions L234A and L235A. In one such embodiment, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. In one embodiment the Fc domain comprises an amino acid substitution at position P329. In a more specific embodiment the amino acid substitution is P329A or P329G, particularly P329G. In one embodiment the Fc domain ses an amino acid substitution at position P329 and a further amino acid substitution at a position selected from E233, L234, L235, N297 and P331. In a more specific embodiment the r amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S. In particular ments the Fc domain comprises amino acid substitutions at positions P329, L234 and L235. In more particular ments the Fc domain comprises the amino acid mutations L234A, L235A and P329G (“P329G LALA”). In one such embodiment, the Fc domain is an IgG1 Fc domain, ularly a human IgG1 Fc domain. The “P329G LALA” ation of amino acid substitutions almost completely abolishes Fcγ receptor binding of a human IgG1 Fc domain, as described in PCT patent application no. , orated herein by reference in its entirety. also describes methods of preparing such mutant Fc domains and methods for determining its properties such as Fc receptor binding or effector functions.

IgG4 antibodies exhibit reduced binding affinity to Fc receptors and reduced effector functions as compared to IgG1 antibodies. Hence, in some ments the Fc domain of the T cell activating ific antigen binding les described is an IgG4 Fc domain, particularly a human IgG4 Fc domain. In one embodiment the IgG4 Fc domain ses amino acid substitutions at position S228, specifically the amino acid substitution S228P. To further reduce its binding affinity to an Fc receptor and/or its or function, in one ment the IgG4 Fc domain comprises an amino acid substitution at on L235, specifically the amino acid substitution L235E. In another embodiment, the IgG4 Fc domain comprises an amino acid substitution at position P329, specifically the amino acid substitution P329G. In a particular embodiment, the IgG4 Fc domain comprises amino acid substitutions at positions S228, L235 and P329, ically amino acid tutions S228P, L235E and P329G. Such IgG4 Fc domain mutants and their Fcγ receptor binding properties are described in PCT patent application no. , incorporated herein by nce in its entirety.

In a particular embodiment the Fc domain exhibiting reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a native IgG1 Fc domain, is a human IgG1 Fc domain comprising the amino acid tutions L234A, L235A and optionally P329G, or a human IgG4 Fc domain sing the amino acid substitutions S228P, L235E and optionally P329G.

In certain embodiments N-glycosylation of the Fc domain has been ated. In one such embodiment the Fc domain comprises an amino acid mutation at position N297, particularly an amino acid substitution replacing asparagine by alanine (N297A) or aspartic acid (N297D).

In addition to the Fc domains described hereinabove and in PCT patent application no.

, Fc domains with d Fc receptor g and/or effector function also include those with substitution of one or more of Fc domain residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056). Such Fc mutants include Fc s with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581).

Mutant Fc domains can be prepared by amino acid deletion, substitution, insertion or modification using genetic or chemical methods well known in the art. Genetic methods may include site-specific nesis of the encoding DNA sequence, PCR, gene synthesis, and the like. The correct nucleotide changes can be verified for e by sequencing.

Binding to Fc receptors can be easily determined e.g. by ELISA, or by Surface Plasmon Resonance (SPR) using standard instrumentation such as a BIAcore instrument (GE Healthcare), and Fc receptors such as may be obtained by recombinant expression. A suitable such binding assay is described herein. Alternatively, binding affinity of Fc domains or cell activating bispecific n g molecules sing an Fc domain for Fc receptors may be evaluated using cell lines known to express particular Fc ors, such as human NK cells expressing FcγIIIa receptor.

Effector function of an Fc domain, or a T cell activating bispecific antigen binding molecule comprising an Fc domain, can be measured by methods known in the art. A suitable assay for measuring ADCC is described herein. Other examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362; Hellstrom et al. Proc Natl Acad Sci USA 83, 7059-7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499- 1502 (1985); U.S. Patent No. 5,821,337; Bruggemann et al., J Exp Med 166, 361 (1987).

Alternatively, non-radioactive assays methods may be employed (see, for example, ACTI™ nonradioactive cytotoxicity assay for flow try (CellTechnology, Inc. Mountain View, CA); and CytoTox 96® non-radioactive cytotoxicity assay (Promega, n, WI)). Useful effector cells for such assays e peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of st may be assessed in vivo, e.g. in a animal model such as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).

In some embodiments, binding of the Fc domain to a complement component, specifically to C1q, is reduced. Accordingly, in some embodiments wherein the Fc domain is engineered to have reduced effector function, said reduced effector function includes reduced CDC. C1q binding assays may be carried out to determine whether the T cell activating bispecific antigen binding molecule is able to bind C1q and hence has CDC activity. See e.g., C1q and C3c binding ELISA in WO 29879 and . To assess complement activation, a CDC assay may be performed (see, for e, Gazzano-Santoro et al., J Immunol s 202, 163 (1996); Cragg et al., Blood 101, 1045-1052 (2003); and Cragg and Glennie, Blood 103, 2738- 2743 (2004)).

Antigen g Moieties The antigen binding molecule described is bispecific, i.e. it comprises at least two antigen binding moieties e of specific binding to two distinct antigenic determinants. According to the description, the antigen binding moieties are Fab molecules (i.e. antigen binding domains composed of a heavy and a light chain, each comprising a variable and a constant region). In one embodiment said Fab molecules are human. In another embodiment said Fab molecules are humanized. In yet another embodiment said Fab molecules comprise human heavy and light chain constant regions.

At least one of the n g moieties is a single chain Fab molecule or a crossover Fab molecule. Such modifications prevent mispairing of heavy and light chains from different Fab molecules, thereby improving the yield and purity of the T cell activating ific antigen binding molecule described in recombinant production. In a particular single chain Fab molecule useful for the T cell activating bispecific antigen binding le described, the C-terminus of the Fab light chain is connected to the N-terminus of the Fab heavy chain by a peptide linker.

The peptide linker allows arrangement of the Fab heavy and light chain to form a functional antigen binding moiety. Peptide linkers le for connecting the Fab heavy and light chain include, for e, -GG (SEQ ID NO: 152) or (SG3)2-(SEG3)4-(SG3)-SG (SEQ ID NO: 153). In a particular ver Fab molecule useful for the T cell activating bispecific antigen g molecule described, the constant regions of the Fab light chain and the Fab heavy chain are exchanged. In r crossover Fab le useful for the T cell activating bispecific antigen g molecule described, the variable regions of the Fab light chain and the Fab heavy chain are exchanged.

In a particular embodiment described, the T cell activating bispecific antigen binding molecule is e of simultaneous binding to a target cell antigen, particularly a tumor cell antigen, and an activating T cell n. In one ment, the T cell activating bispecific antigen binding le is capable of crosslinking a T cell and a target cell by simultaneous binding to a target cell antigen and an activating T cell antigen. In an even more particular ment, such simultaneous binding results in lysis of the target cell, particularly a tumor cell. In one embodiment, such simultaneous binding results in tion of the T cell. In other embodiments, such aneous binding results in a cellular response of a T lymphocyte, particularly a cytotoxic T lymphocyte, selected from the group of: proliferation, differentiation, cytokine secretion, xic effector molecule release, cytotoxic activity, and expression of activation s. In one ment, binding of the T cell activating bispecific antigen binding molecule to the activating T cell antigen without simultaneous binding to the target cell antigen does not result in T cell activation.

In one embodiment, the T cell activating bispecific antigen binding molecule is capable of cting cytotoxic activity of a T cell to a target cell. In a particular embodiment, said re- direction is independent of MHC-mediated peptide antigen tation by the target cell and and/or specificity of the T cell.

Particularly, a T cell according to any of the embodiments described is a cytotoxic T cell. In some embodiments the T cell is a CD4+ or a CD8+ T cell, particularly a CD8+ T cell.

Activating T cell antigen binding moiety The T cell activating bispecific antigen binding molecule described comprises at least one antigen binding moiety capable of binding to an activating T cell antigen (also referred to herein as an “activating T cell antigen binding moiety”). In a particular embodiment, the T cell activating bispecific antigen binding molecule comprises not more than one antigen binding moiety capable of specific binding to an activating T cell antigen. In one embodiment the T cell activating bispecific antigen binding molecule provides monovalent binding to the activating T cell antigen. The activating T cell antigen g moiety can either be a conventional Fab molecule or a modified Fab molecule, i.e. a single chain or crossover Fab molecule. In embodiments where there is more than one antigen binding moiety capable of specific binding to a target cell antigen comprised in the T cell ting bispecific antigen binding molecule, the antigen binding moiety capable of specific binding to an activating T cell n preferably is a modified Fab molecule.

In a particular embodiment the activating T cell antigen is CD3, particularly human CD3 (SEQ ID NO: 265) or cynomolgus CD3 (SEQ ID NO: 266), most particularly human CD3. In a particular embodiment the activating T cell antigen binding moiety is cross-reactive for (i.e. ically binds to) human and cynomolgus CD3. In some embodiments, the activating T cell antigen is the n subunit of CD3.

In one embodiment, the activating T cell antigen binding moiety can compete with monoclonal antibody H2C (described in PCT publication no. WO2008/119567) for binding an epitope of CD3. In another embodiment, the activating T cell antigen binding moiety can compete with monoclonal antibody V9 (described in Rodrigues et al., Int J Cancer Suppl 7, 45-50 (1992) and US patent no. 6,054,297) for binding an e of CD3. In yet another embodiment, the ting T cell n binding moiety can compete with monoclonal dy FN18 (described in Nooij et al., Eur J Immunol 19, 981-984 (1986)) for binding an epitope of CD3. In a particular embodiment, the activating T cell n binding moiety can compete with monoclonal antibody SP34 (described in Pessano et al., EMBO J 4, 337-340 (1985)) for binding an epitope of CD3. In one embodiment, the activating T cell antigen binding moiety binds to the same epitope of CD3 as monoclonal antibody SP34. In one embodiment, the activating T cell antigen binding moiety comprises the heavy chain CDR1 of SEQ ID NO: 163, the heavy chain CDR2 of SEQ ID NO: 165, the heavy chain CDR3 of SEQ ID NO: 167, the light chain CDR1 of SEQ ID NO: 171, the light chain CDR2 of SEQ ID NO: 173, and the light chain CDR3 of SEQ ID NO: 175. In a further ment, the activating T cell antigen binding moiety comprises a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 169 and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 177, or variants thereof that retain functionality.

In a particular ment, the activating T cell antigen binding moiety comprises the heavy chain CDR1 of SEQ ID NO: 249, the heavy chain CDR2 of SEQ ID NO: 251, the heavy chain CDR3 of SEQ ID NO: 253, the light chain CDR1 of SEQ ID NO: 257, the light chain CDR2 of SEQ ID NO: 259, and the light chain CDR3 of SEQ ID NO: 261. In one embodiment, the activating T cell antigen binding moiety can compete for binding an epitope of CD3 with an antigen g moiety comprising the heavy chain CDR1 of SEQ ID NO: 249, the heavy chain CDR2 of SEQ ID NO: 251, the heavy chain CDR3 of SEQ ID NO: 253, the light chain CDR1 of SEQ ID NO: 257, the light chain CDR2 of SEQ ID NO: 259, and the light chain CDR3 of SEQ ID NO: 261. In one embodiment, the ting T cell antigen binding moiety binds to the same epitope of CD3 as an n binding moiety comprising the heavy chain CDR1 of SEQ ID NO: 249, the heavy chain CDR2 of SEQ ID NO: 251, the heavy chain CDR3 of SEQ ID NO: 253, the light chain CDR1 of SEQ ID NO: 257, the light chain CDR2 of SEQ ID NO: 259, and the light chain CDR3 of SEQ ID NO: 261. In a further ment, the activating T cell antigen binding moiety ses a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 255 and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 263, or variants thereof that retain functionality. In one embodiment, the activating T cell antigen binding moiety can compete for binding an epitope of CD3 with an antigen g moiety comprising the heavy chain variable region sequence of SEQ ID NO: 255 and the light chain variable region ce of SEQ ID NO: 263. In one embodiment, the activating T cell antigen binding moiety binds to the same epitope of CD3 as an antigen g moiety comprising the heavy chain variable region sequence of SEQ ID NO: 255 and the light chain variable region sequence of SEQ ID NO: 263. In another embodiment, the activating T cell n binding moiety comprises a humanized version of the heavy chain variable region sequence of SEQ ID NO: 255 and a humanized version of the light chain variable region sequence of SEQ ID NO: 263. In one ment, the activating T cell n binding moiety comprises the heavy chain CDR1 of SEQ ID NO: 249, the heavy chain CDR2 of SEQ ID NO: 251, the heavy chain CDR3 of SEQ ID NO: 253, the light chain CDR1 of SEQ ID NO: 257, the light chain CDR2 of SEQ ID NO: 259, the light chain CDR3 of SEQ ID NO: 261, and human heavy and light chain variable region framework sequences.

Target cell antigen binding moiety The T cell activating bispecific antigen binding molecule described comprises at least one antigen binding moiety capable of binding to a target cell antigen (also referred to herein as an “target cell antigen binding moiety”). In certain embodiments, the T cell activating bispecific antigen binding molecule comprises two antigen binding moieties capable of binding to a target cell antigen. In a particular such embodiment, each of these n binding moieties specifically binds to the same antigenic determinant. In one embodiment, the T cell activating bispecific antigen binding molecule comprises an immunoglobulin molecule e of ic binding to a target cell antigen. In one ment the T cell activating bispecific antigen binding molecule comprises not more than two antigen binding moieties e of binding to a target cell antigen.

The target cell antigen binding moiety is generally a Fab molecule that binds to a specific antigenic determinant and is able to direct the T cell activating bispecific antigen binding molecule to a target site, for example to a specific type of tumor cell that bears the antigenic determinant.

In certain embodiments the target cell antigen binding moiety is directed to an antigen associated with a pathological condition, such as an antigen presented on a tumor cell or on a virus-infected cell. Suitable antigens are cell surface antigens, for example, but not limited to, cell surface receptors. In particular embodiments the antigen is a human antigen. In a specific embodiment the target cell antigen is ed from the group of Fibroblast Activation Protein (FAP), Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP), Epidermal Growth Factor Receptor (EGFR), Carcinoembryonic Antigen (CEA),CD19, CD20 and CD33.

In particular embodiments the T cell activating bispecific antigen binding le comprises at least one n binding moiety that is specific for ma-associated Chondroitin Sulfate Proteoglycan (MCSP). In one embodiment the T cell activating bispecific antigen binding molecule comprises at least one, typically two or more antigen binding moieties that can compete with monoclonal antibody LC007 (see SEQ ID NOs 75 and 83, and European patent ation no. EP 11178393.2, incorporated herein by reference in its ty) for binding to an epitope of MCSP. In one embodiment, the antigen binding moiety that is specific for MCSP ses the heavy chain CDR1 of SEQ ID NO: 69, the heavy chain CDR2 of SEQ ID NO: 71, the heavy chain CDR3 of SEQ ID NO: 73, the light chain CDR1 of SEQ ID NO: 77, the light chain CDR2 of SEQ ID NO: 79, and the light chain CDR3 of SEQ ID NO: 81. In a further embodiment, the antigen binding moiety that is specific for MCSP comprises a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 75 and a light chain le region ce that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 83, or variants thereof that retain functionality. In particular embodiments the T cell activating ific antigen binding le comprises at least one, typically two or more antigen binding moieties that can compete with monoclonal antibody M4-3 ML2 (see SEQ ID NOs 239 and 247, and European patent application no. EP 11178393.2, incorporated herein by reference in its ty) for binding to an epitope of MCSP. In one ment, the antigen binding moiety that is specific for MCSP binds to the same epitope of MCSP as monoclonal antibody M4-3 ML2. In one embodiment, the antigen binding moiety that is specific for MCSP comprises the heavy chain CDR1 of SEQ ID NO: 233, the heavy chain CDR2 of SEQ ID NO: 235, the heavy chain CDR3 of SEQ ID NO: 237, the light chain CDR1 of SEQ ID NO: 241, the light chain CDR2 of SEQ ID NO: 243, and the light chain CDR3 of SEQ ID NO: 245. In a further embodiment, the antigen binding moiety that is specific for MCSP comprises a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, particularly about 98%, 99% or 100%, identical to SEQ ID NO: 239 and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, particularly about 98%, 99% or 100%, identical to SEQ ID NO: 247, or variants thereof that retain functionality. In one embodiment, the antigen binding moiety that is specific for MCSP comprises the heavy and light chain variable region sequences of an affinity matured n of monoclonal antibody M4-3 ML2. In one embodiment, the antigen binding moiety that is specific for MCSP comprises the heavy chain variable region sequence of SEQ ID NO: 239 with one, two, three, four, five, six or seven, particularly two, three, four or five, amino acid substitutions; and the light chain le region sequence of SEQ ID NO: 247 with one, two, three, four, five, six or seven, particularly two, three, four or five, amino acid substitutions. Any amino acid residue within the variable region sequences may be substituted by a different amino acid, including amino acid residues within the CDR regions, provided that binding to MCSP, particularly human MCSP, is preserved. Preferred variants are those having a binding affinity for MCSP at least equal (or er) to the binding ty of the antigen binding moiety comprising the unsubstituted variable region sequences.

In one embodiment the T cell activating bispecific antigen binding molecule ses the polypeptide sequence of SEQ ID NO: 1, the polypeptide sequence of SEQ ID NO: 3 and the polypeptide sequence of SEQ ID NO: 5, or variants thereof that retain functionality. In a further embodiment the T cell activating bispecific antigen binding molecule comprises the polypeptide sequence of SEQ ID NO: 7, the polypeptide sequence of SEQ ID NO: 9 and the polypeptide sequence of SEQ ID NO: 11, or variants f that retain functionality. In yet another embodiment the T cell activating bispecific antigen binding molecule comprises the ptide sequence of SEQ ID NO: 13, the polypeptide ce of SEQ ID NO: 15 and the polypeptide sequence of SEQ ID NO: 5, or variants f that retain functionality. In yet another embodiment the T cell activating bispecific antigen binding molecule comprises the polypeptide sequence of SEQ ID NO: 17, the polypeptide sequence of SEQ ID NO: 19 and the polypeptide sequence of SEQ ID NO: 5, or variants f that retain functionality. In another embodiment the T cell activating bispecific antigen binding molecule comprises the polypeptide sequence of SEQ ID NO: 21, the polypeptide sequence of SEQ ID NO: 23 and the ptide sequence of SEQ ID NO: 5, or variants thereof that retain functionality. In still another embodiment the T cell activating ific antigen g le comprises the polypeptide sequence of SEQ ID NO: 25, the ptide sequence of SEQ ID NO: 27 and the polypeptide sequence of SEQ ID NO: 5, or variants thereof that retain onality. In another embodiment the T cell ting bispecific antigen binding molecule comprises the polypeptide sequence of SEQ ID NO: 29, the polypeptide sequence of SEQ ID NO: 31, the polypeptide sequence of SEQ ID NO: 33, and the polypeptide ce of SEQ ID NO: 5, or variants f that retain functionality.

In another embodiment the T cell activating bispecific antigen binding molecule comprises the polypeptide sequence of SEQ ID NO: 29, the polypeptide sequence of SEQ ID NO: 3, the polypeptide ce of SEQ ID NO: 33, and the ptide sequence of SEQ ID NO: 5, or variants f that retain functionality. In r embodiment the T cell ting bispecific antigen binding molecule comprises the polypeptide sequence of SEQ ID NO: 35, the polypeptide sequence of SEQ ID NO: 3, the polypeptide sequence of SEQ ID NO: 37, and the polypeptide sequence of SEQ ID NO: 5, or variants thereof that retain functionality. In another embodiment the T cell activating bispecific antigen binding le comprises the polypeptide ce of SEQ ID NO: 39, the polypeptide sequence of SEQ ID NO: 3, the polypeptide sequence of SEQ ID NO: 41, and the polypeptide sequence of SEQ ID NO: 5, or variants thereof that retain functionality. In yet another embodiment the T cell activating bispecific antigen binding le comprises the ptide sequence of SEQ ID NO: 29, the polypeptide sequence of SEQ ID NO: 3, the polypeptide sequence of SEQ ID NO: 5 and the polypeptide sequence of SEQ ID NO: 179, or variants thereof that retain functionality. In one embodiment the T cell activating bispecific antigen binding molecule comprises the polypeptide sequence of SEQ ID NO: 5, the polypeptide sequence of SEQ ID NO: 29, the polypeptide sequence of SEQ ID NO: 33 and the polypeptide sequence of SEQ ID NO: 181, or variants thereof that retain functionality. In one embodiment the T cell activating bispecific n binding molecule comprises the polypeptide sequence of SEQ ID NO: 5, the polypeptide sequence of SEQ ID NO: 23, the polypeptide sequence of SEQ ID NO: 183 and the polypeptide ce of SEQ ID NO: 185, or variants thereof that retain functionality. In one embodiment the T cell activating bispecific antigen binding molecule comprises the polypeptide sequence of SEQ ID NO: 5, the polypeptide sequence of SEQ ID NO: 23, the polypeptide sequence of SEQ ID NO: 183 and the polypeptide sequence of SEQ ID NO: 187, or variants thereof that retain functionality. In one embodiment the T cell activating ific antigen binding molecule comprises the polypeptide sequence of SEQ ID NO: 33, the polypeptide sequence of SEQ ID NO: 189, the polypeptide ce of SEQ ID NO: 191 and the polypeptide sequence of SEQ ID NO: 193, or variants thereof that retain functionality. In one embodiment the T cell activating bispecific antigen binding le comprises the polypeptide sequence of SEQ ID NO: 183, the polypeptide ce of SEQ ID NO: 189, the polypeptide sequence of SEQ ID NO: 193 and the polypeptide sequence of SEQ ID NO: 195, or variants thereof that retain functionality. In one embodiment the T cell activating bispecific antigen g molecule comprises the polypeptide sequence of SEQ ID NO: 189, the polypeptide sequence of SEQ ID NO: 193, the polypeptide sequence of SEQ ID NO: 199 and the polypeptide sequence of SEQ ID NO: 201, or variants thereof that retain functionality. In one ment the T cell activating bispecific antigen g molecule comprises the polypeptide sequence of SEQ ID NO: 5, the polypeptide sequence of SEQ ID NO: 23, the polypeptide sequence of SEQ ID NO: 215 and the ptide sequence of SEQ ID NO: 217, or variants thereof that retain functionality. In one embodiment the T cell activating bispecific antigen binding molecule ses the ptide sequence of SEQ ID NO: 5, the polypeptide sequence of SEQ ID NO: 23, the polypeptide sequence of SEQ ID NO: 215 and the polypeptide sequence of SEQ ID NO: 219, or variants thereof that retain functionality.

In a specific embodiment the T cell activating bispecific antigen binding molecule comprises a polypeptide sequence encoded by a cleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group of SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 216, SEQ ID NO: 218 and SEQ ID NO: 220.

In one embodiment the T cell ting bispecific antigen binding molecule comprises at least one antigen binding moiety that is specific for Epidermal Growth Factor Receptor (EGFR). In another ment the T cell activating bispecific antigen binding le comprises at least one, typically two or more antigen binding moieties that can compete with monoclonal antibody GA201 for binding to an epitope of EGFR. See PCT publication , incorporated herein by reference in its entirety. In one embodiment, the n binding moiety that is specific for EGFR comprises the heavy chain CDR1 of SEQ ID NO: 85, the heavy chain CDR2 of SEQ ID NO: 87, the heavy chain CDR3 of SEQ ID NO: 89, the light chain CDR1 of SEQ ID NO: 93, the light chain CDR2 of SEQ ID NO: 95, and the light chain CDR3 of SEQ ID NO: 97. In a further embodiment, the antigen binding moiety that is specific for EGFR comprises a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 91 and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 99, or variants thereof that retain functionality.

In yet another embodiment the T cell activating bispecific antigen binding molecule comprises the polypeptide sequence of SEQ ID NO: 43, the polypeptide sequence of SEQ ID NO: 45 and the ptide sequence of SEQ ID NO: 47, or variants thereof that retain functionality. In a further embodiment the T cell activating bispecific antigen binding molecule comprises the polypeptide sequence of SEQ ID NO: 49, the polypeptide ce of SEQ ID NO: 51 and the polypeptide ce of SEQ ID NO: 11, or variants thereof that retain functionality. In yet another embodiment the T cell activating bispecific antigen binding molecule comprises the polypeptide sequence of SEQ ID NO: 53, the polypeptide sequence of SEQ ID NO: 45 and the ptide sequence of SEQ ID NO: 47, or variants thereof that retain functionality.

In a specific embodiment the T cell activating bispecific antigen g le comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group of SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54 and SEQ ID NO: 12.

In one embodiment the T cell activating bispecific antigen binding molecule comprises at least one antigen binding moiety that is specific for Fibroblast Activation Protein (FAP). In another embodiment the T cell activating bispecific n binding molecule comprises at least one, typically two or more antigen binding moieties that can compete with monoclonal antibody 3F2 for binding to an epitope of FAP. See PCT publication WO 20006, incorporated herein by nce in its entirety. In one embodiment, the antigen binding moiety that is specific for FAP comprises the heavy chain CDR1 of SEQ ID NO: 101, the heavy chain CDR2 of SEQ ID NO: 103, the heavy chain CDR3 of SEQ ID NO: 105, the light chain CDR1 of SEQ ID NO: 109, the light chain CDR2 of SEQ ID NO: 111, and the light chain CDR3 of SEQ ID NO: 113. In a further embodiment, the antigen binding moiety that is specific for FAP comprises a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 107 and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 115, or variants thereof that retain functionality.

In yet another embodiment the T cell activating ific antigen binding molecule comprises the polypeptide sequence of SEQ ID NO: 55, the ptide sequence of SEQ ID NO: 51 and the ptide sequence of SEQ ID NO: 11, or variants thereof that retain functionality. In a further embodiment the T cell activating bispecific antigen binding molecule comprises the polypeptide sequence of SEQ ID NO: 57, the polypeptide sequence of SEQ ID NO: 59 and the polypeptide ce of SEQ ID NO: 61, or variants thereof that retain functionality.

In a specific ment the T cell activating bispecific antigen binding molecule comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group of SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 52 and SEQ ID NO: 12.

In particular embodiments the T cell activating bispecific antigen binding le comprises at least one antigen binding moiety that is specific for Carcinoembryonic n (CEA). In one embodiment the T cell activating bispecific antigen binding molecule comprises at least one, typically two or more antigen binding es that can compete with monoclonal antibody BW431/26 ibed in European patent no. EP 160 897, and Bosslet et al., Int J Cancer 36, 75- 84 (1985)) for g to an epitope of CEA. In one embodiment the T cell activating bispecific antigen binding molecule comprises at least one, typically two or more antigen binding moieties that can compete with monoclonal antibody CH1A1A (see SEQ ID NOs 123 and 131) for g to an epitope of CEA. See PCT patent publication number , incorporated herein by reference in its entirety. In one embodiment, the antigen binding moiety that is specific for CEA binds to the same epitope of CEA as monoclonal dy CH1A1A. In one embodiment, the antigen binding moiety that is specific for CEA ses the heavy chain CDR1 of SEQ ID NO: 117, the heavy chain CDR2 of SEQ ID NO: 119, the heavy chain CDR3 of SEQ ID NO: 121, the light chain CDR1 of SEQ ID NO: 125, the light chain CDR2 of SEQ ID NO: 127, and the light chain CDR3 of SEQ ID NO: 129. In a further embodiment, the antigen binding moiety that is specific for CEA comprises a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, particularly about 98%, 99% or 100%, identical to SEQ ID NO: 123 and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, particularly about 98%, 99% or 100%, identical to SEQ ID NO: 131, or variants thereof that retain functionality. In one embodiment, the antigen binding moiety that is specific for CEA comprises the heavy and light chain variable region sequences of an affinity matured version of monoclonal antibody CH1A1A. In one embodiment, the n binding moiety that is specific for CEA comprises the heavy chain le region sequence of SEQ ID NO: 123 with one, two, three, four, five, six or seven, particularly two, three, four or five, amino acid substitutions; and the light chain variable region sequence of SEQ ID NO: 131 with one, two, three, four, five, six or seven, particularly two, three, four or five, amino acid substitutions. Any amino acid e within the variable region sequences may be substituted by a different amino acid, including amino acid residues within the CDR s, provided that binding to CEA, particularly human CEA, is preserved.

Preferred variants are those having a binding affinity for CEA at least equal (or stronger) to the g affinity of the antigen binding moiety comprising the unsubstituted variable region sequences.

In one embodiment the T cell activating bispecific antigen binding molecule comprises the polypeptide sequence of SEQ ID NO: 63, the polypeptide sequence of SEQ ID NO: 65, the polypeptide ce of SEQ ID NO: 67 and the polypeptide sequence of SEQ ID NO: 33, or variants thereof that retain onality. In one embodiment the T cell activating bispecific antigen binding molecule comprises the ptide sequence of SEQ ID NO: 65, the polypeptide sequence of SEQ ID NO: 67, the polypeptide sequence of SEQ ID NO: 183 and the polypeptide sequence of SEQ ID NO: 197, or variants thereof that retain functionality. In one embodiment the T cell activating bispecific antigen binding molecule comprises the polypeptide sequence of SEQ ID NO: 183, the polypeptide sequence of SEQ ID NO: 203, the ptide sequence of SEQ ID NO: 205 and the ptide sequence of SEQ ID NO: 207, or variants thereof that retain functionality. In one embodiment the T cell activating bispecific antigen binding molecule comprises the polypeptide sequence of SEQ ID NO: 183, the polypeptide sequence of SEQ ID NO: 209, the polypeptide sequence of SEQ ID NO: 211 and the polypeptide sequence of SEQ ID NO: 213, or variants thereof that retain functionality.

In a specific embodiment the T cell activating bispecific antigen binding molecule comprises a polypeptide sequence d by a polynucleotide ce that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a ce selected from the group of SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 34, SEQ ID NO: 184, SEQ ID NO: 198, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212 and SEQ ID NO: 214.

In one embodiment the T cell activating bispecific antigen binding le comprises at least one antigen binding moiety that is specific for CD33. In one embodiment, the n binding moiety that is specific for CD33 comprises the heavy chain CDR1 of SEQ ID NO: 133, the heavy chain CDR2 of SEQ ID NO: 135, the heavy chain CDR3 of SEQ ID NO: 137, the light chain CDR1 of SEQ ID NO: 141, the light chain CDR2 of SEQ ID NO: 143, and the light chain CDR3 of SEQ ID NO: 145. In a further embodiment, the antigen binding moiety that is specific for CD33 ses a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 139 and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 147, or ts thereof that retain functionality.

In one embodiment the T cell activating bispecific antigen binding molecule comprises the polypeptide sequence of SEQ ID NO: 33, the polypeptide sequence of SEQ ID NO: 213, the polypeptide sequence of SEQ ID NO: 221 and the polypeptide sequence of SEQ ID NO: 223, or variants f that retain functionality. In one embodiment the T cell activating bispecific antigen binding molecule comprises the polypeptide sequence of SEQ ID NO: 33, the polypeptide sequence of SEQ ID NO: 221, the polypeptide sequence of SEQ ID NO: 223 and the polypeptide sequence of SEQ ID NO: 225, or ts thereof that retain functionality.

In a specific embodiment the T cell activating bispecific antigen binding molecule comprises a ptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group of SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 34, SEQ ID NO: 214, SEQ ID NO: 222, SEQ ID NO: 224 and SEQ ID NO: 226.

Polynucleotides Also described are isolated polynucleotides encoding a T cell activating bispecific antigen binding molecule as bed herein or a fragment thereof.

Polynucleotides described include those that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences set forth in SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262 and 264, including functional fragments or ts thereof.

The polynucleotides ng T cell activating bispecific antigen g molecules bed may be sed as a single cleotide that encodes the entire T cell activating bispecific antigen binding molecule or as multiple (e.g., two or more) polynucleotides that are co- expressed. Polypeptides encoded by polynucleotides that are co-expressed may ate through, e.g., disulfide bonds or other means to form a onal T cell activating bispecific antigen binding molecule. For example, the light chain portion of an antigen binding moiety may be encoded by a separate polynucleotide from the portion of the T cell activating bispecific antigen binding molecule comprising the heavy chain portion of the antigen binding moiety, an Fc domain subunit and ally (part of) another antigen binding moiety. When co-expressed, the heavy chain polypeptides will associate with the light chain polypeptides to form the n binding moiety. In another example, the portion of the T cell activating bispecific antigen binding molecule comprising one of the two Fc domain subunits and optionally (part of) one or more antigen binding moieties could be encoded by a separate polynucleotide from the portion of the T cell activating bispecific antigen binding molecule comprising the the other of the two Fc domain subunits and optionally (part of) an antigen binding moiety. When co-expressed, the Fc domain subunits will associate to form the Fc domain.

In certain embodiments, an isolated polynucleotide encodes a fragment of a T cell activating bispecific antigen binding molecule comprising a first and a second antigen binding moiety, and an Fc domain consisting of two subunits, wherein the first antigen binding moiety is a single chain Fab molecule. In one embodiment, an isolated polynucleotide described encodes the first antigen binding moiety and a t of the Fc domain. In a more ic embodiment the isolated polynucleotide encodes a polypeptide wherein a single chain Fab molecule shares a carboxy-terminal peptide bond with an Fc domain subunit. In another embodiment, an isolated polynucleotide described encodes the heavy chain of the second antigen binding moiety and a subunit of the Fc domain. In a more specific embodiment the isolated cleotide encodes a ptide wherein a Fab heavy chain shares a carboxy terminal e bond with an Fc domain subunit. In yet another embodiment, an ed polynucleotide described encodes the first antigen binding moiety, the heavy chain of the second antigen binding moiety and a subunit of the Fc domain. In a more specific embodiment, the ed polynucleotide encodes a polypeptide wherein a single chain Fab molecule shares a carboxy-terminal peptide bond with a Fab heavy chain, which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit.

In certain embodiments, an isolated polynucleotide bed encodes a fragment of a T cell activating bispecific antigen binding molecule comprising a first and a second antigen binding moiety, and an Fc domain consisting of two subunits, wherein the first antigen g moiety is a crossover Fab molecule. In one embodiment, an isolated polynucleotide described encodes the heavy chain of the first antigen binding moiety and a subunit of the Fc domain. In a more specific ment the ed polynucleotide s a polypeptide n Fab light chain variable region shares a carboxy terminal peptide bond with a Fab heavy chain constant region, which in turn shares a carboxy-terminal e bond with an Fc domain subunit. In another specific embodiment the isolated polynucleotide encodes a polypeptide wherein Fab heavy chain variable region shares a carboxy terminal peptide bond with a Fab light chain nt region, which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit. In another embodiment, an isolated cleotide described encodes the heavy chain of the second antigen binding moiety and a t of the Fc domain. In a more specific embodiment the isolated polynucleotide encodes a polypeptide wherein a Fab heavy chain shares a carboxy al peptide bond with an Fc domain subunit. In yet another embodiment, an isolated polynucleotide described encodes the heavy chain of the first antigen binding moiety, the heavy chain of the second antigen binding moiety and a subunit of the Fc domain. In a more specific embodiment, the isolated polynucleotide encodes a polypeptide wherein a Fab light chain variable region shares a carboxy-terminal peptide bond with a Fab heavy chain nt region, which in turn shares a carboxy-terminal peptide bond with a Fab heavy chain, which in turn shares a carboxyterminal peptide bond with an Fc domain subunit. In another specific embodiment, the ed polynucleotide encodes a polypeptide wherein a Fab heavy chain variable region shares a carboxy-terminal peptide bond with a Fab light chain constant region, which in turn shares a carboxy-terminal peptide bond with a Fab heavy chain, which in turn shares a y-terminal e bond with an Fc domain subunit. In yet another specific ment the isolated polynucleotide encodes a polypeptide wherein a Fab heavy chain shares a carboxy-terminal peptide bond with a Fab light chain variable region, which in turn shares a carboxy-terminal peptide bond with a Fab heavy chain constant region, which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit. In still another specific embodiment the isolated polynucleotide encodes a polypeptide wherein a Fab heavy chain shares a carboxy-terminal peptide bond with a Fab heavy chain variable region, which in turn shares a carboxy-terminal peptide bond with a Fab light chain constant region, which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit.

In further embodiments, an isolated polynucleotide described encodes the heavy chain of a third antigen binding moiety and a subunit of the Fc domain. In a more specific embodiment the isolated polynucleotide encodes a polypeptide wherein a Fab heavy chain shares a carboxy terminal peptide bond with an Fc domain subunit.

In further ments, an isolated polynucleotide described encodes the light chain of an antigen binding moiety. In some embodiments, the ed polynucleotide s a ptide wherein a Fab light chain variable region shares a y-terminal e bond with a Fab heavy chain constant region. In other embodiments, the isolated polynucleotide encodes a polypeptide wherein a Fab heavy chain variable region shares a carboxy-terminal peptide bond with a Fab light chain constant region. In still other embodiments, an isolated polynucleotide described encodes the light chain of the first antigen g moiety and the light chain of the second antigen binding moiety. In a more specific embodiment, the isolated cleotide encodes a polypeptide wherein a Fab heavy chain variable region shares a carboxy-terminal peptide bond with a Fab light chain constant region, which in turn shares a carboxy-terminal peptide bond with a Fab light chain. In another specific embodiment the isolated cleotide encodes a polypeptide wherein a Fab light chain shares a yterminal peptide bond with a Fab heavy chain variable , which in turn shares a carboxyterminal peptide bond with a Fab light chain nt . In yet another specific embodiment, the isolated polynucleotide encodes a polypeptide wherein a Fab light chain variable region shares a carboxy-terminal peptide bond with a Fab heavy chain constant region, which in turn shares a carboxy-terminal peptide bond with a Fab light chain. In yet r specific embodiment the isolated polynucleotide encodes a polypeptide wherein a Fab light chain shares a carboxy-terminal peptide bond with a Fab light chain variable region, which in turn shares a carboxy-terminal peptide bond with a Fab heavy chain constant region.

In another embodiment, described is an isolated polynucleotide encoding a T cell activating bispecific antigen binding le bed or a fragment thereof, wherein the polynucleotide comprises a sequence that encodes a variable region sequence as shown in SEQ ID NOs 75, 83, 91, 99, 107, 115, 123, 131, 139, 147, 169, 177, 239, 247, 255 and 263. In r embodiment, bed an isolated polynucleotide encoding a T cell activating bispecific antigen binding molecule or fragment thereof, wherein the polynucleotide comprises a sequence that encodes a polypeptide sequence as shown in SEQ ID NOs 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229 and 231. In another embodiment, described is an isolated polynucleotide encoding a T cell activating bispecific antigen binding molecule bed or a fragment thereof, wherein the polynucleotide comprises a ce that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence shown in SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262 or 264. In another embodiment, described is an isolated polynucleotide encoding a T cell activating bispecific antigen binding molecule bed or a fragment thereof, wherein the polynucleotide comprises a nucleic acid sequence shown in SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262 or 264. In another embodiment, described is an isolated polynucleotide encoding a T cell activating bispecific antigen binding molecule described or a fragment thereof, wherein the polynucleotide comprises a sequence that encodes a variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence in SEQ ID NOs 75, 83, 91, 99, 107, 115, 123, 131, 139, 147, 169, 177, 239, 247, 255 or 263. In another embodiment, described is an ed polynucleotide encoding a T cell activating bispecific antigen binding molecule or fragment thereof, wherein the polynucleotide comprises a sequence that encodes a polypeptide ce that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence in SEQ ID NOs 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229 or 231. Described is an isolated polynucleotide encoding a T cell activating ific antigen binding molecule described or a fragment thereof, wherein the polynucleotide comprises a sequence that encodes the variable region sequence of SEQ ID NOs 75, 83, 91, 99, 107, 115, 123, 131, 139, 147, 169, 177, 239, 247, 255 or 263 with vative amino acid substitutions.

Also described is an isolated polynucleotide encoding a T cell activating bispecific antigen binding molecule described or fragment thereof, wherein the polynucleotide comprises a sequence that encodes the polypeptide sequence of SEQ ID NOs 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229 or 231 with vative amino acid substitutions.

In certain embodiments the polynucleotide or nucleic acid is DNA. In other embodiments, a polynucleotide described is RNA, for example, in the form of messenger RNA . RNA bed may be single stranded or double stranded. inant Methods T cell activating bispecific antigen binding molecules described may be obtained, for example, by solid-state e synthesis (e.g. Merrifield solid phase sis) or recombinant production. For recombinant production one or more polynucleotide encoding the T cell ting bispecific n binding le (fragment), e.g., as described above, is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such polynucleotide may be readily isolated and sequenced using conventional procedures. In one embodiment a vector, preferably an expression vector, comprises one or more of the polynucleotides described. Methods which are well known to those skilled in the art can be used to construct expression vectors containing the coding sequence of a T cell activating bispecific antigen binding molecule (fragment) along with appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA ques, synthetic techniques and in vivo recombination/genetic ination. See, for example, the techniques described in Maniatis et al., MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory, N.Y. (1989); and Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR Y, Greene Publishing Associates and Wiley cience, N.Y (1989). The expression vector can be part of a plasmid, virus, or may be a nucleic acid nt. The expression vector includes an sion cassette into which the polynucleotide encoding the T cell activating ific antigen binding molecule (fragment) (i.e. the coding region) is cloned in operable association with a promoter and/or other transcription or translation l elements. As used herein, a "coding region" is a portion of nucleic acid which consists of codons translated into amino acids.

Although a "stop codon" (TAG, TGA, or TAA) is not translated into an amino acid, it may be considered to be part of a coding region, if present, but any ng sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, 5' and 3' untranslated regions, and the like, are not part of a coding region. Two or more coding regions can be present in a single polynucleotide construct, e.g. on a single , or in separate polynucleotide constructs, e.g. on separate (different) vectors. Furthermore, any vector may contain a single coding , or may comprise two or more coding regions, e.g. a vector described may encode one or more polypeptides, which are post- or co-translationally separated into the final proteins via proteolytic cleavage. In on, a vector, cleotide, or nucleic acid bed may encode heterologous coding regions, either fused or unfused to a polynucleotide encoding the T cell activating bispecific antigen binding le (fragment) bed, or variant or derivative thereof. Heterologous coding regions include without limitation specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain. An operable association is when a coding region for a gene product, e.g. a polypeptide, is ated with one or more regulatory sequences in such a way as to place expression of the gene product under the influence or control of the regulatory sequence(s). Two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) are bly associated" if induction of er function results in the transcription of mRNA encoding the desired gene product and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression tory sequences to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed. Thus, a promoter region would be operably associated with a nucleic acid encoding a polypeptide if the promoter was capable of effecting transcription of that nucleic acid. The promoter may be a cell-specific er that directs substantial transcription of the DNA only in predetermined cells. Other transcription control elements, besides a promoter, for example enhancers, operators, sors, and transcription termination signals, can be operably associated with the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcription control regions are disclosed herein. A variety of transcription control regions are known to those skilled in the art. These include, without limitation, transcription l regions, which on in vertebrate cells, such as, but not limited to, promoter and enhancer segments from galoviruses (e.g. the immediate early promoter, in conjunction with intron-A), simian virus 40 (e.g. the early er), and retroviruses (such as, e.g. Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth e and rabbit â-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include -specific promoters and enhancers as well as inducible ers (e.g. promoters ble tetracyclins). Similarly, a variety of translation control ts are known to those of ry skill in the art. These include, but are not limited to ribosome binding sites, translation tion and termination codons, and elements derived from viral systems (particularly an al ribosome entry site, or IRES, also referred to as a CITE sequence). The expression cassette may also e other features such as an origin of replication, and/or some integration elements such as retroviral long terminal s (LTRs), or adeno-associated viral (AAV) inverted terminal repeats (ITRs).

Polynucleotide and nucleic acid coding regions described may be associated with additional coding regions which encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide described. For example, if secretion of the T cell activating bispecific antigen binding molecule is desired, DNA encoding a signal sequence may be placed upstream of the nucleic acid encoding a T cell activating bispecific antigen binding molecule described or a fragment thereof. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence which is cleaved from the mature protein once export of the g protein chain across the rough endoplasmic reticulum has been initiated. Those of ordinary skill in the art are aware that polypeptides secreted by rate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the translated polypeptide to e a secreted or "mature" form of the polypeptide. In certain embodiments, the native signal peptide, e.g. an immunoglobulin heavy chain or light chain signal peptide is used, or a functional derivative of that sequence that retains the ability to direct the secretion of the polypeptide that is operably associated with it.

Alternatively, a heterologous mammalian signal peptide, or a functional derivative thereof, may be used. For e, the wild-type leader sequence may be substituted with the leader sequence of human tissue plasminogen activator (TPA) or mouse β-glucuronidase. Exemplary amino acid and polynucleotide sequences of secretory signal peptides are given in SEQ ID NOs 154-162.

DNA encoding a short protein sequence that could be used to facilitate later purification (e.g. a histidine tag) or assist in labeling the T cell activating bispecific antigen binding molecule may be included within or at the ends of the T cell activating bispecific antigen binding molecule (fragment) encoding polynucleotide.

Also described is a host cell sing one or more polynucleotides described herein. In certain embodiments bed is a host cell comprising one or more vectors described . The polynucleotides and vectors may incorporate any of the features, singly or in combination, described herein in relation to polynucleotides and vectors, respectively. In one such ment a host cell comprises (e.g. has been ormed or transfected with) a vector comprising a cleotide that encodes (part of) a T cell activating bispecific antigen binding molecule described. As used herein, the term "host cell" refers to any kind of cellular system which can be engineered to generate the T cell activating bispecific antigen g molecules described or fragments thereof. Host cells suitable for replicating and for supporting expression of T cell activating bispecific antigen binding molecules are well known in the art. Such cells may be transfected or transduced as riate with the particular expression vector and large quantities of vector containing cells can be grown for seeding large scale fermenters to obtain sufficient quantities of the T cell ting bispecific antigen binding molecule for clinical applications. Suitable host cells include prokaryotic microorganisms, such as E. coli, or various eukaryotic cells, such as Chinese hamster ovary cells (CHO), insect cells, or the like. For example, polypeptides may be produced in bacteria in particular when glycosylation is not needed. After expression, the polypeptide may be ed from the bacterial cell paste in a soluble fraction and can be further purified. In addition to yotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or sion hosts for polypeptide-encoding s, including fungi and yeast strains whose glycosylation pathways have been “humanized”, resulting in the production of a polypeptide with a partially or fully human glycosylation pattern.

See Gerngross, Nat Biotech 22, 414 (2004), and Li et al., Nat Biotech 24, 210-215 (2006).

Suitable host cells for the expression of (glycosylated) polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). es of ebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera erda cells. Plant cell cultures can also be ed as hosts. See e.g. US Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing BODIESTM technology for producing antibodies in transgenic plants). Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are d to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human nic kidney line (293 or 293T cells as described, e.g., in Graham et al., J Gen Virol 36, 59 (1977)), baby hamster kidney cells (BHK), mouse sertoli cells (TM4 cells as described, e.g., in Mather, Biol Reprod 23, 243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT 060562), TRI cells (as described, e.g., in Mather et al., Annals N.Y. Acad Sci 383, 44-68 (1982)), MRC 5 cells, and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including dhfr- CHO cells b et al., Proc Natl Acad Sci USA 77, 4216 (1980)); and myeloma cell lines such as YO, NS0, P3X63 and Sp2/0. For a review of certain mammalian host cell lines suitable for protein production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). Host cells include cultured cells, e.g., mammalian cultured cells, yeast cells, insect cells, bacterial cells and plant cells, to name only a few, but also cells comprised within a transgenic animal, transgenic plant or ed plant or animal tissue. In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell, such as a Chinese Hamster Ovary (CHO) cell, a human embryonic kidney (HEK) cell or a lymphoid cell (e.g., Y0, NS0, Sp20 cell).

Standard technologies are known in the art to s foreign genes in these systems. Cells expressing a polypeptide comprising either the heavy or the light chain of an antigen binding domain such as an dy, may be engineered so as to also express the other of the antibody chains such that the expressed product is an antibody that has both a heavy and a light chain.

In one embodiment, bed is a method of producing a T cell activating bispecific antigen binding molecule described, wherein the method comprises culturing a host cell comprising a cleotide encoding the T cell activating bispecific antigen binding molecule, as described herein, under ions suitable for sion of the T cell activating bispecific antigen binding molecule, and recovering the T cell activating bispecific antigen binding molecule from the host cell (or host cell culture medium).

The components of the T cell activating bispecific antigen binding molecule are genetically fused to each other. T cell ting ific antigen binding molecule can be designed such that its components are fused ly to each other or indirectly through a linker sequence. The composition and length of the linker may be determined in accordance with methods well known in the art and may be tested for efficacy. Examples of linker sequences between different components of T cell activating bispecific antigen binding molecules are found in the sequences described herein. Additional sequences may also be ed to incorporate a cleavage site to te the individual components of the fusion if desired, for example an endopeptidase recognition sequence.

In certain embodiments the one or more n g moieties of the T cell activating bispecific antigen binding molecules se at least an antibody variable region e of binding an nic inant. Variable regions can form part of and be derived from naturally or non-naturally ing dies and fragments thereof. Methods to produce polyclonal antibodies and monoclonal antibodies are well known in the art (see e.g. Harlow and Lane, "Antibodies, a laboratory ", Cold Spring Harbor Laboratory, 1988). Non-naturally occurring antibodies can be constructed using solid phase-peptide synthesis, can be produced recombinantly (e.g. as bed in U.S. patent No. 4,186,567) or can be obtained, for example, by screening combinatorial libraries comprising variable heavy chains and variable light chains (see e.g. U.S. Patent. No. 5,969,108 to McCafferty).

Any animal species of antibody, antibody fragment, antigen binding domain or variable region can be used in the T cell activating bispecific antigen binding molecules described. miting antibodies, antibody fragments, antigen binding domains or variable regions useful herein can be of murine, primate, or human origin. If the T cell activating bispecific antigen binding molecule is intended for human use, a ic form of antibody may be used wherein the constant regions of the antibody are from a human. A humanized or fully human form of the dy can also be prepared in accordance with s well known in the art (see e. g. U.S. Patent No. ,565,332 to Winter). Humanization may be achieved by various methods including, but not limited to (a) grafting the non-human (e.g., donor antibody) CDRs onto human (e.g. recipient antibody) framework and constant s with or without retention of critical framework residues (e.g. those that are important for retaining good antigen binding affinity or antibody functions), (b) grafting only the non-human specificity-determining regions (SDRs or a-CDRs; the residues critical for the antibody-antigen ction) onto human framework and constant regions, or (c) lanting the entire non-human variable domains, but "cloaking" them with a human-like section by replacement of surface residues. Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front Biosci 13, 1619-1633 (2008), and are further described, e.g., in Riechmann et al., Nature 332, 323-329 (1988); Queen et al., Proc Natl Acad Sci USA 86, 10029-10033 (1989); US Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Jones et al., Nature 321, 522-525 (1986); Morrison et al., Proc Natl Acad Sci 81, 6851-6855 ; Morrison and Oi, Adv Immunol 44, 65-92 (1988); Verhoeyen et al., Science 239, 536 (1988); Padlan, Molec Immun 31(3), 169-217 (1994); Kashmiri et al., Methods 36, 25-34 (2005) (describing SDR (a-CDR) grafting); , Mol Immunol 28, 489-498 (1991) (describing “resurfacing”); Dall’Acqua et al., Methods 36, 43-60 (2005) (describing “FR shuffling”); and Osbourn et al., Methods 36, 61-68 (2005) and Klimka et al., Br J Cancer 83, 252-260 (2000) (describing the “guided selection” approach to FR ing).

Human antibodies and human variable regions can be produced using various techniques known in the art. Human antibodies are described lly in van Dijk and van de Winkel, Curr Opin Pharmacol 5, 368-74 (2001) and g, Curr Opin Immunol 20, 450-459 (2008). Human variable regions can form part of and be derived from human monoclonal antibodies made by the hybridoma method (see e.g. Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, . Human antibodies and human variable regions may also be prepared by stering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge (see e.g. Lonberg, Nat Biotech 23, 1117-1125 (2005). Human dies and human variable regions may also be generated by ing Fv clone le region sequences selected from derived phage display libraries (see e.g., Hoogenboom et al. in Methods in Molecular Biology 178, 1-37 (O’Brien et al., ed., Human Press, Totowa, NJ, 2001); and McCafferty et al., Nature 348, 552-554; Clackson et al., Nature 352, 624-628 (1991)).

Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments.

In certain embodiments, the antigen binding moieties useful herein are engineered to have enhanced binding affinity according to, for example, the methods disclosed in U.S. Pat. Appl.

Publ. No. 2004/0132066, the entire contents of which are hereby incorporated by reference. The ability of the T cell activating bispecific antigen binding molecule described to bind to a specific antigenic determinant can be ed either through an enzyme-linked immunosorbent assay (ELISA) or other techniques ar to one of skill in the art, e.g. surface plasmon resonance technique (analyzed on a BIACORE T100 system) (Liljeblad, et al., Glyco J 17, 323-329 (2000)), and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). Competition assays may be used to identify an antibody, antibody nt, n binding domain or variable domain that competes with a reference antibody for binding to a particular antigen, e.g. an antibody that competes with the V9 antibody for binding to CD3. In certain embodiments, such a competing antibody binds to the same epitope (e.g. a linear or a mational epitope) that is bound by the nce dy. Detailed ary methods for mapping an epitope to which an antibody binds are provided in Morris (1996) “Epitope Mapping Protocols,” in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ). In an exemplary competition assay, immobilized antigen (e.g. CD3) is incubated in a solution comprising a first labeled antibody that binds to the antigen (e.g. V9 antibody) and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for g to the antigen. The second antibody may be present in a oma supernatant. As a control, immobilized antigen is incubated in a solution comprising the first d antibody but not the second led antibody. After incubation under conditions permissive for binding of the first antibody to the antigen, excess unbound antibody is removed, and the amount of label associated with lized antigen is measured. If the amount of label associated with immobilized antigen is substantially reduced in the test sample relative to the control sample, then that indicates that the second antibody is competing with the first antibody for binding to the antigen. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

T cell activating bispecific antigen binding molecules prepared as described herein may be purified by art-known techniques such as high performance liquid chromatography, ion ge chromatography, gel electrophoresis, affinity tography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity etc., and will be apparent to those having skill in the art. For affinity chromatography purification an antibody, ligand, receptor or antigen can be used to which the T cell activating bispecific n binding molecule binds. For example, for affinity chromatography purification of T cell activating bispecific antigen binding molecules described, a matrix with protein A or n G may be used. Sequential n A or G affinity chromatography and size exclusion chromatography can be used to isolate a T cell activating bispecific antigen binding molecule essentially as described in the Examples. The purity of the T cell activating bispecific antigen binding molecule can be determined by any of a variety of well known ical s including gel electrophoresis, high pressure liquid chromatography, and the like. For example, the heavy chain fusion proteins sed as described in the Examples were shown to be intact and properly led as demonstrated by reducing SDS-PAGE (see e.g. Figure 2). Three bands were resolved at approximately Mr 25,000, Mr 50,000 and Mr 75,000, corresponding to the predicted molecular weights of the T cell activating bispecific antigen binding le light chain, heavy chain and heavy chain/light chain fusion protein.

Assays T cell activating bispecific antigen g molecules bed herein may be identified, screened for, or characterized for their physical/chemical properties and/or biological activities by various assays known in the art.

Affinity assays The affinity of the T cell activating bispecific n binding le for an Fc receptor or a target antigen can be determined in accordance with the methods set forth in the Examples by surface plasmon resonance (SPR), using standard instrumentation such as a BIAcore instrument (GE Healthcare), and receptors or target proteins such as may be obtained by inant expression. Alternatively, binding of T cell activating bispecific antigen binding molecules for different ors or target antigens may be evaluated using cell lines expressing the particular receptor or target antigen, for example by flow cytometry (FACS). A specific illustrative and exemplary embodiment for ing binding affinity is described in the following and in the Examples below.

According to one embodiment, KD is measured by surface plasmon resonance using a E® T100 machine (GE Healthcare) at 25 °C.

To analyze the interaction between the Fc-portion and Fc receptors, His-tagged recombinant Fcreceptor is captured by an anti-Penta His antibody (Qiagen) immobilized on CM5 chips and the bispecific constructs are used as analytes. Briefly, carboxymethylated dextran biosensor chips (CM5, GE Healthcare) are activated with N-ethyl-N’-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier’s instructions.

Anti Penta-His antibody is diluted with 10 mM sodium acetate, pH 5.0, to 40 μg/ml before injection at a flow rate of 5 μl/min to achieve approximately 6500 se units (RU) of coupled protein. Following the injection of the ligand, 1 M ethanolamine is injected to block unreacted groups. Subsequently the Fc-receptor is captured for 60 s at 4 or 10 nM. For c measurements, four-fold serial dilutions of the bispecific construct (range between 500 nM and 4000 nM) are ed in HBS-EP (GE Healthcare, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05 % Surfactant P20, pH 7.4) at 25 °C at a flow rate of 30 μl/min for 120 s.

To determine the affinity to the target antigen, bispecific ucts are captured by an anti human Fab specific antibody (GE care) that is lized on an ted CM5-sensor chip surface as described for the anti Penta-His antibody. The final amount of coupled protein is is approximately 12000 RU. The bispecific constructs are captured for 90 s at 300 nM. The target ns are passed through the flow cells for 180 s at a concentration range from 250 to 1000 nM with a flowrate of 30 μl/min. The dissociation is monitored for 180 s.

Bulk refractive index ences are corrected for by cting the response obtained on reference flow cell. The steady state response was used to derive the dissociation constant KD by non-linear curve fitting of the Langmuir binding isotherm. Association rates (kon) and iation rates (koff) are calculated using a simple one-to-one Langmuir binding model (BIACORE® T100 Evaluation Software version 1.1.1) by simultaneously fitting the association and dissociation grams. The equilibrium dissociation constant (KD) is calculated as the ratio koff/kon. See, e.g., Chen et al., J Mol Biol 293, 865-881 .

Activity assays Biological activity of the T cell activating bispecific antigen binding molecules described can be measured by various assays as described in the Examples. Biological activities may for example include the induction of eration of T cells, the induction of signaling in T cells, the induction of expression of activation markers in T cells, the induction of cytokine secretion by T cells, the induction of lysis of target cells such as tumor cells, and the induction of tumor regression and/or the improvement of survival.

Compositions, Formulations, and Routes of Administration Also described are pharmaceutical compositions comprising any of the T cell ting bispecific antigen binding molecules described herein, e.g., for use in any of the below therapeutic methods. In one embodiment, a pharmaceutical composition ses any of the T cell activating bispecific antigen g molecules described herein and a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical composition comprises any of the T cell activating bispecific antigen binding molecules described herein and at least one additional therapeutic agent, e.g., as described below.

Further described is a method of producing a T cell activating bispecific antigen binding molecule described in a form suitable for stration in vivo, the method comprising (a) obtaining a T cell activating bispecific antigen binding le described, and (b) formulating the T cell ting bispecific antigen binding molecule with at least one pharmaceutically acceptable carrier, whereby a preparation of T cell activating bispecific antigen binding molecule is formulated for administration in vivo.

Pharmaceutical compositions described comprise a therapeutically ive amount of one or more T cell activating bispecific n g molecule ved or sed in a pharmaceutically acceptable carrier. The phrases "pharmaceutical or pharmacologically acceptable" refers to molecular entities and itions that are generally non-toxic to recipients at the dosages and concentrations employed, i.e. do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of a pharmaceutical composition that contains at least one T cell activating bispecific antigen binding molecule and optionally an additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference. Moreover, for animal (e.g., human) stration, it will be understood that preparations should meet sterility, pyrogenicity, l safety and purity standards as required by FDA Office of ical Standards or ponding authorities in other countries. Preferred compositions are lyophilized formulations or aqueous solutions. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g. antibacterial agents, ngal agents), ic agents, tion delaying agents, salts, preservatives, antioxidants, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegration agents, ants, sweetening agents, flavoring agents, dyes, such like materials and ations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, orated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

The composition may se different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection. T cell activating bispecific antigen binding molecules bed (and any additional therapeutic agent) can be administered intravenously, intradermally, intraarterially, eritoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrasplenically, intrarenally, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, ectally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctivally, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, lly, locally, by inhalation (e.g. aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g. liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference). eral administration, in particular intravenous injection, is most commonly used for administering polypeptide molecules such as the T cell activating bispecific antigen binding molecules described.

Parenteral compositions include those designed for administration by injection, e.g. subcutaneous, ermal, intralesional, intravenous, rterial intramuscular, hecal or intraperitoneal injection. For injection, the T cell activating bispecific antigen binding molecules described may be ated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' on, Ringer's solution, or physiological saline buffer. The solution may contain formulatory agents such as suspending, izing and/or dispersing agents.

Alternatively, the T cell activating bispecific antigen binding molecules may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. Sterile injectable ons are prepared by incorporating the T cell activating bispecific antigen binding molecules described in the required amount in the riate solvent with various of the other ingredients enumerated below, as required. Sterility may be readily lished, e.g., by filtration through sterile filtration nes. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and/or the other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsion, the preferred methods of preparation are vacuum-drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium thereof. The liquid medium should be suitably buffered if necessary and the liquid diluent first rendered isotonic prior to injection with sufficient saline or glucose. The composition must be stable under the conditions of cture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less that 0.5 ng/mg protein.

Suitable ceutically able carriers include, but are not d to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; honium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, n, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, gine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including e, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein xes); and/or non-ionic surfactants such as polyethylene glycol (PEG). s injection suspensions may contain compounds which increase the viscosity of the suspension, such as sodium carboxymethyl ose, sorbitol, dextran, or the like. Optionally, the suspension may also contain le stabilizers or agents which increase the lity of the compounds to allow for the preparation of highly concentrated solutions. Additionally, sions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles e fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl cleats or triglycerides, or liposomes.

Active ingredients may be entrapped in microcapsules prepared, for e, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatinmicrocapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin pheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (18th Ed. Mack Printing Company, 1990). Sustained-release preparations may be prepared. le examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the ptide, which matrices are in the form of shaped articles, e.g. films, or microcapsules. In particular embodiments, prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin or combinations thereof.

In addition to the compositions described previously, the T cell activating bispecific antigen binding molecules may also be ated as a depot preparation. Such long acting formulations may be administered by implantation (for example aneously or intramuscularly) or by uscular injection. Thus, for example, the T cell activating bispecific antigen binding molecules may be formulated with le polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for e, as a sparingly soluble salt.

Pharmaceutical compositions comprising the T cell activating bispecific antigen binding molecules bed may be ctured by means of conventional , dissolving, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the proteins into preparations that can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

The T cell activating bispecific antigen binding molecules may be formulated into a composition in a free acid or base, neutral or salt form. Pharmaceutically acceptable salts are salts that substantially retain the biological ty of the free acid or base. These include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for e, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from nic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine. Pharmaceutical salts tend to be more soluble in aqueous and other protic solvents than are the corresponding free base forms.

Therapeutic s and Compositions Any of the T cell ting bispecific antigen binding les bed herein may be used in therapeutic methods. T cell activating bispecific antigen binding molecules described can be used as immunotherapeutic agents, for example in the treatment of cancers.

For use in therapeutic methods, T cell activating bispecific antigen binding molecules described would be formulated, dosed, and stered in a fashion consistent with good l practice.

Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of stration, the scheduling of administration, and other factors known to medical practitioners.

Described are T cell activating bispecific antigen binding molecules described for use as a medicament. Aso described are T cell activating bispecific antigen binding molecules described for use in treating a disease. In certain ments, T cell activating bispecific antigen binding molecules described for use in a method of treatment are described. In one embodiment, described is a T cell activating bispecific antigen binding molecule as described herein for use in the treatment of a disease in an individual in need thereof. In certain embodiments, described is a T cell activating bispecific antigen binding le for use in a method of treating an individual having a disease comprising administering to the individual a therapeutically ive amount of the T cell ting bispecific antigen binding molecule. In certain embodiments the disease to be treated is a proliferative disorder. In a ular ment the disease is cancer.

In certain embodiments the method further comprises administering to the individual a therapeutically effective amount of at least one onal therapeutic agent, e.g., an anti-cancer agent if the disease to be treated is cancer. In further embodiments, described is a T cell activating bispecific antigen binding molecule as bed herein for use in inducing lysis of a target cell, particularly a tumor cell. In certain embodiments, described is a T cell activating bispecific antigen binding molecule for use in a method of inducing lysis of a target cell, particularly a tumor cell, in an individual comprising administering to the individual an effective amount of the T cell activating bispecific antigen g molecule to induce lysis of a target cell. An “individual” according to any of the above embodiments is a mammal, preferably a human.

Also described is the use of a T cell activating bispecific antigen binding molecule described in the manufacture or preparation of a ment. In one embodiment the medicament is for the ent of a e in an individual in need thereof. In a further embodiment, the medicament is for use in a method of treating a disease comprising administering to an individual having the e a therapeutically effective amount of the medicament. In n embodiments the disease to be treated is a proliferative disorder. In a particular embodiment the disease is cancer.

In one embodiment, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., an anti-cancer agent if the disease to be treated is cancer. In a further embodiment, the ment is for inducing lysis of a target cell, particularly a tumor cell. In still a further embodiment, the medicament is for use in a method of inducing lysis of a target cell, particularly a tumor cell, in an individual comprising administering to the individual an effective amount of the ment to induce lysis of a target cell. An “individual” according to any of the above embodiments may be a mammal, preferably a human.

Also described is a method for treating a disease. In one embodiment, the method comprises administering to an individual having such disease a therapeutically effective amount of a T cell activating bispecific antigen binding molecule described. In one embodiment a composition is administered to said invididual, comprising the T cell activating bispecific n binding molecule described in a pharmaceutically acceptable form. In certain embodiments the disease to be treated is a proliferative er. In a ular embodiment the disease is cancer. In certain ments the method further comprises administering to the individual a therapeutically effective amount of at least one additional eutic agent, e.g., an anti-cancer agent if the e to be treated is cancer. An “individual” according to any of the above embodiments may be a mammal, preferably a human.

Also described is a method for inducing lysis of a target cell, particularly a tumor cell. In one embodiment the method comprises contacting a target cell with a T cell activating bispecific antigen binding molecule described in the presence of a T cell, particularly a cytotoxic T cell. In a further aspect, a method for inducing lysis of a target cell, particularly a tumor cell, in an individual is described. In one such embodiment, the method comprises administering to the dual an effective amount of a T cell activating bispecific n binding molecule to induce lysis of a target cell. In one embodiment, an “individual” is a human.

In certain embodiments the disease to be treated is a erative disorder, particularly cancer.

Non-limiting examples of cancers include r cancer, brain cancer, head and neck , pancreatic cancer, lung , breast cancer, n cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon , colorectal cancer, rectal cancer, gastric , prostate , blood cancer, skin cancer, squamous cell carcinoma, bone , and kidney cancer. Other cell proliferation disorders that can be treated using a T cell activating bispecific antigen binding molecule described include, but are not limited to neoplasms located in the: abdomen, bone, , digestive system, liver, pancreas, peritoneum, endocrine glands al, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, ic region, and urogenital system. Also included are pre-cancerous conditions or s and cancer metastases. In certain embodiments the cancer is chosen from the group consisting of renal cell cancer, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, head and neck cancer. A skilled artisan readily recognizes that in many cases the T cell activating bispecific antigen binding molecule may not provide a cure but may only provide l benefit. In some embodiments, a physiological change having some benefit is also considered therapeutically beneficial. Thus, in some embodiments, an amount of T cell activating bispecific antigen binding molecule that es a physiological change is considered an "effective amount" or a peutically effective amount". The subject, patient, or dual in need of treatment is typically a mammal, more specifically a human.

In some ments, an effective amount of a T cell activating bispecific antigen binding molecule described is administered to a cell. In other embodiments, a therapeutically effective amount of a T cell activating bispecific antigen binding molecule described is administered to an individual for the treatment of disease.

For the prevention or treatment of disease, the appropriate dosage of a T cell activating bispecific antigen binding molecule described (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the route of administration, the body weight of the t, the type of T cell activating bispecific antigen binding molecule, the severity and course of the disease, whether the T cell activating bispecific n binding molecule is administered for tive or therapeutic purposes, previous or concurrent therapeutic interventions, the patient's clinical history and response to the T cell activating bispecific n binding molecule, and the discretion of the attending physician. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject. Various dosing schedules including but not limited to single or multiple administrations over s time-points, bolus stration, and pulse infusion are contemplated herein.

The T cell activating bispecific antigen binding molecule is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 µg/kg to 15 mg/kg (e.g. 0.1 mg/kg – 10 mg/kg) of T cell activating bispecific antigen binding molecule can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. One typical daily dosage might range from about 1 µg/kg to 100 mg/kg or more, ing on the factors mentioned above. For repeated administrations over several days or longer, depending on the ion, the ent would generally be sustained until a desired suppression of disease symptoms occurs. One exemplary dosage of the T cell activating bispecific antigen binding molecule would be in the range from about 0.005 mg/kg to about 10 mg/kg. In other nonlimiting examples, a dose may also comprise from about 1 microgram/kg body weight, about 5 microgram/kg body , about 10 microgram/kg body weight, about 50 microgram/kg body weight, about 100 microgram/kg body weight, about 200 microgram/kg body weight, about 350 microgram/kg body weight, about 500 microgram/kg body weight, about 1 milligram/kg body weight, about 5 milligram/kg body , about 10 milligram/kg body weight, about 50 ram/kg body weight, about 100 milligram/kg body weight, about 200 milligram/kg body weight, about 350 ram/kg body weight, about 500 milligram/kg body weight, to about 1000 mg/kg body weight or more per administration, and any range derivable therein. In nonlimiting examples of a derivable range from the numbers listed herein, a range of about 5 mg/kg body weight to about 100 mg/kg body weight, about 5 microgram/kg body weight to about 500 milligram/kg body weight, etc., can be stered, based on the numbers bed above.

Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 5.0 mg/kg or 10 mg/kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, e.g. every week or every three weeks (e.g. such that the patient receives from about two to about twenty, or e.g. about six doses of the T cell activating bispecific antigen binding molecule). An initial higher loading dose, followed by one or more lower doses may be administered. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

The T cell activating bispecific antigen binding molecules described will generally be used in an amount ive to achieve the intended purpose. For use to treat or prevent a disease condition, the T cell activating bispecific antigen binding molecules described, or pharmaceutical compositions thereof, are administered or applied in a eutically effective amount.

Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed sure provided herein.

For systemic administration, a therapeutically effective dose can be estimated initially from in vitro assays, such as cell e assays. A dose can then be formulated in animal models to achieve a circulating concentration range that includes the IC50 as determined in cell culture.

Such information can be used to more accurately determine useful doses in humans.

Initial dosages can also be estimated from in vivo data, e.g., animal models, using techniques that are well known in the art. One having ordinary skill in the art could readily optimize administration to humans based on animal data.

Dosage amount and interval may be adjusted individually to provide plasma levels of the T cell activating bispecific antigen binding molecules which are sufficient to maintain therapeutic effect. Usual patient dosages for administration by injection range from about 0.1 to 50 mg/kg/day, typically from about 0.5 to 1 mg/kg/day. eutically effective plasma levels may be achieved by administering multiple doses each day. Levels in plasma may be measured, for example, by HPLC.

In cases of local administration or ive uptake, the effective local tration of the T cell activating bispecific n binding molecules may not be related to plasma concentration. One having skill in the art will be able to optimize eutically effective local dosages without undue experimentation.

A therapeutically effective dose of the T cell activating bispecific antigen binding molecules described herein will lly provide therapeutic benefit without causing substantial toxicity.

Toxicity and therapeutic efficacy of a T cell activating bispecific antigen binding molecule can be determined by standard pharmaceutical procedures in cell culture or mental s.

Cell e assays and animal studies can be used to determine the LD50 (the dose lethal to 50% of a population) and the ED50 (the dose eutically effective in 50% of a population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50/ED50. T cell ting bispecific antigen binding molecules that exhibit large eutic indices are preferred. In one embodiment, the T cell activating bispecific antigen g molecule described exhibits a high eutic index. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosages suitable for use in humans. The dosage lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range ing upon a variety of factors, e.g., the dosage form employed, the route of administration utilized, the condition of the subject, and the like. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition (see, e.g., Fingl et al., 1975, in: The Pharmacological Basis of Therapeutics, Ch. 1, p. 1, incorporated herein by reference in its entirety).

The attending physician for patients treated with T cell activating ific antigen binding molecules described would know how and when to terminate, interrupt, or adjust administration due to toxicity, organ dysfunction, and the like. Conversely, the attending physician would also know to adjust ent to higher levels if the clinical response were not adequate (precluding ty). The magnitude of an administered dose in the management of the disorder of interest will vary with the severity of the condition to be treated, with the route of administration, and the like. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency will also vary according to the age, body weight, and response of the individual patient.

Other Agents and Treatments The T cell activating bispecific antigen binding molecules described may be administered in combination with one or more other agents in therapy. For instance, a T cell activating bispecific antigen binding molecule described may be co-administered with at least one additional eutic agent. The term "therapeutic agent” encompasses any agent administered to treat a m or disease in an individual in need of such treatment. Such additional therapeutic agent may comprise any active ingredients suitable for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. In certain embodiments, an onal therapeutic agent is an immunomodulatory agent, a cytostatic agent, an inhibitor of cell adhesion, a cytotoxic agent, an activator of cell apoptosis, or an agent that increases the sensitivity of cells to tic inducers. In a particular embodiment, the additional therapeutic agent is an anti-cancer agent, for e a microtubule disruptor, an tabolite, a topoisomerase inhibitor, a DNA intercalator, an alkylating agent, a hormonal therapy, a kinase tor, a receptor antagonist, an tor of tumor cell apoptosis, or an antiangiogenic agent.

Such other agents are suitably present in combination in amounts that are effective for the purpose intended. The effective amount of such other agents s on the amount of T cell activating bispecific n binding molecule used, the type of er or treatment, and other factors discussed above. The T cell activating bispecific antigen binding molecules are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate.

Such ation therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate compositions), and separate administration, in which case, administration of the T cell activating bispecific n binding molecule described can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent and/or adjuvant. T cell activating bispecific antigen binding molecules described can also be used in combination with radiation therapy.

Articles of cture Also described is an article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the disorders described above. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or c. The container holds a ition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper able by a rmic injection needle). At least one active agent in the composition is a T cell activating bispecific n g molecule bed. The label or package insert indicates that the composition is used for treating the condition of choice. Moreover, the article of manufacture may se (a) a first container with a composition contained therein, wherein the composition comprises a T cell activating bispecific antigen binding molecule described; and (b) a second container with a composition contained therein, wherein the composition comprises a r cytotoxic or otherwise therapeutic agent. The article of manufacture in this embodiment may further comprise a package insert indicating that the compositions can be used to treat a particular condition. Alternatively, or onally, the article of manufacture may further comprise a second (or third) container sing a ceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, ts, filters, needles, and syringes.

Examples The following are es of methods and compositions described. It is understood that various other embodiments may be practiced, given the general description provided above.

General methods Recombinant DNA Techniques Standard methods were used to manipulate DNA as described in Sambrook et al., Molecular cloning: A tory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. The lar biological reagents were used according to the manufacturers’ instructions. General information regarding the nucleotide sequences of human globulins light and heavy chains is given in: Kabat, E.A. et al., (1991) Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication No. 2.

DNA Sequencing DNA sequences were determined by double strand cing.

Gene Synthesis Desired gene segments where required were either generated by PCR using appropriate templates or were synthesized by Geneart AG (Regensburg, Germany) from synthetic oligonucleotides and PCR ts by automated gene synthesis. In cases where no exact gene sequence was available, oligonucleotide primers were designed based on sequences from closest homologues and the genes were isolated by RT-PCR from RNA originating from the appropriate tissue. The gene segments flanked by singular restriction endonuclease cleavage sites were cloned into standard cloning / cing vectors. The plasmid DNA was purified from transformed bacteria and concentration determined by UV spectroscopy. The DNA sequence of the subcloned gene fragments was confirmed by DNA sequencing. Gene segments were designed with suitable restriction sites to allow sub-cloning into the respective expression vectors. All constructs were ed with a 5’-end DNA sequence coding for a leader peptide which targets proteins for secretion in eukaryotic cells. SEQ ID NOs 2 give exemplary leader peptides and polynucleotide sequences encoding them, respectively. ion of primary human pan T cells from PBMCs Peripheral blood mononuclear cells (PBMCs) were prepared by Histopaque density centrifugation from enriched cyte preparations (buffy coats) obtained from local blood banks or from fresh blood from healthy human . Briefly, blood was diluted with sterile PBS and carefully layered over a Histopaque gradient , H8889). After centrifugation for minutes at 450 x g at room temperature (brake switched off), part of the plasma above the PBMC containing interphase was discarded. The PBMCs were transferred into new 50 ml Falcon tubes and tubes were filled up with PBS to a total volume of 50 ml. The mixture was centrifuged at room temperature for 10 minutes at 400 x g (brake switched on). The supernatant was discarded and the PBMC pellet washed twice with sterile PBS (centrifugation steps at 4°C for 10 minutes at 350 x g). The resulting PBMC population was counted automatically (ViCell) and stored in RPMI1640 medium, containing 10% FCS and 1% L-alanyl-L-glutamine (Biochrom, K0302) at 37°C, 5% CO2 in the incubator until assay start.

T cell ment from PBMCs was performed using the Pan T Cell Isolation Kit II (Miltenyi Biotec #130156), according to the manufacturer’s instructions. Briefly, the cell pellets were diluted in 40 µl cold buffer per 10 million cells (PBS with 0.5% BSA, 2 mM EDTA, sterile filtered) and incubated with 10 µl Biotin-Antibody Cocktail per 10 million cells for 10 min at 4°C. 30 µl cold buffer and 20 µl Anti-Biotin magnetic beads per 10 million cells were added, and the mixture incubated for another 15 min at 4°C. Cells were washed by adding 10-20x the current volume and a uent centrifugation step at 300 x g for 10 min. Up to 100 n cells were resuspended in 500 µl buffer. Magnetic separation of unlabeled human pan T cells was performed using LS columns (Miltenyi Biotec #130401) according to the cturer’s instructions. The resulting T cell population was counted automatically (ViCell) and stored in AIM-V medium at 37°C, 5% CO2 in the tor until assay start (not longer than 24 h).

Isolation of primary human naive T cells from PBMCs Peripheral blood mononuclar cells (PBMCs) were ed by Histopaque density centrifugation from ed lymphocyte preparations (buffy coats) obtained from local blood banks or from fresh blood from healthy human donors. T-cell enrichment from PBMCs was performed using the Naive CD8+ T cell isolation Kit from Miltenyi Biotec (#130244), ing to the manufacturer’s instructions, but skipping the last isolation step of CD8+ T cells (also see description for the isolation of primary human pan T cells).

Isolation of murine pan T cells from splenocytes Spleens were isolated from C57BL/6 mice, transferred into a MACS C-tube (Miltenyi h #130237) containing MACS buffer (PBS + 0.5% BSA + 2 mM EDTA) and dissociated with the GentleMACS Dissociator to obtain single-cell suspensions according to the manufacturer’s instructions. The cell suspension was passed through a paration filter to remove remaining undissociated tissue particles. After fugation at 400 x g for 4 min at 4°C, ACK Lysis Buffer was added to lyse red blood cells (incubation for 5 min at room temperature).

The ing cells were washed with MACS buffer twice, counted and used for the isolation of murine pan T cells. The negative (magnetic) selection was performed using the Pan T Cell Isolation Kit from Miltenyi Biotec (#130861), following the manufacturer’s instructions.

The resulting T cell population was tically counted (ViCell) and immediately used for further assays.

Isolation of primary cynomolgus PBMCs from heparinized blood Peripheral blood mononuclar cells (PBMCs) were prepared by density centrifugation from fresh blood from healthy cynomolgus , as follows: nized blood was diluted 1:3 with sterile PBS, and Lymphoprep medium (Axon Lab #1114545) was d to 90% with sterile PBS. Two volumes of the diluted blood were layered over one volume of the diluted density gradient and the PBMC on was separated by centrifugation for 30 min at 520 x g, without brake, at room temperature. The PBMC band was transferred into a fresh 50 ml Falcon tube and washed with sterile PBS by centrifugation for 10 min at 400 x g at 4°C. One low-speed centrifugation was performed to remove the platelets (15 min at 150 x g, 4°C), and the resulting PBMC population was automatically counted (ViCell) and immediately used for further .

Target cells For the assessment of argeting bispecific antigen g molecules, the following tumor cell lines were used: the human melanoma cell line WM266-4 (ATCC 676), derived from a metastatic site of a ant melanoma and expressing high levels of human MCSP; and the human melanoma cell line MV-3 (a kind gift from The Radboud University Nijmegen Medical Centre), expressing medium levels of human MCSP.

For the assessment of CEA-targeting bispecific antigen g molecules, the following tumor cell lines were used: the human gastric cancer cell line MKN45 (DSMZ #ACC 409), expressing very high levels of human CEA; the human female Caucasian colon adenocarcinoma cell line LS-174T (ECACC #87060401), expressing medium to low levels of human CEA; the human epithelioid pancreatic carcinoma cell line Panc-1 (ATCC #CRL-1469), expressing (very) low levels of human CEA; and a murine colon carcinoma cell line MC38-huCEA, that was engineered in-house to stably express human CEA.

In on, a human T cell leukaemia cell line, Jurkat (ATCC #TIB-152), was used to assess binding of different bispecific constructs to human CD3 on cells.

Example 1 Preparation, purification and characterization of bispecific antigen binding molecules The heavy and light chain variable region sequences were subcloned in frame with either the constant heavy chain or the constant light chain pre-inserted into the respective recipient mammalian sion vector. The antibody expression was driven by an MPSV promoter and a synthetic polyA signal sequence is located at the 3’ end of the CDS. In addition each vector contained an EBV OriP sequence.

The molecules were produced by co-transfecting HEK293 EBNA cells with the mammalian expression vectors. Exponentially growing HEK293 EBNA cells were transfected using the calcium phosphate method. Alternatively, HEK293 EBNA cells growing in sion were transfected using polyethylenimine (PEI). For preparation of “1+1 IgG scFab, one armed / one armed inverted” constructs, cells were ected with the corresponding expression vectors in a 1:1:1 ratio (“vector heavy chain” : r light chain” : “vector heavy chain-scFab”). For preparation of “2+1 IgG scFab” constructs, cells were transfected with the corresponding sion vectors in a 1:2:1 ratio (“vector heavy chain” : “vector light chain” : “vector heavy chain-scFab”). For ation of “1+1 IgG Crossfab” constructs, cells were transfected with the corresponding expression vectors in a 1:1:1:1 ratio (“vector second heavy chain” : “vector first light chain” : “vector light chain ab” : “vector first heavy chain-heavy chain Crossfab”).

For preparation of “2+1 IgG Crossfab” constructs cells were transfected with the corresponding expression vectors in a 1:2:1:1 ratio (“vector second heavy chain” : “vector light chain” : “vector first heavy chain-heavy chain Crossfab)” : “vector light chain Crossfab”. For preparation of the “2+1 IgG Crossfab, linked light chain” construct, cells were transfected with the ponding expression vectors in a 1:1:1:1 ratio (“vector heavy chain” : “vector light chain” : r heavy chain (CrossFab-Fab-Fc)” : “vector linked light chain”). For preparation of the “1+1 CrossMab” construct, cells were transfected with the corresponding expression s in a 1:1:1:1 ratio (“vector first heavy chain” : “vector second heavy chain” : “vector first light chain” : r second light chain”). For preparation of the “1+1 IgG Crossfab light chain fusion ” construct, cells were transfected with the corresponding expression vectors in a 1:1:1:1 ratio (“vector first heavy chain” : r second heavy chain” : r light chain Crossfab” : “vector second light For transfection using calcium phosphate cells were grown as adherent monolayer cultures in T- flasks using DMEM culture medium supplemented with 10 % (v/v) FCS, and transfected when they were between 50 and 80 % confluent. For the transfection of a T150 flask, 15 million cells were seeded 24 hours before transfection in 25 ml DMEM culture medium supplemented with FCS (at 10% v/v final), and cells were placed at 37°C in an incubator with a 5% CO2 atmosphere overnight. For each T150 flask to be transfected, a solution of DNA, CaCl2 and water was prepared by mixing 94 µg total plasmid vector DNA divided in the corresponding ratio, water to a final volume of 469 µl and 469 µl of a 1 M CaCl2 solution. To this solution, 938 µl of a 50 mM HEPES, 280 mM NaCl, 1.5 mM Na2HPO4 solution at pH 7.05 were added, mixed immediately for 10 s and left to stand at room ature for 20 s. The suspension was diluted with 10 ml of DMEM supplemented with 2 % (v/v) FCS, and added to the T150 in place of the existing medium. Subsequently, additional 13 ml of transfection medium were added. The cells were incubated at 37°C, 5% CO2 for about 17 to 20 hours, then medium was replaced with 25 ml DMEM, 10 % FCS. The conditioned e medium was harvested approximately 7 days postmedia exchange by centrifugation for 15 min at 210 x g, sterile filtered (0.22  m filter), supplemented with sodium azide to a final concentration of 0.01 % (w/v), and kept at 4°C.

For transfection using polyethylenimine (PEI) HEK293 EBNA cells were cultivated in suspension in serum free CD CHO culture . For the production in 500 ml shake flasks, 400 million HEK293 EBNA cells were seeded 24 hours before transfection. For transfection cells were fuged for 5 min at 210 x g, and supernatant was replaced by 20 ml pre-warmed CD CHO medium. Expression vectors were mixed in 20 ml CD CHO medium to a final amount of 200 μg DNA. After addition of 540 μl PEI, the mixture was vortexed for 15 s and subsequently incubated for 10 min at room ature. Afterwards cells were mixed with the DNA/PEI solution, transferred to a 500 ml shake flask and incubated for 3 hours at 37°C in an incubator with a 5% CO2 atmosphere. After the incubation time 160 ml F17 medium was added and cells were cultivated for 24 hours. One day after transfection 1 mM valproic acid and 7% Feed 1 (Lonza) were added. After a cultivation of 7 days, supernatant was collected for purification by centrifugation for 15 min at 210 x g, the solution was sterile filtered (0.22 μm filter), supplemented with sodium azide to a final concentration of 0.01 % w/v, and kept at 4°C.

The secreted proteins were ed from cell e supernatants by Protein A affinity chromatography, followed by a size exclusion chromatography step.

For affinity chromatography supernatant was loaded on a HiTrap ProteinA HP column (CV = 5 ml, GE Healthcare) equilibrated with 25 ml 20 mM sodium phosphate, 20 mM sodium e, pH 7.5 or 40 ml 20 mM sodium ate, 20 mM sodium e, 0.5 M sodium chloride, pH 7.5. Unbound n was removed by washing with at least ten column volumes 20 mM sodium phosphate, 20 mM sodium citrate, 0.5 M sodium chloride pH 7.5, followed by an additional wash step using six column volumes 10 mM sodium phosphate, 20 mM sodium citrate, 0.5 M sodium de pH 5.45. Subsequently, the column was washed with 20 ml 10 mM MES, 100 mM sodium chloride, pH 5.0, and target protein was eluted in six column volumes 20 mM sodium citrate, 100 mM sodium chloride, 100 mM glycine, pH 3.0. Alternatively, target protein was eluted using a gradient over 20 column volumes from 20 mM sodium citrate, 0.5 M sodium chloride, pH 7.5 to 20 mM sodium citrate, 0.5 M sodium chloride, pH 2.5. The protein on was neutralized by adding 1/10 of 0.5 M sodium phosphate, pH 8. The target protein was concentrated and filtrated prior to loading on a HiLoad Superdex 200 column (GE Healthcare) equilibrated with 25 mM potassium phosphate, 125 mM sodium chloride, 100 mM glycine solution of pH 6.7. For the purification of 1+1 IgG ab the column was equilibrated with 20 mM ine, 140 mM sodium chloride solution of pH 6.0.

The protein concentration of purified n samples was determined by measuring the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence. Purity and molecular weight of the bispecific constructs were analyzed by SDS-PAGE in the presence and absence of a reducing agent (5 mM 1,4-dithiotreitol) and staining with Coomassie (SimpleBlue™ ain from Invitrogen) using the NuPAGE® Pre- Cast gel system (Invitrogen, USA) was used according to the manufacturer’s instructions (4-12% cetate gels or 4-12% Bis-Tris). Alternatively, purity and molecular weight of molecules were analyzed by CE-SDS analyses in the presence and e of a reducing agent, using the Caliper p GXII system (Caliper Lifescience) according to the manufacturer’s instructions.

The aggregate content of the protein samples was analyzed using a Superdex 200 10/300GL analytical size-exclusion chromatography column (GE care) in 2 mM MOPS, 150 mM NaCl, 0.02% (w/v) NaN3, pH 7.3 running buffer at 25°C. Alternatively, the ate content of antibody samples was analyzed using a TSKgel G3000 SW XL analytical size-exclusion column (Tosoh) in 25 mM K2HPO4, 125 mM NaCl, 200 mM L-arginine monohydrocloride, 0.02% (w/v) NaN3, pH 6.7 g buffer at 25°C.

Figures 2-14 show the results of the SDS PAGE and analytical size exclusion chromatography and Table 2A shows the yields, aggregate content after Protein A, and final monomer content of the preparations of the different bispecific constructs.

Figure 47 shows the result of the CE-SDS analyses of the anti-CD3/anti-MCSP bispecific “2+1 IgG Crossfab, linked light chain” construct (see SEQ ID NOs 3, 5, 29 and 179). 2 µg sample was used for analyses. Figure 48 shows the result of the analytical size exclusion tography of the final product (20 µg sample injected).

Figure 54 shows the results of the CE-SDS and SDS PAGE analyses of various constructs, and Table 2A shows the yields, aggregate content after n A and final monomer t of the preparations of the different bispecific constructs.

TABLE 2A. Yields, aggregate content after Protein A and final r content.

Construct Yield Aggregate HMW LMW Monomer [mg/l] content after [%] [%] [%] Protein A [%] MCSP 2+1 IgG Crossfab; VH/VL 12.8 2.2 0 0 100 exchange (LC007/V9) (SEQ ID NOs 3, 5, 29, 33) 2+1 IgG Crossfab; VH/VL 3.2 5.7 0.4 0 99.6 exchange (LC007/FN18) (SEQ ID NOs 3, 5, 35, 37) 2+1 IgG scFab, P329G LALA 11.9 23 0.3 0 99.7 (SEQ ID NOs 5, 21, 23) 2+1 IgG scFab, LALA 9 23 0 0 100 (SEQ ID NOs 5, 17, 19) 2+1 IgG scFab, P329G LALA 12.9 32.7 0 0 100 N297D (SEQ ID NOs 5, 25, 27) 2+1 IgG scFab, wt 15.5 31.8 0 0 100 (SEQ ID NOs 5, 13, 15) 1+1 IgG scFab 7 24.5 0 0 100 (SEQ ID NOs 5, 21, 213) 1+1 IgG scFab “one armed” 7.6 43.7 2.3 0 97.7 (SEQ ID NOs 1, 3, 5) 1+1 IgG scFab “one armed 1 27 7.1 9.1 83.8 inverted” (SEQ ID NOs 7, 9, 11) 1+1 IgG Crossfab; VH/VL 9.8 0 0 0 100 exchange (LC007/V9) (SEQ ID NOs 5, 29, 31, 33) 2+1 IgG Crossfab, linked light 0.54 40 1.4 0 98.6 chain; VL/VH ge (LC007/V9) (SEQ ID NOs 3, 5, 29, 179) 1+1 IgG Crossfab; VL/VH 6.61 8.5 0 0 100 exchange (LC007/V9) (SEQ ID NOs 5, 29, 33, 181) 1+1 CrossMab; CL/CH1 exchange 6.91 10.5 1.3 1.7 97 (LC00/V9) (SEQ ID NOs 5, 23, 183, 185) 2+1 IgG ab, inverted; 9.45 6.1 0.8 0 99.2 CL/CH1 exchange (LC007/V9) (SEQ ID NOs 5, 23, 183, 187) 2+1 IgG Crossfab; VL/VH 36.6 0 9.5 35.3 55.2 exchange (M4-3 ML2/V9) (SEQ ID NOs 33, 189, 191, 193) 2+1 IgG Crossfab; CL/CH1 2.62 12 2.8 0 97.2 exchange (M4-3 ML2/V9) (SEQ ID NOs 183, 189, 193, 195) 2+1 IgG ab; CL/CH1 29.75 0 0 0 100 exchange (M4-3 ML2/H2C) (SEQ ID NOs 189, 193, 199, 201) 2+1 IgG Crossfab; CL/CH1 1.2 0 1.25 1.65 97.1 exchange (LC007/anti-CD3) (SEQ ID NOs 5, 23, 215, 217) 2+1 IgG Crossfab, inverted; 7.82 0.5 0 0 100 CL/CH1 exchange (LC007/anti- CD3) (SEQ ID NOs 5, 23, 215, 219) EGFR 2+1 IgG scFab 5.2 53 0 30 70 (SEQ ID NOs 45, 47, 53) 1+1 IgG scFab 3.4 66.6 0 1.6 98.4 (SEQ ID NOs 47, 53, 213) 1+1 IgG scFab “one armed” 9.05 60.8 0 0 100 (SEQ ID NOs 43, 45, 47) 1+1 IgG scFab “one armed 3.87 58.8 0 0 100 inverted” (SEQ ID NOs 11, 49, 51) 2+1 IgG scFab 12.57 53 0 0 100 (SEQ ID NOs 57, 59, 61) 1+1 IgG scFab 17.95 41 0.4 0 99.6 (SEQ ID NOs 57, 61, 213) 1+1 IgG scFab “one armed 2.44 69 0.6 0 99.4 ed” (SEQ ID NOs 11, 51, 55) 2+1 IgG Crossfab, inverted; VL/VH 0.34 13 4.4 0 95.6 exchange (CH1A1A/V9) (SEQ ID NOs 33, 63, 65, 67) 2+1 IgG Crossfab, inverted; 12.7 43 0 0 100 CL/CH1 exchange (CH1A1A/V9) (SEQ ID NOs 65, 67, 183, 197) 2+1 IgG Crossfab, inverted; 7.1 20 0 0 100 CL/CH1 exchange (431/26/V9) (SEQ ID NOs 183, 203, 205, 207) 1+1 IgG-Crossfab light chain fusion 7.85 27 4.3 3.2 92.5 (CH1A1A/V9) (SEQ ID NOs 183, 209, 211, 213) As controls, bispecific antigen binding molecules were generated in the prior art tandem scFv format (“(scFv)2”) and by fusing a tandem scFv to an Fc domain (“(scFv)2-Fc”). The molecules were produced in HEK293-EBNA cells and purified by Protein A affinity chromatography followed by a size exclusion chromatographic step in an analogous manner as described above for the bispecific antigen g molecules described. Due to high ate formation, some of the samples had to be further purified by applying eluted and concentrated samples from the HiLoad Superdex 200 column (GE Healthcare) to a Superdex 10/300 GL column (GE Healthcare) brated with 20 mM histidine, 140 mM sodium chloride, pH 6.7 in order to obtain protein with high monomer t. Subsequently, protein concentration, purity and molecular weight, and ate content were determined as bed above.

Yields, aggregate content after the first purification step, and final r content for the control molecules is shown in Table 2B. Comparison of the aggregate content after the first purification step (Protein A) indicates the superior ity of the IgG Crossfab and IgG scFab constructs compared to the “(scFv)2-Fc” and the disulfide bridge-stabilized “(dsscFv)2-Fc” molecules.

TABLE 2B. Yields, aggregate content after Protein A and final monomer t.

Construct Yield Aggregates after Final [mg/l] ProteinA [%] HMW LMW Monomer [%] [%] [%] (scFv)2-Fc 76.5 40 0.5 0 99.5 (antiMCSP/anti huCD3) v)2-Fc 2.65 48 7.3 8.0 84.7 (antiMCSP/anti huCD3) Thermal stability of the proteins was monitored by Dynamic Light Scattering (DLS). 30  g of ed protein sample with a protein concentration of 1 mg/ml was applied in duplicate to a Dynapro plate reader (Wyatt Technology Corporation; USA). The temperature was ramped from to75°C at 0.05°C/min, with the radius and total scattering intensity being ted. The results are shown in Figure 15 and Table 2C. For the “(scFv)2-Fc” (antiMCSP/anti huCD3) molecule two aggregation points were observed, at 49°C and 68°C. The “(dsscFv)2-Fc” construct has an sed aggregation temperature (57°C) as a result of the introduced disulfide bridge (Figure 15A, Table 2C). Both, the “2+1 IgG scFab” and the “2+1 IgG Crossfab” constructs are aggregating at temperatures higher than 60°C, demonstrating their superior l stability as compared to the “(scFv)2-Fc” and Fv)2-Fc” s (Figure 15B, Table 2C).

TABLE 2C. Thermal stability determined by dynamic light scattering.

Construct Tagg [°C] 2+1 IgG scFab (LC007/V9) 68 2+1 IgG Crossfab (LC007/V9) 65 Fc-(scFv)2 (LC007/V9) 49/68 Fc-(dsscFv)2 (LC007/V9) 57 Example 2 Surface Plasmon resonance analysis of Fc receptor and target antigen binding Method All surface plasmon resonance (SPR) experiments are performed on a Biacore T100 at 25°C with HBS-EP as g buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20, Biacore, Freiburg/Germany).

Analysis of FcR binding of different Fc-variants The assay setup is shown in Figure 16A. For analyzing interaction of different Fc-variants with human FcγRIIIa-V158 and murine FcγRIV direct ng of around 6,500 resonance units (RU) of the anti-Penta His antibody (Qiagen) is med on a CM5 chip at pH 5.0 using the standard amine coupling kit (Biacore, Freiburg/Germany). HuFcγRIIIa-V158-K6H6 and muFcγRIV- aviHis-biotin are captured for 60 s at 4 and 10 nM respectively.

Constructs with different Fc-mutations are passed h the flow cells for 120 s at a concentration of 1000 nM with a flow rate of 30 μl/min. The dissociation is monitored for 220 s.

Bulk refractive index differences are corrected for by subtracting the response obtained in a reference flow cell. Here, the Fc-variants are flown over a surface with immobilized anti-Penta His antibody but on which HBS-EP has been injected rather than HuFcγRIIIa-V158-K6H6 or IV-aviHis-biotin. Affinity for human FcγRIIIa-V158 and murine FcγRIV was determined for wild-type Fc using a concentration range from 500 – 4000 nM.

The steady state response was used to derive the dissociation constant KD by non-linear curve fitting of the Langmuir binding isotherm. Kinetic constants were derived using the Biacore T100 tion Software (vAA, e AB, a/Sweden), to fit rate equations for 1:1 Langmuir binding by numerical integration.

Result The interaction of Fc variants with human FcγRIIIa and murine FcγRIV was monitored by surface plasmon resonance. Binding to captured IIIa-V158-K6H6 and muFcγRIV- aviHis-biotin is significantly reduced for all analyzed Fc mutants as compared to the construct with a wild-type (wt) Fc domain.

The Fc mutants with the lowest binding to the human Fcγ-receptor were P329G L234A L235A (LALA) and P329G LALA N297D. The LALA on alone was not enough to abrogate binding to huFcγRIIIa-V158-K6H6. The Fc variant carrying only the LALA mutation had a residual binding affinity to human FcγRIIIa of 2.100 nM, while the wt Fc bound the human FcγRIIIa receptor with an affinity of 600 nM (Table 3). Both KD values were derived by 1:1 binding model, using a single tration. ty to human FcγRIIIa-V158 and murine FcγRIV could only be analyzed for wt Fc. KD values are listed in Table 3. Binding to the murine FcγRIV was almost completely eliminated for all analyzed Fc mutants.

TABLE 3. Affinity of Fc-variants to the human FcγRIIIa-V158 and murine FcγRIV.

KD in nM human FcγRIIIa-V158 murine FcγRIV T = 25°C kinetic steady state kinetic steady state Fc-wt 600* (1200) 3470 576 1500 (SEQ ID NOs 5, 13, 15) Fc-LALA 2130* n.d. n.d.

(SEQ ID NOs 5, 17, 19) Fc-P329G LALA n.d. n.d.

(SEQ ID NOs 5, 21, 23) Fc-P329G LALA N297D n.d. n.d.

(SEQ ID NOs 5, 25, 27) *determined using one concentration (1000 nM) Analysis of simultaneous binding to tumor antigen and CD3 Analysis of simultaneous binding of the T-cell bispecific constructs to the tumor antigen and the human CD3ε was performed by direct coupling of 1650 resonance units (RU) of biotinylated D3 domain of MCSP on a sensor chip SA using the standard coupling procedure. Human EGFR was immobilized using standard amino coupling procedure. 8000 RU were immobilized on a CM5 sensor chip at pH 5.5. The assay setup is shown in Figure 16B.

Different T-cell bispecific constructs were captured for 60 s at 200 nM. Human CD3γ(G4S)5CD3ε–AcTev–Fc(knob)–Avi/Fc(hole) was subsequently passed at a concentration of 2000 nM and a flow rate of 40 μl/min for 60 s. Bulk refractive index differences were corrected for by subtracting the response obtained on a nce flow cell where the recombinant CD3ε was flown over a surface with immobilized D3 domain of MCSP or EGFR without captured T- cell bispecific constructs.

Result Simultaneous binding to both tumor antigen and human CD3ε was ed by surface plasmon resonance (Figure 17, Figure 18). All constructs were able to bind the tumor antigen and the CD3 simultaneously. For most of the constructs the binding level (RU) after injection of human CD3ε was higher than the binding level achieved after injection of the construct alone ting that both tumor antigen and the human CD3ε were bound to the construct.

Example 3 Binding of bispecific constructs to the respective target antigen on cells Binding of the different bispecific ucts to CD3 on Jurkat cells (ATCC 52), and the respective tumor n on target cells, was determined by FACS. Briefly, cells were harvested, counted and checked for ity. 0.15 – 0.2 million cells per well (in PBS containing 0.1% BSA; 90 µl) were plated in a bottom 96-well plate and incubated with the indicated tration of the bispecific constructs and corresponding IgG controls (10 µl) for 30 min at 4°C. For a better comparison, all constructs and IgG controls were normalized to same molarity.

After the incubation, cells were centrifuged (5 min, 350 x g), washed with 150 µl PBS containing 0.1% BSA, resuspended and incubated for further 30 min at 4°C with 12 µl/well of a FITC-or PE-conjugated secondary antibody. Bound constructs were detected using a FACSCantoII (Software FACS Diva). The “(scFv)2” molecule was detected using a FITC- conjugated anti-His antibody (Lucerna, 45F-Z). For all other les, a FITC- or PE- conjugated AffiniPure F(ab’)2 Fragment goat uman IgG Fcγ Fragment Specific (Jackson Immuno Research Lab # 109098 / g solution 1:20, or #109170 / working solution 1:80, respectively) was used. Cells were washed by addition of 120 µl/well PBS containing 0.1% BSA and centrifugation at 350 x g for 5 min. A second washing step was performed with 150 µl/well PBS containing 0.1% BSA. Unless otherwise indicated, cells were fixed with 100 µl/well fixation buffer (BD #554655) for 15 min at 4°C in the dark, centrifuged for 6 min at 400 x g and kept in 200 µl/well PBS containing 0.1% BSA until the samples were measured with FACS CantoII. EC50 values were calculated using the GraphPad Prism re.

In a first experiment, different bispecific constructs targeting human MCSP and human CD3 were analyzed by flow cytometry for binding to human CD3 expressed on Jurkat, human T cell leukaemia cells, or to human MCSP on Colo-38 human melanoma cells.

Results are presented in Figure 19-21, which show the mean fluorescence intensity of cells that were incubated with the bispecific molecule, l IgG, the secondary antibody only, or left untreated.

As shown in Figure 19, for both n binding moieties of the “(scFv)2” molecule, i.e. CD3 e 191A) and MCSP (Figure 19B), a clear binding signal is observed compared to the control samples.

The “2+1 IgG scFab” molecule (SEQ ID NOs 5, 17, 19) shows good binding to huMCSP on Colo-38 cells (Figure 20A). The CD3 moiety binds CD3 slightly better than the reference antihuman CD3 IgG (Figure 20B).

As depicted in Figure 21A, the two “1+1” constructs show comparable binding s to human CD3 on cells. The reference anti-human CD3 IgG gives a slightly weaker signal. In addition, both constructs tested (“1+1 IgG scFab, one-armed” (SEQ ID NOs 1, 3, 5) and “1+1 IgG scFab, med ed” (SEQ ID NOs 7, 9, 11)) show able g to human MCSP on cells (Figure 21B). The binding signal obtained with the reference anti-human MCSP IgG is slightly weaker.

In another experiment, the ed “2+1 IgG scFab” bispecific construct (SEQ ID NOs 5, 17, 19) and the corresponding anti human MCSP IgG were analyzed by flow cytometry for dosedependent binding to human MCSP on Colo-38 human melanoma cells, to determine whether the bispecific construct binds to MCSP via one or both of its “arms”. As depicted in Figure 22, the “2+1 IgG scFab” construct shows the same binding pattern as the MCSP IgG.

In yet another experiment, the binding of CD3/CEA “2+1 IgG Crossfab, ed” bispecific constructs with either a VL/VH (see SEQ ID NOs 33, 63, 65, 67) or a CL/CH1 exchange (see SEQ ID NOs 66, 67, 183, 197) in the Crossfab fragment to human CD3, expressed by Jurkat cells, or to human CEA, expressed by LS-174T cells, was assessed. As a control, the equivalent maximum concentration of the corresponding IgGs and the background staining due to the labeled 2ndary antibody (goat anti-human FITC-conjugated AffiniPure F(ab’)2 Fragment, Fcγ Fragment-specific, Jackson Immuno Research Lab # 109098) were assessed as well. As illustrated in Figure 55, both ucts show good binding to human CEA, as well as to human CD3 on cells. The ated EC50 values were 4.6 and 3.9 nM (CD3), and 9.3 and 6.7 nM (CEA) for the “2+1 IgG Crossfab, inverted (VL/VH)” and the “2+1 IgG Crossfab, inverted (CL/CH1)” constructs, respectively.

In another experiment, the binding of CD3/MCSP “2+1 IgG ab” (see SEQ ID NOs 3, 5, 29, 33) and “2+1 IgG Crossfab, inverted” (see SEQ ID NOs 5, 23, 183, 187) constructs to human CD3, expressed by Jurkat cells, or to human MCSP, expressed by WM266-4 cells, was assessed.

Figure 56 shows that, while binding of both constructs to MCSP on cells was comparably good, the binding of the “inverted” construct to CD3 was reduced compared to the other construct. The calculated EC50 values were 6.1 and 1.66 nM (CD3), and 0.57 and 0.95 nM (MCSP) for the “2+1 IgG Crossfab, inverted” and the “2+1 IgG Crossfab” constructs, tively.

In a further ment, binding of the “1+1 IgG Crossfab light chain (LC) fusion” construct (SEQ ID NOs 183, 209, 211, 213) to human CD3, expressed by Jurkat cells, and to human CEA, expressed by LS-174T cells was ined. As a l, the equivalent maximum concentration of the corresponding D3 and anti-CEA IgGs and the background staining due to the d 2ndary antibody (goat anti-human FITC-conjugated AffiniPure F(ab’)2 Fragment, Fcγ Fragment-specific, Jackson Immuno Research Lab #109098) were assessed as well. As depicted in Figure 57, the binding of the “1+1 IgG Crossfab LC fusion” to CEA appears to be greatly reduced, whereas the binding to CD3 was at least able to the reference IgG.

In a final experiment, binding of the “2+1 IgG Crossfab” (SEQ ID NOs 5, 23, 215, 217) and the “2+1 IgG ab, inverted” (SEQ ID NOs 5, 23, 215, 219) constructs to human CD3, expressed by Jurkat cells, and to human MCSP, expressed by WM266-4 tumor cells was determined. As depicted in Figure 58 the g to human CD3 was reduced for the “2+1 IgG Crossfab, inverted” compared to the other construct, but the binding to human MCSP was comparably good. The calculated EC50 values were 10.3 and 32.0 nM (CD3), and 3.1 and 3.4 nM (MCSP) for the “2+1 IgG Crossfab” and the “2+1 IgG Crossfab, inverted” uct, respectively.

Example 4 FACS analysis of surface tion markers on primary human T cells upon engagement of bispecific constructs The purified huMCSP-huCD3-targeting bispecific “2+1 IgG scFab” (SEQ ID NOs 5, 17, 19) and “(scFv)2” molecules were tested by flow cytometry for their ial to up-regulate the early surface activation marker CD69, or the late activation marker CD25 on CD8+ T cells in the ce of human MCSP-expressing tumor cells.

Briefly, MCSP-positive Colo-38 cells were harvested with Cell Dissociation buffer, counted and checked for viability. Cells were adjusted to 0.3 x 106 (viable) cells per ml in AIM-V medium, 100 µl of this cell suspension per well were pipetted into a bottom 96-well plate (as indicated). 50 µl of the (diluted) bispecific construct were added to the cell-containing wells to obtain a final concentration of 1 nM. Human PBMC effector cells were isolated from fresh blood of a healthy donor and adjusted to 6 x 106 (viable) cells per ml in AIM-V medium. 50 µl of this cell suspension was added per well of the assay plate (see above) to obtain a final E:T ratio of :1. To analyze whether the bispecific constructs are able to activate T cells exclusively in the presence of target cells expressing the tumor antigen huMCSP, wells were included that contained 1 nM of the respective bispecific molecules, as well as PBMCs, but no target cells.

After incubation for 15 h (CD69), or 24 h (CD25) at 37°C, 5% CO2, cells were centrifuged (5 min, 350 x g) and washed twice with 150 l PBS containing 0.1% BSA. Surface staining for CD8 (mouse IgG1,κ; clone HIT8a; BD #555635), CD69 (mouse IgG1; clone L78; BD 0) and CD25 (mouse IgG1,ĸ; clone M-A251; BD #555434) was med at 4°C for 30 min, according to the supplier’s suggestions. Cells were washed twice with 150 µl/well PBS containing 0.1% BSA and fixed for 15 min at 4°C, using 100 µl/well fixation buffer (BD #554655). After centrifugation, the s were resuspended in 200 µl/well PBS with 0.1% BSA and analyzed using a FACS I machine (Software FACS Diva).

Figure 23 depicts the expression level of the early activation marker CD69 (A), or the late activation marker CD25 (B) on CD8+ T cells after 15 hours or 24 hours incubation, respectively.

Both constructs induce up-regulation of both activation markers exclusively in the presence of target cells. The “(scFv)2” molecule seems to be slightly more active in this assay than the “2+1 IgG scFab” construct.

The ed huMCSP-huCD3-targeting bispecific “2+1 IgG scFab” and “(scFv)2” molecules were further tested by flow cytometry for their ial to up-regulate the late tion marker CD25 on CD8+ T cells or CD4+ T cells in the presence of human MCSP-expressing tumor cells.

Experimental procedures were as described above, using human pan T effector cells at an E:T ratio of 5:1 and an incubation time of five days.

Figure 24 shows that both constructs induce up-regulation of CD25 exclusively in the presence of target cells on both, CD8+ (A) as well as CD4+ (B) T cells. The “2+1 IgG scFab” construct seems to induce less up-regulation of CD25 in this assay, compared to the “(scFv)2” molecule. In general, the up-regulation of CD25 is more nced on CD8+ than on CD4+ T cells.

In another experiment, purified “2+1 IgG Crossfab” targeting cynomolgus CD3 and human MCSP (SEQ ID NOs 3, 5, 35, 37) was analyzed for its potential to up-regulate the surface activation marker CD25 on CD8+ T cells in the presence of tumor target cells. Briefly, human MCSP-expressing MV-3 tumor target cells were harvested with Cell Dissociation Buffer, washed and resuspendend in DMEM containing 2% FCS and 1% GlutaMax. 30 000 cells per well were plated in a round-bottom 96-well plate and the respective antibody dilution was added at the indicated concentrations (Figure 25). The bispecific uct and the different IgG controls were adjusted to the same molarity. Cynomolgus PBMC effector cells, isolated from blood of two healthy animals, were added to obtain a final E:T ratio of 3:1. After an incubation for 43 h at 37°C, 5% CO2, the cells were centrifuged at 350 x g for 5 min and washed twice with PBS, containing 0.1% BSA. Surface staining for CD8 (Miltenyi Biotech #130601) and CD25 (BD 8) was performed according to the er’s suggestions. Cells were washed twice with 150 µl/well PBS containing 0.1% BSA and fixed for 15 min at 4°C, using 100 µl/well fixation buffer (BD #554655). After centrifugation, the samples were resuspended in 200 µl/well PBS with 0.1% BSA and analyzed using a FACS I machine (Software FACS Diva).

As depicted in Figure 25, the ific construct induces tration-dependent up-regulation of CD25 on CD8+ T cells only in the presence of target cells. The anti cyno CD3 IgG (clone FN- 18) is also able to induce up-regulation of CD25 on CD8+ T cells, without being crosslinked (see data obtained with cyno Nestor). There is no hyperactivation of cyno T cells with the maximal tration of the bispecific construct (in the absence of target cells).

In another experiment, the CD3-MCSP “2+1 IgG Crossfab, linked light chain” (see SEQ ID NOs 3, 5, 29, 179) was compared to the CD3-MCSP “2+1 IgG Crossfab” (see SEQ ID NOs 3, 5, 29, 33) for its ial to up-regulate the early activation marker CD69 or the late activation marker CD25 on CD8+ T cells in the presence of tumor target cells. Primary human PBMCs (isolated as described above) were incubated with the indicated concentrations of bispecific constructs for at least 22 h in the presence or absence of MCSP-positive Colo38 target cells. Briefly, 0.3 million primary human PBMCs were plated per well of a flat-bottom 96-well plate, containing the ositive target cells (or medium). The final effector to target cell (E:T) ratio was 10:1.

The cells were incubated with the ted concentration of the bispecific constructs and controls for the indicated incubation times at 37°C, 5% CO2. The or cells were stained for CD8, and CD69 or CD25 and analyzed by FACS CantoII.

Figure 53 shows the result of this ment. There were no significant differences detected for CD69 (A) or CD25 up-regulation (B) between the two 2+1 IgG Crossfab molecules (with or without the linked light chain).

In yet r experiment, the CD3/MCSP “2+1 IgG ab” (see SEQ ID NOs 3, 5, 29, 33) and “1+1 IgG Crossfab” (see SEQ ID NOs 5, 29, 33, 181) constructs were compared to the “1+1 CrossMab” construct (see SEQ ID NOs 5, 23, 183, 185) for their potential to up-regulate CD69 or CD25 on CD4+ or CD8+ T cells in the presence of tumor target cells. The assay was performed as described above, in the presence of absence of human MCSP expressing MV-3 tumor cells, with an incubation time of 24 h.

As shown in Figure 59, the “1+1 IgG Crossfab” and “2+1 IgG Crossfab” ucts d more pronounced upregulation of activation markers than the “1+1 CrossMab” molecule.

In a final experiment, the CD3/MCSP “2+1 IgG Crossfab” (see SEQ ID NOs 5, 23, 215, 217) and “2+1 IgG Crossfab, inverted” (see SEQ ID NOs 5, 23, 215, 219) constructs were assessed for their potential to up-regulate CD25 on CD4+ or CD8+ T cells from two different cynomolgus monkeys in the presence of tumor target cells. The assay was performed as described above, in the presence of absence of human MCSP expressing MV-3 tumor cells, with an E:T ratio of 3:1 and an incubation time of about 41 h.

As shown in Figure 60, both constructs were able to up-regulate CD25 on CD4+ and CD8+ T cells in a concentration-dependent manner, without significant difference between the two formats. Control samples without antibody and without target cells gave a comparable signal to the s with dy but no targets (not .

Example 5 Interferon-γ secretion upon activation of human pan T cells with CD3 bispecific constructs Purified “2+1 IgG scFab” targeting human MCSP and human CD3 (SEQ ID NOs 5, 17, 19) was analyzed for its potential to induce T cell activation in the ce of human MCSP-positive U- 87MG cells, measured by the release of human interferon (IFN)-γ into the supernatant. As controls, anti-human MCSP and anti-human CD3 IgGs were used, ed to the same molarity.

Briefly, huMCSP-expressing U-87MG glioblastoma astrocytoma target cells (ECACC 02) were harvested with Cell Dissociation Buffer, washed and resuspendend in AIM-V medium (Invitrogen #12055-091). 20 000 cells per well were plated in a round-bottom 96-wellplate and the respective antibody dilution was added to obtain a final concentration of 1 nM.

Human pan T effector cells, isolated from Buffy Coat, were added to obtain a final E:T ratio of :1. After an overnight incubation of 18.5 h at 37°C, 5% CO2, the assay plate was centrifuged for min at 350 x g and the supernatant was transferred into a fresh l plate. Human IFN-γ levels in the supernatant were measured by ELISA, according to the manufacturer’s instructions (BD OptEIA human IFN-γ ELISA Kit II from Becton Dickinson, #550612).

As ed in Figure 26, the reference IgGs show no to weak induction of IFN-γ secretion, whereas the “2+1 IgG scFab” construct is able to activate human T cells to secrete IFN-γ.

Example 6 Re-directed T cell cytotoxicity mediated by cross-linked bispecific constructs targeting CD3 on T cells and MCSP or EGFR on tumor cells (LDH release assay) In a first series of experiments, bispecific constructs targeting CD3 and MCSP were analyzed for their ial to induce T cell-mediated apoptosis in tumor target cells upon crosslinkage of the construct via binding of the antigen binding es to their tive target antigens on cells es 27-38).

In one experiment purified “2+1 IgG scFab” (SEQ ID NOs 5, 21, 23) and “2+1 IgG Crossfab” (SEQ ID NOs 3, 5, 29, 33) constructs targeting human CD3 and human MCSP, and the corresponding “(scFv)2” molecule, were compared. Briefly, huMCSP-expressing MDA-MB-435 human melanoma target cells were harvested with Cell Dissociation Buffer, washed and resuspendend in AIM-V medium (Invitrogen # 12055-091). 30 000 cells per well were plated in a round-bottom 96-well plate and the respective dilution of the construct was added at the indicated tration. All constructs and corresponding l IgGs were adjusted to the same molarity. Human pan T effector cells were added to obtain a final E:T ratio of 5:1. As a positive control for the activation of human pan T cells, 1 µg/ml PHA-M (Sigma #L8902; mixture of tins isolated from Phaseolus vulgaris) was used. For ization, maximal lysis of the target cells (= 100%) was determined by tion of the target cells with a final concentration of 1% Triton X-100. Minimal lysis (= 0%) refers to target cells co-incubated with effector cells, but without any construct or dy. After an ght incubation of 20 h at 37°C, 5% CO2, LDH release of apoptotic/necrotic target cells into the supernatant was measured with the LDH detection kit (Roche Applied Science, #11 644 793 001), according to the manufacturer’s instructions.

As depicted in Figure 27, both “2+1” constructs induce apoptosis in target cells comparable to the “(scFv)2” molecule.

Further, purified “2+1 IgG Crossfab” (SEQ ID NOs 3, 5, 29, 33) and “2+1 IgG scFab” constructs differing in their Fc domain, as well as the “(scFv)2” molecule, were compared. The different mutations in the Fc domain (L234A+L235A (LALA), P329G and/or N297D, as indicated) reduce or abolish the (NK) effector cell function d by constructs containing a ype (wt) Fc domain. Experimental ures were as described above.

Figure 28 shows that all constructs induce apoptosis in target cells able to the “(scFv)2” molecule.

Figure 29 shows the result of a comparison of the purified “2+1 IgG scFab” (SEQ ID NOs 5, 17, 19) and the “(scFv)2” molecule for their potential to induce T cell-mediated apoptosis in tumor target cells. Experimental procedures were as decribed above, using huMCSP-expressing Colo- 38 human melanoma target cells at an E:T ratio of 5:1, and an overnight incubation of 18.5 h. As depicted in the figure, the “2+1 IgG scFab” construct shows comparable cytotoxic activity to the “(scFv)2” molecule.

Similarly, Figure 30 shows the result of a comparison of the purified “2+1 IgG scFab” construct (SEQ ID NOs 5, 17, 19)and the “(scFv)2” molecule, using huMCSP-expressing Colo-38 human melanoma target cells at an E:T ratio of 5:1 and an incubation time of 18 h. As depicted in the figure, the “2+1 IgG scFab” construct shows comparable cytotoxic activity to the (scFv)2 molecule.

Figure 31 shows the result of a comparison of the purified “2+1 IgG scFab” uct (SEQ ID NOs 5, 17, 19) and the “(scFv)2” molecule, using huMCSP-expressing MDA-MB-435 human melanoma target cells at an E:T ratio of 5:1 and an overnight incubation of 23.5 h. As depicted in the , the construct induces apoptosis in target cells comparably to the “(scFv)2” molecule.

The “2+1 IgG scFab” construct shows d efficacy at the highest concentrations.

Furthermore, different bispecific constructs that are monovalent for both targets, human CD3 and human MCSP, as well as the corresponding “(scFv)2” molecule were analyzed for their potential to induce T cell-mediated apoptosis. Figure 32 shows the results for the “1+1 IgG scFab, one-armed” (SEQ ID NOs 1, 3, 5) and “1+1 IgG scFab, one-armed inverted” (SEQ ID NOs 7, 9, 11) constructs, using huMCSP-expressing Colo-38 human melanoma target cells at an E:T ratio of 5:1, and an incubation time of 19 h. As depicted in the figure, both “1+1” ucts are less active than the “(scFv)2” molecule, with the “1+1 IgG scFab, one-armed” molecule being superior to the “1+1 IgG scFab, one-armed inverted” molecule in this assay.

Figure 33 shows the results for the “1+1 IgG scFab” construct (SEQ ID NOs 5, 21, 213), using huMCSP-expressing Colo-38 human ma target cells at an E:T ratio of 5:1, and an incubation time of 20 h. As ed in the figure, the “1+1 IgG scFab” construct is less cytotoxic than the “(scFv)2” le.

In a further experiment the ed “2+1 IgG Crossfab” (SEQ ID NOs 3, 5, 29, 33), the “1+1 IgG Crossfab” (SEQ ID NOs 5, 29, 31, 33) and the “(scFv)2” molecule were analyzed for their potential to induce T cell-mediated apoptosis in tumor target cells upon crosslinkage of the construct via binding of both target antigens, CD3 and MCSP, on cells. huMCSP-expressing MDA-MB-435 human melanoma cells were used as target cells, the E:T ratio was 5:1, and the incubation time 20 h. The results are shown in Figure 34. The “2+1 IgG Crossfab” construct induces apoptosis in target cells ably to the “(scFv)2” molecule. The comparison of the mono- and bivalent “IgG Crossfab” formats clearly shows that the bivalent one is much more potent.

In yet another experiment, the purified “2+1 IgG Crossfab” (SEQ ID NOs 3, 5, 29, 33) uct was analyzed for its potential to induce T cell-mediated apoptosis in different (tumor) target cells. Briefly, MCSP-positive Colo-38 tumor target cells, mesenchymal stem cells (derived from bone marrow, Lonza #PT-2501 or adipose tissue, Invitrogen #R7788-115) or pericytes (from placenta; PromoCell #C-12980), as indicated, were harvested with Cell Dissociation Buffer, washed and resuspendend in AIM-V medium rogen #12055-091). 30 000 cells per well were plated in a round-bottom l plate and the respective antibody dilution was added at the ted concentrations. Human PBMC effector cells isolated from fresh blood of a healthy donor were added to obtain a final E:T ratio of 25:1. After an incubation of 4 h at 37°C, 5% CO2, LDH release of apoptotic/necrotic target cells into the supernatant was measured with the LDH detection kit (Roche d Science, #11 644 793 001), according to the manufacturer’s instructions.

As depicted in Figure 35, significant T-cell mediated cytotoxicity could be observed only with Colo-38 cells. This result is in line with 8 cells expressing significant levels of MCSP, whereas mesenchymal stem cells and tes s MCSP only very weakly.

The purified “2+1 IgG scFab” (SEQ ID NOs 5, 17, 19) construct and the “(scFv)2” molecule were also compared to a glycoengineered uman MCSP IgG antibody, having a reduced proportion of fucosylated N-glycans in its Fc domain (MCSP GlycoMab). For this experiment huMCSP-expressing Colo-38 human melanoma target cells and human PBMC effector cells were used, either at a fixed E:T ratio of 25:1 (Figure 36A), or at different E:T ratios from 20:1 to 1:10 e 36B). The different molecules were used at the concentrations ted in Figure 36A, or at a fixed concentration of 1667 pM (Figure 36B). Read-out was done after 21 h incubation. As depicted in Figure 36 A and B, both bispecific constructs show a higher potency than the MSCP GlycoMab.

In r experiment, purified “2+1 IgG Crossfab” targeting cynomolgus CD3 and human MCSP (SEQ ID NOs 3, 5, 35, 37) was analyzed. Briefly, human MCSP-expressing MV-3 tumor target cells were harvested with Cell iation Buffer, washed and resuspendend in DMEM containing 2% FCS and 1% GlutaMax. 30 000 cells per well were plated in a round-bottom 96- well plate and the respective dilution of construct or reference IgG was added at the concentrations indicated. The bispecific construct and the different IgG ls were adjusted to the same molarity. Cynomolgus PBMC effector cells, isolated from blood of healthy cynomolgus, were added to obtain a final E:T ratio of 3:1. After incubation for 24 h or 43 h at 37°C, 5% CO2, LDH release of apoptotic/necrotic target cells into the supernatant was measured with the LDH detection kit (Roche Applied Science, #11 644 793 001), according to the manufacturer’s instructions.

As depicted in Figure 37, the bispecific construct induces concentration-dependent LDH release from target cells. The effect is er after 43 h than after 24 h. The anti-cynoCD3 IgG (clone FN-18) is also able to induce LDH e of target cells without being inked.

Figure 38 shows the result of a comparison of the purified “2+1 IgG Crossfab” (SEQ ID NOs 3, , 29, 33) and the “(scFv)2“ construct, using MCSP-expressing human melanoma cell line (MV- 3) as target cells and human PBMCs as effector cells with an E:T ratio of 10:1 and an incubation time of 26 h. As depicted in the figure, the “2+1 IgG Crossfab” construct is more potent in terms of EC50 than the “(scFv)2“ molecule.

In a second series of experiments, bispecific constructs ing CD3 and EGFR were analyzed for their potential to induce T cell-mediated apoptosis in tumor target cells upon crosslinkage of the construct via binding of the antigen g moieties to their tive target antigens on cells (Figures 39-41).

In one experiment purified “2+1 IgG scFab” (SEQ ID NOs 45, 47, 53) and “1+1 IgG scFab” (SEQ ID NOs 47, 53, 213) constructs targeting CD3 and EGFR, and the corresponding )2” le, were compared. Briefly, human EGFR-expressing LS-174T tumor target cells were harvested with trypsin, washed and resuspendend in AIM-V medium (Invitrogen # 12055-091). 000 cells per well were plated in a round-bottom 96-well-plate and the tive antibody dilution was added at the indicated concentrations. All constructs and controls were adjusted to the same molarity. Human pan T effector cells were added to obtain a final E:T ratio of 5:1. As a positive control for the activation of human pan T cells, 1 µg/ml PHA-M (Sigma #L8902) was used. For ization, maximal lysis of the target cells (= 100%) was determined by incubation of the target cells with a final concentration of 1% Triton X-100. Minimal lysis (= 0%) refers to target cells ubated with effector cells, but without any construct or antibody. After an overnight incubation of 18 h at 37°C, 5% CO2, LDH release of apoptotic/necrotic target cells into the supernatant was measured with the LDH detection kit (Roche Applied Science, #11 644 793 001), according to the manufacturer’s instructions.

As depicted in Figure 39, the “2+1 IgG scFab” construct shows comparable cytotoxic activity to the “(scFv)2” le, whereas the “1+1 IgG scFab” construct is less active.

In r experiment the purified “1+1 IgG scFab, one-armed” (SEQ ID NOs 43, 45, 47), “1+1 IgG scFab, one-armed inverted” (SEQ ID NOs 11, 49, 51), “1+1 IgG scFab” (SEQ ID NOs 47, 53, 213), and the “(scFv)2” molecule were compared. Experimental conditions were as described above, except for the incubation time which was 21 h.

As depicted in Figure 40, the “1+1 IgG scFab” construct shows a slightly lower cytotoxic activity than the “(scFv)2” molecule in this assay. Both “1+1 IgG scFab, one-armed (inverted)” constructs are clearly less active than the “(scFv)2” molecule.

In yet a r experiment the purified “1+1 IgG scFab, one-armed” (SEQ ID NO 43, 45, 47) and “1+1 IgG scFab, one-armed inverted” (SEQ ID NOs 11, 49, 51) constructs and the “(scFv)2” molecule were ed. The incubation time in this experiment was 16 h, and the result is depicted in Figure 41. ted with human pan T cells, both “1+1 IgG scFab, one-armed (inverted)” ucts are less active than the “(scFv)2” molecule, but show concentrationdependent release of LDH from target cells (Figure 41A). Upon co-cultivation of the LS-174T tumor cells with naive T cells isolated from PBMCs, the constructs had only a basal activity – the most active among them being the “(scFv)2” molecule (Figure 41B).

In a r experiment, purified “1+1 IgG scFab, one-armed inverted” (SEQ ID NOs 11, 51, 55), “1+1 IgG scFab” (57, 61, 213), and “2+1 IgG scFab” (57, 59, 61) targeting CD3 and Fibroblast Activation Protein (FAP), and the corresponding “(scFv)2” molecule were analyzed for their potential to induce T cell-mediated apoptosis in human FAP-expressing fibroblasts GM05389 cells upon inkage of the construct via binding of both targeting moieties to their respective target antigens on the cells. Briefly, human GM05389 target cells were harvested with trypsin on the day before, washed and resuspendend in AIM-V medium (Invitrogen #12055-091). 000 cells per well were plated in a round-bottom 96-well plate and incubated overnight at 37°C, 5% CO2 to allow the cells to recover and adhere. The next day, the cells were centrifuged, the supernatant was ded and fresh medium, as well as the respective dilution of the constructs or reference IgGs was added at the indicated concentrations. All constructs and controls were adjusted to the same molarity. Human pan T effector cells were added to obtain a final E:T ratio of 5:1. As a positive control for the activation of human pan T cells, 5 µg/ml PHA-M (Sigma #L8902) was used. For normalization, maximal lysis of the target cells (= 100%) was determined by incubation of the target cells with a final concentration of 1% Triton X-100.

Minimal lysis (= 0%) refers to target cells co-incubated with effector cells, but without any construct or dy. After an additional overnight tion of 18 h at 37°C, 5% CO2, LDH release of tic/necrotic target cells into the atant was measured with the LDH detection kit (Roche Applied Science, #11 644 793 001), according to the cturer’s instructions.

As depicted in Figure 42, the “2+1 IgG scFab” construct shows comparable cytotoxic ty to the “(scFv)2” molecule in terms of EC50 values. The “1+1 IgG scFab, one-armed inverted” construct is less active than the other ucts tested in this assay.

In another set of experiments, the CD3/MCSP “2+1 IgG Crossfab, linked light chain” (see SEQ ID NOs 3, 5, 29, 179) was compared to the CD3/MCSP “2+1 IgG Crossfab” (see SEQ ID NOs 3, , 29, 33). Briefly, target cells (human Colo-38, human MV-3 or WM266-4 melanoma cells) were ted with Cell Dissociation Buffer on the day of the assay (or with trypsin one day before the assay was started), washed and resuspended in the riate cell culture medium (RPMI1640, including 2% FCS and 1% Glutamax). 20 000 - 30 000 cells per well were plated in a flat-bottom 96-well plate and the respective antibody on was added as indicated (triplicates). PBMCs as effector cells were added to obtain a final or-to-target cell (E:T) ratio of 10:1. All constructs and ls were adjusted to the same molarity, incubation time was 22 h. Detection of LDH release and normalization was done as described above.

Figure 49 to 52 show the result of four assays performed with MV-3 melanoma cells (Figure 49), Colo-38 cells (Figure 50 and 51) or WM266-4 cells (Figure 52). As shown in Figure 49, the construct with the linked light chain was less potent compared to the one without the linked light chain in the assay with MV-3 cells as target cells. As shown in Figure 50 and 51, the construct with the linked light chain was more potent compared to the one without the linked light chain in the assays with high MCSP sing Colo-38 cells as target cells. Finally, as shown in Figure 52, there was no significant difference between the two constructs when high MCSP-expressing WM266-4 cells were used as target cells.

In another experiment, two CEA-targeting “2+1 IgG Crossfab, inverted” constructs were compared, wherein in the Crossfab fragment either the V regions (VL/VH, see SEQ ID NOs 33, 63, 65, 67) or the C regions (CL/CH1, see SEQ ID NOs 65, 67, 183, 197) were exchanged. The assay was performed as described above, using human PBMCs as effector cells and human CEA-expressing target cells. Target cells (MKN-45 or LS-174T tumor cells) were harvested with trypsin-EDTA (LuBiosciences #25300-096), washed and resuspendend in RPMI1640 (Invitrogen #42404042), including 1% Glutamax (LuBiosciences #35050087) and 2% FCS. 30 000 cells per well were plated in a round-bottom l plate and the ific constructs were added at the indicated concentrations. All constructs and controls were adjusted to the same molarity. Human PBMC effector cells were added to obtain a final E:T ratio of 10:1, incubation time was 28 h. EC50 values were calculated using the GraphPad Prism 5 software.

As shown in Figure 61, the construct with the CL/CH1 exchange shows slightly better activity on both target cell lines than the uct with the VL/VH exchange. Calculated EC50 values were 115 and 243 pM on MKN-45 cells, and 673 and 955 pM on LS-174T cells, for the CL/CH1-exchange construct and the VL/VH-exchange construct, respectively.

Similarly, two MCSP-targeting “2+1 IgG Crossfab” constructs were compared, wherein in the Crossfab fragment either the V regions (VL/VH, see SEQ ID NOs 33, 189, 191, 193) or the C regions (CL/CH1, see SEQ ID NOs 183, 189, 193, 195) were exchanged. The assay was performed as described above, using human PBMCs as effector cells and human MCSP- expressing target cells. Target cells (WM266-4) were harvested with Cell Dissociation Buffer (LuBiosciences #13151014), washed and resuspendend in RPMI1640 (Invitrogen #42404042), including 1% Glutamax (LuBiosciences #35050087) and 2% FCS. 30 000 cells per well were plated in a round-bottom l plate and the constructs were added at the ted concentrations. All constructs and ls were adjusted to the same molarity. Human PBMC effector cells were added to obtain a final E:T ratio of 10:1, tion time was 26 h. EC50 values were calculated using the ad Prism 5 software.

As depicted in Figure 62, the two constructs show comparable activity, the construct with the CL/CH1 exchange having a slightly lower EC50 value (12.9 pM for the CL/CH1-exchange construct, compared to 16.8 pM for the VL/VH-exchange construct).

Figure 63 shows the result of a similar assay, performed with human MCSP-expressing MV-3 target cells. Again, both constructs show comparable activity, the construct with the CL/CH1 exchange having a slightly lower EC50 value (approximately 11.7 pM for the CL/CH1-exchange construct, ed to approximately 82.2 pM for the VL/VH-exchange construct). Exact EC50 values could not be calculated, since the g curves did not reach a plateau at high concentrations of the compounds.

In a further experiment, the CD3/MCSP “2+1 IgG ab” (see SEQ ID NOs 3, 5, 29, 33) and “1+1 IgG Crossfab” (see SEQ ID NOs 5, 29, 33, 181) constructs were compared to the SP “1+1 CrossMab” (see SEQ ID NOs 5, 23, 183, 185). The assay was performed as described above, using human PBMCs as or cells and WM266-4 or MV-3 target cells (E:T ratio = 10:1) and an incubation time of 21 h.

As shown in Figure 64, the “2+1 IgG Crossfab” construct is the most potent molecule in this assay, followed by the “1+1 IgG Crossfab” and the “1+1 CrossMab”. This ranking is even more pronounced with MV-3 cells, expressing medium levels of MCSP, compared to high MCSP sing WM266-4 cells. The calculated EC50 values on MV-3 cells were 9.2, 40.9 and 88.4 pM, on WM266-4 cells 33.1, 28.4 and 53.9 pM, for the “2+1 IgG Crossfab”, the “1+1 IgG Crossfab” and the “1+1 CrossMab”, respectively.

In a further experiment, ent concentrations of the “1+1 IgG Crossfab LC fusion” construct (SEQ ID NOs 183, 209, 211, 213) were tested, using MKN-45 or LS-174T tumor target cells and human PBMC effector cells at an E:T ratio of 10:1 and an incubation time of 28 hours. As shown in Figure 65, the “1+1 IgG Crossfab LC fusion“ construct induced apoptosis in MKN-45 target cells with a calculated EC50 of 213 pM, whereas the calculated EC50 is 1.56 nM with LS-174T cells, showing the influence of the ent tumor antigen expression levels on the potency of the bispecific constructs within a certain period of time.

In yet another experiment, the “1+1 IgG Crossfab LC fusion” construct (SEQ ID NOs 183, 209, 211, 213) was compared to a untargeted “2+1 IgG Crossfab” molecule. MC38-huCEA tumor cells and human PBMCs (E:T ratio = 10:1) and an incubation time of 24 hours were used. As shown in Figure 66, the “1+1 IgG Crossfab LC fusion” construct d apoptosis of target cells in a concentration-dependent manner, with a calculated EC50 value of approximately 3.2 nM. In st, the eted “2+1 IgG Crossfab” showed antigen-independent T cell-mediated g of target cells only at the highest concentration.

In a final experiment, the “2+1 IgG Crossfab (V9)” (SEQ ID NOs 3, 5, 29, 33), the “2+1 IgG Crossfab, inverted (V9)” (SEQ ID NOs 5, 23, 183, 187), the “2+1 IgG Crossfab (anti-CD3)” (SEQ ID NOs 5, 23, 215, 217), the “2+1 IgG Crossfab, inverted (anti-CD3)” (SEQ ID NOs 5, 23, 215, 219) were compared, using human MCSP-positive MV-3 or WM266-4 tumor cells and human PBMCs (E:T ratio = 10:1), and an incubation time of about 24 hours. As depicted in Figure 67, the T cell-mediated killing of the “2+1 IgG Crossfab, inverted” ucts seems to be slightly er or at least equal to the one induced by the “2+1 IgG Crossfabt” constructs for both CD3 binders. The calculated EC50 values were as follows: EC50 [pM] 2+1 IgG ab 2+1 IgG Crossfab 2+1 IgG Crossfab 2+1 IgG Crossfab, (V9) inverted (V9) (anti-CD3) inverted (anti-CD3) MV-3 10.0 4.1 11.0 3.0 WM266-4 12.4 3.7 11.3 7.1 Example 7 CD107a/b assay Purified “2+1 IgG scFab” uct (SEQ ID NOs 5, 17, 19) and the “(scFv)2” molecule, both targeting human MCSP and human CD3, were tested by flow cytometry for their potential to upregulate CD107a and intracellular perforin levels in the presence or absence of human MCSP- expressing tumor cells.

Briefly, on day one, 30 000 8 tumor target cells per well were plated in a round-bottom 96-well plate and incubated overnight at 37°C, 5% CO2 to let them adhere. Primary human pan T cells were isolated on day 1 or day 2 from Buffy Coat, as described.

On day two, 0.15 million effector cells per well were added to obtain a final E:T ratio of 5:1.

FITC-conjugated CD107a/b antibodies, as well as the different ific constructs and controls are added. The different bispecific molecules and dies were adjusted to same molarities to obtain a final concentration of 9.43 nM. Following a 1 h incubation step at 37°C, 5% CO2, monensin was added to inhibit secretion, but also to neutralize the pH within endosomes and lysosomes. After an additional incubation time of 5 h, cells were stained at 4°C for 30 min for surface CD8 expression. Cells were washed with staining buffer (PBS / 0.1% BSA), fixed and permeabilized for 20 min using the BD Cytofix/Cytoperm Plus Kit with BD Golgi Stop (BD Biosciences #554715). Cells were washed twice using 1 x BD Perm/Wash buffer, and intracellular staining for perforin was performed at 4°C for 30 min. After a final g step with 1 x BD Perm/Wash , cells were resuspended in PBS / 0.1% BSA and analyzed on FACS CantoII (all antibodies were purchased from BD Biosciences or BioLegend).

Gates were set either on all CD107a/b positive, in-positive or double-positive cells, as indicated (Figure 43). The “2+1 IgG scFab” uct was able to activate T cells and upregulate CD107a/b and intracellular in levels only in the presence of target cells (Figure 43A), whereas the “(scFv)2” molecule shows (weak) induction of tion of T cells also in the e of target cells (Figure 43B). The bivalent reference anti-CD3 IgG results in a lower level of activation compared to the “(scFv)2” molecule or the other bispecific construct.

Example 8 Proliferation assay The purified “2+1 IgG scFab” (SEQ ID NOs 5, 17, 19) and “(scFv)2” molecules, both targeting human CD3 and human MCSP, were tested by flow cytometry for their potential to induce proliferation of CD8+ or CD4+ T cells in the presence and absence of human MCSP-expressing tumor cells.

Briefly, freshly isolated human pan T cells were adjusted to 1 million cells per ml in warm PBS and stained with 1 µM CFSE at room temperature for 10 minutes. The staining volume was doubled by addition of RPMI1640 , containing 10% FCS and 1% GlutaMax. After incubation at room temperature for further 20 min, the cells were washed three times with prewarmed medium to remove ing CFSE. ositive Colo-38 cells were harvested with Cell Dissociation buffer, counted and checked for viability. Cells were adjusted to 0.2 x 106 (viable) cells per ml in AIM-V medium, 100 µl of this cell suspension were pipetted per well into a round-bottom l plate (as indicated). 50 µl of the (diluted) bispecific constructs were added to the cell-containing wells to obtain a final concentration of 1 nM. CFSE-stained human pan T effector cells were adjusted to 2 x 106 e) cells per ml in AIM-V medium. 50 µl of this cell suspension was added per well of the assay plate (see above) to obtain a final E:T ratio of 5:1. To analyze whether the ific constructs are able to activate T cells only in the presence of target cells, expressing the tumor antigen huMCSP, wells were included that contained 1 nM of the respective bispecific molecules as well as PBMCs, but no target cells.

After incubation for five days at 37°C, 5% CO2, cells were centrifuged (5 min, 350 x g) and washed twice with 150 µl/well PBS, including 0.1% BSA. Surface staining for CD8 (mouse IgG1,κ; clone HIT8a; BD #555635), CD4 (mouse IgG1,κ; clone RPA-T4 ; BD #560649), or CD25 (mouse IgG1,ĸ; clone M-A251; BD #555434) was performed at 4°C for 30 min, ing to the supplier’s suggestions. Cells were washed twice with 150 µl/well PBS containing 0.1% BSA, resuspended in 200 µl/well PBS with 0.1% BSA, and analyzed using a FACS CantoII machine (Software FACS Diva). The relative proliferation level was determined by setting a gate around the non-proliferating cells and using the cell number of this gate relative to the overall measured cell number as the reference.

Figure 44 shows that all constructs induce proliferation of CD8+ T cells (A) or CD4+ T cells (B) only in the ce of target cells, comparably to the “(scFv)2” molecule. In l, activated CD8+ T cells proliferate more than activated CD4+ T cells in this assay.

Example 9 Cytokine release assay The purified “2+1 IgG scFab” construct (SEQ ID NOs 5, 17, 19) and the “(scFv)2”molecule, both targeting human MCSP and human CD3, were analyzed for their ability to induce T cell- mediated de novo secretion of cytokines in the presence or absence of tumor target cells.

Briefly, human PBMCs were isolated from Buffy Coats and 0.3 million cells were plated per well into a bottom 96-well plate. Colo-38 tumor target cells, expressing human MCSP, were added to obtain a final E:T-ratio of 10:1. Bispecific ucts and IgG controls were added at 1 nM final concentration and the cells were incubated for 24 h at 37°C, 5% CO2. The next day, the cells were centrifuged for 5 min at 350 x g and the supernatant was transferred into a new deep-well 96-well-plate for the subsequent is. The CBA analysis was performed according to manufacturer’s instructions for FACS CantoII, using the Human Th1/Th2 Cytokine Kit II (BD #551809).

Figure 45 shows levels of the different ne measured in the supernatant. In the presence of target cells the main cytokine secreted upon T cell activation is IFN-γ. The “(scFv)2” molecule induces a slightly higher level of IFN-γ than the “2+1 IgG scFab” construct. The same tendency might be found for human TNF, but the overall levels of this cytokine were much lower compared to IFN-γ. There was no significant secretion of Th2 cytokines (IL-10 and IL-4) upon tion of T cells in the presence (or absence) of target cells. In the absence of Colo-38 target cells, only very weak induction of TNF secretion was observed, which was highest in samples d with the “(scFv)2” molecule.

In a second experiment, the following purified bispecific constructs targeting human MCSP and human CD3 were analyzed: the “2+1 IgG Crossfab” construct (SEQ ID NOs 3, 5, 29, 33), the “(scFv)2” molecule, as well as different “2+1 IgG scFab” les comprising either a wild- type or a mutated (LALA, P329G and/or N297D, as indicated) Fc domain. Briefly, 280 µl whole blood from a healthy donor were plated per well of a deep-well 96-well plate. 30 000 Colo-38 tumor target cells, expressing human MCSP, as well as the different bispecific constructs and IgG controls were added at 1 nM final concentration. The cells were incubated for 24 h at 37°C, % CO2 and then centrifuged for 5 min at 350 x g. The supernatant was transferred into a new ell 96-well-plate for the subsequent analysis. The CBA analysis was performed according to manufacturer’s instructions for FACS CantoII, using the combination of the following CBA Flex Sets: human granzyme B (BD #560304), human IFN-γ Flex Set (BD #558269), human TNF Flex Set (BD #558273), human IL-10 Flex Set (BD #558274), human IL-6 Flex Set (BD #558276), human IL-4 Flex Set (BD #558272), human IL-2 Flex Set (BD #558270).

Figure 46 shows the levels of the ent ne measured in the supernatant. The main ne secreted in the presence of Colo-38 tumor cells was IL-6, ed by IFN-γ. In addition, also the levels of granzyme B strongly increased upon activation of T cells in the presence of target cells. In general, the “(scFv)2” molecule d higher levels of ne secretion in the presence of target cells (Figure 46, A and B). There was no significant secretion of Th2 nes (IL-10 and IL-4) upon activation of T cells in the ce (or absence) of target cells.

In this assay, there was a weak secretion of IFN-γ, induced by different “2+1 IgG scFab” constructs, even in the absence of target cells (Figure 46, C and D). Under these conditions, no significant differences could be observed between “2+1 IgG scFab” constructs with a wild-type or a mutated Fc domain.

* * * Although the foregoing invention has been described in some detail by way of illustration and example for purposes of y of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are sly incorporated in their entirety by reference.

Claims (33)

Claims

1. A T cell activating bispecific antigen binding molecule comprising a first and a second antigen g moiety, one of which is a Fab molecule capable of specific g to CD3 and the other 5 one of which is a Fab molecule capable of specific binding to a target cell antigen, and an Fc domain composed of a first and a second subunit capable of stable association; wherein the first antigen binding moiety is a crossover Fab le wherein either the le or the constant regions of the Fab light chain and the Fab heavy chain are exchanged; 10 wherein (i) the second antigen binding moiety is fused at the inus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety and the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain, or (ii) the first antigen binding moiety is fused at the C- terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen 15 binding moiety and the second n binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or the second subunit of the Fc domain; and wherein the T cell activating bispecific antigen g molecule comprises not more than one antigen binding moiety capable of ic binding to CD3.

2. The T cell activating bispecific antigen binding molecule of claim 1, wherein the Fab light 20 chain of the first n binding moiety and the Fab light chain of the second antigen binding moiety are fused to each other, optionally via a peptide linker.

3. The T cell activating bispecific antigen binding molecule of claim 1 or 2, comprising a third antigen binding moiety which is a Fab molecule capable of specific binding to a target cell antigen. 25

4. The T cell activating bispecific antigen binding molecule of claim 3, wherein the third antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain.

5. The T cell activating bispecific antigen binding molecule of claim 3 or 4, wherein the second and the third antigen g moiety are each fused at the C-terminus of the Fab heavy chain to 30 the inus of one of the subunits of the Fc domain, and the first n binding moiety is fused at the inus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding moiety.

6. The T cell activating bispecific antigen binding molecule of claim 3 or 4, wherein the first and the third antigen binding moiety are each fused at the C-terminus of the Fab heavy chain to the 5 N-terminus of one of the subunits of the Fc domain, and the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety.

7. The T cell activating bispecific antigen binding molecule of claim 5, wherein the second and the third n binding moiety and the Fc domain are part of an immunoglobulin molecule, 10 particularly an IgG class immunoglobulin.

8. The T cell activating ific antigen binding molecule of any one of the preceding claims, wherein the Fc domain is an IgG, specifically an IgG1 or IgG4, Fc domain.

9. The T cell activating bispecific antigen binding molecule of any one of the ing claims, wherein the Fc domain is a human Fc domain. 15

10. The T cell activating bispecific antigen binding molecule of any one of the ing claims, wherein the Fc domain ses a modification promoting the association of the first and the second subunit of the Fc domain, wherein (a) in the CH3 domain of the first subunit of the Fc domain an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance 20 within the CH3 domain of the first subunit which is positionable in a cavity within the CH3 domain of the second t, and in the CH3 domain of the second subunit of the Fc domain an amino acid e is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first t is positionable; or 25 (b) at the interface of the two Fc domain subunits one or more amino acid residues is/are replaced by charged amino acid residues so that homodimer ion becomes electrostatically unfavorable but heterodimerization electrostatically favorable.

11. The T cell activating bispecific antigen binding molecule of any one of the preceding claims, wherein the Fc domain comprises one or more amino acid substitution that reduces binding to an Fc receptor and/or effector function, wherein said one or more amino acid substitution is at a position ed from the group of E233, L234, L235, N297, P331 and P329 (EU numbering).

12. The T cell activating bispecific antigen g molecule of claim 11, wherein said one or more amino acid substitution is at one or more position selected from the group of L234, L235, 5 and P329 (EU ing).

13. The T cell ting bispecific antigen binding molecule of any one of the preceding claims, wherein each subunit of the Fc domain comprises three amino acid substitutions that reduce binding to an activating Fc receptor and/or effector function wherein said amino acid substitutions are L234A, L235A and P329G (EU ing). 10

14. The T cell activating bispecific antigen binding molecule of any one of claims 11 to 13, wherein the Fc receptor is an Fcγ receptor.

15. The T cell activating bispecific antigen binding molecule of any one of claims 11 to 13, wherein the or function is antibody-dependent cell-mediated cytotoxicity (ADCC).

16. The T cell activating ific antigen binding molecule of any one of the preceding claims, 15 wherein the target cell antigen is selected from the group consisting of: Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP), Epidermal Growth Factor Receptor (EGFR), CD19, CD20, CD33, Carcinoembryonic n (CEA) and Fibroblast Activation n (FAP).

17. An isolated polynucleotide encoding the T cell activating bispecific antigen binding molecule of any one of claims 1 to 16. 20

18. A vector, comprising the isolated polynucleotide of claim 17.

19. The vector of claim 18, wherein the vector is an expression vector.

20. A host cell comprising the isolated polynucleotide of claim 17 or the vector of claim 18 or claim 19, wherein the host cell is not a human cell within a human.

21. A method of producing the T cell activating ific n binding molecule of any one 25 of claims 1 to 16, comprising the steps of a) culturing the host cell of claim 20 under ions suitable for the expression of the T cell activating bispecific antigen binding molecule and b) recovering the T cell activating bispecific antigen binding molecule.

22. A pharmaceutical composition comprising the T cell activating bispecific n binding molecule of any one of claims 1 to 16 and a pharmaceutically able carrier.

23. Use of the T cell activating bispecific antigen g molecule of any one of claims 1 to 16 for the manufacture of a medicament for the treatment of a disease in an individual in need 5 thereof.

24. The use of claim 23, wherein said disease is cancer.

25. An in vitro method for ng lysis of a target cell, comprising contacting a target cell with the T cell activating bispecific antigen binding molecule of any one of claims 1-16 in the presence of a T cell. 10

26. A T cell activating bispecific antigen binding le as claimed in any one of claims 1 to 16, substantially as herein described with reference to any example thereof.

27. An isolated polynucleotide as claimed in claim 17, substantially as herein described with reference to any example thereof.

28. A vector as claimed in claim 18 or claim 19, substantially as herein bed with reference 15 to any example thereof.

29. A host cell as claimed in claim 20, substantially as herein described with reference to any example thereof.

30. A method as claimed in claim 21, substantially as herein bed with reference to any example thereof. 20

31. A pharmaceutical composition as claimed in claim 22, ntially as herein described with reference to any example thereof.

32. Use as claimed in claim 23 or claim 24, substantially as herein described with reference to any example thereof.

33. An in vitro method as d in claim 25, substantially as herein described with reference to 25 any example thereof.