NZ721138B2

NZ721138B2 - Bispecific t cell activating antigen binding molecules

Info

Publication number: NZ721138B2
Application number: NZ721138A
Authority: NZ
Inventors: Oliver Ast; Peter Bruenker; Tanja Fauti; Anne Freimosergrundschober; Christiane Jaeger; Christian Klein; Ekkehard Moessner; Pablo Umana; Grundschober Anne Freimoser
Original assignee: Roche Glycart Ag
Priority date: 2011-08-23
Filing date: 2012-08-21
Publication date: 2020-05-29

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. e 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

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

Background The ive destruction of an individual cell or a ic cell type is often desirable in a variety 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 system, however they cannot be activated by the effector mechanism mediated by the Fc domain of conventional therapeutic antibodies.

In this regard, ific antibodies designed to bind with one "arm" to a surface n on target cells, and with the second "arm" to an activating, invariant component of the T cell receptor (TCR) complex, have become of st 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 nt for normal MHC-restricted tion of CTLs. In this t it is crucial that CTLs are only activated when a target cell is presenting 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 investigated. 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 les wherein two scFv molecules are fused by a flexible linker. Further ific formats being evaluated for T cell engagement e ies (Holliger et al., Prot Eng 9, 299-305 (1996)) and derivatives thereof, such as tandem diabodies (Kipriyanov et al., J Mol Biol 293, 41-66 (1999)). A more recent pment are the so-called DART (dual affinity retargeting) molecules, which are based on the diabody format but feature a C-terminal disulfide bridge for additional ization (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 trials, ent a larger sized format (reviewed in Seimetz et al., Cancer Treat Rev 36, 458-467 (2010)).

The variety of formats that are being developed shows the great potential attributed to T cell re- direction and activation in immunotherapy. The task of generating bispecific antibodies suitable therefor is, however, by no means trivial, but involves a number of nges that have to be met related to efficacy, toxicity, applicability and produceability of the antibodies.

Small constructs such as, for example, BiTE molecules – while being able to efficiently ink or and target cells – have a very short serum half life requiring them to be stered to patients by continuous infusion. IgG-like formats on the other hand – while having the great benefit of a long half life – suffer from ty associated with the native effector functions inherent to IgG molecules. Their immunogenic potential constitutes another unfavorable feature of IgG-like bispecific antibodies, especially non-human formats, for successful therapeutic development. y, 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 ses the yield of the correctly assembled construct and results in a number of non-functional side products from which the desired bispecific antibody may be difficult to separate.

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

In this specification where reference has been made to patent specifications, other external documents, 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 external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, 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 practice the invention as defined in the claims of this ation.

Summary of the Invention In a first , the invention provides 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 CD3 and the other one of which is a Fab molecule capable of ic binding to a target cell n, 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 molecule 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 g moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second t of the Fc domain, or (ii) the first n binding moiety is fused at the C- us 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 antigen binding moiety capable of ic binding to CD3.

In a second aspect, the invention provides an isolated polynucleotide ng 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 isolated polynucleotide of the second aspect or the vector of the third , n 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 binding In a sixth aspect, the invention provides a pharmaceutical composition comprising the T cell activating bispecific antigen g molecule of the first aspect and a pharmaceutically able carrier.

In a seventh aspect, the ion 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 dual 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 tion Broadly 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 le capable of specific binding to an activating T cell antigen 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 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 constant 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 g to an ting T cell antigen is present in the T cell activating bispecific n binding molecule (i.e. the T cell ting bispecific antigen binding molecule provides monovalent binding to the activating T cell antigen). In particular embodiments, the first antigen binding moiety is a crossover Fab molecule. In even more particular ments, the first antigen binding moiety is a crossover Fab molecule wherein the constant regions of the Fab light chain and the Fab heavy chain are ged.

In some embodiments, the first and the second antigen g moiety of the T cell activating bispecific antigen binding molecule are fused to each other, optionally via a peptide linker. In one such ment, the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the inus of the Fab heavy chain of the first antigen binding moiety. In another 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 g moiety. 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 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 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, 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 ment, the second antigen binding moiety of the T cell activating bispecific antigen binding molecule 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 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 t of the Fc domain.

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

In certain embodiments, the T cell activating bispecific antigen g molecule comprises a third antigen binding moiety which is a Fab molecule capable of specific binding to a target cell antigen. In one such 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 n binding moiety of the T cell activating antigen g le are each fused at the C-terminus of the Fab heavy chain to the N- us 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 . In another particular embodiment, the first and the third antigen binding moiety of the T cell activating n 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 C-terminus 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 ment the second and the third antigen g moiety and the Fc domain are part of an immunoglobulin molecule. In a particular embodiment the globulin 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 ular embodiment, the Fc domain is an IgG Fc domain. In a specific embodiment, the Fc domain is an IgG1 Fc domain. In another 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 numbering). In particular embodiments the Fc domain is a human Fc .

In particular embodiments the Fc domain comprises a modification 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 protuberance within the CH3 domain of the first subunit is positionable.

In a particular embodiment the Fc domain ts 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 ses one or more amino acid substitution that reduces binding to an Fc receptor and/or effector function. In one ment, 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 ing). In particular 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 domain, particularly a human IgG1 Fc domain. In other embodiments, each subunit of the Fc domain comprises two amino acid tutions that reduce binding to an Fc receptor and/or effector function wherein 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 ment the Fc receptor is a human Fc or. In one embodiment, the Fc receptor is an activating 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 n 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 antigen is selected from the group consisting of: Melanomaassociated Chondroitin Sulfate Proteoglycan (MCSP), Epidermal Growth Factor Receptor (EGFR), Carcinoembryonic n (CEA), Fibroblast Activation Protein (FAP), CD19, CD20 and CD33.

Also described is an isolated polynucleotide encoding a T cell ting bispecific antigen g molecule described or a nt thereof. Also described are ptides encoded by the polynucleotidesdescribed. Further described is an expression vector comprising the isolated polynucleotide i described, and a host cell comprising the isolated polynucleotide or the expression vector described. In some embodiments the host cell is a eukaryotic cell, ularly 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 activating bispecific antigen binding molecule and b) recovering the T cell activating bispecific antigen g le. 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 g molecule and pharmaceutical composition described. Described 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 g molecule or a pharmaceutical composition described for use in the ent of a disease in an individual in need thereof. In a specific embodiment the disease 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 ent of a disease in an individual in need thereof; as well as a method of treating a disease in an dual, comprising administering to said individual a therapeutically effective amount of a composition comprising the T cell activating ific antigen binding molecule described 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, particularly a human.

Also described is a method for inducing lysis of a target cell, particularly a tumor cell, comprising ting a target cell with a T cell activating ific antigen binding molecule bed in the presence of a T cell, particularly a cytotoxic T cell.

Brief Description of the Drawings FIGURE 1. Exemplary configurations of the T cell activating bispecific n 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 , while the "1+1 IgG scFab, one armed ed" le 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 Crossfab" molecule. (F) Illustration of the "2+1 IgG ab" molecule. (G) Illustration of the "2+1 IgG Crossfab" molecule with alternative order of Crossfab and Fab components ("inverted"). (H) Illustration of the "1+1 IgG Crossfab light chain (LC) fusion" molecule. (I) ration 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 Crossfab, 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" (anti-MCSP/anti-huCD3) (see SEQ ID NOs 7, 9, 11), non reduced (C) and reduced (D).

FIGURE 3. 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 "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. ical 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" (anti-EGFR/anti-huCD3) (see SEQ ID NOs 11, 49, 51) (B).

FIGURE 6. (A, B) SDS PAGE (4-12% Bis/Tris, NuPage Invitrogen, Coomassie-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% Bis/Tris, NuPage ogen, 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" 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 d (lane 3).

FIGURE 8. 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 (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 d (A) and reduced (B). (C) ical 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, Coomassie-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" (anti-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, e) 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 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-huCD3).

FIGURE 13. (A, B) SDS PAGE (4-12% Bis/Tris, NuPage Invitrogen, Coomassie-stained) of "2+1 IgG Crossfab" (anti-MCSP/anti-cyCD3) (see SEQ ID NOs 3, 5, 35, 37), non d (A) and reduced (B). (C) Analytical size ion 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 ogen, 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 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, inverted" (anti-CEA/anti-huCD3).

FIGURE 15. (A) Thermal stability of "(scFv)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- MCSP/anti-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 interaction of various Fc-mutants with human Ia, and for (B) simultaneous binding of T cell bespecific ucts with tumor target and human CD3γ(G4S)5CD3ε–AcTev–Fc(knob)–Avi/Fc(hole).

FIGURE 17. Simultaneous 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 ific ucts 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 inverted" (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) ed by FACS. Mean fluorescence intensity compared to untreated cells and cells stained with the secondary antibody only is depicted.

FIGURE 20. Binding of the "2+1 IgG scFab, LALA" (see SEQ ID NOs 5, 17, 19) uct (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 anti-CD3 IgG (as indicated), untreated cells, and cells stained with the ary 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) ed by FACS. Mean scence intensity compared to cells treated with the reference anti-CD3 or anti-MCSP IgG (as indicated), untreated cells, and cells stained with the secondary antibody only is depicted.

FIGURE 22. Dose dependent g 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 ed 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 indicated (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 incubation, respectively.

FIGURE 24. Surface expression level of the late activation marker CD25 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 indicated (E:T ratio = 5:1). Depicted is the expression 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 tion marker CD25 on cynomolgus CD8+ T cells from two different animals (cyno Nestor, cyno Nobu) after 43 hours incubation with the indicated concentrations 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 ls, 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 presence of U87MG tumor cells (E:T ratio = 5:1). As controls, the corresponding anti-CD3 and anti-MCSP IgGs were administered.

FIGURE 27. 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 20 hours by different trations of the "2+1 IgG scFab" (see SEQ ID NOs 5, 21, 23), "2+1 IgG ab" (see SEQ ID NOs 3, 5, 29, 33) and "(scFv)2" bispecific molecules and ponding IgGs.

FIGURE 28. Killing (as measured by LDH release) of MDA-MB-435 tumor cells upon ure with human pan T cells (E:T ratio = 5:1), and activation for 20 hours by different concentrations of the ific constructs and corresponding IgGs. "2+1 IgG scFab" constructs 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), d with CD3-MCSP 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 tion for 23.5 hours by different concentrations 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 e) 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 ponding IgGs.

FIGURE 33. 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 "1+1 IgG scFab" CD3-MCSP bispecific construct (see SEQ ID NOs 5, 21, 213) or )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 tion for 21 hours by different concentrations of the bispecific constructs and corresponding IgGs. The CD3-MCSP ific "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 tissue, or pericytes from placenta; as indicated) 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 tion of 21h, upon co-culture with human PBMCs and different CD3- MCSP bispecific constructs ("2+1 IgG scFab, LALA" (see SEQ ID NOs 5, 17, 19) and "(scFv)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 ty. PHA-M served as a control for (unphysiologic) T cell activation.

FIGURE 38. Killing (as measured by LDH release) of huMCSP-positive MV-3 ma 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 ositive LS-174T tumor cells upon co-culture with human pan T cells (E:T ratio = 5:1), treated with different CD3-EGFR bispecific constructs ("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. 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), d with different 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), "1+1 IgG scFab" (see SEQ ID NOs 47, 53, 213) and "(scFv)2") or reference IgGs for 21 hours.

FIGURE 41. Killing (as measured by LDH release) of ositive 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 ed by LDH release) of sitive 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 perforin 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 presence (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 n levels by preventing protein transport. Gates were set either on all CD107a/b positive, in-positive or -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 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 or e 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 ent cytokines 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 different cytokines measured in the atant 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 e (C, D) of Colo-38 tumor cells for 24 hours. Among the bispecific ucts were different "2+1 IgG scFab" constructs having either a ype 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 analyses. 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 ent CD3-MCSP bispecific constructs for ~ 44 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 50. Killing (as measured by LDH release) of ositive Colo-38 tumor cells upon co-culture by human PBMCs (E:T ratio = 10:1), d 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 healthy volunteers.

FIGURE 51. 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 ent 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 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 healthy 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 incubation 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 ce or absence of human MCSP-expressing Colo-38 tumor target cells (E:T ratio = 10:1).

FIGURE 54. CE-SDS analyses. (A) Electropherogram shown as SDS-PAGE of 1+1 IgG Crossfab; VL/VH ge (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 exchange (LC007/V9) (see SEQ ID NOs 5, 23, 183, 185): a) d, b) non-reduced. (C) opherogram shown as SDS-PAGE of 2+1 IgG Crossfab, inverted; CL/CH1 exchange (LC007/V9) (see SEQ ID NOs 5, 23, 183, 187): a) d, 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 GE of 2+1 IgG ab; CL/CH1 exchange (M4-3 ML2/V9) (see SEQ ID NOs 183, 189, 193, 195): a) reduced, b) non-reduced. (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 exchange (M4-3 ML2/H2C) (see SEQ ID NOs 189, 193, 199, 201): a) reduced, b) nonreduced.

(H) Electropherogram shown as GE of 2+1 IgG Crossfab, inverted; CL/CH1 exchange (431/26/V9) (see SEQ ID NOs 183, 203, 205, 207): a) reduced, 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 ogen, Coomassie-stained) of "1+1 IgG Crossfab" (anti-CD33/anti-huCD3) (see SEQ ID NOs 33, 213, 221, 223), reduced (left) and non-reduced (right). (M) SDS PAGE (4- 12% is, NuPage Invitrogen, Coomassie-stained) of "2+1 IgG ab" (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 Invitrogen, 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, sed 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 nt, Fcγ Fragment-specific, Jackson Immuno Research Lab # 109098) were assessed as well.

FIGURE 56. g of bispecific 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, expressed 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 Crossfab" (see SEQ ID NOs 3, 5, 29, 33) constructs. The assay was performed in the presence or absence 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 presence or absence of huMCSP- ve 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, ed" (see SEQ ID NOs 5, 23, 215, 219) for ~41 hours.

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

FIGURE 62. Killing (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 ent concentrations of the "2+1 IgG Crossfab )" (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 tion for 21 hours by different 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 Crossfab" (see SEQ ID NOs 5, 29, 33, 181), as indicated.

FIGURE 65. Killing (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 ed by LDH release) of uCEA tumor cells upon coculture with human PBMCs (E:T ratio = 10:1) and activation for 24 hours by different concentrations of the "1+1 IgG Crossfab LC " (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 ab (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.

Detailed Description 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 broadest sense to a molecule that specifically binds an antigenic determinant. Examples of antigen binding les 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 ses 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 denotes the presence of a specified number of antigen g 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) n binding site specific for the antigen in the antigen 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 ction with the antigen. For example, the antigen g site of an antibody comprises amino acid residues from the complementarity determining regions . A native immunoglobulin molecule typically has two antigen binding sites, a Fab molecule typically has a single antigen 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 inant. In another embodiment an antigen binding moiety is able to activate ing through its target antigen, for example a T cell or x antigen. Antigen binding es include antibodies and fragments thereof as further defined herein. Particular antigen binding moieties e an antigen binding domain of an antibody, comprising an antibody heavy chain variable region and an antibody light chain variable region. In certain embodiments, the antigen binding moieties may comprise antibody constant regions as further defined 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 determinant" is synonymous with "antigen" and "epitope," and refers to a site (e.g. a contiguous stretch of amino acids or a conformational uration made up of different regions of non-contiguous amino acids) on a polypeptide macromolecule to which an antigen binding moiety binds, forming an antigen binding moiety-antigen complex.

Useful antigenic determinants can be found, for example, on the surfaces of tumor cells, on the es 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 ellular matrix (ECM). The proteins referred to as antigens herein (e.g. MCSP, FAP, CEA, EGFR, CD33, CD3) can be any native form the ns from any vertebrate source, including mammals such as primates (e.g. ) and rodents (e.g. mice and rats), unless otherwise indicated. In a particular ment the antigen is a human protein. 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 variants or c variants. Exemplary human proteins useful as antigens include, but are not limited to: Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP), also known as Chondroitin 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, , NCBI Accession no. NP_004451); Carcinoembroynic antigen (CEA), also known as oembryonic antigen-related cell adhesion molecule 5 (UniProt no. P06731 (version 119), NCBI RefSeq no. NP_004354.2); CD33, also known as gp67 or Siglec-3 (UniProt no. P20138, NCBI Accession nos. NP_001076087, NP_001171079); Epidermal Growth Factor or (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 ce; or UniProt no.

Q95LI5 (version 49), NCBI GenBank no. BAB71849.1, SEQ ID NO: 266 for the cynomolgus [Macaca fascicularis] ce). In certain embodiments the T cell activating bispecific antigen g molecule described binds to an epitope of an activating T cell antigen or a target cell antigen that is conserved among the activating 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 interactions. The y of an antigen binding moiety to bind to a specific antigenic determinant can be measured either through an enzymelinked immunosorbent assay ) or other techniques familiar to one of skill in the art, e.g. surface plasmon nce (SPR) technique zed 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 n binding moiety to an unrelated protein is less than about 10% of the binding of the antigen g moiety to the antigen as measured, e.g., by SPR. In certain embodiments, an antigen binding moiety that binds to the antigen, or an n g molecule comprising 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).

"Affinity" refers to the strength of the sum total of non-covalent interactions between a single g site of a molecule (e.g., a receptor) and its binding partner (e.g., a ligand). Unless indicated otherwise, as used herein, "binding affinity" refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., an n binding moiety and an antigen, or a or 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 ties may comprise different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured by well established methods known in the art, ing those described herein. A particular method for measuring affinity is Surface n 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 clarity the term es also reduction of the affinity to zero (or below the detection limit of the analytic method), i.e. complete abolishment of the interaction. Conversely, "increased binding" refers to an increase in binding affinity for the respective interaction.

An ating T cell antigen" as used herein refers to an antigenic determinant expressed 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 tion by triggering the signaling e of the T cell receptor 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, particularly a cytotoxic T lymphocyte, selected from: proliferation, differentiation, cytokine secretion, cytotoxic effector molecule e, 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 bed 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 t to antigen binding moieties etc., are used for ience of distinguishing when there is more than one of each type of moiety. Use of these terms is not intended to confer a ic order or orientation of the T cell activating bispecific antigen binding molecule unless explicitly so stated.

A "Fab molecule" 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 "fused" 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 e-chain" refers to a molecule comprising amino acid monomers linearly linked by peptide bonds. In certain embodiments, one of the antigen binding moieties is a single-chain Fab le, i.e. a Fab molecule wherein the Fab light chain and the Fab heavy chain are connected by a e 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 referred to herein as the "heavy chain" of the crossover Fab molecule. Conversely, in a ver Fab molecule wherein the constant s of the Fab light chain and the Fab heavy chain are ged, the peptide chain comprising the heavy chain variable region is referred to herein as the "heavy chain" of the crossover Fab molecule.

The term oglobulin molecule" refers to a protein having the structure of a lly occurring antibody. For example, immunoglobulins of the IgG class are tetrameric glycoproteins of about 150,000 daltons, composed of two light chains and two heavy chains that are disulfide-bonded. From N- to C-terminus, 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 . 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, ed by a constant 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 (IgG3), γ4 (IgG4), α1 (IgA1) and α2 . 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 immunoglobulin 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 limited to monoclonal antibodies, polyclonal antibodies, and dy fragments so long as they exhibit the desired antigen-binding ty.

An "antibody fragment" refers to a molecule other than an intact antibody 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, -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 discussion 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 nbinding sites that may be bivalent 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 comprising 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 ments, 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). Antibody fragments can be made by s ques, including but not limited to lytic digestion of an intact antibody as well as production by recombinant host cells (e.g. E. coli or phage), as described The term en binding " refers to the part of an antibody that comprises the area which specifically binds to and is mentary to part or all of an antigen. An antigen binding domain may be provided by, for example, one or more antibody variable domains (also called antibody variable regions). Particularly, an antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

The term "variable " 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, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. n and Co., page 91 (2007). A single VH or VL domain may be ient to confer antigen-binding specificity.

The term "hypervariable " or "HVR", as used herein, refers to each of the regions of an dy variable domain which are hypervariable 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 ining regions (CDRs), the latter being of highest sequence variability and/or involved 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 ed to as "complementarity determining regions" , and these terms are used herein interchangeably in nce to portions 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, Sequences of Proteins of Immunological Interest (1983) and by Chothia et al., J Mol Biol 196:901-917 (1987), where the definitions include pping 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 f 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 variable 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, "Sequence of ns of Immunological Interest" (1983). Unless otherwise specified, references to the ing of specific amino acid residue positions in an antibody variable region are according to the Kabat numbering system.

The polypeptide sequences of the sequence g (i.e., SEQ ID NOs 1, 3, 5, 7, 9, 11, 13, 15 etc.) are not numbered according to the Kabat numbering system. r, it is well within the ordinary skill of one in the art to convert the numbering of the sequences of the ce 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 consists of four FR s: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences lly appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The "class" of an antibody or globulin refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and l of these may be further divided into subclasses pes), 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, respectively.

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 includes native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an IgG heavy chain might vary ly, 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 7) of the Fc region may or may not be present.

Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of ns of Immunological Interest, 5th Ed. Public Health Service, National Institutes of , Bethesda, MD, 1991. A "subunit" of an Fc domain as used herein refers to one of the two polypeptides forming 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 ts the association of a polypeptide comprising the Fc domain subunit with an identical ptide 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), n the modifications are complementary to each other so as to promote association of the two Fc domain ts. For example, 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 ostatically favorable, respectively. Thus, (hetero)dimerization occurs between a polypeptide comprising the first Fc domain subunit and a polypeptide comprising the second Fc domain subunit, which might be non-identical in the sense that further components fused to each of the ts (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 association comprises a separate amino acid mutation, specifically an amino acid substitution, in each of the two subunits of the Fc .

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

As used herein, the terms "engineer, ered, engineering", are considered to include any manipulation of the peptide backbone or the ranslational modifications of a naturally occurring or recombinant polypeptide or fragment thereof. Engineering includes modifications of the amino acid sequence, of the glycosylation n, 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 uct, provided that the final construct possesses the desired characteristics, e.g., reduced binding to an Fc or, or increased ation with another peptide. Amino acid sequence deletions and ions include 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 particularly preferred.

Amino acid substitutions include replacement by non-naturally occurring amino acids or by naturally occurring amino acid derivatives of the twenty rd amino acids (e.g. 4- hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using c 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 methods other than genetic engineering, such as chemical cation, may also be useful. Various designations may be used herein to indicate 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 t.

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 products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known ting/blocking , proteolytic cleavage, or modification by turally occurring amino acids. A ptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily ated from a designated c acid sequence. It may be generated in any manner, including by chemical synthesis. A polypeptide described 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 ure 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 "isolated" polypeptide or a variant, or derivative f is ed 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. Recombinantly 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 ce identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference ptide 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 es of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for ce, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can ine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes , however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The 2 sequence comparison computer 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 ered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San sco, 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 ce comparison parameters are set by the 2 program and do not vary. In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity 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 comprises a certain % amino acid sequence ty 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 m 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 ce 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 aph using the ALIGN-2 computer program.

The term "polynucleotide" refers to an ed nucleic acid molecule or construct, e.g. messenger RNA (mRNA), virally-derived RNA, or plasmid DNA (pDNA). A polynucleotide may comprise a conventional phosphodiester bond or a nventional 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 recombinant polynucleotide encoding a polypeptide ned in a vector is considered isolated for the es of the presentdescription. Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution. An isolated polynucleotide includes a polynucleotide molecule contained in cells that ordinarily contain the polynucleotide 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 double-stranded forms.

Isolated polynucleotides or nucleic acids according to the present description further include such molecules ed synthetically. In addition, a cleotide 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 c acid or cleotide having a nucleotide sequence at least, for example, 95% "identical" to a reference nucleotide sequence described, it is intended that the nucleotide ce of the polynucleotide is identical to the reference sequence except that the polynucleotide ce may include up to five point mutations per each 100 nucleotides of the reference nucleotide ce. 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 tides in the reference sequence may be deleted or substituted with another tide, 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 nucleotide sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference ce. As a practical matter, whether any particular cleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% cal to a nucleotide sequence described can be determined conventionally using known computer programs, such as the ones discussed above for polypeptides (e.g. ALIGN-2).

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

Typically, the recombinant expression cassette portion of an expression vector es, among other sequences, a nucleic acid sequence to be transcribed and a promoter. In certain ments, the sion cassette comprises polynucleotide sequences that encode bispecific antigen binding molecules described or fragments f.

The term "vector" or "expression vector" is mous 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 ly associated in a target cell. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been uced. The expression vector described comprises an sion cassette. Expression vectors 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 ed by the ar transcription and/or translation machinery. In one embodiment, the expression vector described comprises an expression cassette that comprises polynucleotide sequences that encode bispecific antigen 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, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom without regard to the number of passages.

Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are ed 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 cultured cells, e.g. mammalian cultured cells, such as CHO cells, BHK cells, NS0 cells, SP2/0 cells, YO a 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 engagement by an Fc domain of an dy s signaling events that stimulate the receptor-bearing cell to perform effector functions. Human activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa , and FcαRI (CD89).

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism leading to the lysis of antibody-coated target cells by immune effector cells. The target cells are cells to which antibodies or derivatives thereof comprising an Fc region specifically bind, lly via the protein part that is N-terminal to the Fc region. As used herein, the term "reduced ADCC" is d 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 surrounding the target cells, by the mechanism of ADCC defined above, and/or an increase 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 ism of ADCC. The reduction in ADCC is relative to the ADCC ed by the same dy produced by the same type of host cells, using the same standard production, purification, formulation and storage methods (which are known to those skilled in the art), but that has not been engineered. For example the reduction in ADCC mediated by an antibody comprising 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 domain. Suitable assays to measure ADCC are well known in the art (see e.g. PCT publication no. or PCT patent application no. 2012/055393).

An "effective " of an agent refers to the amount that is ary 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 ition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A eutically 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. s 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 ient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.

A "pharmaceutically acceptable carrier" refers to an ingredient in a ceutical 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 ions thereof such as "treat" or "treating") refers to al 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. ble effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and ion 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 e.

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

The term "comprising" as used in this ication means "consisting at least in part of". When interpreting each statement in this ication that includes the term "comprising", features other than that or those prefaced 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 e of specific binding to an activating T cell n and the other one of which is a Fab molecule capable of ic 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 wherein the Fab light chain and the Fab heavy chain are connected by a peptide linker, or (b) a crossover Fab molecule n either the variable or the constant regions of the Fab light chain and the Fab heavy chain are exchanged.

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

In some embodiments, 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.

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 moiety. In a specific such embodiment, the T cell activating bispecific antigen binding molecule essentially consists of a first and a second antigen g 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 N-terminus of the Fab heavy chain of the second antigen binding , and 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 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 ver Fab le, the Fab light chain of the first n binding moiety and the Fab light chain of the second antigen g moiety may additionally be fused to each other.

In an alternative such 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 t of the Fc domain. In a specific such embodiment, the T cell activating bispecific antigen binding molecule ially consists of a first and a second antigen g moiety, an Fc domain composed of a first and a second subunit, and optionally one or more peptide s, 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 ment, 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 binding moiety. 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 ally 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 antigen 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 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 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 s, 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 n binding 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 particular of these embodiments, the first n binding moiety is capable of specific g to an activating T cell antigen. In other embodiments, the first n g 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 example, (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 ary e 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). onally, s may comprise (a n 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 additional peptide linker.

A T cell activating bispecific antigen binding molecule with a single antigen binding moiety e 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 antigen 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 enhance internalization of the target cell antigen, thereby reducing its availablity.

In many other cases, however, it will be advantageous to have a T cell ting bispecific antigen binding molecule comprising two or more antigen binding es specific for a target cell antigen (see es 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 binding 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 binding moiety is capable of specific binding to the same target cell antigen as the first or second antigen binding moiety. In a particular embodiment, the first antigen binding moiety is capable of specific g to an activating T cell antigen, and the second and third antigen binding moieties are capable of specific binding to a target cell antigen.

In one embodiment, the third antigen binding moiety is fused at the inus of the Fab heavy chain to the inus of the first or second subunit of the Fc . 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 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 g moiety may additionally be fused to each other.

The second and the third antigen binding moiety may be fused to the Fc domain directly or through a peptide linker. In a particular embodiment the second and the third antigen g moiety are each fused to the Fc domain through an immunoglobulin hinge . In a specific embodiment, the immunoglobulin hinge region is a human IgG1 hinge region. In one ment the second and the third antigen 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 particular ment the globulin is an IgG1 subclass immunoglobulin. In another embodiment the globulin 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 n binding molecule essentially consists of an immunoglobulin molecule capable of ic binding to a target cell antigen, and an antigen binding moiety capable of specific binding to an activating T cell antigen n the n binding moiety is a single chain Fab molecule or a crossover Fab molecule, ularly a ver Fab molecule, fused to the inus 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 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. In a specific such embodiment, the T cell activating bispecific antigen binding molecule essentially consists of a first, a second and a third antigen binding moiety, an Fc domain composed of a first and a second subunit, and optionally 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 n 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 n the third antigen binding moiety is fused at the C-terminus 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 crossover Fab le. 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 some of the T cell activating bispecific antigen binding molecule described, the Fab light chain of the first antigen g moiety and the Fab light chain of the second antigen g moiety are fused to each other, ally via a linker e. 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 antigen binding 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 les described.

In certain embodiments 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 ptide wherein a second Fab heavy chain shares a carboxyterminal peptide bond with an Fc domain subunit 1-CH2-CH3(-CH4)). In some embodiments the T cell activating bispecific antigen binding molecule further comprises a second Fab light chain polypeptide ). In certain embodiments the polypeptides are covalently linked, e.g., by a disulfide bond.

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

In certain ments the T cell activating bispecific antigen binding molecule comprises a polypeptide n 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 an Fc domain subunit (VL-CH1-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 ments the T cell activating bispecific antigen binding molecule further ses a polypeptide wherein a Fab heavy chain variable region shares a y-terminal peptide bond with a Fab light chain constant region (VH-CL) and a Fab light chain polypeptide ). In certain embodiments the polypeptides are covalently linked, e.g., by a disulfide bond.

In alternative embodiments the T cell activating bispecific antigen binding molecule comprises a ptide 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 nt region is replaced by a light chain constant region), which in turn shares a y-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 binding molecule r comprises a polypeptide wherein a Fab light chain le 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 ments the ptides are covalently linked, e.g., by a disulfide bond.

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

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

In one embodiment, the T cell ting ific antigen g 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-CL-VL-CH1), 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 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 ses 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) (VL-CL-VH-CL), a polypeptide wherein a second Fab heavy chain shares a carboxy-terminal peptide bond with an Fc domain subunit 1-CH2-CH3(-CH4)), and a ptide wherein a first Fab light chain variable region shares a carboxy-terminal peptide bond with a first Fab heavy chain nt 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 ptide wherein a third Fab heavy chain shares a carboxy-terminal peptide 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, components of the T cell activating ific n binding molecule (e.g. antigen g moiety, Fc domain) may be fused directly or through various linkers, particularly peptide linkers comprising one or more amino acids, typically about 2-20 amino acids, that are bed herein or are known in the art. Suitable, nonimmunogenic peptide linkers include, for example, (G4S)n, (SG4)n, (G4S)n or )n peptide linkers, wherein n is generally a number n 1 and 10, typically between 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 molecule. 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 antigen binding le is an IgG Fc domain. In a particular embodiment 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 substitution reduces in vivo Fab arm exchange of IgG4 antibodies (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 ). 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 cations promoting heterodimerization T cell activating ific 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 ression of these polypeptides and subsequent zation leads to several 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 ing 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 extensive n-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 modification 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" cation in one of the two ts of the Fc domain and a "hole" modification in the other one of the two subunits of the Fc domain.

The knob-into-hole technology is described e.g. in US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the method involves introducing a protuberance ") at the interface of a first ptide 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 e heterodimer formation and hinder homodimer formation. Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g. tyrosine or tryptophan). satory 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).

Accordingly, in a particular ment, 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 positionable 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 e 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 subunit is positionable.

The erance 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 t of the Fc domain the ine residue at position 366 is replaced with a tryptophan residue ), 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 ).

In yet a further embodiment, in the first subunit of the Fc domain additionally the serine residue at position 354 is replaced with a cysteine residue (S354C), and in the second t of the Fc domain additionally the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C). Introduction of these two cysteine residues results in formation of a disulfide 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 g moiety capable 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 t of the Fc domain ising the "knob" modification). Without wishing to be bound by theory, fusion of the antigen binding moiety e of binding to an activating T cell antigen to the knob-containing t of the Fc domain will er) minimize the generation of antigen binding molecules comprising two antigen binding moieties capable of binding to an activating T cell n (steric clash of two knob-containing polypeptides).

In an alternative embodiment a modification ing association of the first and the second subunit of the Fc domain comprises a modification mediating electrostatic steering effects, e.g. as described in PCT publication . Generally, this method es 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 ble.

Fc domain modifications reducing Fc receptor g and/or effector function The Fc domain confers to the T cell activating bispecific antigen binding le favorable pharmacokinetic properties, including a long serum half-life which contributes 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 activating bispecific antigen binding molecule to cells expressing Fc receptors 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 systemic administration. Activation of (Fc or-bearing) immune cells other than T cells may even reduce efficacy of the T cell activating bispecific antigen binding 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 activating bispecific antigen binding molecules described exhibits reduced binding affinity to an Fc receptor 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 antigen binding molecule comprising said Fc domain) ts 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 ting 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 n g molecule 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 ment the Fc receptor is a human Fc receptor. In one embodiment the Fc receptor is an activating Fc receptor. In a ic embodiment the Fc receptor is an activating human Fcγ receptor, more ically human FcγRIIIa, FcγRI or FcγRIIa, most specifically 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 g affinity to al Fc or (FcRn), as compared to a native IgG1 Fc domain domain. Substantially similar binding to FcRn is achieved when the Fc domain (or the T cell ting bispecific n binding molecule comprising said Fc domain) exhibits r 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 bispecific antigen binding molecule comprising 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 particular embodiments, the Fc domain of the T cell activating bispecific antigen binding molecule comprises one or more amino acid on that s the binding affinity of the Fc domain to an Fc or and/or effector function. 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 mutation reduces the binding affinity of the Fc domain to an Fc receptor. In one embodiment the amino acid mutation reduces the binding affinity of the Fc domain to an Fc receptor by at least , at least , or at least d. In embodiments where there is more than one amino acid on that reduces the binding affinity of the Fc domain to the Fc receptor, the ation of these amino acid mutations may reduce the binding affinity 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 ting bispecific antigen binding molecule sing 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 antigen binding molecule comprising a non-engineered Fc domain. In a particular embodiment the Fc receptor is an Fcγ receptor. In some embodiments the Fc receptor 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. ably, binding to each of these receptors is reduced. In some embodiments binding affinity to a complement component, specifically binding affinity to C1q, is also reduced. In one embodiment binding affinity to neonatal Fc receptor (FcRn) is not reduced. Substantially similar binding to FcRn, i.e. preservation of the binding ty of the Fc domain to said receptor, is achieved when the Fc domain (or the T cell activating bispecific antigen binding molecule comprising said Fc ) exhibits greater than about 70% of the binding affinity of a non-engineered form of the Fc domain (or the T cell ting bispecific antigen binding molecule comprising said gineered form of the Fc domain) to FcRn. The Fc domain, or T cell activating ific antigen binding 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 activating bispecific antigen binding molecule is engineered to have reduced effector on, as compared to a non-engineered Fc domain. The reduced effector function can include, but is not limited to, one or more of the following: d complement dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated xicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to tes, reduced binding to polymorphonuclear cells, reduced direct signaling inducing apoptosis, reduced crosslinking of target-bound antibodies, reduced dendritic cell maturation, or reduced T cell g. In one embodiment the reduced effector function is one or more selected from the group of d CDC, reduced ADCC, reduced ADCP, and reduced cytokine secretion. In a particular ment 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 molecule comprising a non-engineered Fc domain).

In one ment the amino acid mutation that reduces the g affinity of the Fc domain to an Fc receptor and/or effector function is an amino acid substitution. In one ment 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 , particularly a human IgG1 Fc domain. In one embodiment the Fc domain comprises an amino acid substitution at on P329. In a more specific embodiment the amino acid substitution is P329A or P329G, particularly P329G. In one embodiment the Fc domain comprises an amino acid tution 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 further amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S. In particular embodiments the Fc domain comprises amino acid substitutions at positions P329, L234 and L235. In more particular embodiments the Fc domain ses the amino acid mutations L234A, L235A and P329G ("P329G . In one such ment, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. The "P329G LALA" combination of amino acid substitutions almost completely abolishes Fcγ receptor binding of a human IgG1 Fc , as described in PCT patent application no. , incorporated 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 d effector ons as compared to IgG1 antibodies. Hence, in some ments the Fc domain of the T cell activating bispecific antigen binding molecules described is an IgG4 Fc domain, particularly a human IgG4 Fc domain. In one embodiment the IgG4 Fc domain comprises 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 embodiment the IgG4 Fc domain comprises an amino acid substitution at position L235, specifically the amino acid substitution L235E. In another ment, 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, specifically amino acid substitutions 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 reference 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 substitutions L234A, L235A and optionally P329G, or a human IgG4 Fc domain comprising the amino acid substitutions S228P, L235E and optionally P329G.

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

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

, Fc domains with reduced Fc receptor binding and/or or 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. 056). Such Fc mutants include Fc mutants 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, tution, insertion or modification using c or chemical methods well known in the art. Genetic methods may e site-specific mutagenesis of the encoding DNA sequence, PCR, gene synthesis, and the like. The correct tide 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 care), and Fc receptors such as may be obtained by recombinant expression. A suitable such binding assay is described . Alternatively, binding affinity of Fc domains or cell activating bispecific n binding molecules comprising an Fc domain for Fc receptors may be evaluated using cell lines known to express particular Fc receptors, 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 ed by methods known in the art. A suitable assay for measuring ADCC is described . 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. 337; Bruggemann et al., J Exp Med 166, 1351-1361 (1987).

Alternatively, non-radioactive assays methods may be employed (see, for example, ACTI™ nonradioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest 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 .

In some embodiments, binding of the Fc domain to a complement component, specifically to C1q, is reduced. Accordingly, in some embodiments n the Fc domain is engineered to have reduced effector function, said reduced or function includes reduced CDC. C1q binding assays may be d 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 and . To assess complement activation, a CDC assay may be performed (see, for e, Gazzano-Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et al., Blood 101, 1045-1052 (2003); and Cragg and e, Blood 103, 2738- 2743 (2004)).

Antigen Binding Moieties The antigen binding molecule bed is ific, i.e. it ses at least two antigen binding moieties capable of specific binding to two distinct antigenic determinants. According to the description, the antigen binding moieties are Fab les (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 les 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 binding 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 molecule described, the C-terminus of the Fab light chain is connected to the N-terminus of the Fab heavy chain by a e linker.

The peptide linker allows arrangement of the Fab heavy and light chain to form a functional antigen g moiety. Peptide linkers suitable for connecting the Fab heavy and light chain include, for example, -GG (SEQ ID NO: 152) or (SG3)2-(SEG3)4-(SG3)-SG (SEQ ID NO: 153). In a particular crossover Fab molecule useful for the T cell activating bispecific antigen binding molecule described, the constant regions of the Fab light chain and the Fab heavy chain are exchanged. In another crossover Fab molecule useful for the T cell activating ific antigen binding 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 g molecule is capable of simultaneous binding to a target cell antigen, particularly a tumor cell antigen, and an ting T cell antigen. In one embodiment, the T cell activating bispecific antigen binding molecule 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 ular embodiment, such simultaneous binding results in lysis of the target cell, particularly a tumor cell. In one embodiment, such simultaneous binding results in activation of the T cell. In other ments, such simultaneous binding results in a cellular response of a T lymphocyte, particularly a cytotoxic T lymphocyte, selected from the group of: proliferation, differentiation, cytokine ion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers. In one embodiment, binding of the T cell activating ific 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 ting bispecific antigen g molecule is capable of redirecting cytotoxic activity of a T cell to a target cell. In a particular embodiment, said re- ion is independent of MHC-mediated peptide n presentation by the target cell and and/or icity 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 ting bispecific antigen binding molecule described comprises at least one antigen binding moiety capable of binding to an activating T cell n (also referred to herein as an "activating T cell antigen binding moiety"). In a ular embodiment, the T cell activating bispecific antigen binding molecule ses not more than one antigen binding moiety capable of specific binding to an activating T cell antigen. In one embodiment the T cell activating ific antigen binding molecule provides monovalent binding to the activating T cell antigen. The activating T cell antigen binding 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 activating bispecific antigen binding molecule, the antigen binding moiety capable of specific binding to an activating T cell antigen 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. specifically binds to) human and cynomolgus CD3. In some embodiments, the activating T cell antigen is the epsilon subunit of CD3.

In one embodiment, the activating T cell antigen binding moiety can compete with monoclonal antibody H2C ibed 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 epitope of CD3. In yet another embodiment, the activating T cell antigen binding moiety can compete with monoclonal antibody 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 antigen 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 ment, the activating T cell antigen binding moiety binds to the same epitope of CD3 as monoclonal antibody SP34. In one ment, the activating T cell n binding moiety ses 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 embodiment, the ting 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% cal to SEQ ID NO: 169 and a light chain le region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% cal to SEQ ID NO: 177, or ts f that retain functionality.

In a particular embodiment, the activating T cell antigen g 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 activating T cell antigen binding moiety binds to the same epitope of CD3 as an antigen 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 comprises a heavy chain le 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 onality. 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 sequence 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 binding moiety comprising the heavy chain variable region sequence of SEQ ID NO: 255 and the light chain le region sequence of SEQ ID NO: 263. In r embodiment, the activating T cell antigen 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 embodiment, 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, 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 e of binding to a target cell n (also ed to herein as an "target cell antigen binding moiety"). In certain embodiments, the T cell activating bispecific antigen binding molecule comprises two antigen g moieties capable of binding to a target cell antigen. In a particular such embodiment, each of these antigen binding moieties specifically binds to the same antigenic determinant. In one embodiment, the T cell activating bispecific antigen binding molecule comprises an immunoglobulin molecule capable of specific binding to a target cell antigen. In one embodiment the T cell activating bispecific antigen binding molecule ses not more than two antigen binding moieties e of binding to a target cell antigen.

The target cell antigen binding moiety is lly 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 n 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 ific antigen binding molecule comprises at least one n binding moiety that is specific for Melanoma-associated oitin Sulfate Proteoglycan (MCSP). In one embodiment the T cell ting 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 application no. EP 11178393.2, incorporated herein by reference in its entirety) for binding to an e of MCSP. In one embodiment, the antigen binding moiety that is specific for MCSP comprises 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 ce 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 sequence 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 bispecific antigen binding molecule 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 ation no. EP 11178393.2, incorporated herein by reference in its entirety) for binding to an e of MCSP. In one embodiment, 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 n binding moiety that is specific for MCSP ses 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 le 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 variable 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 g to MCSP, particularly human MCSP, is preserved. Preferred variants are those having a binding affinity for MCSP at least equal (or stronger) to the binding affinity of the antigen binding moiety comprising the unsubstituted variable region sequences.

In one embodiment the T cell activating ific antigen binding molecule comprises 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 thereof that retain onality. In yet another embodiment the T cell activating bispecific n binding molecule comprises the polypeptide 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 thereof that retain functionality. In yet another embodiment the T cell activating bispecific antigen g molecule comprises the ptide 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 thereof that retain functionality. In another ment the T cell activating bispecific n binding molecule comprises the polypeptide sequence of SEQ ID NO: 21, the polypeptide sequence of SEQ ID NO: 23 and the polypeptide ce of SEQ ID NO: 5, or variants thereof that retain functionality. In still another embodiment the T cell ting bispecific antigen g molecule comprises the polypeptide sequence of SEQ ID NO: 25, the polypeptide sequence of SEQ ID NO: 27 and the polypeptide ce of SEQ ID NO: 5, or variants thereof that retain functionality. In another embodiment the T cell activating bispecific antigen binding molecule ses 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 ptide sequence of SEQ ID NO: 5, or ts thereof 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 sequence of SEQ ID NO: 33, and the polypeptide sequence of SEQ ID NO: 5, or variants thereof that retain functionality. In r embodiment the T cell activating 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 molecule comprises the polypeptide sequence of SEQ ID NO: 39, the ptide 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 molecule comprises the polypeptide sequence of SEQ ID NO: 29, the polypeptide ce 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 n 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 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 ce of SEQ ID NO: 185, or variants f 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 ce 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 bispecific antigen binding molecule comprises the polypeptide ce of SEQ ID NO: 33, the polypeptide sequence of SEQ ID NO: 189, the polypeptide sequence of SEQ ID NO: 191 and the polypeptide ce of SEQ ID NO: 193, or variants thereof that retain functionality. In one embodiment the T cell activating bispecific n binding molecule ses the polypeptide sequence of SEQ ID NO: 183, the polypeptide sequence 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 ses 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 ce of SEQ ID NO: 201, 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: 215 and the polypeptide ce of SEQ ID NO: 217, 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: 215 and the polypeptide ce of SEQ ID NO: 219, or variants thereof that retain functionality.

In a specific embodiment the T cell ting bispecific antigen g 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: 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 activating bispecific antigen binding le comprises at least one antigen binding moiety that is specific for Epidermal Growth Factor Receptor (EGFR). In another embodiment the T cell activating bispecific antigen g molecule 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 g 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 ic for EGFR 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: 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 ce of SEQ ID NO: 43, the polypeptide sequence of SEQ ID NO: 45 and the polypeptide sequence of SEQ ID NO: 47, or variants thereof that retain functionality. In a r embodiment the T cell activating bispecific antigen binding molecule comprises the polypeptide sequence of SEQ ID NO: 49, the polypeptide sequence of SEQ ID NO: 51 and the polypeptide sequence of SEQ ID NO: 11, 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: 53, the polypeptide sequence of SEQ ID NO: 45 and the polypeptide sequence of SEQ ID NO: 47, 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 polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a ce ed 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 ment the T cell activating ific 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 antigen 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 , incorporated herein by reference in its entirety. In one embodiment, the antigen binding moiety that is ic 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 le 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 le 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 bispecific antigen binding molecule comprises the polypeptide sequence of SEQ ID NO: 55, the polypeptide sequence of SEQ ID NO: 51 and the polypeptide ce of SEQ ID NO: 11, or variants thereof that retain onality. In a further embodiment the T cell activating bispecific antigen binding le ses the polypeptide sequence of SEQ ID NO: 57, the polypeptide sequence of SEQ ID NO: 59 and the polypeptide sequence of SEQ ID NO: 61, 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 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 molecule comprises at least one antigen binding moiety that is specific for oembryonic Antigen (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 BW431/26 (described in European patent no. EP 160 897, and Bosslet et al., Int J Cancer 36, 75- 84 (1985)) for binding 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 onal antibody CH1A1A (see SEQ ID NOs 123 and 131) for binding to an epitope of CEA. See PCT patent publication number , incorporated herein by reference in its entirety. In one embodiment, the n binding moiety that is specific for CEA binds to the same epitope of CEA as onal antibody CH1A1A. In one embodiment, the antigen binding moiety that is specific for CEA comprises 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 le 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%, cal to SEQ ID NO: 131, or variants f 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 onal antibody . In one embodiment, the antigen binding moiety that is specific for CEA comprises the heavy chain variable 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 residue within the le region sequences may be substituted by a different amino acid, ing amino acid residues within the CDR regions, ed that g to CEA, particularly human CEA, is preserved.

Preferred variants are those having a binding affinity for CEA at least equal (or stronger) to the binding affinity of the antigen g 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: 63, the polypeptide sequence of SEQ ID NO: 65, the polypeptide sequence of SEQ ID NO: 67 and the ptide sequence of SEQ ID NO: 33, 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: 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 polypeptide sequence of SEQ ID NO: 205 and the polypeptide sequence of SEQ ID NO: 207, or variants thereof that retain onality. In one embodiment the T cell activating bispecific antigen binding le 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 ptide ce encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% cal to a sequence 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 ment the T cell activating bispecific antigen binding molecule comprises at least one antigen binding moiety that is specific for CD33. In one embodiment, the n binding moiety that is ic 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 ic for CD33 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: 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 variants thereof that retain functionality.

In one embodiment the T cell activating ific n binding molecule comprises the polypeptide sequence of SEQ ID NO: 33, the ptide 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 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: 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 ses a polypeptide sequence d 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: 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 described herein or a fragment thereof.

Polynucleotides bed include those that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% cal 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 encoding T cell activating bispecific antigen binding molecules described may be expressed as a single polynucleotide that encodes the entire T cell activating bispecific antigen binding le or as multiple (e.g., two or more) polynucleotides that are co- expressed. Polypeptides d by polynucleotides that are co-expressed may associate through, e.g., disulfide bonds or other means to form a functional 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 optionally (part of) another antigen g moiety. When co-expressed, the heavy chain polypeptides will associate with the light chain polypeptides to form the antigen binding . In another example, the portion of the T cell activating bispecific antigen g molecule sing 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 g molecule sing 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 ific antigen binding molecule comprising a first and a second antigen binding moiety, and an Fc domain consisting of two subunits, n the first antigen g 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 ed cleotide encodes a polypeptide wherein a Fab heavy chain shares a carboxy terminal peptide bond with an Fc domain subunit. In yet another embodiment, an isolated 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 isolated 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 described 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 crossover Fab molecule. In one embodiment, an isolated polynucleotide described s the heavy chain of the first antigen binding moiety and a subunit of the Fc domain. In a more specific embodiment the isolated polynucleotide encodes a polypeptide wherein Fab light chain variable region shares a y 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 r specific embodiment the isolated polynucleotide encodes a polypeptide n Fab heavy chain variable region shares a carboxy al peptide bond with a Fab light chain constant region, which in turn shares a carboxy-terminal e 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 polynucleotide encodes a polypeptide wherein a Fab heavy chain shares a carboxy terminal peptide bond with an Fc domain subunit. In yet another embodiment, an isolated polynucleotide described s the heavy chain of the first antigen binding moiety, the heavy chain of the second antigen binding moiety and a subunit of the Fc . In a more specific embodiment, the isolated polynucleotide encodes a polypeptide wherein a Fab light chain le 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 heavy chain, which in turn shares a carboxyterminal peptide bond with an Fc domain subunit. In r ic embodiment, the isolated polynucleotide encodes a polypeptide wherein a Fab heavy chain variable region shares a y-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 carboxy-terminal peptide bond with an Fc domain subunit. In yet another specific embodiment the isolated polynucleotide encodes a polypeptide wherein a Fab heavy chain shares a carboxy-terminal peptide bond with a Fab light chain le region, which in turn shares a y-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 , which in turn 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 t.

In further embodiments, an isolated polynucleotide described encodes the heavy chain of a third 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 terminal peptide bond with an Fc domain subunit.

In further embodiments, an ed polynucleotide described encodes the light chain of an antigen binding moiety. In some embodiments, 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. 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 nt region. In still other embodiments, an isolated polynucleotide described encodes the light chain of the first antigen binding moiety and the light chain of the second antigen binding moiety. In a more ic embodiment, the isolated polynucleotide encodes a polypeptide wherein a Fab heavy chain le 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 ic embodiment the isolated cleotide encodes a polypeptide wherein a Fab light chain shares a carboxyterminal peptide bond with a Fab heavy chain variable region, which in turn shares a carboxyterminal peptide bond with a Fab light chain constant region. In yet another specific embodiment, the isolated cleotide 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 ptide 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 molecule bed or a fragment thereof, wherein the polynucleotide comprises a sequence that s a variable region ce as shown in SEQ ID NOs 75, 83, 91, 99, 107, 115, 123, 131, 139, 147, 169, 177, 239, 247, 255 and 263. In another embodiment, described an isolated polynucleotide encoding a T cell activating bispecific antigen binding molecule or fragment thereof, n 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 r 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 sequence 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 described or a fragment thereof, n 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, bed is an isolated polynucleotide encoding a T cell activating bispecific antigen binding molecule described or a fragment thereof, n 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 ce 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 isolated cleotide encoding a T cell activating bispecific antigen binding molecule or fragment thereof, wherein the polynucleotide comprises a sequence that encodes a polypeptide sequence 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 bispecific antigen binding molecule described or a fragment f, 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 conservative amino acid substitutions.

Also described is an ed polynucleotide encoding a T cell activating bispecific antigen binding molecule described or fragment thereof, wherein the polynucleotide comprises a sequence that encodes the ptide ce 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 conservative amino acid substitutions.

In certain embodiments the polynucleotide or c acid is DNA. In other embodiments, a polynucleotide described is RNA, for example, in the form of ger RNA (mRNA). RNA described 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 peptide synthesis (e.g. Merrifield solid phase synthesis) or recombinant production. For recombinant production one or more polynucleotide encoding the T cell activating bispecific antigen binding molecule (fragment), e.g., as described above, is isolated and inserted into one or more vectors for further cloning and/or sion in a host cell. Such polynucleotide may be readily isolated and sequenced using conventional procedures. In one embodiment a vector, preferably an expression , comprises one or more of the cleotides described. Methods which are well known to those d 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 riate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo ination/genetic recombination. See, for example, the techniques described in Maniatis et al., LAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory, N.Y. (1989); and Ausubel et al., CURRENT OLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and Wiley Interscience, N.Y (1989). The expression vector can be part of a plasmid, virus, or may be a nucleic acid fragment. The expression vector es an expression cassette into which the polynucleotide encoding the T cell activating bispecific antigen g molecule (fragment) (i.e. the coding region) is cloned in operable association with a promoter and/or other transcription or translation control elements. As used herein, a "coding region" is a portion of c 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 flanking 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 cleotide construct, e.g. on a single vector, or in separate polynucleotide constructs, e.g. on separate (different) vectors. Furthermore, any vector may contain a single coding region, or may comprise two or more coding regions, e.g. a vector described may encode one or more polypeptides, which are post- or nslationally separated into the final proteins via proteolytic cleavage. In addition, a vector, polynucleotide, or nucleic acid described may encode heterologous coding regions, either fused or unfused to a polynucleotide encoding the T cell activating bispecific antigen g molecule ent) described, or variant or derivative thereof. Heterologous coding regions include t 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 associated 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 nts (such as a polypeptide coding region and a er associated therewith) are "operably associated" if induction of promoter 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 sion regulatory sequences to direct the expression of the gene product or interfere with the ability of the DNA template to be ribed. Thus, a promoter region would be operably associated with a c acid encoding a polypeptide if the promoter was capable of effecting transcription of that nucleic acid. The promoter may be a cell-specific promoter that directs substantial ription of the DNA only in predetermined cells. Other transcription l elements, s a promoter, for example enhancers, operators, sors, and transcription termination signals, can be operably associated with the polynucleotide to direct pecific 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 control regions, which function in vertebrate cells, such as, but not limited to, promoter and er segments from cytomegaloviruses (e.g. the immediate early promoter, in conjunction with intron-A), simian virus 40 (e.g. the early promoter), and iruses (such as, e.g. Rous a virus). Other ription control regions include those d from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit â-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable ription control regions include tissue-specific promoters and enhancers as well as inducible promoters (e.g. promoters inducible tetracyclins). Similarly, a variety of translation control elements are known to those of ordinary skill in the art. These include, but are not limited to ribosome binding sites, translation initiation and termination codons, and elements derived from viral systems (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence). The expression cassette may also include other features such as an origin of replication, and/or chromosome integration elements such as iral long terminal repeats (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 bed. For example, if secretion of the T cell activating bispecific antigen binding molecule is d, DNA encoding a signal sequence may be placed am of the nucleic acid encoding a T cell activating bispecific antigen binding molecule described or a fragment thereof. ing to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence which is cleaved from the mature n once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Those of ordinary skill in the art are aware that polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the translated polypeptide to produce a secreted or "mature" form of the ptide. In n embodiments, the native signal peptide, e.g. an immunoglobulin heavy chain or light chain signal peptide is used, or a functional derivative of that ce 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 example, the wild-type leader sequence may be tuted 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 ent) encoding polynucleotide.

Also described is a host cell comprising one or more polynucleotides described herein. In certain embodiments described is a host cell comprising one or more vectors described . The cleotides and vectors may incorporate any of the features, singly or in combination, described herein in relation to polynucleotides and vectors, respectively. In one such embodiment a host cell comprises (e.g. has been transformed or transfected with) a vector comprising a polynucleotide that encodes (part of) a T cell activating bispecific antigen g 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 ting ific antigen binding molecules described or fragments f. Host cells suitable for ating and for supporting expression of T cell activating ific antigen binding molecules are well known in the art. Such cells may be transfected or transduced as appropriate with the particular expression vector and large quantities of vector containing cells can be grown for seeding large scale fermenters to obtain ient quantities of the T cell activating bispecific antigen binding molecule for clinical applications. Suitable host cells include yotic microorganisms, such as E. coli, or various eukaryotic cells, such as Chinese r 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 isolated from the bacterial cell paste in a soluble fraction and can be r purified. In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or sion hosts for polypeptide-encoding vectors, including fungi and yeast strains whose glycosylation ys have been "humanized", resulting in the production of a polypeptide with a partially or fully human glycosylation pattern.

See Gerngross, Nat h 22, 1409-1414 (2004), and Li et al., Nat h 24, 210-215 (2006).

Suitable host cells for the expression of (glycosylated) polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, ularly for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be utilized as hosts. See e.g. US Patent Nos. 5,959,177, 6,040,498, 6,420,548, 978, and 6,417,429 (describing PLANTIBODIESTM technology for ing antibodies in transgenic plants). Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in sion may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 ); human nic kidney line (293 or 293T cells as described, e.g., in Graham et al., J Gen Virol 36, 59 ), 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 (Urlaub 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 le for n 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, enic plant or cultured 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 express foreign genes in these s. Cells expressing a polypeptide comprising either the heavy or the light chain of an antigen binding domain such as an antibody, 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, described is a method of producing a T cell activating ific antigen binding molecule described, wherein the method comprises culturing a host cell comprising a polynucleotide encoding the T cell activating bispecific antigen g molecule, as described herein, under conditions suitable for expression 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 bispecific antigen binding molecule can be designed such that its components are fused directly to each other or indirectly through a linker sequence. The composition and length of the linker may be ined in accordance with methods well known in the art and may be tested for cy. Examples of linker sequences between different components of T cell activating ific antigen binding molecules are found in the sequences described herein. Additional sequences may also be included to orate a cleavage site to separate the individual components of the fusion if desired, for example an endopeptidase ition sequence.

In certain embodiments the one or more antigen binding es of the T cell activating bispecific antigen binding les comprise at least an antibody variable region e of binding an antigenic determinant. Variable regions can form part of and be derived from naturally or non-naturally occurring antibodies and fragments thereof. Methods to e polyclonal antibodies and monoclonal antibodies are well known in the art (see e.g. Harlow and Lane, "Antibodies, a laboratory manual", Cold Spring Harbor Laboratory, 1988). Non-naturally occurring antibodies can be constructed using solid phase-peptide synthesis, can be produced recombinantly (e.g. as described 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. 108 to McCafferty).

Any animal species of antibody, antibody fragment, n binding domain or variable region can be used in the T cell activating bispecific antigen binding molecules described. Non-limiting antibodies, dy fragments, antigen binding domains or variable regions useful herein can be of murine, primate, or human origin. If the T cell ting bispecific antigen binding molecule is intended for human use, a chimeric 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 methods 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 d to (a) grafting the non-human (e.g., donor antibody) CDRs onto human (e.g. recipient antibody) framework and constant regions with or without retention of critical framework residues (e.g. those that are important for retaining good antigen binding affinity or antibody functions), (b) ng only the non-human specificity-determining regions (SDRs or a-CDRs; the residues critical for the antibody-antigen interaction) onto human framework and constant regions, or (c) transplanting the entire non-human le 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 ; Queen et al., Proc Natl Acad Sci USA 86, 10029-10033 (1989); US Patent Nos. 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 (1984); Morrison and Oi, Adv Immunol 44, 65-92 (1988); Verhoeyen et al., Science 239, 1534-1536 (1988); Padlan, Molec Immun 31(3), 169-217 (1994); Kashmiri et al., Methods 36, 25-34 (2005) (describing SDR (a-CDR) grafting); Padlan, Mol Immunol 28, 8 (1991) ibing "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 shuffling).

Human antibodies and human variable s can be produced using various techniques known in the art. Human antibodies are described generally 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 s can form part of and be derived from human monoclonal antibodies made by the hybridoma method (see e.g. Monoclonal dy Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)). Human antibodies and human variable regions may also be prepared by stering an gen to a transgenic animal that has been modified to e 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 isolating Fv clone variable region sequences selected from human-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 erty 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. 132066, the entire contents of which are hereby incorporated by reference. The ability of the T cell activating ific antigen binding molecule described to bind to a specific antigenic determinant can be measured either through an enzyme-linked immunosorbent assay (ELISA) or other ques familiar to one of skill in the art, e.g. surface n nce 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 fragment, antigen binding domain or variable domain that competes with a reference dy 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 conformational epitope) that is bound by the reference antibody. Detailed exemplary s 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 dy) and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to the antigen. The second dy may be present in a hybridoma supernatant. As a l, immobilized antigen is incubated in a on comprising the first labeled antibody but not the second unlabeled dy. 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 immobilized 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 dy 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 g molecules prepared as described herein may be ed by art-known techniques such as high performance liquid chromatography, ion ge chromatography, gel electrophoresis, affinity chromatography, 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 nt to those having skill in the art. For affinity chromatography purification an antibody, , receptor or antigen can be used to which the T cell activating bispecific antigen binding le binds. For example, for affinity chromatography purification of T cell activating bispecific antigen binding molecules described, a matrix with protein A or protein G may be used. Sequential Protein A or G ty 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 ined by any of a variety of well known analytical methods including gel electrophoresis, high pressure liquid chromatography, and the like. For example, the heavy chain fusion proteins expressed as described in the Examples were shown to be intact and properly assembled 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 ific antigen binding molecule light chain, heavy chain and heavy light chain fusion protein.

Assays T cell ting bispecific antigen binding molecules described herein may be identified, screened for, or characterized for their physical/chemical properties and/or biological ties by various assays known in the art. ty assays The ty of the T cell activating bispecific antigen binding molecule for an Fc receptor or a target antigen can be ined in accordance with the s 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 recombinant expression. Alternatively, binding of T cell activating bispecific antigen binding molecules for different ors or target ns may be ted using cell lines expressing the particular receptor or target antigen, for example by flow cytometry (FACS). A specific illustrative and exemplary embodiment for measuring binding affinity is described in the following and in the Examples below.

According to one embodiment, KD is measured by surface plasmon nce 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 kinetic measurements, four-fold serial dilutions of the bispecific construct (range between 500 nM and 4000 nM) are injected 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 constructs are captured by an anti human Fab specific antibody (GE Healthcare) that is immobilized on an activated CM5-sensor chip surface as described for the anti Penta-His dy. The final amount of coupled protein is is approximately 12000 RU. The bispecific constructs are ed for 90 s at 300 nM. The target antigens are passed through the flow cells for 180 s at a tration range from 250 to 1000 nM with a flowrate of 30 μl/min. The dissociation is monitored for 180 s.

Bulk refractive index differences are corrected for by subtracting the response obtained on nce flow cell. The steady state response was used to derive the dissociation constant KD by non-linear curve fitting of the ir binding isotherm. Association rates (kon) and dissociation rates (koff) are ated using a simple one-to-one Langmuir binding model (BIACORE® T100 Evaluation Software version 1.1.1) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (KD) is ated as the ratio koff/kon. See, e.g., Chen et al., J Mol Biol 293, 1 (1999). ty assays Biological activity of the T cell activating bispecific antigen binding molecules described can be measured by s assays as described in the Examples. ical activities may for example include the induction of proliferation of T cells, the induction of signaling in T cells, the induction of expression of activation markers in T cells, the induction of cytokine ion 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 activating bispecific n binding molecules described herein, e.g., for use in any of the below therapeutic methods. In one embodiment, a pharmaceutical composition comprises any of the T cell activating bispecific antigen binding molecules described herein and a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical ition comprises any of the T cell activating bispecific n 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 administration in vivo, the method comprising (a) obtaining a T cell activating bispecific antigen g molecule described, and (b) formulating the T cell activating bispecific antigen binding molecule with at least one ceutically 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 effective amount of one or more T cell activating ific antigen binding molecule dissolved or sed in a pharmaceutically acceptable carrier. The phrases "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that are generally xic to recipients at the dosages and concentrations employed, i.e. do not produce an adverse, allergic or other untoward on when administered to an animal, such as, for example, a human, as appropriate. The preparation of a ceutical composition that contains at least one T cell activating bispecific antigen g molecule and ally 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 ng Company, 1990, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet ity, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards or corresponding 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 , ngal agents), isotonic agents, absorption delaying agents, salts, preservatives, idants, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegration agents, lubricants, sweetening , flavoring agents, dyes, such like materials and combinations 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, incorporated 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 comprise different types of carriers depending on r it is to be administered in solid, liquid or aerosol form, and whether it need to be e for such routes of administration as injection. T cell activating bispecific antigen binding molecules described (and any additional therapeutic agent) can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, ranially, intraarticularly, intraprostatically, intrasplenically, intrarenally, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctivally, esicularlly, mucosally, intrapericardially, intraumbilically, cularally, orally, topically, locally, by inhalation (e.g. aerosol inhalation), injection, infusion, uous infusion, localized perfusion bathing target cells ly, 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). Parenteral administration, in particular intravenous injection, is most commonly used for administering polypeptide molecules such as the T cell ting bispecific antigen binding les described.

Parenteral compositions include those designed for administration by injection, e.g. aneous, intradermal, intralesional, intravenous, intraarterial intramuscular, intrathecal or intraperitoneal injection. For injection, the T cell activating bispecific antigen binding molecules described may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, 's solution, or physiological saline buffer. The solution may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. atively, the T cell ting bispecific antigen binding molecules may be in powder form for tution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. Sterile injectable ons are prepared by orating the T cell activating bispecific antigen binding molecules described in the required amount in the appropriate solvent with various of the other ingredients enumerated below, as required. Sterility may be readily accomplished, e.g., by filtration h sterile filtration membranes. Generally, sions are ed 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 s for the preparation of sterile injectable solutions, suspensions or emulsion, the preferred methods of ation are -drying or freeze-drying techniques which yield a powder of the active ingredient plus any onal desired ient 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 manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that xin contamination should be kept minimally at a safe level, for example, less that 0.5 ng/mg protein.

Suitable pharmaceutically acceptable carriers include, but are not limited 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; benzethonium 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; ns, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates ing glucose, 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). Aqueous injection sions may contain compounds which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, dextran, or the like. ally, the suspension may also contain le stabilizers or agents which se the solubility of the compounds to allow for the preparation of highly concentrated solutions. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic ts or vehicles include fatty oils such as sesame oil, or tic fatty acid esters, such as ethyl cleats or triglycerides, or liposomes.

Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatinmicrocapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug ry systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (18th Ed. Mack Printing Company, 1990). Sustained-release ations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers ning the ptide, which matrices are in the form of shaped articles, e.g. films, or microcapsules. In particular embodiments, prolonged tion of an injectable composition can be brought about by the use in the compositions of agents delaying tion, 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 formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the T cell activating bispecific antigen binding molecules may be formulated with suitable polymeric or hydrophobic als (for example as an emulsion in an acceptable oil) or ion exchange resins, or as gly soluble derivatives, for e, as a sparingly soluble salt.

Pharmaceutical compositions comprising the T cell activating bispecific n binding molecules described may be manufactured by means of tional mixing, dissolving, emulsifying, encapsulating, entrapping or lizing processes. ceutical 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 able salts are salts that substantially retain the biological activity 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 inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric ides; 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. eutic Methods and Compositions Any of the T cell activating bispecific antigen binding molecules described 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 medical 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 administration, the scheduling of administration, and other factors known to medical tioners.

Described are T cell activating bispecific antigen g molecules bed for use as a medicament. Aso described are T cell activating bispecific antigen binding les described for use in treating a disease. In certain ments, T cell ting 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, bed is a T cell activating bispecific antigen g molecule for use in a method of treating an individual having a disease comprising administering to the individual a therapeutically effective amount of the T cell activating bispecific antigen binding molecule. In certain embodiments the disease to be treated is a proliferative disorder. In a particular embodiment the disease is .

In certain embodiments the method further comprises administering to the dual 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 le as described herein for use in inducing lysis of a target cell, particularly a tumor cell. In certain embodiments, bed is a T cell activating ific 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 binding molecule to induce lysis of a target cell. An idual" according to any of the above embodiments is a mammal, preferably a human.

Also bed is the use of a T cell activating bispecific antigen binding molecule described in the manufacture or preparation of a medicament. In one embodiment the medicament is for the treatment of a disease 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 disease a therapeutically effective amount of the medicament. In n embodiments the e to be treated is a proliferative disorder. In a particular embodiment the disease is cancer.

In one ment, the method further comprises administering to the dual a eutically 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 medicament is for ng 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 medicament to induce lysis of a target cell. An "individual" according to any of the above embodiments may be a mammal, ably a human.

Also described is a method for treating a e. In one embodiment, the method comprises administering to an individual having such disease a therapeutically effective amount of a T cell activating bispecific antigen g molecule described. In one embodiment a composition is administered to said invididual, comprising the T cell activating bispecific antigen binding molecule described in a pharmaceutically acceptable form. In certain embodiments the disease to be treated is a proliferative disorder. In a particular embodiment the disease is cancer. In certain embodiments 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. An idual" 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 g molecule described in the presence of a T cell, ularly 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 bed. In one such embodiment, the method comprises administering to the individual an ive amount of a T cell activating bispecific antigen 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 proliferative disorder, particularly cancer.

Non-limiting examples of cancers include bladder cancer, brain , head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, e cancer, cervical cancer, endometrial cancer, esophageal , colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, us cell carcinoma, bone cancer, and kidney cancer. Other cell proliferation disorders that can be treated using a T cell ting bispecific antigen binding le described include, but are not limited to neoplasms located in the: abdomen, bone, breast, ive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous system (central and eral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic region, and urogenital system. Also included are pre-cancerous conditions or lesions and cancer metastases. In certain embodiments the cancer is chosen from the group consisting of renal cell cancer, skin cancer, lung , 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 le may not provide a cure but may only provide partial benefit. In some embodiments, a logical change having some t is also ered eutically beneficial. Thus, in some embodiments, an amount of T cell activating bispecific n binding molecule that es a physiological change is considered an "effective amount" or a "therapeutically effective amount". The subject, patient, or individual in need of treatment is typically a mammal, more specifically a human.

In some embodiments, an effective amount of a T cell activating bispecific antigen binding le described is administered to a cell. In other embodiments, a therapeutically effective amount of a T cell activating bispecific antigen binding molecule described is stered 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 patient, the type of T cell activating bispecific antigen binding molecule, the severity and course of the disease, whether the T cell activating bispecific antigen binding molecule is administered for preventive or therapeutic purposes, previous or concurrent therapeutic interventions, the patient's clinical y and response to the T cell activating bispecific antigen binding molecule, and the discretion of the ing physician. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate ) for the individual subject. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, 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 ents. Depending on the type and severity of the e, 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, depending on the factors mentioned above. For repeated administrations over l days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs. One exemplary dosage of the T cell activating ific antigen g 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 weight, 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 weight, about 10 milligram/kg body , about 50 milligram/kg body , about 100 milligram/kg body weight, about 200 milligram/kg body weight, about 350 milligram/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 administered, based on the numbers described 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 f) 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 n binding molecule). An l higher loading dose, followed by one or more lower doses may be administered. However, other dosage regimens may be useful. The progress of this y is easily monitored by conventional techniques and .

The T cell ting bispecific antigen binding molecules described will generally be used in an amount effective to achieve the ed 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 therapeutically ive amount.

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

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

Such information can be used to more accurately determine useful doses in humans. l 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 ed individually to provide plasma levels of the T cell activating bispecific antigen binding molecules which are sufficient to maintain therapeutic . 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. Therapeutically effective plasma levels may be achieved by administering multiple doses each day. Levels in plasma may be measured, for e, by HPLC.

In cases of local administration or selective uptake, the effective local tration of the T cell activating bispecific antigen 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 mentation.

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

Toxicity and eutic cy of a T cell activating bispecific antigen binding molecule can be determined by standard pharmaceutical ures in cell e or experimental animals.

Cell culture assays and animal studies can be used to ine the LD50 (the dose lethal to 50% of a population) and the ED50 (the dose therapeutically ive 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 activating bispecific antigen binding molecules that exhibit large therapeutic indices are preferred. In one embodiment, the T cell activating bispecific antigen binding molecule described exhibits a high therapeutic index. The data ed from cell culture assays and animal studies can be used in ating 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 depending 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 ing physician for patients treated with T cell activating bispecific 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 treatment to higher levels if the clinical response were not adequate (precluding toxicity). The magnitude of an administered dose in the management of the er 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 s 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 stered in combination with one or more other agents in therapy. For instance, a T cell activating bispecific antigen binding le described may be co-administered with at least one additional therapeutic agent. The term "therapeutic agent" encompasses any agent administered to treat a symptom or disease in an individual in need of such treatment. Such onal eutic agent may comprise any active ingredients suitable for the particular tion being treated, preferably those with complementary activities that do not adversely affect each other. In n embodiments, an additional therapeutic agent is an immunomodulatory agent, a atic agent, an inhibitor of cell adhesion, a cytotoxic agent, an activator of cell sis, or an agent that increases the sensitivity of cells to apoptotic inducers. In a particular embodiment, the additional therapeutic agent is an ancer agent, for example a microtubule disruptor, an antimetabolite, a topoisomerase inhibitor, a DNA intercalator, an alkylating agent, a hormonal therapy, a kinase inhibitor, a receptor antagonist, an activator 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 depends on the amount of T cell activating ific antigen binding molecule used, the type of disorder or treatment, and other factors sed above. The T cell ting 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 combination therapies noted above encompass combined administration (where two or more therapeutic agents are ed in the same or separate compositions), and separate administration, in which case, administration of the T cell activating bispecific antigen binding molecule described can occur prior to, aneously, 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 y.

Articles of Manufacture 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, s, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition 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 hypodermic injection ). At least one active agent in the composition is a T cell activating bispecific antigen binding molecule described. The label or e insert indicates that the composition is used for treating the condition of choice. Moreover, the article of manufacture may comprise (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 further xic 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 r comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further e other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and es.

Examples The ing are examples of s and compositions described. It is tood that various other embodiments may be practiced, given the general description provided above.

General methods Recombinant DNA Techniques Standard s were used to manipulate DNA as described in ok et al., Molecular cloning: A laboratory ; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. The molecular biological reagents were used according to the manufacturers’ instructions. General ation regarding the nucleotide sequences of human immunoglobulins light and heavy chains is given in: Kabat, E.A. et al., (1991) Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication No. 91-3242.

DNA Sequencing DNA sequences were ined by double strand sequencing.

Gene Synthesis Desired gene segments where required were either generated by PCR using appropriate templates or were synthesized by Geneart AG (Regensburg, y) from tic oligonucleotides and PCR products 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 d by singular restriction endonuclease cleavage sites were cloned into standard cloning / sequencing 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 cing. Gene segments were ed with suitable restriction sites to allow sub-cloning into the respective expression vectors. All constructs were designed 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.

Isolation of primary human pan T cells from PBMCs Peripheral blood mononuclear cells (PBMCs) were prepared by Histopaque density centrifugation from enriched lymphocyte 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 (Sigma, . 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 e was centrifuged at room temperature for 10 minutes at 400 x g (brake switched on). The supernatant was ded 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 enrichment from PBMCs was performed using the Pan T Cell Isolation Kit II (Miltenyi Biotec #130156), according to the manufacturer’s instructions. y, 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 ted 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 t volume and a subsequent centrifugation step at 300 x g for 10 min. Up to 100 million 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 manufacturer’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 ) were prepared by Histopaque density centrifugation from enriched lymphocyte ations (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), according to the manufacturer’s instructions, but skipping the last isolation step of CD8+ T cells (also see description for the ion of primary human pan T cells).

Isolation of murine pan T cells from splenocytes s were ed from C57BL/6 mice, transferred into a MACS C-tube (Miltenyi Biotech #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 pre-separation filter to remove ing 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 remaining 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 automatically counted (ViCell) and immediately used for further assays.

Isolation of primary cynomolgus PBMCs from heparinized blood Peripheral blood mononuclar cells (PBMCs) were prepared by y centrifugation from fresh blood from healthy cynomolgus donors, as follows: Heparinized blood was diluted 1:3 with sterile PBS, and Lymphoprep medium (Axon Lab #1114545) was diluted to 90% with sterile PBS. Two volumes of the diluted blood were layered over one volume of the diluted density gradient and the PBMC fraction 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 e 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 tically counted l) and immediately used for further assays.

Target cells For the assessment of MCSP-targeting bispecific antigen binding molecules, the following tumor cell lines were used: the human melanoma cell line WM266-4 (ATCC #CRL-1676), 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 binding molecules, the following tumor cell lines were used: the human gastric cancer cell line MKN45 (DSMZ #ACC 409), sing 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 s 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 ation, purification and characterization of ific n 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 expression vector. The dy expression was driven by an MPSV promoter and a synthetic polyA signal ce 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 ected using the calcium phosphate method. Alternatively, HEK293 EBNA cells growing in suspension were transfected using polyethylenimine (PEI). For preparation of "1+1 IgG scFab, one armed / one armed ed" constructs, cells were transfected with the corresponding expression vectors in a 1:1:1 ratio ("vector heavy chain" : "vector light chain" : "vector heavy chain-scFab"). For preparation of "2+1 IgG scFab" constructs, cells were transfected with the corresponding expression vectors in a 1:2:1 ratio ("vector heavy chain" : r light chain" : "vector heavy chain-scFab"). For preparation 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 Crossfab" : "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 corresponding expression s 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 vectors in a 1:1:1:1 ratio or first heavy chain" : "vector second heavy chain" : "vector first light chain" : "vector second light chain"). For preparation of the "1+1 IgG Crossfab light chain fusion " uct, cells were transfected with the corresponding expression vectors in a 1:1:1:1 ratio or first heavy chain" : "vector second heavy chain" : "vector light chain Crossfab" : "vector second light chain").

For transfection using m phosphate cells were grown as adherent monolayer cultures in T- flasks using DMEM culture medium supplemented with 10 % (v/v) FCS, and ected when they were between 50 and 80 % confluent. For the transfection of a T150 flask, 15 n cells were seeded 24 hours before transfection in 25 ml DMEM culture medium mented 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 sion 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 medium. For the production in 500 ml shake flasks, 400 million HEK293 EBNA cells were seeded 24 hours before transfection. For transfection cells were centrifuged for 5 min at 210 x g, and supernatant was replaced by 20 ml rmed CD CHO medium. Expression s 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 ted for 10 min at room temperature. Afterwards cells were mixed with the DNA/PEI on, 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 tion 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 purified 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 phosphate, 20 mM sodium citrate, 0.5 M sodium chloride, pH 7.5. Unbound protein 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 e, 0.5 M sodium chloride 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. atively, 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 solution 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 ate, 125 mM sodium chloride, 100 mM glycine solution of pH 6.7. For the purification of 1+1 IgG Crossfab the column was equilibrated with 20 mM ine, 140 mM sodium chloride solution of pH 6.0.

The protein concentration of purified protein 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 ific constructs were ed by SDS-PAGE in the presence and absence of a reducing agent (5 mM 1,4-dithiotreitol) and staining with Coomassie (SimpleBlue™ SafeStain from Invitrogen) using the NuPAGE® Pre- Cast gel system (Invitrogen, USA) was used according to the manufacturer’s instructions (4-12% Tris-Acetate gels or 4-12% Bis-Tris). atively, purity and molecular weight of molecules were analyzed by CE-SDS analyses in the presence and absence of a reducing agent, using the Caliper LabChip 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 xclusion chromatography column (GE Healthcare) in 2 mM MOPS, 150 mM NaCl, 0.02% (w/v) NaN3, pH 7.3 running buffer at 25°C. Alternatively, the aggregate content of dy samples was ed 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 ical size exclusion chromatography and Table 2A shows the yields, ate content after Protein A, and final monomer content of the preparations of the different bispecific ucts.

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 chromatography 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 , aggregate content after Protein A and final monomer content 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 /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 exchange (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 Crossfab, 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 ge (M4-3 ML2/V9) (SEQ ID NOs 183, 189, 193, 195) 2+1 IgG Crossfab; CL/CH1 29.75 0 0 0 100 exchange (M4-3 ML2/H2C) (SEQ ID NOs 189, 193, 199, 201) 2+1 IgG ab; 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 inverted" (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 6/V9) (SEQ ID NOs 183, 203, 205, 207) 1+1 ossfab light chain fusion 7.85 27 4.3 3.2 92.5 (CH1A1A/V9) (SEQ ID NOs 183, 209, 211, 213) As ls, bispecific antigen binding molecules were ted 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 ed in HEK293-EBNA cells and purified by Protein A affinity chromatography followed by a size exclusion tographic step in an analogous manner as described above for the bispecific antigen binding molecules described. Due to high aggregate formation, some of the samples had to be r purified by applying eluted and concentrated samples from the HiLoad Superdex 200 column (GE Healthcare) to a Superdex 10/300 GL column (GE Healthcare) equilibrated with 20 mM histidine, 140 mM sodium chloride, pH 6.7 in order to obtain protein with high monomer content. Subsequently, protein concentration, purity and molecular weight, and aggregate content were determined as described above.

Yields, aggregate content after the first purification step, and final monomer 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 stability of the IgG Crossfab and IgG scFab constructs compared to the "(scFv)2-Fc" and the ide bridge-stabilized "(dsscFv)2-Fc" molecules.

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

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) (dsscFv)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 filtered 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 collected. The results are shown in Figure 15 and Table 2C. For the "(scFv)2-Fc" (antiMCSP/anti huCD3) le two aggregation points were observed, at 49°C and 68°C. The "(dsscFv)2-Fc" construct has an increased ation 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 thermal stability as compared to the "(scFv)2-Fc" and "(dsscFv)2-Fc" formats (Figure 15B, Table 2C).

TABLE 2C. Thermal stability determined by c light ring. uct Tagg [°C] 2+1 IgG scFab (LC007/V9) 68 2+1 IgG Crossfab (LC007/V9) 65 Fv)2 (LC007/V9) 49/68 Fc-(dsscFv)2 (LC007/V9) 57 Surface Plasmon resonance analysis of Fc receptor and target n binding Method All surface plasmon resonance (SPR) experiments are performed on a Biacore T100 at 25°C with HBS-EP as running 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 ing interaction of different Fc-variants with human FcγRIIIa-V158 and murine FcγRIV direct coupling of around 6,500 resonance units (RU) of the anti-Penta His antibody (Qiagen) is performed on a CM5 chip at pH 5.0 using the standard amine ng kit (Biacore, Freiburg/Germany). HuFcγRIIIa-V158-K6H6 and muFcγRIVbiotin are captured for 60 s at 4 and 10 nM respectively.

Constructs with different Fc-mutations are passed through 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 muFcγRIV-aviHis-biotin. Affinity for human Ia-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 near curve fitting of the Langmuir binding isotherm. Kinetic constants were derived using the Biacore T100 Evaluation Software (vAA, Biacore AB, Uppsala/Sweden), to fit rate ons for 1:1 Langmuir binding by numerical ation.

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

The Fc s with the lowest binding to the human Fcγ-receptor were P329G L234A L235A (LALA) and P329G LALA N297D. The LALA mutation alone was not enough to abrogate binding to huFcγRIIIa-V158-K6H6. The Fc t 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 concentration.

Affinity to human FcγRIIIa-V158 and murine FcγRIV could only be ed 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 .

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 ure. 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 uently 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 cting the se obtained on a reference flow cell where the recombinant CD3ε was flown over a surface with immobilized D3 domain of MCSP or EGFR without captured T- cell ific constructs.

Result Simultaneous g to both tumor antigen and human CD3ε was analyzed 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 ion of the construct alone reflecting that both tumor antigen and the human CD3ε were bound to the construct.

Example 3 Binding of bispecific constructs to the tive target antigen on cells Binding of the different bispecific constructs to CD3 on Jurkat cells (ATCC #TIB-152), and the respective tumor antigen on target cells, was determined by FACS. Briefly, cells were harvested, counted and checked for viability. 0.15 – 0.2 million cells per well (in PBS ning 0.1% BSA; 90 µl) were plated in a round-bottom 96-well plate and incubated with the indicated concentration 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 ed using a FACSCantoII (Software FACS Diva). The "(scFv)2" molecule was detected using a FITC- conjugated anti-His antibody (Lucerna, #RHIS-45F-Z). For all other molecules, a FITC- or PE- conjugated AffiniPure F(ab’)2 Fragment goat anti-human IgG Fcγ Fragment Specific (Jackson Immuno Research Lab # 109098 / working 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 on buffer (BD #554655) for 15 min at 4°C in the dark, centrifuged for 6 min at 400 x g and kept in 200 l PBS containing 0.1% BSA until the samples were ed with FACS CantoII. EC50 values were calculated using the GraphPad Prism software.

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 mia 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, control IgG, the secondary antibody only, or left untreated.

As shown in Figure 19, for both antigen binding moieties of the "(scFv)2" molecule, i.e. CD3 (Figure 191A) and MCSP e 19B), a clear binding signal is observed ed 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 signals to human CD3 on cells. The reference anti-human CD3 IgG gives a slightly weaker . In addition, both constructs tested ("1+1 IgG scFab, one-armed" (SEQ ID NOs 1, 3, 5) and "1+1 IgG scFab, one-armed ed" (SEQ ID NOs 7, 9, 11)) show able binding to human MCSP on cells e 21B). The binding signal ed with the reference anti-human MCSP IgG is slightly weaker.

In another experiment, the purified "2+1 IgG scFab" ific 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 . 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, inverted" 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 nt 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 ng 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 constructs show good g to human CEA, as well as to human CD3 on cells. The calculated EC50 values were 4.6 and 3.9 nM (CD3), and 9.3 and 6.7 nM (CEA) for the "2+1 IgG Crossfab, ed (VL/VH)" and the "2+1 IgG Crossfab, inverted (CL/CH1)" constructs, respectively.

In another experiment, the binding of CD3/MCSP "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) 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 ed 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 experiment, 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 determined. As a l, the equivalent maximum concentration of the corresponding anti-CD3 and anti-CEA IgGs and the background ng 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 depicted in Figure 57, the binding of the "1+1 IgG Crossfab LC fusion" to CEA s to be greatly reduced, whereas the binding to CD3 was at least comparable 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 Crossfab, 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 ed in Figure 58 the binding to human CD3 was d for the "2+1 IgG Crossfab, inverted" ed 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" construct, respectively.

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

Briefly, MCSP-positive Colo-38 cells were ted 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 round-bottom 96-well plate (as indicated). 50 µl of the (diluted) bispecific construct were added to the ontaining 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 ific 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 , or 24 h (CD25) at 37°C, 5% CO2, cells were fuged (5 min, 350 x g) and washed twice with 150 µl/well PBS containing 0.1% BSA. Surface staining for CD8 (mouse IgG1,κ; clone HIT8a; BD #555635), CD69 (mouse IgG1; clone L78; BD #340560) and CD25 (mouse IgG1,ĸ; clone M-A251; BD #555434) was performed at 4°C for 30 min, according to the supplier’s tions. 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 e (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" uct.

The purified huMCSP-huCD3-targeting bispecific "2+1 IgG scFab" and "(scFv)2" molecules were further tested by flow cytometry for their potential to up-regulate the late activation 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 tion 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 )2" molecule. In general, the up-regulation of CD25 is more pronounced 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 endend 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 construct and the different IgG controls were ed to the same ty. 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 #557138) was performed 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 samples were resuspended in 200 µl/well PBS with 0.1% BSA and analyzed using a FACS CantoII e are FACS Diva).

As depicted in Figure 25, the bispecific construct induces concentration-dependent up-regulation of CD25 on CD8+ T cells only in the ce 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 ment, 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 potential 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 ted trations 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 MCSP-positive target cells (or medium). The final effector to target cell (E:T) ratio was 10:1.

The cells were incubated with the indicated concentration of the bispecific constructs and controls for the indicated incubation times at 37°C, 5% CO2. The effector 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 ed 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 another experiment, the CD3/MCSP "2+1 IgG Crossfab" (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 ab" constructs induced more pronounced upregulation of activation markers than the "1+1 CrossMab" le.

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 samples with antibody but no targets (not shown). e 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 γ into the supernatant. As ls, anti-human MCSP and anti-human CD3 IgGs were used, adjusted to the same molarity.

Briefly, -expressing U-87MG glioblastoma astrocytoma target cells (ECACC 89081402) were ted 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 tration of 1 nM.

Human pan T or 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 96-well 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 depicted in Figure 26, the reference IgGs show no to weak induction of IFN-γ ion, whereas the "2+1 IgG scFab" construct is able to te 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 potential to induce T cell-mediated apoptosis in tumor target cells upon crosslinkage of the construct via binding of the antigen binding es to their respective target antigens on cells (Figures .

In one experiment ed "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 concentration. All ucts and corresponding control 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 ; mixture of isolectins isolated from Phaseolus is) was used. For normalization, l 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 antibody. After an overnight incubation of 20 h at 37°C, 5% CO2, LDH release of apoptotic/necrotic target cells into the atant 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 ing in their Fc , as well as the )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 induced by constructs containing a wild-type (wt) Fc domain. Experimental procedures were as described above.

Figure 28 shows that all constructs induce apoptosis in target cells comparable 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. mental 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 ty 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 figure, the construct induces apoptosis in target cells comparably to the "(scFv)2" molecule.

The "2+1 IgG scFab" construct shows reduced cy at the highest trations.

Furthermore, different bispecific constructs that are monovalent for both s, 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 ma target cells at an E:T ratio of 5:1, and an tion time of 19 h. As depicted in the figure, both "1+1" constructs 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 8 human melanoma target cells at an E:T ratio of 5:1, and an incubation time of 20 h. As depicted in the figure, the "1+1 IgG scFab" construct is less xic than the "(scFv)2" molecule.

In a r experiment the purified "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 ediated apoptosis in tumor target cells upon crosslinkage of the construct via g 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 comparably to the "(scFv)2" molecule. The ison of the mono- and bivalent "IgG Crossfab" formats y shows that the bivalent one is much more potent.

In yet another experiment, the purified "2+1 IgG ab" (SEQ ID NOs 3, 5, 29, 33) construct was analyzed for its potential to induce T ediated sis in different ) target cells. Briefly, MCSP-positive Colo-38 tumor target cells, hymal stem cells (derived from bone marrow, Lonza #PT-2501 or adipose tissue, Invitrogen #R7788-115) or tes (from placenta; PromoCell #C-12980), as indicated, 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 l plate and the respective antibody dilution was added at the indicated 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 Applied 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 Colo-38 cells expressing significant levels of MCSP, s mesenchymal stem cells and pericytes express MCSP only very weakly.

The purified "2+1 IgG scFab" (SEQ ID NOs 5, 17, 19) construct and the "(scFv)2" le were also compared to a glycoengineered anti-human MCSP IgG antibody, having a reduced tion 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 (Figure 36B). The different molecules were used at the concentrations indicated 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 ab.

In another 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 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 dilution of construct or reference IgG was added at the concentrations indicated. The ific construct and the ent IgG controls 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), ing to the manufacturer’s instructions.

As depicted in Figure 37, the bispecific construct induces concentration-dependent LDH release from target cells. The effect is stronger after 43 h than after 24 h. The anti-cynoCD3 IgG (clone FN-18) is also able to induce LDH release of target cells t being crosslinked.

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 g of the antigen binding moieties to their respective target antigens on cells (Figures .

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 "(scFv)2" le, were compared. Briefly, human EGFR-expressing LS-174T tumor target cells were harvested with trypsin, washed and resuspendend in AIM-V medium rogen # 12055-091). 000 cells per well were plated in a round-bottom 96-well-plate and the respective antibody dilution was added at the indicated concentrations. All constructs and controls were ed 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 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 t 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" molecule, whereas the "1+1 IgG scFab" uct is less active.

In another 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 tion time which was 21 h.

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

In yet a further experiment the ed "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 compared. The incubation time in this experiment was 16 h, and the result is depicted in Figure 41. Incubated with human pan T cells, both "1+1 IgG scFab, one-armed (inverted)" constructs 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 further experiment, purified "1+1 IgG scFab, one-armed ed" (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 ial to induce T cell-mediated apoptosis in human FAP-expressing fibroblasts GM05389 cells upon crosslinkage of the construct via binding of both ing moieties to their respective target ns 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 l 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 atant was discarded and fresh medium, as well as the respective on of the constructs or reference IgGs was added at the indicated concentrations. All constructs and controls were adjusted to the same ty. 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, l 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 antibody. After an additional 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 42, the "2+1 IgG scFab" construct shows able cytotoxic activity to the )2" molecule in terms of EC50 values. The "1+1 IgG scFab, one-armed inverted" uct is less active than the other constructs tested in this assay.

In another set of experiments, the CD3/MCSP "2+1 IgG ab, 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 harvested 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 appropriate 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 dy dilution was added as indicated (triplicates). PBMCs as effector cells were added to obtain a final effector-to-target cell (E:T) ratio of 10:1. All constructs and controls were adjusted to the same molarity, tion 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 e 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 uct with the linked light chain was more potent compared to the one without the linked light chain in the assays with high MCSP expressing Colo-38 cells as target cells. Finally, as shown in Figure 52, there was no significant difference between the two constructs when high xpressing 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 ged. 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 96-well plate and the bispecific 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 uct with the CL/CH1 exchange shows ly better activity on both target cell lines than the construct 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 uct, 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 iation Buffer (LuBiosciences #13151014), washed and resuspendend in RPMI1640 (Invitrogen #42404042), ing 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 indicated concentrations. All constructs and controls were adjusted to the same molarity. Human PBMC or cells were added to obtain a final E:T ratio of 10:1, incubation time was 26 h. EC50 values were calculated using the GraphPad Prism 5 software.

As depicted in Figure 62, the two constructs show comparable ty, 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, compared to approximately 82.2 pM for the VL/VH-exchange uct). 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 Crossfab" (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 CD3/MCSP "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" uct is the most potent molecule in this assay, followed by the "1+1 IgG ab" and the "1+1 CrossMab". This ranking is even more pronounced with MV-3 cells, expressing medium levels of MCSP, compared to high MCSP expressing 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", tively.

In a further experiment, different 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 tion 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 different tumor antigen sion 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. uCEA 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" uct induced apoptosis of target cells in a concentration-dependent manner, with a calculated EC50 value of approximately 3.2 nM. In contrast, the untargeted "2+1 IgG Crossfab" showed antigen-independent T cell-mediated killing 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 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" constructs seems to be slightly stronger 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 s: EC50 [pM] 2+1 IgG Crossfab 2+1 IgG Crossfab 2+1 IgG Crossfab 2+1 IgG Crossfab, (V9) inverted (V9) (anti-CD3) ed (anti-CD3) MV-3 10.0 4.1 11.0 3.0 4 12.4 3.7 11.3 7.1 Example 7 CD107a/b assay 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 tested by flow cytometry for their potential to late CD107a and intracellular perforin levels in the presence or absence of human MCSP- expressing tumor cells.

Briefly, on day one, 30 000 Colo-38 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 bispecific constructs and ls are added. The ent bispecific molecules and antibodies 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 med at 4°C for 30 min. After a final washing step with 1 x BD Perm/Wash buffer, cells were resuspended in PBS / 0.1% BSA and analyzed on FACS CantoII (all dies were purchased from BD Biosciences or BioLegend).

Gates were set either on all CD107a/b positive, in-positive or double-positive cells, as indicated e 43). The "2+1 IgG scFab" construct was able to activate T cells and upregulate CD107a/b and intracellular perforin levels only in the presence of target cells (Figure 43A), whereas the "(scFv)2" molecule shows (weak) induction of activation of T cells also in the absence of target cells e 43B). The bivalent reference D3 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 n 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 medium, containing 10% FCS and 1% ax. After incubation at room temperature for further 20 min, the cells were washed three times with prewarmed medium to remove remaining CFSE. MCSP-positive 8 cells were harvested with Cell Dissociation buffer, counted and checked for ity. 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 96-well 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 (viable) cells per ml in AIM-V medium. 50 µl of this cell sion was added per well of the assay plate (see above) to obtain a final E:T ratio of 5:1. To analyze whether the bispecific 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 tive 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, ing 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, according 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 e (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 eration of CD8+ T cells (A) or CD4+ T cells (B) only in the presence of target cells, comparably to the "(scFv)2" molecule. In general, activated CD8+ T cells proliferate more than activated CD4+ T cells in this assay.

Example 9 Cytokine e 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 e of tumor target cells.

Briefly, human PBMCs were isolated from Buffy Coats and 0.3 n cells were plated per well into a round-bottom 96-well plate. Colo-38 tumor target cells, expressing human MCSP, were added to obtain a final E:T-ratio of 10:1. ific constructs and IgG controls were added at 1 nM final tration 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 erred into a new deep-well 96-well-plate for the subsequent analysis. 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 cytokine ed 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 cy 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 activation 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 ed, which was highest in samples treated with the "(scFv)2" molecule.

In a second experiment, the following purified ific 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" molecules comprising either a wild- type or a d (LALA, P329G and/or N297D, as indicated) Fc domain. y, 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 atant was transferred into a new deep-well 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 4), human IL-6 Flex Set (BD #558276), human IL-4 Flex Set (BD #558272), human IL-2 Flex Set (BD 0).

Figure 46 shows the levels of the different cytokine measured in the supernatant. The main cytokine secreted in the presence of Colo-38 tumor cells was IL-6, followed 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 induced higher levels of cytokine secretion in the presence of target cells (Figure 46, A and B). There was no icant secretion of Th2 cytokines (IL-10 and IL-4) upon activation of T cells in the presence (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 ences could be ed 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 sures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference.

Claims

1. 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 CD3 and the other 5 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 crossover Fab molecule wherein either the variable or the nt 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 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 15 binding moiety and the second antigen binding moiety is fused at the inus 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 le comprises not more than one antigen binding moiety e of specific binding to CD3.

2. The T cell activating bispecific antigen binding molecule of claim 1, wherein the Fab light 20 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 peptide linker.

3. The T cell ting bispecific antigen g 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 ific 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 inus of the first or second subunit of the Fc domain.

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

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 ts 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 g molecule of claim 5, wherein the second and the third antigen binding moiety and the Fc domain are part of an immunoglobulin molecule, 10 particularly an IgG class immunoglobulin.

8. The T cell activating bispecific antigen binding molecule of any one of the ing , 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 preceding 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 preceding claims, wherein the Fc domain comprises a modification promoting the association of the first and the second subunit of the Fc , 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 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 onable; or 25 (b) at the interface of the two Fc domain subunits one or more amino acid residues is/are ed by charged amino acid residues so that homodimer formation becomes electrostatically unfavorable but heterodimerization electrostatically favorable.

11. The T cell ting 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 selected from the group of E233, L234, L235, N297, P331 and P329 (EU numbering).

12. The T cell activating bispecific antigen binding 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 numbering).

13. The T cell activating bispecific n g molecule of any one of the preceding claims, wherein each t 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 numbering). 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 or.

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

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

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

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

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

20. A host cell comprising the ed 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 bispecific antigen binding molecule of any one 25 of claims 1 to 16, comprising the steps of a) culturing the host cell of claim 20 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 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 acceptable carrier.

23. Use of the T cell activating bispecific antigen binding molecule of any one of claims 1 to 16 for the manufacture of a ment 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 molecule as claimed in any one of claims 1 to 16, substantially as herein bed with reference to any example thereof.

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

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

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

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

31. A pharmaceutical composition as claimed in claim 22, substantially 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 e thereof.

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