US20160208019A1 - T cell activating bispecific antigen binding molecules - Google Patents

T cell activating bispecific antigen binding molecules Download PDF

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US20160208019A1
US20160208019A1 US14/947,657 US201514947657A US2016208019A1 US 20160208019 A1 US20160208019 A1 US 20160208019A1 US 201514947657 A US201514947657 A US 201514947657A US 2016208019 A1 US2016208019 A1 US 2016208019A1
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antigen binding
activating bispecific
cell
bispecific antigen
seq
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Marina Bacac
Peter Bruenker
Anne Freimoser-Grundschober
Ralf Hosse
Christian Klein
Ekkehard Moessner
Pablo Umana
Tina Weinzierl
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Hoffmann La Roche Inc
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Hoffmann La Roche Inc
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Assigned to ROCHE GLYCART AG reassignment ROCHE GLYCART AG ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: UMANA, PABLO, FREIMOSER-GRUNDSCHOBER, ANNE, HOSSE, RALF, KLEIN, CHRISTIAN, MOESSNER, EKKEHARD, WEINZIERL, Tina, BACAC, MARINA, BRUENKER, PETER
Assigned to F. HOFFMANN-LA ROCHE AG reassignment F. HOFFMANN-LA ROCHE AG ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: ROCHE GLYCART AG
Assigned to HOFFMANN-LA ROCHE INC. reassignment HOFFMANN-LA ROCHE INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: F. HOFFMANN-LA ROCHE AG
Publication of US20160208019A1 publication Critical patent/US20160208019A1/en
Priority to US16/016,209 priority Critical patent/US20190031784A1/en
Priority to US17/580,248 priority patent/US20220220224A1/en
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    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
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    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
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    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present invention generally relates to bispecific antigen binding molecules for activating T cells, in particular to bispecific antibodies targeting CD3 and Folate Receptor 1 (FolR1).
  • the present invention relates to polynucleotides encoding such bispecific 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.
  • the selective destruction of an individual cell or a specific 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.
  • 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.
  • bispecific antibodies designed to bind with one “arm” to a surface antigen on target cells, and with the second “arm” to an activating, invariant component of the T cell receptor (TCR) complex, have become of interest in recent years.
  • TCR T cell receptor
  • 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.
  • the immune response is re-directed to the target cells and is independent of peptide antigen presentation by the target cell or the specificity of the T cell as would be relevant for normal MHC-restricted activation of CTLs.
  • CTLs are only activated when a target cell is presenting the bispecific antibody to them, i.e. the immunological synapse is mimicked.
  • bispecific antibodies that do not require lymphocyte preconditioning or co-stimulation in order to elicit efficient lysis of target cells.
  • FOLR1 is expressed on epithelial tumor cells of various origins, e.g., ovarian cancer, lung cancer, breast cancer, renal cancer, colorectal cancer, endometrial cancer.
  • ovarian cancer ovarian cancer
  • lung cancer breast cancer
  • renal cancer colorectal cancer
  • endometrial cancer endometrial cancer.
  • therapeutic antibodies such as farletuzumab, antibody drug conjugates, or adoptive T cell therapy for imaging of tumors
  • the present invention provides bispecific antigen binding molecules designed for targeted T cell activation, particularly, bispecific antigen binding molecules suitable as effective and safe therapeutics that can be readily produced and dosed.
  • the invention provides a T cell activating bispecific antigen binding molecule comprising
  • the T cell activating bispecific antigen binding molecule comprises a first antigen binding moiety that comprises a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 36 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 31.
  • the T cell activating bispecific antigen binding molecule additionally comprises (iii) a third antigen binding moiety capable of specific binding to FolR1.
  • the second and third antigen binding moiety capable of specific binding to FolR1 comprise identical heavy chain complementarity determining region (CDR) and light chain CDR sequences.
  • the third antigen binding moiety is identical to the second antigen binding moiety.
  • the T cell activating bispecific antigen binding molecule of the above embodiments at least one of the second and third antigen binding moiety is a Fab molecule.
  • the T cell activating bispecific antigen binding molecule of the above embodiments additionally comprises an Fc domain composed of a first and a second subunit capable of stable association.
  • the first antigen binding moiety and the second antigen binding moiety are each connected at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain.
  • a third 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, optionally via a peptide linker.
  • the antigen binding moiety capable of specific binding to Folate Receptor 1 comprises at least one heavy chain complementarity determining region (CDR) amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 and at least one light chain CDR selected from the group of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34.
  • the antigen binding moiety capable of specific binding to Folate Receptor 1 (FolR1) comprises a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 15 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 31.
  • the antigen binding moiety capable of specific binding to Folate Receptor 1 comprises at least one heavy chain complementarity determining region (CDR) amino acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 56 and SEQ ID NO: 57 and at least one light chain CDR selected from the group of SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 65.
  • the antigen binding moiety capable of specific binding to Folate Receptor 1 (FolR1) comprises a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 55 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 64.
  • the antigen binding moiety capable of specific binding to Folate Receptor 1 comprises at least one heavy chain complementarity determining region (CDR) amino acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 50 and at least one light chain CDR selected from the group of SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54.
  • CDR heavy chain complementarity determining region
  • the antigen binding moiety capable of specific binding to FolR1 comprises (a) a complementarity determining region heavy chain 1 (CDR-H1) amino acid sequences of SEQ ID NO: 8; (b) a CDR-H2 amino acid sequence of SEQ ID NO: 9; (c) a CDR-H3 amino acid sequence of SEQ ID NO: 50; (d) a complementarity determining region light chain 1 (CDR-L1) amino acid sequence of SEQ ID NO: 52; (e) a CDR-L2 amino acid sequence of SEQ ID NO: 53, and (f) a CDR-L3 amino acid sequence of SEQ ID NO: 54.
  • the antigen binding moiety capable of specific binding to FolR1 comprises a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 49 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 51.
  • the antigen binding moiety capable of specific binding to Folate Receptor 1 comprises at least one heavy chain complementarity determining region (CDR) amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 275 and SEQ ID NO: 315 and at least one light chain CDR selected from the group of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34.
  • CDR heavy chain complementarity determining region
  • the antigen binding moiety capable of specific binding to FolR1 comprises (a) a complementarity determining region heavy chain 1 (CDR-H1) amino acid sequences of SEQ ID NO: 16; (b) a CDR-H2 amino acid sequence of SEQ ID NO: 275; (c) a CDR-H3 amino acid sequence of SEQ ID NO: 315; (d) a complementarity determining region light chain 1 (CDR-L1) amino acid sequence of SEQ ID NO: 32; (e) a CDR-L2 amino acid sequence of SEQ ID NO: 33, and (f) a CDR-L3 amino acid sequence of SEQ ID NO: 34.
  • the antigen binding moiety capable of specific binding to FolR1 comprises a variable heavy chain domain (VH) comprising an amino acid sequence of SEQ ID NO: 274 and a variable light chain domain (VL) comprising an amino acid sequence of SEQ ID NO: 31.
  • VH variable heavy chain domain
  • VL variable light chain domain
  • the T cell activating bispecific antigen binding molecule of the above embodiments binds to a human FolR1. In one embodiment, the T cell activating bispecific antigen binding molecule of the above embodiments binds to a human FolR1 and a cynomolgus monkey FolR1. In one embodiment, the T cell activating bispecific antigen binding molecule of the above embodiments binds to a human FolR1, a cynomolgus monkey FolR1 and a murine FolR1. In one embodiment, the T cell activating bispecific antigen binding molecule of the above embodiments binds to a human FolR1, a cynomolgus monkey FolR1 and not a murine FolR1.
  • 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.
  • the T cell activating bispecific antigen binding molecule of any of the above embodiments comprises not more than one antigen binding moiety capable of specific binding to CD3.
  • the first and the second antigen binding moiety and the Fc domain are part of an immunoglobulin molecule.
  • the Fc domain is an IgG class immunoglobulin, specifically an IgG 1 or IgG 4 , Fc domain.
  • the Fc domain is a human Fc domain.
  • the Fc domain comprises a modification promoting the association of the first and the second subunit of the Fc domain.
  • an amino acid residue 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 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 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 Fc domain comprises at least one amino acid substitution that reduces binding to an Fc receptor and/or effector function, as compared to a native IgG 1 Fc domain.
  • each subunit of the Fc domain comprises three amino acid substitutions that reduce binding to an activating Fc receptor and/or effector function wherein said amino acid substitutions are L234A, L235A and P329G (Kabat numbering).
  • the Fc receptor is an Fc ⁇ receptor.
  • the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC).
  • the T cell activating bispecific antigen binding molecule induces proliferation of a human CD3 positive T cell in vitro.
  • the T cell activating bispecific antigen binding molecule induces human peripheral blood mononuclear cell mediated killing of a FolR1-expressing human tumor cell in vitro.
  • the T cell activating bispecific antigen binding molecule induces T cell mediated killing of a FolR1-expressing human tumor cell in vitro.
  • the T cell is a CD8 positive T cell.
  • the FolR1-expressing human tumor cell is a Hela, Skov-3, HT-29, or HRCEpiC cell.
  • the T cell activating bispecific antigen binding molecule induces T cell mediated killing of the FolR1-expressing human tumor cell in vitro with an EC50 of between about 36 pM and about 39573 pM after 24 hours. In one embodiment, the T cell activating bispecific antigen binding molecule induces T cell mediated killing of the FolR1-expressing tumor cell in vitro with an EC50 of about 36 pM after 24 hours. In one embodiment, the T cell activating bispecific antigen binding molecule induces T cell mediated killing of the FolR1-expressing tumor cell in vitro with an EC50 of about 178.4 pM after 24 hours. In one embodiment, the T cell activating bispecific antigen binding molecule induces T cell mediated killing of the FolR1-expressing tumor cell in vitro with an EC50 of about 134.5 pM or greater after 48 hours.
  • the T cell activating bispecific antigen binding molecule of any of the above embodiments induces upregulation of cell surface expression of at least one of CD25 and CD69 on the T cell as measured by flow cytometry.
  • the T cell is a CD4 positive T cell or a CD8 positive T cell.
  • the T cell activating bispecific antigen binding molecule of any of the above embodiments binds human FolR1 with an apparent K D of about 5.36 pM to about 4 nM.
  • the T cell activating bispecific antigen binding molecule binds human and cynomolgus FolR1 with an apparent K D of about 4 nM.
  • the T cell activating bispecific antigen binding molecule binds murine FolR1 with an apparent K D of about 1.5 nM. In one embodiment, the T cell activating bispecific antigen binding molecule binds human FolR1 with a monovalent binding K D of at least about 1000 nM.
  • the T cell activating bispecific antigen binding molecule of any of the above embodiments is specific for FolR1 and does not bind to FolR2 or FolR3. In one embodiment, the T cell activating bispecific antigen binding molecule of any of the above embodiments has an affinity (monovalent binding) of 1 ⁇ M or greater. In one embodiment, the affinity is around 1.4 ⁇ M for human FolR1. In one embodiment, the T cell activating bispecific antigen binding molecule of any of the above embodiments has an avidity (bivalent binding) of about 1-100 nM or lower. In one embodiment, the avidity is about 10 nM or less. In one embodiment, the avidity is 10 nM.
  • the T cell activating bispecific antigen binding molecule of any of the above embodiments binds to FolR1 expressed on a human tumor cell. In one embodiment, the T cell activating bispecific antigen binding molecule of any of the above embodiments binds to a conformational epitope on human FolR1. In one embodiment, the T cell activating bispecific antigen binding molecule of any of the above embodiments does not bind to human Folate Receptor 2 (FolR2) or to human Folate Receptor 3 (FolR3).
  • the antigen binding moiety binds to a FolR1 polypeptide comprising the amino acids 25 to 234 of human FolR1 (SEQ ID NO:227).
  • the FolR1 antigen binding moiety binds to a FolR1 polypeptide comprising the amino acid sequence of SEQ ID NOs:227, 230 and 231, and wherein the FolR1 antigen binding moiety does not bind to a FolR polypeptide comprising the amino acid sequence of SEQ ID NOs:228 and 229.
  • the invention provides for a bispecific antibody comprising a) a first antigen-binding site that competes for binding to human FolR1 with a reference antibody comprising a variable heavy chain domain (VH) of SEQ ID NO: 49 and a variable light chain domain of SEQ ID NO: 51; and b) a second antigen-binding site that competes for binding to human CD3 with a reference antibody comprising a variable heavy chain domain (VH) of SEQ ID NO: 36 and a variable light chain domain of SEQ ID NO: 31, wherein binding competition is measured using a surface plasmon resonance assay.
  • the invention provides for a bispecific antibody comprising a) a first antigen-binding site that competes for binding to human FolR1 with a reference antibody comprising a variable heavy chain domain (VH) of SEQ ID NO: 274 and a variable light chain domain of SEQ ID NO: 31; and b) a second antigen-binding site that competes for binding to human CD3 with a reference antibody comprising a variable heavy chain domain (VH) of SEQ ID NO: 36 and a variable light chain domain of SEQ ID NO: 31, wherein binding competition is measured using a surface plasmon resonance assay.
  • the invention provides for a T cell activating bispecific antigen binding molecule comprising a first antigen binding moiety capable of specific binding to CD3, and a second antigen binding moiety capable of specific binding to Folate Receptor 1 (FolR1), wherein the T cell activating bispecific antigen binding molecule binds to the same epitope on human FolR1 as a first reference antibody comprising a variable heavy chain domain (VH) of SEQ ID NO: 49 and a variable light chain domain of SEQ ID NO: 51; and wherein the T cell activating bispecific antigen binding molecule binds to the same epitope on human CD3 as a second reference antibody comprising a variable heavy chain domain (VH) of SEQ ID NO: 36 and a variable light chain domain of SEQ ID NO: 31.
  • VH variable heavy chain domain
  • the invention provides for a T cell activating bispecific antigen binding molecule comprising a first antigen binding moiety capable of specific binding to CD3, and a second antigen binding moiety capable of specific binding to Folate Receptor 1 (FolR1), wherein the T cell activating bispecific antigen binding molecule binds to the same epitope on human FolR1 as a first reference antibody comprising a variable heavy chain domain (VH) of SEQ ID NO: 274 and a variable light chain domain (VL) of SEQ ID NO: 31; and wherein the T cell activating bispecific antigen binding molecule binds to the same epitope on human CD3 as a second reference antibody comprising a variable heavy chain domain (VH) of SEQ ID NO: 36 and a variable light chain domain (VL) of SEQ ID NO: 31.
  • VH variable heavy chain domain
  • VL variable light chain domain
  • the invention relates to an antibody or an antigen-binding fragment thereof that competes for binding to human FolR1 with an antibody that comprises a variable heavy chain domain (VH) of SEQ ID NO: 274 and a variable light chain domain of SEQ ID NO: 31, wherein binding competition is measured using a surface plasmon resonance assay.
  • VH variable heavy chain domain
  • the invention provides for a T cell activating bispecific antigen binding molecule, wherein the antigen binding molecule comprises a first, second, third, fourth and fifth polypeptide chain that form a first, a second and a third antigen binding moiety, wherein the first antigen binding moiety is capable of binding CD3 and the second and the third antigen binding moiety each are capable of binding Folate Receptor 1 (FolR1), wherein a) the first and the second polypeptide chain comprise, in amino (N)-terminal to carboxyl (C)-terminal direction, VLD1 and CLD1; b) the third polypeptide chain comprises, in N-terminal to C-terminal direction, VLD2 and CH1D2; c) the fourth polypeptide chain comprises, in N-terminal to C-terminal direction, VHD1, CH1D1, CH2D1 and CH3D1; d) the fifth polypeptide chain comprises VHD1, CH1D1, VHD2, CLD2, CH2D2 and CH3D1;
  • the third polypeptide chain and VHD2 and CLD2 of the fifth polypeptide chain form the first antigen binding moiety capable of binding CD3;
  • the first polypeptide chain and VHD1 and CH1D1 of the fourth polypeptide chain form the second binding moiety capable of binding to FolR1;
  • the second polypeptide chain and VHD1 and CH1D1 of the fifth polypeptide chain form the third binding moiety capable of binding to FolR1.
  • CH2D1, CH3D1, CH2D2 and CH3D2 form an Fc domain of an IgG class immunoglobulin.
  • the Fc domain is a human Fc domain.
  • the Fc domain comprises a modification promoting the association of the first and the second subunit of the Fc domain.
  • CH3D2 comprises an amino acid residue having a larger side chain volume, which is positionable in a cavity within CH3D1.
  • the Fc domain comprises at least one amino acid substitution that reduces binding to an Fc receptor and/or effector function, as compared to a native IgG 1 Fc domain.
  • each subunit of the Fc domain comprises three amino acid substitutions that reduce at least one of binding to an activating Fc receptor and effector function wherein said amino acid substitutions are L234A, L235A and P329G according to Kabat numbering.
  • the Fc receptor is an Fc ⁇ receptor.
  • the T cell activating bispecific antigen binding molecule induces proliferation of a human CD3 positive T cell in vitro. In one of the above embodiments, the T cell activating bispecific antigen binding molecule induces human peripheral blood mononuclear cell mediated killing of a FolR1-expressing human tumor cell in vitro. In one of the above embodiments, the T cell activating bispecific antigen binding molecule induces T cell mediated killing of a FolR1-expressing human tumor cell in vitro. In one such embodiment, the FolR1-expressing human tumor cell is a Hela, Skov-3, HT-29, or HRCEpiC cell.
  • the T cell activating bispecific antigen binding molecule induces T cell mediated killing of the FolR1-expressing tumor cell in vitro with an EC50 of between about 36 pM and about 39573 pM after 24 hours. In one of the above embodiments, the T cell activating bispecific antigen binding molecule induces T cell mediated killing of the FolR1-expressing tumor cell in vitro with an EC50 of about 36 pM after 24 hours. In one of the above embodiments, the T cell activating bispecific antigen binding molecule induces T cell mediated killing of the FolR1-expressing tumor cell in vitro with an EC50 of about 178.4 pM after 24 hours.
  • the T cell activating bispecific antigen binding molecule induces T cell mediated killing of the FolR1-expressing tumor cell in vitro with an EC50 of about 134.5 pM or greater after 48 hours. In one of the above embodiments, the T cell activating bispecific antigen binding molecule induces upregulation of cell surface expression of at least one of CD25 and CD69 on the T cell as measured by flow cytometry. In one such embodiments, the T cell is a CD4 positive T cell or a CD8 positive T cell. In one of the above embodiments, wherein the T cell activating bispecific antigen binding molecule binds human FolR1 with an apparent K D of about 5.36 pM to about 4 nM.
  • the T cell activating bispecific antigen binding molecule binds human and cynomolgus FolR1 with an apparent K D of about 4 nM. In one of the above embodiments, the T cell activating bispecific antigen binding molecule binds murine FolR1 with an apparent K D of about 1.5 nM. In one of the above embodiments, the T cell activating bispecific antigen binding molecule binds human FolR1 with a monovalent binding K D of at least about 1000 nM. In one of the above embodiments, the T cell activating bispecific antigen binding molecule binds to FolR1 expressed on a human tumor cell.
  • the T cell activating bispecific antigen binding molecule binds to a conformational epitope on human FolR1. In one of the above embodiments, the T cell activating bispecific antigen binding molecule does not bind to human Folate Receptor 2 (FolR2) or to human Folate Receptor 3 (FolR3). In one of the above embodiments, the antigen binding moiety binds to a FolR1 polypeptide comprising the amino acids 25 to 234 of human FolR1 (SEQ ID NO:227).
  • the FolR1 antigen binding moiety binds to a FolR1 polypeptide comprising the amino acid sequence of SEQ ID NOs:227, 230 and 231, and wherein the FolR1 antigen binding moiety does not bind to a FolR polypeptide comprising the amino acid sequence of SEQ ID NOs:228 and 229.
  • the T cell activating bispecific antigen binding molecule is a humanized or a chimeric molecule.
  • VHD2 and CH1D1 are linked through a peptide linker.
  • the first and second polypeptide chain comprise the amino acid sequence of SEQ ID NO:230.
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:86.
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:309.
  • the fifth polypeptide chain comprises the amino acid sequence of SEQ ID NO:308.
  • the first and second polypeptide chain comprise the amino acid sequence of SEQ ID NO:230; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:86; the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:309; and the fifth polypeptide chain comprise the amino acid sequence of SEQ ID NO:308.
  • the invention provides for a T cell activating bispecific antigen binding molecule comprising the amino acid sequence of SEQ ID NO:308.
  • the T cell activating bispecific antigen binding molecule of the above embodiment further comprises the amino acid sequence of SEQ ID NO:230 and of SEQ ID NO:86.
  • the invention provides for an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:308. In one aspect, the invention provides for an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:309.
  • the invention provides for a T cell activating bispecific antigen binding molecule comprising the amino acid sequence of SEQ ID NO:276.
  • the T cell activating bispecific antigen binding molecule of the above embodiment further comprises the amino acid sequence of SEQ ID NO:277 and of SEQ ID NO:35.
  • the invention provides for an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:277. In one aspect, the invention provides for an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:276.
  • the invention provides for an isolated polynucleotide encoding the T cell activating bispecific antigen binding molecule of any one of the embodiments disclosed herein. In one embodiment, the invention provides for an isolated polynucleotide encoding a T cell activating bispecific antigen binding molecule comprising the nucleotide sequence of SEQ ID NO:169. In one embodiment, the invention provides for an isolated polynucleotide encoding a T cell activating bispecific antigen binding molecule comprising the nucleotide sequence of SEQ ID NO:246.
  • the invention provides for an isolated polynucleotide encoding a T cell activating bispecific antigen binding molecule comprising the nucleotide sequence of SEQ ID NO:247. In one embodiment, the invention provides for an isolated polynucleotide encoding a T cell activating bispecific antigen binding molecule comprising the nucleotide sequence of SEQ ID NO:97. In one embodiment, the invention provides for an isolated polynucleotide encoding a T cell activating bispecific antigen binding molecule comprising the nucleotide sequence of SEQ ID NO:198.
  • the invention provides for an isolated polynucleotide encoding a T cell activating bispecific antigen binding molecule comprising the nucleotide sequence of SEQ ID NO:287. In one embodiment, the invention provides for an isolated polynucleotide encoding a T cell activating bispecific antigen binding molecule comprising the nucleotide sequence of SEQ ID NO:288. In one embodiment, the invention provides for an isolated polynucleotide encoding a T cell activating bispecific antigen binding molecule comprising the nucleotide sequence of SEQ ID NO:289.
  • the invention provides for an isolated polypeptide encoded by the polynucleotide of the above embodiment.
  • the invention provides for a vector, particularly an expression vector, comprising the polynucleotide encoding the T cell activating bispecific antigen binding molecule of any one of the embodiments disclosed herein.
  • the invention provides for a host cell comprising a polynucleotide or a vector of any of the embodiments disclosed herein.
  • the invention provides for a method of producing the T cell activating bispecific antigen binding molecule capable of specific binding to CD3 and a target cell antigen, comprising the steps of a) culturing the host cell of the above embodiments 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 molecule.
  • the invention provides for T cell activating bispecific antigen binding molecule produced by the method of the above embodiment.
  • the invention provides for a pharmaceutical composition comprising the T cell activating bispecific antigen binding molecule of any one of the above embodiments and a pharmaceutically acceptable carrier. In one aspect, the invention provides for the T cell activating bispecific antigen binding molecule of any one of the above embodiments or the pharmaceutical composition of any of the above embodiments for use as a medicament.
  • the invention provides for the T cell activating bispecific antigen binding molecule of any one of the above embodiments or the pharmaceutical composition of any one of the above embodiments for use in the treatment of a disease in an individual in need thereof.
  • the disease is cancer.
  • the invention provides for a use of the T cell activating bispecific antigen binding molecule of any one of the above embodiments for the manufacture of a medicament for the treatment of a disease in an individual in need thereof.
  • the invention provides for a method of treating a disease in an individual, comprising administering to said individual a therapeutically effective amount of a composition comprising the T cell activating bispecific antigen binding molecule of any one of the above embodiments in a pharmaceutically acceptable form.
  • said disease is a cancer.
  • the invention provides for a method for inducing lysis of a target cell, comprising contacting a target cell with the T cell activating bispecific antigen binding molecule of any one of the above embodiments in the presence of a T cell.
  • the invention provides for a the invention as described hereinbefore.
  • FIGS. 1A-I illustrate exemplary configurations of the T cell activating bispecific antigen binding molecules (TCBs) of the invention.
  • All constructs except the kappa-lambda format in ( FIG. 1I ) have P329G LALA mutations and comprise knob-into-hole Fc fragments with knob-into-hole modifications.
  • FIG. 1A Illustration of the “FolR1 TCB 2+1 inverted (common light chain)”.
  • the FolR1 binder is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain comprising the knob modification.
  • FIG. 1B Illustration of the “FolR1 TCB 1+1 head-to-tail (common light chain)”. These constructs are not crossed and have two times the same VLCL light chain.
  • FIG. 1C Illustration of the “FolR1 TCB 1+1 classical (common light chain)”. These constructs are not crossed and have two times the same VLCL light chain.
  • FIG. 1D Illustration of the “FolR1 TCB 2+1 classical (common light chain)”. The CD3 binder is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain comprising the knob modification. These constructs are not crossed and have three times the same VLCL light chain.
  • FIG. 1C Illustration of the “FolR1 TCB 1+1 classical (common light chain)”. These constructs are not crossed and have two times the same VLCL light chain.
  • FIG. 1D Illustration of the “FolR1 TCB 2+1 classical (common light chain
  • FIG. 1E Illustration of the “FolR1 TCB 2+1 crossfab classical”. These constructs comprise a Ck-VH chain for the CD3 binder instead of the conventional CH1-VH chain.
  • the CD3 binder is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain comprising the knob modification.
  • FIG. 1F Illustration of the “FolR1 TCB 2+1 crossfab inverted”.
  • These constructs comprise a Ck-VH chain for the CD3 binder instead of the conventional CH1-VH chain.
  • the FolR1 binder is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain comprising the knob modification.
  • FIG. 1G Illustration of the “FolR1 TCB 1+1 crossfab head-to-tail”. These constructs comprise a Ck-VH chain for the CD3 binder instead of the conventional CH1-VH chain.
  • FIG. 1H Illustration of the “FolR1 TCB 1+1 crossfab classical”. These constructs comprise a Ck-VH chain for the CD3 binder instead of the conventional CH1-VH chain.
  • FIG. 1I illustrates the CD3/FolR1 kappa-lambda antibody format. These constructs comprise a crossed common light chain VLCH1 and one crossed VHCL chain specific for CD3 and one crossed VHCL chain specific for FolR1.
  • FIGS. 2A-C depict graphs summarizing Binding of FoLR1 IgG binders to HeLa cells. Binding of newly generated FolR1 binders to FolR1 expressed on HeLa cells were determined by flow cytometry. Bound antibodies were detected with a fluorescently labeled anti-human secondary antibody.
  • FIGS. 3A-B depict graphs summarizing specificity of FolR1 binders for FolR1. Binding of FolR1 IgGs to HEK cells transiently transfected with either FolR1 or FolR2 was analyzed by flow cytometry to identify clones which bind specifically to FolR1 and not to FolR2. The antibodies were detected with a fluorescently labeled anti-human secondary antibody.
  • FIGS. 4A-B depict graphs summarizing cross-reactivity of FolR1 binders to cyFoLR1.
  • Cross-reactivity of the FolR1 antibodies to cyno FolR1 was addressed on HEK cells transiently transfected with cyFolR1 by flow cytometry. The antibodies were detected with a fluorescently labeled anti-human secondary antibody.
  • FIG. 5 depicts a graph illustrating internalization of FolR1 TCBs after binding. Internalization of the four FolR1 TCBs after binding to FolR1 was tested on HeLa cells. Remaining FolR1 TCBs on the surface were detected with a fluorescently labeled anti-human secondary antibody after indicated time points of incubation at 37° C. Percentage of internalization was calculated.
  • FIGS. 6A-E depict graphs summarizing binding of FolR1 IgGs to cells with different FolR1 expression levels. Binding of 9D11, 16D5 and Mov19 IgG to tumor cells with different FolR1 expression levels was analyzed by flow cytometry. DP47 IgG was included as isotype control and MKN-45 were included as FolR1 negative cell line. The antibodies were detected with a fluorescently labeled anti-human secondary antibody.
  • FIGS. 7A-L depict graphs summarizing T cell mediated killing of HT-29 and SKOV3 cells.
  • FolR1 TCBs were used to test T cell mediated killing of HT-29 and SKOV3 tumor cells and upregulation of activation marker on T cells upon killing.
  • FIGS. 7A-D T cell mediated killing of HT-29 and SKOV3 cells in the presence of 9D11 FolR1 TCB and 16D5 FolR1 TCB was measured by LDH release after 24 h and 48 h. DP47 TCB was included as negative control. After 48 h incubation upregulation of the activation marker CD25 and CD69 on CD8 T cells and CD4 T cells upon killing of SKOV3 ( FIGS. 7E-H ) or HT-29 ( FIG. 7I-L ) tumor cells was assessed by flow cytometry.
  • FIG. 8 depicts a graph showing absence of anti-FolR1 binding to erythrocytes.
  • Erythrocytes were gated as CD235a positive population and binding of 9D11 IgG, 16D5 IgG, Mov19 IgG and DP47 IgG to this population was determined by flow cytometry.
  • the antibodies were detected with a fluorescently labeled anti-human secondary antibody.
  • FIGS. 9A-D depict graphs summarizing activation marker upregulation in whole blood.
  • FIG. 10 Binding of 9D11 TCB a-glyco variants to HeLa cells. Binding of 9D11 FolR1 TCB a-glyco variants to Hela cells was compared to binding of the original 9D11 TCB on HeLa cells. The antibodies were detected with a fluorescently labeled anti-human secondary antibody and binding was determined by flow cytometry.
  • FIGS. 11A-F depict graphs summarizing T cell mediated killing with 9D11 FolR1 TCB a-glyco variants of tumor cells.
  • 9D11 FolR1 TCB a-glyco variants were used to test T cell mediated killing of ( FIG. 11A-D ) SKOV3, MKN-45 (as FolR1 negative control) and ( FIG. 11E-F ) HT-29 tumor cells in comparison to killing with the original 9D11 FolR1 TCB.
  • FIGS. 12A-X depict graphs summarizing T cell mediated killing of primary epithelial cells.
  • Primary epithelial cells with very low levels of FolR1 were used to test T cell mediated killing with 16D5 FolR1 TCB and 9D11 FolR1 TCB, DP47 TCB was included as a negative control and HT29 cells were included as positive control.
  • FIGS. 12A-H LDH release of human retinal pigment (HRP), human renal cortical (HRC), human bronchial (HB) and of HT29 cells was determined after 24 h and 48 h.
  • CD25 and CD69 activation marker upregulation on CD4 T cells and CD8 T cells upon killing of FIGS. 12I-L ) HRP, ( FIGS. 12M-P ) HRC, ( FIGS. 12Q-T ) HB and ( FIGS. 12 U-X) HT29 was determined after 48 h by flow cytometry.
  • FIGS. 13A-C show a comparison of different TCB formats with 16D5.
  • Four different TCB formats containing the FolR1 binder 16D5 were compared in FIG. 13A binding to HeLa cells, in FIG. 14 B T cell mediated killing of SKOV3 cells after 24 h and 48 h and in FIG. 14C CD25 and CD69 activation marker upregulation on CD4 T cells and CD8 T cells 48 h after killing.
  • FIGS. 14A-C depict a comparison of different TCB formats with 9D11.
  • Three different TCB formats containing the FolR1 binder 9D11 were compared in A) binding to HeLa cells, in B) T cell mediated killing of SKOV3 cells after 24 h and 48 h and in C) CD25 and CD69 activation marker upregulation on CD4 T cells and CD8 T cells 48 h after killing.
  • FIG. 15 depicts a PK-profile of FOLR1 TCB in NOG mice for three different doses.
  • FIG. 16 illustrates an experimental protocol for efficacy study with FOLR1 TCB.
  • FIGS. 17A-B depict tumor growth curves.
  • FIG. 17A Mean values and SEM of tumor volumes in the different treatment groups.
  • FIG. 17B Tumor growth of single mice in all treatment groups. TGI (tumor growth inhibition) give the percentage of the Mean tumor volume compared to vehicle group.
  • FIG. 18 shows tumor weights at study termination.
  • FIGS. 19A-B show FACS analysis of tumor infiltrating T-cells at study day 32.
  • FIG. 19A Tumor single cells suspensions were stained with anti-human CD3/CD4/CD8 and analyzed by flow cytometry.
  • FIG. 19B Mean values and SEM of T-cell counts per mg tumor tissue in different treatment groups.
  • FIGS. 20A-B show FACS analysis for T-cell activation/degranulation and cytokine secretion at study day 32.
  • FIGS. 21A-B show percent tumor lysis.
  • SKOV3 cells were incubated with PBMCs in the presence of either kappa lambda FoLR1 TCB or DP47 TCB. After 24 h ( FIG. 21A ) and 48 h ( FIG. 21B ) killing of tumor cells was determined by measuring LDH release.
  • FIGS. 22A-D show CD25 and CD69 upregulation on CD4 T cells.
  • SKOV3 cells were incubated with PBMCs in the presence of either kappa lambda FoLR1 TCB or DP47 TCB. After 48 h CD25 and CD69 upregulation on CD4 T cells ( FIG. 22A-B ) and CD8 T cells ( FIG. 22C-D ) was measured by flow cytometry.
  • DP47 TCB was included as non-binding control.
  • FIG. 27 depicts a table summarizing quantification of FolR1 binding sites on various normal and cancer cells lines.
  • FIGS. 28A-B show binding of 16D5 TCB and its corresponding CD3 deamidation variants 16D5 TCB N100A and 16D5 TCB S100aA and 9D11 TCB and its demidation variants 9D11 TCB N100A and 9D11 TCB S100aA to human CD3 expressed on Jurkat cells.
  • DP47 TCB was included as non-binding control.
  • DP47 TCB was included as non-binding control.
  • FIGS. 31A-C show mean fluorescence intensity and tumor cell lysis.
  • FIGS. 32A-E shows binding of 36F2 TCB, 16D5 TCB and 16D5 HC/LC variants to human FolR1 expressed on Hela cells.
  • FIG. 33 shows binding of 36F2 TCB, 16D5 TCB and the two 16D5 affinity reduced variants 16D5 W96Y/D52E TCB and 16D5 G49S/S93A TCB to human FolR1 on Hela cells.
  • FIGS. 34A-E show binding of 36F2 TCB, 16D5 TCB and 16D5 HC/LC variants to human FolR1 expressed on HT-29 cells.
  • FIGS. 35A-D show binding of intermediate FolR1 binders (6E10 TCB, 14B1 TCB and 9C7 TCB), 16D5 TCB and 36F2 TCB to HEK293T cells expressing either human or mouse FolR1 or FolR2.
  • FIG. 36A-F show T-cell killing of Hela (high FolR1 expression), SKov-3 (medium FolR1 expression) and HT-29 (low FolR1 expression) human tumor cells induced by intermediate FolR1 binders (6E10 TCB, 14B1 TCB and 9C7 TCB), 16D5 TCB and 36F2 TCB after 24 h (A-C) and 48 h (D-F) of incubation.
  • HT-29 cells low FolR1 expression
  • DP47 TCB served as non-binding control.
  • FIGS. 39A-B show single dose PK of FOLR1 TCB constructs in female NOG mice.
  • FIGS. 40A-G show in vivo efficacy of FOLR1 TCB constructs (16D5, 16D5 G49S/S93A and 16D5 W96Y/D52E) after human PBMC transfer in Hela-bearing NOG mice.
  • FIG. 41 shows that Farletuzumab (dark green, second from the top) and Mov19 (grey, top) are able to bind on huFolR1 that is captured on 16D5, demonstrating that the 16D5 series binders recognize an epitope distinct from that recognized by either Farletuzumab or Mov19.
  • antigen binding molecule refers in its broadest sense to a molecule that specifically binds an antigenic determinant.
  • antigen binding molecules are immunoglobulins and derivatives, e.g. fragments, thereof.
  • bispecific means that the antigen binding molecule is able to specifically bind to at least two distinct antigenic determinants.
  • a bispecific antigen binding molecule comprises at least two antigen binding sites, each of which is specific for a different antigenic determinant.
  • the bispecific antigen binding molecule is capable of simultaneously binding two antigenic determinants, particularly two antigenic determinants expressed on two distinct cells.
  • valent denotes the presence of a specified number of antigen binding sites in an antigen binding molecule.
  • monovalent binding to an antigen denotes the presence of one (and not more than one) antigen binding site specific for the antigen in the antigen binding molecule.
  • an “antigen binding site” refers to the site, i.e. one or more amino acid residues, of an antigen binding molecule which provides interaction with the antigen.
  • the antigen binding site of an antibody comprises amino acid residues from the complementarity determining regions (CDRs).
  • CDRs complementarity determining regions
  • a native immunoglobulin molecule typically has two antigen binding sites, a Fab molecule typically has a single antigen binding site.
  • an antigen binding moiety refers to a polypeptide molecule that specifically binds to an antigenic determinant.
  • 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 antigenic determinant.
  • an antigen binding moiety is able to activate signaling through its target antigen, for example a T cell receptor complex antigen.
  • Antigen binding moieties include antibodies and fragments thereof as further defined herein. Particular antigen binding moieties include an antigen binding domain of an antibody, comprising an antibody heavy chain variable region and an antibody light chain variable region.
  • 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: ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ .
  • Useful light chain constant regions include any of the two isotypes: ⁇ and ⁇ .
  • antigenic determinant is synonymous with “antigen” and “epitope,” and refers to a site (e.g. a contiguous stretch of amino acids or a conformational configuration made up of 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 surfaces of virus-infected cells, on the surfaces of other diseased cells, on the surface of immune cells, free in blood serum, and/or in the extracellular matrix (ECM).
  • ECM extracellular matrix
  • the proteins referred to as antigens herein can be any native form the proteins from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g. mice and rats), unless otherwise indicated.
  • the antigen is a human protein.
  • the term encompasses the “full-length”, 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 allelic variants.
  • Exemplary human proteins useful as antigens include, but are not limited to: FolR1 (Folate receptor alpha (FRA); Folate binding protein (FBP); human FolR1 UniProt no.: P15328; murine FolR1 UniProt no.: P35846; cynomolgus FolR1 UniProt no.: G7PR14) and CD3, particularly the epsilon subunit of CD3 (see UniProt no. P07766 (version 130), NCBI RefSeq no. NP_000724.1, SEQ ID NO:150 for the human sequence; or UniProt no. Q95LI5 (version 49), NCBI GenBank no.
  • the T cell activating bispecific antigen binding molecule of the invention binds to an epitope of CD3 or a target cell antigen that is conserved among the CD3 or target antigen from different species. In certain embodiments the T cell activating bispecific antigen binding molecule of the invention binds to CD3 and FolR1, but does not bind to FolR2 (Folate receptor beta; FRB; human FolR2 UniProt no.: P14207) or FolR3 (Folate receptor gamma; human FolR3 UniProt no.: P41439).
  • FolR2 FolR2
  • FolR3 FolR3 (Folate receptor gamma; human FolR3 UniProt no.: P41439).
  • ELISA enzyme-linked immunosorbent assay
  • SPR surface plasmon resonance
  • an antigen binding moiety that binds to the antigen, or an antigen binding molecule comprising that antigen binding moiety has a dissociation constant (K D ) of ⁇ 1 ⁇ M, ⁇ 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).
  • K D dissociation constant
  • Binding affinity refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., an antigen binding moiety and an antigen, or a receptor and its ligand).
  • the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (K D ), which is the ratio of dissociation and association rate constants (k off and k on , respectively).
  • affinities may comprise different rate constants, as long as the ratio of the rate constants remains the same.
  • Affinity can be measured by well-established methods known in the art, including those described herein.
  • a particular method for measuring affinity is Surface Plasmon Resonance (SPR).
  • Reduced binding for example reduced binding to an Fc receptor, refers to a decrease in affinity for the respective interaction, as measured for example by SPR.
  • the term includes also reduction of the affinity to zero (or below the detection limit of the analytic method), i.e. complete abolishment of the interaction.
  • increased binding refers to an increase in binding affinity for the respective interaction.
  • T cell activation 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 release, cytotoxic activity, and expression of activation markers.
  • the T cell activating bispecific antigen binding molecules of the invention are capable of inducing T cell activation. Suitable assays to measure T cell activation are known in the art described herein.
  • target cell antigen 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.
  • target cell antigen refers to Folate Receptor 1.
  • first and second with respect to antigen binding moieties etc., are used for convenience of distinguishing when there is more than one of each type of moiety. Use of these terms is not intended to confer a specific 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.
  • Fab molecules having identical VLCL light chains refers to binders that share one light chain but still have separate specificities, e.g., can bind CD3 or FolR1.
  • the T-cell activating bispecific molecules comprise at least two Fab molecules having identical VLCL light chains. The corresponding heavy chains are remodeled and confer specific binding to the T cell activating bispecific antigen CD3 and the target cell antigen FolR1, respectively.
  • 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.
  • single-chain refers to a molecule comprising amino acid monomers linearly linked by peptide bonds.
  • one of the antigen binding moieties is a single-chain Fab molecule, i.e. a Fab molecule wherein the Fab light chain and the Fab heavy chain are connected by a peptide linker to form a single peptide chain.
  • 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.
  • 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.
  • the peptide chain comprising the heavy chain constant region is referred to herein as the “heavy chain” of the crossover Fab molecule.
  • CrossFab an antibody that comprises one or more CrossFabs.
  • a “conventional” Fab molecule is meant a Fab molecule in its natural format, i.e. comprising a heavy chain composed of the heavy chain variable and constant regions (VH-CH1), and a light chain composed of the light chain variable and constant regions (VL-CL).
  • immunoglobulin molecule refers to a protein having the structure of a naturally occurring antibody.
  • immunoglobulins of the IgG class are heterotetrameric 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 region.
  • VH variable region
  • CH1, CH2, and CH3 constant domains
  • each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain, also called a light chain constant region.
  • VL variable region
  • the heavy chain of an immunoglobulin may be assigned to one of five types, called ⁇ (IgA), ⁇ (IgD), ⁇ (IgE), ⁇ (IgG), or ⁇ (IgM), some of which may be further divided into subtypes, e.g. ⁇ 1 (IgG 1 ), ⁇ 2 (IgG 2 ), ⁇ 3 (IgG 3 ), ⁇ 4 (IgG 4 ), ⁇ 1 (IgA 1 ) and ⁇ 2 (IgA 2 ).
  • the light chain of an immunoglobulin may be assigned to one of two types, called kappa ( ⁇ ) and lambda ( ⁇ ), 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.
  • antibody herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, and antibody fragments so long as they exhibit the desired antigen-binding activity.
  • 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.
  • antibody fragments include but are not limited to Fv, Fab, Fab′, Fab′-SH, F(ab′) 2 , diabodies, linear antibodies, single-chain antibody molecules (e.g. scFv), and single-domain antibodies.
  • scFv single-chain antibody molecules
  • Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific.
  • 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.
  • a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, Mass.; see e.g. U.S. Pat. No. 6,248,516 B1).
  • Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g. E. coli or phage), as described herein.
  • an antigen binding domain refers to the part of an antibody that comprises the area which specifically binds to and is complementary to part or all of an antigen.
  • An antigen binding domain may be provided by, for example, one or more antibody variable domains (also called antibody variable regions).
  • an antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).
  • variable region 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, 6 th ed., W.H. Freeman and Co., page 91 (2007).
  • a single VH or VL domain may be sufficient to confer antigen-binding specificity.
  • hypervariable region refers to each of the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops (“hypervariable loops”).
  • native four-chain antibodies comprise six HVRs; three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3).
  • HVRs generally comprise amino acid residues from the hypervariable loops and/or from the complementarity determining regions (CDRs), the latter being of highest sequence variability and/or 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 are also referred to as “complementarity determining regions” (CDRs), and these terms are used herein interchangeably in reference to portions of the variable region that form the antigen binding regions.
  • CDRs complementarity determining 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 overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein.
  • Kabat et al. also defined a numbering system for variable region sequences that is applicable to any antibody.
  • Kabat numbering refers to the numbering system set forth by Kabat et al., U.S. Dept. of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983). Unless otherwise specified, references to the numbering of specific amino acid residue positions in an antibody variable region are according to the Kabat numbering system.
  • the polypeptide sequences of the sequence listing are not numbered according to the Kabat numbering system. However, it is well within the ordinary skill of one in the art to convert the numbering of the sequences of the Sequence Listing to Kabat numbering.
  • “Framework” or “FR” refers to variable domain residues other than hypervariable region (HVR) residues.
  • the FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3 (L3)-FR4.
  • the “class” of an antibody or immunoglobulin refers to the type of constant domain or constant region possessed by its heavy chain.
  • the heavy chain constant domains that correspond to the different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • 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.
  • the boundaries of the Fc region of an IgG heavy chain might vary slightly, the human IgG heavy chain Fc region is usually defined to extend from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain.
  • the C-terminal lysine (Lys447) of the Fc region may or may not be present.
  • 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.
  • a subunit of an IgG Fc domain comprises an IgG CH2 and an IgG CH3 constant domain.
  • a “modification promoting the association of the first and the second subunit of the Fc domain” is a manipulation of the peptide backbone or the post-translational modifications of an Fc domain subunit that reduces or prevents the association of a polypeptide comprising the Fc domain subunit with an identical polypeptide to form a homodimer.
  • a modification promoting association as used herein particularly includes separate modifications made to each of the two Fc domain subunits desired to associate (i.e. the first and the second subunit of the Fc domain), wherein the modifications are complementary to each other so as to promote association of the two Fc domain subunits.
  • a modification promoting association may alter the structure or charge of one or both of the Fc domain subunits so as to make their association sterically or electrostatically favorable, respectively.
  • (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 subunits (e.g. antigen binding moieties) are not the same.
  • the modification promoting association comprises an amino acid mutation in the Fc domain, specifically an amino acid substitution.
  • the modification promoting association comprises a separate amino acid mutation, specifically an amino acid substitution, in each of the two subunits of the Fc domain.
  • effector functions refers to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype.
  • 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.
  • engine engineered, engineering
  • engineering includes modifications of the amino acid sequence, of the glycosylation pattern, or of the side chain group of individual amino acids, as well as combinations of these approaches.
  • amino acid mutation as used herein is meant to encompass amino acid substitutions, deletions, insertions, and modifications. Any combination of substitution, deletion, insertion, and modification can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., reduced binding to an Fc receptor, or increased association with another peptide.
  • Amino acid sequence deletions and insertions include amino- and/or carboxy-terminal deletions and insertions of amino acids.
  • Particular amino acid mutations are amino acid substitutions.
  • 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 standard amino acids (e.g. 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine).
  • Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis 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 modification, 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, G 329 , P329G, or Pro329Gly.
  • polypeptide refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds).
  • polypeptide refers to any chain of two or more amino acids, and does not refer to a specific length of the product.
  • 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.
  • 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 protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids.
  • a polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It may be generated in any manner, including by chemical synthesis.
  • a polypeptide of the invention may be of a size of about 3 or more, 5 or more, 10 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 three-dimensional structure, although they do not necessarily have such structure. Polypeptides with a defined three-dimensional structure are referred to as folded, and polypeptides which do not possess a defined three-dimensional structure, but rather can adopt a large number of different conformations, and are referred to as unfolded.
  • an “isolated” polypeptide or a variant, or derivative thereof is intended a polypeptide that is not in its natural milieu. No particular level of purification is required.
  • an isolated 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 the invention, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.
  • Percent (%) amino acid sequence identity with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2.
  • the ALIGN-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 registered under U.S. Copyright Registration No. TXU510087.
  • the ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from the source code.
  • the ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
  • % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows:
  • polynucleotide refers to an isolated nucleic acid molecule or construct, e.g. messenger RNA (mRNA), virally-derived RNA, or plasmid DNA (pDNA).
  • mRNA messenger RNA
  • pDNA virally-derived RNA
  • a polynucleotide may comprise a conventional phosphodiester bond or a non-conventional bond (e.g. an amide bond, such as found in peptide nucleic acids (PNA).
  • PNA peptide nucleic acids
  • nucleic acid molecule refers to any one or more nucleic acid segments, e.g. DNA or RNA fragments, present in a polynucleotide.
  • isolated nucleic acid molecule or polynucleotide is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment.
  • a recombinant polynucleotide encoding a polypeptide contained in a vector is considered isolated for the purposes of the present invention.
  • 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 of the present invention, as well as positive and negative strand forms, and double-stranded forms. Isolated polynucleotides or nucleic acids according to the present invention further include such molecules produced synthetically.
  • a polynucleotide or a nucleic acid may be or may include a regulatory element such as a promoter, ribosome binding site, or a transcription terminator.
  • a nucleic acid or polynucleotide having a nucleotide sequence at least, for example, 95% “identical” to a reference nucleotide sequence of the present invention it is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence.
  • a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
  • These alterations of the reference sequence may occur at the 5′ or 3′ terminal positions of the reference 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 sequence.
  • any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the present invention can be determined conventionally using known computer programs, such as the ones discussed above for polypeptides (e.g. ALIGN-2).
  • 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, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment.
  • the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter.
  • the expression cassette of the invention comprises polynucleotide sequences that encode bispecific antigen binding molecules of the invention or fragments thereof.
  • vector or “expression vector” is synonymous with “expression construct” and refers to a DNA molecule that is used to introduce and direct the expression of a specific gene to which it is operably associated in a target cell.
  • the term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced.
  • the expression vector of the present invention comprises an expression 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 produced by the cellular transcription and/or translation machinery.
  • the expression vector of the invention comprises an expression cassette that comprises polynucleotide sequences that encode bispecific antigen binding molecules of the invention or fragments thereof.
  • host cell refers 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 included herein.
  • a host cell is any type of cellular system that can be used to generate the bispecific antigen binding molecules of the present invention.
  • Host cells include cultured cells, e.g.
  • mammalian cultured cells such as CHO cells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, insect cells, and plant cells, to name only a few, but also cells comprised within a transgenic animal, transgenic plant or cultured plant or animal tissue.
  • an “activating Fc receptor” is an Fc receptor that following engagement by an Fc domain of an antibody elicits signaling events that stimulate the receptor-bearing cell to perform effector functions.
  • Human activating Fc receptors include Fc ⁇ RIIIa (CD16a), Fc ⁇ RI (CD64), Fc ⁇ RIIa (CD32), and Fc ⁇ RI (CD89).
  • Antibody-dependent cell-mediated cytotoxicity 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, generally via the protein part that is N-terminal to the Fc region.
  • reduced ADCC is defined as either a reduction in the number of target cells that are lysed in a given time, at a given concentration of antibody in the medium 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 mechanism of ADCC.
  • the reduction in ADCC is relative to the ADCC mediated by the same antibody produced by the same type of host cells, using the same standard production, purification, formulation and storage methods (which are known to those skilled in the art), but that has not been engineered.
  • 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. WO 2006/082515 or PCT publication no. WO 2012/130831).
  • an “effective amount” of an agent refers to the amount that is necessary to result in a physiological change in the cell or tissue to which it is administered.
  • a “therapeutically effective amount” of an agent refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
  • a therapeutically effective amount of an agent for example eliminates, decreases, delays, minimizes or prevents adverse effects of a disease.
  • mammals include, but are not limited to, domesticated animals (e.g. cows, sheep, cats, dogs, and horses), primates (e.g. humans and non-human primates such as monkeys), rabbits, and rodents (e.g. mice and rats). Particularly, the individual or subject is a human.
  • domesticated animals e.g. cows, sheep, cats, dogs, and horses
  • primates e.g. humans and non-human primates such as monkeys
  • rabbits e.g. mice and rats
  • rodents e.g. mice and rats
  • composition refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
  • a “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical composition, other than an active ingredient, which is nontoxic to a subject.
  • a pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
  • treatment refers to clinical intervention in an attempt to alter the natural course of a disease in the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of 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 remission or improved prognosis.
  • T cell activating bispecific antigen binding molecules of the invention are used to delay development of a disease or to slow the progression of a disease.
  • antagonist is used in the broadest sense, and includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of a native polypeptide disclosed herein.
  • agonist is used in the broadest sense and includes any molecule that induces a biological activity of a native polypeptide disclosed herein.
  • Suitable agonist or antagonist molecules specifically include agonist or antagonist antibodies or antibody fragments, including engineered antibody fragments, fragments or amino acid sequence variants of native polypeptides, peptides, antisense oligonucleotides, small organic molecules, etc.
  • Methods for identifying agonists or antagonists of a polypeptide may comprise contacting a polypeptide with a candidate agonist or antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the polypeptide.
  • package insert is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.
  • the T cell activating bispecific antigen binding molecule of the invention is bispecific, i.e. it comprises at least two antigen binding moieties capable of specific binding to two distinct antigenic determinants, i.e. to CD3 and to FolR1.
  • the antigen binding moieties are Fab molecules (i.e. antigen binding domains composed of a heavy and a light chain, each comprising a variable and a constant region).
  • said Fab molecules are human.
  • said Fab molecules are humanized.
  • said Fab molecules comprise human heavy and light chain constant regions.
  • the T cell activating bispecific antigen binding molecule of the invention is capable of simultaneous binding to the target cell antigen FolR1 and CD3.
  • the T cell activating bispecific antigen binding molecule is capable of crosslinking a T cell and a FolR1 expressing target cell by simultaneous binding to the target cell antigen FolR1 and CD3.
  • simultaneous binding results in lysis of the FolR1 expressing target cell, particularly a FolR1 expressing tumor cell.
  • such simultaneous binding results in activation of the T cell.
  • 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 secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers.
  • a T lymphocyte particularly a cytotoxic T lymphocyte, selected from the group of: proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers.
  • binding of the T cell activating bispecific antigen binding molecule to CD3 without simultaneous binding to the target cell antigen FolR1 does not result in T cell activation.
  • the T cell activating bispecific antigen binding molecule is capable of re-directing cytotoxic activity of a T cell to a FolR1 expressing target cell.
  • said re-direction is independent of MHC-mediated peptide antigen presentation by the target cell and and/or specificity of the T cell.
  • a T cell according to some of the embodiments of the invention is a cytotoxic T cell.
  • the T cell is a CD4 + or a CD8 + T cell, particularly a CD8 + T cell.
  • the T cell activating bispecific antigen binding molecule of the invention comprises at least one antigen binding moiety capable of binding to CD3 (also referred to herein as an “CD3 antigen binding moiety” or “first antigen binding moiety”).
  • the T cell activating bispecific antigen binding molecule comprises not more than one antigen binding moiety capable of specific binding to CD3.
  • the T cell activating bispecific antigen binding molecule provides monovalent binding to CD3.
  • CD3 is human CD3 or cynomolgus CD3, most particularly human CD3.
  • the CD3 antigen binding moiety is cross-reactive for (i.e. specifically binds to) human and cynomolgus CD3.
  • the first antigen binding moiety is capable of specific binding to the epsilon subunit of CD3 (see UniProt no. P07766 (version 130), NCBI RefSeq no. NP_000724.1, SEQ ID NO:150 for the human sequence; UniProt no. Q95LI5 (version 49), NCBI GenBank no. BAB71849.1, for the cynomolgus [ Macaca fascicularis ] sequence).
  • the CD3 antigen binding moiety comprises at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39 and at least one light chain CDR selected from the group of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34.
  • CDR heavy chain complementarity determining region
  • the CD3 antigen binding moiety comprises the heavy chain CDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO: 38, the heavy chain CDR3 of SEQ ID NO:39, the light chain CDR1 of SEQ ID NO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3 of SEQ ID NO:34.
  • the CD3 antigen binding moiety comprises a variable heavy chain comprising an amino acid sequence of: SEQ ID NO: 36 and a variable light chain comprising an amino acid sequence of: SEQ ID NO: 31.
  • the CD3 antigen binding moiety comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 36 and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 31.
  • the T cell activating bispecific antigen binding molecule of the invention comprises at least one antigen binding moiety capable of binding to the target cell antigen FolR1 (also referred to herein as an “FolR1 binding moiety” or “second” or “third” antigen binding moiety).
  • the antigen binding moiety capable of binding to the target cell antigen FolR1 does not bind to FolR2 or FolR3.
  • the FolR1 antigen binding moiety is cross-reactive for (i.e. specifically binds to) human and cynomolgus FolR1.
  • the T cell activating bispecific antigen binding molecule comprises two antigen binding moieties capable of binding to the target cell antigen FolR1.
  • each of these antigen binding moieties specifically binds to the same antigenic determinant. In an even more particular embodiment, all of these antigen binding moieties are identical. In one embodiment the T cell activating bispecific antigen binding molecule comprises not more than two antigen binding moieties capable of binding to FolR1.
  • the FolR1 binding moiety is generally a Fab molecule that specifically binds to FolR1 and is able to direct the T cell activating bispecific antigen binding molecule to which it is connected to a target site, for example to a specific type of tumor cell that expresses FolR1.
  • the present invention provides a T cell activating bispecific antigen binding molecule comprising
  • the first antigen binding moiety which is a Fab molecule capable of specific binding to CD3 comprises a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 36 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 31.
  • the second and third antigen binding moiety capable of specific binding to FolR1 comprise identical heavy chain complementarity determining region (CDR) and light chain CDR sequences.
  • the third antigen binding moiety is identical to the second antigen binding moiety.
  • the T cell activating bispecific antigen binding molecule of any of the above embodiments additionally comprises an Fc domain composed of a first and a second subunit capable of stable association.
  • first antigen binding moiety and the second antigen binding moiety are each fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain.
  • the third 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, optionally via a peptide linker.
  • not more than one antigen binding moiety capable of specific binding to CD3 is present in the T cell activating bispecific antigen binding molecule (i.e. the T cell activating bispecific antigen binding molecule provides monovalent binding to CD3).
  • the inventors of the present invention generated a bispecific antibody wherein the binding moieties share a common light chain that retains the specificity and efficacy of the parent monospecific antibody for CD3 and can bind a second antigen (e.g., FolR1) using the same light chain.
  • a bispecific molecule with a common light chain that retains the binding properties of the parent antibody is not straight-forward as the common CDRs of the hybrid light chain have to effectuate the binding specificity for both targets.
  • the present 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 specific binding to FolR1, wherein the first and the second Fab molecule have identical VLCL light chains.
  • said identical light chain (VLCL) comprises the light chain CDRs of SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34.
  • said identical light chain (VLCL) comprises SEQ ID NO. 35.
  • a first antigen binding moiety which is a Fab molecule capable of specific binding to CD3, and which comprises at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39 and at least one light chain CDR selected from the group of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34;
  • CDR heavy chain complementarity determining region
  • a second antigen binding moiety which is a Fab molecule capable of specific binding to Folate Receptor 1 (FolR1) and which comprises at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 and at least one light chain CDR selected from the group of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34.
  • CDR heavy chain complementarity determining region
  • the CD3 antigen binding moiety comprises the heavy chain CDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO: 38, the heavy chain CDR3 of SEQ ID NO:39, the light chain CDR1 of SEQ ID NO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3 of SEQ ID NO:34 and the FolR1 antigen binding moiety comprises the heavy chain CDR1 of SEQ ID NO: 16, the heavy chain CDR2 of SEQ ID NO: 17, the heavy chain CDR3 of SEQ ID NO:18, the light chain CDR1 of SEQ ID NO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3 of SEQ ID NO:34.
  • a first antigen binding moiety which is a Fab molecule capable of specific binding to CD3 comprising a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 36 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 31.
  • a second antigen binding moiety which is a Fab molecule capable of specific binding to Folate Receptor 1 (FolR1) comprising a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 15 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 31.
  • a first antigen binding moiety which is a Fab molecule capable of specific binding to CD3, and which comprises at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39 and at least one light chain CDR selected from the group of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34;
  • CDR heavy chain complementarity determining region
  • a second antigen binding moiety which is a Fab molecule capable of specific binding to Folate Receptor 1 (FolR1) and which comprises at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 275 and SEQ ID NO: 315 and at least one light chain CDR selected from the group of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34.
  • CDR heavy chain complementarity determining region
  • a first antigen binding moiety which is a Fab molecule capable of specific binding to CD3, and which comprises the heavy chain complementarity determining region (CDR) amino acid sequences of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39, and the light chain CDR amino acid sequences of SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34;
  • CDR complementarity determining region
  • a second antigen binding moiety which is a Fab molecule capable of specific binding to Folate Receptor 1 (FolR1) and which comprises the heavy chain complementarity determining region (CDR) amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 275 and SEQ ID NO: 315, and the light chain CDR amino acid sequences of SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34.
  • FolR1 Folate Receptor 1
  • a first antigen binding moiety which is a Fab molecule capable of specific binding to CD3 comprising a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 36 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 31;
  • a second antigen binding moiety which is a Fab molecule capable of specific binding to Folate Receptor 1 (FolR1) comprising a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 274 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 31.
  • a first antigen binding moiety which is a Fab molecule capable of specific binding to CD3 comprising a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 36 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 31.
  • a second antigen binding moiety which is a Fab molecule capable of specific binding to Folate Receptor 1 (FolR1) comprising a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 15 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 31.
  • the antigen binding moiety that is specific for FolR1 comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:15 and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 31 or variants thereof that retain functionality.
  • the T cell activating bispecific antigen binding molecule comprises a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 36, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:15, and a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 31.
  • the second and third antigen binding moiety capable of specific binding to FolR1 comprise identical heavy chain complementarity determining region (CDR) and light chain CDR sequences.
  • the third antigen binding moiety is identical to the second antigen binding moiety.
  • a first antigen binding moiety which is a Fab molecule capable of specific binding to CD3, and which comprises at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39 and at least one light chain CDR selected from the group of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34;
  • CDR heavy chain complementarity determining region
  • a second antigen binding moiety which is a Fab molecule capable of specific binding to Folate Receptor 1 (FolR1) and which comprises at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 and at least one light chain CDR selected from the group of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34.
  • CDR heavy chain complementarity determining region
  • a third antigen binding moiety which is a Fab molecule capable of specific binding to Folate Receptor 1 (FolR1) and which comprises at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 and at least one light chain CDR selected from the group of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34.
  • CDR heavy chain complementarity determining region
  • the CD3 antigen binding moiety comprises the heavy chain CDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO: 38, the heavy chain CDR3 of SEQ ID NO:39, the light chain CDR1 of SEQ ID NO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3 of SEQ ID NO:34 and the FolR1 antigen binding moiety comprises the heavy chain CDR1 of SEQ ID NO: 16, the heavy chain CDR2 of SEQ ID NO: 17, the heavy chain CDR3 of SEQ ID NO:18, the light chain CDR1 of SEQ ID NO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3 of SEQ ID NO:34.
  • a first antigen binding moiety which is a Fab molecule capable of specific binding to CD3 comprising a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 36 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 31.
  • a second antigen binding moiety which is a Fab molecule capable of specific binding to Folate Receptor 1 (FolR1) comprising a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 15 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 31.
  • a third antigen binding moiety which is a Fab molecule capable of specific binding to Folate Receptor 1 (FolR1) comprising a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 15 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 31.
  • the invention relates to bispecific molecules wherein at least two binding moieties have identical light chains and corresponding remodeled heavy chains that confer the specific binding to the T cell activating antigen CD3 and the target cell antigen FolR1, respectively.
  • the use of this so-called ‘common light chain’ principle i.e. combining two binders that share one light chain but still have separate specificities, prevents light chain mispairing.
  • there are less side products during production facilitating the homogenous preparation of T cell activating bispecific antigen binding molecules.
  • T cell activating bispecific antigen binding molecule can be fused to each other in a variety of configurations. Exemplary configurations are depicted in FIGS. 1A-I and are further described below.
  • said T cell activating bispecific antigen binding molecule further comprises an Fc domain composed of a first and a second subunit capable of stable association.
  • Fc domain composed of a first and a second subunit capable of stable association.
  • the inventors of the present invention generated a second bispecific antibody format wherein one of the binding moieties is a crossover Fab fragment.
  • a monovalent bispecific antibody is provided, wherein one of the Fab fragments of an IgG molecule is replaced by a crossover Fab fragment.
  • Crossover Fab fragments are Fab fragments wherein either the variable regions or the constant regions of the heavy and light chain are exchanged.
  • Bispecific antibody formats comprising crossover Fab fragments have been described, for example, in WO2009080252, WO2009080253, WO2009080251, WO2009080254, WO2010/136172, WO2010/145792 and WO2013/026831.
  • 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.
  • Such modification prevent mispairing of heavy and light chains from different Fab molecules, thereby improving the yield and purity of the T cell activating bispecific antigen binding molecule of the invention in recombinant production.
  • the variable regions of the Fab light chain and the Fab heavy chain are exchanged.
  • the constant regions of the Fab light chain and the Fab heavy chain are exchanged.
  • a first antigen binding moiety which is a crossover Fab molecule capable of specific binding to CD3, comprising at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39 and at least one light chain CDR selected from the group of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34;
  • CDR heavy chain complementarity determining region
  • a second antigen binding moiety capable of specific binding to Folate Receptor 1 comprising at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 56 and SEQ ID NO: 57 and at least one light chain CDR selected from the group of SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 65.
  • CDR Folate Receptor 1
  • the CD3 antigen binding moiety comprises the heavy chain CDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO: 38, the heavy chain CDR3 of SEQ ID NO:39, the light chain CDR1 of SEQ ID NO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3 of SEQ ID NO:34 and the FolR1 antigen binding moiety comprises the heavy chain CDR1 of SEQ ID NO: 8, the heavy chain CDR2 of SEQ ID NO: 56, the heavy chain CDR3 of SEQ ID NO:57, the light chain CDR1 of SEQ ID NO: 59, the light chain CDR2 of SEQ ID NO: 60, and the light chain CDR3 of SEQ ID NO:65.
  • the second antigen binding moiety is a conventional Fab molecule.
  • a first antigen binding moiety which is a crossover Fab molecule capable of specific binding to CD3 comprising a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 36 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 31.
  • a second antigen binding moiety which is a Fab molecule capable of specific binding to Folate Receptor 1 (FolR1) comprising a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 55 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 64.
  • the second antigen binding moiety is a conventional Fab molecule.
  • the antigen binding moiety that is specific for FolR1 comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:55 and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 64 or variants thereof that retain functionality.
  • the T cell activating bispecific antigen binding molecule comprises a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 36, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 31, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:55, and a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 64.
  • the third antigen binding moiety is a conventional Fab molecule. In one embodiment, the third antigen binding moiety is a crossover Fab molecule.
  • the second and third antigen binding moiety capable of specific binding to FolR1 comprise identical heavy chain complementarity determining region (CDR) and light chain CDR sequences.
  • the third antigen binding moiety is identical to the second antigen binding moiety.
  • a first antigen binding moiety which is a crossover Fab molecule capable of specific binding to CD3, comprising at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39 and at least one light chain CDR selected from the group of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34;
  • CDR heavy chain complementarity determining region
  • a second antigen binding moiety capable of specific binding to Folate Receptor 1 comprising at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 56 and SEQ ID NO: 57 and at least one light chain CDR selected from the group of SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 65.
  • CDR Folate Receptor 1
  • a third antigen binding moiety capable of specific binding to Folate Receptor 1 comprising at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 56 and SEQ ID NO: 57 and at least one light chain CDR selected from the group of SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 65.
  • CDR Folate Receptor 1
  • the CD3 antigen binding moiety comprises the heavy chain CDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO: 38, the heavy chain CDR3 of SEQ ID NO:39, the light chain CDR1 of SEQ ID NO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3 of SEQ ID NO:34 and the FolR1 antigen binding moiety comprises the heavy chain CDR1 of SEQ ID NO: 8, the heavy chain CDR2 of SEQ ID NO: 56, the heavy chain CDR3 of SEQ ID NO:57, the light chain CDR1 of SEQ ID NO: 59, the light chain CDR2 of SEQ ID NO: 60, and the light chain CDR3 of SEQ ID NO:65.
  • the second antigen binding moiety and the third antigen binding moiety are both a conventional Fab molecule.
  • a first antigen binding moiety which is a crossover Fab molecule capable of specific binding to CD3 comprising a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 36 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 31.
  • a second antigen binding moiety which is a Fab molecule capable of specific binding to Folate Receptor 1 (FolR1) comprising a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 55 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 64.
  • a third antigen binding moiety which is a Fab molecule capable of specific binding to Folate Receptor 1 (FolR1) comprising a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 55 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 64.
  • the second antigen binding moiety and the third antigen binding moiety are both a conventional Fab molecule.
  • a first antigen binding moiety which is a crossover Fab molecule capable of specific binding to CD3, comprising at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39 and at least one light chain CDR selected from the group of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34;
  • CDR heavy chain complementarity determining region
  • a second antigen binding moiety capable of specific binding to Folate Receptor 1 comprising at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 50 and at least one light chain CDR selected from the group of SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54.
  • CDR heavy chain complementarity determining region
  • the CD3 antigen binding moiety comprises the heavy chain CDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO: 38, the heavy chain CDR3 of SEQ ID NO:39, the light chain CDR1 of SEQ ID NO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3 of SEQ ID NO:34 and the FolR1 antigen binding moiety comprises the heavy chain CDR1 of SEQ ID NO: 8, the heavy chain CDR2 of SEQ ID NO: 9, the heavy chain CDR3 of SEQ ID NO:50, the light chain CDR1 of SEQ ID NO: 52, the light chain CDR2 of SEQ ID NO: 53, and the light chain CDR3 of SEQ ID NO:54.
  • the second antigen binding moiety is a conventional Fab molecule. In one embodiment, the second antigen binding moiety is a crossover Fab molecule.
  • a second antigen binding moiety which is a Fab molecule capable of specific binding to Folate Receptor 1 (FolR1) comprising a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 49 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 51.
  • the second antigen binding moiety is a conventional Fab molecule. In one embodiment, the second antigen binding moiety is a crossover Fab molecule.
  • the antigen binding moiety that is specific for FolR1 comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:49 and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 51 or variants thereof that retain functionality.
  • the T cell activating bispecific antigen binding molecule comprises a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 36, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 31, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:49, and a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 51.
  • the third antigen binding moiety is a conventional Fab molecule. In one embodiment, the second antigen binding moiety is a crossover Fab molecule.
  • the second and third antigen binding moiety capable of specific binding to FolR1 comprise identical heavy chain complementarity determining region (CDR) and light chain CDR sequences.
  • the third antigen binding moiety is identical to the second antigen binding moiety.
  • a first antigen binding moiety which is a crossover Fab molecule capable of specific binding to CD3, comprising at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39 and at least one light chain CDR selected from the group of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34;
  • CDR heavy chain complementarity determining region
  • a second antigen binding moiety capable of specific binding to Folate Receptor 1 comprising at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 49 and at least one light chain CDR selected from the group of SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54.
  • CDR heavy chain complementarity determining region
  • a third antigen binding moiety capable of specific binding to Folate Receptor 1 comprising at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 50 and at least one light chain CDR selected from the group of SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54.
  • CDR heavy chain complementarity determining region
  • the CD3 antigen binding moiety comprises the heavy chain CDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO: 38, the heavy chain CDR3 of SEQ ID NO:39, the light chain CDR1 of SEQ ID NO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3 of SEQ ID NO:34 and the FolR1 antigen binding moiety comprises the heavy chain CDR1 of SEQ ID NO: 8, the heavy chain CDR2 of SEQ ID NO: 9, the heavy chain CDR3 of SEQ ID NO:50, the light chain CDR1 of SEQ ID NO: 52, the light chain CDR2 of SEQ ID NO: 53, and the light chain CDR3 of SEQ ID NO:54.
  • the second antigen binding moiety and the third antigen binding moiety are both a conventional Fab molecule.
  • a first antigen binding moiety which is a crossover Fab molecule capable of specific binding to CD3 comprising a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 36 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 31.
  • a second antigen binding moiety which is a Fab molecule capable of specific binding to Folate Receptor 1 (FolR1) comprising a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 49 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 51.
  • a third antigen binding moiety which is a Fab molecule capable of specific binding to Folate Receptor 1 (FolR1) comprising a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 49 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 51.
  • the second antigen binding moiety and the third antigen binding moiety are both a conventional Fab molecule.
  • the invention relates to bispecific molecules wherein two binding moieties confer specific binding to FolR1 and one binding moiety confers specificity to the T cell activating antigen CD3.
  • One of the heavy chains is modified to ensure proper pairing of the heavy and light chains, thus eliminating the need for a common light chain approach.
  • the presence of two FolR1 binding sites enables appropriate engagement with the target antigen FolR1 and the activation of T cells.
  • T cell activating bispecific antigen binding molecule can be fused to each other in a variety of configurations. Exemplary configurations are depicted in FIGS. 1A-I and are further described below.
  • said T cell activating bispecific antigen binding molecule further comprises an Fc domain composed of a first and a second subunit capable of stable association.
  • Fc domain composed of a first and a second subunit capable of stable association.
  • the T cell activating bispecific antigen binding molecules comprise at least two Fab fragments having identical light chains (VLCL) and having different heavy chains (VHCL) which confer the specificities to two different antigens, i.e. one Fab fragment is capable of specific binding to a T cell activating antigen CD3 and the other Fab fragment is capable of specific binding to the target cell antigen FolR1.
  • VLCL identical light chains
  • VHCL variable heavy chains
  • the T cell activating bispecific antigen binding molecule comprises at least two antigen binding moieties (Fab molecules), one of which is a crossover Fab molecule and one of which is a conventional Fab molecule.
  • Fab molecules antigen binding moieties
  • the first antigen binding moiety capable of specific binding to CD3 is a crossover Fab molecule
  • the second antigen binding moiety capable of specific binding to FolR is a conventional Fab molecule.
  • T cell activating bispecific antigen binding molecule can be fused to each other in a variety of configurations. Exemplary configurations are depicted in FIGS. 1A-I .
  • the first and second antigen binding moiety are each 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 essentially consists of a first and a second antigen binding moiety, an Fc domain composed of a first and a second subunit, and optionally one or more peptide linkers, wherein the first and second antigen binding moiety are each 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 first and second antigen binding moiety both are Fab fragments and have identical light chains (VLCL).
  • the first antigen binding moiety capable of specific binding to CD3 is a crossover Fab molecule and the second antigen binding moiety capable of specific binding to FolR is a conventional Fab molecule.
  • 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 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.
  • the T cell activating bispecific antigen binding molecule essentially consists of a first and a second antigen binding moiety, an Fc domain composed of a first and a second subunit, and optionally one or more peptide linkers, wherein the first 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 first and second antigen binding moiety both are Fab fragments and have identical light chains (VLCL).
  • the first antigen binding moiety capable of specific binding to CD3 is a crossover Fab molecule and the second antigen binding moiety capable of specific binding to FolR is a conventional Fab molecule.
  • the Fab light chain of the first antigen binding moiety and the Fab light chain of the second antigen binding moiety may additionally be fused to each other.
  • the 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.
  • 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 T cell activating bispecific antigen binding molecule essentially consists of a first and a second antigen binding moiety, an Fc domain composed of a first and a second subunit, and optionally one or more peptide linkers, wherein the 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.
  • the first and second antigen binding moiety both are Fab fragments and have identical light chains (VLCL).
  • the first antigen binding moiety capable of specific binding to CD3 is a crossover Fab molecule and the second antigen binding moiety capable of specific binding to FolR is a conventional Fab molecule.
  • the Fab light chain of the first antigen binding moiety and the Fab light chain of the second antigen binding moiety may additionally be fused to each other.
  • the 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, (G 4 S) n (SEQ ID NO: 300), (SG 4 ) n (SEQ ID NO: 301), (G 4 S) n (SEQ ID NO: 300) or G 4 (SG 4 ) n (SEQ ID NO: 302) 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 (G 4 S) 2 (SEQ ID NO: 303).
  • An exemplary peptide linker suitable for connecting the Fab heavy chains of the first and the second antigen binding moiety is EPKSC(D)-(G 4 S) 2 (SEQ ID NOS 304 and 305).
  • linkers may comprise (a portion of) an immunoglobulin hinge region. Particularly where an antigen binding moiety is fused to the N-terminus of an Fc domain subunit, it may be fused via an immunoglobulin hinge region or a portion thereof, with or without an additional peptide linker.
  • T cell activating bispecific antigen binding molecule comprising two binding moieties specific for the target cell antigen FolR have superior characteristics compared to T cell activating bispecific antigen binding molecule comprising only one binding moiety specific for the target cell antigen FolR.
  • the T cell activating bispecific antigen binding molecule of the invention further comprises a third antigen binding moiety which is a Fab molecule capable of specific binding to FolR.
  • the second and third antigen binding moiety capable of specific binding to FolR1 comprise identical heavy chain complementarity determining region (CDR) and light chain CDR sequences.
  • the third antigen binding moiety is identical to the second antigen binding moiety (i.e. they comprise the same amino acid sequences).
  • the first and second antigen binding moiety are each fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain and the third antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus to the N-terminus of the Fab heavy chain of the first antigen binding moiety.
  • 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 first and second antigen binding moiety are each fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain and the third 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 first, second and third antigen binding moiety are conventional Fab fragments and have identical light chains (VLCL).
  • the first antigen binding moiety capable of specific binding to CD3 is a crossover Fab molecule and the second and third antigen binding moiety capable of specific binding to FolR is a conventional Fab molecule.
  • the Fab light chain of the first antigen binding moiety and the Fab light chain of the third antigen binding moiety may additionally be fused to each other.
  • the T cell activating bispecific antigen binding molecule of the invention comprises five polypeptide chains that form a first, a second and a third antigen binding moiety wherein the first antigen binding moiety is capable of binding CD3 and the second and the third antigen binding moiety each are capable of binding Folate Receptor 1 (FolR1).
  • the first and the second polypeptide chain comprise, in amino (N)-terminal to carboxyl (C)-terminal direction, a first light chain variable domain (VLD1) and a first light chain constant domain (CLD1).
  • the third polypeptide chain comprises, in N-terminal to C-terminal direction, second light chain variable domain (VLD2) and a second heavy chain constant domain 1 (CH1D2).
  • the fourth polypeptide chain comprises, in N-terminal to C-terminal direction, a first heavy chain variable domain (VHD1), a first heavy chain constant domain 1 (CH1D1), a first heavy chain constant domain 2 (CH2D1) and a first heavy chain constant domain 3 (CH3D1).
  • the fifth polypeptide chain comprises VHD1, CH1D1, a second heavy chain variable domain (VHD2), a second light chain constant domain (CLD2), a second heavy chain constant domain 2 (CH2D2) and a second heavy chain constant domain 3 (CH3D2).
  • the third polypeptide chain and VHD2 and CLD2 of the fifth polypeptide chain form the first antigen binding moiety capable of binding CD3.
  • the second polypeptide chain and VHD1 and CH1D1 of the fifth polypeptide chain form the third binding moiety capable of binding to FolR1.
  • the first polypeptide chain and VHD1 and CH1D1 of the fourth polypeptide chain form the second binding moiety capable of binding to FolR1.
  • 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 the first or second subunit 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.
  • 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 and third antigen binding moiety are each fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit 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 third antigen binding moiety.
  • the first, second and third antigen binding moiety are conventional Fab fragments and have identical light chains (VLCL).
  • the first antigen binding moiety capable of specific binding to CD3 is a crossover Fab molecule and the second and third antigen binding moiety capable of specific binding to FolR is a conventional Fab molecule.
  • the Fab light chain of the first antigen binding moiety and the Fab light chain of the second antigen binding moiety may additionally be fused to each other.
  • the antigen binding moieties may be fused to the Fc domain directly or through a peptide linker.
  • the antigen binding moieties are each fused to the Fc domain through an immunoglobulin hinge region.
  • the immunoglobulin hinge region is a human IgG 1 hinge region.
  • the first and the second antigen binding moiety and the Fc domain are part of an immunoglobulin molecule.
  • the immunoglobulin molecule is an IgG class immunoglobulin.
  • the immunoglobulin is an IgG 1 subclass immunoglobulin.
  • the immunoglobulin is an IgG 4 subclass immunoglobulin.
  • the immunoglobulin is a human immunoglobulin.
  • the immunoglobulin is a chimeric immunoglobulin or a humanized immunoglobulin.
  • said T cell activating bispecific antigen binding molecule the first and the second antigen binding moiety and the Fc domain are part of an immunoglobulin molecule, and the third 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, wherein the first, second and third antigen binding moiety are conventional Fab fragments and have identical light chains (VLCL), wherein the first antigen binding moiety capable of specific binding to CD3 comprises at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39 and at least one light chain CDR selected from the group of SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34; and the second and the third antigen binding moiety capable of specific binding to FolR1 comprise at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO:
  • said T cell activating bispecific antigen binding molecule the first and the second antigen binding moiety and the Fc domain are part of an immunoglobulin molecule, and the third 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, wherein the first, second and third antigen binding moiety are conventional Fab fragments and have identical light chains (VLCL), wherein the first antigen binding moiety capable of specific binding to CD3 comprises a variable heavy chain comprising a sequence of SEQ ID NO: 36, a variable light chain comprising a sequence of SEQ ID NO: 31; and the second and the third antigen binding moiety capable of specific binding to FolR1 comprise a variable heavy chain comprising a sequence of SEQ ID NO: 15, a variable light chain comprising a sequence of SEQ ID NO: 31.
  • said T cell activating bispecific antigen binding molecule the first and the second antigen binding moiety and the Fc domain are part of an immunoglobulin molecule, and the third 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 capable of specific binding to CD3 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, comprising at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39 and at least one light chain CDR selected from the group of SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34; and the second and the third antigen binding moiety capable of specific binding to FolR1 comprise at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SDR) selected from
  • said T cell activating bispecific antigen binding molecule the first and the second antigen binding moiety and the Fc domain are part of an immunoglobulin molecule, and the third 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 capable of specific binding to CD3 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 the first antigen binding moiety capable of specific binding to CD3 comprises a variable heavy chain comprising a sequence of SEQ ID NO: 36, a variable light chain comprising a sequence of SEQ ID NO: 31; and the second and the third antigen binding moiety capable of specific binding to FolR1 comprise a variable heavy chain comprising a sequence of SEQ ID NO: 55, a variable light chain comprising a sequence of SEQ ID NO: 65.
  • the T cell activating bispecific antigen binding molecule is monovalent for each antigen.
  • the T cell activating bispecific antigen binding molecule can bind to human CD3 and human folate receptor alpha (FolR1) and was made without employing a hetero-dimerization approach, such as, e.g., knob-into-hole technology.
  • the molecule can be produced by employing a common light chain library and CrossMab technology.
  • the variable region of the CD3 binder is fused to the CH1 domain of a standard human IgG1 antibody to form the VLVH crossed molecule (fused to Fc) which is common for both specificities.
  • VHCL To generate the crossed counterparts (VHCL), a CD3 specific variable heavy chain domain is fused to a constant human ⁇ light chain whereas a variable heavy chain domain specific for human FolR1 (e.g., isolated from a common light chain library) is fused to a constant human ⁇ light chain.
  • the resulting desired molecule with correctly paired chains comprises both kappa and lambda light chains or fragments thereof. Consequently, this desired bispecific molecule species can be purified from mispaired or homodimeric species with sequential purification steps selecting for kappa and lambda light chain, in either sequence.
  • purification of the desired bispecific antibody employs subsequent purification steps with KappaSelect and LambdaFabSelect columns (GE Healthcare) to remove undesired homodimeric antibodies.
  • 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.
  • 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.
  • the T cell activating bispecific antigen binding molecule of the invention comprises not more than one Fc domain.
  • the Fc domain of the T cell activating bispecific antigen binding molecule is an IgG Fc domain.
  • the Fc domain is an IgG 1 Fc domain.
  • the Fc domain is an IgG 4 Fc domain.
  • the Fc domain is an IgG 4 Fc domain comprising an amino acid substitution at position S228 (Kabat numbering), particularly the amino acid substitution S228P. This amino acid substitution reduces in vivo Fab arm exchange of IgG 4 antibodies (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)).
  • the Fc domain is human. An exemplary sequence of a human IgG 1 Fc region is given in SEQ ID NO:245.
  • T cell activating bispecific antigen binding molecules comprise different antigen binding moieties, fused to one or the other of the two subunits of the Fc domain, thus the two subunits of the Fc domain are typically comprised in two non-identical polypeptide chains. Recombinant co-expression of these polypeptides and subsequent dimerization leads to 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 promoting the association of the desired polypeptides.
  • the Fc domain of the T cell activating bispecific antigen binding molecule comprises a modification promoting the association of the first and the second subunit of the Fc domain.
  • the site of most extensive protein-protein interaction between the two subunits of a human IgG Fc domain is in the CH3 domain of the Fc domain.
  • said modification is in the CH3 domain of the Fc domain.
  • said modification is a so-called “knob-into-hole” modification, comprising a “knob” modification in one of the two subunits of the Fc domain and a “hole” modification in the other one of the two subunits of the Fc domain.
  • the knob-into-hole technology is described e.g. in U.S. Pat. No. 5,731,168; U.S. Pat. No. 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001).
  • the method involves introducing a protuberance (“knob”) at the interface of a first polypeptide and a corresponding cavity (“hole”) in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote 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.
  • Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine).
  • 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 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.
  • the protuberance and cavity can be made by altering the nucleic acid encoding the polypeptides, e.g. by site-specific mutagenesis, or by peptide synthesis.
  • the threonine residue at position 366 in the CH3 domain of the first subunit of the Fc domain the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the CH3 domain of the second subunit of the Fc domain the tyrosine residue at position 407 is replaced with a valine residue (Y407V).
  • the threonine residue at position 366 in the second subunit of the Fc domain additionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A).
  • the serine residue at position 354 is replaced with a cysteine residue (S354C)
  • the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C).
  • the antigen binding moiety capable of binding to CD3 is fused (optionally via the antigen binding moiety capable of binding to FolR1 on a target cell antigen) to the first subunit of the Fc domain (comprising the “knob” modification).
  • fusion of the antigen binding moiety capable of binding to CD3 to the knob-containing subunit of the Fc domain will (further) minimize the generation of antigen binding molecules comprising two antigen binding moieties capable of binding to CD3 (steric clash of two knob-containing polypeptides).
  • a modification promoting 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 WO 2009/089004.
  • this method involves replacement of one or more amino acid residues at the interface of the two Fc domain subunits by charged amino acid residues so that homodimer formation becomes electrostatically unfavorable but heterodimerization electrostatically favorable.
  • the Fc domain confers to the T cell activating bispecific antigen binding molecule 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 receptor-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.
  • the Fc domain of the T cell activating bispecific antigen binding molecules according to the invention exhibits reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a native IgG 1 Fc domain.
  • the Fc domain (or the T cell activating bispecific antigen binding molecule comprising said Fc domain) exhibits less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% of the binding affinity to an Fc receptor, as compared to a native IgG 1 Fc domain (or a T cell activating bispecific antigen binding molecule comprising a native IgG 1 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 IgG 1 Fc domain domain (or a T cell activating bispecific antigen binding molecule comprising a native IgG 1 Fc domain).
  • the Fc domain domain (or the T cell activating bispecific antigen binding molecule comprising said Fc domain) does not substantially bind to an Fc receptor and/or induce effector function.
  • the Fc receptor is an Fc ⁇ receptor.
  • the Fc receptor is a human Fc receptor.
  • the Fc receptor is an activating Fc receptor.
  • the Fc receptor is an activating human Fc ⁇ receptor, more specifically human Fc ⁇ RIIIa, Fc ⁇ RI or Fc ⁇ RIIa, most specifically human Fc ⁇ RIIIa.
  • 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.
  • the Fc domain domain exhibits substantially similar binding affinity to neonatal Fc receptor (FcRn), as compared to a native IgG 1 Fc domain domain.
  • FcRn neonatal Fc receptor
  • Substantially similar binding to FcRn is achieved when the Fc domain (or the T cell activating bispecific antigen binding molecule comprising said Fc domain) exhibits greater than about 70%, particularly greater than about 80%, more particularly greater than about 90% of the binding affinity of a native IgG 1 Fc domain (or the T cell activating bispecific antigen binding molecule comprising a native IgG 1 Fc domain) to FcRn.
  • 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.
  • the Fc domain of the T cell activating bispecific antigen binding molecule comprises one or more amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor and/or effector function. Typically, the same one or more amino acid mutation is present in each of the two subunits of the Fc domain.
  • the amino acid mutation reduces the binding affinity of the Fc domain to an Fc receptor.
  • the amino acid mutation reduces the binding affinity of the Fc domain to an Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold.
  • the combination 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.
  • the T cell activating bispecific antigen binding molecule comprising an engineered Fc domain exhibits less than 20%, particularly less than 10%, more particularly less than 5% of the binding affinity to an Fc receptor as compared to a T cell activating bispecific antigen binding molecule comprising a non-engineered Fc domain.
  • the Fc receptor is an Fc ⁇ receptor.
  • the Fc receptor is a human Fc receptor.
  • the Fc receptor is an activating Fc receptor.
  • the Fc receptor is an activating human Fc ⁇ receptor, more specifically human Fc ⁇ RIIIa, Fc ⁇ RI or Fc ⁇ RIIa, most specifically human Fc ⁇ RIIIa.
  • binding to each of these receptors is reduced.
  • binding affinity to a complement component, specifically binding affinity to C1q is also reduced.
  • binding affinity to neonatal Fc receptor (FcRn) is not reduced. Substantially similar binding to FcRn, i.e.
  • the Fc domain (or the T cell activating bispecific antigen binding molecule comprising said Fc domain) exhibits greater than about 70% of the binding affinity of a non-engineered form of the Fc domain (or the T cell activating bispecific antigen binding molecule comprising said non-engineered form of the Fc domain) to FcRn.
  • the Fc domain, or T cell activating bispecific antigen binding molecules of the invention comprising said Fc domain may exhibit greater than about 80% and even greater than about 90% of such affinity.
  • the Fc domain of the T cell activating bispecific antigen binding molecule is engineered to have reduced effector function, 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: reduced complement dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (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 monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling inducing apoptosis, reduced crosslinking of target-bound antibodies, reduced dendritic cell maturation, or reduced T cell priming.
  • CDC complement dependent cytotoxicity
  • ADCC reduced antibody-dependent cell-mediated cytotoxicity
  • ADCP reduced antibody-dependent cellular phagocytosis
  • reduced immune complex-mediated antigen uptake by antigen-presenting cells reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling inducing
  • the reduced effector function is one or more selected from the group of reduced CDC, reduced ADCC, reduced ADCP, and reduced cytokine secretion. In a particular embodiment the reduced effector function is reduced ADCC. In one embodiment the reduced ADCC is less than 20% of the ADCC induced by a non-engineered Fc domain (or a T cell activating bispecific antigen binding molecule comprising a non-engineered Fc domain).
  • the amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor and/or effector function is an amino acid substitution.
  • the Fc domain comprises an amino acid substitution at a position selected from the group of E233, L234, L235, N297, P331 and P329.
  • the Fc domain comprises an amino acid substitution at a position selected from the group of L234, L235 and P329.
  • the Fc domain comprises the amino acid substitutions L234A and L235A.
  • the Fc domain is an IgG 1 Fc domain, particularly a human IgG 1 Fc domain.
  • the Fc domain comprises an amino acid substitution at position P329.
  • the amino acid substitution is P329A or P329G, particularly P329G.
  • the Fc domain comprises an amino acid substitution at position P329 and a further amino acid substitution at a position selected from E233, L234, L235, N297 and P331.
  • the further amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S.
  • the Fc domain comprises amino acid substitutions at positions P329, L234 and L235.
  • the Fc domain comprises the amino acid mutations L234A, L235A and P329G (“P329G LALA”).
  • the Fc domain is an IgG 1 Fc domain, particularly a human IgG 1 Fc domain.
  • the “P329G LALA” combination of amino acid substitutions almost completely abolishes Fc ⁇ receptor binding of a human IgG 1 Fc domain, as described in PCT publication no. WO 2012/130831, incorporated herein by reference in its entirety.
  • WO 2012/130831 also describes methods of preparing such mutant Fc domains and methods for determining its properties such as Fc receptor binding or effector functions.
  • the Fc domain of the T cell activating bispecific antigen binding molecules of the invention is an IgG 4 Fc domain, particularly a human IgG 4 Fc domain.
  • the IgG 4 Fc domain comprises amino acid substitutions at position S228, specifically the amino acid substitution S228P.
  • the IgG 4 Fc domain comprises an amino acid substitution at position L235, specifically the amino acid substitution L235E.
  • the IgG 4 Fc domain comprises an amino acid substitution at position P329, specifically the amino acid substitution P329G.
  • the IgG 4 Fc domain comprises amino acid substitutions at positions S228, L235 and P329, specifically amino acid substitutions S228P, L235E and P329G.
  • IgG 4 Fc domain mutants and their Fc ⁇ receptor binding properties are described in PCT publication no. WO 2012/130831, incorporated herein by reference in its entirety.
  • the Fc domain exhibiting reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a native IgG 1 Fc domain is a human IgG 1 Fc domain comprising the amino acid substitutions L234A, L235A and optionally P329G, or a human IgG 4 Fc domain comprising the amino acid substitutions S228P, L235E and optionally P329G.
  • N-glycosylation of the Fc domain has been eliminated.
  • the Fc domain comprises an amino acid mutation at position N297, particularly an amino acid substitution replacing asparagine by alanine (N297A) or aspartic acid (N297D).
  • Fc domains with reduced Fc receptor binding and/or effector function also include those with substitution of one or more of Fc domain residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. No. 6,737,056).
  • 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 (U.S. Pat. No. 7,332,581).
  • Mutant Fc domains can be prepared by amino acid deletion, substitution, insertion or modification using genetic or chemical methods well known in the art. Genetic methods may include site-specific mutagenesis of the encoding DNA sequence, PCR, gene synthesis, and the like. The correct nucleotide changes can be verified for example by sequencing.
  • Binding to Fc receptors can be easily determined e.g. by ELISA, or by Surface Plasmon Resonance (SPR) using standard instrumentation such as a BIAcore instrument (GE Healthcare), and Fc receptors such as may be obtained by recombinant expression. A suitable such binding assay is described herein. Alternatively, binding affinity of Fc domains or cell activating bispecific antigen 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 Fc ⁇ IIIa receptor.
  • Effector function of an Fc domain, or a T cell activating bispecific antigen binding molecule comprising an Fc domain can be measured by methods known in the art.
  • a suitable assay for measuring ADCC is described herein.
  • Other examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. 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. Pat. No. 5,821,337; Bruggemann et al., J Exp Med 166, 1351-1361 (1987).
  • non-radioactive assays methods may be employed (see, for example, ACTITM non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, Calif.); and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wis.)).
  • Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
  • PBMC peripheral blood mononuclear cells
  • NK Natural Killer
  • 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 (1998).
  • binding of the Fc domain to a complement component, specifically to C1q is reduced.
  • said reduced effector function includes reduced CDC.
  • C1q binding assays may be carried out to determine whether the T cell activating bispecific antigen binding molecule is able to bind C1q and hence has CDC activity. See e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402.
  • a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et al., Blood 101, 1045-1052 (2003); and Cragg and Glennie, Blood 103, 2738-2743 (2004)).
  • One of skill in the art can appreciate the advantageous efficiency of a molecule that selectively distinguishes between cancerous and non-cancerous, healthy cells.
  • One way to accomplish this goal is by appropriate target selection. Markers expressed exclusively on tumor cells can be employed to selectively target effector molecules or cells to tumor cells while sparing normal cells that do not express such marker. However, in some instances, so called tumor cell markers are also expressed in normal tissue, albeit at lower levels. This expression in normal tissue raises the possibility of toxicity. Thus, there was a need in the art for molecules that can more selectively target tumor cells.
  • the invention described herein provides for T cell activating bispecific antigen binding molecules that selectively target FolR1-positive tumor cells and not normal, non-cancerous cells that express FolR1 at low levels or not at all.
  • the T cell activating bispecific antigen binding molecule comprises at least two, preferably two, FolR1 binding moieties of relatively low affinity that confer an avidity effect which allows for differentiation between high and low FolR1 expressing cells. Because tumor cells express FolR1 at high or intermediate levels, this embodiment of the invention selectively binds to, and/or induces killing of, tumor cells and not normal, non-cancerous cells that express FolR1 at low levels or not at all. In one embodiment, the T cell activating bispecific antigen binding molecule is in the 2+1 inverted format.
  • the T cell activating bispecific antigen binding molecule induces T cell mediated killing of FolR1-positive tumor cells and not non-tumor cells and comprises a CD3 antigen binding moiety that comprises the heavy chain CDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO: 38, the heavy chain CDR3 of SEQ ID NO:39, the light chain CDR1 of SEQ ID NO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3 of SEQ ID NO:34 and two FolR1 antigen binding moieties that each comprise the heavy chain CDR1 of SEQ ID NO: 8, the heavy chain CDR2 of SEQ ID NO: 9, the heavy chain CDR3 of SEQ ID NO:50, the light chain CDR1 of SEQ ID NO: 52, the light chain CDR2 of SEQ ID NO: 53, and the light chain CDR3 of SEQ ID NO:54.
  • a CD3 antigen binding moiety that comprises the heavy chain CDR1 of SEQ ID NO:
  • the T cell activating bispecific antigen binding molecule does not induce killing of a normal cells having less than about 1000 copies of FolR1 its surface.
  • the invention does not require chemical cross linking or a hybrid approach to be produced. Accordingly, in one embodiment, the invention provides for T cell activating bispecific antigen binding molecule capable of production in CHO cells. In one embodiment, the T cell activating bispecific antigen binding molecule comprises humanized and human polypeptides. In one embodiment, the T cell activating bispecific antigen binding molecule does not cause FcgR crosslinking.
  • the T cell activating bispecific antigen binding molecule is capable of production in CHO cells and comprises a CD3 antigen binding moiety that comprises the heavy chain CDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO: 38, the heavy chain CDR3 of SEQ ID NO:39, the light chain CDR1 of SEQ ID NO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3 of SEQ ID NO:34 and two FolR1 antigen binding moieties that each comprise the heavy chain CDR1 of SEQ ID NO: 8, the heavy chain CDR2 of SEQ ID NO: 9, the heavy chain CDR3 of SEQ ID NO:50, the light chain CDR1 of SEQ ID NO: 52, the light chain CDR2 of SEQ ID NO: 53, and the light chain CDR3 of SEQ ID NO:54.
  • a CD3 antigen binding moiety that comprises the heavy chain CDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO: 38
  • some embodiments contemplated herein include T cell activating bispecific antigen binding molecules having two binding moieties that confer specific binding to FolR1 and one binding moiety that confers specificity to the T cell activating antigen CD3, wherein each individual FolR1 binding moiety engages the antigen with low affinity. Because the molecule comprises two antigen binding moieties that confer binding to FolR1, the overall avidity of the molecule, nevertheless, provides effective binding to FolR1-expressing target cells and activation of T cells to induce T cell effector function.
  • the T cell activating bispecific antigen binding molecules have a monovalent binding affinity to huFolR1 in the micromolar range and an avidity to huFolR1 in the nanomolar range.
  • the T cell activating bispecific antigen binding molecule binds human FolR1 with an apparent K D of about 10 nM to about 40 nM. In one embodiment, the T cell activating bispecific antigen binding molecule binds human FolR1 with an apparent K D of about 10 nM. In one embodiment, the T cell activating bispecific antigen binding molecule binds human and cynomolgus FolR1 with an apparent K D of about 10 nM and about 30 nM, respectively. In one embodiment, the T cell activating bispecific antigen binding molecule binds human FolR1 with a monovalent binding K D of at least about 1000 nM.
  • the T cell activating bispecific antigen binding molecule binds human FolR1 with a monovalent binding K D of about 1400 nM. In one embodiment, the T cell activating bispecific antigen binding molecule binds human FolR1 with a monovalent binding K D of about 1400 nM and to cynomolgus FolR1 with a monovalent binding K D of about 5600 nM. In one embodiment, the T cell activating bispecific antigen binding molecule binds human FolR1 with an apparent K D of about 10 nM and with a monovalent binding K D of about 1400 nM.
  • the T cell activating bispecific antigen binding molecule binds human FolR1 with an apparent K D of about 5.36 pM to about 4 nM. In one embodiment, the T cell activating bispecific antigen binding molecule binds human and cynomolgus FolR1 with an apparent K D of about 4 nM. In one embodiment, the T cell activating bispecific antigen binding molecule binds murine FolR1 with an apparent K D of about 1.5 nM. In one embodiment, the T cell activating bispecific antigen binding molecule binds human FolR1 with a monovalent binding K D of at least about 1000 nM.
  • the T cell activating bispecific antigen binding molecule binds human and cynomolgus FolR1 with an apparent K D of about 4 nM, binds murine FolR1 with an apparent K D of about 1.5 nM, and comprises a CD3 antigen binding moiety that comprises the heavy chain CDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO: 38, the heavy chain CDR3 of SEQ ID NO:39, the light chain CDR1 of SEQ ID NO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3 of SEQ ID NO:34 and two FolR1 antigen binding moieties that each comprise the heavy chain CDR1 of SEQ ID NO: 8, the heavy chain CDR2 of SEQ ID NO: 9, the heavy chain CDR3 of SEQ ID NO:50, the light chain CDR1 of SEQ ID NO: 52, the light chain CDR2 of SEQ ID NO: 53, and the light chain CDR3 of SEQ
  • the T cell activating bispecific antigen binding molecule binds human FolR1 with a monovalent binding K D of at least about 1000 nM and comprises a CD3 antigen binding moiety that comprises the heavy chain CDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO: 38, the heavy chain CDR3 of SEQ ID NO:39, the light chain CDR1 of SEQ ID NO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3 of SEQ ID NO:34 and two FolR1 antigen binding moieties that each comprise the heavy chain CDR1 of SEQ ID NO: 8, the heavy chain CDR2 of SEQ ID NO: 9, the heavy chain CDR3 of SEQ ID NO:50, the light chain CDR1 of SEQ ID NO: 52, the light chain CDR2 of SEQ ID NO: 53, and the light chain CDR3 of SEQ ID NO:54.
  • a CD3 antigen binding moiety that comprises the heavy chain CDR1 of SEQ ID NO:
  • the T cell activating bispecific antigen binding molecules contemplated herein can induce T cell effector function, e.g., cell surface marker expression, cytokine production, T cell mediated killing.
  • the T cell activating bispecific antigen binding molecule induces T cell mediated killing of the FolR1-expressing target cell, such as a human tumor cell, in vitro.
  • the T cell is a CD8 positive T cell.
  • FolR1-expressing human tumor cells include but are not limited to Hela, Skov-3, HT-29, and HRCEpiC cells. Other FolR1 positive human cancer cells that can be used for in vitro testing are readily available to the skilled artisan.
  • the T cell activating bispecific antigen binding molecule induces T cell mediated killing of the FolR1-expressing human tumor cell in vitro with an EC50 of between about 36 pM and about 39573 pM after 24 hours.
  • T cell activating bispecific antigen binding molecules that induce T cell mediated killing of the FolR1-expressing tumor cell in vitro with an EC50 of about 36 pM after 24 hours.
  • the T cell activating bispecific antigen binding molecule induces T cell mediated killing of the FolR1-expressing tumor cell in vitro with an EC50 of about 178.4 pM after 24 hours.
  • the T cell activating bispecific antigen binding molecule induces T cell mediated killing of the FolR1-expressing tumor cell in vitro with an EC50 of about 134.5 pM or greater after 48 hours.
  • the EC50 can be measure by methods known in the art, for example by methods disclosed herein by the examples.
  • the T cell activating bispecific antigen binding molecule of any of the above embodiments induces upregulation of cell surface expression of at least one of CD25 and CD69 on the T cell as measured by flow cytometry.
  • the T cell is a CD4 positive T cell or a CD8 positive T cell.
  • the T cell activating bispecific antigen binding molecule of any of the above embodiments binds to FolR1 expressed on a human tumor cell. In one embodiment, the T cell activating bispecific antigen binding molecule of any of the above embodiments binds to a conformational epitope on human FolR1. In one embodiment, the T cell activating bispecific antigen binding molecule of any of the above embodiments does not bind to human Folate Receptor 2 (FolR2) or to human Folate Receptor 3 (FolR3).
  • the antigen binding moiety binds to a FolR1 polypeptide comprising the amino acids 25 to 234 of human FolR1 (SEQ ID NO:227).
  • the FolR1 antigen binding moiety binds to a FolR1 polypeptide comprising the amino acid sequence of SEQ ID NO:227, to a FolR1 polypeptide comprising the amino acid sequence of SEQ ID NO:230 and to a FolR1 polypeptide comprising the amino acid sequence of SEQ ID NO:231, and wherein the FolR1 antigen binding moiety does not bind to a FolR polypeptide comprising the amino acid sequence of SEQ ID NOs:228 or 229.
  • the T cell activating bispecific antigen binding molecule comprises a FolR1 antigen binding moiety that binds to a FolR1 polypeptide comprising the amino acid sequence of SEQ ID NOs:227, 230 and 231, and wherein the FolR1 antigen binding moiety does not bind to a FolR polypeptide comprising the amino acid sequence of SEQ ID NOs:228 or 229, and comprises a CD3 antigen binding moiety that comprises the heavy chain CDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO: 38, the heavy chain CDR3 of SEQ ID NO:39, the light chain CDR1 of SEQ ID NO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3 of SEQ ID NO:34 and two FolR1 antigen binding moieties that each comprise the heavy chain CDR1 of SEQ ID NO: 8, the heavy chain CDR2 of SEQ ID NO: 9, the heavy chain CDR3 of SEQ ID NO:50
  • the FolR1 antigen binding moiety binds to a FolR1 polypeptide comprising the amino acid sequence of SEQ ID NO:227 and to a FolR1 polypeptide comprising the amino acid sequence of SEQ ID NO:231, and wherein the FolR1 antigen binding moiety does not bind to a FolR polypeptide comprising the amino acid sequence of SEQ ID NOs:228, 229 or 230.
  • the T cell activating bispecific antigen binding molecule comprises a FolR1 antigen binding moiety that binds to a FolR1 polypeptide comprising the amino acid sequence of SEQ ID NO:227 and to a FolR1 polypeptide comprising the amino acid sequence of SEQ ID NO:231, and wherein the FolR1 antigen binding moiety does not bind to a FolR polypeptide comprising the amino acid sequence of SEQ ID NOs:228, 229 or 230, and comprises a CD3 antigen binding moiety that comprises the heavy chain CDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO: 38, the heavy chain CDR3 of SEQ ID NO:39, the light chain CDR1 of SEQ ID NO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3 of SEQ ID NO:34 and two FolR1 antigen binding moieties that each comprise the heavy chain CDR1 of SEQ ID NO: 16, the heavy chain CDR2 of
  • the T cell activating bispecific antigen binding molecules contemplated herein can have agonist, antagonist or neutral effect.
  • agonist effect include induction or enhancement of signaling through the FolR1 upon engagement by the FolR1 binding moiety with the FolR1 receptor on the target cell.
  • antagonist activity include abrogation or reduction of signaling through the FolR1 upon engagement by the FolR1 binding moiety with the FolR1 receptor on the target cell. This can, for example, occur by blocking or reducing the interaction between folate with FolR1. Sequence variants of the embodiments disclosed herein having lower affinity while retaining the above described biological properties are specifically contemplated.
  • the invention also pertains to immunoconjugates comprising a T cell activating bispecific antigen binding molecule conjugated to a cytotoxic agent such as a chemotherapeutic agent, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • a cytotoxic agent such as a chemotherapeutic agent, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • the invention further provides isolated polynucleotides encoding a T cell activating bispecific antigen binding molecule as described herein or a fragment thereof.
  • Polynucleotides of the invention include those that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences set forth in SEQ ID NOs:151-226 including functional fragments or variants thereof.
  • the polynucleotides encoding T cell activating bispecific antigen binding molecules of the invention may be expressed as a single polynucleotide that encodes the entire T cell activating bispecific antigen binding molecule or as multiple (e.g., two or more) polynucleotides that are co-expressed. Polypeptides encoded by polynucleotides that are co-expressed may associate through, e.g., disulfide bonds or other means to form a functional T cell activating bispecific antigen binding molecule.
  • 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 binding moiety.
  • the heavy chain polypeptides When co-expressed, the heavy chain polypeptides will associate with the light chain polypeptides to form the antigen binding moiety.
  • the portion of the T cell activating bispecific antigen binding molecule comprising one of the two Fc domain subunits and optionally (part of) one or more antigen binding moieties could be encoded by a separate polynucleotide from the portion of the T cell activating bispecific antigen binding molecule comprising the the other of the two Fc domain subunits and optionally (part of) an antigen binding moiety. When co-expressed, the Fc domain subunits will associate to form the Fc domain.
  • the isolated polynucleotide encodes the entire T cell activating bispecific antigen binding molecule according to the invention as described herein. In other embodiments, the isolated polynucleotide encodes a polypeptides comprised in the T cell activating bispecific antigen binding molecule according to the invention as described herein.
  • the present invention is directed to an isolated polynucleotide encoding a T cell activating bispecific antigen binding molecule of the invention or a fragment thereof, wherein the polynucleotide comprises a sequence that encodes a variable region sequence as shown in SEQ ID NOs 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182 and 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223.
  • the present invention is directed to an isolated polynucleotide encoding a T cell activating bispecific antigen binding molecule or fragment thereof, wherein the polynucleotide comprises a sequence that encodes a polypeptide sequence as shown in SEQ ID NOs:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 1, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,
  • the invention is further directed to an isolated polynucleotide encoding a T cell activating bispecific antigen binding molecule of the invention 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 97, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 20
  • the invention is directed to an isolated polynucleotide encoding a T cell activating bispecific antigen binding molecule of the invention or a fragment thereof, wherein the polynucleotide comprises a nucleic acid sequence shown in SEQ ID NOs 97, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 12, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222,
  • the invention is directed to an isolated polynucleotide encoding a T cell activating bispecific antigen binding molecule of the invention or a fragment thereof, wherein the polynucleotide comprises a sequence that encodes a variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence in SEQ ID NOs 1, 2, 3, 4, 5, 6, 7, 11, 13, 15, 19, 21, 12, 25, 27, 29, 31, 36, 41, 45, 49, 51, 55, 58, 62, 64, 66, 68, 70, 72, 74, 76, 78, 82, 113, 114, 115, 116, 117, 118, 119, 12, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135.
  • the invention is directed to an isolated polynucleotide encoding a T cell activating bispecific antigen binding molecule or fragment thereof, wherein the polynucleotide comprises a sequence that encodes a polypeptide comprising one or more sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence in SEQ ID NOs: 8, 9, 50, 37, 38, and 39.
  • the invention encompasses an isolated polynucleotide encoding a T cell activating bispecific antigen binding molecule of the invention or a fragment thereof, wherein the polynucleotide comprises a sequence that encodes the variable region sequence of SEQ ID NOs 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182 and 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223 with conservative amino acid substitutions.
  • the invention also encompasses an isolated polynucleotide encoding a T cell activating bispecific antigen binding molecule of the invention or fragment thereof, wherein the polynucleotide comprises a sequence that encodes the polypeptide sequence of SEQ ID NOs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 1, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96
  • RNA for example, in the form of messenger RNA (mRNA).
  • mRNA messenger RNA
  • RNA of the present invention may be single stranded or double stranded.
  • T cell activating bispecific antigen binding molecules of the invention may be obtained, for example, by solid-state peptide synthesis (e.g. Merrifield solid phase synthesis) or recombinant production.
  • solid-state peptide synthesis e.g. Merrifield solid phase synthesis
  • Such polynucleotide may be readily isolated and sequenced using conventional procedures.
  • a vector, preferably an expression vector, comprising one or more of the polynucleotides of the invention is provided.
  • the expression vector can be part of a plasmid, virus, or may be a nucleic acid fragment.
  • the expression vector includes an expression cassette into which the polynucleotide encoding the T cell activating bispecific antigen binding molecule (fragment) (i.e. the coding region) is cloned in operable association with a promoter and/or other transcription or translation control elements.
  • a “coding region” is a portion of nucleic acid which consists of codons translated into amino acids.
  • 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 polynucleotide construct, e.g. on a single vector, or in separate polynucleotide constructs, e.g. on separate (different) vectors.
  • any vector may contain a single coding region, or may comprise two or more coding regions, e.g.
  • a vector of the present invention may encode one or more polypeptides, which are post- or co-translationally separated into the final proteins via proteolytic cleavage.
  • a vector, polynucleotide, or nucleic acid of the invention may encode heterologous coding regions, either fused or unfused to a polynucleotide encoding the T cell activating bispecific antigen binding molecule (fragment) of the invention, or variant or derivative thereof.
  • Heterologous coding regions include without limitation specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain. An operable association is when a coding region for a gene product, e.g.
  • a polypeptide is 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 fragments (such as a polypeptide coding region and a promoter 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 expression regulatory sequences to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed.
  • a promoter region would be operably associated with a nucleic acid encoding a polypeptide if the promoter was capable of effecting transcription of that nucleic acid.
  • the promoter may be a cell-specific promoter that directs substantial transcription of the DNA only in predetermined cells.
  • Other transcription control elements besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide to direct cell-specific transcription.
  • Suitable promoters and other transcription control regions are disclosed herein.
  • a variety of transcription control regions are known to those skilled in the art. These include, without limitation, transcription control regions, which function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (e.g. the immediate early promoter, in conjunction with intron-A), simian virus 40 (e.g.
  • transcription control regions include those derived 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 transcription 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.
  • the expression cassette may also include other features such as an origin of replication, and/or chromosome integration elements such as retroviral long terminal repeats (LTRs), or adeno-associated viral (AAV) inverted terminal repeats (ITRs).
  • LTRs retroviral long terminal repeats
  • AAV adeno-associated viral
  • Polynucleotide and nucleic acid coding regions of the present invention may be associated with additional coding regions which encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide of the present invention.
  • additional coding regions which encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide of the present invention.
  • DNA encoding a signal sequence may be placed upstream of the nucleic acid encoding a T cell activating bispecific antigen binding molecule of the invention or a fragment thereof.
  • proteins secreted by mammalian cells have a signal peptide or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated.
  • 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 polypeptide.
  • the native signal peptide e.g.
  • an immunoglobulin heavy chain or light chain signal peptide is used, or a functional derivative of that sequence that retains the ability to direct the secretion of the polypeptide that is operably associated with it.
  • a heterologous mammalian signal peptide, or a functional derivative thereof may be used.
  • the wild-type leader sequence may be substituted with the leader sequence of human tissue plasminogen activator (TPA) or mouse ⁇ -glucuronidase.
  • DNA encoding a short protein sequence that could be used to facilitate later purification (e.g. a histidine tag) or assist in labeling the T cell activating bispecific antigen binding molecule may be included within or at the ends of the T cell activating bispecific antigen binding molecule (fragment) encoding polynucleotide.
  • a host cell comprising one or more polynucleotides of the invention.
  • a host cell comprising one or more vectors of the invention.
  • the polynucleotides and vectors may incorporate any of the features, singly or in combination, described herein in relation to polynucleotides and vectors, respectively.
  • 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 binding molecule of the invention.
  • the term “host cell” refers to any kind of cellular system which can be engineered to generate the T cell activating bispecific antigen binding molecules of the invention or fragments thereof.
  • Host cells suitable for replicating and for supporting expression of T cell activating bispecific antigen binding molecules are well known in the art. Such cells may be transfected or transduced as appropriate with the particular expression vector and large quantities of vector containing cells can be grown for seeding large scale fermenters to obtain sufficient quantities of the T cell activating bispecific antigen binding molecule for clinical applications.
  • Suitable host cells include prokaryotic microorganisms, such as E. coli , or various eukaryotic cells, such as Chinese hamster ovary cells (CHO), insect cells, or the like.
  • 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 further purified.
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for polypeptide-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been “humanized”, resulting in the production of a polypeptide with a partially or fully human glycosylation pattern. See Gerngross, Nat Biotech 22, 1409-1414 (2004), and Li et al., Nat Biotech 24, 210-215 (2006).
  • Suitable host cells for the expression of (glycosylated) polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells.
  • baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.
  • Plant cell cultures can also be utilized as hosts. See e.g. U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIESTM technology for producing antibodies in transgenic plants).
  • Vertebrate cells may also be used as hosts.
  • mammalian cell lines that are adapted to grow in suspension may be useful.
  • 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 BBL 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.
  • MRC 5 cells MRC 5 cells
  • 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.
  • CHO Chinese hamster ovary
  • dhfr ⁇ CHO cells Urlaub et al., Proc Natl Acad Sci USA 77, 4216 (1980)
  • myeloma cell lines such as YO, NS0, P3X63 and Sp2/0.
  • Yazaki and Wu Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J.), pp. 255-268 (2003).
  • Host cells include cultured cells, e.g., mammalian cultured cells, yeast cells, insect cells, bacterial cells and plant cells, to name only a few, but also cells comprised within a transgenic animal, transgenic plant or cultured plant or animal tissue.
  • 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).
  • CHO Chinese Hamster Ovary
  • HEK human embryonic kidney
  • a lymphoid cell e.g., Y0, NS0, Sp20 cell.
  • 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.
  • a method of producing a T cell activating bispecific antigen binding molecule according to the invention comprises culturing a host cell comprising a polynucleotide encoding the T cell activating bispecific antigen binding molecule, as provided 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).
  • T cell activating bispecific antigen binding molecule The components of the T cell activating bispecific antigen binding molecule are genetically fused to each other.
  • T cell activating 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 determined in accordance with methods well known in the art and may be tested for efficacy. Examples of linker sequences between different components of T cell activating bispecific antigen binding molecules are found in the sequences provided herein. Additional sequences may also be included to incorporate a cleavage site to separate the individual components of the fusion if desired, for example an endopeptidase recognition sequence.
  • the one or more antigen binding moieties of the T cell activating bispecific antigen binding molecules comprise at least an antibody variable region capable 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 produce 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. Pat. 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. Pat. No. 5,969,108, McCafferty).
  • any animal species of antibody, antibody fragment, antigen binding domain or variable region can be used in the T cell activating bispecific antigen binding molecules of the invention.
  • Non-limiting antibodies, antibody fragments, antigen binding domains or variable regions useful in the present invention can be of murine, primate, or human origin. If the T cell activating 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 antibody can also be prepared in accordance with methods well known in the art (see e. g. U.S. Pat. No. 5,565,332 to Winter).
  • Humanization may be achieved by various methods including, but not limited to (a) grafting the non-human (e.g., donor antibody) CDRs onto human (e.g. recipient antibody) framework and constant 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) grafting 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 variable domains, but “cloaking” them with a human-like section by replacement of surface residues.
  • 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)
  • SDRs or a-CDRs the residues critical for the antibody-antigen interaction
  • Human antibodies and human variable regions 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 Lonberg, Curr Opin Immunol 20, 450-459 (2008). Human variable regions can form part of and be derived from human monoclonal antibodies made by the hybridoma method (see e.g. Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)). Human antibodies and human variable regions may also be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge (see e.g.
  • Human antibodies 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, N.J., 2001); and McCafferty et al., Nature 348, 552-554; Clackson et al., Nature 352, 624-628 (1991)). Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments.
  • scFv single-chain Fv
  • the antigen binding moieties useful in the present invention are engineered to have enhanced binding affinity according to, for example, the methods disclosed in U.S. Pat. Appl. Publ. No. 2004/0132066, the entire contents of which are hereby incorporated by reference.
  • the ability of the T cell activating bispecific antigen binding molecule of the invention to bind to a specific antigenic determinant can be measured either through an enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to one of skill in the art, e.g.
  • Competition assays may be used to identify an antibody, antibody fragment, antigen binding domain or variable domain that competes with a reference antibody for binding to a particular antigen, e.g. an antibody that competes with the V9 antibody for binding to CD3.
  • a competing antibody binds to the same epitope (e.g. a linear or a conformational epitope) that is bound by the reference antibody.
  • immobilized antigen e.g. CD3
  • a first labeled antibody that binds to the antigen (e.g. V9 antibody, described in U.S. Pat. No. 6,054,297)
  • a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to the antigen.
  • the second antibody may be present in a hybridoma supernatant.
  • immobilized antigen is incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody. After incubation under conditions permissive for binding of the first antibody to the antigen, excess unbound antibody is removed, and the amount of label associated with 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 antibody for binding to the antigen. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).
  • T cell activating bispecific antigen binding molecules prepared as described herein may be purified by art-known techniques such as high performance liquid chromatography, ion exchange 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 apparent to those having skill in the art.
  • affinity chromatography purification an antibody, ligand, receptor or antigen can be used to which the T cell activating bispecific antigen binding molecule binds.
  • a matrix with protein A or protein G may be used for affinity chromatography purification of T cell activating bispecific antigen binding molecules of the invention.
  • Sequential Protein A or G affinity chromatography and size exclusion chromatography can be used to isolate a T cell activating bispecific antigen binding molecule essentially as described in the Examples.
  • the purity of the T cell activating bispecific antigen binding molecule can be determined by any of a variety of well known analytical methods including gel electrophoresis, high pressure liquid chromatography, and the like.
  • 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. FIG. 2 ). Three bands were resolved at approximately Mr 25,000, Mr 50,000 and Mr 75,000, corresponding to the predicted molecular weights of the T cell activating bispecific antigen binding molecule light chain, heavy chain and heavy chain/light chain fusion protein.
  • T cell activating bispecific antigen binding molecules provided herein may be identified, screened for, or characterized for their physical/chemical properties and/or biological activities by various assays known in the art.
  • the affinity of the T cell activating bispecific antigen binding molecule for an Fc receptor or a target antigen can be determined in accordance with the methods set forth in the Examples by surface plasmon resonance (SPR), using standard instrumentation such as a BIAcore instrument (GE Healthcare), and receptors or target proteins such as may be obtained by recombinant expression.
  • SPR surface plasmon resonance
  • GE Healthcare BIAcore instrument
  • receptors or target proteins such as may be obtained by recombinant expression.
  • binding of T cell activating bispecific antigen binding molecules for different receptors or target antigens may be evaluated using cell lines expressing the particular receptor or target antigen, for example by flow cytometry (FACS).
  • FACS flow cytometry
  • K D is measured by surface plasmon resonance using a BIACORE® T100 machine (GE Healthcare) at 25° C.
  • CM5 chips To analyze the interaction between the Fc-portion and Fc receptors, His-tagged recombinant Fc-receptor is captured by an anti-Penta His antibody (“Penta His” disclosed as SEQ ID NO: 306) (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 (NETS) according to the supplier's instructions.
  • EDC N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride
  • NETS N-hydroxysuccinimide
  • Penta-His antibody (“Penta-His” disclosed as SEQ ID NO: 306) is diluted with 10 mM sodium acetate, pH 5.0, to 40 ⁇ g/ml before injection at a flow rate of 5 ⁇ l/min to achieve approximately 6500 response 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.
  • HBS-EP GE Healthcare, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Surfactant P20, pH 7.4
  • HBS-EP GE Healthcare, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Surfactant P20, pH 7.4
  • 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 antibody (“Penta-His” disclosed as SEQ ID NO: 306).
  • the final amount of coupled protein is approximately 12000 RU.
  • the bispecific constructs are captured for 90 s at 300 nM.
  • the target antigens are passed through the flow cells for 180 s at a concentration range from 250 to 1000 nM with a flowrate of 30 ⁇ l/min. The dissociation is monitored for 180 s.
  • Biological activity of the T cell activating bispecific antigen binding molecules of the invention can be measured by various assays as described in the Examples. Biological 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 secretion by T cells, the induction of lysis of target cells such as tumor cells, and the induction of tumor regression and/or the improvement of survival.
  • compositions Compositions, Formulations, and Routes of Administration
  • the invention provides pharmaceutical compositions comprising any of the T cell activating bispecific antigen binding molecules provided herein, e.g., for use in any of the below therapeutic methods.
  • a pharmaceutical composition comprises any of the T cell activating bispecific antigen binding molecules provided herein and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition comprises any of the T cell activating bispecific antigen binding molecules provided herein and at least one additional therapeutic agent, e.g., as described below.
  • a method of producing a T cell activating bispecific antigen binding molecule of the invention in a form suitable for administration in vivo comprising (a) obtaining a T cell activating bispecific antigen binding molecule according to the invention, and (b) formulating the T cell activating bispecific antigen binding molecule with at least one pharmaceutically acceptable carrier, whereby a preparation of T cell activating bispecific antigen binding molecule is formulated for administration in vivo.
  • compositions of the present invention comprise a therapeutically effective amount of one or more T cell activating bispecific antigen binding molecule dissolved or dispersed in a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable refers to molecular entities and compositions that are generally non-toxic to recipients at the dosages and concentrations employed, i.e. do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate.
  • the preparation of a pharmaceutical composition that contains at least one T cell activating bispecific antigen binding molecule and optionally an additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed.
  • compositions are lyophilized formulations or aqueous solutions.
  • pharmaceutically acceptable carrier includes any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g.
  • antibacterial agents antifungal agents
  • isotonic agents absorption delaying agents, salts, preservatives, antioxidants, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, 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.
  • T cell activating bispecific antigen binding molecules of the present invention can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrasplenically, intrarenally, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctivally, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, topically, locally, by inhalation (e.g.
  • aerosol inhalation injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g. liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference).
  • Parenteral administration in particular intravenous injection, is most commonly used for administering polypeptide molecules such as the T cell activating bispecific antigen binding molecules of the invention.
  • compositions include those designed for administration by injection, e.g. subcutaneous, intradermal, intralesional, intravenous, intraarterial intramuscular, intrathecal or intraperitoneal injection.
  • the T cell activating bispecific antigen binding molecules of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer.
  • the solution may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the T cell activating bispecific antigen binding molecules may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • Sterile injectable solutions are prepared by incorporating the T cell activating bispecific antigen binding molecules of the invention 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 through sterile filtration membranes. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and/or the other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsion, the preferred methods of preparation are vacuum-drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium thereof.
  • the liquid medium should be suitably buffered if necessary and the liquid diluent first rendered isotonic prior to injection with sufficient saline or glucose.
  • the composition must be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less that 0.5 ng/mg protein.
  • Suitable 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; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides
  • Aqueous injection suspensions may contain compounds which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, dextran, or the like.
  • the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • suspensions of the active compounds may be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl cleats or triglycerides, or liposomes.
  • Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • Sustained-release preparations may be prepared.
  • sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, which matrices are in the form of shaped articles, e.g. films, or microcapsules.
  • prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin or combinations thereof
  • 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.
  • the T cell activating bispecific antigen binding molecules may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • compositions comprising the T cell activating bispecific antigen binding molecules of the invention may be manufactured by means of conventional mixing, dissolving, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • Pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the proteins into preparations that can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the T cell activating bispecific antigen binding molecules may be formulated into a composition in a free acid or base, neutral or salt form.
  • Pharmaceutically acceptable salts are salts that substantially retain the biological 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 example, 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 hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine. Pharmaceutical salts tend to be more soluble in aqueous and other protic solvents than are the corresponding free base forms.
  • T cell activating bispecific antigen binding molecules may be used in therapeutic methods.
  • T cell activating bispecific antigen binding molecules of the invention can be used as immunotherapeutic agents, for example in the treatment of cancers.
  • T cell activating bispecific antigen binding molecules of the invention would be formulated, dosed, and administered 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 practitioners.
  • T cell activating bispecific antigen binding molecules of the invention for use as a medicament are provided.
  • T cell activating bispecific antigen binding molecules of the invention for use in treating a disease are provided.
  • T cell activating bispecific antigen binding molecules of the invention for use in a method of treatment are provided.
  • the invention provides 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.
  • the invention provides a T cell activating bispecific antigen binding 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.
  • the disease to be treated is a proliferative disorder.
  • the disease is cancer.
  • 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.
  • the invention provides a T cell activating bispecific antigen binding molecule as described herein for use in inducing lysis of a target cell, particularly a tumor cell.
  • the invention provides a T cell activating bispecific antigen binding molecule for use in a method of inducing lysis of a target cell, particularly a tumor cell, in an individual comprising administering to the individual an effective amount of the T cell activating bispecific antigen binding molecule to induce lysis of a target cell.
  • An “individual” according to any of the above embodiments is a mammal, preferably a human.
  • the invention provides for the use of a T cell activating bispecific antigen binding molecule of the invention in the manufacture or preparation of a medicament.
  • the medicament is for the treatment of a disease in an individual in need thereof.
  • 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.
  • the disease to be treated is a proliferative disorder.
  • the disease is cancer.
  • 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.
  • the medicament is for inducing lysis of a target cell, particularly a tumor cell.
  • 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, preferably a human.
  • the invention provides a method for treating a disease.
  • the method comprises administering to an individual having such disease a therapeutically effective amount of a T cell activating bispecific antigen binding molecule of the invention.
  • a composition is administered to said individual, comprising the T cell activating bispecific antigen binding molecule of the invention in a pharmaceutically acceptable form.
  • the disease to be treated is a proliferative disorder.
  • the disease is cancer.
  • 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 “individual” according to any of the above embodiments may be a mammal, preferably a human.
  • the invention provides a method for inducing lysis of a target cell, particularly a tumor cell.
  • the method comprises contacting a target cell with a T cell activating bispecific antigen binding molecule of the invention in the presence of a T cell, particularly a cytotoxic T cell.
  • a method for inducing lysis of a target cell, particularly a tumor cell, in an individual is provided.
  • the method comprises administering to the individual an effective amount of a T cell activating bispecific antigen binding molecule to induce lysis of a target cell.
  • an “individual” is a human.
  • the disease to be treated is a proliferative disorder, particularly cancer.
  • cancers include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer.
  • the cancer is chosen from the group consisting of renal cell cancer, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, head and neck cancer.
  • T cell activating bispecific antigen binding molecule may not provide a cure but may only provide partial benefit.
  • a physiological change having some benefit is also considered therapeutically beneficial.
  • an amount of T cell activating bispecific antigen binding molecule that provides 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.
  • an effective amount of a T cell activating bispecific antigen binding molecule of the invention is administered to a cell. In other embodiments, a therapeutically effective amount of a T cell activating bispecific antigen binding molecule of the invention is administered to an individual for the treatment of disease.
  • the appropriate dosage of a T cell activating bispecific antigen binding molecule of the invention (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 history and response to the T cell activating bispecific antigen binding molecule, and the discretion of the attending physician.
  • the practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.
  • Various dosing schedules including but not limited to single or multiple administrations over 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 treatments.
  • about 1 ⁇ g/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 ⁇ g/kg to 100 mg/kg or more, depending on the factors mentioned above.
  • the treatment would generally be sustained until a desired suppression of disease symptoms occurs.
  • 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 weight, about 50 milligram/kg body weight, 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.
  • 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.
  • one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 5.0 mg/kg or 10 mg/kg (or any combination thereof) may be administered to the patient.
  • Such doses may be administered intermittently, e.g. every week or every three weeks (e.g. such that the patient receives from about two to about twenty, or e.g. about six doses of the T cell activating bispecific antigen binding molecule).
  • An initial higher loading dose, followed by one or more lower doses may be administered.
  • other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
  • the T cell activating bispecific antigen binding molecules of the invention will generally be used in an amount effective to achieve the intended purpose.
  • the T cell activating bispecific antigen binding molecules of the invention, or pharmaceutical compositions thereof are administered or applied in a therapeutically effective amount. Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.
  • 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 includes the IC 50 as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.
  • Initial dosages can also be estimated from in vivo data, e.g., animal models, using techniques that are well known in the art. One having ordinary skill in the art could readily optimize administration to humans based on animal data.
  • Dosage amount and interval may be adjusted individually to provide plasma levels of the T cell activating bispecific antigen binding molecules which are sufficient to maintain therapeutic effect.
  • Usual patient dosages for administration by injection range from about 0.1 to 50 mg/kg/day, typically from about 0.5 to 1 mg/kg/day.
  • Therapeutically effective plasma levels may be achieved by administering multiple doses each day. Levels in plasma may be measured, for example, by HPLC.
  • the effective local concentration 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 therapeutically effective local dosages without undue experimentation.
  • a therapeutically effective dose of the T cell activating bispecific antigen binding molecules described herein will generally provide therapeutic benefit without causing substantial toxicity.
  • Toxicity and therapeutic efficacy of a T cell activating bispecific antigen binding molecule can be determined by standard pharmaceutical procedures in cell culture or experimental animals. Cell culture assays and animal studies can be used to determine the LD 50 (the dose lethal to 50% of a population) and the ED 50 (the dose therapeutically effective in 50% of a population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD 50 /ED 50 . T cell activating bispecific antigen binding molecules that exhibit large therapeutic indices are preferred.
  • the T cell activating bispecific antigen binding molecule according to the present invention exhibits a high therapeutic index.
  • the data obtained from cell culture assays and animal studies can be used in formulating a range of dosages suitable for use in humans.
  • the dosage lies preferably within a range of circulating concentrations that include the ED 50 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.
  • the attending physician for patients treated with T cell activating bispecific antigen binding molecules of the invention 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 disorder of interest will vary with the severity of the condition to be treated, with the route of administration, and the like. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency will also vary according to the age, body weight, and response of the individual patient.
  • the T cell activating bispecific antigen binding molecules of the invention may be administered in combination with one or more other agents in therapy.
  • a T cell activating bispecific antigen binding molecule of the invention may be co-administered with at least one additional therapeutic agent.
  • therapeutic agent encompasses any agent administered to treat a symptom or disease in an individual in need of such treatment.
  • additional therapeutic agent may comprise any active ingredients suitable for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
  • an additional therapeutic agent is an immunomodulatory agent, a cytostatic agent, an inhibitor of cell adhesion, a cytotoxic agent, an activator of cell apoptosis, or an agent that increases the sensitivity of cells to apoptotic inducers.
  • the additional therapeutic agent is an anti-cancer 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 bispecific antigen binding molecule used, the type of disorder or treatment, and other factors discussed above.
  • the T cell activating bispecific antigen binding molecules are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate.
  • combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate compositions), and separate administration, in which case, administration of the T cell activating bispecific antigen binding molecule of the invention can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent and/or adjuvant.
  • T cell activating bispecific antigen binding molecules of the invention can also be used in combination with radiation therapy.
  • the invention provides for a bispecific antibody comprising a) a first antigen-binding site that comprises a variable heavy chain domain (VH) of SEQ ID NO: 274 and a variable light chain domain of SEQ ID NO: 31; and b) a second antigen-binding site that comprises a variable heavy chain domain (VH) of SEQ ID NO: 36 and a variable light chain domain of SEQ ID NO: 31 for use in combination with an antibody to PD-L1 or FAP-4-1BBL.
  • the bispecific antibody further comprises a third antigen-binding site that comprises a variable heavy chain domain (VH) of SEQ ID NO: 274 and a variable light chain domain of SEQ ID NO: 31.
  • an article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the disorders described above comprises a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc.
  • the containers may be formed from a variety of materials such as glass or 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 pierceable by a hypodermic injection needle).
  • At least one active agent in the composition is a T cell activating bispecific antigen binding molecule of the invention.
  • the label or package insert indicates that the composition is used for treating the condition of choice.
  • 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 of the invention; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent.
  • the article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition.
  • the article of manufacture may further 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 include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
  • BWFI bacteriostatic water for injection
  • phosphate-buffered saline such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution.
  • BWFI bacteriostatic water for injection
  • phosphate-buffered saline such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution.
  • BWFI bacteriostatic water for injection
  • Ringer's solution such as phosphate
  • Desired gene segments where required were either generated by PCR using appropriate templates or were synthesized by Geneart AG (Regensburg, Germany) from synthetic oligonucleotides and PCR 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 flanked 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 sequencing. Gene segments were designed 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.
  • PBMCs Peripheral blood mononuclear cells
  • enriched lymphocyte preparations obtained from local blood banks or from fresh blood from healthy human donors. Briefly, blood was diluted with sterile PBS and carefully layered over a Histopaque gradient (Sigma, H8889). After centrifugation for 30 minutes at 450 ⁇ g at room temperature (brake switched off), part of the plasma above the PBMC containing interphase was discarded. The PBMCs were transferred into new 50 ml Falcon tubes and tubes were filled up with PBS to a total volume of 50 ml. The mixture was centrifuged at room temperature for 10 minutes at 400 ⁇ g (brake switched on).
  • the supernatant was discarded and the PBMC pellet washed twice with sterile PBS (centrifugation steps at 4° C. for 10 minutes at 350 ⁇ 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% CO 2 in the incubator until assay start.
  • T cell enrichment from PBMCs was performed using the Pan T Cell Isolation Kit II (Miltenyi Biotec #130-091-156), according to the manufacturer's instructions. Briefly, the cell pellets were diluted in 40 ⁇ l cold buffer per 10 million cells (PBS with 0.5% BSA, 2 mM EDTA, sterile filtered) and incubated with 10 ⁇ l Biotin-Antibody Cocktail per 10 million cells for 10 min at 4° C. 30 ⁇ l cold buffer and 20 ⁇ l 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 10-20 ⁇ the current volume and a subsequent centrifugation step at 300 ⁇ g for 10 min.
  • PBMCs Peripheral blood mononuclar cells
  • enriched lymphocyte preparations 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 (#130-093-244), according to the manufacturer's instructions, but skipping the last isolation step of CD8 + T cells (also see description for the isolation of primary human pan T cells).
  • Spleens were isolated from C57BL/6 mice, transferred into a GentleMACS C-tube (Miltenyi Biotech #130-093-237) 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 remaining undissociated tissue particles. After centrifugation at 400 ⁇ 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 (#130-090-861), following the manufacturer's instructions.
  • the resulting T cell population was automatically counted (ViCell) and immediately used for further assays.
  • PBMCs Peripheral blood mononuclar cells
  • Heparinized blood was diluted 1:3 with sterile PBS
  • 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 ⁇ g, without brake, at room temperature.
  • the PBMC band was transferred into a fresh 50 ml Falcon tube and washed with sterile PBS by centrifugation for 10 min at 400 ⁇ g at 4° C.
  • One low-speed centrifugation was performed to remove the platelets (15 min at 150 ⁇ g, 4° C.), and the resulting PBMC population was automatically counted (ViCell) and immediately used for further assays.
  • the following antigens and screening tools were generated as monovalent Fc fusion proteins (the extracellular domain of the antigen linked to the hinge region of Fc-knob which is co-expressed with an Fc-hole molecule).
  • the antigen genes were synthesized (Geneart, Regensburg, Germany) based on sequences obtained from GenBank or SwissProt and inserted into expression vectors to generate fusion proteins with Fc-knob with a C-terminal Avi-tag for in vivo or in vitro biotinylation. In vivo biotinylation was achieved by co-expression of the bacterial birA gene encoding a bacterial biotin ligase during production. Expression of all genes was under control of a chimeric MPSV promoter on a plasmid containing an oriP element for stable maintenance of the plasmids in EBNA containing cell lines.
  • HEK293 EBNA cells were seeded 24 hours before transfection.
  • transfection cells were centrifuged for 5 minutes at 210 g, and supernatant was replaced by pre-warmed CD CHO medium.
  • Expression vectors were resuspended in 20 mL of CD CHO medium containing 200 ⁇ g of vector DNA. After addition of 540 ⁇ L of polyethylenimine (PEI), the solution was mixed for 15 seconds and incubated for 10 minutes at room temperature. Afterwards, cells were mixed with the DNA/PEI solution, transferred to a 500 mL shake flask and incubated for 3 hours at 37° C. in an incubator with a 5% CO 2 atmosphere.
  • PEI polyethylenimine
  • F17 medium After the incubation, 160 mL of F17 medium was added and cells were cultured for 24 hours. One day after transfection, 1 mM valproic acid and 7% Feed 1 (Lonza) were added to the culture. The production medium was also supplemented with 100 ⁇ M biotin. After 7 days of culturing, the cell supernatant was collected by spinning down cells for 15 min at 210 g. The solution was sterile filtered (0.22 ⁇ m filter), supplemented with sodium azide to a final concentration of 0.01% (w/v), and kept at 4° C.
  • Secreted proteins were purified from cell culture supernatants by affinity chromatography using Protein A, followed by size exclusion chromatography.
  • pH of collected fractions was adjusted by adding 1/10 (v/v) of 0.5 M sodium phosphate, pH 8.0.
  • the protein was concentrated and filtered prior to loading on a HiLoad Superdex 200 column (GE Healthcare) equilibrated with 20 mM histidine, 140 mM sodium chloride, pH 6.0.
  • the protein concentration 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 FolR1-Fc-fusion was analyzed by SDS capillary electrophoresis in the presence and absence of a reducing agent following the manufacturer instructions (instrument Caliper LabChipGX, Perkin Elmer).
  • the aggregate content of samples was analyzed using a TSKgel G3000 SW XL analytical size-exclusion column (Tosoh) equilibrated in 25 mM K2HPO4, 125 mM NaCl, 200 mM L-arginine monohydrochloride, 0.02% (w/v) NaN3, pH 6.7 running buffer at 25° C.
  • Purified antigen-Fc-fusion proteins were analyzed by surface plasmon resonance assays using commercially available antibodies to confirm correct and natural conformation of the antigens (data not shown).
  • the T cell activating bispecific molecules described herein comprise at least one CD3 binding moiety.
  • This moiety can be generated by immunizing laboratory animals, screening phage library or using known anti-CD3 antibodies.
  • the common light chain with CD3 ⁇ specificity was generated by humanizing the light chain of a murine parental anti-CD3 ⁇ antibody (CH2527).
  • CH2527 murine parental anti-CD3 ⁇ antibody
  • acceptor framework sequences are selected by aligning the sequence of the donor to a collection of potential acceptor sequences and choosing one that has either reasonable homology to the donor, or shows similar amino acids at some positions critical for structure and activity.
  • the search for the antibody acceptor framework was performed by aligning the mouse VL-domain sequence of the parental antibody to a collection of human germline sequences and choosing the human sequence that showed high sequence identity.
  • a good match in terms of framework sequence homology was found in a rather infrequent human light chain belonging to the V-domain family 7 of the lambda type, more precisely, hVL_7_46 (IMGT nomenclature, GenBank Acc No. Z73674).
  • This infrequent human light chain was subsequently chosen as acceptor framework for humanization of the light chain of CH2527.
  • the three complementarity determining regions (CDRs) of the mouse light chain variable domain were grafted onto this acceptor framework. Since the framework 4 region is not part of the variable region of the germline V-gene, the alignment for this region (J-element) was done individually. Hence the IGLJ3-02 sequence was chosen for humanization of this light chain.
  • Humanized VL variants were assessed as chimera in a 2+1 classical format (FIG. 1D), i.e. humanized light chain V-domains were paired with murine heavy chain V-domains. SPR assessment was carried out on a ProteOn XPR36 instrument (Bio-Rad). More precisely, the variants were captured directly from the culture supernatant on an anti-Fab derivatized GLM sensorchip (Goat Anti-Human IgG, F(ab′)2 Fragment Specific, Jackson ImmunoResearch) in vertical orientation.
  • the light chain V-domain variant that was chosen for the humanized lead molecule is VL7_46-13.
  • the degree of humanness i.e. the sequence homology of the humanized V-domain to the human germline V-domain sequence was determined.
  • the overall sequence identity with the closest human germline homolog is 65% before humanization and 80% afterwards. Omitting the CDR regions, the sequence identity is 92% to the closest human germline homolog.
  • VL7_46-13 is the only humanized VL variant out of a panel of 13 variants that showed comparable binding to the parental murine antibody and also retained its cross-reactivity to cynomolgus CD3 ⁇ .
  • the humanized VL-domain VL7_46-13 based on an infrequent human germline belonging to the V-domain family 7 of the lambda type and retaining affinity and specificity for CD3 ⁇ , is also suitable to be used as a common light chain in phage-displayed antibody libraries of the Fab-format and enables successful selection for novel specificities which greatly facilitates the generation and production of bispecific molecules binding to CD3 ⁇ and e.g. a tumor target and sharing the same ‘common’ light chain.
  • the design of the randomization included the CDR-H1, CDR-H2, and CDR-H3.
  • CDR-H1 and CDR-H2 a “soft” randomization strategy was chosen, and the randomization oligonucleotides were such that the codon for the amino acid of the germ-line sequence was present at 50%. All other amino acids, except cysteine, were summing up for the remaining 50%.
  • CDR-H3 where no germ-line amino acid is present due to the presence of the genetic D-element, oligonucleotides were designed that allow for the usage of randomized inserts between the V-element and the J-element of 4 to 9 amino acids in length. Those oligonucleotides contained in their randomized part e.g. The three amino acids G/Y/S are present to 15% each, those amino acids A/D/T/R/P/L/V/N/W/F/I/E are present to 4.6% each.
  • the light chain is derived from the human sequence hVK1-39, and is used in an unmodified and non-randomized fashion. This will ensure that the same light chain can be used for other projects without additional modifications.
  • Elution of phage particles is done by addition of 1 ml 100 mM TEA (triethylamine) for 10 min and neutralization by addition of 500 ul 1M Tris/HCl pH 7.4 and 5.
  • Re-infection of exponentially growing E. coli TG1 bacteria, infection with helper phage VCSM13 and subsequent PEG/NaCl precipitation of phagemid particles is applied in subsequent selection rounds. Selections are carried out over 3-5 rounds using either constant or decreasing (from 10A-7M to 2 ⁇ 10 ⁇ 9M) antigen concentrations.
  • capture of antigen/phage complexes is performed using neutravidin plates instead of streptavidin beads. All binding reactions are supplemented either with 100 nM bovine serum albumin, or with non-fat milk powder in order to compete for unwanted clones arising from mere sticky binding of the antibodies to the plastic support.
  • Specific binders are defined as signals ca. 5 ⁇ higher than background and are identified by ELISA. Specific binders are identified by ELISA as follows: 100 ⁇ l of 10 nM biotinylated antigen per well are coated on neutravidin plates. Fab-containing bacterial supernatants are added and binding Fabs are detected via their Flag-tags by using an anti-Flag/HRP secondary antibody.
  • ELISA-positive clones are bacterially expressed as soluble Fab fragments in 96-well format and supernatants are subjected to a kinetic screening experiment by SPR-analysis using ProteOn XPR36 (BioRad).
  • Clones expressing Fabs with the highest affinity constants are identified and the corresponding phagemids are sequenced.
  • the Fab sequences are amplified via PCR from the phagemid and cloned via appropriate restriction sites into human IgG1 expression vectors for mammalian production.
  • an antibody library for the display on M13 phage is described.
  • a multi framework library for the heavy chain with one constant (or “common”) light chain was designed for the generation of Fc-containing, but FcgR binding inactive T cell bispecific antibodies of IgG1 P329G LALA or IgG4 SPLE PG isotype in which one or two Fab recognize a tumor surface antigen expressed on a tumor cell whereas the remaining Fab arm of the antibody recognizes CD3e on a T cell.
  • an antibody library for the display on M13 phage is described.
  • a multi framework library for the heavy chain with one constant (or “common”) light chain is designed solely for the generation of Fc-containing, but FcgR binding inactive T cell bispecific antibodies of IgG1 P329G LALA or IgG4 SPLE PG isotype.
  • the antibodies 16A3, 15A1, 18D3, 19E5, 19A4, 15H7, 15B6, 16D5, 15E12, 21D1, 16F12, 21A5, 21G8, 19H3, 20G6, and 20H7 comprising the common light chain VL7_46-13 with CD3 ⁇ specificity were obtained by phage display selections against different species (human, cynomolgus and murine) of FolR1.
  • Clones 16A3, 15A1, 18D3, 19E5, 19A4, 15H7, 15B6, 21D1, 16F12, 19H3, 20G6, and 20H7 were selected from a sub-library in which the common light chain was paired with a heavy chain repertoire based on the human germline VH1_46.
  • CDR3 of VH1_46 has been randomized based on 6 different CDR3 lengths.
  • Clones 16D5, 15E12, 21A5, and 21G8 were selected from a sub-library in which the common light chain was paired with a heavy chain repertoire based on the human germline VH3_15.
  • CDR3 of VH3_15 has been randomized based on 6 different CDR3 lengths.
  • Clones exhibiting signals on human FolR1 and being negative on human IgG were short-listed for further analyses and were also tested in a similar fashion against the remaining two species of FolR1. They were bacterially expressed in a 0.5 liter culture volume, affinity purified and further characterized by SPR-analysis using BioRad's ProteOn XPR36 biosensor.
  • K D Affinities (K D ) of selected clones were measured by surface plasmon resonance (SPR) using a ProteOn XPR36 instrument (Biorad) at 25° C. with biotinylated human, cynomolgus, and murine FolR1 as well as human FolR2 and FolR3 (negative controls) immobilized on NLC chips by neutravidin capture.
  • Immobilization of antigens (ligand) Recombinant antigens were diluted with PBST (10 mM phosphate, 150 mM sodium chloride pH 7.4, 0.005% Tween 20) to 10 ⁇ g/ml, then injected at 30 ⁇ l/minute in vertical orientation.
  • KD Equilibrium dissociation constants
  • the antibodies 11F8, 36F2, 9D11, 5D9, 6B6, and 14E4 were obtained by phage display selections based on generic multi-framework sub-libraries against different species (human, cynomolgus and murine) of FolR1.
  • different VL-domains with randomized CDR3 (3 different lengths) are paired with different VH-domains with randomized CDR3 (6 different lengths).
  • the selected clones are of the following VL/VH pairings: 11F8 (Vk_1_5/VH_1_69), 36F2 (Vk_3_20/VH_1_46), 9D11 (Vk2D_28/VH1_46), 5D9 (Vk3_20/VH1_46), 6B6 (Vk3_20/VH1_46), and 14E4 (Vk3_20/VH3_23).
  • Fab-containing bacterial supernatants were added and binding Fabs were detected via their Flag-tags using an anti-Flag/HRP secondary antibody.
  • Clones exhibiting signals on human FolR1 and being negative on human IgG were short-listed for further analyses and were also tested in a similar fashion against the remaining two species of FolR1. They were bacterially expressed in a 0.5 liter culture volume, affinity purified and further characterized by SPR-analysis using BioRad's ProteOn XPR36 biosensor.
  • K D Affinities (K D ) of selected clones were measured by surface plasmon resonance (SPR) using a ProteOn XPR36 instrument (Biorad) at 25° C. with biotinylated human, cynomolgus, and murine FolR1 as well as human FolR2 and FolR3 (negative controls) immobilized on NLC chips by neutravidin capture.
  • Immobilization of antigens (ligand) Recombinant antigens were diluted with PBST (10 mM phosphate, 150 mM sodium chloride pH 7.4, 0.005% Tween 20) to 10 ⁇ g/ml, then injected at 30 ⁇ l/minute in vertical orientation.
  • K D Equilibrium dissociation constants (K D ) for anti-FolR1 antibodies (Fab-format) selected by phage display from generic multi-framework sub-libraries.
  • K D (nM) Clone huFolR1 cyFolR1 muFolR1 huFolR2 huFolR3 11F8 632 794 1200 no binding no binding 36F2 1810 1640 737 no binding no binding 9D11 8.64 5.29 no binding no binding no binding 5D9 8.6 5.9 no binding no binding 6B6 14.5 9.4 no binding no binding no binding 14E4 no binding no binding 6.09 no binding no binding no binding
  • the antibodies isolated from a common light chain- or Fab-library were converted into the corresponding human IgG1 format.
  • the variable heavy and variable light chains of unique FolR1 binders from phage display were amplified by standard PCR reactions using the Fab clones as the template.
  • the PCR products were purified and inserted (either by restriction endonuclease and ligase based cloning, or by ‘recombineering’ using the InFusion kit from Invitrogen) into suitable expression vectors in which they are fused to the appropriate human constant heavy or human constant light chain.
  • the expression cassettes in these vectors consist of a chimeric MPSV promoter and a synthetic polyadenylation site.
  • the plasmids contain the oriP region from the Epstein Barr virus for the stable maintenance of the plasmids in HEK293 cells harboring the EBV nuclear antigen (EBNA).
  • EBNA EBV nuclear antigen
  • HEK293 EBNA cells are cultivated in suspension serum free in CD CHO culture medium. For the production in 500 ml shake flask 400 million HEK293 EBNA cells are seeded 24 hours before transfection (for alternative scales all amounts were adjusted accordingly). For transfection cells are centrifuged for 5 min by 210 ⁇ g, supernatant is replaced by pre-warmed 20 ml CD CHO medium. Expression vectors are mixed in 20 ml CD CHO medium to a final amount of 200 ⁇ g DNA.
  • PEI solution After addition of 540 ⁇ l PEI solution is vortexed for 15 s and subsequently incubated for 10 min at room temperature. Afterwards cells are mixed with the DNA/PEI solution, transferred to a 500 ml shake flask and incubated for 3 hours by 37° C. in an incubator with a 5% CO2 atmosphere. After incubation time 160 ml F17 medium is added and cell are cultivated for 24 hours. One day after transfection 1 mM valporic acid and 7% Feed 1 is added.
  • Purity and molecular weight of the constructs were analyzed by SDS capillary electrophoresis in the presence and absence of a reducing agent following the manufacturer instructions (instrument Caliper LabChipGX, Perkin Elmer). Purified proteins were frozen in liquid N 2 and stored at ⁇ 80° C.
  • binders were converted into a T-cell bispecific format.
  • the FolR1:CD3 binding moieties are arranged in a 2:1 order with the FolR1 Fabs being located at the N-terminus.
  • the CD3 binding part was generated as a CrossFab (CH1C ⁇ crossing) while for the clones from the common light chain library no crossing was necessary.
  • IgG TCB Yield Monomer Yield Monomer # Clone Library [mg/L] [%] [mg/L ⁇ [%] 1 11F8 Fab 8.03 96.26 — — 2 14E4 Fab 8.90 98.12 — — 3 15B6 CLC 7.72 100.00 — — 4 15E12 CLC 6.19 100.00 — — 5 15H7 CLC 8.94 100.00 — — 6 16A3 CLC 0.60 n.d.
  • T-cell bispecific formats were prepared for one common light chain binder (16D5) and three formats for one binder from the Fab library (9D11) to compare their killing properties in vitro.
  • the standard format is the 2+1 inverted format as already described (FolR1:CD3 binding moieties arranged in a 2:1 order with the FolR1 Fabs located at the N-terminus).
  • the FolR1:CD3 binding moieties are arranged in a 2:1 order with the CD3 Fab being located at the N-terminus.
  • Two monovalent formats were also prepared.
  • the 1+1 head-to-tail has the FolR1:CD3 binding moieties arranged in a 1:1 order on the same arm of the molecule with the FolR1 Fab located at the N-terminus.
  • the FolR1:CD3 binding moieties are present once, each on one arm of the molecule.
  • the IgGs were injected for 60 seconds at a concentration of 500 nM. IgGs binding to huFolR2 and huFolR3 were rejected for lack of specificity. Most of the binders are only crossreactive between human and cyno FolR1, additional crossreactivity to murine FolR1 went most of the time hand in hand with loss of specificity.
  • Recombinant biotinylated monomeric Fc fusions of human, cynomolgus and murine Folate Receptor 1 (FolR1-Fc) were directly coupled on a SA chip using the standard coupling instruction (Biacore, Freiburg/Germany).
  • the immobilization level was about 300-400 RU.
  • the anti-FolR1 IgGs or T cell bispecifics were passed at a concentration range from 2.1 to 500 nM with a flow of 30 ⁇ L/minutes through the flow cells over 180 seconds. The dissociation was monitored for 600 seconds. Bulk refractive index differences were corrected for by subtracting the response obtained on reference flow cell immobilized with recombinant biotinylated IL2 receptor Fc fusion.
  • folate (Sigma F7876) was added in the HBS-EP running buffer at a concentration of 2.3 ⁇ M.
  • the binding curves resulting from the bivalent binding of the IgGs or T cell bispecifics were approximated to a 1:1 Langmuir binding and fitted with that model (which is not correct, but gives an idea of the avidity).
  • the apparent avidity constants for the interactions were derived from the rate constants of the fitting using the Bia Evaluation software (GE Healthcare).
  • the dissociation phase was monitored for 240 s and triggered by switching from the sample solution to HBS-EP.
  • the chip surface was regenerated after every cycle using a double injection of 60 sec 10 mM Glycine-HCl pH 2.1 or pH 1.5. Bulk refractive index differences were corrected for by subtracting the response obtained on the reference flow cell 1.
  • the affinity constants for the interactions were derived from the rate constants by fitting to a 1:1 Langmuir binding using the Bia Evaluation software (GE Healthcare).
  • the chip surface was regenerated after every cycle using a double injection of 60 sec 10 mM Glycine-HCl pH 2.1. Bulk refractive index differences were corrected for by subtracting the response obtained on the reference flow cell 1.
  • the affinity constants for the interactions were derived from the rate constants by fitting to a 1:1 Langmuir binding using the Bia Evaluation software (GE Healthcare).
  • the CD3 binding part is identical for all constructs and the affinity is similar for the measured T cell bispecifics (KD range between 60 and 90 nM).
  • the anti-FolR1 T cell bispecifics were injected for 60 s at 500 nM with a flow of 30 ⁇ L/minutes through the flow cells, followed by an injection of hu CD ⁇ -Fc for 60 s at 500 nM. Bulk refractive index differences were corrected for by subtracting the response obtained on reference flow cell immobilized with recombinant biotinylated IL2 receptor Fc fusion.
  • the four T cell bispecifics tested (16D5 TCB, 21A5 TCB, 51C7 TCB and 45D2 TCB) were able to bind simultaneously to Folate Receptor 1 and human CD3 as expected.
  • the anti-FolR1 IgGs or T cell bispecifics were directly immobilized on a CM5 chip at pH 5.0 using the standard amine coupling kit (GE Healthcare), with a final response around 700 RU.
  • 500 nM huFolR1-Fc was then captured for 60 s, followed by 500 nM of the different binders for 30 s.
  • the surface was regenerated with two injections of 10 mM glycine pH 2 for 30 s each. It is assessed if the different binders can bind to huFolR1 captured on immobilized binders (Table 12).
  • FolR1 binders in the IgG formats were screened by surface plasmon resonance (SPR) and by in vitro assay on cells to select the best candidates.
  • the anti-FolR1 IgGs were analyzed by SPR to characterize their crossreactivity (to human, murine and cynomolgus FolR1) and specificity (to human FolR1, human FolR2, human FolR3). Unspecific binding to human FolR2 and 3 was considered an exclusion factor. Binding and specificity to human FolR1 was confirmed on cells. Some binders did not bind on cells expressing FolR1 even though they recognized the recombinant human FolR1 in SPR. Aggregation temperature was determined but was not an exclusion factor because the selected binders were all stable. Selected binders were tested in a polyreactivity ELISA to check for unspecific binding, which led to the exclusion of four binders.
  • New FolR1 binders were generated via Phage Display using either a Fab library or a common light chain library using the CD3 light chain. The identified binders were converted into a human IgG1 format and binding to FolR1 high expressing HeLa cells was addressed. As reference molecule the human FolR1 binder Mov19 was included. Most of the binders tested in this assay showed intermediate to good binding to FolR1 with some clones binding equally well as Mov19 (see FIG. 2). The clones 16A3, 18D3, 15H7, 15B6, 21D1, 14E4 and 16F12 were excluded because binding to FolR1 on cells could not be confirmed by flow cytometry.
  • HEK cells were transiently transfected with either human FolR1 or human FolR2 to address specificity.
  • the clones 36F2 and 9D11 derived from the Fab library and the clones 16D5 and 21A5 derived from the CLC library bind specifically to human FolR1 and not to human FolR2 (see FIGS. 3A-B ). All the other tested clones showed at least some binding to human FolR2 (see FIGS. 3A-B ). Therefore these clones were excluded from further characterization.
  • Mov19 FolR1 TCB was included in all studies. These FolR1 TCBs were then used to compare induction of internalization after binding to FolR1 on HeLa cells. All three tested clones are internalized upon binding to FolR1 comparable to internalization upon binding of Mov19 FoLR1 TCB ( FIG. 5 ). 21A5 FolR1 TCB was discontinued due to signs of polyreactivity.
  • the FolR1 TCBs were used to determine T cell mediated killing of tumor cells expressing FoLR1.
  • a panel of potential target cell lines was used to determine FoLR1 binding sites by Qifikit analysis.
  • the used panel of tumor cells contains FolR1 high, intermediate and low expressing tumor cells and a FolR1 negative cell line.
  • Binding of the three different FoLR1 TCBs (containing 9D11, 16D5 and Mov19 binders) to this panel of tumor cell lines was determined showing that the FoLR1 TCBs bind specifically to FolR1 expressing tumor cells and not to a FoLR1 negative tumor cell line.
  • the amount of bound construct is proportional to the FolR1 expression level and there is still good binding of the constructs to the FolR1 low cell line HT-29 detectable.
  • the intermediate expressing cell line SKOV3 and the low expressing cell line HT-29 were further on used to test T cell mediated killing and T cell activation using 16D5 TCB and 9D11 TCB; DP47 TCB was included as negative control. Both cell lines were killed in the presence of already very low levels of 16D5 TCB and 9D11 TCB and there was no difference in activity between both TCBs even though 9D11 TCB binds stronger to FolR1 than 16D5 TCB. Overall killing of SKOV3 cells was higher compared to HT-29 which reflects the higher expression levels of FolR1 on SKOV3 cells ( FIGS. 7A-D ).
  • the four T cell bispecifics were transiently produced in HEK293 EBNA cells using a PEI mediated transfection procedure for the required vectors as described below.
  • HEK293 EBNA cells were cultivated in suspension serum free in CD CHO culture medium.
  • 400 million HEK293 EBNA cells were seeded 24 hours before transfection (for alternative scales all amounts were adjusted accordingly).
  • For transfection cells were centrifuged for 5 min by 210 ⁇ g, supernatant was replaced by pre-warmed 20 ml CD CHO medium.
  • Expression vectors were mixed in 20 ml CD CHO medium to a final amount of 200 ⁇ g DNA.
  • PEI solution After addition of 540 ⁇ l PEI solution was vortexed for 15 s and subsequently incubated for 10 min at room temperature. Afterwards cells were mixed with the DNA/PEI solution, transferred to a 500 ml shake flask and incubated for 3 hours by 37° C. in an incubator with a 5% CO2 atmosphere. After incubation time 160 ml F17 medium was added and cell were cultivated for 24 hours. One day after transfection 1 mM valporic acid and 7% Feed 1 was added.
  • Fractions containing the protein of interest were pooled and the pH of the solution was gently adjusted to pH 6.0 (using 2 M Tris pH 8.0). Samples were concentrated to 1 ml using ultra-concentrators (Vivaspin 15R 30.000 MWCO HY, Sartorius) and subsequently applied to a SuperdexTM 200 10/300 GL (GE Healthcare) equilibrated with 20 mM Histidine, pH 6.0, 140 mM NaCl, 0.01% Tween-20. The aggregate content of eluted fractions was analyzed by analytical size exclusion chromatography.
  • Purity and molecular weight of the constructs were analyzed by SDS capillary electrophoresis in the presence and absence of a reducing agent following the manufacturer instructions (instrument Caliper LabChipGX, Perkin Elmer). Purified proteins were frozen in liquid N 2 and stored at ⁇ 80° C.
  • N100S N95S
  • N100Q N95Q
  • T102A T97A
  • T102N T97N
  • FolR1 is expressed at low levels on primary epithelial cells. Here we wanted to test if these levels are sufficient to induce T cell mediated killing in the presence of the FolR1 TCBs. To test this we used primary human bronchial epithelial cells, primary human choroid plexus epithelial cell, primary human renal cortical epithelial cells and primary human retinal pigment epithelial cells. As positive control either FolR1 positive SKOV3 cells or HT-29 cells were included. First we verified FolR1 expression on the used primary cells and determined the amount of FolR1 binding sites on these cells.
  • Bronchial epithelial cells, renal cortical epithelial cells and retinal pigment epithelial cells express very low but significant levels of FolR1 compared to the levels expressed on tumor cells.
  • the choroid plexus epithelial cells do not express significant levels of FolR1.
  • the primary epithelial cells that demonstrated FolR1 expression on the surface were used to address the question if these cells can be killed by T cells in the presence of FoLR1 TCBs. No significant levels of killing could be measured but induction of T cell activation in the presence of retinal pigment epithelial cells, bronchial epithelial cells and renal cortical cells resulting in upregulation of CD25 and CD69 was detected. The strongest activation is seen with retinal pigment epithelial cells resulting in upregulation of CD25 and CD69 both on CD4 + T cells and CD8 + T cells.
  • T cells In the presence of bronchial epithelial cells lower activation of T cells is induced with upregulation of CD69 on CD4 + T cells and CD8 + T cells but very low upregulation of CD25 only on CD4 + T cells but not on CD8 + T cells.
  • the lowest activation of T cells is obtained in the presence of renal epithelial cells with no upregulation of CD25 on CD4 T + cells and CD8 + T cells and CD69 been only upregulated on CD8 + T cells ( FIGS. 12A-X ).
  • TCB 2+1 inverted format is the most active format with the selected FolR1 binder
  • different formats containing either 16D5 or 9D11 were produced and compared in target cell binding, T cell mediated killing and T cell activation.
  • the 16D5 binder was tested in the TCB 2+1 inverted ( FIG. 1A ), TCB 2+1 classical (FIG. 1D), TCB 1+1 classical ( FIG. 1C ) and TCB 1+1 head-to-tail ( FIG. 1B ) format
  • the 9D11 binder was tested in the TCB 2+1 inverted ( FIG. 1A ), TCB 1+1 classical ( FIG. 1C ) and TCB 1+1 head-to-tail ( FIG. 1B ) format.
  • HeLa cells were obtained from ATCC and cultured in DMEM with 10% FCS and 2 mM Glutamine
  • SKOV3 HBV-77
  • OVCAR5 were obtained from NCI and cultured in RPMI with 10% FCS and 2 mM Glutamine
  • HT-29 ACC-299
  • MKN-45 ACC-409
  • HRCEpiC Human Renal Cortical Epithelial Cells (HRCEpiC), Catalog Number 4110, were cultured in Epithelial Cell Medium (EpiCM, Cat. No. 4101, ScienCell).
  • HRCEpiC Human Choroid Plexus Epithelial Cells (HCPEpiC), Catalog Number 1310 were cultured in Epithelial Cell Medium (EpiCM, Cat. No. 4101, ScienCell).
  • Target cells as indicated were harvested with Cell Dissociation Buffer, washed with PBS and resuspended in FACS buffer. The antibody staining was performed in a 96well round bottom plate. Therefore 200′000 cells were seeded per well. The plate was centrifuged for 4 min at 400 g and the supernatant was removed. The test antibodies were diluted in FACS buffer and 20 ⁇ l of the antibody solution were added to the cells for 30 min at 4° C.
  • the cells were washed twice with FACS buffer before addition of the diluted secondary antibody (FITC conjugated AffiniPure F(ab′)2 fragment goat anti-human IgG, Fcg Fragment, Jackson ImmunoResearch #109-096-098 or PE-conjugated AffiniPure F(ab′)2 Fragment goat anti-human IgG Fcg Fragment Specific, Jackson ImmunoResearch #109-116-170. After 30 min incubation on 4° C. unbound secondary antibody was washed away. Before measurement the cells were resuspended in 200 ⁇ l FACS buffer and analyzed by flow cytometry using BD Canto II or BD Fortessa.
  • the cells were harvested and the viability was determined.
  • the cells were re-suspended in fresh cold medium at 2 Mio cells per ml and the cell suspension was transferred in a 15 ml falcon tube for each antibody.
  • the antibodies that should be tested for internalization were added with a final concentration of 20 ⁇ g per ml to the cells.
  • the tubes were incubated for 45 min in the cold room on a shaker. After incubation the cells were washed three times with cold PBS to remove unbound antibodies. 0.2 Mio cells per well were transfer to the FACS plate as time point zero.
  • the labeled cells were re-suspended in warm medium and incubated at 37° C.
  • Mio cells per well were transferred in cold PBS, washed in plated on the FACS plate. To detect the constructs that remain on the surface the cells were stained with PE-labeled anti-human Fc secondary antibody. Therefore 20 ⁇ l of the diluted antibody were added per well and the plate was incubated for 30 min at 4° C. Then the cells were washed twice to remove unbound antibodies and then fixed with 1% PFA to prevent any further internalization. The fluorescence was measured using BD FACS CantoII.
  • QIFIKIT® contains a series of beads, 10 ⁇ m in diameter and coated with different, but well-defined quantities of mouse Mab molecules (high-affinity anti-human CD5, Clone CRIS-1, isotype IgG2a).
  • the beads mimic cells with different antigen densities which have been labeled with a primary mouse Mab, isotype IgG. Briefly, cells were labeled with primary mouse monoclonal antibody directed against the antigen of interest. In a separate test well, cells were labeled with irrelevant mouse monoclonal antibody (isotype control). Then, cells, Set-Up Beads and Calibration Beads were labeled with a fluorescein-conjugated anti-mouse secondary antibody included in the kit.
  • the primary antibody used for labeling of the cells has to be used at saturating concentration.
  • the primary antibody may be of any mouse IgG isotype. Under these conditions, the number of bound primary antibody molecules corresponds to the number of antigenic sites present on the cell surface.
  • the secondary antibody is also used at saturating concentration. Consequently, the fluorescence is correlated with the number of bound primary antibody molecules on the cells and on the beads.
  • Target cells were harvested with Trypsin/EDTA, counted and viability was checked. The cells were resuspended in their respective medium with a final concentration of 300′000 cells per ml. Then 100 ⁇ l of the target cell suspension was transferred into each well of a 96-flat bottom plate. The plate was incubated overnight at 37° C. in the incubator to allow adherence of the cells to the plate. On the next day PBMCs were isolated from whole blood from healthy donors. The blood was diluted 2:1 with PBS and overlayed on 15 ml Histopaque-1077 (#10771, Sigma-Aldrich) in Leucosep tubes and centrifuged for 30 min at 450 g without break.
  • the band containing the cells was collected with a 10 ml pipette and transferred into 50 ml tubes.
  • the tubes were filled up with PBS until 50 ml and centrifuged (400 g, 10 min, room temperature). The supernatant was removed and the pellet resuspended in PBS. After centrifugation (300 g, 10 min, room temperature), supernatants were discarded, 2 tubes were pooled and the washing step was repeated (this time centrifugation 350 ⁇ g, 10 min, room temperature). Afterwards the cells were resuspended and the pellets pooled in 50 ml PBS for cell counting.
  • Activation marker upregulation on T cells after tumor cell killing was measured by flow cytometry. Briefly PBMCs were harvested, transferred into a 96 well round bottom plate and stained with CD4 PE-Cy7 (#3557852, BD Bioscience), CD8 FITC (#555634, BD Bioscience), CD25 APC (#555434, BD Bioscience), CD69 PE (#310906, BioLegend) antibodies diluted in FACS buffer. After 30 min incubation at 4° C. the cells were washed twice with FACS buffer. Before measuring the fluorescence using BD Canto II the cells were resuspended in 200 ⁇ l FACS buffer.
  • 280 ⁇ l of fresh blood were added into a 96 well conical deep well plate. Then 20 ⁇ l of the diluted TCBs were added to the blood and mixed well by shaking the plate. After 24 h incubation at 37° C. in an incubator the blood was mixed and 35 ⁇ l were transferred to a 96well round bottom plate. Then 20 ⁇ l of the antibody staining mix were added consisting of CD4 PE-Cy7 (#3557852, BD Bioscience), CD8 FITC (#555634, BD Bioscience), CD25 APC (#555434, BD Bioscience), CD69 PE (#310906, BioLegend) and CD45 V500 (#560777, BD Horizon) and incubated for 15 min in the dark at room temperature. Before measuring 200 ⁇ l of the freshly prepared BD FACS lysing solution (#349202, BD FCAS) was added to the blood. After 15 min incubation at room temperature the cells were measured with BD Fortessa.
  • mice Female NOD/Shi-scid/IL-2R ⁇ null (NOG) mice, age 6-7 weeks at start of the experiment (bred at Taconic, Denmark) were maintained under specific-pathogen-free condition with daily cycles of 12 h light/12 h darkness according to committed guidelines (GV-Solas; Felasa; TierschG). The experimental study protocol was reviewed and approved by local government (P 2011/128). After arrival, animals were maintained for one week to get accustomed to the new environment and for observation. Continuous health monitoring was carried out on a regular basis.
  • mice were injected i.v. with 10/1/0.1 ⁇ g/mouse of the FOLR1 TCB whereas 3 mice were bled per group and time point. All mice were injected with a total volume of 200 ⁇ l of the appropriate solution. To obtain the proper amount of the FOLR1 TCB per 200 the stock solutions were diluted with PBS when necessary. Serum samples were collected 5 min, 1 h, 3 h, 8 h, 24 h, 48 h, 72 h, 96 h and 168 h after therapy injection.
  • FIG. 15 shows that the 16D5 FOLR1 TCB shows typical and dose proportional IgG-like PK properties in NOG mice with slow clearance.
  • the FOLR1 TCB was tested in the human ovarian carcinoma cell line Skov3, injected s.c. into PBMC engrafted NOG mice.
  • the Skov3 ovarian carcinoma cells were obtained from ATCC (HTB-77).
  • the tumor cell line was cultured in RPMI containing 10% FCS (Gibco) at 37° C. in a water-saturated atmosphere at 5% CO 2 .
  • Passage 35 was used for transplantation, at a viability >95%.
  • 5 ⁇ 10 6 cells per animal were injected s.c. into the right flank of the animals in a total of 100 ⁇ l of RPMI cell culture medium (Gibco).
  • mice Female NOD/Shi-scid/IL-2R ⁇ null (NOG) mice, age 6-7 weeks at start of the experiment (bred at Taconic, Denmark) were maintained under specific-pathogen-free condition with daily cycles of 12 h light/12 h darkness according to committed guidelines (GV-Solas; Felasa; TierschG). The experimental study protocol was reviewed and approved by local government (P 2011/128). After arrival, animals were maintained for one week to get accustomed to the new environment and for observation. Continuous health monitoring was carried out on a regular basis.
  • mice were injected s.c. on study day 0 with 5 ⁇ 10 6 of the Skov3.
  • human PBMC of a healthy donor were isolated via the Ficoll method and 10 ⁇ 10 6 cells were injected i.p. into the tumor-bearing mice.
  • the mice in the vehicle group were injected with PBS. To obtain the proper amount of TCB per 200 the stock solutions were diluted with PBS when necessary. Tumor growth was measured once weekly using a caliper ( FIG. 17 ) and tumor volume was calculated as followed:
  • mice per treatment group were sacrificed at study day 32, tumors were removed and single cell suspensions were prepared through an enzymatic digestion with Collagenase V, Dispase II and DNAse for subsequent FACS-analysis ( FIGS. 19 and 20 ).
  • Single cells where either used directly for staining of extracellular antigens and activation markers or were re-stimulated using 5 ng/ml PMA and 500 ng/ml Ionomycin in the presence of a protein transport inhibitor Monensin for 5 h in normal culture medium. After re-stimulation, cells were stained for surface antigens, followed by a fixation and permeabilization step.
  • TNF- ⁇ , IFN- ⁇ and IL-2 producing as well as perforin + /granzym-B + CD4 + and CD8 + T-cells were detected in FOLR1 TCB treated tumors.
  • Tumor infiltrating T-cells treated with FOLR1 TCB also showed higher degranulation rates compared to control groups.
  • a bispecific antibody (monovalent for each antigen) that simultaneously can bind to human CD3 and human folate receptor alpha (FolR1) without using any hetero-dimerization approach (e.g. knob-into-hole technology)
  • a combination of a common light chain library with the so-called CrossMab technology was applied:
  • the variable region of the humanized CD3 binder (CH2527_VL7_46/13) was fused to the CH1 domain of a standard human IgG1 antibody to form the VLVH crossed molecule (fused to Fc) which is common for both specificities.
  • VHCL a CD3 specific variable heavy chain domain (CH2527_VH_23/12) was fused to a constant human ⁇ light chain whereas a variable heavy chain domain specific for human FolR1 (clone 16D5, isolated from common light chain library) was fused to a constant human ⁇ light chain.
  • This enables the purification of the desired bispecific antibody by applying subsequent purification steps with KappaSelect and LambdaFab Select columns (GE Healthcare) to remove undesired homodimeric antibodies.
  • HEK293 EBNA cells were transfected with plasmids encoding the respective genes using a standard polyethlenimine (PEI) based method.
  • PEI polyethlenimine
  • the used plasmid ratio of the three expression vectors was 1:1:1.
  • Transfected cells were cultivated for 7 days before supernatants were harvested for purification.
  • the bispecific FolR1/CD3-kappa-lambda antibodies were produced and purified as follows.
  • HEK293 EBNA cells were cultivated in suspension serum free in CD CHO culture medium. For the production in 500 ml shake flask 400 million HEK293 EBNA cells were seeded 24 hours before transfection (for alternative scales all amounts were adjusted accordingly). For transfection cells were centrifuged for 5 min by 210 ⁇ g, supernatant is replaced by pre-warmed 20 ml CD CHO medium. Expression vectors were mixed in 20 ml CD CHO medium to a final amount of 200 ⁇ g DNA.
  • PEI solution After addition of 540 ⁇ l PEI solution is vortexed for 15 s and subsequently incubated for 10 min at room temperature. Afterwards cells were mixed with the DNA/PEI solution, transferred to a 500 ml shake flask and incubated for 3 hours by 37° C. in an incubator with a 5% CO2 atmosphere. After incubation time 160 ml F17 medium was added and cell were cultivated for 24 hours. One day after transfection 1 mM valporic acid and 7% Feed 1 was added.
  • the kappa-lambda bispecific antibody was purified in three steps, using an affinity step specific for kappa light chains, followed by an affinity step specific for lambda light chains and finally by a size exclusion chromatography step for removal of aggregates.
  • the protein was eluted using a pH gradient to buffer B (50 mM Tris, 100 mM glycine, 150 mM NaCl, pH 2.0) over 25 cv.
  • buffer B 50 mM Tris, 100 mM glycine, 150 mM NaCl, pH 2.0
  • Fractions containing the protein of interest were pooled and the pH of the solution was adjusted to pH 8.0 (using 2 M Tris pH 8.0).
  • the protein was eluted using a pH gradient to buffer B (50 mM Tris, 100 mM glycine, 150 mM NaCl, pH 2.0) over 25 cv.
  • buffer B 50 mM Tris, 100 mM glycine, 150 mM NaCl, pH 2.0
  • Fractions containing the protein of interest were pooled and the pH of the solution was adjusted to pH 8.0 (using 2 M Tris pH 8.0).
  • This solution was concentrated using ultra-concentrators (Vivaspin 15R 30.000 MWCO HY, Sartorius) and subsequently applied to a SuperdexTM 200 10/300 GL (GE Healthcare) equilibrated with 20 mM Histidine, pH 6.0, 140 mM NaCl, 0.01% Tween-20.
  • the pooled fractions after size exclusion were again concentrated using ultra-concentrators (Vivaspin 15R 30.000 MWCO HY, Sartorius).
  • the protein concentration 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 constructs were analyzed by SDS capillary electrophoresis in the presence and absence of a reducing agent following the manufacturer instructions (instrument Caliper LabChipGX, Perkin Elmer). Only small amounts of protein could be purified with a final yield of 0.17 mg/L.
  • T cell mediated killing of SKOV3 cells was determined after 24 h and 48 h by LDH release. After 48 h the T cells were harvested and CD69 and CD25 upregulation on CD4 T cells and CD8 T cells was measured by flow cytometry.
  • the kappa lambda FolR1 construct induces killing of SKOV3 cells in a concentration dependent manner which is accompanied by CD69 and CD25 upregulation both on CD4 T cells and on CD8 T cells.
  • SKOV3 cells were incubated with PBMCs in the presence of either kappa lambda FoLR1 TCB or DP47 TCB. After 24 h and 48 h killing of tumor cells was determined by measuring LDH release ( FIG. 21 ). SKOV3 cells were incubated with PBMCs in the presence of either kappa lambda FoLR1 TCB or DP47 TCB. After 48 h CD25 and CD69 upregulation on CD4 T cells and CD8 T cells was measured by flow cytometry ( FIG. 22 ).
  • Binding of anti-FolR1 16D5 in different monovalent or bivalent T-cell bispecific formats and of anti-FolR1 36F2 as IgG or as T-cell bispecific to recombinant human, cynomolgus and murine folate receptor 1 (all as Fc fusions) was assessed by surface plasmon resonance (SPR). All SPR experiments were performed on a Biacore T200 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, GE Healthcare).
  • Recombinant biotinylated monomeric Fc fusions of human, cynomolgus and murine Folate Receptor 1 (FolR1-Fc) were directly coupled on a SA chip using the standard coupling instruction (Biacore, GE Healthcare).
  • the immobilization level was about 300-400 RU.
  • the anti-FolR1 IgGs or T cell bispecifics were passed at a concentration range from 3.7 to 900 nM with a flow of 30 ⁇ L/minutes through the flow cells over 180 seconds. The dissociation was monitored for 240 or 600 seconds.
  • the chip surface was regenerated after every cycle using a double injection of 30 sec 10 mM Glycine-HCl pH 2.
  • the chip surface was regenerated after every cycle using a double injection of 60 sec 10 mM Glycine-HCl pH 1.5. Bulk refractive index differences were corrected for by subtracting the response obtained on the reference flow cell 1.
  • the affinity constants for the interactions were derived from the rate constants by fitting to a 1:1 Langmuir binding using the Bia Evaluation software (GE Healthcare).
  • the affinity (monovalent binding) to human and cyno FolR1-Fc of 36F2 TCB is similar and around 1000 nM for both, whereas the affinity to murine FolR1-Fc is slightly better and around 300 nM.
  • the 36F2 can be used in murine and primate models, there is no need for a surrogate.
  • the avidity (apparent KD) of 36F2 TCB to human FolR1 is around 30 times lower than the affinity of the 16D5 TCB to human FolR1.
  • 36F2 TCB is in the low nanomolar range
  • 16D5 TCB is in the low picomolar range (1000 fold difference).
  • FolR1 is expressed on tumor cells overexpressed, at intermittent and high levels, on the surface of cancer cells in a spectrum of epithelial malignancies, including ovarian, breast, renal, colorectal, lung and other solid cancers and is also expressed on the apical surface of a limited subset of polarized epithelial cells in normal tissue.
  • These non-tumorous, normal cells express FolR1 only at low levels, and include, e.g., bronchiolal epithelial cells on alveolar surface, renal cortical luminal border of tubular cells, retinal pigment epithelium (basolateral membrane) and choroid plexus.
  • 16D5 TCB binds to normal tissues cells expressing low amounts of FolR1 which results in their T cell mediated killing. This might, at least in part, account for limited tolerance observed at 10 ⁇ g/kg in cynomolgus monkeys.
  • the inventors wanted to determine if lowering the affinity of the T cell bispecific molecule could increase the differentiation between high and low target density tissues and, thereby, lower toxicity by making use of bivalent binding and avidity.
  • Low affinity binders are ordinarily not selected as suitable candidates for further analysis because low affinity is often associated with low potency and efficacy. Nevertheless, the low affinity FolR1 binder 36F2 was developed in several formats and characterized for its biological properties.
  • the avidity effect (difference between monovalent and bivalent binding) is around 250 fold (1000 nM versus 4 nM).
  • affinity defined the interaction and with 1000 nM led to a low potency of the TCB.
  • molecule's avidity comes into play and with 4 nM led to a high activity of the TCB (see Example 32).
  • the inventors generated monovalent formats of 16D5 and low affinity variant of 16D5 (affinity about 10-40 nM) in a bivalent format.
  • the 16D5 binder used in a monovalent format (1+1) has an affinity of about 50 nM.
  • the differentiation between high and low target density tissues can be better achieved by taking advantage of the avidity effect.
  • PBMCs peripheral blood mononuclear cells
  • Fresh blood was diluted with sterile PBS and layered over Histopaque gradient (Sigma, #H8889). After centrifugation (450 ⁇ g, 30 minutes, room temperature), the plasma above the PBMC-containing interphase was discarded and PBMCs transferred in a new falcon tube subsequently filled with 50 ml of PBS. The mixture was centrifuged (400 ⁇ g, 10 minutes, room temperature), the supernatant discarded and the PBMC pellet washed twice with sterile PBS (centrifugation steps 350 ⁇ g, 10 minutes).
  • 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 cell incubator until further use (no longer than 24 h).
  • the antibody was added at the indicated concentrations (range of 0.005 pM-5 nM in triplicates).
  • PBMCs were added to target cells at final E:T ratio of 10:1.
  • DP47 TCB antibody was included as negative control. Human PBMCs were used as effectors and the killing was detected at 24 h of incubation with the bispecific antibody.
  • PBMCs Peripheral blood mononuclear cells
  • Histopaque density centrifugation of enriched lymphocyte preparations (buffy coats) obtained from healthy human donors. Fresh blood was diluted with sterile PBS and layered over Histopaque gradient (Sigma, #H8889). After centrifugation (450 ⁇ g, 30 minutes, room temperature), the plasma above the PBMC-containing interphase was discarded and PBMCs transferred in a new falcon tube subsequently filled with 50 ml of PBS.
  • the mixture was centrifuged (400 ⁇ g, 10 minutes, room temperature), the supernatant discarded and the PBMC pellet washed twice with sterile PBS (centrifugation steps 350 ⁇ g, 10 minutes).
  • 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 cell incubator until further use (no longer than 24 h).
  • the antibody was added at the indicated concentrations (range of 0.01 pM-100 nM in triplicates).
  • PBMCs were added to target cells at final E:T ratio of 10:1.
  • this data shows that using the 36F2 FolR1 binder in the bivalent 2+1 TCB format widens the therapeutic window compared to the 16D5 FOLR1 TCB ( FIG. 24A-C ).
  • the 36F2 FOLR1 TCB differentiates between high and low expressing cells which is of special importance to reduce toxicity as the cells of some normal, non-tumorous tissues express very low levels of FolR1 (approximately less than 1000 copies per cell). Consistent with this observation, the results discussed in Example 35 below show that 36F2 TCB does not induce T-cell killing of primary cells ( FIGS. 26A-D ) whereas for 16D5 TCB some killing can be observed on HRCEpiC and HRPEpiC cells after 48 h of incubation ( FIGS. 26B and C).
  • 36F2 TCB This important characteristic of 36F2 TCB allows for dosing for the treatment of FolR1-positive tumors so that it mediates potent killing of tumor tissues with high or medium FOLR1 expression, but not of normal tissues with low (partially polarized) expression. Notably, this characteristic appears to be mediated by the avidity of 36F2 TCB in the bivalent 2+1 inverted format, as it was not observed when using the 1+1 monovalent formats carrying the same low affinity 36F2 binder.
  • 36F2 TCB in the bivalent 2+1 format comprises FolR1 binding moieties of relatively low affinity but it possesses an avidity effect which allows for differentiation between high and low FolR1 expressing cells. Because tumor cells express FolR1 at high or intermediate levels, this TCB selectively binds to tumor cells and not normal, non-cancerous cells that express FolR1 at low levels or not at all.
  • the 36F2 TCB in the bivalent 2+1 inverted format also has the advantage that it does not require chemical cross linking or other hybrid approach. This makes it suitable for manufacture of a medicament to treat patients, for example patients having FolR1-positive cancerous tumors.
  • the 36F2 TCB in the bivalent 2+1 inverted format can be produced using standard CHO processes with low aggregates.
  • the 36F2 TCB in the bivalent 2+1 comprises human and humanized sequences making it superior to molecules that employ rat and murine polypeptides that are highly immunogenic when administered to humans.
  • the 36F2 TCB in the bivalent 2+1 format was engineered to abolish FcgR binding and, as such, does not cause FcgR crosslinking and infusion reactions, further enhancing its safety when administered to patients.
  • a further advantage of the 36F2 TCB in the bivalent 2+1 format and other embodiments disclosed herein is that their clinical development does not require the use of surrogate molecules as they bind to human, cynomous and murine FolR1. As such, the molecules disclosed herein recognize a different epitope than antibodies to FolR1 previously described that do not recognize FolR1 from all three species.
  • Table 29 shows a comparison of EC50 values of 16D5 TCB and 36F2 TCB on the different cell lines tested. Out of the obtained EC50 values the delta (EC50 of 16D5 TCB minus EC50 of 36F2 TCB) and the x-fold difference (EC50 of 16D5 TCB divided by the EC50 of 36F2 TCB) was calculated.
  • Tables 30 and 31 show the comparisons of the EC50 values of 16D5 TCB vs 16D5 TCB HT (Table 30) and 16D5 TCB vs 16D5 TCB 1+1 (Table 31) as well as the corresponding deltas (EC50 of 16D5 TCB minus EC50 of 16D5 TCB HT/1+1) and the x-fold differences (EC50 of 16D5 TCB divided by the EC50 of 16D5 TCB HT/1+1).
  • Activation of CD8 + and CD4 + T cells after T-cell killing of FolR1-expressing Hela, SKov-3 and HT-29 tumor cells mediated by 36F2 TCB and 16D5 TCB was assessed by FACS analysis using antibodies recognizing the T cell activation markers CD25 (late activation marker) and CD69 (early activation marker).
  • DP47 TCB was included as non-binding control.
  • the antibody and the killing assay conditions were essentially as described above (Example 32) using the same antibody concentration range (0.01 pM-100 nM in triplicates), E:T ratio 10:1 and an incubation time of 48 h.
  • PBMCs were transferred to a round-bottom 96-well plate, centrifuged at 400 ⁇ g for 4 min and washed twice with PBS containing 0.1% BSA.
  • Cells were washed twice with 150 ⁇ l/well PBS containing 0.1% BSA. After centrifugation, the samples were resuspended in 200 ⁇ l/well PBS 0.1% for the FACS measurement. Samples were analyzed at BD FACS Canto II.
  • 36F2 TCB induced a target-specific up-regulation of activation markers (CD25, CD69) on CD8+ and CD4+ T cells after killing of Hela ( FIG. 25A ) and SKov-3 ( FIG. 25B ) cells.
  • activation markers CD25, CD69
  • FIG. 25A a target-specific up-regulation of activation markers
  • FIG. 25B a target-specific up-regulation of activation markers
  • T-cell killing mediated by 36F2 TCB and 16D5 TCB was assessed on primary cells (Human Renal Cortical Epithelial Cells (HRCEpiC) (ScienCell Research Laboratories; Cat No 4110) and Human Retinal Pigment Epithelial Cells (HRPEpiC) (ScienCell Research Laboratories; Cat No 6540)).
  • HRCEpiC Human Renal Cortical Epithelial Cells
  • HRPEpiC Human Retinal Pigment Epithelial Cells
  • HT-29 cells low FolR1
  • DP47 TCB served as non-binding control.
  • Human PBMCs were used as effectors and the killing was detected at 24 h and 48 h of incubation with the bispecific antibodies.
  • PBMCs Peripheral blood mononuclear cells
  • Histopaque density centrifugation of enriched lymphocyte preparations (buffy coats) obtained from healthy human donors. Fresh blood was diluted with sterile PBS and layered over Histopaque gradient (Sigma, #H8889). After centrifugation (450 ⁇ g, 30 minutes, room temperature), the plasma above the PBMC-containing interphase was discarded and PBMCs transferred in a new falcon tube subsequently filled with 50 ml of PBS.
  • the mixture was centrifuged (400 ⁇ g, 10 minutes, room temperature), the supernatant discarded and the PBMC pellet washed twice with sterile PBS (centrifugation steps 350 ⁇ g, 10 minutes).
  • 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 cell incubator until further use (no longer than 24 h).
  • the antibody was added at the indicated concentrations (range of 0.01 pM-10 nM in triplicates).
  • PBMCs were added to target cells at final E:T ratio of 10:1.
  • the “untargeted TCB” was used as a control in the above experiments.
  • the bispecific antibody engages CD3e but does not bind to any other antigen and therefore cannot crosslink T cells to any target cells (and subsequently cannot induce any killing). It was therefore used as negative control in the assays to monitor any unspecific T cell activation.
  • This untargeted TCB was prepared as described in WO2014/131712. In brief, the variable region of heavy and light chain DNA sequences have been subcloned in frame with either the constant heavy chain or the constant light chain pre-inserted into the respective recipient mammalian expression vector. The antibody expression was driven by an MPSV promoter and carries a synthetic polyA signal sequence at the 3′ end of the CDS. In addition each vector contains an EBV OriP sequence.
  • the molecule was produced by co-transfecting HEK293-EBNA cells with the mammalian expression vectors using polyethylenimine. The cells were transfected with the corresponding expression vectors in a 1:2:1:1 ratio (“vector heavy chain Fc(hole)”: “vector light chain”: “vector light chain Crossfab”: “vector heavy chain Fc(knob)-FabCrossfab”).
  • HEK293 EBNA cells were cultivated in suspension serum free in CD CHO culture medium. For the production in 500 ml shake flask 400 million HEK293 EBNA cells were seeded 24 hours before transfection. For transfection cells were centrifuged for 5 min by 210 ⁇ g, supernatant is replaced by pre-warmed 20 ml CD CHO medium. Expression vectors were mixed in 20 ml CD CHO medium to a final amount of 200 g DNA. After addition of 540 ⁇ l PEI solution was vortexed for 15 s and subsequently incubated for 10 min at room temperature.
  • cells were mixed with the DNA/PEI solution, transferred to a 500 ml shake flask and incubated for 3 hours by 37° C. in an incubator with a 5% CO2 atmosphere. After incubation time 160 ml F17 medium was added and cell were cultivated for 24 hours. One day after transfection 1 mM valporic acid and 7% Feed 1 was added. After 7 days cultivation supernatant was collected for purification by centrifugation for 15 min at 210 ⁇ g, the solution was sterile filtered (0.22 ⁇ m filter) and sodium azide in a final concentration of 0.01% w/v was added, and kept at 4° C.
  • the secreted protein was purified from cell culture supernatants by affinity chromatography using ProteinA.
  • Target protein was eluted during a gradient over 20 column volume 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.
  • Protein solution was neutralized by adding 1/10 of 0.5 M sodium phosphate, pH 8.
  • Target protein was concentrated and filtrated prior loading on a HiLoad Superdex 200 column (GE Healthcare) equilibrated with 20 mM Histidine, 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.
  • the aggregate content of antibody samples was analyzed using a TSKgel G3000 SW XL analytical size-exclusion column (Tosoh) in 25 mM K2HPO4, 125 mM NaCl, 200 mM L-Arginine Monohydrocloride, 0.02% (w/v) NaN3, pH 6.7 running buffer at 25° C.
  • the binding of 16D5 TCB and the corresponding CD3 deamidation variants 16D5 TCB N100A and 16D5 TCB S100aA and 9D11 TCB and the demidation variants 9D11 TCB N100A and 9D11 TCB S100aA to human CD3 was assessed on a CD3-expressing immortalized T lymphocyte line (Jurkat).
  • results show reduced binding of the deamidation variants N100A and S100aA to CD3 compared to the parental antibodies 16D5 TCB ( FIG. 28A ) and 9D11 TCB ( FIG. 28B ).
  • T-cell killing mediated by 16D5 TCB and the corresponding CD3 deamidation variants 16D5 TCB N100A and 16D5 TCB S100aA and 9D11 TCB and the demidation variants 9D11 TCB N100A and 9D11 TCB S100aA was assessed on SKov-3 (medium FolR1) and HT-29 (low FolR1) cells.
  • Human PBMCs were used as effectors and the killing was detected at 24 h of incubation with the bispecific antibodies. Briefly, target cells were harvested with Trypsin/EDTA, washed, and plated at a density of 25 000 cells/well using flat-bottom 96-well plates. Cells were left to adhere overnight.
  • PBMCs Peripheral blood mononuclear cells
  • enriched lymphocyte preparations obtained from healthy human donors.
  • Fresh blood was diluted with sterile PBS and layered over Histopaque gradient (Sigma, #H8889). After centrifugation (450 ⁇ g, 30 minutes, room temperature), the plasma above the PBMC-containing interphase was discarded and PBMCs transferred in a new falcon tube subsequently filled with 50 ml of PBS. The mixture was centrifuged (400 ⁇ g, 10 minutes, room temperature), the supernatant discarded and the PBMC pellet washed twice with sterile PBS (centrifugation steps 350 ⁇ g, 10 minutes).
  • 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 cell incubator until further use (no longer than 24 h).
  • the antibody was added at the indicated concentrations (range of 0.01 pM-10 nM in triplicates). PBMCs were added to target cells at final E:T ratio of 10:1.
  • Binding of two 16D5 TCBs with CD3 binder variants (N100A or S100aA) to human recombinant CD3 (CD3epsilon-CD3delta heterodimer as Fc fusion) was assessed by surface plasmon resonance (SPR). All SPR experiments were performed on a Biacore T200 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).
  • the chip surface was regenerated after every cycle using a double injection of 60 sec 10 mM Glycine-HCl pH 1.5. Bulk refractive index differences were corrected for by subtracting the response obtained on the reference flow cell 1.
  • the affinity constants for the interactions were derived from the rate constants by fitting to a 1:1 Langmuir binding using the Bia Evaluation software (GE Healthcare).
  • the two CD3 deamidation variants have a slightly reduced affinity compared to the wild-type CD3 binder (CH2527), but the difference is not grave.
  • the two deamidation variants 16D5 TCBs were transiently produced in HEK293 EBNA cells using a PEI mediated transfection procedure for the required vectors as described below.
  • HEK293 EBNA cells are cultivated in suspension serum free in CD CHO culture medium.
  • 400 million HEK293 EBNA cells are seeded 24 hours before transfection (for alternative scales all amounts were adjusted accordingly).
  • For transfection cells are centrifuged for 5 min by 210 ⁇ g, supernatant is replaced by pre-warmed 20 ml CD CHO medium.
  • Expression vectors are mixed in 20 ml CD CHO medium to a final amount of 200 ⁇ g DNA.
  • PEI solution After addition of 540 ⁇ l PEI solution is vortexed for 15 s and subsequently incubated for 10 min at room temperature. Afterwards cells are mixed with the DNA/PEI solution, transferred to a 500 ml shake flask and incubated for 3 hours by 37° C. in an incubator with a 5% CO2 atmosphere. After incubation time 160 ml F17 medium is added and cell are cultivated for 24 hours. One day after transfection 1 mM valporic acid and 7% Feed 1 is added.
  • the two deamidation variants 16D5 TCBs were purified in two steps using standard procedures, such as protein A affinity purification ( ⁇ kta Explorer) and size exclusion chromatography.
  • Purity and molecular weight of the constructs were analyzed by SDS capillary electrophoresis (CE-SDS) in the presence and absence of a reducing agent following the manufacturer instructions (instrument Caliper LabChipGX, Perkin Elmer). Purified proteins were frozen in liquid N 2 and stored at ⁇ 80° C.
  • Both TCBs were produced in good quality, similar to the construct with the wild-type CD3 binder.
  • HEK293 EBNA cells are cultivated in suspension serum free in CD CHO culture medium.
  • HEK293 EBNA cells are seeded 24 hours before transfection (for alternative scales all amounts were adjusted accordingly).
  • transfection cells are centrifuged for 5 min by 210 ⁇ g, supernatant is replaced by pre-warmed 20 ml CD CHO medium.
  • Expression vectors are mixed in 20 ml CD CHO medium to a final amount of 200 ⁇ g DNA.
  • PEI solution After addition of 540 ⁇ l PEI solution is vortexed for 15 s and subsequently incubated for 10 min at room temperature. Afterwards cells are mixed with the DNA/PEI solution, transferred to a 500 ml shake flask and incubated for 3 hours by 37° C. in an incubator with a 5% CO2 atmosphere. After incubation time 160 ml F17 medium is added and cell are cultivated for 24 hours. One day after transfection 1 mM valporic acid and 7% Feed 1 is added.
  • the two IgGs were purified in two steps using standard procedures, such as protein A affinity purification ( ⁇ kta Explorer) and size exclusion chromatography.
  • the 5 ml containing the protein of interest are stored in a loop on the ⁇ kta Explorer and subsequently applied to a HiLoad 16/60 SuperdexTM 200 (GE Healthcare) equilibrated with 20 mM Histidine, pH 6.0, 140 mM NaCl (no TWEEn was used).
  • Fractions containing the IgGs were pooled and concentrated using ultra concentrators (Amicon Ultra-15, 30.000 MWCO, Millipore). The aggregate content of the final pool was analyzed by analytical size exclusion chromatography.
  • Recombinant biotinylated monomeric Fc fusions of human, cynomolgus and murine Folate Receptor 1 (FolR1-Fc) were directly coupled on a SA chip using the standard coupling instruction (Biacore, Freiburg/Germany).
  • the immobilization level was about 160.
  • the anti-FolR1 IgGs or T cell bispecifics were passed at a concentration range from 3.7 to 900 nM with a flow of 30 ⁇ L/minutes through the flow cells over 180 seconds. The dissociation was monitored for 600 seconds. Bulk refractive index differences were corrected for by subtracting the response obtained on reference flow cell immobilized with recombinant biotinylated murine IL2 receptor Fc fusion.
  • the binding curves resulting from the bivalent binding of the IgG or T cell bispecifics were approximated to a 1:1 Langmuir binding (even though it is a 1:2 binding) and fitted with that model to get an apparent KD representing the avidity of the bivalent binding.
  • the apparent avidity constants for the interactions were derived from the rate constants of the fitting using the Bia Evaluation software (GE Healthcare).
  • the chip surface was regenerated after every cycle using a double injection of 60 sec 10 mM Glycine-HCl pH 1.5. Bulk refractive index differences were corrected for by subtracting the response obtained on the reference flow cell 1.
  • the affinity constants for the interactions were derived from the rate constants by fitting to a 1:1 Langmuir binding using the Bia Evaluation software (GE Healthcare).
  • the two 16D5 hot spot variants have similar avidity (bivalent binding) than the wild-type 16D5 binder.
  • the avidity is slightly decreased for the D52dQ variant and this difference is even more visible in affinity (monovalent binding).
  • the twelve IgGs were transiently produced in HEK293 EBNA cells using a PEI mediated transfection procedure for the required vectors as described below.
  • HEK293 EBNA cells are cultivated in suspension serum free in CD CHO culture medium.
  • 400 million HEK293 EBNA cells are seeded 24 hours before transfection (for alternative scales all amounts were adjusted accordingly).
  • For transfection cells are centrifuged for 5 min by 210 ⁇ g, supernatant is replaced by pre-warmed 20 ml CD CHO medium.
  • Expression vectors are mixed in 20 ml CD CHO medium to a final amount of 200 ⁇ g DNA.
  • PEI solution After addition of 540 ⁇ l PEI solution is vortexed for 15 s and subsequently incubated for 10 min at room temperature. Afterwards cells are mixed with the DNA/PEI solution, transferred to a 500 ml shake flask and incubated for 3 hours by 37° C. in an incubator with a 5% CO2 atmosphere. After incubation time 160 ml F17 medium is added and cell are cultivated for 24 hours. One day after transfection 1 mM valporic acid and 7% Feed 1 is added.
  • the 5 ml containing the protein of interest are stored in a loop on the ⁇ kta Explorer and subsequently applied to a HiLoad 16/60 SuperdexTM 200 (GE Healthcare) equilibrated with 20 mM Histidine, pH 6.0, 140 mM NaCl, 0.01% Tween-20.
  • Fractions containing the IgGs were pooled and concentrated using ultra concentrators (Amicon Ultra-15, 30.000 MWCO, Millipore). The aggregate content of the final pool was analyzed by analytical size exclusion chromatography.
  • Recombinant biotinylated monomeric Fc fusions of human, cynomolgus and murine Folate Receptor 1 (FolR1-Fc) were directly coupled on a SA chip using the standard coupling instruction (Biacore, Freiburg/Germany).
  • the immobilization level was about 300.
  • the anti-FolR1 IgGs or T cell bispecifics were passed at a concentration range from 11.1 to 900 nM with a flow of 30 ⁇ L/minutes through the flow cells over 180 seconds. The dissociation was monitored for 240 or 600 seconds.
  • the chip surface was regenerated after every cycle using a double injection of 60 sec 10 mM Glycine-HCl pH 1.5. Bulk refractive index differences were corrected for by subtracting the response obtained on the reference flow cell 1.
  • the affinity constants for the interactions were derived from the rate constants by fitting to a 1:1 Langmuir binding using the Bia Evaluation software (GE Healthcare).
  • 16D5 T-Cell Bispecific with Mutations to Reduce the Affinity to Human and Cynomolgus FolR1 16D5 TCB G49S/S93A, G49S/K53A, W96Y, W96Y/D52E
  • HEK293 EBNA cells Four additional variants of 16D5 TCBs having reduced affinity to FolR1 were transiently produced in HEK293 EBNA cells using a PEI mediated transfection procedure for the required vectors as described below.
  • HEK293 EBNA cells are cultivated in suspension serum free in Excell culture medium containing 6 mM L-Glutamine and 250 mg/l G418 culture medium.
  • 600 ml tubespin flask maximum. working volume 400 mL
  • 600 million HEK293 EBNA cells are seeded 24 hours before transfection.
  • transfection cells are centrifuged for 5 min by 210 ⁇ g, supernatant is replaced by pre-warmed 20 ml CD CHO medium.
  • Expression vectors are mixed in 20 ml CD CHO medium to a final amount of 400 ⁇ g DNA. After addition of 1080 ⁇ l PEI solution (2.7 ⁇ g/ml) is vortexed for 15 s and subsequently incubated for 10 min at room temperature. Afterwards cells are mixed with the DNA/PEI solution, transferred to a 600 ml tubespin flask and incubated for 3 hours by 37° C. in an incubator with a 5% CO2 atmosphere. After incubation time 360 ml Excell+6 mM L-Glutamine+5 g/L Pepsoy+1.0 mM VPA medium is added and cells are cultivated for 24 hours. One day after transfection 7% Feed 7 is added.
  • the reduced affinity variants 16D5 TCBs were purified in two steps using standard procedures, such as protein A affinity purification ( ⁇ kta Explorer) and size exclusion chromatography.
  • Purity and molecular weight of the constructs were analyzed by SDS capillary electrophoresis (CE-SDS) in the presence and absence of a reducing agent following the manufacturer instructions (instrument Caliper LabChipGX, Perkin Elmer). Purified proteins were frozen in liquid N 2 and stored at ⁇ 80° C.
  • the intermediate affinity TCBs were transiently produced in HEK293 EBNA cells using a PEI mediated transfection procedure for the required vectors as described below.
  • HEK293 EBNA cells are cultivated in suspension serum free in Excell culture medium containing 6 mM L-Glutamine and 250 mg/l G418 culture medium.
  • 600 ml tubespin flask max. working volume 400 mL
  • 600 million HEK293 EBNA cells are seeded 24 hours before transfection.
  • For transfection cells are centrifuged for 5 min by 210 ⁇ g, supernatant is replaced by pre-warmed 20 ml CD CHO medium.
  • Expression vectors are mixed in 20 ml CD CHO medium to a final amount of 400 ⁇ g DNA. After addition of 1080 ⁇ l PEI solution (2.7 ⁇ g/ml) is vortexed for 15 s and subsequently incubated for 10 min at room temperature. Afterwards cells are mixed with the DNA/PEI solution, transferred to a 600 ml tubespin flask and incubated for 3 hours by 37° C. in an incubator with a 5% CO2 atmosphere. After incubation time 360 ml Excell+6 mM L-Glutamine+5 g/L Pepsoy+1.0 mM VPA medium is added and cells are cultivated for 24 hours. One day after transfection 7% Feed 7 is added.
  • the intermediate affinity TCBs were purified in two steps using standard procedures, such as protein A affinity purification ( ⁇ kta Explorer) and size exclusion chromatography.
  • Purity and molecular weight of the constructs were analyzed by SDS capillary electrophoresis (CE-SDS) in the presence and absence of a reducing agent following the manufacturer instructions (instrument Caliper LabChipGX, Perkin Elmer). Purified proteins were frozen in liquid N 2 and stored at ⁇ 80° C.
  • FIG. 34A-E The binding of 36F2 TCB, 16D5 TCB and various HC/LC variants ( FIG. 34A-E ) of 16D5 to human FolR1 was assessed on HT-29 cells. Briefly, cells were harvested, counted, checked for viability and resuspended at 2 ⁇ 10 6 cells/ml in FACS buffer (100 ⁇ l PBS 0.1% BSA). 100 ⁇ l of cell suspension (containing 0.2 ⁇ 10 6 cells) was incubated in round-bottom 96-well plates for 30 min at 4° C. with different concentrations of the bispecific antibodies (229 pM-500 nM). After two washing steps with cold PBS 0.1% BSA, samples were re-incubated for further 30 min at 4° C.
  • FACS buffer 100 ⁇ l PBS 0.1% BSA. 100 ⁇ l of cell suspension (containing 0.2 ⁇ 10 6 cells) was incubated in round-bottom 96-well plates for 30 min at 4° C. with 100 nM of the bispecific antibodies. After two washing steps with cold PBS 0.1% BSA, samples were re-incubated for further 30 min at 4° C. with a Fluorescein (FITC) AffiniPure F(ab′) 2 Fragment Goat Anti-Human IgG, Fc ⁇ Fragment Specific secondary antibody (Jackson Immuno Research Lab PE #109-096-098).
  • FITC Fluorescein
  • Binding of anti-FolR1 16D5 reduced affinity variants and additional intermediate affinity binders in the bivalent T-cell bispecific format to recombinant human, cynomolgus and murine folate receptor 1 (all as Fc fusions) was assessed by surface plasmon resonance (SPR). All SPR experiments were performed on a Biacore T200 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, GE Healthcare). The molecules used for affinity and avidity determination are described in Table 45.
  • Figure Name Description reference 16D5 reduced affinity 2 + 1 T-cell bispecific, inverted FIG. 1A variants format (common light chain) 16D5 TCB 16D5 G49S/S93A TCB 16D5 G49S/K53A TCB 16D5 W96Y TCB 16D5 W96Y/D52E TCB Intermediate affinity 2 + 1 T-cell bispecific, inverted FIG. 1F binders format, crossfab 36F2 TCB 6E10 TCB 14B1 TCB 9C7 TCB
  • the anti-FolR1 TCBs were analyzed by single injections (Table 46) to characterize their crossreactivity (to human, murine and cyno FolR1) and specificity (to human FolR1, human FolR2, human FolR3).
  • Recombinant biotinylated monomeric Fc fusions of human, cynomolgus and murine Folate Receptor 1 (FolR1-Fc) or human Folate Receptor 2 and 3 (FolR2-Fc, FolR3-Fc) were directly coupled on a SA chip using the standard coupling instruction (Biacore, Freiburg/Germany). The immobilization level was about 300-400 RU.
  • the TCBs were injected for 60 seconds at a concentration of 500 nM.
  • Recombinant biotinylated monomeric Fc fusions of human, cynomolgus and murine Folate Receptor 1 (FolR1-Fc) were directly coupled on a SA chip using the standard coupling instruction (Biacore, GE Healthcare).
  • the immobilization level was about 200-300 RU.
  • the anti-FolR1 T cell bispecifics were passed at a concentration range from 11.1 to 900 nM (for the 16D5 reduced affinity variants) or 0.2 to 500 nM (for the additional intermediate affinity binders and 36F2) with a flow of 30 ⁇ L/minutes through the flow cells over 180 seconds.
  • the dissociation was monitored for 240 or 600 seconds.
  • the chip surface was regenerated after every cycle using a double injection of 30 sec 10 mM Glycine-HCl pH 1.5. Bulk refractive index differences were corrected for by subtracting the response obtained on reference flow cell immobilized with recombinant biotinylated murine IL2R Fc fusion (unrelated Fc fused receptor).
  • the binding curves resulting from the bivalent binding of the T cell bispecifics were approximated to a 1:1 Langmuir binding (even though it is a 1:2 binding) and fitted with that model to get an apparent KD representing the avidity of the bivalent binding.
  • the apparent avidity constants for the interactions were derived from the rate constants of the fitting using the Bia Evaluation software (GE Healthcare).

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