MOLECULE THAT BINDES THE BIESPECIFIC ANTIGEN THAT ACTIVATES T CELLS, ISOLATED POLYNUCLEOTIDE OR POLY

专利摘要:
molecules that bind to the bispecific antigen that activate t cells, polynucleotide, vector, host cell, method to produce the molecule, pharmaceutical composition and use of the molecule the present invention generally relates to new bispecific antigen binding molecules for cell activation redirecting you to specific target cells. moreover, the present invention relates to polynucleotides that encode these bispecific antigen-binding molecules, and vectors and host cells that comprise these polynucleotides. the invention further relates to methods for producing bispecific antigen-binding molecules of the invention, and methods of using these bispecific antigen-binding molecules in the treatment of disease.
公开号:BR112015015824B1
申请号:R112015015824-2
申请日:2014-02-24
公开日:2020-09-24
发明作者:Marina Bacac；Thomas Hofer；Ralf Hosse；Christiane Jaeger；Christian Klein；Ekkehard Moessner；Pablo Umana；Tina Weinzierl
申请人:Roche Glycart Ag；
IPC主号:

专利说明:

[0001] [001] The present invention generally relates to bispecific antigen binding molecules to activate T cells. In addition, the present invention relates to polynucleotides encoding these bispecific antigen binding molecules, and host vectors and cells that comprise these polynucleotides. The invention further relates to methods for producing bispecific antigen-binding molecules of the invention, and methods of using these bispecific antigen-binding molecules in the treatment of disease. Background of the Invention
[0002] [002] The selective destruction of an individual cell or a specific cell type is often desirable in a variety of clinical situations. For example, it is a primary goal of cancer therapy to specifically destroy tumor cells, while leaving healthy cells and tissues intact and undamaged.
[0003] [003] An attractive way to achieve this is to induce an immune response against the tumor, to cause immune effector cells, such as natural killer cells (Natural Killer - NK) or cytotoxic T lymphocytes (CTLs), to attack and destroy cells tumoral. CTLs are 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.
[0004] [004] In this regard, bispecific antibodies designed to bind with a "arm" to a surface antigen on target cells, and with the second "arm" to an invariant component of activation of the T cell receptor complex (TCR) , have become of interest in recent years. The simultaneous binding of this antibody to both of its targets will force a temporary interaction between the target cell and T cells, causing activation of any cytotoxic T cell and subsequent lysis of the target cell. Then, the immune response is redirected to the target cells and is independent of the presentation of the peptide antigen by the target cell or of the specificity of the T cell, as it could be relevant for normal MHC restricted activation of CTLs. In this context, it is crucial that CTLs are only activated when a target cell is presenting the bispecific antibody for them, that is, the immune synapse is imitated. Bispecific antibodies that do not require preconditioning or co-stimulation in order to obtain efficient lysis of target cells are particularly desirable.
[0005] [005] Various bispecific antibody formats have been developed and their suitability for T cell-mediated immunotherapy has been investigated. Of these, the molecules called BiTE (bispecific T cell engagers, bispecific T cell engager) have been very well characterized and have already shown promise in offices (reviewed in Nagorsen and Bäuerle, Exp Cell Res 317, 1255-1260 (2011)). BiTEs are tandem scFv molecules in which two scFv molecules are fused by a flexible ligand. Additional bispecific formats being evaluated for T cell engagement include diabody (Holliger et al., Prot Eng 9, 299-305 (1996)) and derivatives thereof, such as tandem diabody (Kipriyanov et al., J Mol Biol 293, 41 -66 (1999)). A more recent development are molecules called DART (double affinity redirect), which are based on the diabody shape, but have a C-terminal disulfide bond for further stabilization (Moore et al., Blood 117, 4542-51 (2011)) . The so-called triomabs, which are hybrid mouse / rat IgG molecules and are also currently being evaluated in clinical trials, represent a larger format (reviewed in Seimetz et al., Cancer Treat Rev 36, 458-467 (2010) ).
[0006] [006] The variety of formats being developed shows the great potential attributed to the direction and activation of T cells in immunotherapy. The task of generating suitable bispecific antibodies for this purpose is, however, by non-trivial means, but it involves a series of challenges that have to be met, related to the effectiveness, toxicity, applicability and productivity of the antibodies.
[0007] [007] Small constructs, such as BITE molecules, although being able to efficiently cross-link effector and target cells, have a very short serum half-life, requiring them to be administered to patients by continuous infusion. IgG-like formats, on the other hand, although they have the great advantage of a long half-life, suffer from toxicity associated with native effector functions inherent to IgG molecules. Its immunogenic potential is another unfavorable characteristic of bispecific IgG antibodies, especially non-human formats, for successful therapeutic development. Finally, a major challenge in the general development of bispecific antibodies has been the production of bispecific antibody constructs with clinically sufficient quantity and purity, due to the incompatible pairing of the heavy and light chains of the antibody of different specificities through coexpression, which reduces the performance of the construct correctly assembled and results in a series of non-functional side products, from which the desired bispecific antibody can be difficult to separate.
[0008] [008] Given the difficulties and disadvantages associated with bispecific antibodies currently available to mediate T cell immunotherapy, there remains a need for new and improved formats of these molecules. The present invention provides bispecific antigen-binding molecules designed for activation and redirection of T cells that combine good efficacy and productivity with low toxicity and favorable pharmacokinetic properties. Brief Description of the Invention
[0009] (i) um primeira porção que se liga ao antígeno que é uma molécula Fab capaz de se ligar especificamente a CD3, que compreende pelo menos uma região determinante de complementaridade da cadeia pesada (CDR) selecionada a partir do grupo que consiste em SEQ ID NO: 4, SEQ ID NO: 5 e SEQ ID NO: 6 e pelo menos uma CDR da cadeia leve selecionada a partir do grupo de SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10; (ii) uma segunda porção que se liga ao antígeno que é uma molécula Fab capaz de se ligar especificamente a um antígeno de célula alvo. [009] In a first aspect, the present invention provides a bispecific antigen-binding molecule that activates T cells, which comprises: (i) a first antigen-binding portion that is a Fab molecule capable of specifically binding to CD3, comprising at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO : 4, SEQ ID NO: 5 and SEQ ID NO: 6 and at least one CDR of the light chain selected from the group of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10; (ii) a second portion that binds to the antigen, which is a Fab molecule capable of specifically binding to a target cell antigen.
[0010] [0010] In one embodiment, the first portion that binds to the antigen which is a Fab molecule capable of specifically binding to CD3 and comprises a variable region of the heavy chain that comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group of: SEQ ID NO: 3, SEQ ID NO: 32 and SEQ ID NO: 33 and a variable light chain that comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group of: SEQ ID NO: 7 and SEQ ID NO : 31.
[0011] [0011] In one embodiment, the first portion that binds to the antigen which is a Fab molecule capable of specifically binding to CD3 and comprises a variable region of the heavy chain that comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 3, and a variable region of the light chain comprising an amino acid sequence that is at least about 95%, 96% , 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 7.
[0012] [0012] In a specific embodiment, the second portion that binds to the antigen is able to specifically bind to the Carcinoembryonic Antigen (CEA, CEACAM5) and comprises at least one complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26 and at least one CDR of the light chain selected from the group of SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30.
[0013] [0013] In another specific embodiment, the second antigen-binding portion is able to specifically bind CEA and comprises a variable region of the heavy chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 23, and a variable region of the light chain comprising an amino acid sequence that is at least about 95%, 96%, 97% , 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 27.
[0014] [0014] In another specific embodiment, the second antigen-binding portion is capable of specifically binding to MCSP (CSPG4) and comprises at least one complementarity determining region (CDR) of the heavy chain selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 40 and at least one selected light chain CDR from the group of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 48, SEQ ID NO: 49 and SEQ ID NO : 50.
[0015] [0015] In another specific embodiment, the second portion that binds to the antigen is able to specifically bind to Melanoma Associated Chondroitin Sulfate Proteoglycan (MCSP, CSPG4) and comprises at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 and at least one CDR of the light chain selected from the group of SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20.
[0016] [0016] In another specific embodiment, the second antigen-binding portion is capable of specifically binding to MCSP and comprises a variable region of the heavy chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group of SEQ ID NO: 13, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 39 and SEQ ID NO : 41, and a variable region of the light chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group of SEQ ID NO: 17, SEQ ID NO: 43, SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 51.
[0017] [0017] In another specific embodiment, the second portion that binds to the antigen is able to specifically bind to MCSP and comprises a variable region of the heavy chain that comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 13, and a variable region of the light chain comprising an amino acid sequence that is at least about 95%, 96%, 97% , 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 17.
[0018] [0018] In a specific embodiment, the first portion that binds to the antigen is a cross between the Fab molecule, in which both the variable regions and the constants of the Fab light chain and the Fab heavy chain are exchanged. In an even more specific embodiment, the first portion that binds to the antigen is a cross between the Fab molecule, in which the regions constant on the Fab light chain like the Fab heavy chain are switched.
[0019] [0019] In one embodiment, the second portion that binds to the antigen is a conventional Fab molecule.
[0020] [0020] In an additional specific embodiment, no more than one antigen-binding component capable of specifically binding to CD3 is present in the bispecific antigen-binding molecule that activates T cells (ie, the bispecific antigen-binding molecule that active T cell provides monovalent binding to CD3).
[0021] [0021] In a further embodiment, said bispecific antigen-binding molecule that activates T cells further comprises a third portion that binds to the antigen which is a Fab molecule capable of specifically binding to a target cell antigen. In one embodiment, said third antigen-binding molecule is a conventional Fab molecule. In one embodiment, said third antigen-binding molecule is identical to the second antigen-binding molecule.
[0022] [0022] In a specific embodiment, said bispecific antigen binding molecule that activates T cells further comprises a third portion that binds to the antigen which is a Fab molecule capable of specifically binding to CEA, and comprises at least one determinant region of complementarity (CDR) of the heavy chain selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26 and at least one CDR of the light chain selected from the group of SEQ ID NO : 28, SEQ ID NO: 29 and SEQ ID NO: 30.
[0023] [0023] In a specific embodiment, said bispecific antigen-binding molecule that activates T cells comprises a third portion that binds to the antigen which is a Fab molecule capable of specifically binding to CEA, and comprises a variable region of the heavy chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 23, and a variable region of the light chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 27.
[0024] [0024] In one embodiment, said bispecific antigen-binding molecule that activates T cells further comprises a third portion that binds to the antigen which is a Fab molecule capable of specifically binding to MCSP, and comprises at least one determinant region of complementarity (CDR) of the heavy chain selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 40 and at least one CDR of the light chain selected from the group of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 48, SEQ ID NO: 49 and SEQ ID NO: 50.
[0025] [0025] In a specific embodiment, said bispecific antigen binding molecule that activates T cells further comprises a third portion that binds to the antigen which is a Fab molecule capable of specifically binding to MCSP, and comprises at least one determinant region of complementarity (CDR) of the heavy chain selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 and at least one CDR of the light chain selected from the group of SEQ ID NO : 18, SEQ ID NO: 19 and SEQ ID NO: 20.
[0026] [0026] In one embodiment, said bispecific antigen binding molecule that activates T cells comprises a third portion that binds to the antigen which is a Fab molecule capable of specifically binding to MCSP, and comprises a variable region of the heavy chain that comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence selected from SEQ ID NO: 13, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 39 and SEQ ID NO: 41, and a variable region of the light chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the selected amino acid sequence from the group of SEQ ID NO: 17, SEQ ID NO: 43, SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 51.
[0027] [0027] In a specific embodiment, said bispecific antigen binding molecule that activates T cells comprises a third portion that binds to the antigen which is a Fab molecule capable of specifically binding to MCSP, and comprises a variable region of the heavy chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 13, and a variable region of the light chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 17.
[0028] [0028] In some embodiments, the first and the second portion that binds to the antigen of the bispecific antigen binding molecule that activates T cells are fused together, optionally via a peptide ligand. In this embodiment, the second antigen-binding portion is fused at the C termination of the Fab heavy chain to the N termination of the Fab heavy chain of the first antigen-binding portion. In another embodiment, the first antigen-binding portion is fused at the C termination of the Fab heavy chain to the N termination of the Fab heavy chain of the second antigen-binding portion. In embodiments where both (i) the second antigen-binding portion 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 portion and (ii) the first portion that binds to the antigen is fused at the C termination of the Fab heavy chain to the N termination of the Fab heavy chain of the second portion that binds to the antigen, in addition to the Fab light chain of the first portion that binds to the antigen and the light chain of Fab of the second portion that binds to the antigen can be fused together, optionally via a peptide linker.
[0029] [0029] In one embodiment, said bispecific antigen-binding molecule that activates T cells further comprises (iii) an Fc domain composed of a first and a second subunit capable of stable association.
[0030] [0030] In one embodiment, the second portion that binds to the antigen of the bispecific antigen binding molecule that activates T cells is fused at the C terminus of the Fab heavy chain to the N termination of the first or second subunit of the Fc domain. In another embodiment, the first antigen-binding portion is fused at the C termination of the Fab heavy chain to the N termination of the first or second subunit of the Fc domain. In one embodiment, the first and second portion that binds to the antigen of the bispecific antigen binding molecule that activates T cells are each fused at the C terminus of the Fab heavy chain to the N termination of one of the subunits of the Fc domain .
[0031] [0031] In one embodiment, the third portion that binds to the antigen is fused at the C termination of the Fab heavy chain to the N termination of the first or second subunit of the Fc domain. In a specific embodiment, the second and third portions that bind to the antigen of the antigen binding molecule that activates T cells are each fused at the C terminus of the Fab heavy chain to the N termination of one of the subunits of the Fc domain , and the first antigen-binding portion 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 portion. In another specific embodiment, the first and third portions that bind to the antigen of the antigen binding molecule that activates T cells are each fused at the C terminus of the Fab heavy chain to the N termination of one of the subunits of the Fc domain , and the first antigen-binding portion 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 portion. The components of the bispecific antigen-binding molecule that activates T cells can be fused directly or through suitable peptide ligands. In one embodiment, the first and third antigen-binding components and the Fc domain are part of an immunoglobulin molecule.
[0032] [0032] In a specific embodiment, the immunoglobulin molecule is an immunoglobulin of the IgG class. In an even more specific embodiment, the immunoglobulin is an immunoglobulin of the IgG1 class. In another embodiment, the immunoglobulin is an immunoglobulin of the IgG4 subclass.
[0033] [0033] In a specific embodiment, the Fc domain is an IgG Fc domain. In a specific embodiment, the Fc domain is an IgG1 Fc domain. In another specific embodiment, the Fc domain is an IgG4 Fc domain. In an even more specific embodiment, the Fc domain is an IgG4 Fc domain that comprises the substitution of amino acid S228P (EU numbering). In specific embodiments, the Fc domain is a human Fc domain.
[0034] [0034] In specific projects, the Fc domain comprises a modification that promotes the association of the first and the second subunit of the Fc domain. In this specific embodiment, an amino acid residue in the CH3 domain of the first subunit of the Fc domain is replaced by an amino acid residue that has a larger side chain volume, thereby generating a bulge within the CH3 domain of the first subunit that is positionable in a cavity. within the CH3 domain of the second subunit, and an amino acid residue in the CH3 domain of the second Fc domain subunit is replaced by an amino acid residue that has a smaller side chain volume, thus generating a cavity within the CH3 domain of the second subunit within from which the protrusion within the CH3 domain of the first subunit is positionable.
[0035] [0035] In a specific embodiment, the Fc domain exhibits reduced binding affinity for an Fc receptor and / or reduced effector function, compared to a native IgG1 Fc domain. In certain embodiments, the Fc domain is genetically engineered to have reduced binding affinity for an Fc receptor and / or reduced effector function, compared to a non-genetically engineered Fc domain. In one embodiment, the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor and / or effector function. In one embodiment, one or more amino acid substitutions in the Fc domain that reduce binding to an Fc receptor and / or effector function are in one or more positions selected from the group of L234, L235, P329 (EU numbering). In specific embodiments, each subunit of the Fc domain comprises three amino acid substitutions that reduce binding to an Fc receptor and / or effector function, wherein said amino acid substitutions are L234A, L235A and P329G. In one embodiment, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. In other embodiments, each subunit of the Fc domain comprises two amino acid substitutions that reduce binding to an Fc receptor and / or effector function, wherein said amino acid substitutions are L235E and P329G. In one embodiment, the Fc domain is an IgG4 Fc domain, particularly a human IgG4 Fc domain. In one embodiment, the Fc domain of the bispecific antigen-binding molecule that activates T cells is an IgG4 Fc domain and comprises amino acid substitutions L235E and S228P (SPLE).
[0036] [0036] In one embodiment, the Fc receptor is an Fcү receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activating Fc receptor. In a specific embodiment, the Fc receptor is FcүRIIa, FcүRI and / or FcүRIIIa. In one embodiment, the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC).
[0037] [0037] According to another aspect of the invention, an isolated polynucleotide is provided that encodes a bispecific antigen-binding molecule that activates T cells of the invention or a fragment thereof. The invention also encompasses polypeptides encoded by the polynucleotides of the invention. The invention further provides an expression vector comprising the isolated polynucleotide of the invention, and a host cell comprising an isolated polynucleotide or the expression vector of the invention. In some embodiments, the host cell is a eukaryotic cell, particularly a mammalian cell.
[0038] [0038] In another aspect, a method is provided to produce the bispecific antigen-binding molecules that activate T cells of the invention, comprising the steps of a) culturing the host cell of the invention under conditions suitable for expression of the binding molecule bispecific antigen that activates T cells and b) recovering the bispecific antigen binding molecule that activates T cells. The invention also encompasses a bispecific antigen binding molecule that activates T cells produced by the method of the invention.
[0039] [0039] The invention further provides a pharmaceutical composition comprising the bispecific antigen-binding molecule that activates T cells of the invention and a pharmaceutically acceptable carrier.
[0040] [0040] Also encompassed by the invention are methods of using the bispecific antigen binding molecule that activates T cells and the pharmaceutical composition of the invention. In one aspect, the invention provides a bispecific antigen-binding molecule that activates T cells or a pharmaceutical composition of the invention for use as a medicament. In one aspect, a bispecific antigen-binding molecule that activates T cells or a pharmaceutical composition according to the invention is provided for use in the treatment of a disease in an individual in need thereof. In a specific embodiment, the disease is cancer.
[0041] [0041] The use of a bispecific antigen binding molecule that activates T cells of the invention is also provided for the manufacture of a drug for the treatment of a disease in an individual in need of it, as well as a method of treating a disease in an individual, comprising administering to said individual a therapeutically effective amount of a composition comprising the bispecific antigen binding molecule that activates T cells according to the invention in a pharmaceutically acceptable form. In a specific embodiment, the disease is cancer. In any of the above embodiments, the individual is preferably a mammal, particularly a human.
[0042] [0042] The invention also provides a method for inducing lysis of a target cell, particularly a tumor cell, which comprises placing a target cell in contact with a bispecific antigen binding molecule that activates T cells of the invention in the presence of a T cell , particularly a cytotoxic T cell. Brief Description of the Figures
[0043] [0043] FIGURE 1. Exemplary configurations of the activation of bispecific antigen-binding molecules that activate T cells (TCBs) of the invention. (A) Illustration of the "1 + 1 IgG Crossfab" molecule. (B) Illustration of the "2 + 1 IgG Crossfab" molecule. (C) Illustration of the "2 + 1 IgG Crossfab" molecule with alternative order of the Crossfab and Fab components ("inverted"). (D) Illustration of the "1 + 1 IgG Crossmab" molecule. (E) Illustration of the "2 + 1 IgG Crossfab molecule, linked light chain". (F) Illustration of the "1 + 1 IgG Crossfab molecule, linked light chain". (G) Illustration of the molecule "2 + 1 IgG Crossfab, inverted, linked light chain". (H) Illustration of the "1 + 1 IgG Crossfab molecule, inverted, linked light chain" Black dot: optional modification in the Fc domain that promotes heterodimerization.
[0044] [0044] FIGURE 2. Alignment of affinity matured anti-MCSP clones, compared to the unmatured parental clone (M4-3 ML2).
[0045] [0045] FIGURE 3. Schematic drawing of the MCSP TCB molecule (2 + 1 Crossfab-IgG P329G LALA inverted).
[0046] [0046] FIGURE 4. CE-SDS analysis of MCSP TCB (2 + 1 Crossfab-IgG P329G LALA inverted, SEQ ID NOs: 12, 53, 54 and 55). Electropherogram shown as MCSP TCB SDS-Page: A) not reduced, B) reduced.
[0047] [0047] FIGURE 5. Schematic drawing of the CEA TCB molecule (2 + 1 Crossfab-IgG P329G LALA inverted).
[0048] [0048] FIGURE 6. CE-SDS analysis of CEA TCB molecule (2 + 1 Crossfab-IgG P329G inverted LALA, SEQ ID NOs: 22, 56, 57 and 58). Electropherogram shown as CEA TCB SDS-Page: A) not reduced, B) reduced.
[0049] [0049] FIGURE 7. Binding of MCSP TCB (SEQ ID NOs: 12, 53, 54 and 55) to A375 cells (MCSP +) (A) and Jurkat (CD3 + cells) (B). "TCB not directed": bispecific antibody that engages CD3, but not the second antigen (SEQ ID NOs: 59, 60, 61 and 62).
[0050] [0050] FIGURE 8. Death by T cells induced by MCSP TCB antibody (SEQ ID NOs: 12, 53, 54 and 55) of A375 (high MCSP) (A), MV-3 (average MCSP) (B), HCT -116 (low MCSP) (C) and LS180 (negative MCSP) (D) target cells (E: T = 10: 1, human effector PBMCs, incubation time 24 h). "TCB not directed": bispecific antibody that engages CD3, but not the second antigen (SEQ ID NOs: 59, 60, 61 and 62).
[0051] [0051] FIGURE 9. Suppression of CD25 and CD69 in human CD8 + (A, B) and CD4 + (C, D) T cells after T cell-mediated MV3 melanoma cell death (E: T = 10: 1, incubation of 24 h) induced by MCSP TCB antibody (SEQ ID NOs: 12, 53, 54 and 55). "TCB not directed": bispecific antibody that engages CD3, but not the second antigen (SEQ ID NOs: 59, 60, 61 and 62).
[0052] [0052] FIGURE 10. Secretion of IL-2 (A), IFN-γ (B), TNFα (C), IL-4 (D), IL-10 (E) and Granzyme B (F) by human PBMCs after T cell-mediated MV3 melanoma cell death (E: T = 10: 1, 24 h incubation) induced by MCSP TCB antibody (SEQ ID NOs: 12, 53, 54 and 55). "TCB not directed": bispecific antibody that engages CD3, but not the second antigen (SEQ ID NOs: 59, 60, 61 and 62).
[0053] [0053] FIGURE 11. Binding of CEA TCB (SEQ ID NOs: 22, 56, 57 and 58) to A549 lung adenocarcinoma cells that express CEA (A) and human T lymphocyte and immortalized cynomolgus lines that express CD3 ( Jurkat (B) and HSC-F (C), respectively).
[0054] [0054] FIGURE 12. T cell death induced by CEA TCB (SEQ ID NOs: 22, 56, 57 and 58) of HPAFII cells (high CEA) (A, E), BxPC-3 (mean CEA) (B, F), ASPC-1 (low CEA) (C, G) and HCT-116 (negative CEA) (D, H). E: T = 10: 1, effector human PBMCs, incubation time 24 h (A to D) or 48 h (E to H). "TCB not directed": bispecific antibody that engages CD3, but not the second antigen (SEQ ID NOs: 59, 60, 61 and 62).
[0055] [0055] FIGURE 13. Proliferation of human CD8 + and CD4 + T cells (A to D) and CD25 overload in human CD8 + and CD4 + T cells (E to H) 5 days after HPAFII T cell mediated death (high CEA) (A, E), BxPC-3 (medium CEA) (B, F), ASPC-1 (low CEA) (C, G) and HCT-116 (negative CEA) (D, H) induced by CEA TCB (SEQ ID NOs: 22, 56, 57 and 58). "DP47 TCB”: bispecific antibody that engages CD3, but not the second antigen (SEQ ID NOs: 59, 60, 61 and 62).
[0056] [0056] FIGURE 14. Secretion of IFN-γ (A), TNFα (B), Granzyme B (C), IL-2 (D), IL-6 (E) and IL-10 (F) after cell death MKN45 tumor mediated by T cells (E: T = 10: 1, 48 h incubation) induced by CEA TCB (SEQ ID NOs: 22, 56, 57 and 58). "TCB not directed": bispecific antibody that engages CD3, but not the second antigen (SEQ ID NOs: 59, 60, 61 and 62).
[0057] [0057] FIGURE 15. T cell-mediated death of tumor target cells LS180 that express CEA induced by CEA TCB (SEQ ID NOs: 22, 56, 57 and 58) in the presence of increasing concentrations of shed CEA detected 24 h (A) or 48 h (B) after incubation with CEA TCB and sCEA.
[0058] [0058] FIGURE 16. T-cell-mediated death of A549 cells (lung adenocarcinoma) that overexpress human CEA (A549-hCEA), assessed 21 h (A, B) and 40 h (C, D) after incubation with CEA TCB (SEQ ID NOs: 22, 56, 57 and 58) and human PBMCs (A, C) or cynomolgus PBMCs (B, D) as effector cells.
[0059] [0059] FIGURE 17. T-cell-mediated death of human colorectal cancer cell lines expressing CEA induced by CEA TCB (SEQ ID NOs: 22, 56, 57 and 58) at 0.8 nM (A), 4 nM ( B) and 20 nM (C). (D) correlation between CEA expression and% specific lysis at 20 nm CEA TCB, (E) correlation between CEA expression and EC50 of CEA TCB.
[0060] [0060] FIGURE 18. In vivo antitumor efficacy of CEA TCB (SEQ ID NOs: 22, 56, 57 and 58) in a human colon carcinoma LS174T-fluc2 grafted with human PBMC (E: T ratio 5: 1). The results show the mean and SEM of 12 mice of tumor volume measured by caliper (A and C) and by bioluminescence (Total Flow, B and D) in the different study groups. (A, B) initial treatment starting on day 1, (C, D) delayed treatment starting on day 7. MCSP TCB (SEQ ID NOs: 12, 53, 54 and 55) was used as a negative control.
[0061] [0061] FIGURE 19. In vivo antitumor efficacy of CEA TCB (SEQ ID NOs: 22, 56, 57 and 58) in a human colon carcinoma LS174T-fluc2 co-grafted with human PBMC (E: T ratio 1: 1). The results show the mean and SEM of 10 mice of tumor volume measured by caliper (A) and by bioluminescence (Total Flow, B) in the different study groups. The MCSP TCB (SEQ ID NOs: 12, 53, 54 and 55) was used as a negative control.
[0062] [0062] FIGURE 20. In vivo efficacy of murine CEA TCB in a Panco2-huCEA orthotopic tumor model in immunocompetent huCD3ε / huCEA transgenic mice.
[0063] [0063] FIGURE 21. Thermal stability of CEA TCB. Dynamic Light Scattering measured at a temperature ramp from 25 to 75 ° C to 0.05 ° C / min. The duplicate is shown in gray.
[0064] [0064] FIGURE 22. Thermal stability of MCSP TCB. Dynamic Light Scattering measured at a temperature ramp from 25 to 75 ° C to 0.05 ° C / min. The duplicate is shown as a gray line.
[0065] [0065] FIGURE 23. T-cell mediated death induced by MCSP TCB antibodies (SEQ ID NOs: 12, 53, 54 and 55) and MCSP 1 + 1 CrossMab TCB from tumor target cells (A) A375 (high MCSP), ( B) MV-3 (average MCSP) and (C) HCT-116 (low MCSP). (D) LS180 (MCSP negative tumor cell line) was used as a negative control. Tumor cell death was assessed 24 h (A to D) and 48 h (E to H) after incubation of the target cells with antibodies and effector cells (human PBMCs).
[0066] [0066] FIGURE 24. Suppression of CD25 and CD69 in CD8 + and CD4 + T cells after T cell death of tumor cells expressing MCSP (A375, A to D and MV-3, E to H) mediated by MCSP TCB antibodies (SEQ ID NOs: 12, 53, 54 and 55) and MCSP 1 + 1 CrossMab TCB.
[0067] [0067] FIGURE 25. EC-SDS analysis of DP47 GS TCB (2 + 1 Crossfab-IgG P329G LALA inverted = "TCB not directed" SEQ ID NOs: 59, 60, 61 and 62) containing DP47 GS as non-binding antibody and Humanized CH2527 as anti-CD3 antibody Electropherogram shown as DP47 GS TCB SDS-PAGE: A) not reduced, B) reduced. Detailed Description of the Invention Definitions
[0068] [0068] The terms are used in the present application as generally used in the art, unless otherwise defined below.
[0069] [0069] As used in the present application, the term "antigen-binding molecule" refers in its broadest sense to a molecule that specifically binds to an antigenic determinant. Examples of antigen-binding molecules are immunoglobulins and their derivatives, for example, fragments thereof.
[0070] [0070] The term "bispecific" means that the antigen-binding molecule is able to specifically bind to at least two different antigenic determinants. Typically, a bispecific antigen-binding molecule comprises two antigen-binding sites, each of which is specific to a different antigenic determinant In certain embodiments, the bispecific antigen binding molecule is capable of binding simultaneously to two antigenic determinants, particularly two antigenic determinants expressed in two distinct cells.
[0071] [0071] The term "valiant", as used in this application, denotes the presence of a specified number of antigen-binding sites on an antigen-binding molecule. Thus, the term "monovalent binding to an antigen" indicates the presence of one (and not more than one) antigen specific binding site for the antigen on the antigen binding molecule.
[0072] [0072] An "antigen-binding site" refers to the site, that is, one or more amino acid residues, of an antigen-binding molecule that provides interaction with the antigen. For example, the antigen-binding site of an antibody comprises amino acid residues of complementarity determining regions (CDRs) .A native immunoglobulin molecule typically has two antigen binding sites, a Fab molecule typically has a single antigen binding site.
[0073] [0073] As used in this application, the term "antigen-binding component" refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one embodiment, an antigen-binding component is able to target the entity to which it is attached (for example, a second portion that binds to the antigen) to a target site, for example, to a specific type of tumor cell or stromal tumor bearing the antigenic determinant. In another embodiment, a binding component antigen is able to activate signaling through its target antigen, for example, a T cell antigen receptor complex. Antigen-binding components include antibodies and fragments thereof, as further defined in the present application. include an antigen binding domain of an antibody, which comprises a variable region of the antibody heavy chain and variable region of the antibody light chain. In the embodiments, the antigen-binding components can comprise constant regions of the antibody, as further defined in the present application and known in the art. Useful heavy chain constant regions include any of the five isotypes: α, δ, ε, γ or μ. Useful light chain constant regions include either of the two isotypes: κ and λ.
[0074] [0074] As used in this application, the term "antigenic determinant" is synonymous with "antigen" and "epitope", and refers to a location (for example, a continuous stretch of amino acids or a conformational configuration made up of different regions non-contiguous amino acids) in a polypeptide macromolecule to which an antigen that binds to the component binds, forming a component-antigen complex that binds to the antigen.Useful antigenic determinants can be found, for example, on the surfaces of tumor cells, in surfaces of virus-infected cells, on the surfaces of other diseased cells, on the surface of immune cells, free in the blood serum and / or in the extracellular matrix (ECM) .The proteins referred to as antigens in the present application (for example, MCSP, CEA, CD3) can be any native form of proteins from any vertebrate source, including mammals such as primates (eg, humans) and rodents (eg, mice and rats), unless than otherwise indicated. In a specific embodiment, the antigen is a human protein. Whenever reference is made to a protein produced in the present application, the term encompasses whole, unprocessed protein, as well as any form of protein that results from processing in the cell. The term also encompasses naturally occurring protein variants, for example, splicing variants or allelic variants. Exemplary human proteins useful as antigens include, but are not limited to: Melanoma Associated Chondroitin Sulfate Proteoglycan (MCSP), also known as Proteoglycan 4 Chondroitin Sulfate (CSPG4, UniProt No. Q6UVK1 (version 70), NCBI RefSeq No. NP_001888 .2); Carcinoembryonic Antigen (CEA), also known as cell adhesion molecule 5 related to the carcinoembryonic antigen (CEACAM5, UniProt No. P06731 (version 119), NCBI RefSeq No. NP_004354.2); and CD3, particularly the epsilon subunit of CD3 (see UniProt No. P07766 (version 130), NCBI RefSeq No. NP_000724.1, SEQ ID NO: 103 for the human sequence; or UniProt No. Q95LI5 (version 49), NCBI GenBank No. BAB71849.1, SEQ ID NO: 104 for the sequence of cynomolgus (Macaca fascicularis)). In certain embodiments, the bispecific antigen-binding molecule that activates T cells of the invention binds to a CD3 epitope or to a target cell antigen that is conserved between the CD3 or target antigen of different species. In certain embodiments, the bispecific antigen-binding molecule that activates T cells of the invention binds to CD3, and CEACAM5, but does not bind to CEACAM1 or CEACAM6. By "specific binding" is meant that binding is selective for the antigen and can be broken down from unwanted or non-specific interactions. The ability of an antigen-binding component to bind to a specific antigenic determinant can be measured both through an enzyme-linked immunosorbent assay (ELISA) as well as other techniques familiar to a person skilled in the art, for example, surface plasmon resonance (SPR) technique (analyzed in a BIACORE T100 system) (Liljeblad, et al., Glyco J 17, 323-329 (2000)), and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)) In one embodiment, the measurement of binding of an antigen-binding component to a protein does not related is less than about 10% of the binding of the antigen-binding portion to the antigen as measured, for example, by SPR. In certain embodiments, an antigen-binding component that binds to the antigen, or to a binding molecule to the antigen that co comprises the portion that binds to the antigen, has a dissociation constant (KD) ≤ 1 μΜ, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM or ≤ 0.001 nM (for example, 10-8 M or less, for example, 10-8 M to 10-13 M, for example, 10-9 M to 10-13 M).
[0075] [0075] "Affinity" refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (for example, a receptor) and its binding partner (for example, a ligand). Unless otherwise stated, as used in the present application, "binding affinity" refers to the intrinsic binding affinity that reflects a 1: 1 interaction between members of a binding pair (for example, an antigen binding component and a antigen, or a receptor and its ligand). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD), which is the ratio of the dissociation and association rate constants (koff and kon, respectively). In this way, equivalent affinities can comprise different rate constants, since the ratio of the rate constants remains the same. Affinity can be measured by well-established methods known in the art, including those described in the present application. A specific method for measuring affinity is Plasmonic Surface Resonance (SPR).
[0076] [0076] "Reduced binding", for example, reduced binding to an Fc receptor, refers to a decrease in affinity for the respective interaction, as measured, for example, by SPR. For clarity, the term also includes reduced affinity to zero (or below the detection limit of the analytical method), that is, complete elimination of the interaction, in contrast, "increased binding" refers to an increase in the binding affinity for the respective interaction.
[0077] [0077] "T cell activation", as used in the present application, refers to one or more cellular responses of a T lymphocyte, particularly a cytotoxic T lymphocyte, selected from: proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecule, cytotoxic activity and expression of activation markers.The bispecific antigen-binding molecules that activate T cells of the invention are capable of inducing T cell activation. Suitable assays to measure T cell activation are known in the art and described in this application.
[0078] [0078] A target cell antigen, as used in the present application, 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 tumor stroma cell.
[0079] [0079] As used in the present application, the terms "first" and "second" in relation to antigen binding components, etc., are used for convenience to distinguish when there is more than one of each type of component. The use of these terms is not intended to confer a specific order or orientation of the bispecific antigen-binding molecule that activates T cells, unless explicitly stated.
[0080] [0080] 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.
[0081] [0081] By "fused" is meant that the components (for example, a Fab molecule and a subunit of the Fc domain) are linked by peptide bonds, either directly or through one or more peptide ligands.
[0082] [0082] As used in the present application, the term "single chain" refers to a molecule comprising amino acid monomers linearly linked by peptide bridges. In certain embodiments, one of the antigen-binding components is a single chain Fab molecule , that is, a Fab molecule in which the Fab light chain and the Fab heavy chain are connected by a peptide linker to form a single peptide chain. In this specific embodiment, the C terminus of the Fab light chain is connected to the N termination of the Fab heavy chain into the single chain Fab molecule.
[0083] [0083] By a "crossing" of Fab molecule (also called "Crossfab”) is meant a Fab molecule in which both the variable regions and the constant regions of the Fab heavy and light chains are exchanged, that is, the crossing The Fab molecule comprises a peptide chain composed of the variable region of the light chain and constant region of the heavy chain, and a peptide chain composed of the variable region of the heavy chain and the constant region of the light chain. For clarity, at an intersection of the Fab molecule in which the variable regions of the Fab light chain and the Fab heavy chain are exchanged, the peptide chain comprising the constant region of the heavy chain is referred to in the present application as "heavy chain" the crossing of the Fab molecule. Conversely, at a crossing of the Fab molecule in which the Fab light chain and Fab heavy chain constant regions are switched, the peptide chain comprising the variable region of the heavy chain is referred to in the present application as "heavy chain" of the crossing of the Fab molecule.
[0084] [0084] On the contrary, a "conventional" Fab molecule is understood to mean a Fab molecule in its natural format, that is, which comprises a heavy chain composed of the variable and constant regions of the heavy chain (VH-CH1), and a chain light composed of the variable and constant regions of the light chain (VL-CL).
[0085] [0085] The term "immunoglobulin molecule" refers to a protein that has the structure of a naturally occurring antibody. For example, immunoglobulins of the IgG class are heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy chains that are linked by disulfide. From the termination N to C, each heavy chain has a variable region (VH), also called a heavy variable domain or a heavy chain variable domain, followed by three constant domains ( CH1, CH2 and CH3), also called the constant region of the heavy chain, similarly, from the termination N to C, each light chain has a variable region (VL), also called the light variable domain or a variable domain of the light chain, followed by a light constant domain (CL), also called a light chain constant region.The heavy chain of an immunoglobulin can be assigned to one of five types, called α (IgA), δ (IgD ), ε (IgE), γ (IgG) or μ (IgM), some of which can be further divided into subtypes, for example, γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1) and α2 (IgA2). The light chain of an immunoglobulin can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain. An immunoglobulin essentially consists of two Fab molecules and an Fc domain, linked through the immunoglobulin hinge region.
[0086] [0086] The term "antibody" in the present application is used in the broadest sense and encompasses several antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies and antibody fragments, as long as they exhibit antigen binding activity desired.
[0087] [0087] An "antibody fragment" refers to a molecule other than an intact antibody, which comprises a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, are not limited to Fv, Fab, Fab ', Fab'-SH, F (ab') 2, diabody, linear antibodies, single chain antibody molecules (eg scFv) and single domain antibodies. certain antibody fragments, see Hudson et al., Med. 9: 129-134 (2003). For a review of scFv fragments, see, for example, Plückthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pages 269-315 (1994); see also WO 93/16185; and US patents 5,571,894 and 5,587,458. For discussion of Fab and F (ab ') fragments 2 which comprise epitope residues that bind to rescue receptors and have an increased half-life in vivo, see US patent 5,869,046. pos are antibody fragments with two antigen-binding sites that can be bivalent or bispecific. See, for example, patent EP 404,097; WO 1993/01161; Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al, Proc Natl Acad Sci USA 90, 6444-6448 (1993). Tribodies and tetrabodies are also described in Hudson et al., Nat Med 9, 129-134 (2003). Single domain antibodies are fragments that comprise all or a portion of the variable domain of the heavy chain or all or a portion of the variable domain of the light chain of an antibody. In certain embodiments, a single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, for example, US patent 6,248,516 B1). Antibody fragments can be produced 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 in the present application.
[0088] [0088] The term "antigen binding domain” refers to the part of an antibody that comprises the area that specifically binds, and is complementary to, part or all of an antigen. An antigen binding domain can be provided , for example, by one or more variable domains of the antibody (also called variable regions of the antibody). Particularly, an antigen binding domain comprises a variable region of the antibody light chain (VL) and a variable region of the heavy chain of the antibody. antibody (VH).
[0089] [0089] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to the antigen. The heavy chain and light chain variable domains (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, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen binding specificity.
[0090] [0090] The term "hypervariable region" or "HVR", as used in the present application, refers to each of the regions of an antibody variable domain that are hypervariable in the sequence and / or forms structurally defined as loops ("loops" hypervariable "). Generally, antibodies of four native chains comprise six HVRs, three in VH (H1, H2, H3) and three in VL (L1, L2, L3). HVRs generally comprise amino acid residues from the hypervariable loops and / or "complementarity determining regions" (CDRs), the latter being of higher sequence variability and / or involved in antigen recognition. With the exception of CDR1 in VH, CDRs generally comprise the amino acid residues that form the hypervariable loops. Hypervariable regions (HVRs) are also called "complementarity determining regions" (CDRs), and these terms are used in the present application interchangeably in reference to portions of the variable region to form the antigen-binding regions. This specific region has been described by Kabat et al., US 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 definitions include overlap or subgroups of amino acid residues when compared to each other, however, the application of any 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 in this application. appropriate amino acid residues that encompass CDRs, as defined by each of the references cited above are defined below in Table A. The exact numbers of residues that comprise a Specific CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a specific CDR given the amino acid sequence of the variable region of the antibody.
[0091] [0091] Kabat et al. also defined a variable region sequence numbering system that is applicable to any antibody. Generally, a person skilled in the art can unequivocally assign this "Kabat numbering" system to any variable region sequence, without relying on any experimental data other than the sequence itself. As used in this application, "Kabat numbering" refers to to the numbering system established by Kabat et al., US 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 a variable region of the antibody are in accordance with the numbering system of Kabat.
[0092] [0092] The polypeptide sequences in the sequence list are not numbered according to the Kabat numbering system. However, it is well within the normal skills of a technician on the subject to convert the sequence numbering from the Sequence Listing to the Kabat numbering.
[0093] [0093] "Structure region" or "FR" refers to residues of variable domain, except residues of the hypervariable region (HVR). The RF of a variable domain generally consists of four FR domains: FR1, FR2, FR3 and FR4. Consequently, 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.
[0094] [0094] The "class" of an antibody or immunoglobulin refers to the type of constant domain or constant region of its heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG and IgM, and several of these can, further, be divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.The constant domains of the heavy chain that correspond to the different classes of immunoglobulins are called α, δ, ε, γ and μ , respectively.
[0095] [0095] The term "Fc domain" or "Fc region" in the present application is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. Although the limits of the Fc region of an IgG heavy chain may vary slightly, the Fc region of a human IgG heavy chain is usually defined as extending from Cys226, or Pro230, to the carboxyl termination of the heavy chain. However, C-terminal lysine (Lys447) from the Fc region may or may not be present. Unless otherwise specified in the present application, the numbering of amino acid residues in the Fc region or constant region is in accordance with the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. A “subunit” of an Fc domain, as used in the present application, refers to one of the two polypeptides that form the dimeric Fc domain , that is, a polypeptide comprising the C-terminal constant regions of an immunoglobulin heavy chain, capable of stable self-association. For example, a subunit of an IgG Fc domain comprises an IgG CH2 constant domain and an IgG CH3 domain.
[0096] [0096] A "modification that promotes the association of the first and second subunits of the Fc domain" is a manipulation of the peptide backbone or post-translational modifications of a subunit of the Fc domain that reduces or prevents the association of a polypeptide comprising the subunit of the Fc domain with an identical polypeptide to form a homodimer. A modification that promotes association, as used in the present application, includes particularly separate modifications made for each of the subunits of the Fc domain to be associated (that is, the first and second subunits of the Fc domain), in which the modifications are complementary each other, in order to promote the association of two subunits of the Fc domain. For example, a modification that promotes association can alter the structure or charge of one or both subunits of the Fc domain in order to make its association sterically or electrostatically favorable, respectively. Thus, (hetero) dimerization occurs between a polypeptide comprising the first subunit of the Fc domain and a polypeptide comprising the second subunit of the Fc domain, which may not be identical in the sense that additional components fused in each of the subunits (for example, antigen-binding components) are not the same. In some embodiments, the association-promoting modification comprises an amino acid mutation in the Fc domain, specifically an amino acid substitution. In a specific embodiment, the association-promoting modification comprises a separate amino acid mutation, specifically an amino acid substitution in each of the two subunits of the Fc domain.
[0097] [0097] The term "effector functions" refers to those biological activities attributable to the Fc region of an antibody, which vary according to the antibody's isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytoxicity (CDC), Fc receptor binding, antibody dependent cell mediated cytotoxicity (ADCC), antibody dependent cell phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen perception by antigen presenting cells (eg B cell receptor) and activation of B cells.
[0098] [0098] As used in this application, the terms "genetically engineered, genetically engineered, genetic engineered" are considered to include any manipulation of the peptide skeleton or naturally occurring post-translational modifications, or of a recombinant polypeptide or fragment of Genetic design includes modifications to the amino acid sequence, glycosylation pattern, or side chain group of individual amino acids, as well as combinations of these approaches.
[0099] [0099] The term "amino acid mutation", as used in this application, is intended to encompass amino acid substitutions, deletions, insertions and modifications. Any combination of substitution, deletion, insertion and modification can be made to arrive at the construct end, as long as the final construct has the desired characteristics, for example, reduced binding to an Fc receptor or increased association with another peptide.Delections and insertions of amino acid sequences include deletions of amino and / or carboxy terminal amino acids. specific substitutions are amino acid substitutions. For the purposes of altering, for example, the binding characteristics of an Fc region, non-conservative amino acid substitutions, that is, the replacement of one amino acid with another amino acid that has different chemical and / or chemical properties different structural structures are particularly preferred. Amino acid substitutions include substitution by am non-naturally occurring inoacids or by amino acid derivatives of the twenty standard amino acids occur naturally (eg, 4-hydroxy proline, 3-methyl histidine, ornithine, homoserine, 5-hydroxy lysine). Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods can 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. Several designations can be used in the present application to indicate the same amino acid mutation. For example, a substitution of proline at position 329 of the Fc domain for glycine can be indicated as 329G, G329, G329, P329G or Pro329Gly.
[0100] [00100] As used in the present application, the term "polypeptide" refers to a molecule composed of monomers (amino acids) linearly linked by amide bridges (also known as peptide bridges). The term "polypeptide" refers to any chain of two or more amino acids and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "protein", "chain of amino acids" or any other term used to refer to a chain of two or more amino acids, are included in the definition of "polypeptide" and the term "polypeptide ”Can be used, rather than, or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protection / blocking groups, proteolytic cleavage or modification by occurring amino acids A polypeptide can be derived from a natural biological source or produced by recombinant technology, but it is not necessarily translated from a designated nucleic acid sequence. It can be generated in any way, including chemical synthesis. A polypeptide of the invention can have 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 can have a defined three-dimensional structure, although they do not necessarily have such a structure Polypeptides with a defined three-dimensional structure are ref erected as folded, and polypeptides that do not have a defined three-dimensional structure, but preferably can adopt a large number of different conformations and are referred to as unfolded.
[0101] [00101] An "isolated" polypeptide, a variant or derivative thereof, means a polypeptide that is not in its natural environment. No specific level of purification is required. For example, an isolated polypeptide can be removed from its 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 that have been separated, fractionated, or partially, or substantially purified by any suitable technique.
[0102] [00102] "Percentage (%) of amino acid sequence identity" in relation to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence , after alignment of the sequences and introduction of gaps, if necessary, to achieve the maximum percentage of sequence identity, and not considering any conservative substitution as part of the sequence identity. Alignment, for the purposes of determining the percentage of sequence identity of amino acids, can be achieved in several ways, which are within the technique, for example, with the use of publicly available computer programs, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) computer programs. subject can determine appropriate parameters for sequence alignment, including any algorithms necessary for the Achieve maximum alignment over the entire sequences being compared. For the purposes of the present application, however,% amino acid sequence identity values are obtained using the ALIGN-2 sequence comparison computer program. The ALIGN-2 sequence comparison computer program was developed by Genentech, Inc. and the source code was deposited with the user documentation at the US Copyright Office, Washington DC, 20559, where it is registered under US Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from source code. The ALIGN-2 program must be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are defined by the ALIGN-2 program and do not vary. In situations where ALIGN-2 is used for amino acid sequence comparisons, the% amino acid sequence identity of a given A amino acid sequence for, with or against, a given B amino acid sequence (which can alternatively be formulated as a given sequence of amino acids A which has or comprises a certain% identity of amino acid sequence a, with or against a given sequence of amino acids B) is calculated as follows: 100 times the X / Y fraction
[0103] [00103] where X is the number of amino acid residues punctuated as identical comparisons by the ALIGN-2 sequence alignment program in that A and B program alignment, and where Y is the total number of amino acid residues in B. It is considered that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the% identity of amino acid sequence A with respect to B is not equal to the% identity of amino acid sequence B with respect to to A. Unless specifically stated otherwise, all% amino acid sequence identity values used in this application are obtained as described in the immediately preceding paragraph, using the ALIGN-2 computer program.
[0104] [00104] The term "polynudeotide" refers to an isolated nucleic acid molecule or construct, for example, messenger RNA (mRNA), virus-derived RNA or plasmid DNA (pDNA). A polynucleotide can comprise a conventional phosphodiester bond or an unconventional bond (for example, an amide bond, as found in peptide nucleic acids (PNA)). The term "nucleic acid molecule" refers to any one or more nucleic acid segments, for example, fragments of DNA or RNA, present in a polynucleotide.
[0105] [00105] By "isolated" nucleic acid molecule or polynucleotide is meant a nucleic acid molecule, DNA or RNA, which has been removed from its native environment. For example, a recombinant polynucleotide that encodes a polypeptide contained in a vector is considered to be isolated for the purposes of the present invention. Additional examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified polynucleotides (partially or substantially) in solution. An isolated polynucleotide includes a polynucleotide molecule contained in cells that normally contain the polynucleotide molecule, but the polynucleotide molecule is present outside the chromosome or at a different location on the chromosome than the chromosome. 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 strand forms. Polynucleotides or nucleic acids isolated according to the present invention still include those synthetically produced molecules. In addition, a polynucleotide or nucleic acid can be or can include a regulatory element such as a promoter, ribosome binding site or a transcription terminator.
[0106] [00106] By a nucleic acid or polynucleotide that has a nucleotide sequence, at least, for example, 95% "identical" to a reference nucleotide sequence of the present invention, the nucleotide sequence of the polynucleotide is intended to be identical to the reference sequence, except that the polynucleotide sequence can include up to five mutation points for every 100 nucleotides in the reference nucleotide sequence. In other words, to obtain a polynucleotide that has a nucleotide sequence at least 95% identical to a sequence of reference nucleotides, up to 5% of the nucleotides in the reference sequence can be deleted or replaced by another nucleotide or a number of nucleotides up to 5% of the total nucleotides in the reference sequence can be inserted in the reference sequence. reference sequence can occur at the 5 'or 3' end positions of the reference nucleotide sequence or at which either place between the terminal positions, interspersed either individually between residues in the reference sequence or in one or more contiguous groups in the reference sequence. As a practical matter, whether any specific 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 with the use of known computer programs, such as those discussed above for polypeptides (for example, ALIGN-2).
[0107] [00107] The term "expression cassette" refers to a polynucleotide generated recombinantly or synthetically, with a series of specified nucleic acid elements that allow for the transcription of a specific nucleic acid in a target cell. The expression cassette The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus or nucleic acid fragment Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter In certain embodiments, the expression cassette of the invention comprises polynucleotide sequences that encode antigen-binding molecules of the invention or fragments thereof.
[0108] [00108] The term "vector" or "expression vector" is synonymous with "expression construct" and refers to a DNA molecule that is used to introduce and direct the expression of a specific gene to which it is associated functional 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 it was introduced in. The expression vector of the present invention comprises an expression cassette. of expression allow transcription of large amounts of stable mRNA, since the expression vector is within the target cell, the ribonucleic acid or protein molecule that is encoded by the gene is produced by cell transcription and / or translation machines. embodiment, the expression vector of the invention comprises an expression cassette comprising polynucleotide sequences encoding bispecific antigen-binding molecules of the invention or fr them.
[0109] [00109] The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to the cells into which the exogenous nucleic acid was introduced, including the progeny of these cells. "transformants" and "transformed cells", which include the transformed primary cell and the progeny derived therefrom regardless of the number of passages. The progeny may not be completely identical to a parental cell in the nucleic acid content, but may contain Mutant progenies that have the same biological function or activity as those screened or selected for the originally transformed cell are included in the present application A host cell is any type of cellular system that can be used to generate bispecific antigen-binding molecules Host cells include cultured cells, for example, cultured mammalian cells, such as c CHO cells, BHK cells, NOS cells, Sp2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, insect cells, plant cells, for to name just a few, but also cells comprised within a transgenic animal, transgenic plant or cultivated plant or animal tissue.
[0110] [00110] An "activating Fc receptor" is an Fc receptor that after coupling by an antibody Fc domain produces events that stimulate cells carrying receptors to perform effector functions. Human activating Fc receptors include FcүRIIIa (CD16a), FcүRI ( CD64), FcүRIIa (CD32) and FcαRI (CD89).
[0111] [00111] Antibody dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism that leads to the lysis of antibody-coated target cells by immune effector cells. Target cells are cells to which antibodies or derivatives thereof that comprise an Fc region specifically bind, in general, through part of the protein that is N-terminal to the Fc region. As used in the present application, the term "reduced ADCC" is defined both as a reduction in the number of target cells that are lysed at a given time, at a given concentration of antibodies in the medium around the target cells, by the defined ADCC mechanism above, and / or an increase in the antibody concentration, in the medium around the target cells, necessary to lysis a certain number of target cells in a given time, by the ADCC mechanism. The reduction in ADCC is related to 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 which were not genetically engineered. in ADCC mediated by an antibody that comprises in its Fc domain an amino acid substitution that reduces ADCC, it is relative to ADCC mediated by the same antibody without that amino acid substitution in the Fc domain. Suitable assays for measuring ADCC are well known in the art (see, for example, PCT publication WO 2006/082515 or PCT publication WO 2012/130831).
[0112] [00112] An "effective amount" of an agent refers to the amount that is required to result in a change in the physiological state in the cell or tissue to which it is administered.
[0113] [00113] An "therapeutically effective amount" of an agent, for example, a pharmaceutical formulation, refers to an effective amount in the dosages and for periods of time necessary to achieve the desired therapeutic or prophylactic result. The therapeutically effective amount of a agent, for example, eliminates, decreases, delays, minimizes or prevents adverse effects of a disease.
[0114] [00114] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (for example, cows, sheep, cats, dogs and horses), primates (for example, human and non-human primates such as monkeys), rabbits and rodents (for example, mice and rats) ). In particular, the individual or subject is a human.
[0115] [00115] The term "pharmaceutical composition" refers to a preparation that is in that form to allow the biological activity of an active ingredient contained therein to be effective, and that does not contain additional components that are unacceptably toxic to a subject to whom the formulation would be administered.
[0116] [00116] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition, except an active ingredient, which is not toxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer or preservative.
[0117] [00117] As used in the present application, "treatment" (and grammatical variations of it as "treating" or "treating") refers to clinical intervention in an attempt to alter the natural course of a disease in the individual being treated, and can be performed both for prophylaxis and during the course of clinical pathology. Desired treatment effects include, but are not limited to, preventing the occurrence or recurrence of the disease, relieving symptoms, decreasing any direct or indirect pathological consequences of the disease, prevention of metastasis, decreased rate of disease progression, improvement or alleviation of the disease state and remission or prognosis of improvement.In some embodiments, the bispecific antigen-binding molecules that activate T cells of the invention are used to delay the development of a disease or slow the progression of a disease.
[0118] [00118] The term "package insert" is used to refer to the instructions included as usual in commercial packaging of therapeutic products, which contain information on the indications, use, dosage, administration, combination therapy, against indications and / or warnings regarding the use of these therapeutic products. Detailed Description of Achievements
[0119] (i) um primeira porção que se liga ao antígeno que é uma molécula Fab capaz de se ligar especificamente a CD3, e que compreende pelo menos uma região determinante de complementaridade da cadeia pesada (CDR) selecionada a partir do grupo que consiste em SEQ ID NO: 4, SEQ ID NO: 5 e SEQ ID NO: 6 e pelo menos uma CDR da cadeia leve selecionada a partir do grupo de SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10; (ii) uma segunda porção que se liga ao antígeno que é uma molécula Fab capaz de se ligar especificamente a um antígeno de célula alvo. [00119] In a first aspect, the present invention provides a bispecific antigen-binding molecule that activates T cells, which comprises: (i) a first antigen-binding portion which is a Fab molecule capable of specifically binding to CD3, and which comprises at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 and at least one CDR of the light chain selected from the group of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10; (ii) a second portion that binds to the antigen, which is a Fab molecule capable of specifically binding to a target cell antigen.
[0120] [00120] In one embodiment, the first antigen-binding portion comprises a variable region of the heavy chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100 % identical to an amino acid sequence selected from the group of: SEQ ID NO: 3, SEQ ID NO: 32 and SEQ ID NO: 33, and a variable region of the light chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group of: SEQ ID NO: 7 and SEQ ID NO: 31.
[0121] [00121] In one embodiment, the first antigen-binding portion comprises a variable region of the heavy chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100 % identical to the amino acid sequence of SEQ ID NO: 3, and a variable region of the light chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 7.
[0122] [00122] In a specific embodiment, the second antigen-binding portion is able to specifically bind CEA and comprises at least one complementarity determining region (CDR) of the heavy chain selected from the group consisting of SEQ ID NO : 24, SEQ ID NO: 25 and SEQ ID NO: 26 and at least one CDR of the light chain selected from the group of SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30.
[0123] [00123] In another specific embodiment, the second antigen-binding portion is able to specifically bind CEA and comprises a variable region of the heavy chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 23, and a variable region of the light chain comprising an amino acid sequence that is at least about 95%, 96%, 97% , 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 27.
[0124] [00124] In another specific embodiment, the second antigen-binding portion is capable of specifically binding to MCSP and comprises at least one complementarity determining region (CDR) of the heavy chain selected from the group consisting of SEQ ID NO : 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 40 and at least one CDR of the light chain selected from the group of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 48, SEQ ID NO: 49 and SEQ ID NO: 50.
[0125] [00125] In another specific embodiment, the second antigen-binding portion is capable of specifically binding to MCSP and comprises at least one complementarity determining region (CDR) of the heavy chain selected from the group consisting of SEQ ID NO : 14, SEQ ID NO: 15 and SEQ ID NO: 16 and at least one CDR of the light chain selected from the group of SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20.
[0126] [00126] In another specific embodiment, the second antigen-binding portion is able to specifically bind to MCSP and comprises a variable region of the heavy chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group of SEQ ID NO: 13, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 39 and SEQ ID NO : 41, and a variable region of the light chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group of SEQ ID NO: 17, SEQ ID NO: 43, SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 51.
[0127] [00127] In another specific embodiment, the second antigen-binding portion is capable of specifically binding to MCSP and comprises a variable region of the heavy chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 13, and a variable region of the light chain comprising an amino acid sequence that is at least about 95%, 96%, 97% , 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 17.
[0128] [00128] In one embodiment, the present invention provides a bispecific antigen-binding molecule that activates T cells, which comprises: (i) a first antigen-binding portion that is a Fab molecule capable of specifically binding to CD3, comprising at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO : 4, SEQ ID NO: 5 and SEQ ID NO: 6 and at least one CDR of the light chain selected from the group of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10; (i) a second antigen-binding portion which is a Fab molecule capable of specifically binding CEA, comprising at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO : 24, SEQ ID NO: 25 and SEQ ID NO: 26 and at least one CDR of the light chain selected from the group of SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30.
[0129] [00129] In one embodiment, the present invention provides a bispecific antigen-binding molecule that activates T cells, which comprises: (i) a first antigen-binding portion that is a Fab molecule capable of specifically binding to CD3 that comprises a variable region of the heavy chain that comprises an amino acid sequence that is at least about 95%, 96%, 97 %, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 3, and a variable region of the light chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 7, (i) a second antigen-binding portion which is a Fab molecule capable of specifically binding CEA which comprises a variable region of the heavy chain which comprises an amino acid sequence which is at least about 95%, 96%, 97 %, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 23, and a variable region of the light chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 27.
[0130] [00130] In one embodiment, the present invention provides a bispecific antigen-binding molecule that activates T cells, which comprises: (i) a first antigen-binding portion that is a Fab molecule capable of specifically binding to CD3, comprising at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO : 4, SEQ ID NO: 5 and SEQ ID NO: 6 and at least one CDR of the light chain selected from the group of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10; (i) a second antigen-binding portion that is a Fab molecule capable of specifically binding to MCSP, comprising at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO : 14, SEQ ID NO: 15 and SEQ ID NO: 16 and at least one CDR of the light chain selected from the group of SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20.
[0131] [00131] In one embodiment, the present invention provides a bispecific antigen-binding molecule that activates T cells, which comprises: (i) a first antigen-binding portion that is a Fab molecule capable of specifically binding to CD3 that comprises a variable region of the heavy chain that comprises an amino acid sequence that is at least about 95%, 96%, 97 %, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 3, and a variable region of the light chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 7. (i) a second antigen-binding portion which is a Fab molecule capable of specifically binding CEA which comprises a variable region of the heavy chain which comprises an amino acid sequence which is at least about 95%, 96%, 97 %, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 13, and a variable region of the light chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 17.
[0132] [00132] In a specific embodiment, the first portion that binds to the antigen is a cross between the Fab molecule, in which both the variable regions and the constants of the Fab light chain and the Fab heavy chain are exchanged.
[0133] [00133] In one embodiment, the second portion that binds to the antigen is a conventional Fab molecule.
[0134] [00134] In a specific embodiment, the first portion that binds to the antigen is a cross of the Fab molecule, in which the constant regions of the Fab light chain and the Fab heavy chain are exchanged, and the second portion that binds to the antigen is a conventional Fab molecule. In an additional specific embodiment, the first and second antigen-binding moieties are fused to each other, optionally via a peptide linker.
[0135] [00135] In specific embodiments, the bispecific antigen-binding molecule that activates T cells further comprises an Fc domain composed of a first and a second subunit capable of stable association.
[0136] [00136] In an additional specific embodiment, no more than one antigen-binding component capable of specifically binding to CD3 is present in the bispecific antigen-binding molecule that activates T cells (ie, the bispecific antigen-binding molecule that active T cell provides monovalent binding to CD3). FORMATS OF BIESPECIFIC ANTIGEN BINDING MOLECULES THAT ACTIVATE T CELLS
[0137] [00137] The components of the bispecific antigen-binding molecule that activates T cells can be fused to each other in a variety of configurations. Exemplary configurations are shown in Figures 1, 3 and 5.
[0138] [00138] In specific embodiments, the bispecific antigen-binding molecule that activates T cells comprises an Fc domain composed of a first and a second subunit capable of stable association. In some embodiments, the second antigen-binding portion 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.
[0139] [00139] In one embodiment, the first antigen-binding portion is fused at the C termination of the Fab heavy chain to the N termination of the Fab heavy chain of the second antigen-binding portion. In this specific embodiment, the bispecific antigen-binding molecule that activates T cells essentially consists of a first and a second portion that binds to the antigen, an Fc domain composed of a first and a second subunit, and optionally one or more peptide ligands, wherein the first antigen-binding portion is fused at the C-termination of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding portion, and the second antigen-binding portion is fused at the termination C from the Fab heavy chain to the N-terminus of the first and second subunits of the Fc domain. Optionally, the Fab light chain of the first portion that binds to the antigen and the Fab light chain of the second portion that binds to the antigen can be further fused to each other.
[0140] [00140] In this other embodiment, the first antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. In that specific embodiment, the bispecific antigen-binding molecule that activates T cells essentially consists of a first and a second portion that binds to the antigen, an Fc domain composed of a first and a second subunit, and optionally one or more peptide ligands, wherein the first and second antigen-binding moieties are fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain.
[0141] [00141] In other embodiments, the first antigen-binding portion 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.
[0142] [00142] In this specific embodiment, the second antigen-binding portion is fused at the C termination of the Fab heavy chain to the N termination of the Fab heavy chain of the first antigen-binding portion. In this specific embodiment, the bispecific antigen-binding molecule that activates T cells essentially consists of a first and a second portion that binds to the antigen, an Fc domain composed of a first and a second subunit, and optionally one or more peptide ligands, wherein the second antigen-binding portion is fused at the C-termination of the Fab heavy chain to the N termination of the Fab heavy chain of the first antigen-binding portion, and the first antigen-binding portion is fused at the termination C from the Fab heavy chain to the N-terminus of the first and second subunits of the Fc domain. Optionally, the Fab light chain of the first portion that binds to the antigen and the Fab light chain of the second portion that binds to the antigen can be further fused to each other.
[0143] [00143] The antigen-binding components can be fused to the Fc domain or to each other directly or through a peptide linker, which comprises one or more amino acids, typically about 2 to 20 amino acids. Peptide linkers are known in the art or are described in the present application. Suitable non-immunogenic peptide linkers include, for example, peptide linkers (G4S) n, (SG4) n, (G4S) n. "n" is generally a number between 1 and 10, typically between 2 and 4. A peptide linker particularly suitable for fusing the Fab light chains of the first and second antigen-binding strands is (G4S) 2. An exemplary peptide linker suitable for connecting the Fab heavy chains of the first and second antigen-binding portions is EPKSC (D) - (G4S) 2 (SEQ ID NOs 105 and 106). Additionally, ligands may comprise (a portion de) an immunoglobulin hinge region. Particularly, when an antigen-binding component is fused to the N-terminus of a subcategory of the Fc domain, it can be fused through an immunoglobulin hinge region or a portion thereof, with or without an additional peptide linker.
[0144] [00144] A bispecific antigen binding molecule that activates T cells with a single portion that binds to the antigen capable of binding to a specific target cell antigen (for example, as shown in Figure 1A, 1D, 1F or 1H) is useful, particularly in cases where internalization of the target cell antigen is expected after binding of a high-affinity antigen-binding component. In these cases, the presence of more than one antigen-binding component specific to the target cell antigen can increase the internalization of the target cell antigen, thus reducing its availability.
[0145] [00145] In many cases, however, it will be advantageous to have a bispecific antigen-binding molecule that activates T cells that comprises two or more antigen-binding components for a specific target cell antigen (see examples shown in Figure 1A, 1C , 1E or 1G), for example, to optimize targeting to the target site or to allow cross-linking of target cell antigens.
[0146] Consequently, in certain embodiments, the bispecific antigen-binding molecule that activates T cells of the invention further comprises a third portion that binds to the antigen which is a Fab molecule capable of specifically binding to a target cell antigen. In one embodiment, the third portion that binds to the antigen is a conventional Fab molecule. In one embodiment, the third portion that binds to the antigen is able to specifically bind to the same target cell antigen, as well as the second portion that binds to the antigen. In a specific embodiment, the first antigen-binding portion is capable of specifically binding to CD3, and the second and third antigen-binding components are capable of specifically binding to a target cell antigen. In a specific embodiment, the second and third portions that bind to the antigen are identical (that is, they comprise the same amino acid sequences).
[0147] [00147] In a specific embodiment, the first antigen-binding portion is capable of specifically binding to CD3, and the second and third antigen-binding components are capable of specifically binding to CEA, in which the second and third antigen binding components comprise at least one complementarity determining region (CDR) of the heavy chain selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26 and at least one CDR of light chain selected from the group of SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30.
[0148] [00148] In a specific embodiment, the first antigen-binding portion is capable of specifically binding to CD3, and comprises at least one complementarity determining region (CDR) of the heavy chain selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 and at least one CDR of the light chain selected from the group of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10; and the second and third antigen-binding components are able to specifically bind CEA, wherein the second and third antigen-binding components comprise at least one complementarity determining region (CDR) of the heavy chain selected from the group that consists of SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26 and at least one light chain CDR selected from the group of SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30 .
[0149] [00149] In a specific embodiment, the first antigen-binding portion is capable of specifically binding to CD3, and comprises at least one complementarity determining region (CDR) of the heavy chain selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 and at least one CDR of the light chain selected from the group of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10; and the second and third antigen-binding components are able to specifically bind CEA, wherein the second and third antigen-binding components comprise at least one complementarity determining region (CDR) of the heavy chain selected from the group that consists of SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26 and at least one light chain CDR selected from the group of SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30 .
[0150] [00150] In a specific embodiment, the first antigen-binding portion is capable of specifically binding to CD3 and comprises a variable region of the heavy chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group of: SEQ ID NO: 3, SEQ ID NO: 32 and SEQ ID NO: 33, and a variable region of the light chain that comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group of: SEQ ID NO: 7 and SEQ ID NO : 31, and the second and third portions that bind to the antigen are able to specifically bind CEA, where the second and third portions that bind to the antigen comprise a variable region of the heavy chain that comprises a sequence of amino acids which is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID N O: 23, and a variable region of the light chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 27.
[0151] [00151] In a specific embodiment, the first antigen-binding portion is capable of specifically binding to CD3 and comprises a variable region of the heavy chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 3, and a variable region of the light chain comprising an amino acid sequence that is at least about 95%, 96%, 97% , 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 7, and the second and third portion that bind to the antigen are able to specifically bind to CEA, where the second and third portion antigen-binding comprises a variable region of the heavy chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO : 23, and a variable region of the light chain that comprises an amino acid sequence that is at least about 95% , 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 27.
[0152] [00152] In one embodiment, the first antigen-binding portion is capable of specifically binding to CD3, and the second and third antigen-binding components are capable of specifically binding to MCSP, wherein the second and third components antigen binding agents comprise at least one complementarity determining region (CDR) of the heavy chain selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 40 and at least one light chain CDR selected from the group of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 48, SEQ ID NO: 49 and SEQ ID NO: 50.
[0153] [00153] In a specific embodiment, the first antigen-binding portion is capable of specifically binding to CD3, and comprises at least one complementarity determining region (CDR) of the heavy chain selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 and at least one CDR of the light chain selected from the group of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10; and the second and third antigen binding components are able to specifically bind to MCSP, wherein the second and third antigen binding components comprise at least one complementarity determining region (CDR) of the heavy chain selected from the group that consists of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 40 and at least one chain CDR light selected from the group of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 48, SEQ ID NO: 49 and SEQ ID NO: 50.
[0154] [00154] In one embodiment, the first antigen-binding portion is capable of specifically binding to CD3, and comprises at least one complementarity determining region (CDR) of the heavy chain selected from the group consisting of SEQ ID NO : 4, SEQ ID NO: 5 and SEQ ID NO: 6 and at least one CDR of the light chain selected from the group of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10; and the second and third antigen binding components are able to specifically bind to MCSP, wherein the second and third antigen binding components comprise at least one complementarity determining region (CDR) of the heavy chain selected from the group that consists of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 and at least one CDR of the light chain selected from the group of SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20 .
[0155] [00155] In one embodiment, the first antigen-binding portion is capable of specifically binding to CD3 and comprises a variable region of the heavy chain comprising an amino acid sequence that is at least about 95%, 96%, 97 %, 98%, 99% or 100% identical to an amino acid sequence selected from the group of: SEQ ID NO: 3, SEQ ID NO: 32 and SEQ ID NO: 33, and a variable region of the light chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group of: SEQ ID NO: 7 and SEQ ID NO: 31, and the second and third antigen-binding moieties are able to specifically bind CEA, wherein the second and third antigen-binding moieties comprise a variable region of the heavy chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 13, SEQ I D NO: 34, SEQ ID NO: 36, SEQ ID NO: 39 and SEQ ID NO: 41, and a light chain variable region comprising an amino acid sequence that is at least about 95%, 96%, 97% , 98%, 99% or 100% identical to an amino acid sequence selected from the group of SEQ ID NO: 17, SEQ ID NO: 43, SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 51 .
[0156] [00156] In one embodiment, the first antigen-binding portion is capable of specifically binding to CD3 and comprises a variable region of the heavy chain that comprises an amino acid sequence that is at least about 95%, 96%, 97 %, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 3, and a variable region of the light chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 7, and the second and third portions that bind to the antigen are capable of specifically binding to MCSP, where the second and third portions that binds to the antigen comprise a variable region of the heavy chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 13, and a variable region of the light chain comprising an amino acid sequence that is at least about 95%, 96%, 97% , 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 17.
[0157] [00157] In one embodiment, the third portion that binds to the antigen is fused at the C termination of the Fab heavy chain to the N termination of the first or second subunit of the Fc domain. In a more specific embodiment, the second and third antigen-binding portions are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain, and the first portion that attaches to the antigen is fused at the C termination of the Fab heavy chain to the N termination of the Fab heavy chain of the second antigen-binding portion. Optionally, the Fab light chain of the first portion that binds to the antigen and the Fab light chain of the second portion that binds to the antigen can be further fused to each other.
[0158] [00158] The second and third portions that bind to the antigen can be fused to the Fc domain directly or through a peptide ligand. In a specific embodiment, the second and third portions that bind to the antigen are each fused to the Fc domain through an immunoglobulin hinge region. In a specific embodiment, the immunoglobulin hinge region is a human IgG1 hinge region. In one embodiment, the second, the third portion that binds to the antigen and the Fc domain is part of an immunoglobulin molecule. In a specific embodiment, the immunoglobulin molecule is an immunoglobulin of the IgG class. In an even more specific embodiment, the immunoglobulin is an immunoglobulin of the IgG1 class. In another embodiment, the immunoglobulin is an immunoglobulin of the IgG4 subclass. In an additional specific embodiment, the immunoglobulin is a human immunoglobulin. In other embodiments, the immunoglobulin is a chimeric immunoglobulin or a humanized immunoglobulin. In one embodiment, the bispecific antigen binding molecule that activates T cells essentially consists of an immunoglobulin molecule capable of specifically binding to a target cell antigen, and an antigen binding component capable of specifically binding to CD3, in that the antigen-binding portion is a Fab molecule, particularly a crossed Fab molecule, fused to the N-terminus of one of the immunoglobulin chains, optionally via a peptide linker.
[0159] [00159] In a specific embodiment, the first and third portions that bind to the antigen are each fused at the C terminus of the Fab heavy chain to the N termination of one of the subunits of the Fc domain, and the second portion that is antigen-binding 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 portion. In this specific embodiment, the bispecific antigen-binding molecule that activates T cells essentially consists of a first, a second and a third portion that binds to the antigen, an Fc domain composed of a first and a second subunit, and optionally one or more peptide ligands, where the second antigen-binding portion is fused at the C termination of the Fab heavy chain to the N termination of the Fab heavy chain of the first antigen-binding portion, and the first antigen-binding portion is fused at the C termination of the Fab heavy chain to the N termination of the first and second subunits of the Fc domain, and where the third antigen-binding portion is fused at the C termination of the Fab heavy chain to the N termination of the second subunit of the Fc domain. Optionally, the Fab light chain of the first portion that binds to the antigen and the Fab light chain of the second portion that binds to the antigen can be further fused to each other.
[0160] [00160] In one embodiment, the present invention provides a bispecific antigen-binding molecule that activates T cells, which comprises: (i) a first antigen-binding portion that is a Fab molecule capable of specifically binding to CD3, which comprises the complementarity determining region (CDR) 1 of the heavy chain of SEQ ID NO: 4, the CDR 2 of the chain heavy chain of SEQ ID NO: 5, CDR 3 of the heavy chain of SEQ ID NO: 6, CDR1 of the light chain of SEQ ID NO: 8, CDR2 of the light chain of SEQ ID NO: 9 and CDR3 of the light chain of SEQ ID NO: 10, where the first portion that binds to the antigen is a cross between the Fab molecule, in which both the variable and the constant regions, particularly the constant regions, of the Fab light chain and the Fab heavy chain are exchanged. (i) a second and a third antigen-binding portion, each of which is a Fab molecule capable of specifically binding the CEA comprising CDR 1 of the SEQ ID NO: 24 heavy chain, CDR 2 of heavy chain of SEQ ID NO: 25, CDR 3 of the heavy chain of SEQ ID NO: 26, CDR1 of the light chain of SEQ ID NO: 28, CDR2 of the light chain of SEQ ID NO: 29 and CDR3 of the chain of SEQ ID NO: 30.
[0161] [00161] In one embodiment, the present invention provides a bispecific antigen-binding molecule that activates T cells, which comprises: (i) a first antigen-binding portion that is a Fab molecule capable of specifically binding to CD3 that comprises a variable region of the heavy chain that comprises an amino acid sequence that is at least about 95%, 96%, 97 %, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 3, and a variable region of the light chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 7, where the first portion that binds to the antigen is a cross between the Fab molecule, in which both the variable and the constant regions, particularly the regions constants, Fab light chain and Fab heavy chain are exchanged. (i) a second and a third antigen-binding portion, each of which is a Fab molecule capable of specifically binding CEA comprising a variable region of the heavy chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 23, and a variable region of the light chain comprising an amino acid sequence that is at least about 95% , 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 27.
[0162] [00162] In one embodiment, the present invention provides a bispecific antigen-binding molecule that activates T cells, which comprises: (i) a first antigen-binding portion that is a Fab molecule capable of specifically binding to CD3, which comprises the complementarity determining region (CDR) 1 of the heavy chain of SEQ ID NO: 4, the CDR 2 of the chain heavy chain of SEQ ID NO: 5, CDR 3 of the heavy chain of SEQ ID NO: 6, CDR1 of the light chain of SEQ ID NO: 8, CDR2 of the light chain of SEQ ID NO: 9 and CDR3 of the light chain of SEQ ID NO: 10, where the first portion that binds to the antigen is a cross between the Fab molecule, in which both the variable and the constant regions, particularly the constant regions, of the Fab light chain and the Fab heavy chain are exchanged. (i) a second and a third antigen-binding portion, each of which is a Fab molecule capable of specifically binding to MCSP which comprises the heavy chain CDR 1 of SEQ ID NO: 14, the CDR 2 of the heavy chain of SEQ ID NO: 15, CDR 3 of the heavy chain of SEQ ID NO: 16, CDR1 of the light chain of SEQ ID NO: 18, CDR2 of the light chain of SEQ ID NO: 19 and CDR3 of the chain of SEQ ID NO: 20.
[0163] [00163] In one embodiment, the present invention provides a bispecific antigen-binding molecule that activates T cells, which comprises: (i) a first antigen-binding portion that is a Fab molecule capable of specifically binding to CD3 that comprises a variable region of the heavy chain that comprises an amino acid sequence that is at least about 95%, 96%, 97 %, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 3, and a variable region of the light chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 7, where the first portion that binds to the antigen is a cross between the Fab molecule, in which both the variable and the constant regions, particularly the regions constants, Fab light chain and Fab heavy chain are exchanged. (i) a second and a third antigen-binding moiety, each of which is a Fab molecule capable of specifically binding to MCSP that comprises a variable region of the heavy chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 13, and a variable region of the light chain comprising an amino acid sequence that is at least about 95% , 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 17.
[0164] [00164] The bispecific antigen-binding molecule that activates T cells, according to any of the above four embodiments, may further comprise (iii) an Fc domain composed of a first and a second subunit capable of stable association, in which the second antigen-binding portion 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 portion, and the first antigen-binding portion is fused at the C-termination of the chain Fab heavy chain to the N termination of the first Fc domain subunit, and where the third antigen-binding portion is fused at the C termination of the Fab heavy chain to the N termination of the second Fc domain subunit.
[0165] [00165] In some of the bispecific antigen-binding molecules that activate T cells of the invention, the Fab light chain from the first portion that binds to the antigen and the Fab light chain from the second portion that binds to the antigen are fused one at a time another, optionally via a peptide linker. Depending on the configuration of the first and second antigen-binding portion, the Fab light chain of the first antigen-binding portion can be fused at its C-terminus to the N termination of the Fab light chain of the second antigen-binding portion , or the Fab light chain of the second antigen-binding portion can be fused at its C-terminus to the N termination of the Fab light chain of the first antigen-binding portion. The fusion of the Fab light chains of the first and the second antigen-binding portion further reduces the unsuccessful pairing of the Fab heavy and light chains, and also reduces the number of plasmids necessary for the expression of some of the molecules binding to the bispecific antigens that activate T cells of the invention.
[0166] [00166] In certain embodiments, the bispecific antigen-binding molecule that activates T cells comprises a polypeptide, in which the variable region of the Fab light chain of the first portion that binds to the antigen shares a carboxy-terminal peptide bridge with the region Fab heavy chain constant of the first antigen-binding portion (i.e., a first antigen-binding portion comprises a crossed Fab heavy chain, where the variable region of the heavy chain is replaced by a variable region of the chain light), which in turn shares a peptide bridge with a subunit of the Fc domain (VL (1) -CH1 (1) -CH2-CH3 (-CH4)), and a polypeptide where the Fab heavy chain of the second portion that binds to the antigen shares a carboxy-terminal peptide bridge with a subunit of the Fc domain (VH (2) -CH1 (2) -CH2-CH3 (-CH4)). In some embodiments, the bispecific antigen binding molecule that activates T cells further comprises a polypeptide in which the Fab heavy chain variable region of the first antigen-binding portion shares a carboxy-terminal peptide bridge with the constant region of the chain Fab light chain from the first portion that binds to the antigen (VH (1) -CL (1)) and the Fab light chain polypeptide from the second portion that binds to the antigen (VL (2) -CL (2)). In certain embodiments, the polypeptides are covalently linked, for example, by a disulfide bridge.
[0167] [00167] In alternative embodiments, the bispecific antigen-binding molecule that activates T cells comprises a polypeptide, in which the variable region of the Fab heavy chain of the first portion that binds to the antigen shares a carboxy-terminal peptide bridge with the region Fab light chain constant of the first antigen-binding portion (i.e., the first antigen-binding portion comprises a crossed Fab heavy chain, where the constant region of the heavy chain is replaced by a constant region of the chain light), which in turn shares a peptide bridge with a subunit of the Fc domain (VH (1) -CL (1) -CH2-CH3 (-CH4)), and a polypeptide in which the Fab heavy chain of the second portion that binds to the antigen shares a carboxy-terminal peptide bridge with a subunit of the Fc domain (VH (2) -CH1 (2) -CH2-CH3 (-CH4)). In some embodiments, the bispecific antigen-binding molecule that activates T cells further comprises a polypeptide in which the variable region of the Fab light chain of the first portion that binds to the antigen shares a carboxy-terminal peptide bridge with the constant region of the chain Fab heavy from the first portion that binds to the antigen (VL (1) -CH (1)) and the Fab light chain polypeptide from the second portion that binds to the antigen (VL (2) -CL (2)). In certain embodiments, the polypeptides are covalently linked, for example, by a disulfide bridge.
[0168] [00168] In some embodiments, the bispecific antigen-binding molecule that activates T cells comprises a polypeptide, in which the variable region of the Fab light chain of the first portion that binds to the antigen shares a carboxy-terminal peptide bridge with the region Fab heavy chain constant of the first antigen-binding portion (i.e., the first antigen-binding portion comprises a crossed Fab heavy chain, where the variable region of the heavy chain is replaced by a variable region of the chain light), which in turn shares a peptide bridge with the Fab heavy chain of the second portion that binds to the antigen, which in turn shares a carboxy-terminal peptide bridge with a subunit of the Fc domain (VL (1) -CH1 (1) -VH (2) -CH1 (2) -CH2-CH3 (-CH4)). In other embodiments, the bispecific antigen-binding molecule that activates T cells comprises a polypeptide, in which the variable region of the Fab heavy chain of the first portion that binds to the antigen shares a carboxy-terminal peptide bridge with the constant region of the chain Fab light from the first antigen-binding portion (that is, the first antigen-binding portion comprises a crossed Fab heavy chain, where the heavy chain constant region is replaced by a light chain constant region), which in turn shares a peptide bridge with the Fab heavy chain of the second portion that binds to the antigen, which in turn shares a carboxy-terminal peptide bridge with a subunit of the Fc (VH (1) -CL ( 1) -VH (2) -CH1 (2) -CH2-CH3 (-CH4)). In still other embodiments, the bispecific antigen-binding molecule that activates T cells comprises a polypeptide, in which the Fab heavy chain of the second portion that binds to the antigen shares a carboxy-terminal peptide bridge with the variable region of the light chain of Fab of the first antigen-binding portion, which in turn shares a carboxy-terminal peptide bridge with the Fab heavy chain constant region (ie, the first antigen-binding portion comprises a crossed Fab heavy chain , in which the variable region of the heavy chain is replaced by a variable region of the light chain), which in turn shares a carboxy-terminal peptide bridge with a subunit of the Fc domain (VH (2) -CH1 (2) -VL ( 1) -CH1 (1) -CH2-CH3 (-CH4)). In still other embodiments, the bispecific antigen-binding molecule that activates T cells comprises a polypeptide, in which the Fab heavy chain of the second portion that binds to the antigen shares a carboxy-terminal peptide bridge with the variable region of the Fab heavy chain of the first antigen-binding portion, which in turn shares a carboxy-terminal peptide bridge with the Fab light chain constant region (ie, the first antigen-binding portion comprises a crossed heavy Fab chain, where the constant region of the heavy chain is replaced by a constant region of the light chain), which in turn shares a carboxy-terminal peptide bridge with a subunit of the Fc domain (VH (2) -CH1 (2) -VH (1 ) -CL (1) -CH2-CH3 (-CH4)).
[0169] [00169] In some of these embodiments, the molecule that binds to the bispecific antigen that activates T cells further comprises a cross-linked Fab light chain polypeptide from the first portion that binds to the antigen, wherein the Fab heavy chain variable region of first antigen-binding portion shares a carboxy-terminal peptide bridge with the Fab light chain constant region of the first antigen (VH (1) -CL (1)) antigen and the Fab light chain polypeptide of the second portion that binds to the antigen (VL (2) -CL (2)). In other of these embodiments, the bispecific antigen-binding molecule that activates T cells further comprises a crossed Fab light chain polypeptide, in which the variable Fab light chain region of the first portion that binds to the antigen shares a carboxy peptide bridge -terminal with the Fab heavy chain constant region of the first portion that binds to the antigen (VL (1) -CH (1)) and the Fab light chain polypeptide of the second portion that binds to the antigen (VL (2) ) -CL (2)). In still other of these embodiments, the bispecific antigen-binding molecule that activates T cells further comprises a polypeptide, in which the variable region of the Fab light chain of the first portion that binds to the antigen shares a carboxy-terminal peptide bridge with the region Fab heavy chain constant of the first portion that binds to the antigen, which in turn shares a carboxy-terminal peptide bridge with the Fab light chain polypeptide of the second portion that binds to the (VL (1) -CH1 ( 1) -VL (2) -CL (2)), a polypeptide in which the Fab heavy chain variable region of the first antigen-binding portion shares a carboxy-terminal peptide bridge with the Fab light chain constant region of the first portion that binds to the antigen, which in turn shares a carboxy-terminal peptide bridge with the Fab light chain polypeptide of the second portion that binds to the antigen (VH (1) -CL (1) -VL (2 ) -CL (2)), a polypeptide in which the polypeptide that of the Fab light chain of the second portion that binds to the antigen shares a carboxy-terminal peptide bridge with the variable region of the Fab light chain of the first portion that binds to the antigen, which in turn shares a carboxy-terminal peptide bridge with the Fab heavy chain constant region of the first portion that binds to the antigen (VL (2) -CL (2) -VL (1) -CH1 (1)), or a polypeptide in which the light chain polypeptide of Fab from the second portion that binds to the antigen shares a carboxy-terminal peptide bridge with the variable region of the Fab heavy chain from the first portion that binds to the antigen, which in turn shares a carboxy-terminal peptide bridge with the constant region from Fab light chain of the first antigen-binding portion (VL (2) -CL (2) -VH (1) -CL (1)).
[0170] The bispecific antigen-binding molecule that activates T cells, according to these embodiments, may further comprise (i) a polypeptide of the Fc domain subunit (CH2-CH3 (-CH4)), or (ii) a polypeptide where the Fab heavy chain of a third moiety that binds to the antigen shares a carboxy-terminal peptide bridge with a subunit of the Fc domain (VH (3) -CH1 (3) -CH2-CH3 (-CH4)) and the a third portion Fab light chain polypeptide that binds to the antigen (VL (3) -CL (3)). In certain embodiments, the polypeptides are covalently linked, for example, by a disulfide bridge.
[0171] [00171] According to any of the above embodiments, components of the bispecific antigen-binding molecule that activates T cells (eg, antigen-binding component, Fc domain) can be fused directly or through various ligands, particularly peptide ligands which comprise one or more amino acids, typically about 2 to 20 amino acids, which are described in the present application or are known in the art. Suitable non-immunogenic peptide linkers include, for example, peptide linkers (G4S) n, (SG4) n, (G4S) n or G4 (SG4) n, where n is generally a number between 1 and 10, typically between 2 and 4. Fc Domain
[0172] [00172] The Fc domain of the bispecific antigen binding molecule that activates T cells consists of a pair of polypeptide chains comprising domains of the heavy chain of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, each subunit of which comprises the IgG CH2 and CH3 heavy chain constant domains. The two subunits of the Fc domain are capable of stable association with each other. In one embodiment, the bispecific antigen-binding molecule that activates T cells of the invention comprises no more than one Fc domain.
[0173] [00173] In an embodiment according to the invention, the Fc domain of the bispecific antigen binding molecule that activates T cells is an IgG Fc domain. In a specific embodiment, the Fc domain is an IgG1 Fc domain. In another embodiment, the Fc domain is an IgG4 Fc domain. In a more specific embodiment, the Fc domain is an IgG4 Fc domain that comprises an amino acid substitution at position S228 (KabatEU numbering), particularly the substitution of amino acid S228P. This amino acid substitution reduces the exchange of the Fab arm in vivo of IgG4 antibodies (see, Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). In a further particular embodiment, the Fc domain is human. An exemplary sequence of an IgG1 Fc region is provided in SEQ ID NO: 107. FC DOMAIN MODIFICATIONS THAT PROMOTE HETERODIMERIZATION
[0174] The bispecific antigen-binding molecules that activate T cells, according to the invention, comprise different antigen-binding components, fused to one or the other of the two subunits of the Fc domain, so the two subunits of the Fc domain are typically comprised of the two non-identical polypeptide chains. The recombinant coexpression of these polypeptides and subsequent dimerization leads to several possible combinations of the two polypeptides. In order to improve the yield and purity of the bispecific antigen-binding molecules that activate T cells in recombinant production, it will therefore be advantageous to introduce into the Fc domain of the bispecific antigen-binding molecule that activates T cells a modification that promotes the association of the desired polypeptides.
[0175] Consequently, in specific embodiments of the Fc domain of the bispecific antigen binding molecule that activates T cells, according to the invention, it comprises a modification that promotes the association of the first and the second subunit of the Fc domain. The most extensive protein-protein interaction site between the two subunits of a human IgG Fc domain is in the CH3 domain of the Fc domain. Thus, in one embodiment, said modification is in the CH3 domain of the Fc domain.
[0176] [00176] In a specific embodiment, said modification is also known as a "knob-into-hole" modification, which comprises a modification of a knob in one of the two subunits of the Fc domain and one modification of a hole in the other of the two subunits of the Fc domain.
[0177] [00177] The orifice bulge technology is described, for example, in US patents 5,731,168, US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). The method generally involves introducing a protuberance ("protrusion") at the interface of a first polypeptide and a corresponding cavity ("hole") at the interface of a second polypeptide, so that the protuberance can be positioned in the cavity in order to promote formation of heterodimer and delay the formation of homodimer. Lumps are constructed to replace the small amino acid side chains at the interface of the first polypeptide with larger side chains (for example, tyrosine or tryptophan). Compensatory cavities of identical or similar size to the protuberances are created on the interface of the second polypeptide by replacing large side chains of amino acids with smaller chains (for example, alanine or threonine).
[0178] Consequently, in a specific embodiment, in the CH3 domain of the first subunit of the Fc domain of the bispecific antigen binding molecule that activates T cells, an amino acid residue is replaced by an amino acid residue that has a larger side chain volume , thus generating a bulge within the CH3 domain of the first subunit that is positionable in a cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subcategory of the Fc domain, an amino acid residue is replaced by an amino acid residue that has a smaller side chain volume, thus 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.
[0179] [00179] The bulge and cavity can be made by altering the nucleic acid encoding the polypeptides, for example, by site-specific mutagenesis, or by peptide synthesis.
[0180] [00180] In a specific embodiment, in the CH3 domain of the first subunit of the Fc domain, the threonine residue at position 366 is replaced by a tryptophan residue (T366W), and in the CH3 domain of the second subunit of the Fc domain, the residue of tyrosine at position 407 is replaced by a valine residue (Y407V). In one embodiment, in the second subunit of the Fc domain, in addition the threonine residue at position 366 is replaced by a serine residue (T366S) and the leucine residue at position 368 is replaced by an alanine residue (L368A).
[0181] [00181] Still in an additional realization, in the first subunit of the Fc domain, additionally the serine residue at position 354 is replaced by a cysteine residue (S354C), and in the second subunit of the Fc domain, additionally the tyrosine residue in position 349 is replaced by a cysteine residue (Y349C). The introduction of these two cysteine residues results in the formation of a disulfide bridge between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).
[0182] [00182] In a specific embodiment, the portion that binds to the antigen capable of binding to CD3 is fused (optionally through the portion that binds to the antigen capable of binding to a target cell antigen) to the first subunit of the Fc domain (which comprises the "bulge" modification.) Without sticking to the theory, fusing the portion that binds to the CD3-binding antigen to the bulge containing the Fc domain subunit will minimize the generation of the antigen-binding molecule that comprises two components of antigen binding capable of binding to CD3 (steric shock of two protuberance-containing polypeptides).
[0183] [00183] In an alternative embodiment, a modification that promotes association of the first and second subunits of the Fc domain comprises a modification that mediates electrostatic directional effects, for example as described in PCT publication WO 2009/089004. Generally, this method involves replacing one or more amino acid residues at the interface of the Fc domain subunits with charged amino acid residues, so that homodimer formation becomes electrostatically unfavorable, but electrostatically favorable heterodimerization.
[0184] [00184] Modifications in the Fc domain that reduce the connection to the Fc receptor and / or effector function
[0185] [00185] The Fc domain gives the bispecific antigen-binding molecule that activates T cells favorable pharmacokinetic properties, including a long serum half-life that contributes to good accumulation in the target tissue and a favorable blood-tissue distribution ratio. At the same time, however, it can lead to the undesirable targeting of the bispecific antigen-binding molecule that activates T cells to cells that express Fc receptors instead of the preferred antigen-bearing cells. In addition, the coactivation of Fc receptor signaling pathways can lead to the release of cytokine, which, in combination with the activation properties of T cells and the long half-life of the antigen-binding molecule, results in excessive activation of cytokine and severe side effects through systemic administration. Activation of immune cells (carrying the Fc receptor), except for T cells, may even reduce the effectiveness of the bispecific antigen-binding molecule that activates T cells due to the potential for destruction of T cells, for example, by NK cells.
[0186] Consequently, in specific embodiments, the Fc domain of bispecific antigen-binding molecules that activate T cells, according to the invention, exhibits reduced binding affinity to an Fc receptor and / or reduced effector function, compared to a native IgG1 Fc domain. In this embodiment, the Fc domain (or the bispecific antigen-binding molecule that activates T cells comprising said Fc domain) exhibits less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% binding affinity to an Fc receptor, compared to a native native IgG1 Fc domain (or a bispecific antigen binding molecule that activates T cells that comprises a native IgG1 Fc domain), and / or less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% of the effector function, compared to a native IgG1 Fc domain (or a bispecific antigen binding molecule that activates T cells which comprises a native IgG1 Fc domain). In one embodiment, the Fc domain (or the bispecific antigen binding molecule that activates T cells that comprise said Fc domain) does not substantially bind to an Fc receptor and / or induce effector function. In a specific embodiment, the Fc receptor is an Fcү receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activating Fc receptor. In a specific embodiment, the activation Fc receptor is an activation of human Fcү receptor, more specifically FcүRIIIa, FcүRI or human FcүRIIa, more specifically human FcүRIIIa. In one embodiment, the effector function is one or more among those selected from the group of CDC, ADCC, ADCP and cytokine secretion. In a specific embodiment, the effecting function is ADCC. In one embodiment, the Fc domain exhibits substantially binding affinity to the neonatal Fc receptor (FcRn), as compared to a native IgG1 Fc domain. Substantially similar binding to FcRn is achieved when the Fc domain (or bispecific antigen binding molecule that activates T cells comprising said Fc domain) exhibits more than about 70%, particularly more than about 80%, more particularly more that about 90% of the binding affinity of a native IgG1 Fc domain (or the bispecific antigen binding molecule that activates T cells that comprises a native IgG1 Fc domain) to FcRn.
[0187] [00187] In certain embodiments, the Fc domain is genetically engineered to have reduced binding affinity for an Fc receptor and / or reduced effector function, compared to a non-genetically engineered Fc domain. In specific embodiments, the Fc domain of the bispecific antigen binding molecule that activates T cells comprises one or more amino acid mutations that reduce the binding affinity of the Fc domain to an Fc receptor and / or effector function. Typically, the same or more amino acid mutations are present in each of the two subunits of the Fc domain. In one embodiment, the amino acid mutation reduces the binding affinity of the Fc domain to an Fc receptor. In one embodiment, the amino acid mutation reduces the binding affinity of the Fc domain to an Fc receptor by at least 2 times, at least 5 times or at least 10 times. In embodiments where there is more than one amino acid mutation that reduces the binding affinity of the Fc domain to the Fc receptor, the combination of these amino acid mutations can reduce the binding affinity of the Fc domain to an Fc receptor by at least 10 times 20 times or even at least 50 times. In one embodiment, the bispecific antigen-binding molecule that activates T cells that comprises a genetically engineered Fc domain exhibits less than 20%, particularly less than 10%, more particularly less than 5% of binding affinity to an Fc receptor, in comparison to a bispecific antigen-binding molecule that activates T cells that comprise a non-genetically engineered Fc domain. In a specific embodiment, the Fc receptor is an Fcү receptor. In some embodiments, the Fc receptor is a human Fc receptor. In some embodiments, the Fc receiver is an activating Fc receiver. In a specific embodiment, the activation Fc receptor is an activation of human Fcү receptor, more specifically FcүRIIIa, FcүRI or human FcүRIIa, more specifically human FcүRIIIa. Preferably, the binding to each of these receptors is reduced. In some embodiments, binding affinity to a complement component, specifically binding affinity to C1q, is also reduced. In one embodiment, the binding affinity to the neonatal Fc receptor (FcRn) is not reduced. Substantially similar binding to FcRn, that is, the conservation of binding affinity to the Fc domain to said receptor, is achieved when the Fc domain (or the bispecific antigen binding molecule that activates T cells comprising said Fc domain) exhibits more than about 70% of the binding affinity to a non-genetically engineered form of the Fc domain (or the bispecific antigen-binding molecule that activates T cells that comprises said genetically engineered form of the Fc domain) to the FcRn. The Fc domain or bispecific antigen-binding molecules that activate T cells of the invention that comprise said Fc domain, can exhibit more than about 80% and even more than about 90% of that affinity. In certain embodiments, the Fc domain of the bispecific antigen-binding molecule that activates T cells is genetically engineered to have reduced effector function, compared to a non-genetically engineered Fc domain. The reduced effector function may 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 cell phagocytosis (ADCP) , reduced cytokine secretion, reduced immune complex-mediated absorption by antigen presenting cells, reduced NK cell binding, reduced macrophage binding, reduced monocyte binding, reduced polymorphonuclear cell binding, induction of reduced apoptosis direct signaling, reduced cross-linking of target-linked antibodies, reduced dendritic cell maturation, reduced T-cell preparation. In one embodiment, the reduced effector function is one or more selected from the group among, reduced CDC, reduced ADCC, reduced ADCP and reduced cytokine secretion. In a specific 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-genetically engineered Fc domain (or a bispecific antigen-binding molecule that activates T cells that comprises a non-genetically engineered Fc domain).
[0188] [00188] In one embodiment, the amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor and / or effector function is an amino acid substitution. In one embodiment, the Fc domain comprises an amino acid substitution at a position selected from the group of E233, L234, L235, N297, P331 and P329. In a more specific embodiment, the Fc domain comprises an amino acid substitution at a position selected from the group of L234, L235 and P329. In some embodiments, the Fc domain comprises amino acid substitutions L234A and L235A. In one embodiment, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. In one embodiment, the Fc domain comprises an amino acid substitution at the P329 position. In a more specific embodiment, the amino acid substitution is P329A or P329G, particularly P329G. In one embodiment, the Fc domain comprises an amino acid substitution at position P329 and an additional substitution at a position selected from E233, L234, L235, N297 and P331. In a more specific embodiment, the additional amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S. In specific embodiments, the Fc domain comprises amino acid substitutions at positions P329, L234 and L235. In more specific embodiments, the Fc domain comprises the amino acid mutations L234A, L235A and P329G ("P329G LALA”). In one embodiment, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. The combination " P329G LALA ”of amino acid substitutions almost completely eliminates the binding to the Fcy receptor (as well as the complement) of a human IgG1 Fc domain, as described in PCT publication WO 2012/130831, which is incorporated in this application by reference. WO 2012/130831 also describes methods for preparing these mutant Fc domains and methods for determining their properties as binding to the Fc receptor or effector functions.
[0189] [00189] IgG4 antibodies exhibit reduced binding affinity to Fc receptors and reduced effector functions, compared to IgG1 antibodies. Therefore, in some embodiments the T cell Fc domain that activates bispecific antigen-binding molecules of the invention is an IgG4 domain, particularly a human IgG4 Fc domain. In one embodiment, the IgG4 Fc domain comprises amino acid substitutions at the S228 position, specifically the amino acid substitution S228P. To further reduce its binding affinity to an Fc receptor and / or its effector function, in one embodiment the IgG4 Fc domain comprises an amino acid substitution at the L235 position, specifically the L235E amino acid substitution. In another embodiment, the IgG4 Fc domain comprises an amino acid substitution at the P329 position, specifically the amino acid substitution P329G. In a specific embodiment, the IgG4 Fc domain comprises amino acid substitutions at positions S228, L235 and P329, specifically amino acid substitutions S228P, L235E and P329G. These mutant IgG4 Fc domains and their binding properties to the Fcү receptor are described in PCT publication WO 2012/130831, which is incorporated herein by reference.
[0190] [00190] In a specific embodiment, the Fc domain that exhibits reduced binding affinity to a Fc receptor and / or reduced effector function, compared to a native IgG1 Fc domain, is a human IgG1 Fc domain that comprises the substitutions of amino acids L234A, L235A and optionally P329G, or a human IgG4 Fc domain, comprising amino acid substitutions S228P, L235E and optionally P329G.
[0191] [00191] In certain embodiments, N-glycosylation of the Fc domain has been eliminated. In that embodiment, the Fc domain comprises an amino acid mutation at the N297 position, particularly an amino acid substitution that replaces asparagine with alanine (N297A) or aspartic acid (N297D).
[0192] [00192] In addition to the Fc domains described earlier in this application and in the PCT publication WO 2012/130831, Fc domains with reduced Fc receptor binding and / or effector function also include those with replacement of one or more residues among the residues of the domain Fc 238, 265, 269, 270, 297, 327 and 329 (US patent 6,737,056). These Fc mutants include Fc mutants with substitutions at two or more positions of amino acids 265, 269, 270, 297 and 327, including the also known as Fc mutant "DANA" with substitutions of residues 265 and 297 for alanine (US patent 7,332,581 ).
[0193] [00193] 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-directed mutagenesis of the coding DNA sequence, PCR, gene synthesis and the like. Correct nucleotide changes can be verified, for example, by sequencing.
[0194] [00194] Binding to Fc receptors can be easily determined, for example, by ELISA, or by surface plasmon resonance (SPR) using conventional instruments, such as a BIAcore instrument (GE Healthcare), and Fc receptors, as they can be obtained by recombinant expression. A suitable binding test is described in the present application. Alternatively, the binding affinity of bispecific Fc domains or antigen-binding molecules that comprise an Fc domain for Fc receptors can be assessed with the use of cell lines known to express specific Fc receptors, such as human NK cells that express the receptor FcүIIa.
[0195] [00195] The effector function of an Fc domain, or a bispecific antigen-binding molecule that activates T cells comprising an Fc domain, can be measured by methods known in the art. A suitable assay for measuring ADCC is described in the present application. Other examples of in vitro assays to assess the ADCC activity of a molecule of interest are described in US patent 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); US patent 5,821,337; Bruggemann et al., J Exp Med 166, 1351-1361 (1987). Alternatively, non-radioactive assay methods can be employed (see, for example, ACTI ™ non-radioactive cytotoxicity assay for cytometric flow (CellTechnology, Inc. Mountain View, CA); and CytoTox 96® non-radioactive cytotoxicity assay ( Promega, Madison, WI)). Effector cells useful for these assays include peripheral blood mononuclear cells (PBMC) and natural killer cells (Natural Killer-NK). Alternatively or additionally, the ADCC activity of the molecule of interest can be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).
[0196] [00196] In some embodiments, the binding of the Fc domain to a complement component, specifically C1q, is reduced. Consequently, in some embodiments in which the Fc domain is genetically engineered to have reduced effector function, said effector function includes reduced CDC. C1q binding assays can be performed to determine whether the bispecific antigen binding molecule that activates T cells is able to bind C1q and then have CDC activity. See, for example, C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay can 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)). ANTIGEN BINDING COMPONENTS
[0197] [00197] The antigen binding molecule of the invention is bispecific, that is, it comprises at least two antigen binding components capable of specifically binding two distinct antigenic determinants. According to the invention, the antigen-binding components are Fab molecules (i.e., antigen-binding domains composed of a heavy chain and a light chain, each comprising a variable region and a constant). In one embodiment, said Fab molecules are human. In another embodiment, said Fab molecules are humanized. In yet another embodiment, said Fab molecules comprise constant regions of the human heavy and light chain.
[0198] [00198] At least one of the antigen-binding components is a Fab molecule cross. This modification prevents unsuccessful pairing of the heavy and light chains of different Fab molecules, thus improving the yield and purity of the bispecific antigen-binding molecule that activates T cells of the invention in recombinant production. In a specific crossed Fab molecule useful for the bispecific antigen binding molecule that activates T cells of the invention, the constant regions of the Fab light chain and the Fab heavy chain are switched. In another specific Fab cross molecule useful for the bispecific antigen binding molecule that activates T cells of the invention, the variable regions of the Fab light chain and the Fab heavy chain are switched.
[0199] [00199] In a specific embodiment, according to the invention, the bispecific antigen-binding molecule that activates T cells is capable of simultaneous binding to a target cell antigen, particularly a tumor cell antigen, and CD3. In one embodiment, the bispecific antigen binding molecule that activates T cells is capable of cross-linking a T cell and a target cell by simultaneous binding to a target cell and CD3 antigen. In an even more specific embodiment, this simultaneous binding results in lysis of the target cell, particularly a tumor cell. In one embodiment, this simultaneous binding results in the activation of the T cell. In other embodiments, this simultaneous binding results in a cellular response of a T lymphocyte, especially a cytotoxic T lymphocyte, selected from the group of: proliferation, differentiation, secretion cytokine, release of cytotoxic effector molecule, cytotoxic activity and expression of activation markers. In one embodiment, binding of the binding molecule to the bispecific antigen that activates T cells to CD3 without simultaneous binding to the target cell's antigen does not result in T cell activation.
[0200] [00200] In one embodiment, the bispecific antigen-binding molecule that activates T cells is able to redirect cytotoxic activity from a T cell to a target cell. In a specific embodiment, said redirection is independent of MHC-mediated peptide antigen presentation by the target cell and / or T cell specificity.
[0201] [00201] In particular, a T cell according to any of the embodiments of the invention is a cytotoxic T cell. In some embodiments, the T cell is a CD4 + T cell or a CD8 + T cell, particularly a CD8 + T cell. CD3 CONNECTION COMPONENT
[0202] The bispecific antigen binding molecule that activates T cells of the invention comprises at least one antigen binding component capable of binding to CD3 (also referred to as "CD3 antigen binding component" or "first binding portion antigen ”). In a specific embodiment, the bispecific antigen-binding molecule that activates T cells comprises no more than an antigen-binding component capable of specifically binding to CD3. In one embodiment, the bispecific antigen-binding molecule that activates T cells provides monovalent binding to CD3. The binding to the CD3 antigen is a crossed Fab molecule, that is, a Fab molecule in which both the variable region and the constant of the heavy and light chains are exchanged. In embodiments where there is more than one antigen binding component capable of specifically binding to a target cell antigen comprised in the bispecific antigen binding molecule that activates T cells, the portion that binds to the antigen capable of specifically binding to CD3 it is preferably a crossed Fab molecule and the antigen-binding components capable of specifically binding to a cell antigen are conventional Fab molecules.
[0203] [00203] In a specific embodiment, CD3 is human CD3 (SEQ ID NO: 103) or cynomolgus CD3 (SEQ ID NO: 104), more particularly human CD3. In a specific embodiment, the portion that binds to the antigen is cross-reactive (that is, specifically binds) to human and cynomolgus CD3. In some embodiments, the first portion that binds to the antigen is able to specifically bind to the epsilon subunit of CD3.
[0204] [00204] The portion that binds to the CD3 antigen comprises at least one complementarity determining region (CDR) of the heavy chain selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 and at least one light chain CDR selected from the group of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10.
[0205] [00205] In one embodiment, the CD3 antigen-binding portion comprises CDR1 of the SEQ ID NO: 4 heavy chain, CDR2 of the SEQ ID NO: 5 heavy chain, CDR3 of the SEQ ID NO: 6 heavy chain , CDR1 of the light chain of SEQ ID NO: 8, CDR2 of the light chain of SEQ ID NO: 9 and CDR3 of the light chain of SEQ ID NO: 10.
[0206] [00206] In one embodiment, the portion that binds to the CD3 antigen comprises a variable region sequence of the heavy chain that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to a amino acid sequence selected from the group of: SEQ ID NO: 3, SEQ ID NO: 32 and SEQ ID NO: 33, and a variable region sequence of the light chain that is at least about 95%, 96%, 97 %, 98%, 99% or 100% identical to an amino acid sequence selected from the group of: SEQ ID NO: 7 and SEQ ID NO: 31.
[0207] [00207] In one embodiment, the portion that binds to the CD3 antigen comprises a variable region of the heavy chain that comprises an amino acid sequence selected from the group of: SEQ ID NO: 3, SEQ ID NO: 32 and SEQ ID NO : 33, and a variable region of the light chain comprising an amino acid sequence selected from the group of: SEQ ID NO: 7 and SEQ ID NO: 31.
[0208] [00208] In one embodiment, the portion that binds to the CD3 antigen comprises a variable region sequence of the heavy chain that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 3, and a variable region sequence of the light chain that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 7.
[0209] [00209] In one embodiment, the portion that binds to the CD3 antigen comprises a variable region of the heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a variable region of the light chain comprising the amino acid sequence of SEQ ID NO: 7.
[0210] [00210] In one embodiment, the CD3 antigen-binding portion comprises the heavy chain variable region sequence of SEQ ID NO: 3 and the light chain variable region sequence of SEQ ID NO: 7. TARGET CELL ANTIGEN BINDING COMPONENT
[0211] The bispecific antigen-binding molecule that activates T cells of the invention comprises at least one antigen-binding component capable of binding to a target cell antigen (also cited in this application as an "antigen-binding component of target cell ”or“ second ”or“ third ”antigen binding component.) In certain embodiments, the bispecific antigen binding molecule that activates T cells comprises two antigen binding components capable of binding to a target cell antigen. In this specific embodiment, each of these antigen-binding components binds specifically to the same antigenic determinant.In an even more specific embodiment, all of these antigen-binding components are identical. In one embodiment, the bispecific antigen-binding molecule that activates T cells comprise an immunoglobulin molecule capable of specifically binding to a target cell antigen. In one embodiment, the molecule bispecific antigen binding agent that activates T cells comprises no more than two antigen binding components capable of binding to a target cell antigen.
[0212] [00212] The portion that binds to the target cell antigen is usually a Fab molecule, particularly a conventional Fab molecule that binds to a specific antigenic determinant and is capable of directing the bispecific antigen-binding molecule that activates T cells to a target site, for example, for a specific type of tumor cell that produces the antigenic determinant.
[0213] [00213] In certain embodiments, the portion that binds to the target cell antigen specifically binds to a cell surface antigen. In a specific embodiment, the portion that binds to the target cell antigen specifically binds to a proximal membrane region of a cell surface antigen. In this specific embodiment, the cell surface antigen is Carcinoembryonic Antigen (CEA) and the proximal membrane region is the B3 domain of CEA (residues 208 to 286 SEQ ID NO: 119). In another specific embodiment, the cell surface antigen is Melanoma Associated Chondroitin Sulfate Proteoglycan (MCSP) and the proximal membrane region is the MSCP D3 domain (SEQ ID NO: 118).
[0214] [00214] In certain embodiments, the portion that binds to the target cell antigen is directed to an antigen associated with a pathological condition, such as an antigen presented in a tumor cell or in a tumor cell environment or in a virus-infected cell . Suitable antigens are cell surface antigens, for example, but are not limited to, cell surface receptors. In specific embodiments, the antigen is a human antigen. In a specific embodiment, the target cell antigen is selected from Proteoglycan Chondroitin Sulfate Associated with Melanoma (MCSP, CSPG4) and Carcinoembryonic Antigen (CEA, CEACAM5).
[0215] [00215] In some embodiments, the molecule that binds to the bispecific antigen that activates T cells comprises at least one antigen-binding component that is specific for Melanoma-Associated Chondroitin Sulfate Proteoglycan (MCSP). In one embodiment, the antigen-binding portion that is specific for MCSP comprises at least one complementarity determining region (CDR) of the heavy chain selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 40 and at least one light chain CDR selected from the group of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 48, SEQ ID NO: 49 and SEQ ID NO: 50.
[0216] [00216] In one embodiment, the antigen-binding portion that is specific for MCSP comprises at least one complementarity determining region (CDR) of the heavy chain selected from the group consisting of SEQ ID NO: 14, SEQ ID NO : 15 and SEQ ID NO: 16 and at least one CDR of the light chain selected from the group of SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20.
[0217] [00217] In one embodiment, the antigen-binding portion that is specific for MCSP comprises CDR1 of the heavy chain of SEQ ID NO: 14, CDR2 of the heavy chain of SEQ ID NO: 15, CDR3 of the heavy chain of SEQ ID NO: 16, CDR1 of the SEQ ID NO: 18 light chain, CDR2 of the SEQ ID NO: 19 light chain and CDR3 of the SEQ ID NO: 20 light chain.
[0218] [00218] In an additional embodiment, the antigen-binding portion that is specific for MCSP comprises a variable region sequence of the heavy chain that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group of SEQ ID NO: 13, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 39 and SEQ ID NO: 41 and a variable region sequence of light chain that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group of SEQ ID NO: 17, SEQ ID NO: 43, SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 51.
[0219] [00219] In an additional embodiment, the antigen-binding portion that is specific for MCSP comprises a variable region of the heavy chain that comprises an amino acid sequence selected from the group of SEQ ID NO: 13, SEQ ID NO: 34 , SEQ ID NO: 36, SEQ ID NO: 39 and SEQ ID NO: 41, and a variable region of the light chain comprising an amino acid sequence selected from the group of SEQ ID NO: 17, SEQ ID NO: 43, SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 51.
[0220] [00220] In an additional embodiment, the antigen-binding portion that is specific for MCSP comprises a variable region sequence of the heavy chain that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 13, and a variable region sequence of the light chain that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 17 or variants of it that retain functionality.
[0221] [00221] In one embodiment, the antigen-binding portion that is specific for MCSP comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 13 and a light chain variable region comprising a sequence of amino acids of SEQ ID NO: 17.
[0222] [00222] In one embodiment, the antigen-binding portion that is specific for MCSP comprises the heavy region variable region sequence of SEQ ID NO: 13 and the light chain variable region sequence of SEQ ID NO: 17.
[0223] [00223] In one embodiment, the bispecific antigen binding molecule that activates T cells comprises a sequence of polypeptides that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 12, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 53, a polypeptide sequence that is at least about 95 %, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 54, and a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 55.
[0224] [00224] In specific embodiments, the bispecific antigen binding molecule that activates T cells comprises at least one antigen binding component that is specific for Carcinoembryonic Antigen. In one embodiment, the antigen-binding portion that is specific for CEA comprises at least one complementarity determining region (CDR) of the heavy chain selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26 and at least one CDR of the light chain selected from the group of SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30.
[0225] [00225] In one embodiment, the antigen-binding portion that is specific for CEA comprises the heavy chain CDR1 of SEQ ID NO: 24, the heavy chain CDR2 of SEQ ID NO: 25, the heavy chain CDR3 of SEQ ID NO: 25 SEQ ID NO: 26, CDR1 of the SEQ ID NO: 28 light chain, CDR2 of the SEQ ID NO: 29 light chain and CDR3 of the SEQ ID NO: 30 light chain.
[0226] [00226] In an additional embodiment, the portion that binds to the antigen that is specific for CEA comprises a variable region sequence of the heavy chain that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 23, and a variable region sequence of the light chain that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 27, or variants of it that retain functionality.
[0227] [00227] In one embodiment, the antigen-binding portion that is specific for CEA comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 23 and a light chain variable region comprising a sequence of amino acids of SEQ ID NO: 27.
[0228] [00228] In one embodiment, the antigen-binding portion that is specific for CEA comprises the heavy chain variable region sequence of SEQ ID NO: 23 and the light chain variable region sequence of SEQ ID NO: 27.
[0229] [00229] In one embodiment, the bispecific antigen binding molecule that activates T cells comprises a sequence of polypeptides that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 22, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 56, a polypeptide sequence that is at least about 95 %, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 57, and a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 58. POLYNUCLEOTIDS
[0230] [00230] The invention further provides isolated polynucleotides that encode a bispecific antigen-binding molecule that activates T cells, as described in the present application, or a fragment thereof. In some embodiments, said fragment is an antigen-binding fragment.
[0231] [00231] 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 shown in SEQ ID NOs 63, 64, 65, 66, 67, 68, 69, 70, 71, 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, 97 and 98, including functional fragments or variants thereof.
[0232] [00232] Polynucleotides encoding bispecific antigen binding molecules that activate T cells of the invention can be expressed as a single polynucleotide encoding the bispecific antigen binding molecule that activates whole T cells or as multiple polynucleotides (e.g., two or two more) that are coexpressed. Polypeptides encoded by polynucleotides that are coexpressed can associate through, for example, disulfide bridges or other means to form a bispecific antigen-binding molecule that activates functional T cells. For example, the portion of the light chain of an antigen-binding component can be encoded by a polynucleotide separated from the portion of the bispecific antigen-binding molecule that activates T cells that comprises the portion of the heavy chain of the portion that binds to the antigen, a subunit of the Fc domain and optionally (part of) another portion that binds to the antigen. When coexpressed, the heavy chain polypeptides will associate with the light chain polypeptides to form the portion that binds to the antigen. In another example, the portion of the bispecific antigen-binding molecule that activates T cells, which comprises one of two subunits of the Fc domain and optionally (part of) one or more antigen-binding components, could be encoded by a separate polynucleotide a from the portion of the bispecific antigen-binding molecule that activates T cells that comprise the other of the subunits of the Fc domain and optionally (part of) an antigen-binding component. When coexpressed, the subunits of the fc domain will join to form the Fc domain.
[0233] [00233] In some embodiments, the isolated polynucleotide encodes the entire bispecific antigen-binding molecule that activates T cells, according to the invention, as described in the present application. In other embodiments, the isolated polynucleotide encodes a polypeptide comprised in the bispecific antigen-binding molecule that activates T cells, according to the invention, as described in the present application.
[0234] [00234] In another embodiment, the present invention is directed to an isolated polynucleotide that encodes a bispecific antigen-binding molecule that activates T cells of the invention or a fragment thereof, wherein the polynucleotide comprises a sequence that encodes a region sequence variable, as shown in SEQ ID NOs: 3, 7, 13, 17, 23, 27, 31, 32, 33, 34, 36, 39, 41, 43, 46, 47 or 51. In another embodiment, the present invention is directed to an isolated polynucleotide that encodes a bispecific antigen-binding molecule that activates T cells or a fragment thereof, where the polynucleotide comprises a sequence that encodes a sequence of polypeptides, as shown in SEQ ID NOs: 22, 56, 57 , 58, 12, 53, 54 and 55. In another embodiment, the invention is still directed to a polynucleotide that encodes a bispecific antigen-binding molecule that activates T cells of the invention or a fragment thereof, wherein the polynucleotide comprises a next which is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence shown in SEQ ID NOs: 63, 64, 65, 66, 67 , 68, 69, 70, 71, 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, 97 or 98. In another embodiment, the invention is directed to an isolated polynucleotide that encodes a bispecific antigen-binding molecule that activates T cells of the invention or a fragment thereof, wherein the polynucleotide comprises a nucleic acid sequence shown in SEQ ID NOs: 63, 64, 65, 66, 67, 68, 69, 70, 71, 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, 97 or 98. In another embodiment, the invention is directed to a polynucleotide that encodes a molecule of binding to the bispecific antigen that activates T cells 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 the amino acid sequence in SEQ ID NOs: 3, 7, 13, 17, 23, 27, 31, 32, 33, 34, 36, 39, 41, 43, 46, 47 or 51. In another embodiment, the invention is directed to a polynucleotide that encodes a bispecific antigen-binding molecule that activates T cells or a fragment of even, wherein the polynucleotide comprises a sequence that encodes a sequence of polypeptides that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence in SEQ ID NOs: 22, 56, 57, 58, 12, 53, 54 or 55. The invention encompasses an isolated polynucleotide that encodes a bispecific antigen-binding molecule that activates T cells of the invention or a fragment thereof, wherein the polynucleotide comprises a sequence encoding the variable region sequence of SEQ ID NOs: 3, 7, 13, 17, 23, 27, 31, 32, 33, 34, 36, 39, 41, 43, 46, 47 or 51 with substitutions of conservative amino acids. The invention also encompasses an isolated polynucleotide that encodes a bispecific antigen-binding molecule that activates T cells of the invention or a fragment thereof, wherein the polynucleotide comprises a sequence that encodes the polypeptide sequence of SEQ ID NOs: 22, 56, 57, 58, 12, 53, 54 or 55 with conservative amino acid substitutions.
[0235] [00235] In certain embodiments, the polynucleotide or nucleic acid is DNA. In other embodiments, a polynucleotide of the present invention is RNA, for example, in the form of messenger RNA (mRNA). The RNA of the present invention can be single-stranded or double-stranded. RECOMBINANT METHODS
[0236] [00236] Bispecific antigen-binding molecules that activate T cells of the invention can be obtained, for example, by solid phase peptide synthesis (e.g. Merrifield solid phase synthesis) or recombinant production. For recombinant production, one or more polynucleotides that encode the bispecific antigen-binding molecule that activates T cells (fragment), for example, as described above, are isolated and inserted into one or more vectors for cloning and / or additional expression in a host cell. These polynucleotides can be easily isolated and sequenced using conventional procedures. In one embodiment, a vector is provided, preferably an expression vector, which comprises one or more of the polynucleotides of the invention. Methods that are well known to those skilled in the art can be used to construct expression vectors containing the coding sequence for a bispecific antigen-binding molecule that activates T cells (fragment), along with appropriate transcriptional / translation control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination / genetic recombination. See, for example, the techniques described in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. (1989); and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N.Y (1989). The expression vector can be part of a plasmid, virus, or it can be a fragment of nucleic acid. The expression vector includes an expression cassette in which the polynucleotide encoding the bispecific antigen-binding molecule that activates T cells (fragment) (ie, the coding region) is cloned in functional association with a promoter and / or other elements of transcription or translation control. As used in this application, a "coding region" is a portion of nucleic acid that consists of codons translated into amino acids. Although a "stop codon" (TAG, TGA or TAA) does not translate into an amino acid, it can be considered as part of a coding region, if present, but any of the adjacent sequences, for example, promoters, ribosome binding sites, transcription terminators, introns, 5 'and 3' untranslated regions, and the like, are not part of an encoding region. Two or more coding regions can be present in a single polynucleotide construct, for example, in a single vector or in separate polynucleotide constructs, for example, in separate (different) vectors. Furthermore, any vector can contain a single coding region or can comprise two or more coding regions, for example, a vector of the present invention can encode one or more polypeptides, which are separated after or in translation into final proteins by proteolytic cleavage. In addition, a vector, polynucleotide or nucleic acid of the invention can encode heterologous coding regions, either fused or not fused to a polynucleotide that encodes the bispecific antigen-binding molecule that activates T cells (fragment) of the invention, variant or derived from it . Heterologous coding regions include, without limitation, specialized elements or motifs, such as a signal peptide secretory or a heterologous functional domain. A functional association is when a coding region for a gene product, for example, a polypeptide, is associated with one or more regulatory sequences in order to place the expression of the gene product under the influence or control of the regulatory sequence (s) (s). Two fragments of DNA (such as a polypeptide coding region and a promoter associated with it) are "functionally associated" if induction of the promoter function results in the transcription of mRNA that encodes the desired gene product, and if the nature of the link between the two DNA fragments do not interfere with the ability of the expression of regulatory sequences to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed, so that a promoter region would be operationally associated with a nucleic acid encoding a polypeptide if the promoter was able to transcribe that nucleic acid. The promoter can be a specific cell promoter that directs substantial DNA transcription only in predetermined cells. Other elements of transcription control, in addition to a promoter, for example, enhancers, operators, repressors and transcription termination signals, can be operationally associated with poly nucleotide to direct cell-specific transcription. Suitable promoters and other transcription control regions are disclosed in this application. 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, cytomegalovirus promoters and enhancer segments (for example, the immediate initial promoter, in conjunction with A-intron), viruses 40 of simian (for example, the initial promoter) and retroviruses (such as, for example, Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes, such as actin, heat shock protein, bovine growth hormone and rabbit β-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcriptional control regions include tissue-specific promoters and enhancers, as well as inducible promoters (for example, tetracycline-inducible promoters). Similarly, a variety of translation control elements are generally known to those skilled in the art. These include, but are not limited to, ribosome binding sites, translation initiation and termination codons and elements derived from viral systems (especially an internal ribosome entry site or IRES, also referred to as an ISCED sequence). The expression cassette can also include other features such as a source of replication and / or chromosome integration elements, such as retrovirus long terminal repeats (LTRs), or inverted terminal repeats (ITRs) of adeno-associated viruses (AAV).
[0237] [00237] Polynucleotides and nucleic acid coding regions of the present invention can be associated with additional coding regions that encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide of the present invention. For example, if the secretion of the bispecific antigen-binding molecule that activates T cells is desired, DNA encoding a signal sequence can be placed upstream of the nucleic acid that encodes a bispecific antigen-binding molecule that activates T cells of the invention. , or a fragment thereof. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence that is cleaved from the mature protein, once the export of the growing protein chain through the rough endoplasmic reticulum has started. Those skilled in the art are aware that polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the translated polypeptide to produce a secreted or "mature" form of the polypeptide. native signal peptide, for example, an immunoglobulin heavy chain or light chain signal peptide is used, or a functional derivative of that sequence that maintains the ability to direct the secretion of the polypeptide that is functionally associated with it. heterologous mammalian signal or a functional derivative thereof, for example, the wild type leader sequence can be replaced with the leader sequence of human tissue plasminogen activator (TPA) or mouse β glucuronidase. secretory signal peptide polynucleotides are given in SEQ ID NOs 108 to 116.
[0238] [00238] DNA encoding a short protein sequence that could be used to facilitate further purification (eg, histidine marker), or assist in labeling the bispecific antigen-binding molecule that activates T cells can be included within or in ends of the bispecific antigen-binding molecule that activates T cells (fragment) encoding the polynucleotide.
[0239] [00239] In a further embodiment, a host cell is provided that comprises one or more polynucleotides of the invention. In certain embodiments, a host cell is provided that comprises one or more vectors of the invention. Polynucleotides and vectors can incorporate any of the characteristics, alone or in combination, described in the present application in relation to polynucleotides and vectors, respectively. In that embodiment, a host cell (for example, has been transformed or transfected) comprises a vector comprising a polynucleotide that encodes (part of) a bispecific antigen-binding molecule that activates T cells of the invention. As used in the present application, the term "host cell" refers to any type of cellular system that can be genetically engineered to generate the bispecific antigen-binding molecules that activate T cells of the invention or fragments thereof. replicate and to support the expression of bispecific antigen-binding molecules that activate T cells are well known in the art.These cells can be transfected or transduced, as appropriate, with the specific expression vector, and large amounts of cells containing the vector can be cultured to seed large-scale fermenters to obtain sufficient amounts of the bispecific antigen-binding molecule that activates T cells for clinical applications. Suitable host cells include prokaryotic microorganisms, such as E. coli, or various eukaryotic cells, such as hamster ovary cells. Chinese (CHO), insect cells, or the like. For example, po lipeptides can be produced in bacteria, in particular, when glycosylation is not necessary. After expression, the polypeptide can be isolated from the bacterial cell paste in a soluble fraction and can still be purified. In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeasts are suitable cloning or expression hosts for vectors that encode polypeptides, including fungal and yeast strains, whose glycosylation pathways have been "humanized", resulting in the production of a polypeptide with a pattern of glycosylation partially or fully human, see Gerngross, Nat Biotech 22, 1409-1414 (2004), and Li et al., Nat Biotech 24, 210-215 (2006). Host cells suitable for the expression of (glycosylated) polypeptides are also derived from multicellular organisms (invertebrates and vertebrates) Examples of invertebrate cells include plant and insect cells Numerous baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells. plants can also be used as hosts, see, for example, US patents 5.95 9,177, 6,040,498, PLANTIBODIES ™ for antibody production in transgenic plants). Vertebrate cells can also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension can be useful. Other examples of mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney lineage (293 or 293T cells, as described above, for example, in Graham et al., J Gen Virol 36, 59 (1977)), hamster cub kidney (BHK) cells, Sertoli cells from mouse (TM4 cells, as described, for example, in Mather, Biol. Reprod. 23 243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), cells human cervical carcinoma (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT 060562), TRI cells (as described, for example, in Mather et al., Annals NY Acad Sci 383, 44-68 (1982)), MRC 5 cells and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including CHO dhfr - cells (Urlaub et al., Proc Natl Acad Sci USA 77, 4216 (1980)); and myeloma cell lines such as YO, NS0, P3X63 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for protein production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pages 255 -268 (2003). Host cells include cultured cells, for example, cultured mammalian cells, yeast cells, insect cells, bacterial cells and plant cells, to name just a few, but also cells comprised in a transgenic animal, transgenic plant, cultivated plant or animal tissue . In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell such as, for example, Chinese hamster ovary (CHO) cell, human embryonic kidney cell (HEK), or lymphoid cell (for example, Y0 cell, NS0, Sp20).
[0240] [00240] Standard technologies for expressing foreign genes in these systems are known in the art. Cells that express a polypeptide that comprises both the heavy chain and the light chain of an antigen-binding domain such as an antibody, can be genetically engineered to express other chains of the antibody, so that the expressed product is an antibody that has both a heavy chain and a light chain.
[0241] [00241] In one embodiment, a method is provided to produce a bispecific antigen binding molecule that activates T cells according to the invention, wherein the method comprises culturing a host cell that comprises a polynucleotide that encodes the binding molecule bispecific antigen that activates T cells, as provided in the present application, under conditions suitable for expression of the bispecific antigen that activates T cells, and recover the bispecific antigen binding molecule that activates T cells from the host cell (or host cell culture medium).
[0242] [00242] The components of the bispecific antigen-binding molecule that activates T cells are genetically fused to each other. The bispecific antigen-binding molecule that activates T cells can be designed so that their components are fused directly to each other or indirectly through a ligand sequence. The composition and length of the binder can be determined according to methods well known in the art and can be tested for effectiveness. Examples of linker sequences between different components of bispecific antigen-binding molecules that activate T cells are found in the sequences provided in the present application. Additional sequences can also be included to incorporate a cleavage site to separate components individually from the fusion, if desired, for example, an endopeptidase recognition sequence.
[0243] [00243] In certain embodiments, one or more antigen-binding components of the bispecific antigen-binding molecules that activate T cells comprise at least one variable region of the antibody capable of binding an antigenic determinant. Variable regions can form part of, and can be derived from, naturally occurring or non-naturally occurring antibodies and fragments thereof. Methods for producing polyclonal antibodies and monoclonal antibodies are well known in the art (see, for example, Harlow and Lane, "Antibodies, a laboratory manual", Cold Spring Harbor Laboratory, 1988). Antibodies that do not occur naturally can be constructed using of solid phase peptide synthesis, can be produced recombinantly (for example, as described in US patent 4,186,567) or can be obtained, for example, by screening combinatorial libraries comprising variable heavy chains and variable light chains (see , for example, US patent 5,969,108 to McCafferty).
[0244] [00244] Any species of animal antibody, antibody fragment, antigen binding domain or variable region can be used in the bispecific antigen binding molecules that activate T cells of the invention. Antibodies, antibody fragments, antigen binding domains or variable regions useful in the present invention can be of murine, primate or human origin. If the bispecific antigen-binding molecule that activates T cells is intended for human use, a chimeric form of antibody can be used, in which the antibody's constant regions are from a human. A fully human or humanized form of the antibody can also be prepared according to methods well known in the art (see, for example, US patent 5,565,332 to Winter). Humanization can be accomplished by several methods, including, but not limited to, (a) grafting non-human CDRs (eg, donor antibody) into human structure (eg, recipient antibody) and constant regions, with or without retention of important structural residues (for example, those that are important for maintaining good antigen binding affinity or antibody functions), (b) grafts only in non-human specificity determining regions (SDRs or a-CDRs, important residues for antibody-antigen interaction) in human structure and constant regions, or (c) transplantation of entire non-human variable domains, but "camouflaging" them with a human-like section by replacing surface residues. Humanized antibodies and methods for to manufacture them are reviewed, for example, in Almagro and Fransson, Front Biosci 13, 1619-1633 (2008), and are further described, for example, in Riechmann et al., Nature 332, 323-329 (1988) ; Queen et al., Proc Natl Acad Sci USA 86, 10029-10033 (1989); US patents 5,821,337, 7,527,791, 6,982,321 and 7,087,409; Jones et al., Nature 321, 522-525 (1986); Morrison et al., Proc Natl Acad Sci 81, 6851-6855 (1984); Morrison and Oi, Adv Immunol 44, 65-92 (1988); Verhoeyen et al., Science 239, 1534-1536 (1988); Padlan, Molec Immun 31 (3), 169-217 (1994); Kashmiri et al., Methods 36, 25-34 (2005) (which describes SDR graft (a-CDR)); Padlan, Mol Immunol 28, 489-498 (1991) (which describes "resurfacing”); Dall'Acqua et al., Methods 36, 43-60 (2005) (which describes "FR shuffling"); and Osbourn et al., Methods 36, 61-68 (2005) and Klimka et al., Br J Cancer 83, 252-260 (2000) (which describes the "guided selection" approach to RF shuffling). Human antibodies and human variable regions can be produced using several known techniques.Human antibodies are generally described 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 manufactured by the hybridoma method (see, for example, Monoclonal Antibody Production Techniques and Applications, pages 51 to 63 (Marcel Dekker, Inc., New York) , 1987)). Human antibodies and human variable regions can also be prepared by administering an immunogen to a transgenic animal, which has been modified to produce intact human antibodies or intact antibodies to the human variable regions in response to the antigen challenge ( see, for example Lonberg, Nat. Biotech 23, 1117-1125 (2005). Human antibodies and human variable regions can also be generated by isolating sequences from the variable region of the Fv clone selected from human-derived phage display libraries (see, for example, Hoogenboom et al. In Methods in Molecular Biology 178, 1 -37 (O'Brien et al., Ed., Human Press, Totowa, NJ, 2001); and McCafferty et al., Nature 348, 552-554; Clackson et al., Nature 352, 624-628 (1991) ). The phage typically exhibits antibody fragments, either as single chain Fv fragments (scFv) or as Fab fragments.
[0245] [00245] In certain embodiments, the antigen-binding components useful in the present invention are genetically engineered to have improved binding affinity, for example, according to the methods disclosed in US patent application publication 2004/0132066, the contents of which is incorporated into this application as a reference. The ability of the bispecific antigen-binding molecule that activates T cells of the invention to bind to a specific antigenic determinant can be measured either through an enzyme-linked immunosorbent assay (ELISA) or by other techniques familiar to a person skilled in the art, for example , surface plasmon resonance technique (analyzed in a BIACORE T100 system) (Liljeblad, et al., Glyco J 17, 323-329 (2000)), and traditional binding assays (Heeley, Endocr Res 28, 217-229 ( 2002)). Competition assays can be used to identify an antibody, antibody fragment, antigen binding domain or variable domain that competes with a reference antibody for binding to a specific antigen, for example, an antibody that competes with the V9 antibody for binding the CD3. In certain embodiments, this competition antibody binds to the same epitope (for example, a linear or a conformational epitope) that is bound by a reference antibody. Detailed exemplary methods for mapping an epitope to which an antibody binds are provided in Morris (1996) "Epitope Mapping Protocols" in Methods in Molecular Biology volume 66 (Humana Press, Totowa, NJ). In an exemplary competition trial, the immobilized antigen (for example, CD3) is incubated in a solution that comprises a first labeled antibody that binds to the antigen (for example, V9 antibody, described in US patent 6,054,297) and a second unlabeled antibody being tested as to its ability to compete with the first antibody for binding to the antigen. The second antibody may be present in a hybridoma supernatant. As a control, the immobilized antigen is incubated in a solution comprising the first labeled antibody, but not the second antibody After incubation under permissive conditions for binding the first antibody to the antigen, the excess unbound antibody is removed and the amount of marker associated the with the immobilized antigen is measured. If the amount of marker associated with the immobilized antigen is substantially reduced in the test sample compared to the control sample, then this 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, chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).
[0246] [00246] Bispecific antigen binding molecules that activate T cells prepared as described in the present application can be purified by techniques known 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 specific protein will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, etc., and will be evident to those skilled in the art. For purification by affinity chromatography, an antibody, ligand, receptor or antigen can be used, to which the bispecific antigen-binding molecule that activates T cells binds. For example, for purification by affinity chromatography of bispecific antigen-binding molecules that activate T cells of the invention, a matrix with protein A or protein G can be used. Affinity chromatography with protein A or G sequential and size exclusion chromatography can be used to isolate a bispecific antigen-binding molecule that essentially activates T cells as described in the Examples. The purity of the bispecific antigen binding molecule that activates T cells can be determined by any of a variety of known methods, including gel electrophoresis, high pressure liquid chromatography, and the like. For example, the heavy chain fusion proteins expressed as described in the Examples were shown to be intact and properly assembled as demonstrated by reduction SDS-PAGE (see, for example, Figure 4). Three bands were resolved in approximately Mr 25,000, Mr 50,000 and Mr 75,000, corresponding to the predicted molecular weights of the bispecific antigen binding molecule that activates light chain, heavy chain and heavy chain / light chain fusion protein T cells. ESSAY
[0247] [00247] Bispecific antigen-binding molecules that activate T cells provided in the present application can be identified, selected or characterized by their physical / chemical properties and / or biological activities by various assays known in the art. AFFINITY TESTS
[0248] [00248] The affinity of the bispecific antigen-binding molecule that activates T cells to an Fc receptor or target target can be determined according to the methods presented in the Examples by surface plasmon resonance (SPR), using instrumentation standard as a BIAcore instrument (GE Healthcare), and target receptors or proteins can be obtained by recombinant expression. Alternatively, the binding of bispecific antigen-binding molecules that activate T cells to different target receptors or antigens can be assessed using cell lines that express the specific receptor or target antigen, for example, by flow cytometry (FACS ). A specific and exemplary illustrative embodiment for measuring binding affinity is described below and in the Examples below.
[0249] [00249] According to one embodiment, KD is measured by surface plasmon resonance using a BIACORE® T100 machine (GE Healthcare) at 25 ° C.
[0250] [00250] To analyze the interaction between the Fc portion and Fc receptors, the His-tagged recombinant Fc receptor is captured by an anti-Penta His (Qiagen) antibody immobilized on CM5 chips and the bispecific constructs are used as an analyte. Briefly, carboxymethylated dextran (CM5, GE, Inc.) biosensor chips are activated with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), according to the instructions in the provider. The anti-Penta-His antibody is diluted with 10 mM sodium acetate, pH 5.0, at 40 μg / mL prior to injection at a flow rate of 5 μL / min to obtain approximately 6500 response units (RU) of coupled protein. After injecting the binder, 1M ethanolamine is injected to block unreacted groups. Subsequently, the Fc receptor is captured for 60 s at 4 or 10 nM. For kinetic measurements, four-fold serial dilutions of the bispecific construct (range between 500 nM and 4000 nM) are injected into HBS-EP (GE Healthcare, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% P20 surfactant, pH 7.4) at 25 ° C, at a flow rate of 30 μL / min for 120 s.
[0251] [00251] To determine affinity for the target antigen, bispecific constructs are captured by a specific human anti-Fab antibody (GE Healthcare) that is immobilized on an activated CM5 sensor chip surface, as described for the Penta-His antibody. The final amount of coupled protein is approximately 12,000 RU The bispecific constructs are captured for 90 seconds at 300 nM. The target antigens are passed through the flow cells for 180 s at a concentration range of 250 to 1,000 nm, with a flow rate of 30 μL / min. Dissociation is monitored for 180 s.
[0252] [00252] Differences in the mass refractive index are corrected by subtracting the response obtained in the reference flow cell. The steady-state response was used to obtain the KD dissociation constant by adjusting the nonlinear curve of the Langmuir binding isotherm. The association rates (kon) and dissociation rates (koff) are calculated using a Langmuir one-to-one connection model (BIACORE® T100 Evaluation Computer Program) by simultaneously adjusting the association and dissociation sensors. The equilibrium of the dissociation constant (Kd) is calculated as the koff / kon ratio. See, for example, Chen et al., J Mol Biol 293, 865-881 (1999). ACTIVITY TESTS
[0253] [00253] The biological activity of bispecific antigen-binding molecules that activate T cells of the invention can be measured by various assays, as described in the Examples. Biological activities may, for example, include the induction of T cell proliferation, the induction of T cell signaling, the induction of the expression of T cell activation markers, the induction of cytokine secretion by T cells, the induction of lysis of target cells as tumor cells, and the induction of tumor regression and / or improvement of survival. COMPOSITIONS, FORMULATIONS AND ROUTES OF ADMINISTRATION
[0254] [00254] In a further aspect, the invention provides pharmaceutical compositions comprising any of the bispecific antigen binding molecules that activate T cells provided in the present application, for example, for use in any of the therapeutic methods below. In one embodiment, a pharmaceutical composition comprises any of the bispecific antigen-binding molecules that activate T cells provided in the present application and a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical composition comprises any of the bispecific antigen-binding molecules that activate T cells provided in the present application and at least one additional therapeutic agent, for example, as described below.
[0255] [00255] A method is also provided in the present application to produce a bispecific antigen-binding molecule that activates T cells of the invention in a form suitable for administration in vivo, the method of which comprises (a) obtaining a bispecific antigen-binding molecule that activates T cells according to the invention, and (b) formulating the bispecific antigen binding molecule that activates T cells with at least one pharmaceutically acceptable carrier, whereby a bispecific antigen binding molecule that activates T cells is formulated for in vivo administration.
[0256] [00256] Pharmaceutical compositions of the present invention comprise a therapeutically effective amount of one or more of the bispecific antigen binding molecules that activate dissolved or dispersed T cells in a pharmaceutically acceptable carrier. The phrases "pharmaceutically or pharmacologically acceptable" refer to molecular entities and compositions that are generally non-toxic to receptors at the dosages and concentrations used, that is, they do not produce an adverse, allergic or unpleasant reaction when administered to an animal, such as example, a human, as appropriate. The preparation of a pharmaceutical composition containing at least one bispecific antigen-binding molecule that activates T cells and, optionally, an additional active ingredient will be known to those skilled in the art, in consideration of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated into this application as a reference. In addition, for administration to animals (e.g., human), it will be understood that preparations must meet sterility standards, general safety and purity pyrogenicity as required by the FDA Office of Biological Standards or authority corresponding accessions in other countries. Preferred compositions are lyophilized formulations or aqueous solutions. As used in the present application, "pharmaceutically acceptable carrier" includes any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delay agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrating agents, lubricants, sweetening agents, flavoring agents, dyes, as materials and combinations thereof, as would be known to a person skilled in the art (see, for example , Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pages 1289-1329, incorporated herein by reference.) Except to the extent that any conventional vehicle is incompatible with the active component, its use in therapeutic or pharmaceutical compositions is contemplated.
[0257] [00257] The composition can comprise different types of vehicles, depending on whether it will be administered in solid, liquid or aerosol form and whether it needs to be sterile by these routes of administration, such as injection. Bispecific antigen-binding molecules that activate T cells of the present invention (and any additional therapeutic agent) can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrasplenically, intrarenally, intrapleurally. , intratracheal, intranasal, intravitreal, intravaginal, rectal, topical, intratumoral, intramuscular, intraperitoneal, subcutaneous, subconjunctival, intravesicular, mucosal, intrapericardial, intraumbilical, intraocular, oral, topical, locally, by inhalation (for example, aerosol inhalation ), injection, infusion, continuous infusion, localized infusion that bathes target cells directly, through a catheter, through a wash, in creams, in lipid compositions (for example, liposomes), or by another method or any combination of the background as would generally be known to a person skilled in the art (see, for example, Remington's Sciences, 18th Ed. Mack Printing Company, 1990, incorporated by reference in this application). Parenteral administration by specific intravenous injection is most commonly used to administer polypeptide molecules as the bispecific antigen-binding molecules that activate T cells of the invention.
[0258] [00258] Parenteral compositions include those designed for administration by injection, for example, subcutaneous, intradermal, intralesional, intravenous, intraarterial, intramuscular, intrathecal or intraperitoneal injection. For injection, the bispecific antigen-binding molecules that activate T cells of the invention can 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 formulating agents such as suspension, stabilization and / or dispersing agents. Alternatively, the bispecific antigen-binding molecules that activate T cells may be in powder form for constitution with a suitable vehicle, for example, sterile pyrogenic water, before use. Sterile injectable solutions are prepared by incorporating the bispecific antigen-binding molecules that activate T cells of the invention in the required amount in the appropriate solvent with various other ingredients listed below, as needed. Sterility can be easily accomplished, for example, by filtration through sterile filtration membranes. Generally, dispersions are prepared by incorporating various sterile active ingredients into a sterile vehicle that 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 emulsions, the preferred methods of preparation are vacuum drying or lyophilization techniques that produce an active ingredient powder plus any additional desired ingredient from a previously liquid medium. sterilized-filtered of the same. The liquid medium must be adequately buffered, if necessary, and the first isotonic liquid diluent processed before injection with sufficient saline or glucose. The composition must be stable in the conditions of manufacture and storage and preserved against the action of contamination by microorganisms, such as bacteria and fungi. It is estimated that endotoxin contamination should be minimally maintained at a safe level, for example, less than 0.5 ng / mg of protein. Suitable pharmaceutically acceptable vehicles 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, it alcylates parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol) ; low molecular weight polypeptides (less than about 10 residues); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers like polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugars like sucrose, mannitol, trehalose or sorbitol; formation of counter ions salts such as sodium; metal complexes (for example, Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Aqueous injection suspensions may contain compounds that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, dextran or the like. Optionally, the suspension can also contain suitable stabilizers or agents that increase the solubility of the compounds to allow the preparation of highly concentrated solutions. In addition, suspensions of the active compounds can be prepared as appropriate oil injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or esters of synthetic fatty acids, such as ethyl acetate or triglycerides, or liposomes.
[0259] [00259] The active ingredients can be retained in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly- (methylmethacrylate) microcapsules, respectively, in drug delivery systems colloidal (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. These techniques are disclosed in Remington’s Pharmaceutical Sciences (18th Ed. Mack Printing Company, 1990). Sustained release preparations can be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, whose matrices are in the form of molded articles, for example, films or microcapsules. In specific embodiments, the prolonged absorption of an injectable composition can be caused by the use of compositions of delaying absorption agents such as, for example, aluminum monostearate, gelatin or combinations thereof.
[0260] [00260] In addition to the compositions described above, bispecific antigen-binding molecules that activate T cells can also be formulated as a depot preparation. These long-acting formulations can be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, bispecific antigen-binding molecules that activate T cells can be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins or as poorly soluble derivatives, for example example, as a sparingly soluble salt.
[0261] [00261] Pharmaceutical compositions comprising the bispecific antigen binding molecules that activate T cells of the invention can be manufactured by means of conventional mixing, dissolving, emulsifying, encapsulating, inserting or lyophilizing processes. Pharmaceutical compositions can be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries that facilitate the processing of proteins in preparations that can be used pharmaceutically. The appropriate formulation is dependent on the chosen route of administration.
[0262] [00262] Bispecific antigen-binding molecules that activate T cells can be formulated into a composition with a neutral or salt-free acid or base form. Pharmaceutically acceptable salts are salts that maintain the biological activity of the acid or free base. These include addition salts, for example, those formed with the free amino groups of a protein composition, or that are formed by inorganic acids such as, for example, hydrochloric or phosphoric acids or as organic acids such as acetic, oxalic, tartaric or mandelic. Salts formed with the carboxyl-free groups can also be derived from inorganic bases, such as, for example, sodium, potassium, ammonium, calcium or iron hydroxide; or organic bases like isopropylamine, trimethylamine, histidine or procaine. Pharmaceutical salts tend to be more soluble in aqueous and other protic solvents than are the corresponding free base forms. THERAPEUTIC METHODS AND COMPOSITIONS
[0263] [00263] Any of the bispecific antigen-binding molecules that activate T cells provided in the present application can be used in therapeutic methods. Bispecific antigen-binding molecules that activate T cells of the invention can be used as immunotherapeutic agents, for example, in the treatment of cancer.
[0264] [00264] For use in therapeutic methods, the bispecific antigen-binding molecules that activate T cells of the invention would be formulated, dosed and administered in a manner consistent with good medical practice. Factors for consideration in this context include the specific dysfunction to be treated, the specific mammal to be treated, the clinical condition of each patient, the cause of the dysfunction, the location of distribution of the agent, the method of administration, the administration schedule and other factors known to medical specialists.
[0265] [00265] In one aspect, bispecific antigen-binding molecules are provided that activate T cells of the invention for use as a medicine. In additional aspects, bispecific antigen-binding molecules that activate T cells of the invention are provided for use in the treatment of a disease. In certain embodiments, bispecific antigen-binding molecules are provided that activate T cells of the invention for use in a treatment method. In one embodiment, the invention provides a bispecific antigen-binding molecule that activates T cells as described in the present application for use in treating a disease in an individual in need thereof. In certain embodiments, the invention provides a bispecific antigen-binding molecule that activates T cells for use in a method to treat an individual who has a disease, which comprises administering to the individual a therapeutically effective amount of the bispecific antigen-binding molecule that activates T cells. In certain embodiments, the disease to be treated is a proliferative disorder. In a specific embodiment, the disease is cancer. In certain embodiments, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, for example, an anti-cancer agent, if the disease to be treated is cancer. In further embodiments, the invention provides a bispecific antigen-binding molecule that activates T cells, as described in the present application, for use in inducing lysis of a target cell, particularly a tumor cell. In certain embodiments, the invention provides a bispecific antigen-binding molecule that activates T cells for use in a method to induce lysis of a target cell, particularly a tumor cell, in an individual comprising administering to the individual an effective amount of the molecule of binding to the bispecific antigen that activates T cells to induce lysis of a target cell. An "individual", according to any of the above embodiments, is a mammal, preferably a human.
[0266] [00266] In a further aspect, the invention provides the use of a bispecific antigen-binding molecule that activates T cells of the invention in the manufacture or preparation of a medicament. In one embodiment, the drug is for the treatment of a disease in an individual in need of it. In a further embodiment, the drug is for use in a method for treating a disease which comprises administering to a subject who has the disease, a therapeutically effective amount of the drug. In certain embodiments, the disease to be treated is a proliferative disorder. In a specific embodiment, the disease is cancer. In one embodiment, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, for example, an anti-cancer agent, if the disease to be treated is cancer. In a further embodiment, the drug is to induce lysis of a target cell, particularly a tumor cell. In a still further embodiment, the drug is for use in a method of inducing lysis of a target cell, particularly a tumor cell in an individual, which comprises administering to the individual an effective amount of the drug to induce lysis of a target cell. An "individual", according to any of the above embodiments, can be a mammal, preferably a human.
[0267] [00267] In a further aspect, the invention provides a method for treating a disease. In one embodiment, the method comprises administering to a subject who has this disease a therapeutically effective amount of a bispecific antigen-binding molecule that activates T cells of the invention. In one embodiment, a composition is administered to said individual, which comprises the bispecific antigen binding molecule that activates T cells of the invention in a pharmaceutically acceptable form. In certain embodiments, the disease to be treated is a proliferative disorder. In a specific embodiment, the disease is cancer. In certain embodiments, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, for example, an anti-cancer agent, if the disease to be treated is cancer. An "individual", according to any of the above embodiments, can be a mammal, preferably a human.
[0268] [00268] In a further aspect, the invention provides a method for inducing lysis of a target cell, particularly a tumor cell. In one embodiment, the method comprises placing a target cell in contact with a bispecific antigen-binding molecule that activates T cells of the invention in the presence of a T cell, particularly a cytotoxic T cell. In a further aspect, a method is provided to induce lysis of a target cell, particularly a tumor cell in an individual. In this embodiment, the method comprises administering to the individual an effective amount of a bispecific antigen-binding molecule that activates T cells to induce lysis of a target cell. In one embodiment, an "individual" is a human.
[0269] [00269] In certain embodiments, the disease to be treated is a proliferative dysfunction, particularly cancer. Non-limiting examples of 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, cancer colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer and kidney cancer. Other cell proliferation disorders that can be treated with the use of a bispecific antigen-binding molecule that activates T cells of the present invention include, but are not limited to, neoplasms located in: abdomen, bone, sinus, digestive system, liver, pancreas , peritoneum, endocrine glands (adrenal, parathyroid, pituitary gland, testicles, ovary, thymus, thyroid), eyes, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissues, spleen, thoracic region and urogenital system. Also included are precancerous conditions or injuries and cancer metastasis. In certain embodiments, cancer is chosen from the group consisting of kidney cell cancer, skin cancer, lung cancer, colorectal cancer, breast cancer, brain, head and neck cancer. One skilled in the art readily recognizes that in many cases the bispecific antigen-binding molecule that activates T cells may not provide a cure, but may only provide partial benefit. In some embodiments, a physiological change that has some benefit is also considered to be therapeutically beneficial. Thus, in some embodiments, an amount of bispecific antigen-binding molecule that activates T cells 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.
[0270] [00270] In some embodiments, an effective amount of a bispecific antigen-binding molecule that activates T cells of the invention is administered to a cell. In other embodiments, a therapeutically effective amount of a bispecific antigen-binding molecule that activates T cells of the invention is administered to an individual for the treatment of the disease.
[0271] [00271] For disease prevention or treatment, the appropriate dosage of a bispecific antigen-binding molecule that activates T cells of the invention (when used alone or in combination with one or more additional therapeutic agents) will depend on the type of disease to be treated, the route of administration, the type of bispecific antigen binding molecule that activates T cells, the severity and course of the disease, whether the bispecific antigen binding molecule that activates T cells is administered for preventive or therapeutic purposes, therapeutic interventions previous or competing, the patient's medical history and the response to the bispecific antigen-binding molecule that activates T cells, and the physician's diagnosis. The administering physician will, in any case, determine the concentration of active ingredient (s) in a composition and dose (s) appropriate for the individual patient. Various dosage schedules including, but not limited to, single or multiple administrations at various points in time, bolus administration and pulse infusion are contemplated in the present application.
[0272] [00272] The bispecific antigen-binding molecule that activates T cells is properly administered to the patient from time to time or over a series of treatments. Depending on the type and severity of the disease, about 1 μg / kg to 15 mg / kg (eg 0.1 mg / kg to 10 mg / kg) of bispecific antigen-binding molecule that activates T cells may be a initial candidate dosage to administer to the patient, for example, either by one or more separate administrations or by continuous infusion. A typical daily dosage can range from about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, treatment would generally be continued until a desired suppression of the symptoms of the disease occurs. An exemplary dosage of the bispecific antigen-binding molecule that activates T cells would be in the range of about 0.005 mg / kg to about 10 mg / kg. In other non-limiting examples, a dose can also comprise about 1 microgram / kg of body weight, about 5 micrograms / kg of body weight, about 10 micrograms / kg of body weight, about 50 micrograms / kg of body weight , about 100 micrograms / kg body weight, about 200 micrograms / kg body weight, about 350 micrograms / kg body weight, about 500 micrograms / kg body weight, about 1 milligram / kg body weight , about 5 milligrams / kg of body weight, about 10 milligrams / kg of body weight, about 50 milligrams / kg of body weight, about 100 milligrams / kg of body weight, about 200 milligrams / kg of body weight , about 350 milligrams / kg of body weight, about 500 milligrams / kg of body weight to about 1000 mg / kg of body weight or more per administration and any derivable range therein. In non-limiting examples of a range derivable from the numbers listed in the present application, a range of about 5 mg / kg body weight to about 100 mg / kg body weight, about 5 micrograms / kg body weight to about 500 milligrams / kg of body weight, etc., can be administered based on the numbers described above. In this way, 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) can be administered to the patient. These doses can be administered intermittently, for example, every week or every three weeks (for example, so that the patient receives from two to about twenty or, for example, about six doses of the bispecific antigen binding molecule. that activates T cells). A higher initial loading dose may be administered, followed by one or more lower doses. However, other dosage regimens can be useful. The progress of this therapy is easily monitored by conventional techniques and trials.
[0273] [00273] Bispecific antigen-binding molecules that activate T cells of the invention will generally be used in an effective amount to achieve the intended objective. For use to treat or prevent a disease condition, the bispecific antigen-binding molecules that activate T cells of the invention or pharmaceutical compositions thereof, are administered or applied in a therapeutically effective amount. The determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in view of the detailed disclosure provided in the present application.
[0274] [00274] For systemic administration, a therapeutically effective dose can be estimated initially from in vitro assays, such as cell culture assays. A dose can then be formulated in animal models to obtain a circulating concentration range that includes the IC 50, as determined in cell culture. This information can be used to more accurately determine useful doses in humans.
[0275] [00275] Initial doses can also be estimated from in vivo data, for example, animal models, using techniques that are well known. Generally, a person skilled in the art could easily optimize administration for humans based on animal data.
[0276] [00276] The amount and dosage range can be individually adjusted to provide the plasma levels of the bispecific antigen-binding molecules that activate T cells that are sufficient to maintain the therapeutic effect. Usual dosages for patients for administration by injection in the range of about 0.1 to 50 mg / kg / day, typically about 0.5 to 1 mg / kg / day. Therapeutically effective plasma levels can be achieved by administering multiple doses each day. Plasma levels can be measured, for example, by HPLC.
[0277] [00277] In cases of local administration or selective absorption, the effective local concentration of bispecific antigen-binding molecules that activate T cells cannot be related to plasma concentration. A person skilled in the art will be able to optimize therapeutically effective local dosages without undue experimentation.
[0278] [00278] A therapeutically effective dose of the bispecific antigen binding molecule that activates T cells described in the present application will generally provide therapeutic benefits without causing substantial toxicity. The toxicity and therapeutic efficacy of a bispecific antigen-binding molecule that activates T cells can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. Cell culture assays and animal studies can be used to determine LD50 (the lethal dose for 50% of a population) and ED50 (the therapeutically effective dose in 50% of a population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the LD50 / ED50 ratio. Bispecific antigen-binding molecules that activate T cells that exhibit large therapeutic indicators are preferred. In one embodiment, the bispecific antigen-binding molecule that activates T cells according to the present invention exhibits a high therapeutic index. The data obtained from cell culture assays and animal studies can be used to formulate a range of dosages suitable for use in humans. The dosage is preferably within a range of circulating concentrations that includes ED50 with little or no toxicity. The dosage can vary within this range depending on a variety of factors, for example, the dosage form employed, the route of administration used, the condition of the subject, and the like. The exact formulation, route of administration and dosage can be chosen by each physician in view of the patient's condition (see, for example, Fingl et al., 1975, in: The Pharmacological Basis of Therapeutics, Chapter 1, page 1, fully incorporated this application as a reference).
[0279] [00279] The physician for patients treated with bispecific antigen-binding molecules that activate T cells of the invention will know how and when to terminate, interrupt or adjust administration due to toxicity, organ dysfunction, and the like. Conversely, the doctor will also know how to adjust treatment to higher levels, if the clinical response is not adequate (preventing toxicity). The magnitude of a dose administered in the management of the dysfunction of interest will vary with the severity of the condition being treated, the route of administration, and the like. The severity of the condition can, for example, be assessed, in part, by standard methods of assessing prognosis. In addition, the dose and perhaps the frequency of the dose will also vary according to the age, body weight and response of the individual patient. OTHER AGENTS AND TREATMENTS
[0280] The bispecific antigen-binding molecules that activate T cells of the invention can be administered in combination with one or more other agents in therapy. For example, a bispecific antigen-binding molecule that activates T cells of the invention can be co-administered with at least one additional therapeutic agent. The term "therapeutic agent" includes any agent administered to treat a symptom or disease in an individual in need of such treatment. This additional therapeutic agent may comprise any active ingredients suitable for the specific indication to be treated, preferably those with complementary activities that are not affect each other contrary. In certain embodiments, an additional therapeutic agent is an immunomodulatory agent, a cytostatic agent, a cell adhesion inhibitor, a cytotoxic agent, a cell apoptosis activator, or an agent that increases the sensitivity of cells to inducers In a specific embodiment, the additional therapeutic agent is an anticancer agent, for example, a microtubule disruptor, an antimetabolite, a topoisomerase inhibitor, a DNA interleaver, an alkylating agent, a hormone therapy, a kinase inhibitor, a receptor antagonist, an activator of tumor cell apoptosis s or an antiangiogenic agent.
[0281] [00281] These other agents are suitably present in combination in amounts that are effective for the desired purpose. The effective amount of these other agents depends on the amount of bispecific antigen-binding molecule that activates T cells used, the type of dysfunction or treatment and the other factors discussed above. Bispecific antigen-binding molecules that activate T cells are generally used in the same dosage and with administration routes as described in this application, or about 1 to 99% of the dosages described in this application, or any dosage and by any route that be empirically / clinically determined to be appropriate.
[0282] [00282] These combination therapies noted above encompass combined administration (when two or more therapeutic agents are included in the same composition or separate), and separate administration, in this case, administration of the bispecific antigen-binding molecule that activates T cells of the invention can occur before, simultaneously and / or after the administration of the additional therapeutic agent and / or adjuvant. Bispecific antigen-binding molecules that activate T cells can also be used in combination with radiation therapy. MANUFACTURING ITEMS
[0283] [00283] In another aspect of the invention, an article of manufacture is provided containing materials useful for the treatment, prevention and / or diagnosis of the disorders described above. The article of manufacture comprises a container and a label, or package insert associated with the container. Suitable containers include, for example, bottles, vials, syringes, bags for IV solution, etc. Containers can be manufactured from a variety of materials, such as glass or plastic. The container contains a composition that 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 bottle that has a pierceable cap for a hypodermic injection needle). At least one active agent in the composition is a bispecific antigen-binding molecule that activates T cells of the invention. The label or package insert indicates that the composition is used to treat the recommended condition. In addition, the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises a bispecific antigen-binding molecule that activates T cells of the invention; and (b) a second container with a composition contained therein, wherein the composition comprises an additional cytotoxic agent or otherwise a therapeutic agent. The article of manufacture in that embodiment of the invention may, furthermore, comprise a package insert indicating that the compositions can be used to treat a specific condition. Alternatively or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), buffered saline phosphate, Ringer's solution and dextrose solution. In addition, it may include other materials desirable from a commercial and user perspective, including other buffers, thinners, filters, needles and syringes. EXAMPLES
[0284] [00284] The following are examples of methods and compositions of the invention. It is understood that several other achievements can be practiced, given the general description provided above. GENERAL METHODS RECOMBINANT DNA TECHNIQUES
[0285] [00285] Standard methods were used to manipulate DNA, as described in Sambrook, J., et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biological reagents were used according to manufacturers' instructions. General information related to human immunoglobulin light and heavy chain nucleotide sequences is provided in: Kabat, E.A. et al., (1991) Sequences of Proteins of Immunological Interest, 5th edition, NIH publication No. 91-3242. DNA SEQUENCING
[0286] [00286] DNA sequences were determined by double stranded sequencing. GENE SYNTHESIS
[0287] [00287] The desired gene segments, when necessary, were generated both by PCR using appropriate molds and 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 the sequences of closest homologs and the genes were isolated by RT-PCR from RNA originating from the appropriate tissue. Gene segments flanked by unique restriction endonuclease cleavage sites were cloned into standard cloning vectors or sequencing vectors. The DNA plasmid was purified from transformed bacteria and the concentration determined by UV spectroscopy. The DNA sequence of the subcloned gene fragments was confirmed by DNA sequencing. The gene segments were designed with suitable restriction sites to allow sub-cloning in the respective expression vectors. All constructs were designed with a 5 'end of DNA sequence coding for a leader peptide that directs protein secretion in eukaryotic cells. Leading peptides and exemplary polynucleotide sequences encoding them are represented in SEQ ID NOs: 108 to 116. ISOLATION OF HUMAN PRIMARY PAN T CELLS FROM PBMCS
[0288] [00288] Peripheral blood mononuclear cells (PBMCs) were prepared by Histopaque density centrifugation from enriched lymphocyte preparations (buffy coat) obtained from local blood banks or fresh blood from healthy human donors. Briefly, the blood was diluted with sterile PBS and carefully layered over a Histopaque gradient (Sigma, H8889). After centrifugation for 30 minutes at 450 x g at room temperature (brake off), part of the plasma above the PBMC containing the interphase was discarded. PBMCs were transferred to new 50 ml Falcon tubes that were supplemented with PBS for a total volume of 50 ml. The mixture was centrifuged at room temperature for 10 minutes at 400 x g (brake on). The supernatant was discarded and the PBMC pellet washed twice with sterile PBS (centrifugation steps at 4 ° C for 10 minutes at 350 x g). The resulting PBMC population was counted automatically (ViCell) and stored in RPMI1640 medium, containing 10% FCS and 1% L-alanyl-L-glutamine (Biochrom, K0302) at 37 ° C, 5% CO2 in the incubator until the beginning of the test.
[0289] [00289] The enrichment of T cells from PBMCs was performed using Kit II Pan T Cell Isolation (Miltenyi Biotec n ° 130091-156), according to the manufacturer's instructions. Briefly, cell pellets were diluted in 40 μL of cold buffer per 10 million cells (PBS with 0.5% BSA, 2 mM EDTA, sterile filtrate) and incubated with 10 μL of Biotin-Antibody Cocktail for 10 million cells for 10 minutes at 4 ° C. 30 μL of cold buffer and 20 μL of Anti-Biotin magnetic microspheres per 10 million cells were added, and the mixture was incubated for another 15 minutes at 4 ° C. The cells were washed by adding 10 to 20x the volume of buffer and a subsequent centrifugation step at 300 x g for 10 minutes. More than 100 million cells were resuspended in 500 μL of buffer. Magnetic separation of unmarked human pan T cells was performed using LS columns (Miltenyi Biotec n ° 130-042-401) according to the manufacturer's instructions. The resulting T cell population was counted automatically (ViCell) and stored in AIM-V medium at 37 ° C, 5% CO2 in the incubator until the start of the test (no more than 24 hours). ISOLATION OF PRIMARY HUMAN VIRGIN T CELLS FROM PBMCS
[0290] [00290] Peripheral blood mononuclear cells (PBMCs) were prepared by Histopaque density centrifugation from enriched lymphocyte preparations (buffy coat layers) obtained from local blood banks or fresh blood from healthy human donors. T cell enrichment from PBMCs was performed using the CD8 + Virgin T cell isolation kit obtained from Miltenyi Biotec (n ° 130-093-244), according to the manufacturer's instructions, but skipping the last one isolation step of CD8 + T cells (see also description for isolation of primary human pan T cells). ISOLATION OF PAN T MURIN CELLS FROM SPLENOCYTES
[0291] [00291] The spleens were isolated from C57BL / 6 mice, transferred to a GentleMACS C tube (Miltenyi Biotech n ° 130-093-237) containing MACS buffer (PBS + 0.5% BSA + 2 mM EDTA) and dissociated with GentleMACS dissociator to obtain a single cell suspension according to the manufacturer's instructions. The cell suspension was passed through a pre-separation filter to remove the remaining particles from the undissociated tissue. After centrifugation at 400 x g for 4 minutes at 4 ° C, the ACK lysis buffer was added to lyse red blood cells (incubation for 5 minutes at room temperature). The remaining cells were washed twice with MACS buffer, counted and used for the isolation of murine pan T cells. The negative (magnetic) selection was performed using the Pan T Cell Isolation Kit obtained from Miltenyi Biotec (n ° 130-090-861), following the manufacturer's instructions. The resulting T cell population was counted automatically (ViCell) and immediately used for further assays. ISOLATION OF PRIMARY CYNOMOLGUS PBMCS FROM HEPARINIZED BLOOD
[0292] [00292] Peripheral blood mononuclear cells (PBMCs) were prepared by density centrifugation from fresh blood from cynomolgus donors, as follows: Heparinized blood was diluted 1: 3 with sterile PBS and Lymphoprep medium (Axon Lab n ° 1114545) was diluted to 90% with sterile PBS. Two volumes of the diluted blood were layered over a density gradient volume and the PBMC fraction was separated by centrifugation for 30 minutes at 520 x g, without brake, at room temperature. The PBMC band was transferred to a new 50 mL Falcon tube and washed with sterile PBS by centrifugation for 10 minutes at 400 x g at 4 ° C. A low speed centrifugation was performed to remove the platelets (15 minutes at 150 x g, 4 ° C), and the resulting PBMC population was counted automatically (ViCell) and immediately used for further testing. TARGET CELLS
[0293] [00293] For the evaluation of bispecific antigen-binding molecules that target MCSP, the following tumor cell lines were used: the human melanoma cell line WM266-4 (ATCC No. CRL-1676), derived from a metastatic site malignant melanoma and expressing high levels of human MCSP; the human melanoma cell line MV-3 (kindly provided by The Radboud University Nijmegen Medical Center), which expresses average levels of human MCSP; the human malignant melanoma cell line (primary tumor) A375 (ECACC No. 88113005) expressing high levels of MCSP; the human colon carcinoma cell line HCT-116 (ATCC No. CCL-247) that does not express MCSP; and the Caucasian human colon adenocarcinoma cell line LS180 (ECACC No. 87021202) that does not express MCSP.
[0294] [00294] For the evaluation of bispecific antigen-binding molecules that target CEA, the following tumor cell lines were used: the MKN45 human gastric tumor cell line (DSMZ No. ACC 409), which expresses high levels of human CEA ; the HPAF-II human pancreatic adenocarcinoma cell line (kindly provided by Roche Nutley), which expresses high levels of human CEA; the primary human pancreatic adenocarcinoma cell line BxPC-3 (ECACC No. 93120816) which expresses average levels of human CEA; the cell line of Caucasian females of human colon adenocarcinoma LS-174T (ECACC n ° 87060401), expressing average levels of human CEA; the ASPC-1 human pancreatic adenocarcinoma cell line (ECACC No. 96020930) expressing low levels of human CEA; the Panc-1 human pancreatic epithelioid carcinoma cell line (ATCC No. CRL-1469), expressing (very) low levels of human CEA; the human colon carcinoma cell line HCT-116 (ATCC No. CCL-247) that does not express CEA; the A549-huCEA human basal alveolar epithelial adenocarcionoma cell line, which has been stably transfected internally (in-house) to express human CEA; and the MC38-huCEA murine colon carcinoma cell line, which was designed internally (in-house) to stably express human CEA.
[0295] [00295] In addition, a human T-cell leukemia cell line, Jurkat (ATCC No. TIB-152), was used to assess the binding of different bispecific constructs to human CD3 in the cells. EXAMPLE 1 ANTI-MCSP M4-3 / ML2 ANTIBODY AFFINITY MATURATION
[0296] [00296] Affinity maturation was performed through the oligonucleotide-directed mutagenesis procedure. For this, the M4-3 variant of the heavy chain, and the ML2 variant of the light chain were cloned into a phage vector, similar to those described by Hoogenboom, (Hoogenboom et al., Nucleic Acids Res. 1991, 19, 4133-4137 ). The residues to be randomized were identified by the first generation of a 3D model of that antibody through classical homology modeling and, then, identification of accessible solvent residues from the complementarity determining regions (CDRs) of the heavy and light chain. Oligonucleotides with randomization based on trinucleotide synthesis as shown in Table 1 were purchased from Ella Biotech (Munich, Germany). Three independent sub-libraries were generated using classical PCR, and comprise randomization in CDR-H1 together with CDR-H2, or CDR-L1 together with CDR-L2. CDR-L3 was randomized in a separate approach. The DNA fragments from these libraries were cloned into the phagemid through restriction digestion and ligation, and subsequently electroporated in TG1 bacteria. LIBRARY SELECTION
[0297] The antibody variants thus generated were displayed monovalently from filamentous phage particles as fusions with the M13 gene III product packaged within each particle. The phage-displayed variants were then screened for their biological activities (in this application: binding affinity) and candidates who have one or more improved activities were used for further development. Methods for preparing phage display libraries can be found in Lee et al., J. Mol. Biol. (2004) 340, 1073-1093.
[0298] 1. ligação de aproximadamente 1.012 partículas de fagomídeo de cada biblioteca de maturação de afinidade para hu-MCSP(domínio D3)-avi-his biotinilado 100 nM (SEQ ID NO: 118) por 0,5 hora em um volume total de1 mL, 2. captura de hu-MCSP(domínio D3)-avi-his biotinilado e partículas de fago ligadas especificamente por adição de 5,4 χ 107 micro esferas magnéticas revestidas com estreptavidina por 10 minutos, 3. lavagem de micro esferas com o uso de 1 mL de PBS/Tween-20 5 a 10x e 1 mL de PBS 5 10x, 4. eluição de partículas de fago por adição de 1 mL de TEA (trietilamina) 100 mM por 10 minutos e neutralização por adição de 500 μL de Tris/HCl 1M, pH 7,4 e 5. reinfecção da bactéria E. coli TG1 com crescimento exponencial, a infecção com o fago auxiliar VCSM13 e subsequente precipitação com PEG/NaCl das partículas de fagomídeo a serem usadas em rodadas de seleção subsequentes. As seleções foram realizadas ao longo de 3 a 5 rodadas com o uso de concentrações de antígeno tanto constantes como diminuindo (a partir de 10-7 M para 2 x 10-9 M). Na rodada dois, a captura de complexos antígeno-fago foi realizada com o uso de placas de neutravidina ao invés de microesferas de estreptavidina. Os ligantes específicos foram identificados por ELISA da seguinte forma: 100 μL de hu-MCSP(domínio D3)-avi-his biotinilado 10 nM por poço foram revestidos em placas de neutravidina. Os sobrenadantes contendo Fab bacteriano foram adicionados e as ligações Fabs foram detectadas através de seus marcadores Flag com o uso de anticorpo secundário anti-Flag/HRP. Os clones positivos por ELISA foram expressos em bactérias como fragmentos solúveis de Fab em formato de 96 poços e os sobrenadantes foram sujeitos a um experimento de triagem cinética por análise SPR com o uso de ProteOn XPR36 (BioRad). Os clones que expressam Fabs com as maiores constantes de afinidade foram identificados e os fagomídeos correspondentes foram sequenciados.
[0299] [00299] Figure 2 shows the alignment of the affinity matured anti-MCSP clones compared to the unripened parental clone (M4-3 mL2). Heavy chain randomization was performed only on CDR1 and 2. Light chain randomization was performed on CDR1 and 2, and independent on CDR3.
[0300] [00300] During the selection, some mutations in the structures occurred as F71Y in clone G3 or Y87H in clone E10. PRODUCTION AND PURIFICATION OF HUMAN IGG1
[0301] [00301] The variable regions of the DNA sequences of the heavy and light chain of the matured affinity variants were subcloned in the frame with both the constant heavy chain and the constant light chain pre-inserted in the respective recipient mammalian expression vector. The antibody expression was driven by an MPSV promoter and transports to a synthetic polyA signal sequence at the 3 'end of CDS. In addition, each vector contained an EBV OriP sequence.
[0302] [00302] The molecule was produced by HEK293-EBNA cells cotrasfected with the mammalian expression vectors with the use of polyethyleneimine (PEI). The cells were transfected with the corresponding expression vectors in a 1: 1 ratio. For transfection, HEK293 EBNA cells were cultured in suspension in serum-free CD CHO culture medium. For production in a 500 mL shake flask, 400 million HEK293 EBNA cells were cultured 24 hours before transfection. For transfection, the cells were centrifuged for 5 minutes at 210 x g, the supernatant was replaced with 20 ml of pre-heated CD CHO medium. The expression vectors were mixed in 20 mL of CD CHO medium to a final value of 200 μg of DNA. After adding 540 μL of PEI solution, the mixture was vortexed for 15 seconds and subsequently incubated for 10 minutes at room temperature. Then, the cells were mixed with the DNA / PEI solution, transferred to a 500 mL shaking flask and incubated for 3 hours at 37 ° C in an incubator with a 5% CO2 atmosphere. After the incubation time, 160 ml of F17 medium was added and the cells were cultured for 24 hours. One day after transfection, 1 mM valpoic acid and 7% Feed 17 (Lonza) were added. After 7 days of culture, the supernatant was collected by purification by centrifugation for 15 minutes at 210 xg, the solution was sterilized by filtration (0.22 μm filter) and sodium azide in a final concentration of 0.01% w / v was added, and maintained at 4 ° C.
[0303] [00303] The secreted protein was purified from cell culture supernatants by affinity chromatography using Protein A. The supernatant was loaded onto a HiTrap Protein A HP column (CV = 5 mL, GE Healthcare) equilibrated with 40 mL 20 mM sodium phosphate, 20 mM sodium citrate, 0.5 M sodium chloride, pH 7.5. Unbound protein was removed by washing with at least 10 column volumes of 20 mM sodium phosphate, 20 mM sodium citrate, 0.5 M sodium chloride, pH 7.5. The protein was eluted during a gradient over 20 column volumes of 20 mM sodium citrate, 0.5 M sodium chloride, pH 7.5 20 mM sodium citrate, 0.5 M sodium chloride, pH 2, 5. The protein solution was neutralized by adding 1/10 0.5 M sodium phosphate, pH 8. The target 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 solution, pH 6.0.
[0304] [00304] The protein concentration of the purified protein samples was determined by measuring the optical density (OD) at 280 nm, using a molar extinction coefficient calculated based on the amino acid sequence. The purity and molecular weight of the molecules were analyzed by CE-SDS analysis in the presence and absence of a reducing agent. The Caliper LabChip GXII system (Caliper Life Sciences) was used according to the manufacturer's instructions. 2 μg of sample was used for the analyzes. The aggregated content of antibody samples was analyzed using a TSKgel G3000 SW XL (Tosoh) size exclusion column in 25 mM K2HPO4, 125 mM NaCl, 200 mM L-arginine monohydrochloride, 0.02% (p / v) NaN3, running buffer with pH 6.7 at 25 ° C.
[0305] [00305] KD was measured by surface plasmon resonance using a ProteOn XPR36 (BioRad) machine at 25 ° C with specific capture anti-human F (ab ') 2 fragment (Jackson ImmunoResearch n ° 109-005 -006) immobilized by coupling amine to CM5 chips and subsequent capture of the Fabs from the bacterial supernatant or from purified Fab preparations. Briefly, carboxymethyl dextran (CM5, GE Healthcare) biosensor chips were activated with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The specific capture anti-human F (ab ') 2 fragment was diluted with 10 mM sodium acetate, pH 5.0 at 50 μg / mL before injection at a flow rate of 10 μL / minute to obtain approximately 10,000 response units (RU) of the coupled capture antibody. After the injection of the capture antibody, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, bacterial supernatant Fabs or purified Fabs were injected at a flow rate of 10 μL / minute for 300 seconds and a dissociation of 300 seconds for stabilization of baseline capture. The capture levels were in the range of 100-500 RU. In a subsequent step, the human MCSP analyte (domain D3) -avi-his was injected with either a single concentration or a series of concentrations (depending on the affinity of the clone in a range between 100 nM and 250 pM) diluted in HBS- EP + (GE Healthcare, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% P20 surfactant, pH 7.4) at 25 ° C at a flow rate of 50 μΕ / minute. The sensorchip surface was regenerated by injection of glycine pH 1.5 for 30 seconds at 90 μL / minute followed by injection of NaOH for 20 seconds at the same flow rate. Association rates (kon) and dissociation rates (koff) were calculated using a Langmuir one-to-one connection model (XPR36 Evaluation computer program or Scrubber computer program (BioLogic)) by simultaneous adjustment of the sensorgrams association and dissociation. The equilibrium dissociation constant (KD) was calculated as the koff / kon ratio. These data were used to determine the comparative binding affinity of the matured affinity variants with the parental antibody. Table 3a shows the data generated from these tests.
[0306] [00306] G3, E10, C5 for the light chain, and D6, A7, B7, B8, C1 for the heavy chain were chosen for conversion to human IgG1 format. Since CDR1 and 2 of the light chain were randomized independent of CDR3, the obtained CDRs were combined during the conversion of IgG.
[0307] [00307] IgG affinity formats were measured again with the human MCSP antigen (SEQ ID NO: 118), in addition, also for the cynomolgus homologue (SEQ ID NO: 117).
[0308] [00308] The method used was exactly as described for the Fab fragments, only with the use of purified IgG from mammalian production.
[0309] [00309] Surface plasmon resonance (SPR) experiments to determine the affinity and affinity avidity of mature IgGs were performed in a Biacore T200 at 25 ° C with HBS-EP as running buffer (HEPES 0.01 M pH 7 , 4, 0.15 M NaCl, 3 mM EDTA, 0.005% P20 surfactant, Biacore, Freiburg / Germany).
[0310] [00310] To analyze the avidity of the interaction of different anti-MCSP IgGs for direct coupling of human MCSP D3 and cynomolgus from about 9,500 resonance units (RU) of the anti-Penta His antibody (Qiagen) was performed on a CM5 chip in pH 5.0 using the standard amine coupling kit (Biacore, Freiburg / Germany). The antigens were captured for 60 seconds at 30 nM with 10 μL / minute, respectively. IgGs were passed at a concentration of 0.0064 to 100 nM with a flow rate of 30 μL / minute through the flow of cells over 280 seconds. Dissociation was monitored for 180 seconds. The differences in the mass refractive index were corrected by subtracting the response obtained in the reference flow cell. In the present application, IgGs were eluted on a surface with immobilized anti-Penta His antibody but where HBS-EP was preferably injected into human MCSP D3 or MCSP D3 from cynomolgus.
[0311] [00311] For affinity measurements, IgGs were captured on a CM5 sensorchip surface with immobilized anti-human Fc. The capture of IgG was coupled to the sensorchip surface by direct immobilization of about 9,500 resonance units (UK) at pH 5.0 using a standard amine coupling kit (Biacore, Freiburg / Germany). IgGs are captured for 25 seconds at 10 nM at 30 μL / min. Human and cynomolgus MCSP D3 were passed to a concentration of 2 to 500 nM with a flow rate of 30 μL / minute through cell flow over 120 seconds. Dissociation was monitored for 60 seconds. Association and dissociation for the concentration of 166 and 500 nM were monitored for 1200 and 600 seconds, respectively. The differences in the mass refractive index were corrected by subtracting the response obtained in the reference cell flow. In the present application, the antigens were released on a surface with an immobilized human anti-Fc antibody, but in which HBS-EP was preferably injected into anti-MCSP IgGs.
[0312] [00312] The kinetic constants were derived using the computer program Biacore T200 Evaluation (vAA, Biacore AB, Uppsala / Sweden), to adjust equations of ratio for 1: 1 of Langmuir binding by numerical integration.
[0313] [00313] Greater affinities to human MCSP D3 and cynomolgus were confirmed through measurements of surface plasmon resonance with the use of Biacore T200. In addition, avidity measurements showed up to a 3-fold increase in bivalent bonding (Table 3b)
[0314] [00314] The variable region of heavy chain and light chain DNA sequences have been subcloned into the structure with both the constant heavy chain and the constant light chain pre-inserted into the respective recipient mammalian expression vector. The expression antibody was driven by an MPSV promoter and carries a synthetic polyA signal sequence at the 3 'end of CDS. In addition, each vector contains an EBV OriP sequence.
[0315] [00315] The molecule was produced by cotransfection of HEK293-EBNA cells with mammalian expression vectors using polyethyleneimine (PEI). The cells were transfected with the corresponding expression vectors at a 1: 2: 1: 1 ratio ("heavy chain Fc vector (orifice)": "light chain vector": "light chain Crossfab vector": "Fc vector (bulge) -FabCrossfab of the heavy chain ”).
[0316] [00316] For transfection, HEK293 EBNA cells were cultured in suspension in serum-free CD CHO culture medium. For production in a 500 mL shake flask, 400 million HEK293 EBNA cells were cultured 24 hours before transfection. For transfection, the cells were centrifuged for 5 minutes at 210 x g, the supernatant was replaced with 20 ml of pre-heated CD CHO medium. The expression vectors were mixed in 20 mL of CD CHO medium for a final amount of 200 μg of DNA. After adding 540 μL of PEI solution, the mixture was vortexed for 15 seconds and subsequently incubated for 10 minutes at room temperature. Then the cells were mixed with the DNA / PEI solution, transferred to a 500 mL shaking flask and incubated for 3 hours at 37 ° C in an incubator with a 5% CO2 atmosphere. After the incubation time, 160 ml of F17 medium was added and the cells were cultured for 24 hours. One day after transfection, 1 mM valproic acid and 7% Feed 1 (Lonza) were added. After 7 days of culture, the supernatant was collected for purification by centrifugation for 15 minutes at 210 xg, the solution was sterilized by filtration (0.22 μm filter) and sodium azide in a final concentration of 0.01% w / v was added, and maintained at 4 ° C.
[0317] [00317] The secreted protein was purified from cell culture supernatants by affinity chromatography using Protein A. The supernatant was loaded onto a HiTrap Protein A HP column (CV = 5 mL, GE Healthcare) equilibrated with 40 mL 20 mM sodium phosphate, 20 mM sodium citrate, 0.5 M sodium chloride, pH 7.5. Unbound protein was removed by washing with at least 10 column volumes of 20 mM sodium phosphate, 20 mM sodium citrate, 0.5 M sodium chloride, pH 7.5. The target protein was eluted during a gradient over 20 volumes of the 20 mM sodium citrate column, 0.5 M sodium chloride, pH 7.5 20 mM sodium citrate, 0.5 M sodium chloride, pH 2 , 5. The protein solution was neutralized by adding 1/10 of 0.5 M sodium phosphate, pH 8. The target protein was concentrated and filtered before loading onto a HiLoad Superdex 200 column (GE Healthcare) equilibrated with 20 mM histidine, solution 140 mM sodium chloride pH 6.0
[0318] [00318] The protein concentration of purified protein samples was determined by measuring the optical density (OD) at 280 nm, using a molar extinction coefficient calculated based on the amino acid sequence.
[0319] [00319] The purity and molecular weight of molecules were analyzed by CE-SDS analysis in the presence and absence of a reducing agent. The Caliper LabChip GXII (Caliper Lifescience) system was used according to the manufacturer's instructions. 2 μg of sample was used for the analyzes.
[0320] [00320] The aggregated content of antibody samples was analyzed using an TSKgel G3000 SW XL (Tosoh) size exclusion column in K2HPO425 mM, 125 mM NaCl, 200 mM L-arginine monohydrate, 0.02 NaN3 % (w / v), pH 6.7 and running buffer at 25 ° C.
[0321] [00321] Figure 3 shows a schematic drawing of the MCSP TCB molecule (2 + 1 Crossfab-IgG P329G inverted LALA).
[0322] [00322] Figure 4 and Table 4b show the CE-SDS analyzes of an MCSP TCB molecule (2 + 1 Crossfab-IgG P329G LALA inverted) (SEQ ID NOs: 12, 53, 54 and 55).
[0323] [00323] The variable region of heavy chain and light chain DNA sequences were subcloned in the frame with both the constant heavy chain and the constant light chain pre-inserted in the respective container mammalian expression vector. Antibody expression was driven by an MPSV promoter and carries a synthetic PoliA signal sequence at the 3 'end of CDS. In addition, each vector contained an EBV OriP sequence.
[0324] [00324] The molecule was produced by cotransfection of HEK293 EBNA cells with mammalian expression vectors with the use of polyethyleneimine (PEI). The cells were transfected with the corresponding expression vectors at a ratio of 1: 2: 1: 1 ("heavy chain Fc vector (orifice)": "light chain vector": "light chain Crossfab vector": "vector Heavy chain Fc-FabCrossfab (bulge).
[0325] [00325] For transfection, HEK293 EBNA cells were cultured in suspension in serum-free CD CHO culture medium. For production in 500 ml shake flasks, 400 million HEK293 EBNA cells were cultured 24 hours before transfection. For transfection, the cells were centrifuged for 5 minutes at 210 x g, the supernatant was replaced with 20 ml of pre-heated CD CHO medium. The expression vectors were mixed in 20 mL of CD CHO medium for a final amount of 200 μg of DNA. After adding 540 μL of PEI solution, the mixture was vortexed for 15 seconds and subsequently incubated for 10 minutes at room temperature. Then the cells were mixed with the DNA / PEI solution, transferred to a 500 mL shaking flask and incubated for 3 hours at 37 ° C in an incubator with a 5% CO2 atmosphere. After the incubation time, 160 ml of F17 medium was added and the cells were cultured for 24 hours. One day after transfection, 1 mM valproic acid and 7% Feed 17 (Lonza) were added. After 7 days of culture, the supernatant was collected for purification by centrifugation for 15 minutes at 210 xg, the solution was sterilized by filtration (0.22 Mm filter) and sodium azide in a final concentration of 0.01% w / v was added, and maintained at 4 ° C.
[0326] [00326] The secreted protein was purified from cell culture supernatants by affinity chromatography with the use of Protein A. The supernatant was loaded onto a HiTrap Protein A HP column (CV = 5 mL, GE Healthcare) equilibrated with 40 mL of 20 mM sodium phosphate, 20 mM sodium citrate, 0.5 M sodium chloride, pH 7.5. Unbound protein was removed by washing with at least 10 column volumes of 20 mM sodium phosphate, 20 mM sodium citrate, 0.5 M sodium chloride, pH 7.5. The target protein was eluted during a gradient over 20 column volumes of 20 mM sodium citrate, 0.5 M sodium chloride, pH 7.5 20 mM sodium citrate, 0.5 M sodium chloride, pH 2 , 5. The protein solution was neutralized by adding 1/10 of 0.5 M sodium phosphate, pH 8. The target protein was concentrated and filtered before loading onto a HiLoad Superdex 200 column (GE Healthcare) equilibrated with 20 mM histidine, 140 mM sodium chloride solution, pH 6.0.
[0327] [00327] The protein concentration of purified protein samples was determined by measuring the optical density (OD) at 280 nm, using the molar extinction coefficient calculated based on the amino acid sequence.
[0328] [00328] The purity and molecular weight of the molecules were analyzed by CE-SDS analysis in the presence and absence of a reducing agent. The Caliper LabChip GXII (Caliper Lifescience) system was used according to the manufacturer's instructions. 2 μg sample were used for the analyzes.
[0329] [00329] The aggregated content of antibody samples was analyzed using a TSKgel G3000 SW XL (Tosoh) size exclusion column in K2HPO425 mM, 125 mM NaCl, 200 mM L-arginine monohydrate, 0.02% of (w / v) NaN3, pH 6.7 and running buffer at 25 ° C.
[0330] [00330] Figure 5 shows a schematic drawing of the CEA TCB molecule (2 + 1 Crossfab-IgG P329G LALA inverted).
[0331] [00331] Figure 6 and Table 6 show the CE-SDS analyzes of a CEA TCB molecule (2 + 1 Crossfab-IgG P329G inverted LALA) (SEQ ID NOs: 22, 56, 57 and 58).
[0332] [00332] In an alternative purification method, CEA TCB was captured from fermentation supernatant, collected and clarified by Protein A affinity chromatography (MabSelect SuRe). The eluted Protein A was then subjected to cation exchange chromatography (Poros 50 HS) and subsequently fractionated and analyzed by means of SE-HPLC and capillary electrophoresis. The products containing the fractions were grouped and subjected to hydrophobic interaction chromatography (Butyl-Sepharose 4FF) at room temperature in a link-elution manner. The eluate was then fractionated and analyzed using SE-HPLC and capillary electrophoresis. The products containing the fractions were grouped and anion exchange chromatography (Q-Sepharose FF) in flow mode was performed. The material obtained using this purification method had a monomer content> 98%. EXAMPLE 4 MCSP TCB CONNECTION IN MCSP- AND CELLS THAT EXPRESS CD3
[0333] [00333] MCSP binding to TCB was tested on a human malignant melanoma cell line that expresses MCSP (A375) and on a lymphocyte line of immortalized T cells that express CD3 (Jurkat). Briefly, the cells were collected, counted, checked for viability and resuspended at 2 x 106 cells / mL in FACS buffer (100 μL PBS 0.1% BSA). 100 μL of the cell suspension (containing 0.2 x 10 6 cells) were incubated in 96-well round-bottom plates for 30 minutes at 4 ° C with increasing concentrations of MCSP TCB (2.6 pM at 200 nM), washed twice times with cold 0.1% BSA PBS, incubated again for another 30 minutes at 4 ° C with goat anti-human IgG Fcy Fragment Specific secondary antibody F (ab ') 2 AffiniPure fragment conjugated to PE (Jackson Immuno Research Lab PE n ° 109-116-170), washed twice with cold PBS 0.1% BSA and immediately analyzed by FACS using FACS CantolI (FACS Diva computer program) by gating live for DAPI-negative cells. Binding curves were obtained using GraphPadPrism5 (Figure 7A binding to A375 cells, EC50 = 3381 pM; Figure 7B, binding to Jurkat cells). EXAMPLE 5 T-CELL DEATH INDUCED BY MCSP TCB ANTIBODY
[0334] [00334] MCSP TCB antibody mediated T cell death was assessed using a panel of tumor cell lines expressing different MCSP levels (A375 = high MCSP, MV-3 = average MSCP, HCT-116 = Low MCSP, LS180 = negative MCSP). Briefly, the target cells were collected with trypsin / EDTA, washed, and plated at a density of 25,000 cells / well using flat bottom 96-well plates. The cells were left for adhesion overnight. Peripheral blood mononuclear cells (PBMC) were prepared by Histopaque density centrifugation of enriched lymphocyte preparations (buffy coat) obtained from healthy human donors. The fresh blood was diluted with sterile PBS and layered on a Histopaque gradient (Sigma, No. H8889). After centrifugation (450 xg, 30 minutes, room temperature), the plasma above PBMC containing the interphase was discarded and the PBMCs were transferred to a new tube subsequently filled with 50 mL of PBS. The mixture was centrifuged (400 xg, 10 minutes, room temperature), the supernatant was discarded and the PBMC pellet was washed twice with sterile PBS (centrifugation steps 350 xg, 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 a cell incubator until its use (no more than 24 hours). For the death assay, the antibody was added at the indicated concentrations (range from 1 pM to 10 nM in triplicate. PBMCs were added to target cells in the final effector to the target (E: T) at a rate of 10: 1. of target cells was evaluated after 24 hours of incubation at 37 ° C, 5% CO2 by quantification of LDH released to cell supernatants by cells undergoing apoptosis / necrosis (LDH detection kit, Roche Applied Science, No. 11 644 793 001) The maximum lysis of target cells (= 100%) was obtained by incubating the target cells with 1% Triton X-100. The minimum lysis (= 0%) refers to target cells matched with effector cells without a bispecific construct. results show that MCSP TCB strongly and target-specificly induced the death of MCSP-positive target cell lines without the death of MCSP-negative cell lines (Figure 8, A to D). EC50 values related to the death assays , calculated using GraphPadPrism5 are shown in Table 7.
[0335] [00335] Activation of CD8 + and CD4 + T cells after death of T cells from MV-3 tumor cells expressing MCSP, mediated by the MCSP TCB antibody was assessed by FACS analysis with the use of antibodies that recognize cell activation markers T CD25 (late activation marker) and CD69 (early activation marker). The antibody and death test conditions were essentially as described above (Example 5), using the same range of antibody concentrations (1 pM to 10 nM in triplicates), E: T 10: 1 ratio and a time of 24 hour incubation.
[0336] [00336] After incubation, the PBMCs were transferred to a 96-well round bottom plate, centrifuged at 350 x g for 5 minutes and washed twice with PBS containing 0.1% BSA. Surface staining for CD8 (FITC anti human CD8, BD No. 555634), CD4 (PECy7 anti human CD4, BD No. 557852), CD69 (PE anti human CD9, Biolegend No. 310906) and CD25 (APC anti human CD25 , BD n ° 555434) was carried out in accordance with the suppliers' instructions. The cells were washed twice with 150 μL / well of PBS containing 0.1% BSA and fixed for 15 minutes at 4 ° C with the use of 100 pL / well of fixation buffer (BD No. 554655). After centrifugation, the samples were resuspended in 200 pL / well of PBS containing 0.1% BSA DAPI to exclude dead cells for FACS measurement. The samples were analyzed at BD FACS Fortessa. The results show that MCSP TCB induced a strong and target-specific overload of activation markers (CD25, CD69) in CD8 + T cells (Figure 9 A, B) and CD4 + T cells (Figure 9 C, D) after death. EXAMPLE 7 CYTOKINES SECRETION BY HUMAN EFFECTIVE CELLS AFTER THE DEATH OF TUMOR T CELLS T-CELLS EXPRESSING INDUCED MCSP FOR THE MCSP TCB ANTIBODY
[0337] [00337] Cytokine secretion by human PBMC after the death of tumor T cells expressing MCSP MV-3 induced by MCSP TCB antibody was assessed by FACS analysis of cell supernatants after the death assay.
[0338] [00338] The same antibody was used and the death assay was performed essentially as described (Examples 5 and 6), with use and an E: T ratio of 10: 1 and an incubation time of 24 hours.
[0339] [00339] At the end of the incubation time, the plate was centrifuged for 5 minutes at 350 x g, the supernatant was transferred to a new 96-well plate and stored at -20 ° C until subsequent analysis. Granzyme B, TNFa, IFN-γ, IL-2, IL-4 and IL-10 secreted in cell supernatants were detected using BD CBA Human Soluble Protein Flex Set, according to the manufacturer's instructions in a CantolI FACS . The following kits were used: BD CBA human Granzima B BD CBA human granzima B Flex Set n ° BD 560304; Human BD CBA TNF Flex Set No. BD 558273; Human BD CBA IFN-γ Flex Set No. BD 558269; Human BD CBA IL-2 Flex Set No. BD 558270; Human BD CBA IL-4 Flex Set No. BD 558272; Human BD CBA IL-10 Flex Set No. BD 558274.
[0340] [00340] The results show that MCSP TCB induced the secretion of IL-2, IFN-γ, TNFα, Granzyme B and IL-10 (but not IL-4) after death (Figure 10, A to F).
[0341] Mostrou uma boa ligação às células A375 MCSP-positivas Induziu uma morte forte e alvo-específica de linhagens de células alvo MCSP-positivas e nenhuma morte de linhagens celulares MCSP-negativas. Induziu uma suprarregulação forte e alvo-específica de marcadores de ativação (CD25, CD69) em células T CD8+ e CD4+ após a morte. Induziu secreção de IL-2, IFN-γ, TNFα, Granzima B e IL-10 (mas não de IL-4) após a morte. [00341] Together, these examples show that the bispecific antibody MCSP CD3: Showed good binding to MC75-positive A375 cells It induced a strong, target-specific death of MCSP-positive target cell lines and no death of MCSP-negative cell lines. It induced a strong and target-specific overload of activation markers (CD25, CD69) in CD8 + and CD4 + T cells after death. It induced secretion of IL-2, IFN-γ, TNFα, Granzyme B and IL-10 (but not IL-4) after death.
[0342] [00342] CEA binding to TCB was tested on CEA lung adenocarcinoma cells expressing transfected CEA (A549-huCEA) and immortalized human T lymphocyte and cynomolgus lines that express CD3 (Jurkat and HSC-F, respectively). An undirected TCB (SEQ ID NOs: 59, 60, 61 and 62; see example 24) was used as a control. Briefly, the cells were harvested, counted, checked for viability and resuspended at 2 x 106 cells / mL in FACS buffer (100 μL of PBS 0.1% BSA). 100 μL of cell suspension (containing 0.2 x 10 6 cells) were incubated in a 96-well round bottom plate for 30 min at 4 ° C with increasing concentrations of CEA TCB (61 pM at 1000 nM), washed twice with PBS of cold 0.1% BSA, reincubated for another 30 min at 4 ° C with Fragment F (ab ') 2 goat anti-human IgG Specific Fragment F (ab') 2 AffiniPure conjugated with FITC (Jackson Immuno Research Lab FITC No. 109-096-097), washed twice with cold 0.1% BSA PBS and analyzed immediately by FACS using a FACS CantoII or Fortessa (FACS Diva computer program) by blockage of live negative PI cells. The binding curves were obtained using GraphPadPrism5 (Figure 11A, binding to A549 cells (EC50 6.6 nM); Figure 11B, binding to Jurkat cells; Figure 11C, binding to HSC-F cells). EXAMPLE 9 DEATH MEDIATED BY TUMORAL TARGET CELLS T-CELLS THAT EXPRESS CEA INDUCED BY CEA TCB ANTIBODY
[0343] [00343] T cell-mediated death of target cells induced by CEA TCB antibody was evaluated in human tumor cells HPAFII (high CEA), BxPC-3 (medium CEA) and ASPC-1 (low CEA). HCT-116 (CEA negative tumor cell line) and undirected TCB were used as negative controls. Human PBMCs were used as effectors and death detected 24h and 48h after incubation with bispecific antibody. Briefly, target cells were harvested with Trypsin / EDTA, washed and plated at a density of 25,000 cells / well using 96-well flat-bottom plates. Cells were left to adhere overnight. Peripheral blood mononuclear cells (PMBCs) were prepared by Histopaque density centrifugation of enriched lymphocyte preparations (buffy coat layers) obtained from healthy human donors. Fresh blood was diluted with sterile PBS and layered on the Histopaque gradient (Sigma, No. H8889). After centrifugation (450 xg, 30 minutes, room temperature), the plasma above the interphase containing PBMC was discarded and the PBMCs transferred to a new falcon tube, subsequently filled with 500mL of PBS. The mixture was centrifuged (400 xg, 10 minutes, room temperature), the supernatant discarded and the PMBC pellet washed twice with sterile PBS (350 xg step centrifugation, 10 minutes) The resulting PBMC population was counted automatically (ViCell) and maintained in RPMI1640 medium containing 10% FCS and 1% L-alanyl-L-glutamine (Biochrom, K0302) in the cell incubator (37 ° C, 5% CO2), until later use (no more than 24 h). For the death assay, antibodies were added at the indicated concentrations (range from 6 pM to 100nM in triplicates). PBMCs were added to the target cells in the final 10: 1 E: T ratio. Target cell death was evaluated and after 24h and 48h of incubation by LDH (lactate dehydrogenase) quantification, released in cell supernatants by apoptotic / necrotic cells (LDH detection kit, Roche Applied Science, No. 11 644 793 001) . Maximum target cell lysis (= 100%) was obtained by incubating target cells with 1% Triton X-100. Minimal lysis (= 0%) refers to target cells matched with effector cells without bispecific antibody. The results show that CEA TCB induced a strong and target-specific death of CEA positive target cells (Figure 12, A to H). The EC50 values related to the death tests, calculated using GraphPadPrism5 are provided in Table 8.
[0344] [00344] T cell proliferation and activation was detected 5 days after CEA TCB-mediated death of CEA expressing human tumor cells HPAFII (high CEA), BxPC-3 (medium CEA) and ASPC-1 (low CEA). HCT-116 (CEA negative tumor cell line) and undirected TCB were used as negative controls. The experimental conditions for the proliferation assay were similar to those described in Example 9, but only 10,000 target cells were plated per well of a 96-well flat bottom plate. To assess T cell proliferation, freshly isolated PBMCs were labeled using CFSE (Sigma No. 21888). Briefly, the CFSE stock solution was diluted to obtain a 100 μΜ working solution. 90 x 106 PBMC cells were resuspended in 90 mL of pre-heated PBS and supplemented with 90μL of CFSE working solution. The cells were mixed immediately and incubated for 15 min at 37 ° C. 10mL of pre-heated FCS was added to the cells to stop the reaction. The cells were centrifuged for 10 min at 400 g, resuspended in 50 ml of medium and incubated for 30 min at 37 ° C. After incubation, the cells were washed once with heated medium, counted, resuspended in medium and added to the target cells for the death assay and subsequently measurement of cell proliferation and activation to a 10: 1 E: T. Proliferation was assessed 5 days after death on CD4 and CD8 positive T cells by quantification of CFSE dye dilution. CD25 expression was evaluated in the same subsets of T cells using an anti-human CD25 antibody. Briefly, after centrifugation (400 xg for 4 min), the cells were resuspended with FACS buffer and incubated with 25 μL of the CD4 / CD8 / CD25 antibody mixture diluted for 30 min at 4 ° C (APC / Cy7 n anti-human CD4 ° 317418, APC anti-human CD8 No. 301014, anti-human CD25 PE / Cy7 No. 302612). The cells were then washed three times to remove unbound antibody and finally resuspended in 200 μL of FACS buffer containing propidium iodide (PI) to exclude dead cells for FACS measurement. Fluorescence was measured using BD FACS CantolI. The results show that CEA TCB induced a strong and target-specific proliferation of CD8 + and CD4 + T cells (Figure 13, A to D), as well as their activation, as detected by overloading the CD25 activation marker (Figure 13, E a H). EXAMPLE 11 CYTOKIN SECRETION BY HUMAN EFFECTIVE CELLS AFTER DEATH MEDIATED BY TUMOR CELLS THAT EXPRESS CEA T-CELLS INDUCED BY CEA TCB
[0345] [00345] Cytokine secretion by human PBMCs after T cell-mediated death of MKN45 tumor cells expressing CEA induced by CEA TCB was evaluated by FACS analysis (CBA kit) of cell supernatants 48h after death.
[0346] [00346] The experimental conditions were identical to those described in Example 9. At the end of the incubation time, the plate was centrifuged for 5 minutes at 350 xg, the supernatant transferred to a new 96-well plate and stored at -20 ° C until subsequent analysis. (A) IFN-γ, (B) TNFa, (C) Granzyme B, (D) IL-2, (E) IL-6 and (F) IL-10 secreted in cell supernatants were detected using the BD Set CBA Human Soluble Protein Flex Set, according to the manufacturer's instructions on a FACS CantoII. The following kits were used: CD set CBA human IL-2 BD Flex n ° BD 558270; BD CBA human set Granzyme B BD Flex n ° BD 560304; BD CBA human set TNF Flex No. BD 558273; BD CBA human set IFN-γ Flex No. BD 558269; BD CBA human set IL-4 Flex No. BD 558272; Conjunto Cd human IL-10 Flex No. BD 558274.
[0347] [00347] The results show that CEA TCB mediated death (but not death mediated by targetless TCB control), induced secretion of IFN-γ, TNFα, Granzima B, IL-2, IL-6 and IL-10 (Figure 14, A to F). EXAMPLE 12 MEDIATE DEATH BY TARGET CELL T CELLS IN THE PRESENCE OF GROWING SHED CEA (SCEA) CONCENTRATIONS
[0348] [00348] T cell mediated death of tumor target cells expressing CEA (LS180) induced by the CEA TCB antibody in the presence of increasing concentrations of shed CEA (sCEA 2.5 ng / mL to 5 μg / mL) was evaluated. Human PBMCs were used as effector cells and death was detected 24h and 48h after incubation with bispecific antibody and sCEA. Briefly, target cells were harvested with Trypsin / EDTA, washed and plated at a density of 25,000 cells / well using 96-well round bottom plates. The cells were left to adhere overnight. Peripheral blood mononuclear cells (PMBCs) were prepared by Histopaque density centrifugation of enriched lymphocyte preparations (buffy coat layers) obtained from healthy human donors. Fresh blood was diluted with sterile PBS and layered on the Histopaque gradient (Sigma, No. H8889). After centrifugation (450 x g, 30 minutes, room temperature), the plasma above the interphase containing PBMC was discarded and the PBMCs were transferred to a new Falcon tube, subsequently filled with 500mL of PBS. The mixture was centrifuged (400 xg, 10 minutes, room temperature), the supernatant discarded and the PMBC pellet washed twice with sterile PBS (centrifugation in 350 xg steps, 10 minutes). The resulting PBMC population was counted automatically (ViCell) and maintained in RPMI1640 medium containing 10% FCS and 1% L-alanyl-L-glutamine (Biochrom, K0302) in a cell incubator (37 ° C, 5% CO2), until later use (no more than 24 h). For the death assay, the CEA TCB antibody was used at a fixed concentration of 1 nM and sCEA was enriched in the experiment at a concentration ratio of 2.5 ng to 5 μg / mL. PBMCs were added to the target cells in the final 10: 1 E: T ratio. Target cell death was evaluated and after 24h and 48h of incubation by LDH (lactate dehydrogenase) quantification, released in cell supernatants by apoptotic / necrotic cells (LDH detection kit, Roche Applied Science, No. 11 644 793 001) . Maximum target cell lysis (= 100%) was obtained by incubating target cells with 1% Triton X-100. Minimal lysis (= 0%) refers to target cells matched with effector cells without bispecific antibody. Death mediated by CEA TCB in the absence of sCEA was adjusted to 100% and death obtained in the presence of increasing concentrations of sCEA was normalized for it. The results show that sCEA had only a minor impact on CEA-TCB mediated death of CEA-expressing target cells (Figure 15 A, B). No effect on T cell death was detected up to 0.2 μg / m L of sCEA. SCEA concentrations above 0.2 μg / mL only had a minor impact on overall death (10 to 50% reduction). EXAMPLE 13 DEATH MEDIATED BY T-CELLS OF TARGET CELLS USING HUMAN AND CYNOMOLGUS PBMCS AS EFFECTIVE CELLS
[0349] [00349] T cell mediated death of A549 cells (pulmonary adenocarcinoma) that overexpress human CEA (A549-hCEA), assessed 21 h and 40 h after incubation with CEA TCB antibody and human PBMCs or cynomolgus PBMCs as effector cells was evaluated . Briefly, the target cells were harvested with Trypsin / EDTA, washed and plated at a density of 25,000 cells / well using flat bottom 96-well plates. The cells were left to adhere for several hours. Peripheral blood mononuclear cells (PMBCs) were prepared by Histopaque density centrifugation of enriched lymphocyte preparations (buffy coat layers) obtained from healthy human donors or healthy cynomolgus monkeys. For the latter, a Histopaque-PBS90% density gradient was used. Fresh blood was diluted with sterile PBS and layered on the Histopaque gradient (Sigma, No. H8889). After centrifugation (450 xg, 30 minutes, room temperature for human PBMCs, respectively 520 xg, 30 min, room temperature for cynomolgus PBMCs), the plasma above the PBMC-containing interface was discarded and the PBMCs transferred to a new Falcon tube, subsequently filled with 500mL of PBS. The mixture was centrifuged (400 xg, 10 minutes, room temperature), the supernatant discarded and the PMBC pellet washed twice with sterile PBS (centrifugation in 350 xg steps, 10 minutes) For the preparation of the cynomolgus PBMCs, an additional step of low speed centrifugation was performed at 150 xg for 15 min. The resulting PBMC population was counted automatically (ViCell) and maintained in RPMI1640 medium containing 10% FCS and 1% L-alanyl-L-glutamine (Biochrom, K0302) in a cell incubator (37 ° C, 5% CO2), until later use (up to 4 h). For the death assay, antibodies were added at the indicated concentrations (range from 6 pM to 100 nM in triplicates). PBMCs were added to target cells in the final 10: 1 E: T ratio. Target cell death was assessed and after 21 h and 40 h of incubation by quantification of LDH (lactate dehydrogenase), released in cell supernatants by apoptotic / necrotic cells (LDH detection kit, Roche Applied Science, No. 11 644 793 001 ). Maximum target cell lysis (= 100%) was obtained by incubating target cells with 1% Triton X-100. Minimal lysis (= 0%) refers to target cells matched with effector cells without bispecific antibody. The results show that CEA TCB mediated target-specific death of positive CEA target cells with the use of both human effector cells (Figure 16, A, C) and cynomolgus (Figure 16, B, D) (PBMCs). EC50 values related to 40 h of death, calculated using GraphPadPrism5 are 306 pM for human PBMCs and 102 pM for cynomolgus PBMCs. EXAMPLE 14 T-CELL-MEDIATED DEATH OF CELLULAR LINES OF HUMAN COLORECTAL CANCER EXPRESSING CEA INDUCED BY CEA TCB ANTIBODY
[0350] [00350] T cell-mediated death of human colorectal cancer cell lines expressing CEA 48 h after incubation with human PBMCs and CEA TCB antibody at 0.8 nM, 4 nM was evaluated. In summary, PBMCs were isolated from leukocyte cones obtained from single healthy donors. The cells were diluted with PBS (1:10) and layered on Lymphoprep in 50 ml Falcon tubes. After centrifugation (1800 rpm for 25 min), the PBMC layer was removed from the interface and washed 4x with PBS. PBMCs were counted, frozen in 10% DMSO in FCS under controlled ratio freezing conditions at 40 x 106 cells / mL and stored in liquid nitrogen until further use. For the T cell death assay, tumor cells were plated directly into 96-well plates from frozen stocks. The cells were quickly heated and transferred immediately in the preheated medium, centrifuged and resuspended in complete medium (DMEM, Iscoves or RPMI-1640, all supplemented with FCS 10% and penicillin / streptomycin 1%) and plated at a density of 2, 5 x 104 cells / well. The cells were then incubated at 37 ° C in a humidified 10% CO2 incubator and the medium replaced the next day with 100 μL of RPMI FCS 2% with 1% glutamine and 50 μL of CEA TCB (final concentrations ranging from 6.4 to 20000 pM, 1: 5 titration steps, in duplicate wells for each condition). Freshly thawed PBMCs were used for the assay (thawed from the frozen vials within 2 hours of the start of the assay) and 50 μL (3 x 105) was added to each well to give an effector: target (E: T) ratio of 10: 1 Triton X100 (50 μL of 4%) was added to 150 μL of target cells to obtain maximum release values. The plates were incubated at 37 ° C for 48 h and the death activity determined using the Lactose Dehydrogenase Cytotoxicity Detection Kit (Roche) in accordance with the manufacturer's instructions. The percentage of specific cell lysis was calculated as (sample release - spontaneous release) / (maximum release - spontaneous release) x 100. Figure 17, A to C, shows the correlation between CEA expression (receptor copy number) quantified using QIFIKIT, see below) and the% death for 31 colorectal cancer cell lines (listed on the x-axis). Figure 17 shows the correlation between CEA expression and% specific lysis in 20 nM CEA TCB (Spearman correlation = 0.7289, p <0.0001, n = 31), indicating that tumor cells showing numbers high copies of cEA receptor (> 50,000) are efficiently lysed by CEA TCB, while a group of cells showing low number of copies of CEA receptor (<10,000) is not being lysed by CEA TCB under the same experimental conditions. Figure 17, E, shows the correlation between the expression of CEA and EC50 of CEA TCB. Although the correlation is not statistically significant (Spearman correlation = -0.3994, p = 0.1006, R2 = 0.1358), the graph clearly shows a better CEA TCB power pattern (for example, lower EC50 values ) on tumor cell lines expressing high numbers of CEA receptor copies.
[0351] [00351] For the analysis of CEA surface expression in cancer cell lines, Qifikit (DakoCytomation, Glostrup, Denmark) was used to calibrate the fluorescent signals and determine the number of binding sites per cell. The cells were incubated on ice for 30 min with a mouse anti-human CEACAM5 monoclonal antibody (0.5 μg for 5 x 105 cells, clone: CI-P83-1, sc-23928, Santa Cruz), washed twice with PBS1X-BSA 0.1% followed by a 45 min incubation with goat anti-mouse antibody conjugated to polyclonal fluorescein isothiocyanate, supplied with Qifikit. Dead cells were excluded from the analysis using 4 ', 6-diamidino-2-phenylindola (DAPI) staining. The samples were analyzed on a CyAn ™ ADP Analyzer (Beckman Coulter). All mean fluorescence intensities (MFIs) were obtained after data analysis using the Summit 4.3 computer program. These MFIs were used to determine the relative number of antibody binding sites in cell lines (referred to as CEA copy numbers in the results), using the equation obtained from the calibration curve (Qifikit calibration microspheres).
[0352] [00352] Colorectal cancer cell lines used for T cell death assays and CEA surface expression quantitation were seeded from Cryovials. The method used to keep the stock frozen was as described in Bracht et al. (Bracht et al. (2010), Br J Cancer 103, 340-346). EXAMPLE 15 IN VIVO ANTI-TUMORAL EFFECTIVENESS OF CEA TCB IN A HUMAN COLON CARCINOMA LS174T-FLUC2 COUNTED WITH HUMAN PBMC (E: T 5: 1)
[0353] [00353] NOG mice (NOD / Shi-scid / IL-2RYnull) (n = 12) were injected subcutaneously with 1 x 106 LS174T-fluc2 cells pre-mixed with human PBMC in a total volume of 100 μL in PBS, ratio E : T 5: 1. LS174T-fluc2 cells were genetically engineered to express luciferase, which allows the monitoring of tumor progression by bioluminescence (BLI) in a non-invasive and highly sensitive way. To assess the effects of premature and late treatment, mice received intravenous injections twice a week or 0.5 or 2.5 mg / kg CEA TCB, starting on day 1 (premature treatment) or on day 7 (late treatment) , after subcutaneous (sc) grafting of tumor cells / PBMCs. As a control, a group of mice received twice a week intravenous injections of 2.5 mg / kg from a control TCB that had the same shape as the CEA TCB (in this case, the MCSP TCB served as an untargeted control, insofar as the LS174T-fluc2 cells do not express MCSP), and an extra control group received only PBS (vehicle), starting on day 1. The tumor volume was measured once a week by digital caliper. In addition, the mice were injected intraperitoneally once a week with Luciferin D and the light bioluminescent emission of live tumor cells was measured with IVIS Spectrum (Perkin Elmer). The treatment was administered up to 19 days after the inoculation of tumor cells, which corresponds to the day of the end of the study. The results of the experiment are shown in Figure 18 A to D. The results show the average and SEM of 12 mice of tumor volume measured by caliper (A and C) and by bioluminescence (Total Flux, B and D) in the different groups of Study ((A, B) premature treatment, (C, D), broken treatment). EXAMPLE 16 IN VIVO ANTI-TUMORAL EFFECTIVENESS OF CEA TCB IN A HUMAN COLON CARCINOMA LS174T-FLUC2 COUNTED WITH HUMAN PBMC (E: T 1: 1)
[0354] [00354] NOG mice (NOD / Shi-scid / IL-2RYnull) (n = 10) were injected subcutaneously with 1 x 106 LS174T-fluc2 cells (see Example 15), premixed with human PBMC in a total volume of 100 μL in PBS, E: T ratio 1: 1. To assess the effects of early and late treatment, mice received intravenous injections (iv) twice a week of 2.5 mg / kg CEA TCB, starting on day 1 (initial treatment) or day 7 (late treatment) after treatment. inoculation of tumor cells. As a control, a group of mice received twice a week intravenous injections of 2.5 mg / kg of MCSP TCB (see also Example 15) and an extra control group received only PBS (vehicle), starting on day 1. The Tumor volume was measured once a week with a digital caliper. In addition, the mice were injected intraperitoneally once a week with Luciferin D and the light bioluminescent emission of live tumor cells was measured with IVIS Spectrum (Perkin Elmer). The treatment was administered up to 23 days after the inoculation of tumor cells, which corresponds to the day of the end of the study. The results of the experiment are shown in Figure 19. The results show the mean and SEM of the tumor volume measured by caliper (A), as well as by bioluminescence (B) in the different study groups (n = 10). EXAMPLE 17 IN VIVO EFFECTIVENESS OF MURINIZED CEA TCB IN A PANCO2-HUCEA ORTOTOPIC TUMOR MODEL IN HUCD3E / HUCEA TRANSGENIC MICE IMMUNOCOMPETENTS
[0355] [00355] Transgenic huCD3ε / huCEA mice (n = 10) received an intra-pancreatic injection of 2 x 105 Panco2-huCEA cells in a total volume of 10 μL in PBS. Insofar as murine cells do not express CEA, the Panco2 murine pancreatic carcinoma cell line was genetically engineered to overexpress human CEA as the target antigen for CEA TCB. Mice were injected twice a week intravenously with CEA TCB or PBS as a control group (vehicle) and survival was monitored. The animals were monitored daily for clinical symptoms and detection of adverse effects. The termination criteria for animals were visible diseases: dirty skin, arched back, respiratory problems, impaired locomotion. The result as overall survival is shown in Figure 20. The result shows the percentage of animals surviving by time point. The significance of the treatment group for the PBS control group was compared with the use of a paired Student's test (p = 0.078). EXAMPLE 18 AFFINITY OF CEA TCB FOR CEA AND CD3 BY PLASMOMIC SURFACE RESONANCE (SPR)
[0356] [00356] Surface plasmon resonance experiments were performed in a Biacore T100 at 25 ° C with HBS-EP as running buffer (HEPES 0.01 M, pH 7.4, NaCl 0.15 M, EDTA 3 mM, P20 surfactant 0.005%, Biacore, Freiburg / Germany).
[0357] [00357] For affinity measurements, the CEA TCB was captured on a surface of the CM5 sensorchip with immobilized anti-human Fab (GE Healthcare n ° 28-9583-25). The capture lgG was coupled to the sensorchip surface by direct immobilization of approximately 10,000 resonance units (UK), with pH 5.0 using the standard amine coupling kit (Biacore, Freiburg / Germany).
[0358] [00358] To analyze the interaction with CD3ε stalk-Fc (protuberance) -Avi / CD3δ-stalk-Fc (orifice) (SEQ ID NOs: 120 and 121, respectively), CEA TCB was captured for 30 s at 50 nM with 10 μL / min CD3ε / CD3δ was passed at a concentration of 0.68 to 500 nM with a flow rate of 30 μL / min through the flow cells over 360 s. Dissociation was monitored for 360 s.
[0359] [00359] The Kp value of the interaction between CEA TCB and the recombinant human target tumor NABA-avi-his antigen (containing the human CEA B3 domain (CEACAM5) surrounded by human CEACAM1 N, A1 and A2 domains with a marker termination C avi 6his; see SEQ ID NO: 119) was determined by capturing the TCB molecule for 40 s at 10 μL / min. The antigen flowed over the cell flow for 240 s in a concentration range from 0.68 to 500 nM at a flow rate of 30 μL / min. Dissociation was measured over 240 s.
[0360] [00360] The differences in the mass refractive index were corrected by subtracting the response obtained in a reference cell flow. In the present application the antigens flowed on a surface with immobilized anti-human Fab antibody, but on which HBS-EP was injected instead of CEA.
[0361] [00361] The kinetic constants were derived using the computer program Biacore T200 Evaluation (vAA, Biacore AB, Uppsala / Sweden) to adapt the ratio equations for the Langmiur 1: 1 link by numerical integration. The half-life (t1 / 2) of the interaction was calculated using the following formula: t1 / 2 = ln2 / koff.
[0362] [00362] The CEA TCB binds to the target tumor and CD3ε / CD3δ in the nM range with KD values of 62 nM for human NABA and 75.3 nM for human CD3ε / CD3δ. The half-life of the monovalent bond is up to 5.3 minutes for NABA, the half-life of the CD3ε / CD3δ bond is 5.7 minutes. The kinetic values are summarized in Table 9.
[0363] [00363] Surface plasmon resonance experiments were performed in a Biacore T100 at 25 ° C with HBS-EP as running buffer (HEPES 0.01 M, pH 7.4, NaCl 0.15 M, EDTA 3 mM, P20 surfactant 0.005%, Biacore, Freiburg / Germany).
[0364] [00364] For affinity measurements, the MCSP TCB was captured on a surface of the CM5 sensorchip with immobilized anti-human Fab (GE Healthcare n ° 28-9583-25). The capture lgG was coupled to the sensorchip surface by direct immobilization of about 7,500 resonance units (UK), with pH 5.0 using the standard amine coupling kit (Biacore, Freiburg / Germany). MCSP TCB was captured for 60 s at 30 nM with 10 μL / min. Human and cynomolgus MCSP D3 (see SEQ ID NOs: 118 and 117, respectively) were passed to a concentration of 0.024 to 50 nM with a flow rate of 30 μL / min through the flow of cells over 90 s. The concentration range for human and cynomolgus CD3ε stalk-Fc (bulge) -Avi / CD3δ-stalk-Fc (bore) was 1.17-600 nM. As the interaction with murine MCSP D3 (SEQ ID NO: 122 was expected to be weak, the concentration range for this antigen was chosen between 3.9 and 500 nM. Dissociation for all interactions was monitored for 120 s The differences in the mass refractive index were corrected by subtracting the response obtained in a reference flow cell In this application the antigens flowed on a surface with immobilized anti-human Fab antibody, but on which HBS-EP was injected instead of MCSP TCB.
[0365] [00365] The kinetic constants were derived using the computer program Biacore T200 Evaluation (vAA, Biacore AB, Uppsala / Sweden) to adapt the Langmiur 1: 1 link ratio equations by numerical integration. The interaction for the MCSP TCB with the murine MCSP D3 was determined at steady state. The half-life (t1 / 2) of the interaction was calculated using the following formula: t1 / 2 = ln2 / koff.
[0366] [00366] P MCSP TCB binds to the target tumor in the pM range with kD values of 0.15 nM for human antigen and 0.12 nM for cynomolgus. The CD3ε / CD3δ recombinant is bound by the MCSP TCB with a KD value of 78 nM (human) and 104 nM (cynomolgus). The half-life of the monovalent bond is up to 260 minutes for the target tumor and 2.9 minutes for CD3e / CD3d. At affinity maturation, the MCSP antibody obtained some binding to the recombinant murine MCSP D3. The kD value for this interaction is in the range of mM (1.6 mM). The kinetic values are summarized in Table 10.
[0367] [00367] The thermal stability of CEA TCB was monitored by Dynamic Light Scattering (DLS). 30 μg of filtered protein sample with a protein concentration of 0.5 mg / mL was applied to a Dynapro plate reader (Wyatt Technology Corporation; USA). The temperature was increased progressively from 25 to 75 ° C at 0.05 ° C / min, with the radius and the total dispersion intensity being collected.
[0368] [00368] The result is shown in Figure 21. The aggregation temperature of CEA TCB was measured at 55 ° C. EXAMPLE 21 MCSP TCB THERMAL STABILITY
[0369] [00369] The thermal stability of MCSP TCB was monitored by Dynamic Light Scattering (DLS). 30 μg of filtered protein sample with a protein concentration of 0.5 mg / mL was applied to a Dynapro plate reader (Wyatt Technology Corporation; USA). The temperature was increased progressively from 25 to 75 ° C at 0.05 ° C / min, with the radius and the total dispersion intensity being collected.
[0370] [00370] The result is shown in Figure 22. The aggregation temperature of MCSP TCB was measured at 55 ° C. EXAMPLE 22 DEATH MEDIATED BY TUMOR CELL T-CELLS THAT EXPRESS MCSP INDUCED BY ANTIBODIES MCSP TCB AND MCSP 1 + 1 CROSSMAB
[0371] [00371] T cell-mediated death of target cells induced by MCSP TCB and MCSP 1 +1 CrossMab TCB antibodies (a bispecific antibody that activates T cells, which have the same CD3 and MCSP binding sequences as the MCSP TCB, with the Molecular shape shown in Figure 1 D) was evaluated on tumor target cells A375 (high MCSP), MC-3 (medium MCSP) and HCT-116 (low MCSP). LS180 (MCSP negative tumor cell line) was used as a negative control. Tumor cell death was assessed 24 h and 48 h after incubation of target cells with antibodies and effector cells (human PBMCs). Briefly, target cells were harvested with Trypsin / EDTA, washed and plated at a density of 25,000 cells / well using 96-well round bottom plates. The cells were left to adhere overnight. Peripheral blood mononuclear cells (PMBCs) were prepared by Histopaque density centrifugation of enriched lymphocyte preparations (buffy coat layers) obtained from healthy human donors. Fresh blood was diluted with sterile PBS and layered on the Histopaque gradient (Sigma, No. H8889). After centrifugation (450 xg, 30 minutes, room temperature), the plasma above the interphase containing PBMC was discarded and the PBMCs were transferred to a new Falcon tube, subsequently filled with 500mL of PBS. The mixture was centrifuged (400 xg, 10 minutes, room temperature), the supernatant discarded and the PMBC pellet washed twice with sterile PBS (350 xg step centrifugation, 10 minutes) The resulting PBMC population was counted automatically (ViCell) and maintained in RPMI1640 medium containing 10% FCS and 1% L-alanyl-L-glutamine (Biochrom, K0302) in the cell incubator (37 ° C, 5% CO2), until later use (no more than 24 h). For the death assay, antibodies were added at the indicated concentrations (range 0.01 pM to 100nM in triplicates). PBMCs were added to target cells in the final 10: 1 E: T ratio. Target cell death was evaluated and after 24h and 48h of incubation by LDH (lactate dehydrogenase) quantification, released in cell supernatants by apoptotic / necrotic cells (LDH detection kit, Roche Applied Science, No. 11 644 793 001) . Maximum target cell lysis (= 100%) was obtained by incubating target cells with 1% Triton X-100. Minimal lysis (= 0%) refers to target cells matched with effector cells without bispecific antibody. The results show that the MCSP TCB antibody is more potent than the MCSP 1 +1 CrossMab TCB in that it induced stronger death of MCSP positive target cells at both time points and in all tumor target cells (Figure 23 AH). The EC50 values related to the death tests, calculated using GraphPadPrism5 are provided in Table 11.
[0372] [00372] The activation of CD8 + and CD4 + T cells after death by T cells of tumor cells expressing MCSP (A375 and MV-3) mediated by the MCSP antibodies TCB and MCSP 1 + 1 CrossMab was evaluated by FACS analysis using antibodies recognizing T cell activation markers CD25 (late activation marker) and CD 69 (initial activation marker). The antibody and death assay conditions were essentially as described above (Example 22), using the same antibody concentration range (0.01 pM to 10 nM in triplicates), E: T ratio 10: 1 and a 48 h incubation time.
[0373] [00373] After incubation, PBMCs were transferred to a 96-well round bottom plate, centrifuged at 350 x g for 5 min and washed twice with PBS containing 0.1% BSA. Surface staining for CD8 (anti-human CDIT FITC, BD No. 555634), CD4 (anti-human CD4 PECy7, BD No. 557852), CD69 (CD69anti-human PE, Biolegend No. 310906) and CD25 (anti-CD25 - human APC, BD n ° 555434) was carried out according to the suppliers' instructions. The cells were washed twice with 150 μL / well with PBS containing 0.1% BSA and fixed for 15 min at 4 ° C using 100 pL / well of fixation buffer (BD No. 554655). After centrifugation, the samples were resuspended in 200 pL / well of PBS with 0.1% BSA containing DAPI to exclude dead cells for FACS measurement. The samples were analyzed in BD FACS Fortessa. The results show that MCSP TCB induced a strong and specific overloading of activation markers (CD25, CD69) in CD8 + T cells (Figure 24 A, B (for A375 cells) and E, F (for MV-3 cells)) and CD4 + T cells (Figure 24 C, D (for A375 cells) and G, H (for MV-3 cells)) after death. As for death results, T cell activation was stronger with MCSP TCB than with MCSP 1 + 1 CrossMab. EXAMPLE 24 PREPARATION OF DP47 GS TCB (2 + 1 CROSSFAB-IGG P329G LALA INVERTED = “NON-DIRECTED TCB”) CONTAINING DP47 GS AS NON-BINDING ANTIBODY AND HUMANIZED CH2527 AS ANTI CD3 ANTIBODY
[0374] [00374] The "undirected TCB" was used as a control in the above experiments. The bispecific antibody employs CD3ε, but does not bind to any other antigen and therefore cannot cross-link T cells to any other target cells (and subsequently cannot induce any death.) It was therefore used as a negative control in assays to monitor any non-specific T cell activation.
[0375] [00375] The variable region of DNA sequences of the heavy and light chains was subcloned in the structure, either with the constant heavy chain or the constant light chain pre-inserted in the respective mammalian expression vector. Antibody expression is driven by an MPSV promoter and carries a synthetic PoliA signal sequence at the 3 'end of the CDS. In addition, each vector contains an OriP EBV sequence.
[0376] [00376] The molecule was produced by cotransfection of HEK293 EBNA cells with mammalian expression vectors using polyethylenimine (PEI). The cells were transfected with the corresponding expression vectors in a 1: 2: 1: 1 ratio ("Fc of the vector heavy chain (orifice)": "vector light chain": "Crossfab vector light chain": "chain heavy vector Fc (bulge) -FabCrossfab ”).
[0377] [00377] For transfection, HEK293 EBNA cells were cultured in serum-free suspension in CHO CD culture medium. For 500 ml shake flask production, 400 million HEK293 EBNA cells were seeded 24 hours before transfection. For transfection, the cells were centrifuged for 5 min at 210 x g, the supernatant replaced with 20 ml of preheated CHO CD medium. The expression vectors were mixed in 2 ml of CHO CD medium for a final amount of 200 μg DNA. After adding 540 μL of PEI solution, the mixture was vortexed for 15 s and subsequently incubated for 10 min at room temperature. Subsequently, the cells were mixed with the DNA / PEI solution, transferred to a 500 mL shaking flask and incubated for 3 hours at 37 ° C in an incubator with a 5% CO2 atmosphere. After the incubation time, 160 mL of the F17 medium was added and the cells were cultured for 24 hours. One day after transfection, 1 mM valproic acid and 7% Feed 17 (Lonza) was added. After 7 days of culture, the supernatant was collected for purification by centrifugation for 15 min at 210 xg, the solution was sterilized by filtration (0.22 μm filter) and sodium azide in a final concentration of 0.01% p / v was added and maintained at 4 ° C.
[0378] [00378] The secreted protein was purified from cell culture supernatants by affinity chromatography using Protein A. The supernatant was loaded onto a HiTrap Protein A column (CV = 5 mL, GE Healthcare), equilibrated with 40mL of 20 mM sodium phosphate, 20 mM sodium citrate, 0.5 M sodium chloride, pH 7.5. Unbound protein was removed by washing with at least 10 column volumes of 20 mM sodium phosphate, 20 mM sodium citrate, 0.5 M sodium chloride, pH 7.5. The target protein was eluted during a gradient over 20 column volumes of 20 mM sodium citrate, 0.5 M sodium chloride, pH 7.5 for 20 mM sodium citrate, 0.5 M sodium chloride, pH 2.5. The protein solution was neutralized by adding 1/10 of 0.5 M sodium phosphate, pH 8. The target protein was concentrated and filtered before loading onto a HiLoad Superdex column (GE Healthcare), equilibrated with 20 mM histidine, solution of 140 mM sodium chloride pH 6.0.
[0379] [00379] The protein concentration of purified protein samples was determined by measuring the optical density (OD) at 280nm, using the molar extinction coefficient calculated based on the amino acid sequence.
[0380] [00380] The purity and molecular weight of molecules were analyzed by CE-SDS analysis in the presence and absence of reducing agent. The Caliper LabChip GXII system (Caliper lifescience) was used according to the manufacturer's instructions. 2 μg of sample was used for the analyzes.
[0381] [00381] The aggregated content of antibody samples was analyzed using a TSKgel G3000 SW XL (Tosoh) analytical size exclusion column in 25 mM K2HPO4, 125 mM NaCl, 200 mM L-arginine monohydrochloride, NaN3 0, 02% (w / v), running buffer pH 6.7 at 25 ° C.
[0382] [00382] Figure 25 and Table 13 show the CE-SDS analyzes of DP47 GS TCB (2 + 1 Crossfab-IgG P329G inverted LALA) containing DP47 GS as non-binding antibody and humanized CH2527 as anti-CD3 antibody. (SEQ ID NOs: 59, 60, 61 and 62).
[0383] [00383] Although the aforementioned invention has been described in some detail by way of illustration and examples for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patents and scientific literature cited in this application are expressly incorporated in their entirety as a reference.

权利要求:
Claims (26)
[0001]
MOLECULE THAT BINS TO THE BIESPECIFIC ANTIGEN THAT ACTIVATES T CELLS, characterized by understanding: (i) a first antigen-binding portion that is a Fab molecule capable of specifically binding to CD3, which comprises the complementarity determining region (CDR) 1 of the heavy chain of SEQ ID NO: 4, the CDR 2 of the chain heavy chain of SEQ ID NO: 5, CDR 3 of the heavy chain of SEQ ID NO: 6, CDR1 of the light chain of SEQ ID NO: 8, CDR2 of the light chain of SEQ ID NO: 9 and CDR3 of the light chain of SEQ ID NO: 10, where the first portion that binds to the antigen is a crossed Fab molecule, in which both the variable and constant regions, particularly the constant regions, of the Fab light chain and the Fab heavy chain are exchanged; (ii) a second and a third antigen-binding portion, each of which is a Fab molecule capable of specifically binding the CEA comprising CDR 1 of the heavy chain of SEQ ID NO: 24, CDR 2 of the chain heavy chain of SEQ ID NO: 25, CDR 3 of the heavy chain of SEQ ID NO: 26, CDR1 of the light chain of SEQ ID NO: 28, CDR2 of the light chain of SEQ ID NO: 29 and CDR3 of the light chain SEQ ID NO: 30; and (iii) an Fc domain composed of a first and a second subunit capable of stable association, wherein the second antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminal of the Fab heavy chain of the first antigen, and the first antigen binding portion is fused at the C terminal of the Fab heavy chain end to the N terminal of the first Fc domain subunit, and the third antigen binding portion is fused at the C terminal of the Fab heavy chain to the N-terminal of the second subunit of the Fc domain.
[0002]
MOLECULE according to claim 1, characterized in that the first antigen-binding portion comprises a variable region of the heavy chain comprising the amino acid sequence of SEQ ID NO: 3, and a variable region of the light chain comprising the sequence of amino acids of SEQ ID NO: 7.
[0003]
MOLECULE according to any one of claims 1 to 2, characterized by the second portion and the third portion that bind to the antigen, each comprising a variable region of heavy chain comprising the amino acid sequence of SEQ ID NO: 23, and a variable region of the light chain comprising the amino acid sequence of SEQ ID NO: 27.
[0004]
MOLECULE according to any one of claims 1 to 3, characterized in that the first antigen binding portion is a crossed Fab molecule in which the constant regions of the Fab light chain and the Fab heavy chain are exchanged, and which comprises a variable region of heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a variable light chain region comprising the amino acid sequence of SEQ ID NO: 7, wherein each of the second and third antigen-binding portions is a Fab molecule conventional which comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 27.
[0005]
MOLECULE according to any one of claims 1 to 4, characterized by comprising: (i) a first antigen-binding portion which is a Fab molecule capable of specifically binding to CD3 which comprises a variable region of the heavy chain comprising the amino acid sequence of SEQ ID NO: 3, and a variable region of the chain light comprising the amino acid sequence of SEQ ID NO: 7, in which the first portion that binds to the antigen is a cross between the Fab molecule, in which both the variable and the constant regions, particularly the constant regions, of the light chain of Fab and Fab heavy chain are exchanged; (ii) a second and a third portion that bind to the antigen, each of which is a Fab molecule capable of specifically binding to CEA comprising a variable region of the heavy chain comprising the amino acid sequence of SEQ ID NO: 23 , and a variable region of the light chain comprising the amino acid sequence of SEQ ID NO: 27, and (iii) an Fc domain composed of a first and a second subunit capable of stable association, wherein the second antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminal of the Fab heavy chain of the first antigen, and the first antigen binding portion is fused at the C terminal of the Fab heavy chain end to the N terminal of the first Fc domain subunit, and the third antigen binding portion is fused at the C terminal of the Fab heavy chain to the N-terminal of the second subunit of the Fc domain.
[0006]
MOLECULE according to any one of claims 1 to 5, characterized in that it comprises no more than a portion that binds to the antigen capable of specifically binding to CD3.
[0007]
MOLECULE according to any one of claims 1 to 6, characterized in that the Fc domain is an IgG, specifically an IgG1 Fc domain.
[0008]
MOLECULE according to any one of claims 1 to 7, characterized in that the Fc domain is a human Fc domain.
[0009]
MOLECULE according to any one of claims 1 to 8, characterized in that the Fc domain comprises a modification that promotes the association of the first and second subunits of the Fc domain.
[0010]
MOLECULE according to any one of claims 1 to 9, characterized in that, in the CH3 domain of the first subunit of the Fc domain, an amino acid residue is replaced by an amino acid residue that has a larger side chain volume, thus generating a bulge within the CH3 domain of the first subunit that 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 by an amino acid residue that has a side chain volume smaller, thus 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.
[0011]
MOLECULE according to any one of claims 1 to 10, characterized in that, in the CH3 domain of the first Fc domain subunit, the threonine residue of position 366 is replaced by a tryptophan residue (T366W) and, in the CH3 domain of the second subunit in the Fc domain, the tyrosine residue of position 407 is replaced by a valine residue (Y407V); and where optionally (a) in the second subunit of the Fc domain, in addition, the threonine residue of position 366 is replaced by serine residue (T336S) and the leucine residue of position 368 is replaced by an alanine residue (L368A); and / or (b) in the first subunit of the Fc domain, in addition, the serine residue of position 354 is replaced with a cysteine residue (S354C), and in the second subunit of the Fc domain, in addition, the tyrosine residue of position 349 is replaced by a cysteine residue (Y349C) (EU numbering according to Kabat).
[0012]
MOLECULE according to any one of claims 1a to 11, characterized in that the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor, particularly an Fcy receptor, and / or effector function, particularly an antibody dependent mediated cell by cytotoxicity (ADCC), wherein said one or more amino acid substitutions are in one or more positions selected from the group of L234, L235 and P329 (EU number according to Kabat).
[0013]
MOLECULE according to any one of claims 1 to 12, characterized in that each subunit of the Fc domain comprises said amino acid substitutions L234A, L235A and P329G (EU numbering according to Kabat).
[0014]
MOLECULE according to any one of claims 1 to 13, characterized in that it comprises a SED ID NO: 22 polypeptide sequence, a SED ID NO: 56 polypeptide sequence, a SED ID NO: 57 polypeptide sequence and polypeptide sequence of SED ID NO: 58.
[0015]
POLYNUCLEOTIDE ISOLATED OR POLYNUCLEOTIDE PLURALITY, characterized by encoding the molecule that binds to the bispecific antigen that activates T cells, as defined in any one of claims 1 to 14 comprising (i) the sequences of SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 72, or degenerate sequences thereof, which encode the CDRs of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, respectively, and (ii) the sequences of SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 91 and SEQ ID NO: 92, or degenerate sequences thereof, which encode the CDRs of SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, respectively.
[0016]
VECTOR, particularly an expression vector, characterized in that it comprises the polynucleotide, as defined in claim 15.
[0017]
METHOD TO PRODUCE A MOLECULE, which binds to the bispecific antigen that activates T cells, capable of specifically binding to CD3 and CEA, characterized by comprising the steps of a) culturing a host cell that comprises the polynucleotide, as defined in claim 15 , or the vector, as defined in claim 16, under conditions suitable for the expression of the bispecific antigen binding molecule that activates T cells and b) recovering the bispecific antigen binding molecule that activates T cells.
[0018]
PHARMACEUTICAL COMPOSITION, characterized by comprising the molecule that binds to the bispecific antigen that activates T cells, as defined in any one of claims 1 to 14, and a pharmaceutically acceptable carrier.
[0019]
MOLECULE according to any one of claims 1 to 14, characterized in that it is for use as a medicament.
[0020]
PHARMACEUTICAL COMPOSITION, according to claim 18, characterized in that it is for use as a medicine.
[0021]
MOLECULE according to any one of claims 1 to 14, characterized in that it is for use in the treatment of a disease in an individual in need of it.
[0022]
MOLECULE, according to claim 21, characterized by the disease being cancer.
[0023]
PHARMACEUTICAL COMPOSITION, according to claim 18, characterized for being for use in the treatment of a disease in an individual in need of it.
[0024]
PHARMACEUTICAL COMPOSITION, according to claim 23, characterized by the disease being cancer.
[0025]
USE OF THE MOLECULE, which binds to the bispecific antigen that activates T cells, as defined in any one of claims 1 to 14, characterized by being for the manufacture of a medicine for the treatment of a disease in an individual in need of it.
[0026]
USE, according to claim 25, characterized by said disease being cancer.

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法律状态:
2018-01-16| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2019-05-21| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2020-05-12| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

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2020-08-11| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-09-24| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 24/02/2014, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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EP13156686|2013-02-26|

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EP13156686.1|2013-02-26|

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PCT/EP2014/053490|WO2014131712A1|2013-02-26|2014-02-24|Bispecific t cell activating antigen binding molecules|

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