WO2014012085A2 - Bispecific asymmetric heterodimers comprising anti-cd3 constructs - Google Patents
Bispecific asymmetric heterodimers comprising anti-cd3 constructs Download PDFInfo
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
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- A61P31/12—Antivirals
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- A61P33/00—Antiparasitic agents
- A61P33/14—Ectoparasiticides, e.g. scabicides
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- A61P35/00—Antineoplastic agents
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- A61P35/00—Antineoplastic agents
- A61P35/02—Antineoplastic agents specific for leukemia
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- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
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- C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
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- C07K2317/35—Valency
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- C07K2317/52—Constant or Fc region; Isotype
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- C07K2317/524—CH2 domain
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- C07K2317/526—CH3 domain
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- C07K2317/52—Constant or Fc region; Isotype
- C07K2317/528—CH4 domain
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- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
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- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
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- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/71—Decreased effector function due to an Fc-modification
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- C07K2317/72—Increased effector function due to an Fc-modification
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- C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
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- C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
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- C07K2317/74—Inducing cell proliferation
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- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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- C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
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- C07K2319/00—Fusion polypeptide
- C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
Definitions
- the field of the invention is the rational design of multispecific scaffolds comprising a CD3 binding domain for custom development of biotherapeutics.
- bispecific antibodies and other fused multispecific therapeutics exhibit dual or multiple target specificities and an opportunity to create drugs with novel modes of action.
- the development of such multivalent and multispecific therapeutic proteins with favorable pharmacokinetics and functional activity has been a challenge.
- T cells thymus derived cells
- B cells bone marrow derived cells
- T cells exhibit immunological specificity and are directly involved in cell-mediated immune responses (such as graft rejection).
- T cells act against or in response to a variety of foreign structures (antigens). In many instances these foreign antigens are expressed on host cells as a result of infection. However, foreign antigens can also come from the host having been altered by neoplasia or infection.
- T cell activation is a complex phenomenon that depends on the participation of a variety of cell surface molecules expressed on the responding WSGF
- the antigen-specific T cell population For example, the antigen-specific T cell population.
- the antigen-specific T cell population For example, the antigen-specific T cell population.
- CD3 gamma and delta
- multispecific heteromultimers comprising a CD3 binding domain.
- a bispecific asymmetric heterodimer comprising anti-CD3 constructs.
- isolated multispecific heteromultimer constructs comprising: a first polypeptide construct comprising a first heavy chain polypeptide and a CD3 binding polypeptide construct that binds to a CD3 complex on at least one CD3 expressing cell; a second polypeptide construct comprising a second heavy chain polypeptide which is different from said first heavy chain polypeptide, and an antigen binding polypeptide construct that binds to a target antigen on at least one B cell; wherein: at least one of said CD3 binding polypeptide construct and said antigen binding polypeptide construct comprises a single chain Fv region; wherein said multispecific heteromultimer construct simultaneously engages said at least one B cell and said at least one CD3 expressing cell such that the CD3 expressing cell is activated, thereby inducing killing of the B cell; and said first and second heavy chain polypeptides form a heterodimeric Fc region comprising a variant immunoglobulin CH3 region comprising at least one amino acid mutation that promotes the formation of said hetero
- said stable mammalian cell is transfected at least a first DNA
- the first or second polypeptide construct is devoid of at least one of immunoglobulin light chain, and immunoglobulin first constant (CH1 ) region.
- the isolated heteromultimer constructs described herein wherein the heterodimer Fc region comprises a variant CH2 domain or hinge comprising amino acid modifications that prevents functionally effective binding to all the Fcgamma receptors.
- the isolated multispecific heteromultimer described herein wherein wherien said variant CH2 domain or hinge comprising amino acid modification also prevents functionally effective binding to complement proteins (C1 q complex).
- the heterodimer Fc region comprises a variant CH2 domain or hinge comprising amino acid modifications that enhance binding to the FcyRllb receptor.
- heteromultimer construct comprising: a first polypeptide construct comprising a first heavy chain polypeptide and a CD3 binding polypeptide construct that binds to a CD3 complex on at least one CD3 expressing cell; a second polypeptide construct comprising a second heavy chain polypeptide which is different from said first heavy chain polypeptide, and an antigen binding polypeptide construct that binds to a target antigen on at least one B cell;
- At least one of said CD3 binding polypeptide construct and said antigen binding polypeptide construct optionally comprises a single chain Fv region; said first and second heavy chain polypeptides form a heterodimeric Fc region comprising a variant immunoglobulin CH3 region comprising at least one amino acid mutation that promotes the formation of said heterodimeric Fc, wherein: said heterodimeric Fc is formed with stability at least comparable to a
- said expression product comprises greater than 70% of said multispecific heteromultimer, and less than 10% monomers or homodimers of said first or second polypeptide constructs; and said multispecific heteromultimer construct binds said at least one B cell with a valency greater than one, and said multispecific heteromultimer simultaneously engages said at least one B cell and said at least one CD3 expressing cell such that the CD3 expressing cell is activated, thereby inducing killing of the B cell.
- said multispecific heteromultimer construct binds said at least one B cell with a valency of two.
- an isolated multispecific heteromultimer construct comprising: a first polypeptide construct comprising a first heavy chain polypeptide and a CD3 binding polypeptide construct that binds to a CD3 complex on at least one CD3 expressing cell; a second polypeptide construct comprising a second heavy chain polypeptide which is different from said first heavy chain polypeptide, and a steric modulator construct which exhibits negligible receptor binding; wherein: said multispecific heteromultimer construct simultaneously engages at least one B cell and said at least one CD3 expressing cell such that the CD3 expressing cell is activated, thereby inducing killing of the B cell; and said first and second heavy chain polypeptides form a heterodimeric Fc region comprising a variant immunoglobulin CH3 region comprising at least one amino acid mutation that promotes the formation of said heterodimeric Fc, wherein: said heterodimeric Fc is formed with stability at least comparable to a native homodimeric Fc, and said heterodimeric F
- first polypeptide construct comprising a first heavy chain polypeptide and a CD3 binding polypeptide construct that binds to a CD3 complex on at least one CD3 expressing cell; a second polypeptide construct comprising a second heavy chain polypeptide which is different from said first heavy chain polypeptide, and wherein said second polypeptide construct does not comprise an antigen binding polypeptide construct; wherein: said multispecific heteromultimer construct simultaneously engages at least one B cell and said at least one CD3 expressing cell such that the CD3 expressing cell is activated, thereby inducing killing of the B cell; and said first and second heavy chain polypeptides form a heterodimeric Fc region comprising a variant immunoglobulin CH3 region comprising at least one amino acid mutation that promotes the formation of said heterodimeric Fc, wherein: said heterodimeric
- Fc is formed with stability at least comparable to a native homodimeric Fc, and said heterodimeric Fc is formed with purity such that when said multispecific heteromultimer construct is coexpressed from a stable mammalian cell in an expression product, said expression product comprises at least about 75% of said multispecific heteromultimer, and less than 10% monomers or
- heterodimer Fc region comprises a variant CH2 domain comprising amino acid modifications to promote selective binding of a
- the variant CH2 domain selectively binds Fcgammallb receptor greater than to wild-type CH2 domain. In certain embodiments, the variant CH2 domain selectively binds atleast one of
- Fcgammallla and Fcgammalla receptor greater than a wild-type CH2 domain.
- the heterodimer Fc region is formed with a purity greater than about 90%. In certain embodiments, the heterodimer Fc region is formed with a purity of about 95%
- the variant CH3 sequence of the first heavy chain polypeptide comprises the amino acid modifications L351Y, F405A, and Y407V
- the variant CH3 sequence of the second transporter polypeptide comprises the amino acid modifications T366L, K392M, and T394W
- the variant CH3 sequence of the first heavy chain polypeptide comprises the amino acid modifications L351Y, F405A, and Y407V
- the variant CH3 sequence of the second heavy chain polypeptide comprises the amino acid modifications T366L, K392L, and T394W.
- the variant CH3 sequence of the first heavy chain polypeptide comprises the amino acid modifications T350V, L351Y, F405A, and Y407V
- the variant CH3 sequence of the second heavy chain polypeptide comprises the amino acid modifications T350V, T366L, K392M, and T394W
- the variant CH3 sequence of the first heavy chain polypeptide comprises the amino acid modifications T350V, L351Y, F405A, and Y407V
- the variant CH3 sequence of the second heavy chain polypeptide comprises the amino acid modifications T350V, T366L, K392L, and T394W.
- the variant CH3 sequence of the first heavy chain polypeptide comprises the amino acid modifications T366L, N390R, K392R, and T394W
- the variant CH3 sequence of the second heavy chain polypeptide comprises the amino acid modifications L351 Y, S400E, F405A, and Y407V
- the variant CH3 sequence of the first heavy chain polypeptide comprises the amino acid modifications T350V, T366L, N390R, K392R, and T394W
- the variant CH3 sequence of the second heavy chain polypeptide comprises the amino acid modifications T350V, L351Y, S400E, F405A, and Y407V.
- heterodimer Fc is aglycosylated.
- the isolated multispecific heteromultimer described herein wherein the antigen binding polypeptide construct that binds to a target antigen on at least one B cell comprises at least one target antigen binding domain derived from an antibody, a fibronectin, an affibody, anticalin, cysteine knot protein, DARPin, avimer, Kunitz domain or variant or derivative thereof.
- said antibody is a heavy chain antibody devoid of light chains.
- said antigen binding polypeptide construct comprises at least one CD19 binding domain.
- said antigen binding polypeptide construct comprises at least one CD20 binding domain.
- an isolated multispecific heteromultimer construct comprising: a first polypeptide construct comprising a first transporter polypeptide fused to at least one CD3 binding polypeptide construct that binds to a CD3 complex on at least one CD3 expressing cell; a second polypeptide construct comprising a second transporter polypeptide which is different from said first transporter polypeptide, fused to at least one antigen binding polypeptide construct that binds to a target antigen on at least one B cell;
- first and second transporter polypeptides are derived from a protein by segmentation of said protein, each transporter polypeptide comprising an amino acid sequence with at least 90% identity to a segment of said protein, and wherein said transporter polypeptides self-assemble to form a quasi-native structure of said monomeric protein.
- each transporter polypeptide is an albumin derivative.
- said albumin is human serum albumin.
- least one transporter polypeptide is an allo-
- isolated multispecific heteromultimer constructs comprising: a first polypeptide construct comprising a first transporter polypeptide fused to at least one CD3 binding polypeptide construct that binds to a CD3 complex on at least one CD3 expressing cell; a second polypeptide construct comprising a second transporter polypeptide which is different from said first transporter polypeptide, fused to at least one antigen binding polypeptide construct that binds to a target antigen on at least one B cell;
- first and second transporter polypeptides are obtained by segmentation of albumin, and each transporter polypeptide comprising an amino acid sequence with at least 90% identity to a segment of albumin such that said transporter polypeptides self-assemble to form quasi-native albumin, and wherein said first cargo polypeptide does not have any binding domain present in said second cargo polypeptide.
- a heteromultimer described herein wherein said multispecific heteromultimer construct simultaneously engages said at least one B cell and said at least one CD3 expressing cell such that the CD3 expressing cell is activated, thereby inducing killing of the B cell.
- said antigen binding polypeptide construct that binds to a target antigen on at least one B cell comprises at least one target antigen binding domain derived from an antibody, a fibronectin, an affibody, anticalin, cysteine knot protein, DARPin, avimer, Kunitz domain or variant or derivative thereof.
- the antigen binding polypeptide construct comprises at least one CD19 binding domain.
- a multispecific heteromultimer described herein wherein said antigen binding polypeptide construct comprises at least one CD20 binding domain.
- the at least one CD3 binding polypeptide construct comprises at least one CD3 binding domain derived from a CD3 specific
- the at least one CD3 binding domain comprises at least one amino acid modification that reduces immunogenicity as compared to a corresponding CD3 binding domain not comprising said modification.
- the isolated multispecific heteromultimer described herein wherein said at least one CD3 binding domain comprises at least one amino acid modification that increases its stability as measured by T m , as compared to a corresponding CD3 binding domain not comprising said modification.
- the at least one CD3 binding polypeptide construct comprises at least one CD3 binding domain derived from a CD3 specific antibody is a heavy chain antibody devoid of light chains.
- the at least one CD3 binding polypeptide construct comprises at least one CD3 binding domain derived from a non-antibody protein scaffold domain.
- the isolated heteromutlimer construct described herein wherein at least one of said first and second polypeptide constructs further comprises a single-chain Fv polypeptide. In certain embodiments, is provided the isolated heteromutlimer construct described herein, wherein at least one of said first and second polypeptide constructs further comprises a single-chain Fab polypeptide.
- the isolated heteromutlimer construct described herein where in the CD3 expressing cell is a T-cell.
- the isolated heteromultimer described herein wherein said heteromultimer binds to the T-cell with sufficient affinity and decorates the T cell at sufficient capacity that induces the T-cell to display B cell killing activity when the T cell and the B cell are bridged.
- CD3 expressing cell is a human cell.
- the CD3 expressing cell is a human cell.
- CD3 expressing cell is a non-human, mammalian cell.
- the mammalian cell is a primate cell.
- the primate is a monkey.
- the CD3 binding polypeptide binds to CD3 constructs across multiple species which include at least one or more of human, rat, mouse and monkey.
- the at least one B cell is associated with a disease.
- the disease is a cancer selected from a carcinoma, a sarcoma, leukaemia, lymphoma and glioma.
- the cancer is at least one of squamous cell carcinoma, adenocarcinoma, transition cell carcinoma, osteosarcoma and soft tissue sarcoma.
- the at least one B cell is an autoimmune reactive cell that is a lymphoid or myeloid cell.
- heteromultimer further comprises at least one binding domain that binds at least one of: EpCAM, EGFR, IGFR, HER-2 neu, HER-3, HER-4, PSMA, CEA, MUC-1 (mucin), MUC2, MUC3, MUC4, MUC5, MUC7, CCR4, CCR5, CD19, CD20, CD33, CD30, ganglioside GD3, 9-0-Acetyl-GD3, GM2, Poly SA, GD2, Carboanhydrase IX (MN/CA IX), CD44v6, Sonic Hedgehog (Shh), Wue-1 , Plasma Cell Antigen, (membrane-bound), Melanoma
- MCSP Chondroitin Sulfate Proteoglycan
- CCR8 TNF-alpha precursor
- STEAP mesothelin, A33 Antigen, Prostate Stem Cell Antigen (PSCA), Ly-6; desmoglein 4, E-cadherin neoepitope, Fetal Acetylcholine Receptor, CD25, CA19-9 marker, CA-125 marker and Muellerian Inhibitory Substance (MIS) Receptor type II, sTn (sialylated Tn antigen; TAG-72), FAP (fibroblast activation antigen), endosialin, LG, SAS, EPHA4 CD63, CD3 BsAb immunocytokines TNF, IFNy, IL-2, and TRAIL.
- MIS Muellerian Inhibitory Substance
- heteromultimer construct described herein wherein said heteromultimer optionally comprises at least one linker.
- said at least one linker is a polypeptide comprising from about 1 to about 100 amino acids.
- multispecific heteromultimer described herein comprising at least a first DNA sequence encoding said first polypeptide construct and at least a second DNA sequence encoding said second polypeptide construct.
- the method comprising: transfecting at least one mammalian cell with: at least a first DNA sequence encoding said first polypeptide construct and at least a second DNA sequence encoding said second polypeptide construct, such that said at least one first DNA sequence, said at least one second DNA sequence are transfected in said at least one mammalian cell in a pre-determined ratio to generate stable mammalian cells; culturing said stable mammalian cells to produce said expression product comprising said multispecific heteromultimer.
- said mammalian cell is selected from the group consisting of a VERO, HeLa, HEK, NS0, Chinese Hamster Ovary (CHO), W138, BHK, COS-7, Caco-2 and MDCK cell, and subclasses and variants thereof.
- composition comprising a
- multispecific heteromultimer described herein and a suitable excipient.
- a process for the production of said pharmaceutical composition comprising: culturing a host cell under conditions allowing the expression of a heteromultimer as defined herein; recovering the produced heteromultimer from the culture; and producing the pharmaceutical
- a proliferative disease a minimal residual cancer, a tumorous disease, an inflammatory disease, an immunological disorder, an autoimmune disease, an infectious disease, viral disease, allergic reactions, parasitic
- said method comprising administering to a subject in need of such a prevention, treatment or amelioration a pharmaceutical composition described herein.
- a method of treating cancer in a mammal in need thereof comprising administering to the mammal a composition comprising an effective amount of the pharmaceutical composition described herein, optionally in combination with other pharmaceutically active molecules.
- said cancer is a solid tumor.
- said solid tumor is one or more of sarcoma, carcinoma, and lymphoma.
- the cancer is a hematological cancer.
- the cancer is one or more of B-cell lymphoma, non-Hodgkin's lymphoma, and leukemia.
- a method of treating cancer cells comprising providing to said cell a composition comprising a heteromultimer described herein.
- said heteromultimer is provided in conjugation with another therapeutic agent.
- composition comprising an effective amount of the pharmaceutical composition described herein.
- a method of treating a cancer cell regressive after treatment with blinatumomab comprising providing to said cancer cell a composition comprising an effective amount of the pharmaceutical composition described herein.
- a method of treating an individual suffering from a disease characterized by expression of B cells comprising providing to said individual an effective amount of a composition comprising an effective amount of the pharmaceutical composition described herein.
- said disease is not responsive to treatment with at least one of an anti-CD19 antibody and an anti-CD20 antibody.
- said mammal in need thereof, comprising administering to said mammal a composition comprising an effective amount of the pharmaceutical composition provided herein.
- said autoimmune condition is one or more of multiple sclerosis, rheumatoid arthritis, lupus erytematosus, psoriatic arthritis, psoriasis, vasculitis, uveitis, Crohn's disease, and type 1 diabetes.
- a method of treating an inflammatory condition in a mammal in need thereof comprising administering to said mammal a composition comprising an effective amount of the pharmaceutical composition comprising an heteromultimer provided herein.
- kits comprising a heteromultimer as defined herein, and instructions for use thereof.
- a heteromultimer construct comprising: a first monomer comprising a first transporter polypeptide fused to a first cargo polypeptide that comprises at least one HER2 binding domain; a second monomer comprising a second transporter polypeptide which is different from said first transporter polypeptide, fused to a second cargo polypeptide that comprises at least one HER3 binding domain; wherein said first cargo polypeptide does not have any binding domain present in said second cargo polypeptide; wherein said first and second transporter polypeptide form a heterodimeric Fc region comprising a variant immunoglobulin CH3 region comprising at least one amino acid mutation that promotes the formation of said heterodimer with stability comparable to a native homodimeric Fc.
- a heteromultimer construct comprising: a first monomer comprising a first transporter polypeptide fused to a first cargo polypeptide that comprises at least one HER2 binding domain; a second monomer comprising a second transporter polypeptide which is different from said first transporter polypeptide, fused to a second cargo polypeptide polypeptide that comprises at least one HER3 binding domain; wherein said first and second transporter polypeptides are obtained by segmentation of
- a pharmaceutical composition comprising an insolated multispecific heteromultimer as defined herein, and a suitable excipient. Also provided is a process for the production of such a
- composition said process comprising: culturing a host cell under conditions allowing the expression of a heteromultimer as defined herein; recovering the produced heteromultimer from the culture; and producing the pharmaceutical composition.
- host cells comprising nucleic acid encoding a heteromultimer described herein.
- the nucleic acid encoding the first monomeric protein and the nucleic acid encoding the second monomeric protein are present in a single vector.
- the nucleic acid encoding the first monomeric protein and the nucleic acid encoding the second monomeric protein are present in separate vectors.
- kits comprising a heteromultimer as defined herein, and instructions for use thereof.
- Fig. 1 A-B Fig. 1 A depicts exemplary schematic representation of heteromultimer constructs provided herein.
- the Immunoglobulin based Anti-CD3 x CD19 constructs demonstrate different aspects of the heteromultimers for instance the cartoon shows the first and second
- polypeptide constructs wherein the first polypeptide construct comprises a CH3 binding construct (black) and the second polypeptide construct comprises an antigen binding construct (blue).
- the antigen binding construct is absent or replaced by a steric modulating construct. Also shown is the Fc heteromultimer formed by the variant CH3 regions of the first and
- FIG. 1 B shows the abil
- Fig. 2 demonstrates that a heteromultimer described herein (v873) is able to selectively bind and bridge to CD3-expressing Jurkat T cells (lower right panel) and to CD19-expressing Raji B cells (upper right panel).
- Figure 2 also demonstrates that the one-armed anti-CD3 antibody specifically binds to Jurkat T cells (lower middle panels)and does not cross-react to CD19 expressing B cells, (upper middle panels)and that the one-armed anti-CD19 antibody specifically binds to Raji B cells (upper left panel) and does not cross- react to Jurkat T cells (lower left panel).
- Fig. 3A-3B Fig. 3A depict the ability of a heteromultimer described herein (v873) to redirect IL-2 activated PBMC to kill target Raji B cells from 3 donors.
- Fig. 3B demonstrates that a heteromultimer described herein is able to mediate higher redirected T-cell cytotoxicity than a construct lacking the heterodimeric Fc in one of the donors.
- Fig. 4 shows that heteromultimers described herein are able to bind to CD3-expressing Jurkat T-cells and to CD19-expressing Raji B-cells.
- Fig. 5A-5B Fig. 5A indicates that at the concentration tested, v1093, which is a heteromultimer described herein, was able to bridge 31 % of total cells, and v873 another heteromultimer construct described herein was able to bridge 25% of total cells.
- Fig. 5B demonstrates that v1093 is able to bridge Jurkat T cells and Raji B cells to a greater extent than v221 and similar to v891 and v873.
- Fig. 6 shows antibody therapeutics that can be provided along with a heteromultimer described herein for treatment of certain indications.
- Fig. 7 shows an SDS-PAGE demonstratin
- heteromultimer constructs described herein are expressed transiently in
- CH03E7 cells with a cell viability of > 80 % CH03E7 cells with a cell viability of > 80 %.
- Fig. 8 shows that a heteromultimer described herein (v873) induces are higher % cytotoxicity to target B cells when compared to negative control human lgG1 (G1 ) when comparing across individual donors.
- Fig. 9A-9B Fig. 9A shows that human IgG (hlgG) does not bind to Jurkat T-cells and has low level binding to Raji B-cells.
- Figure 9A also shows that anti-CD19 one arm constructs bind selectively to the Raji B-cells and does not cross-react to Jurkat T cells.
- Fig. 9B demonstrates FACS assay shows that v873-a heteromultimer described herein, binds selectively to Jurkat T-cells and to Raji B-cells.
- Fig. 10 shows that v873-a heteromultimer described herein does not bind to the K562 cell line, which does not express CD19 or CD3.
- Fig. 11 shows that v873-a heteromultimer described herein does not bind to mouse lymphoid cells which does not express CD19 or CD3.
- Fig 12.A-B show FACS binding curves of heteromultimer constructs described herein (v873, v875) and the construct lacking a heterodimeric Fc (v891 ), to CD3 expressing HPB-ALL and CD3 expressing Jurkat T cells, and to CD19 expressing Raji B cells.
- Fig. 13A-B Fig. 13A illustrates the FACS binding curves for heteromultimer constructs v875, v1379, v1380, v1381 , and control v891 binding to CD19 expressing Raji cells tested in 0.1 to 300 nM range.
- Fig. 13B illustrates the FACS binding curve for heteromultimer constructs v875, v1379, v1380, binding to HBP-ALL T cells tested in 0.1 to 300 nM range.
- Fig. 14 indicates that heteomultimer constructs described herein (v875 and v891 ) facilitate comparable bridging between Raji B-cells and Jurkat T-cells.
- Use of the control human IgG resulted in 2.5% bridging between Raji and Jurkat cells, while v875 facilitated bridging of 22.9% of total cells, and v891 facilitated bridging of 14.5% of total cells.
- Fig. 15A-15B Fig. 15A shows the amounl
- Fig. 15B shows the amount of bridging using a 15:1 ratio of T-cells to B-cells, with heteromultimer concentrations ranging from 0.3 nM to 3 nM.
- E:T ratios (1 :1 and 15:1 ) tested with v875 resulted in similar total T cell-B cell bridging when expressed as fold over background.
- Fig 16A-16E depicts the ability of v875, v1379 and v1380 to mediate antibody dependent B cell cytoxicity by redirected CD4+ and CD8+ T cell towards Raji B cells.
- Fig. 16 B-16E depict representations of the data in Fig. 16A normalized to human IgG, for v875 (Fig. 16B and Fig. 16C), and v1379 and v1380 (Fig. 16D and Fig. 16E), and include % cytotoxicity indicated at each test antibody concentration.
- Fig. 17A-17B Fig. 17A illustrates that Fc blocking of IL-2 activated PBMC results in a minor (v875) or no (v873) reduction in the % cytotoxicity of target Raji B cells.
- Figure 17B illustrates that Fc blocking of resting PBMC results in a reduction in the % cytotoxicity of target Raji B cells for v875 and v873.
- Fig. 18A-18B Fig. 18A illustrates that Fc blocking of IL-2 activated PBMC results in a reduction in the % cytotoxicity of target Raji B cells at all antibody concentrations tested for v875 and v873.
- Fig. 18B illustrates that Fc blocking of resting PBMC results in a reduction in the % cytotoxicity of target Raji B cells at all antibody concentrations tested for v875 and v873.
- Fig. 19A-19B Fig. 19A shows that v875 and v873 elicit >30% cytotoxicity to target Raji B cells with IL-2 activiated CD8+ T cells as effectors, and maximal target cell killing is seen at the 3 nM concentration.
- Fig. 19B shows that v875 and v873 elicit dose dependent (>20%) cytotoxicity to target Raji B cells with resting CD8+ T cells as effectors.
- Fig. 20A-20B Fig. 20A shows the target Raji B cell cytotoxicty of v875 with IL-2 activated CD4+ and CD8+ T cells.
- Fig. 20B shows the target Raji B cell cytotoxicty of v875 with resting CD4+ and CD8+ T cells.
- Fig. 21 shows, relative to untreated media
- PBMC and total IL-2 activated PBMC activated PBMC.
- Fig. 22 shows, relative to untreated media and human IgG controls, v875 has a more selective B cell killing by sparing more autologous T cells compared to v873 and v891.
- Fig. 23A-23D shows the effects of v875 on the viability of CD20+, CD4+, CD8+ subsets in IL-2 activated cell cultures.
- Fig. 23B shows the effects of v875 on the viability of CD20+, CD4+, CD8+ subsets resting cell cultures.
- Fig. 23C shows the effects of v1379 and v1380 on the viability of CD20+, CD4+, CD8+ subsets in IL-2 activated cell cultures.
- Fig. 23D shows shows the effects of v1379 and v1380 on the viability of CD20+, CD4+, CD8+ subsets resting cell cultures.
- Fig. 24A-24B Results of antibody mediated LDH release in resting effector and Raji B cells shown in Fig. 24A. Results of antibody mediated LDH release in activiated effector are shown in Figure 24B.
- Fig. 25A-25D Fig 25A illustrates the mediation of ADCC by rituximab and by a heteromultimer described herein with a WT Fc (v875) (ca. 40% max cell lysis).
- Fig 25B shows that v1379 which is a heteromultimer described herein with a WT Fc can mediate ADCC while v1380, with a
- FIG. 25C-25D show the results of the CDC assay with v1380 and v1379 (Fig. 25C) and v875 (Fig. 25D) of target Daudi B cells with comparisons to positive control Rituximab.
- Fig. 26 shows that at 0.3 nM, v875 and v1380 do not induce PBMC proliferation compared to human IgG.
- the lower panel of Fig. 26 shows the results of the 100 nM antibody concentrations, and shows that v875, v1380 and v891 induce higher cell proliferation relative to human IgG.
- Fig. 26 (lower panel) also shows that at 100 nM, v875 has a similar proliferative index compared to anti-CD3 OKT3 in all four PBMC populations.
- Fig. 27A-27E show that v1380 (L234AJJ
- Fig. 27A-27E The results from the cytokine release assay as shown in Fig. 27A-27E include summary plots of PBMC supernatant TNFa (Fig. 27A) INFy (Fig. 27B), IL-2 (Fig. 27C), IL-4 (Fig. 27D), and IL-10 (Fig. 27E) levels following incubation with test items at 0.3 nM concentrations for 4 days (graph y-axis represents log cytokine levels in pg per mL from 4 donors).
- Fig. 28A-28B show the results from the average stimulation index induced by v875 at 0.3nM (Fig. 28A) and 100 nM (Fig. 28B) concentrations on purified CD8+ T cells in the absence or presence of purified CD19+ B cells at 4 days incubation time-point.
- Fig. 29A-29B show the results from the average stimulation index induced by v1380 at 0.3nM (Fig. 29A) and 100nM (Fig. 29B) concentrations on purified CD8+ T cells in the absence or presence of purified CD19+ B cells at 4 days incubation time-point.
- Fig. 30A-30C show the results from the T:B cell bridging
- FIG. 30A shows a direct comparison of human IgG and v875 at 200X magnification and illustrates a higher amount of bridging visible between Raji B cell and Jurkat T cells compared to human IgG.
- Figure 30B and Figure 30C show two fields of view for v875 ( Figure 30B) and human IgG ( Figure 30C) at 400X magnification.
- Fig. 31 A-31 B shows the SDS-PAGE analysis and relative purity of v875, v1380, v1379 and v891 following protein A and SEC purification, and following 47 day storage at 4°C.
- Fig. 31 B shows the SDS-PAGE analysis and relative purity of additional exemplary hetermultimers including v875, v1653, v1654, v1655, v1656, v1660, v1800, and v1802 following protein A and SEC purification.
- Fig. 32 shows the LC-MS results of the Max Ent. molecular weight profiles for v875.
- Fig. 33A-33C show DSC results for heter
- Fig. 34A-C show ability of heteromultimers described herein to bridge Raji B and Jurkat T cells (B:T), as well RajkRaji B cell bridging (B:B) and JurkatJurkat T cell bridging (T:T) assessed by FACS.
- Fig. 34A shows the amount of T:B, B:B and T:T bridging of v875, v1379, v1380, v891 , v1381 , commercial OKT3 and human IgG over three experimental replicates.
- Fig. 34A shows the amount of T:B, B:B and T:T bridging of v875, v1379, v1380, v891 , v1381 , commercial OKT3 and human IgG over three experimental replicates.
- 34B shows the amount of T:B, B:B and T:T bridging of variants with engineered anti-CD3 warheads for stability enhancement (v1653, v1654, v1655, v1656, v1660, v1800, v1802) and v875, and human IgG.
- stability enhancement v1653, v1654, v1655, v1656, v1660, v1800, v1802
- v875 human IgG.
- 34C shows the amount of T:B, B:B and T:T bridging of Fc knock-out variants that have either engineered anti-CD3 warheads for stability enhancement (v1666), or have human/cynomolgous monkey cross-reactive anti-CD3 and anti-CD19 scFvs (v4541 , v4543, v4545, v4548) commercial OKT3 anti-CD3 control, v2176 anti- CD19 control and human IgG negative control, and all variants mediate low T:T bridging.
- engineered anti-CD3 warheads for stability enhancement v1666)
- human/cynomolgous monkey cross-reactive anti-CD3 and anti-CD19 scFvs v4541 , v4543, v4545, v4548
- Fig. 35 illustrates binding of heteromultimers described herein to human CD3 (top panel) and binding to the cynomologous CD3 receptor (bottom panel) as determined by ELISA.
- Fig. 36A-36B illustrates affinity on linear and log scales
- Fig. 37 depicts the binding of variant 1090 compared to the control 1087 in MALME-3M cells and indicates that v1090 has similar binding as v1087 to target MALME-3M cells.
- any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
- “about” means ⁇ 10% of the indicated range, value, sequence, or structure, unless otherwise indicated.
- the terms “a” and “an” as used herein refer to “one or more” of the enumerated components unless otherwise indicated or dictated by its context. The use of the alternative (e.g., "or”) should be understood to mean either one, both, or any combination thereof of the alternatives.
- amino acid names and atom names e.g.
- polypeptide peptide
- protein protein
- polypeptide peptide
- peptide protein
- the terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non-naturally encoded amino acid.
- the terms encompass amino acid chains of any length, including full length proteins, wherein the amino acid residues are linked by covalent peptide bonds.
- nucleotide sequence is intended to indicate a
- nucleotide sequence may be of genomic, cDNA, RNA, semisynthetic or synthetic origin, or any combination thereof.
- PCR polymerase chain reaction
- PCR generally refers to a method for amplification of a desired nucleotide sequence in vitro, as described, for example, in U.S. Pat. No. 4,683,195.
- the PCR method involves repeated cycles of primer extension synthesis, using oligonucleotide primers capable of hybridising preferentially to a template nucleic acid.
- Transformation and “transfection” are used interchangeably to refer to the process of introducing DNA into a cell.
- amino acid refers to naturally occurring and non- naturally occurring amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids
- Naturally encoded amino acids are the 20 comr
- arginine asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, praline, serine, threonine, tryptophan, tyrosine, and valine) and pyrrolysine and selenocysteine.
- Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, such as, homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium.
- Such analogs have modified R groups (such as, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
- Reference to an amino acid includes, for example, naturally occurring proteogenic L-amino acids; D-amino acids, chemically modified amino acids such as amino acid variants and derivatives; naturally occurring non-proteogenic amino acids such as ⁇ -alanine, ornithine, etc.; and chemically synthesized compounds having properties known in the art to be characteristic of amino acids.
- non-naturally occurring amino acids include, but are not limited to, a-methyl amino acids (e.g.
- a-methyl alanine D- amino acids, histidine-like amino acids (e.g., 2-amino-histidine, ⁇ -hydroxy- histidine, homohistidine), amino acids having an extra methylene in the side chain (“homo" amino acids), and amino acids in which a carboxylic acid functional group in the side chain is replaced with a sulfonic acid group (e.g., cysteic acid).
- non-natural amino acids including synthetic non-native amino acids, substituted amino acids, or one or more D-amino acids into the proteins of the present invention may be advantageous in a number of different ways.
- D-amino acid-containing peptides, etc. exhibit increased stability in vitro or in vivo compared to L-amino acid-containing counterparts.
- the construction of peptides, etc., incorporating D-amino acids can be particularly useful when greater intracellular stability is desired or required. More specifically, D-peptides, etc., are resistant to endogenous peptidases and proteases, thereby providing improved bioavailability of the molecule, and
- peptides, etc. cannot be processed efficiently for major histocompatibility complex class ll-restricted presentation to T helper cells, and are therefore, less likely to induce humoral immune responses in the whole organism.
- Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the lUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
- Constantly modified variants applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, “conservatively modified variants” refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
- nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
- silent variations are one species of conservatively modified variations.
- Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
- each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule.
- each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.
- amino acid sequences one of ordinary skill in the art will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a
- nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same. Sequences are “substantially identical” if they have a percentage of amino acid residues or nucleotides that are the same (i.e., about 50% identity, about 55% identity, 60% identity, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% identity over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms (or other algorithms available to persons of ordinary skill in the art) or by manual alignment and visual inspection. This definition also refers to the complement of a test sequence. The identity can exist over a region that is at least about 50 amino acids or nucleotides that are the same. Sequences are “substantially identical” if they have a percentage of amino acid residues or nucleotides that are the same (i.e., about 50% identity, about 55% identity, 60% identity,
- a polynucleotide encoding a polypeptide of the present invention may be obtained by a process comprising the steps of screening a library under stringent hybridization conditions with a labeled probe having a polynucleotide sequence of the invention or a fragment thereof, and isolating full-length cDNA and genomic clones containing said polynucleotide sequence.
- a derivative, or a variant of a polypeptide is said to share
- the derivative or variant is at least 75% the same as that of either the peptide or a fragment of the peptide having the same number of amino acid residues as the derivative. . In certain embodiments, the derivative or variant is at least 85% the same as that of either the peptide or a fragment of the peptide having the same number of amino acid residues as the derivative. In certain embodiments, the amino acid sequence of the derivative is at least 90% the same as the peptide or a fragment of the peptide having the same number of amino acid residues as the derivative.
- the amino acid sequence of the derivative is at least 95% the same as the peptide or a fragment of the peptide having the same number of amino acid residues as the derivative. In certain embodiments, the derivative or variant is at least 99% the same as that of either the peptide or a fragment of the peptide having the same number of amino acid residues as the derivative.
- bispecific is intended to include any agent, e.g., heteromultimer, monomer, protein, peptide, or protein or peptide complex, which has two different binding specificities.
- agent e.g., heteromultimer, monomer, protein, peptide, or protein or peptide complex, which has two different binding specificities.
- the molecule may bind to, or interact with, (a) a cell surface target molecule and (b) an Fc receptor on the surface of an effector cell.
- the monomer is bispecific formed by attaching to the same transporter polypeptide, two cargo molecules with different binding specificities.
- the heteromultimer is itself bispecific formed by attaching to the transporter polypeptides, at least two cargo molecules with different specificities.
- multispecific or “heterospecific" is intended to include any agent, e.g., a protein, peptide, or protein or peptide complex, which has more than two different binding specificities.
- the molecule may bind to, or interact with, (a) a cell surface target molecule such as but not limited to cell surface antigens, (b) an Fc receptor on the surface of an effector cell, and optionally (c) at least one other component.
- a cell surface target molecule such as but not limited to cell surface antigens
- an Fc receptor on the surface of an effector cell and optionally (c) at least one other component.
- embodiments of the heteromultimers described herein are inclusive of, but not limited to, bispecific, trispecific, tetraspecific, and other multispecific molecules.
- these molecules are directed to cell surface antigens, such as CD30, and to other targets, such as Fc receptors on effector cells.
- isolated heteromultimer means a heteromultimer that has been identified and separated and/or recovered from a component of its natural cell culture environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the heteromultimer, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.
- the phrase "selectively (or specifically) hybridizes to” refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (including but not limited to, total cellular or library DNA or RNA).
- an “antibody” or “immunoglobulin” refers to a polypeptide substantially encoded by an immunoglobulin gene or
- immunoglobulin genes or fragments thereof, which specifically bind and recognize an analyte (antigen).
- the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes.
- Light chains are classified as either kappa or lambda.
- Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively.
- An exemplary immunoglobulin (antibody) structural unit is composed of two pairs of polypeptide chains, each pair having one "light"
- variable light chain VL
- variable heavy chain VH
- the lgG1 heavy chain comprises of the VH, CH1 , CH2 and CH3 domains respectively from the N to C-terminus.
- the light chain comprises of the VL and CL domains from N to C terminus.
- the lgG1 heavy chain comprises a hinge between the CH1 and CH2 domains.
- the immunoglobulin constructs comprise at least one
- immunoglobulin domain from IgG, IgM, IgA, IgD, or IgE connected to a therapeutic polypeptide.
- the immunoglobulin domain from IgG, IgM, IgA, IgD, or IgE connected to a therapeutic polypeptide.
- the immunoglobulin domain from IgG, IgM, IgA, IgD, or IgE connected to a therapeutic polypeptide.
- the immunoglobulin based construct such as a diabody, or a nanobody.
- the immunoglobulin constructs described herein comprise at least one immunoglobulin domain from a heavy chain antibody such as a camelid antibody.
- the immunoglobulin constructs provided herein comprise at least one immunoglobulin domain from a mammalian antibody such as a bovine antibody, a human antibody, a camelid antibody, a mouse antibody or any chimeric antibody.
- antigenic determinant is synonymous with “antigen” and “epitope,” and refers to a site (e.g. a contiguous stretch of amino acids or a conformational configuration made up of different regions of non-contiguous amino acids) on a polypeptide macromolecule to which an antigen binding moiety binds, forming an antigen binding moiety-antigen complex.
- Useful antigenic determinants can be found, for example, on the surfaces of tumor cells, on the surfaces of virus-infected cells, on the surfaces of other diseased cells, on the surface of immune cells, free in blood serum, and/or in the extracellular matrix (ECM).
- ECM extracellular matrix
- MCSP, FAP, CEA, EGFR, CD33, CD3 can be any native form the proteins from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g. mice and rats), unless otherwise indicated.
- the antigen is a human protein.
- the term encompasses the "full-length", unprocessed protein as well as any form of the protein that results from processing in the cell.
- the term also encompasses naturally occurring variants of the protein, e.g. splice variants or allelic variants.
- Exemplary human proteins useful as antigens include, but are not limited to: Melanoma-associated
- MCSP Chondroitin Sulfate Proteoglycan
- Proteoglycan 4 (UniProt no. Q6UVK1 (version 70), NCBI RefSeq no. NP
- FAP Fibroblast Activation Protein
- Carcinoembroynic antigen also known as Carcinoembryonic antigen-
- CD33 also known as gp67 or Siglec-3 (UniProt no. P20138, NCBI Accession nos. NP 001076087, NP 001 171079); Epidermal Growth Factor Receptor (EGFR), also known as ErbB-1 or Herl (UniProt no. P0053, NCBI Accession nos. NP 958439, NP 958440), and CD3, particularly the epsilon subunit of CD3 (see UniProt no. P07766 (version 130), NCBI RefSeq no. NP 000724.1 , SEQ ID NO: 265 for the human sequence; or UniProt no.
- the T cell activating bispecific antigen binding molecule of the invention binds to an epitope of an activating T cell antigen or a target cell antigen that is conserved among the activating T cell antigen or target antigen from different species.
- binding is selective for the antigen and can be discriminated from unwanted or nonspecific interactions.
- the ability of an antigen binding moiety to bind to a specific antigenic determinant can be measured either through an enzyme- linked immunosorbent assay (ELISA) or other techniques familiar to one of skill in the art, e.g. surface plasmon resonance (SPR) technique (analyzed on a BIAcore instrument) (Liljeblad et al, Glyco J 17, 323-329 (2000)), and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)).
- ELISA enzyme- linked immunosorbent assay
- SPR surface plasmon resonance
- an antigen binding moiety that binds to the antigen, or an antigen binding molecule comprising that antigen binding moiety has a dissociation constant (K D ) of ⁇ 1 ⁇ , ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, ⁇ 0.1 nM, ⁇ 0.01 nM, or ⁇ 0.001 nM (e.g. 10 ⁇ 8 M or less, e.g. from 10 ⁇ 8 M to 10 "13 M, e.g., from 10 "9 M to 10 "13 M).
- K D dissociation constant
- Affinity refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., a receptor) and its binding partner (e.g., a ligand). Unless indicated otherwise, as used herein,
- binding affinity refers to intrinsic binding affinity which reflects a 1 : 1
- the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (K D ), which is the ratio of dissociation and association rate constants (k 0ff and k on , respectively).
- K D dissociation constant
- equivalent affinities may comprise different rate constants, as long as the ratio of the rate constants remains the same.
- Affinity can be measured by well established methods known in the art, including those described herein. A particular method for measuring affinity is Surface Plasmon Resonance (SPR).
- Reduced binding for example reduced binding to an Fc receptor, refers to a decrease in affinity for the respective interaction, as measured for example by SPR.
- the term includes also reduction of the affinity to zero (or below the detection limit of the analytic method), i.e. complete abolishment of the interaction.
- increased binding refers to an increase in binding affinity for the respective interaction.
- an "activating T cell antigen” as used herein refers to an antigenic determinant expressed on the surface of a T lymphocyte, particularly a cytotoxic T lymphocyte, which is capable of inducing T cell activation upon interaction with an antigen binding molecule. Specifically, interaction of an antigen binding molecule with an activating T cell antigen may induce T cell activation by triggering the signaling cascade of the T cell receptor complex. In a particular embodiment the activating T cell antigen is CD3.
- T cell activation refers to one or more cellular response of a T lymphocyte, particularly a cytotoxic T lymphocyte, selected from: proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers.
- T cell activating bispecific antigen binding molecules of the invention are capable of inducing T cell activation.
- Suitable assays to measure T cell activation are known in the art described herein.
- a "target cell antigen” as used herein refers to an antigenic determinant presented on the surface of a target cell, for example a B cell in a
- 31 WSGF tumor such as a cancer cell or a cell of the tumor stror
- first and second with respect to antigen binding moieties etc., are used for convenience of distinguishing when there is more than one of each type of moiety. Use of these terms is not intended to confer a specific order or orientation of the T cell activating bispecific antigen binding molecule unless explicitly so stated.
- a “Fab molecule” refers to a protein consisting of the VH and CH1 domain of the heavy chain (the “Fab heavy chain”) and the VL and CL domain of the light chain (the “Fab light chain”) of an immunoglobulin.
- fused is meant that the components (e.g. a Fab molecule and an Fc domain subunit) are linked by peptide bonds, either directly or via one or more peptide linkers.
- single-chain refers to a molecule comprising amino acid monomers linearly linked by peptide bonds.
- one of the antigen binding moieties is a single-chain Fab molecule, i.e. a Fab molecule wherein the Fab light chain and the Fab heavy chain are connected by a peptide linker to form a single peptide chain.
- the C-terminus of the Fab light chain is connected to the N-terminus of the Fab heavy chain in the single-chain Fab molecule.
- one of the antigen binding moieties is a single- chain Fv molecule
- crossover Fab molecule also termed “Crossfab” is meant a crossover Fab molecule
- the crossover Fab molecule comprises a peptide chain composed of the light chain variable region and the heavy chain constant region, and a peptide chain composed of the heavy chain variable region and the light chain constant region.
- the crossover Fab molecule comprises a peptide chain composed of the light chain variable region and the heavy chain constant region, and a peptide chain composed of the heavy chain variable region and the light chain constant region.
- peptide chain comprising the heavy chain variable region is referred to herein as the "heavy chain” of the crossover Fab molecule.
- FR refers to variable domain residues other than hypervariable region (HVR) residues.
- the FR of a variable domain generally consists of four FR domains: FR1 , FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1 -H1 (L1 )-FR2-H2(L2)-FR3-H3(L3)-FR4.
- the "class" of an antibody or immunoglobulin refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE,
- IgG, and IgM may be further divided into subclasses (isotypes), e.g., IgGi, lgG 2 , lgG 3 , lgG , IgAi, and lgA 2 .
- immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
- Fc domain or "Fc region” herein is used to define a C- terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
- the term includes native sequence Fc regions and variant Fc regions.
- the boundaries of the Fc region of an IgG heavy chain might vary slightly, the human IgG heavy chain Fc region is usually defined to extend from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain.
- the C-terminal lysine (Lys447) of the Fc region may or may not be present.
- numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health
- a "subunit" of an Fc domain as used herein refers to one of the two polypeptides forming the dimeric Fc domain, i.e. a polypeptide comprising C-terminal constant regions of an immunoglobulin heavy chain, capable of stable self-association.
- a subunit of an IgG Fc domain comprises ar
- a "modification promoting the association of the first and the second subunit of the Fc domain” is a manipulation of the peptide backbone or the post-translational modifications of an Fc domain subunit that reduces or prevents the association of a polypeptide comprising the Fc domain subunit with an identical polypeptide to form a homodimer.
- a modification promoting association as used herein particularly includes separate modifications made to each of the two Fc domain subunits desired to associate (i.e. the first and the second subunit of the Fc domain), wherein the promote association of the two Fc domain subunits and the formation of heterodimers.
- a modification promoting association may alter the structure or charge of one or both of the Fc domain subunits so as to make their
- effector functions refers to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype.
- antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody- dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (AD CP), cytokine secretion, immune complex-mediated antigen uptake by antigen presenting cells, down regulation of cell surface receptors (e.g. B cell receptor), and B cell activation.
- albumin refers collectively to albumin protein or amino acid sequence, or an albumin segment or variant, having one or more functional activities (e.g., biological activities) of albumin.
- albumin refers to human albumin or segments thereof (see for example, EP 201 239, EP 322 094 WO 97/24445, WO95/23857) especially the mature form of human albumin, or albumin from other vertebrates, or segments thereof, or analogs or variants of these molecules or fragments thereof.
- albumin refers to a truncated version of albumin.
- quadsi-albumin refers to a heter
- the monomeric polypeptides are "segments" that preferentially associate as heteromultimeric pairs to form a quasi-protein.
- the quasi-albumin has 90% of the activity of the whole albumin. In some embodiments, the quasi-albumin has 75% of the activity of whole- albumin. In an embodiment, the quasi-albumin has 50% of the activity of whole albumin. In some embodiments, the quasi-albumin has 50-75% of the activity of whole albumin. In an embodiment, quasi-albumin has 80% of the activity of whole albumin. In some embodiments, the quasi-albumin has 90% of the structure of whole albumin as determined by molecular modeling. In some embodiments, the quasi-albumin has 80% of the structure of whole albumin as determined by molecular modeling. In some embodiments, the quasi-albumin has 70% of the structure of whole albumin as determined by molecular modeling. In some embodiments, the quasi-albumin has 50% of the structure of whole albumin as determined by molecular modeling. In some embodiments, the quasi-albumin has 50%-75% of the structure of whole albumin as determined by molecular modeling.
- HSA human serum albumin
- HA human albumin
- albumin and serum albumin are broader, and encompass human serum albumin (and fragments and variants thereof) as well as albumin from other species (and fragments and variants thereof).
- each albumin-based construct described herein is based on a variant of normal HA.
- variants includes insertions, deletions and substitutions, either conservative or non conservative, where such changes do not substantially alter one or more of the oncotic, useful ligand-binding and non-immunogenic properties of albumin, or the active
- the isolated heteromultimeric constructs described herein include naturally occurring polymorphic variants of human albumin and fragments of human albumin, for example those fragments disclosed in EP 322 094 (namely HA (Pn), where n is 369 to 419).
- the albumin is derived from any vertebrate, especially any mammal that includes but is not limited to human, cow, sheep, rat, mouse, rabbit, horse, dog or pig. In certain embodiments, the albumin is derived from non-mammalian albumins including, but are not limited to hen and salmon.
- an "alloalbumin” is a genetic variant of albumin.
- the alloalbumin is human alloalbumin (HAA).
- HAA human alloalbumin
- Alloalbumins that differ in electrophoretic mobility from albumin have been identified through population genetics surveys in the course of clinical electrophoresis, or in blood donor surveys. As markers of mutation and migration, alloalbumins are of interest to geneticists, biochemists, and anthropologists, but most of these
- Table 1 List of substitutions comprised by various alloalbumins as compared to HA of SEQ ID NO: 1. Thermostability, half-life information and other HAAs are provided in Krogh-hansen et al. Biochim Biophys Acta 1747, 81 -88(
- segmentation refers to a precise internal splice of the original protein sequence which results in “segments” of the protein sequence that preferentially associate as heteromultimers to form a quasi-protein.
- proteins and/or 'quasi-native structures' present the native protein like functional and structural characteristics. Proteins are naturally dynamics molecules and display an ensemble of structural configurations although we ascribe a native structure to it, such as the one obtained by X-ray
- the reference native protein in this case is the protein from which the transporter polypeptide is derived and the reference native structure is the structure of the monomeric protein from which the transporter polypeptide is derived.
- the reference native structure is the structure of the monomeric protein from which the transporter polypeptide is derived.
- two or more different polypeptides self-assemble to form a heteromultimeric structural and exhibit functional characteristics like a native protein which itself is a monomeric entity.
- heteromultimer constructs comprising transporter polypeptides derived from albumin that self-assemble to form a heteromultimer that exhibits native albumin like functional characteristics such as FcRn binding and structural characteristics. These heteromultimers are referred to as being quasi-native.
- CD3 complex as described herein is a complex of at least five membrane-bound polypeptides in mature T-lymphocytes that are non- covalently associated with one another and with the T-cell receptor.
- the CD3 complex includes the gamma, delta, epsilon, zeta, and eta chains (also referred to as gamma, delta, epsilon, zeta, and eta chains (also referred to CD3 complex).
- CD19 has proved to be a very useful target.
- CD19 is expressed in the whole B lineage from the pro B cell to the mature B cell, it is not shed, is uniformly expressed on all lymphoma cells, and is absent from stem cells.
- compositions containing a multispecific heteromultimeric construct described herein can be administered for prophylactic, enhancing, and/or therapeutic treatments.
- engineered, engineered, engineering are considered to include any manipulation of the peptide backbone or the post- translational modifications of a naturally occurring or recombinant polypeptide or fragment thereof.
- Engineering includes modifications of the amino acid sequence, of the glycosylation pattern, or of the side chain group of individual amino acids, as well as combinations of these approaches.
- the engineered proteins are expressed and produced by standard molecular biology
- isolated nucleic acid molecule or polynucleotide is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment.
- a recombinant polynucleotide encoding a polypeptide contained in a vector is considered isolated.
- Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in
- An isolated polynucleotide includes a polynucleotide molecule contained in cells that ordinarily contain the polynucleotide molecule, but the polynucleotide molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
- Isolated RNA molecules include in vivo or in vitro RNA transcripts, as well as positive and negative strand forms, and double-stranded forms. Isolated polynucleotides or nucleic acids described herein, further include such molecules produced synthetically.
- a polynucleotide or a nucleic acid in certain embodiments, include a regulatory element such as a promoter, ribosome binding site, or a transcription terminator.
- nucleic acid or polynucleotide having a nucleotide sequence at least, for example, 95% "identical" to a reference nucleotide sequence of the present invention it is intended that the nucleotide sequence of the
- polynucleotide is identical to the reference sequence except that the
- polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence.
- up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
- These alterations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions,
- any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the present invention can be determined conventionally using known computer programs, such as the ones discussed above for polypeptides (e.g. ALIGN-2).
- expression cassette refers to a
- the recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment.
- the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter.
- the expression cassette of the invention comprises polynucleotide sequences that encode bispecific antigen binding molecules of the invention or fragments thereof.
- vector or "expression vector” is synonymous with
- expression construct refers to a DNA molecule that is used to introduce and direct the expression of a specific gene to which it is operably associated in a target cell.
- the term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced.
- the expression vector of the present invention comprises an expression cassette. Expression vectors allow transcription of large amounts of stable mRNA. Once the expression vector is inside the target cell, the ribonucleic acid molecule or protein that is encoded by the gene is produced by the cellular transcription and/or translation machinery.
- the expression vector of the invention comprises an expression cassette that comprises polynucleotide sequences that encode bispecific antigen binding molecules of the invention or fragments thereof.
- host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
- Host cells include
- transformants and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages.
- progeny are not completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the
- Host cells include cultured cells, e.g. mammalian cultured cells, such as CHO cells, BHK cells, NSO cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, insect cells, and plant cells, to name only a few, but also cells comprised within a transgenic animal, transgenic plant or cultured plant or animal tissue.
- mammalian cultured cells such as CHO cells, BHK cells, NSO cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, insect cells, and plant cells, to name only a few, but also cells comprised within a transgenic animal, transgenic plant or cultured plant or animal tissue.
- An "activating Fc receptor” is an Fc receptor that following engagement by an Fc domain of an antibody elicits signaling events that stimulate the receptor-bearing cell to perform effector functions.
- Human activating Fc receptors include FcyRllla (CD 16a), FcyRI (CD64), and FcyRlla (CD32).
- Antibody-dependent cell-mediated cytotoxicity is an immune mechanism leading to the lysis of antibody-coated target cells by immune effector cells.
- the target cells are cells to which antibodies or derivatives thereof comprising an Fc region specifically bind, generally via the protein part that is N-terminal to the Fc region.
- ADCC antibody-dependent cell-mediated cytotoxicity
- reduced ADCC is defined as either a reduction in the number of target cells that are lysed in a given time, at a given concentration of antibody in the medium surrounding the target cells, by the mechanism of ADCC defined above, and/or an increase in the concentration of antibody in the medium surrounding the target cells, required to achieve the lysis of a given number of target cells in a given time, by the mechanism of ADCC.
- ADCC is relative to the ADCC mediated by the same antibody produced by the same type of host cells, using the same standard production, purification, formulation and storage methods (which are known to those skilled in the art), but that has not been engineered.
- the reduction in ADCC mediated by an antibody comprising in its Fc domain an amino acid substitution that reduces ADCC is relative to the ADCC mediated by the same antibody without this amino acid substitution in the Fc domain.
- heteromultimer described herein refers to the amount that is necessary to result in a physiological change in the cell or tissue to which it is administered.
- a "therapeutically effective amount" of an agent e.g. a
- composition comprising a multispecific heteromultimer described herein, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
- a therapeutically effective amount of an agent for example eliminates, decreases, delays, minimizes or prevents adverse effects of a disease.
- mammals include, but are not limited to, domesticated animals (e.g. cows, sheep, cats, dogs, and horses), primates (e.g. humans and non-human primates such as monkeys), rabbits, and rodents (e.g. mice and rats). Particularly, the individual or subject is a human.
- domesticated animals e.g. cows, sheep, cats, dogs, and horses
- primates e.g. humans and non-human primates such as monkeys
- rabbits e.g. mice and rats
- rodents e.g. mice and rats
- composition refers to a preparation which is in such form as to permit the biological activity of a multispecific heteromultimer construct contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
- a "pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical composition, other than an active ingredient, which is nontoxic to a subject.
- a pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
- treatment refers to clinical intervention in an attempt to alter the natural course of a disease in the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology.
- Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or
- multispecific heteromultimer constructs described herein are used to delay development of a disease or to slow the progression of a disease.
- instructions is used to refer to instructions customarily included in commercial packages of therapeutic products that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.
- cross-species binding means binding of a binding domain described herein to the same target molecule in humans and other organisims for instance, but not restricted to non-chimpanzee primates.
- cross-species binding or “interspecies binding” is to be understood as an interspecies reactivity to the same molecule "X” (i.e. the homolog) expressed in different species, but not to a molecule other than "X”.
- macaque CD3 epsilon can be determined, for instance, by FACS analysis.
- the FACS analysis is carried out in a way that the respective monoclonal antibody is tested for binding to human and non-chimpanzee primate cells, e.g.
- macaque PSCA, CD19, C-MET, Endosialin, EpCAM, IGF-1 R or FAPa can be determined, for instance, by FACS analysis.
- the FACS analysis is carried out in a way that the respective monoclonal antibody is tested for binding to human and non-chimpanzee primate cells, e.g. macaque cells,
- Endosialin, EpCAM, IGF-1 R or FAPa antigens were endosialin, EpCAM, IGF-1 R or FAPa antigens, respectively.
- isolated multispecific heteromultimer constructs comprising a first polypeptide construct comprising a first heavy chain polypeptide and a CD3 binding polypeptide construct that binds to a CD3 complex on at least one CD3 expressing cell; and a second polypeptide construct comprising a second heavy chain polypeptide which is different from said first heavy chain polypeptide, and an antigen binding polypeptide construct that binds to a target antigen on at least one B cell, wherein:at least one of said
- CD3 binding polypeptide construct and said antigen binding polypeptide construct comprises a single chain Fv region; said multispecific heteromultimer construct simultaneously engages said at least one B cell and said at least one
- CD3 expressing cell such that the CD3 expressing cell is activated, thereby inducing killing of the B cell; and said first and second heavy chain polypeptides form a heterodimeric Fc region comprising a variant immunoglobulin CH3 region comprising at least one amino acid mutation that promotes the formation of said heterodimeric Fc which is formed with stability at least comparable to a native homodimeric Fc, and with purity such that when said multispecific heteromultimer construct is expressed from a mammalian cell in an expression product, said expression product comprises at least about 70% of said multispecific heteromultimer, and less than 10% monomers or homodimers of said first or second polypeptide constructs.
- the expression product comprises at least about 75% of said multispecific heteromultimer, and less than 15% monomers or homodimers of said first or second polypeptide constructs. In certain embodiments, the expression product comprises at least about 80% of said multispecific heteromultimer, and less than 10% monomers or homodimers of said first or second polypeptide
- expression product comprises at least about 90% of said multispecific heteromultimer, and less than 10% monomers or homodimers of said first or second polypeptide constructs.
- [00173] is the isolated multispecific heteromultimer construct, wherein said first or second polypeptide construct is devoid of at least one of immunoglobulin light chain, and immunoglobulin first constant (CH1 ) region.
- an isolated multispecific heteromultimer construct comprising: a first polypeptide construct comprising a first heavy chain polypeptide and a CD3 binding polypeptide construct that binds to a CD3 complex on at least one CD3 expressing cell; a second polypeptide construct comprising a second heavy chain polypeptide which is different from said first heavy chain polypeptide, and wherein said second polypeptide construct does not comprise an antigen binding polypeptide construct that binds to a target antigen on a B cell; wherein: said multispecific heteromultimer construct simultaneously engages at least one B cell and said at least one CD3 expressing cell such that the CD3 expressing cell is activated, thereby inducing killing of the B cell; and said first and second heavy chain polypeptides form a heterodimeric Fc region comprising a variant
- immunoglobulin CH3 region comprising at least one amino acid mutation that promotes the formation of said heterodimeric Fc, wherein: said heterodimeric
- Fc is formed with stability at least comparable to a native homodimeric Fc, and said heterodimeric Fc is formed with purity such that when said multispecific heteromultimer construct is expressed from a stable mammalian cell in an expression product, said expression product comprises at least about 75% of said multispecific heteromultimer, and less than 10% monomers or
- the heterodimeric Fc interacts with cell surface receptors such as
- an isolated multispecific heteromultimer construct comprising: a first polypeptide construct comprising a first heavy chain polypeptide and a CD3 binding polypeptide construct that binds to a CD3 complex on at least one CD3 expressing cell; a second polypeptide construct comprising a second heavy chain polypeptide which is different from said first heavy chain polypeptide, and a steric modulator construct which exhibits negligible receptor binding; wherein: said multispecific heteromultimer construct simultaneously engages at least one B cell and said at least one CD3 expressing cell such that the CD3 expressing cell is activated, thereby inducing killing of the B cell; and said first and second heavy chain polypeptides form a heterodimeric Fc region comprising a variant immunoglobulin CH3 region comprising at least one amino acid mutation that promotes the formation of said heterodimeric Fc, wherein: said heterodimeric Fc is formed with stability at least comparable to a native homodimeric Fc, and said heterodimeric F
- the steric modulator construct is a polypeptide sequence that helps modulate sterical features of the multispecific
- heteromultimer as the multimer binds to T and/or B cells.
- the steric modulator construct comprises a polypeptide domain that is designed de-novo.
- the steric modulator construct comprises polypeptide domains obtained by engineering a known
- the steric modulator construct comprises an engineered Fab region or fragment thereof which is engineered to remove binding properties.
- an isolated multispecific heteromultimer construct comprising: a first polypeptide construct comprising a first heavy chain polypeptide and a CD3 binding polypeptide construct that binds to a CD3 complex on at least one CD3 expressing cell; a second polypeptide construct comprising a second heavy chain polypeptide which is different from said first heavy chain polypeptide, and wherein said second polypeptide construct does not comprise an antigen binding polypeptide construct; wherein: said multispecific heteromultimer construct simultaneously engages at least one B cell and said at least one CD3 expressing cell such that the CD3 expressing cell is activated, thereby inducing killing of the B cell; and said first and second heavy chain polypeptides form a heterodimeric Fc region comprising a variant immunoglobulin CH3 region comprising at least one amino acid mutation that promotes the formation of said heterodimeric Fc, wherein: said heterodimeric
- Fc is formed with stability at least comparable to a native homodimeric Fc, and said heterodimeric Fc is formed with purity such that when said multispecific heteromultimer construct is coexpressed from a stable mammalian cell in an expression product, said expression product comprises at least about 75% of said multispecific heteromultimer, and less than 10% monomers or
- heterodimer Fc region comprises a variant CH2 domain or hinge comprising amino acid modifications that prevents functionally effective binding to all the Fcgamma receptors.
- the multispecific heteromultimer constructs that bind at least one B cell with a valency greater than one, and simultaneously engage said at least one B cell and at least one CD3 expressing cell such that the CD3 expressing cell is activated, thereby inducing killing of the B cell.
- binding polypeptide construct that binds to a CD3 complex on at least one CD3 expressing cell; and a second polypeptide construct comprising a second heavy chain polypeptide which is different from said first heavy chain polypeptide, and an antigen binding polypeptide construct that binds to a target antigen on at least one B cell; wherein: at least one of said CD3 binding polypeptide construct and said antigen binding polypeptide construct optionally comprises a single chain Fv region; said first and second heavy chain polypeptides form a heterodimeric Fc region comprising a variant
- immunoglobulin CH3 region comprising at least one amino acid mutation that promotes the formation of said heterodimeric Fc with stability at least comparable to a native homodimeric Fc, and with purity such that when said multispecific heteromultimer construct is expressed from a mammalian cell in an expression product, said expression product comprises greater than 75% of said multispecific heteromultimer, and less than 10% monomers or
- multispecific heteromultimer construct is capable of interacting with the B-cell via the said antigen binding polypeptide construct on the second heavy chain as well as interaction via the said heterodimeric Fc with FcgRllb receptors on the B-cell to show valency greater than one during B-cell engagement.
- heterodimer Fc region comprises a variant CH2 domain comprising amino acid modifications to promote selective binding of a Fcgamma receptor.
- the heteromultimer wherein the variant CH2 domain selectively binds at least one of Fcgammallla and Fcgammallb receptor as compared to wild-type CH2 domain.
- [00181] is an isolated multispecific heteromultimer construct described herein wherein the heterodimer Fc is glycosylated.
- [00182] is an isolated multispecific heteromultimer described herein, wherein the heterodimer Fc is afucosylated.
- Provded herein are multispecific heteromultimer comstructs that comprise different antigen binding moieties, fused to one or the other of the two subunits of the Fc domain, thus the two subunits of the Fc domain are typically comprised of two non-identical polypeptide chains.
- polypeptides is modified to promote the association of the desired polypeptides.
- the first and second heavy chain are identical to each other.
- polypeptides of the heteromultimer constructs described herein form a heterodimeric Fc region comprising a variant immunoglobulin CH3 region comprising at least one amino acid mutation that promotes the formation of said heterodimeric Fc which is formed with stability at least comparable to a native homodimeric Fc, and with purity such that when said multispecific heteromultimer construct is coexpressed from a mammalian cell as an expression product, said expression product comprises at least about 75% of said multispecific heteromultimer, and less than 10% monomers or
- the first and second heavy chain are identical to each other.
- polypeptides of the heteromultimer constructs described herein form a heterodimeric Fc region comprising a variant immunoglobulin CH3 region comprising at least one amino acid mutation that promotes the formation of said heterodimeric Fc which is formed with stability at least comparable to a native homodimeric Fc, and with purity such that when said multispecific heteromultimer construct is co-expressed from a mammalian cell as an expression product, said expression product comprises at least about 90% of said multispecific heteromultimer, and less than 10% monomers or
- the first and secon are identical to each other.
- polypeptides of the heteromultimer constructs described herein form a heterodimeric Fc region comprising a variant immunoglobulin CH3 region comprising at least one amino acid mutation that promotes the formation of said heterodimeric Fc which is formed with stability at least comparable to a native homodimeric Fc, and with purity such that when said multispecific heteromultimer construct is co-expressed from a mammalian cell as an expression product, said expression product comprises at least about 95% of said multispecific heteromultimer, and less than 10% monomers or
- the isolated multispecific heteromultimer provided herein, wherein the variant CH3 domain has a melting temperature (Tm) of about 73°C or greater.
- [00191] in certain embodiments is the isolated multispecific heteromultimer described herein, wherein the heterodimer Fc region is formed with a purity greater than about 78%.
- the isolated multispecific heteromultimer described herein wherein the heterodimer Fc region is formed with a purity of at least about 78% or greater and the Tm is at least about 75°C.
- the isolated multispecific heteromultimer described herein wherein the heterodimer Fc region is formed with a purity of at least about 75% and the Tm is about 75°C or greater.
- isolated multispecific heteromultimer constructs wherein: a) the variant CH3 sequence of the first heavy chain polypeptide comprises the amino acid modifications L351 Y,
- the variant CH3 sequence of the second transporter polypeptide comprises the amino acid modifications T366L, K392M, and
- the variant CH3 sequence of the first heavy chain polypeptide comprises the amino acid modifications L351Y, F405A, and Y407V
- the variant CH3 sequence of the second heavy chain polypeptide comprises the amino acid modifications T366L, K392L, and T394W
- the variant CH3 sequence of the second heavy chain polypeptide comprises the amino acid modifications T350V, T366L, K392M, and T394W; d) the variant CH3 sequence of the first heavy chain polypeptide comprises the amino acid modifications T350V, L351Y, F405A, and Y407V, and the variant CH3 sequence of the second heavy chain polypeptide comprises the amino acid modifications T350V, T366L, K392L, and T394W; e) the variant CH3 sequence of the first heavy chain polypeptide comprises the amino acid modifications T366L, N390R, K392R, and T394W, and the variant CH3 sequence of the second heavy chain polypeptide comprises the amino acid modifications L351 Y, S400E, F405A, and Y407V; or f) the variant CH3 sequence of the first heavy chain polypeptide comprises the amino acid modifications T350V, T366L
- the Fc regions of the heteromultimer constructs described herein comprises a variant CH2 domain comprising amino acid modifications to promote selective binding of a Fcgamma receptor.
- the isolated multispecific heteromultimer described herein comprise a variant CH2 domain that selectively binds a Fcgammallb receptor with an affinity greater than that of the wild-type CH2 domain.
- the isolated multispecific heteromultimer described herein comprise a variant CH2 domain that selectively binds a FcgammallA and/or FcgammalllA receptor with an affinity greater than that of the wild-type CH2 domain.
- the Fc regions of the heteromultimer constructs described herein exhibit reduced binding affinity to an Fc receptor
- the Fc region exhibits less than 50%, alternatively less than 20%, alternatively less than 10% and in some embodiments, less than 5% of the binding affinity to an Fc receptor, as compared to a native lgG1 Fc region, and/or less than 50%, alternatively less than 20%, alternatively less than 10% and in some embodiments less than 5% of the effector function, as compared to a native lgG1 Fc region.
- the Fc region of a heteromutlimer construct described herein does not substantially bind to an Fc receptor or induce appreciable effector function.
- the Fc receptor is an Fey receptor.
- the Fc receptor is a mammalian Fc receptor.
- the mammalian Fc receptor is a human Fc receptor.
- the Fc receptor is an activating Fc receptor.
- the Fc receptor is an activating human Fey receptor, more specifically human FcyRllla, FcyRI or FcyRlla, most specifically human
- the effector function is one or more function selected from the group consisting of CDC, ADCC, ADCP, and cytokine secretion. In a particular embodiment the effector function is ADCC.
- the Fc region exhibits binding affinity to neonatal Fc receptor (FcRn). In certain embodiments, the FcRn binding affinity is substantially similar to that of a native lgG1 Fc. In some embodiments, substantially similar binding to FcRn is achieved when the Fc region of a heteromultimer construct described herein exhibits greater than about 70%, or in some embodiments greater than about 80%, and in some particular embodiments greater than about 90% of the binding affinity of a native lgG1 Fc domain to FcRn.
- the Fc region of a heteromultimer construct described herein is engineered to have reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a non-engineered Fc domain.
- the engineered mutations are present in the lower hinge and CH2 domain.
- the Fc region of a heteromultimer construct described herein is engineered to have reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a non-engineered Fc domain.
- the engineered mutations are present in the lower hinge and CH2 domain.
- the Fc region of a heteromultimer construct described herein is engineered to have reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a non-engineered Fc domain.
- the engineered mutations are present in the lower hinge and CH2 domain.
- 55 WSGF heteromultimer described herein comprises one or mc
- the same one or more amino acid mutation is present in each of the two subunits of the Fc region. In some embodiments, different amino acid mutations are introduced in each of the two subunits of the Fc region. In one embodiment the amino acid mutation reduces the binding affinity of the Fc region to an Fc receptor. In one embodiment the amino acid mutation reduces the binding affinity of the Fc region to an Fc receptor by at least 2-fold, or in some embodiments at least 5-fold, or in an embodiment at least 10-fold.
- the combination of these amino acid mutations reduces the binding affinity of the Fc region to an Fc receptor by at least 10-fold, or in some embodiments at least 20-fold, or in certain embodiments at least 50-fold.
- the binding affinity of the Fc region for the Fc receptor is reduced to an extent where there is no longer any detectable binding for the mutant Fc for the Fc receptor in standard binding assay such as using the SPR instrument.
- the heteromultimer construct described herein comprising an engineered Fc domain exhibits less than 20%, and in certain embodiments less than 10%, and in select embodiments less than 5% of the binding affinity to an Fc receptor as compared to a corresponding construct comprising an Fc domain which is not engineered to reduce binding to an Fc receptor.
- the Fc receptor is an Fey receptor.
- the Fc receptor is a human Fc receptor.
- the Fc receptor is an activating Fc receptor.
- the Fc receptor is an activating human Fey receptor which in certain embodiments is one of human FcyRllla, FcyRI and FcyRlla.
- binding to each of these receptors is reduced.
- binding affinity to a complement component for instance, but not restricted to C1 q, is also reduced.
- binding affinity to a complement component for instance, but not restricted to C1 q
- FcRn 56 WSGF neonatal Fc receptor
- the reduced effector function can include, but is not limited to, one or more of the following: reduced complement dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen- presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling inducing apoptosis, reduced crosslinking of target- bound antibodies, reduced dendritic cell maturation, or reduced T cell priming.
- CDC reduced complement dependent cytotoxicity
- ADCC reduced antibody-dependent cell-mediated cytotoxicity
- ADCP reduced antibody-dependent cellular phagocytosis
- reduced immune complex-mediated antigen uptake by antigen- presenting cells reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling
- the reduced effector function is one or more selected from the group of reduced CDC, reduced ADCC, reduced ADCP, and reduced cytokine secretion. In certain embodiments the reduced effector function is reduced ADCC. In one embodiment the reduced ADCC is less than 20% of the ADCC induced by a non-engineered Fc domain (or a T cell activating bispecific antigen binding molecule comprising a non-engineered Fc domain). In another embodiment the reduced ADCC is less than 50% of the ADCC induced by a non-engineered Fc domain (or a T cell activating bispecific antigen binding molecule comprising a non-engineered Fc domain). In a further embodiment the reduced ADCC is less than 10% of the ADCC induced by a non-engineered Fc domain (or a T cell activating bispecific antigen binding molecule comprising a non-engineered Fc domain).
- heterodimer Fc region comprises a variant CH2 domain comprising amino acid modifications to promote selective binding of a Fcgamma receptor as described herein.
- the multispecific heteromultimer construct comprises a variant CH2 region that binds at least one B cell such that the heteromultimer construct binds B cells with a valency greater than one.
- isolated multispecific heteromultimer constructs comprising: a first polypeptide construct comprising a first transporter polypeptide fused to at least one CD3 binding polypeptide construct that binds to a CD3 complex on at least one CD3 expressing cell; a second polypeptide construct comprising a second transporter polypeptide which is different from said first transporter polypeptide, fused to at least one antigen binding polypeptide construct that binds to a target antigen on at least one B cell;
- each transporter polypeptide comprises an amino acid sequence with at least 85% identity to a segment of the protein.
- each transporter polypeptide comprises an amino acid sequence with at least 80% identity to a segment of the protein.
- each transporter polypeptide comprises an amino acid sequence with at least 95% identity to a segment of the protein.
- each transporter polypeptide comprises an amino acid sequence with at least 99% identity to a segment of the protein.
- each transporter polypeptide is an albumin
- embidments is an albumin based i
- each transporter polypeptide is an allo-albumin derivative. In certain embodiments is an isolated multispecific heteromultimer described herein wherein each transporter polypeptide is derived from a different alloalbumin. In some embodiments, each transporter polypeptide comprises an amino acid sequence with at least 75% identity to a segment of albumin. In some embodiments, each transporter polypeptide comprises an amino acid sequence with at least 80% identity to a segment of albumin. In some embodiments, each transporter polypeptide comprises an amino acid sequence with at least 90% identity to a segment of albumin. In some other embodiments, each transporter polypeptide comprises an amino acid sequence with at least 95% identity to a segment of albumin. In some other embodiments, each transporter polypeptide comprises an amino acid sequence with at least 99% identity to a segment of albumin.
- an albumin based isolated multispecific heteromultimer construct comprising: a first monomer comprising a first transporter polypeptide fused to at least one CD3 binding polypeptide construct that binds to a CD3 complex on at least one CD3 expressing cell; a second polypeptide construct comprising a second transporter polypeptide which is different from said first transporter polypeptide, fused to at least one antigen binding polypeptide construct that binds to a target antigen on at least one B cell; wherein said first and second transporter polypeptides are obtained by segmentation of albumin, and each transporter polypeptide comprising an amino acid sequence with at least 90% identity to a segment of albumin such that said transporter polypeptides self-assemble to form quasi-native albumin, and wherein said first cargo polypeptide does not have any binding domain present in said second cargo polypeptide.
- albumin based multispecific heteromultimer constructs as described above, wherein said first transporter polypeptide comprising at least one mutation selected from A194C, L198C, W214C,
- polypeptide comprises at least one mutation selected from L331 C, A335C,
- V343C, L346C, A350C, V455C, and N458C are examples of V343C, L346C, A350C, V455C, and N458C.
- multispecific heteromultimer constructs described herein wherein said multispecific heteromultimer construct simultaneously engages said at least one B cell and said at least one CD3 expressing cell such that the CD3 expressing cell is activated, thereby inducing killing of the B cell.
- heteromultimer constructs comprising transporter polypeptides derived from albumin that self-assemble to form a heteromultimer that exhibits native albumin like functional characteristics such as FcRn binding and structural characteristics.
- the tumor cells are from a solid tumor. In some embodiments, the
- heteromultimer constructs described herein home to tumor cells and
- heteromultimer constructs described herein home to at least one tumor cell, bind simultaneously to said at least one tumor cell and and at least one T-cell in a manner that results in the lysis of said tumor cell.
- the heteromultimer constructs described herein home to at least one tumor cell, bind simultaneously to said at least one tumor cell and and at least one T-cell such that the binding to said tumor cell is with a higher valency than the binding to said T-cell, and causes the lysis of said tumor cell.
- CD3 complex binding polypeptide constructs [00212] CD3 complex binding polypeptide constructs:
- heteromultimer construct comprises at least one CD3 binding polypeptide construct that binds to a CD3 complex on at least one CD3 expressing cell.
- the at least one CD3 binding polypeptide construct comprises at least one CD3 binding polypeptide construct that binds to a CD3 complex on at least one CD3 expressing cell.
- 60 WSGF comprises at least one CD3 binding domain from a Ct
- the at least one CD3 binding domain comprises at least one amino acid modification that reduces immunogenicity as compared to a corresponding CD3 binding domain not comprising said modification.
- the at least one CD3 binding domain comprises at least one amino acid modification that increases its stability as measured by T m , as compared to a corresponding CD3 binding domain not comprising said modification. In some embodiments, there is about a 3 degree increase in the T m as compared to the native CD3 binding domain not comprising said at least one modification.
- the at least one CD3 binding polypeptide construct described herein comprises at least one CD3 binding domain from a CD3 specific antibody wherein said CD3 specific antibody is a heavy chain antibody devoid of light chains.
- the at least one CD3 binding polypeptide construct described herein comprises at least one CD3 binding domain derived from a non-antibody protein scaffold domain.
- the CD3 binding polypeptide constructs are CD3 binding Fab constructs (i.e. antigen binding constructs comprising a heavy and a light chain, each comprising a variable and a constant region).
- said Fab construct is mammalian. In one embodiment said
- Fab construct is human. In another embodiment said Fab construct is humanized. In yet another embodiment said Fab contruct comprises at least
- said Fab construct is a single chain Fab (scFab).
- the CD3 binding polypeptide constructs comprise CD3 binding scFab constructs wherein the C-terminus of the Fab light chain is connected to the N-terminus of the Fab heavy chain by a peptide linker.
- the peptide linker allows arrangement of the Fab heavy and light chain to form a functional CD3 binding moiety.
- the peptide linkers suitable for connecting the Fab heavy and light chain include sequences comprising glycine-serine linkers for instance, but not limited to (G m S) n -GG, (SGn)m, (SEGn)m, wherein m and n are between 0-20.
- the scFab construct is a cross-over construct wherein the constant regions of the Fab light chain and the Fab heavy chain are exchanged.
- the variable regions of the Fab light chain and the Fab heavy chain are exchanged.
- the CD3 binding polypeptide constructs comprise CD3 binding Fv constructs (i.e. antigen binding constructs comprising a heavy and a light chain, each comprising a variable region).
- said Fv construct is mammalian.
- said Fv construct is human.
- said Fv construct is humanized.
- said Fv contruct comprises at least one of human heavy and light chain variable regions.
- said Fv construct is a single chain Fv (scFv).
- the CD3 binding polypeptide construct of a multispecific heteromultimer construct described herein bind to at least one component of the CD3 complex.
- the CD3 binding polypeptide construct binds to at least one of CD3 epsilon, CD3 gamma, CD3 delta or CD3 zeta of the CD3 complex.
- the CD3 binding polypeptide construct binds the CD3epsilon domain.
- binding polypeptide construct binds a human CD3 complex.
- the CD3 binding polypeptide construct exhibits cross- species binding to a least one member of the CD3 complex.
- multispecific heteromultimer constructs comprising at least one CD3 binding polypeptide construct that binds to a CD3 complex on at least one CD3 expressing cell, where in the CD3 expressing cell is a T-cell.
- the CD3 expressing cell is a human cell.
- the CD3 expressing cell is a non-human, mammalian cell.
- the T cell is a cytotoxic T cell. In some embodiments the T cell is a CD4 + or a CD8 + T cell.
- the construct is capable of activating and redirecting cytotoxic activity of a T cell to a target cell such as a B cell.
- said redirection is independent of MHC-mediated peptide antigen presentation by the target cell and and/or specificity of the T cell.
- hetromultimer constructs that are capable of simultaneous binding to a B cell antigen for instance a tumor cell antigen, and an activating T cell antigen.
- the heteromultimer construct is capable of crosslinking a T cell and a target B cell by simultaneous binding to a B cell antigen for instance CD19 or CD20 and an activating T cell antigen for instance CD3.
- the simultaneous binding results in lysis of a target B cell, for instance a tumor cell. In one embodiment, such simultaneous binding results in activation of the T cell.
- such simultaneous binding results in a cellular response of a T lymphocyte, for instance a cytotoxic T lymphocyte, selected from the group of: proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers.
- a T lymphocyte for instance a cytotoxic T lymphocyte, selected from the group of: proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers.
- the antigen binding polypeptide construct binds to a target antigen on at least one B cell.
- the antigen binding polypeptide construct binds at least one member of a B cell CD21 -CD19-CD81 complex.
- the antigen binding polypeptide construct comprises at least one CD19 binding domain or fragment thereof.
- the antigen binding polypeptide construct comprises at least one CD20 binding domain.
- the at least one antigen binding domain is a CD19 or CD20 binding domain which is obtained from a CD19 or CD20 specific antibody, a nanobody, fibronectin, affibody, anticalin, cysteine knot protein, DARPin, avimer, Kunitz domain or variant or derivative thereof.
- the at least one antigen binding polypeptide construct described herein comprises at least one antigen binding domain which is a CD19 or CD20 binding domain from an antibody which is a heavy chain antibody devoid of light chains.
- the at least one antigen binding domain is a CD19 or CD20 binding domain that comprises at least one amino acid modification that reduces immunogenicity as compared to a corresponding antigen binding domain not comprising said modification.
- the at least one antigen binding domain is a CD19 or CD20 binding domain comprising at least one amino acid modification that increases its stability as measured by T m , as compared to a corresponding domain not comprising said modification.
- polypeptide construct is a Fab construct that binds at least one of CD19 and
- said Fab construct is mammalian. In one embodiment said Fab construct is human. In another embodiment said Fab construct is humanized. In yet another embodiment said Fab contruct comprises at least one of human heavy and light chain constant regions. In a further embodiment said Fab construct is a single chain Fab (scFab).
- construct comprises a scFab construct wherein the C-terminus of the Fab light chain is connected to the N-terminus of the Fab heavy chain by a peptide linker.
- the peptide linker allows arrangement of the Fab heavy and light chain to form a functional CD19 and/or CD20 binding moiety.
- the peptide linkers suitable for connecting the Fab heavy and light chain include sequences comprising glycine-serine linkers for instance, but not limited to (G m S) n -GG, (SG n ) m , (SEG n ) m , wherein m and n are between 0-20.
- the scFab construct is a cross-over construct wherein the constant regions of the Fab light chain and the Fab heavy chain are exchanged. In another embodiment of a cross-over Fab, the variable regions of the Fab light chain and the Fab heavy chain are exchanged.
- polypeptide construct is a Fv construct that binds at least one of CD19 and CD20 on a B cell.
- said Fv construct is mammalian.
- said Fv construct is human.
- said Fv construct is humanized.
- said Fv contruct comprises at least one of human heavy and light chain variable regions.
- said Fv construct is a single chain Fv (scFv).
- the antigen binding polypeptide construct exhibits cross-species binding to a least one antigen expressed on the surface of a B cell.
- the antigen binding polypeptide construct of a multispecific heteromultimer construct described herein bind to at least one of mammalian CD19 and CD20.
- binding polypeptide construct binds a human CD19 or CD20.
- hetromultimer constructs that are capable of simultaneous binding to a B cell antigen for instance a tumor cell antigen, and an activating T cell antigen.
- the heteromultimer construct is capable of crosslinking a T cell and a target B cell by simultaneous binding to a B cell antigen for instance CD19 or CD20 and an activating T cell antigen for instance CD3.
- the disease comprises at least one antigen binding polypeptide construct that binds to a target antigen such as a CD19 or CD20 on at least one B cell associated with a disease.
- the disease is a cancer selected from a carcinoma, a sarcoma, leukaemia, lymphoma and glioma.
- the cancer is at least one of squamous cell carcinoma, adenocarcinoma, transition cell carcinoma, osteosarcoma and soft tissue sarcoma.
- the at least one B cell is an autoimmune reactive cell that is a lymphoid or myeloid cell.
- an albumin or immunoglobulin based multispecific heteromultimer construct described herein further comprises at least one binding domain that binds at least one of: EpCAM, EGFR, IGFR, HER-2 neu, HER-3, HER-4, PSMA, CEA, MUC-1 (mucin), MUC2, MUC3, MUC4, MUC5, MUC7, CCR4, CCR5, CD19, CD20, CD33, CD30, ganglioside GD3, 9-0-Acetyl-GD3, GM2, Poly SA, GD2, Carboanhydrase IX (MN/CA IX), CD44v6, Sonic Hedgehog (Shh), Wue-1 , Plasma Cell Antigen, (membrane- bound), Melanoma Chondroitin Sulfate Proteoglycan (MCSP), CCR8, TNF- alpha precursor, STEAP, mesothelin, A33 Antigen, Prostate Stem Cell Antigen (PSCA), Ly-6;
- multispecific heteromultimer constructs described herein which are differentially modified during or after translation.
- the modification is at least one of: glycosylation,
- the heteromultimer construct is chemically modified by known techniques, including but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH 4 ; acetylation, formylation, oxidation, reduction; and metabolic synthesis in the presence of tunicamycin.
- Additional post-translational modifications of heteromultimers described herein include, for example, N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O- linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of procaryotic host cell expression.
- the heteromultimer constructs described herein are modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein.
- examples of suitable enzyme labels include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;
- examples of suitable prosthetic group complexes include streptavidin biotin and avidin/biotin;
- examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol;
- bioluminescent materials include luciferase, luciferin, and aequorin
- suitable radioactive material include iodine, carbon, sulfur, tritium, indium, technetium, thallium, gallium, palladium, molybdenum, xenon, fluorine.
- heteromultimer constructs described herein are attached to macrocyclic chelators that associate with radiometal ions.
- heteromultimer constructs described herein are modified by either natural processes, such as post-translational
- polypeptides from heteromultimers described herein are branched, for example, as a result of ubiquitination, and in some embodiments are cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides are a result from posttranslation natural processes or made by synthetic methods.
- Modifications include acetylation, acylation, ADP- ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
- heteromultimeric constructs described herein are attached to solid supports, which are particularly useful for immunoassays or purification of polypeptides that are bound by, that bind to, or associate with albumin fusion proteins of the invention.
- solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
- RNA for example, in the form of messenger RNA (mRNA).
- RNA of the present invention may be single stranded or double stranded.
- a set of expression vectors for expressing a multispecific heteromultimer construct described herein which comprises a first and a second polypeptide construct, said set comprising at least a first DNA sequence encoding said first polypeptide construct and at least a second DNA sequence encoding said second polypeptide construct.
- polynucleotide sequences encoding a heteromultimer construct described herein or a polypeptide construct thereof with sequence as provided herein.
- a polynucleotide comprising a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%,
- nucleotide sequence shown in Figure 98%, or 99% identical to a nucleotide sequence shown in Figure .
- kits for producing an expression product containing a multispecific heteromultimer construct as described herein, in stable mammalian cells comprising: transfecting at least one mammalian cell with: at least a first DNA sequence encoding said first polypeptide construct and at least a second DNA sequence encoding said second polypeptide construct, such that said at least one first DNA sequence, said at least one second DNA sequence are transfected in said at least one
- said predetermined ratio of the at least one first DNA sequence: at least one second DNA sequence is about 1 :1 . In certain other embodiments, said predetermined ratio of the at least one first DNA sequence: at least one second DNA sequence is skewed towards a larger amount of the one first DNA sequence such as about 2:1. In yet other embodiments, said predetermined ratio of the at least one first DNA sequence: at least one second DNA sequence is skewed towards a larger amount of the one first DNA sequence such as about 1 :2.
- the mammalian cell is selected from the group consisting of a VERO, HeLa, HEK, NSO, Chinese Hamster Ovary (CHO), W138, BHK, COS-7, Caco-2 and MDCK cell, and subclasses and variants thereof.
- heteromultimers produced as recombinant molecules by secretion from yeast, a microorganism such as a bacterium, or a human or animal cell line.
- the polypeptides are secreted from the host cells.
- Embodiments include a cell, such as a yeast cell transformed to express a heteromultimer protein described herein.
- a cell such as a yeast cell transformed to express a heteromultimer protein described herein.
- transformed host cells themselves, are provided culture of those cells, preferably a monoclonal (clonally homogeneous) culture, or a culture derived from a monoclonal culture, in a nutrient medium. If the polypeptide is secreted, the medium will contain the polypeptide, with the cells, or without the cells if they have been filtered or centrifuged away.
- Many expression systems are known and may be used, including bacteria (for example E. coli and Bacillus subtilis), yeasts (for example Saccharomyces cerevisiae, Kluyveromyces lactis and Pichia pastoris, filamentous fungi (for example Aspergillus), plant cells, animal cells and insect cells.
- a heteromultimer described herein is produced in conventional ways, for example from a coding sequence inserted in the host chromosome or on a free plasmid.
- the yeasts are transformed with a coding sequence for the
- Successfully transformed cells i.e., cells that contain a DNA construct of the present invention
- cells resulting from the introduction of an expression construct can be grown to produce the desired polypeptide.
- Cells can be harvested and lysed and their DNA content examined for the presence of the DNA using a method such as that described by Southern (1975) J. Mol. Biol. 98, 503 or Berent et al. (1985) Biotech. 3, 208.
- the presence of the protein in the supernatant can be detected using antibodies.
- Useful yeast plasmid vectors include pRS403-406 and pRS413-416 and are generally available from Stratagene Cloning Systems, La Jolla, Calif. 92037, USA.
- Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (Yips) and incorporate the yeast selectable markers HIS3, 7RP1 , LEU2 and URA3.
- Plasmids pRS413-416 are Yeast Centromere plasmids (Ycps).
- a variety of methods have been developed to operably link DNA to vectors via complementary cohesive termini. For instance, complementary homopolymer tracts can be added to the DNA segment to be inserted to the vector DNA. The vector and DNA segment are then joined by hydrogen bonding between the complementary honmopolymeric tails to form
- Synthetic linkers containing one or more restriction sites provide an alternative method of joining the DNA segment to vectors.
- the DNA segment, generated by endonuclease restriction digestion, is treated with bacteriophage
- T4 DNA polymerase or E. coli DNA polymerase 1 enzymes that remove protruding, _single-stranded termini with their 3' 5'-exonucleolytic activities, and fill in recessed 3'-ends with their polymerizing activities.
- the products of the reaction are DNA segments carrying polymeric linker sequences at their ends. These DNA segments are then cleaved with the appropriate restriction enzyme and ligated to an expression vector that has been cleaved with an enzyme that produces termini compatible with those of the DNA segment.
- Exemplary genera of yeast contemplated to be useful in the practice of the present invention as hosts for expressing the albumin, fusion proteins are Pichua (formerly classified as Hansenula), Saccharomyces, Kluyveromyces, Aspergillus, Candida, Torulopsis, Torulaspora,
- Schizosaccharomyces Citeromyces, Pachysolen, Zygosaccharomyces, Debaromyces, Trichoderma, Cephalosporium, Humicola, Mucor, Neurospora, Yarrowia, Metschunikowia, Rhodosporidium, Leucosporidium, Botryoascus, Sporidiobolus, Endomycopsis, and the like.
- Preferred genera are those selected from the group consisting of Saccharomyces, Schizosaccharomyces, Kluyveromyces, Pichia and Torulaspora. Examples of Saccharomyces spp. are S. cerevisiae, S. italicus and S. rouxii.
- Kluyveromyces spp. are K. fragilis, K. lactis and K. marxianus.
- a suitable Torulaspora species is T. delbrueckii.
- Examples of Pichia (Hansenula) spp. are P. angusta (formerly H. polymorpha), P. anomala
- Exemplary species of Saccharomyces useful for the synthesis of heteromultimer constructs described herein include S. cerevisiae, S. italicus, S. diastaticus, and Zygosaccharomyces rouxii.
- Kluyveromyces include K. fragilis and K. lactis.
- Hansenula include H. polymorpha (now Pichia angust
- Pichia anomala and Pichia capsulata. Additional preferred exemplary species of Pichia include P. pastoris. Preferred exemplary species of Aspergillusinclude
- Yarrowia include Y. lipolytica.
- Many preferred yeast species are available from the ATCC.
- the following preferred yeast species are available from the ATCC and are useful in the expression of albumin fusion proteins: Saccharomyces cerevisiae, Hansen, teleomorph strain BY4743 yap3 mutant (ATCC Accession
- Suitable promoters for S. cerevisiae include those associated with the PGKI gene, GAL1 or GALI O genes, CYCI, PH05, TRP1 , ADH1 , ADH2, the genes for glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, triose phosphate isomerase,
- phosphoglucose isomerase glucokinase
- alpha-mating factor pheromone [a mating factor pheromone]
- the PRBI promoter the GUT2 promoter
- the GPDI promoter the GPDI promoter
- hybrid promoters involving hybrids of parts of 5' regulatory regions with parts of 5' regulatory regions of other promoters or with upstream activation sites (e.g. the promoter of EP-A-258 067).
- pombe are the thiamine-repressible promoter from the nmt gene as described by Maundrell (1990) J. Biol. Chem. 265, 10857-10864 and the glucose repressible jbpl gene promoter as described by Hoffman & Winston (1990) Genetics 124, 807-816.
- Suitable promoters include AOX1 and AOX2.
- Gleeson et al. (1986) J. Gen. Microbiol. 132, 3459-3465 include information on Hansenula vectors and transformation, suitable promoters being MOX1 and FMD1 ; whilst EP 361 991 , Fleer et al. (1991 ) and other publications from Rhone-Poulenc Rorer teach how to express foreign proteins in Kluyveromyces spp., a suitable promoter being PGKI.
- the transcription termination signal is preferably the 3' flanking sequence of a eukaryotic gene which contains proper signals for transcription termination and polyadenylation.
- Suitable 3' flanking sequences may, for example, be those of the gene naturally linked to the expression control sequence used, i.e. may correspond to the promoter. Alternatively, they may be different in which case the termination signal of the S. cerevisiae ADHI gene is preferred.
- the desired heteromultimer protein is initially expressed with a secretion leader sequence, which may be any leader effective in the yeast chosen.
- leader sequence useful in S. cerevisiae include that from the mating factor alpha polypeptide (MFa-1 ) and the hybrid leaders of EP-A- 387 319. Such leaders (or signals) are cleaved by the yeast before the mature albumin is released into the surrounding medium. Further such leaders include those of S. cerevisiae invertase (SUC2) disclosed in JP 62-096086 (granted as 91 1036516), acid phosphatase (PH05), the pre-sequence of MFa-1 , 0
- MEL1 carlsbergensis a-galactosidase
- K lactis killer toxin
- Candida glucoarnylase
- vectors containing polynucleotides encoding a heteromultimer construct described herein, host cells, and the production of the heteromultimer proteins by synthetic and recombinant techniques may be, for example, a phage, plasmid, viral, or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.
- heteromultimer proteins described herein are joined to a vector containing a selectable marker for propagation in a host.
- a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
- the polynucleotide insert is operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the
- E. coli lac, trp, phoA and rac promoters the SV40 early and late promoters and promoters of retroviral LTRs, to name a few.
- Other suitable promoters will be known to the skilled artisan.
- the expression constructs will further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation.
- the coding portion of the transcripts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
- the expression vectors will preferably include at least one selectable marker.
- markers include dihydrofolate reductase, G418, glutamine synthase, or neomycin resistance for eukaryotic cell culture, and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria.
- Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella
- WSGF typhimurium cells
- fungal cells such as yeast cells
- insect cells such as Drosophila S2 and Spodoptera Sf9 cells
- animal cells such as CHO, COS, NSO, 293, and Bowes melanoma cells
- plant cells Appropriate culture mediums and conditions for the above-described host cells are known in the art.
- vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNH8A, pNH16a, pNH18A; pNH46A, available from Stratagene Cloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia Biotech, Inc.
- preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.
- Preferred expression vectors for use in yeast systems include, but are not limited to pYES2, pYD1 , pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1 , pPIC3.5K, pPIC9K, and PAO815 (all available from Invitrogen, Carlbad, CA).
- Other suitable vectors will be readily apparent to the skilled artisan.
- polynucleotides encoding a multispecific heteromultimer construct described herein are fused to signal sequences that will direct the localization of a protein of the invention to particular
- a prokaryotic or eukaryotic cell For example, in E. coli, one may wish to direct the expression of the protein to the periplasmic space.
- signal sequences or proteins (or fragments thereof) to which the heteromultimeric proteins are fused in order to direct the expression of the polypeptide to the periplasmic space of bacteria include, but are not limited to, the pelB signal sequence, the maltose binding protein (MBP) signal sequence, MBP, the ompA signal sequence, the signal sequence of the periplasmic E. coli heat-labile enterotoxin B-subunit, and the signal sequence of alkaline phosphatase.
- MBP maltose binding protein
- ompA signal sequence the signal sequence of the periplasmic E. coli heat-labile enterotoxin B-sub
- polynucleotides albumin fusion proteins of the invention may be fused to the pelB pectate lyase signal sequence to increase the efficiency of expression and purification of such polypeptides in Gram-negative bacteria. See, U.S. Pat. Nos. 5,576,195 and 5,846,818, the contents of which are herein incorporated by reference in their entireties.
- Examples of signal peptides that are fused to a heteromultimeric protein in order to direct its secretion in mammalian cells include, but are not limited to, the MPIF-1 signal sequence (e.g., amino acids 1 -21 of GenBank
- a suitable signal sequence that may be used in conjunction with baculoviral expression systems is the gp67 signal sequence (e.g., amino acids 1 -19 of GenBank Accession Number AAA72759).
- Vectors which use glutamine synthase (GS) or DHFR as the selectable markers can be amplified in the presence of the drugs methionine sulphoximine or methotrexate, respectively.
- An advantage of glutamine synthase based vectors are the availably of cell lines (e.g., the murine myeloma cell line, NSO) which are glutamine synthase negative.
- Glutamine synthase expression systems can also function in glutamine synthase expressing cells (e.g., Chinese Hamster Ovary (CHO) cells) by providing additional inhibitor to prevent the functioning of the endogenous gene.
- a glutamine synthase expression system and components thereof are detailed in
- glutamine synthase expression vectors can be obtained from Lonza Biologies, Inc. (Portsmouth, N.H.). Expression and production of monoclonal antibodies using a GS expression system in murine myeloma cells is described in Bebbington et al., Bio/technology 10:169(1992)
- host cells containing vector constructs described herein and additionally host cells containing nucleotide sequences that are operably associated with one or more heterologous control regions (e.g., promoter and/or enhancer) using techniques known of in the art.
- the host cell can be a higher eukaryotic cell, such as a mammalian cell (e.g., a human derived cell), or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.
- a host strain may be chosen which modulates the expression of the inserted gene sequences, or modifies and processes the gene product in the specific fashion desired.
- Expression from certain promoters can be elevated in the presence of certain inducers; thus expression of the genetically engineered polypeptide may be controlled.
- different host cells have characteristics and specific mechanisms for the translational and post-translational processing and modification (e.g., phosphorylation, cleavage) of proteins. Appropriate cell lines can be chosen to ensure the desired modifications and processing of the foreign protein expressed.
- nucleic acids and nucleic acid constructs of the invention into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986). It is specifically contemplated that the polypeptides of the present invention may in fact be expressed by a host cell lacking a recombinant vector.
- the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., the coding sequence corresponding to a Cargo
- Cargo polypeptide and/or to include genetic material.
- the genetic material operably associated with the endogenous polynucleotide may activate, alter, and/or amplify endogenous polynucleotides.
- heterologous polynucleotides e.g., polynucleotides encoding an albumin protein, or a fragment or variant thereof
- heterologous control regions e.g., promoter and/or enhancer
- endogenous polynucleotide sequences encoding a Therapeutic protein via homologous recombination (see, e.g., U.S. Pat. No. 5,641 ,670, issued Jun. 24, 1997; International
- Heteromultimer proteins described herein can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography such as with protein A, hydroxylapatite chromatography, hydrophobic charge interaction
- HPLC high performance liquid chromatography
- heteromultimer proteins of the invention are purified using Anion Exchange Chromatography including, but not limited to, chromatography on Q-sepharose, DEAE sepharose, poros HQ, poros DEAF, Toyopearl Q, Toyopearl QAE, Toyopearl DEAE, Resource/Source Q and DEAE, Fractogel Q and DEAE columns.
- Anion Exchange Chromatography including, but not limited to, chromatography on Q-sepharose, DEAE sepharose, poros HQ, poros DEAF, Toyopearl Q, Toyopearl QAE, Toyopearl DEAE, Resource/Source Q and DEAE, Fractogel Q and DEAE columns.
- the proteins described herein are purified using Cation Exchange Chromatography including, but not limited to, SP- sepharose, CM sepharose, poros HS, poros CM, Toyopearl SP, Toyopearl CM,
- heteromultimer proteins described herein can be chemically synthesized using techniques known in the art (e.g., see Creighton, 1983, Proteins: Structures and Molecular Principles, W. H. Freeman & Co., N.Y and Hunkapiller et al., Nature, 310:105-1 1 1 (1984)). For example, a
- polypeptide corresponding to a fragment of a polypeptide can be synthesized by use of a peptide synthesizer.
- nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the polypeptide sequence.
- Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids,
- 2,4diaminobutyric acid alpha-amino isobutyric acid, 4aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, ⁇ -alanine, fluoro-amino acids, designer amino acids such as ⁇ -methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general.
- the amino acid can be D (dextrorotary) or L (levorotary).
- heteromultimer constructs described herein can be assayed for functional activity (e.g., biological activity) using or routinely modifying assays known in the art, as well as assays described herein.
- immunoassays known in the art can be used, including but not limited to, competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme
- antibody binding is detected by detecting a label on the primary antibody.
- the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody.
- the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.
- a binding partner e.g., a receptor or a ligand
- heteromultimer described herein is assayed, e.g., by means well-known in the art, such as, for example, reducing and non-reducing gel chromatography, protein affinity chromatography, and affinity blotting. See generally, Phizicky et al., Microbiol. Rev. 59:94-123 (1995).
- the ability of physiological correlates of a heteromultimeric protein to bind to a substrate(s) of antigen binding polypeptide constructs of the heteromultimers described herein can be routinely assayed using techniques known in the art.
- heteromultimers described herein are directed to antibody-based therapies which involve administering heteromultimers described comprising cargo polypeptide(s) which is an antibody, a fragment or variant of an antibody, to a patient for treating one or more of the disclosed diseases, disorders, or conditions.
- Therapeutic compounds described herein include, but are not limited to, heteromultimers described herein, nucleic acids encoding heteromultimers described herein.
- a proliferative disease a minimal residual cancer
- a tumorous disease a tumorous disease
- an inflammatory disease an inflammatory disease
- immunological disorder an autoimmune disease, an infectious disease, viral disease, allergic reactions, parasitic reactions, graft-versus-host diseases or host-versus-graft diseases or cell malignancies, said method comprising administering to a subject in need of such a prevention, treatment or
- the cancer is a solid tumor.
- the solid tumor is one or more of sarcoma, carcinoma, and lymphoma.
- the cancer is a hematological cancer.
- the cancer is one or more of B-cell lymphoma, non-Hodgkin's lymphoma, and leukemia.
- a method of treating cancer cells comprising providing to said cell a composition comprising a heteromultimer construct described herein. In some embodiments, the method further comprising providing said heteromultimer in conjugation with another therapeutic agent.
- a method of treating a cancer non-responsive to blinatumomab in a mammal in need thereof comprising administering to the mammal a composition comprising an effective amount of the pharmaceutical composition comprising a heteromultimer construct described herein.
- Aamethod of treating a cancer cell regressive after treatment with blinatumomab comprising providing to said cancer cell a composition comprising an effective amount of the pharmaceutical composition comprising a heteromultimer construct described herein.
- composition comprising an effective amount of the pharmaceutical composition comprising a
- the disease is not responsive to treatment with at least one of an anti-CD19 antibody and an anti-CD20 antibody.
- the disease is a cancer or autoimmune condition resistant to CD19 or CD20 lytic antibodies
- a method of treating an autoimmune condition in a mammal in need thereof comprising administering to said mammal a composition comprising an effective amount of the pharmaceutical composition described herein.
- the autoimmune condition is one or more of multiple sclerosis, rheumatoid arthritis, lupus erytematosus, psoriatic arthritis, psoriasis, vasculitis, uveitis, Crohn's disease, and type 1 diabetes.
- a method of treating an inflammatory condition in a mammal in need thereof comprising administering to said mammal a composition comprising an effective amount of the pharmaceutical composition comprising an heteromultimer described herein.
- heteromultimers described herein, comprising at least a fragment or variant of an antibody may be administered alone or in combination with other types of treatments (e.g., radiation therapy, chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents).
- treatments e.g., radiation therapy, chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents.
- administration of products of a species origin or species reactivity in the case of antibodies
- human antibodies, fragments derivatives, analogs, or nucleic acids are administered to a human patient for therapy or prophylaxis.
- Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid.
- the nucleic acids produce their encoded protein that mediates a therapeutic effect. Any of the methods for gene therapy available in the art can be used.
- bispecific single chain antibodies described in the art have great therapeutic potential for the treatment of malignant diseases, most of these bispecific molecules are limited in that they are species specific and recognize only human antigen, and— due to genetic similarity— likely the chimpanzee counterpart.
- the advantage of the present invention is the provision of a bispecific single chain antibody comprising a binding domain exhibiting cross-species specificity to human and non-chimpanzee primate of the CD3 epsilon chain.
- heteromultimers or pharmaceutical compositions described herein are tested in vitro, and then in vivo for the desired therapeutic or prophylactic activity, prior to use in humans.
- in vitro assays which can be used to determine whether administration of a specific compound is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in
- the heteromultimer is substantially purified (e.g., substantially free from substances that limit its effect or produce undesired side-effects).
- the subject is an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and in certain embodiments, a mammal, and most preferably human.
- Various delivery systems are known and can be used to administer a heteromultimer formulation described herein, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu and
- nucleic acid as part of a retroviral or other vector, etc.
- Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
- the compounds or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
- intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir.
- Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
- heteromultimers, or compositions described herein locally to the area in need of treatment may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non- porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
- care must be taken to use materials to which the protein does not absorb.
- the heteromultimers or composition can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527- 1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)
- the heteromultimers or composition can be delivered in a controlled release system.
- a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321 :574 (1989)).
- polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla.
- a controlled release system can be placed in proximity of the therapeutic target, e.g., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 1 15-138 (1984)).
- the nucleic acid in a specific embodiment comprising a nucleic acid encoding a heteromultimer decribed herein, can be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Pat. No.
- a nucleic acid can be introduced intracellular ⁇ and incorporated within host cell DNA for expression, by homologous recombination.
- compositions comprise a therapeutically effective amount of a compound, and a pharmaceutically acceptable carrier.
- a pharmaceutically acceptable carrier for example
- carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
- Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
- Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
- the composition if
- 87 WSGF desired can also contain minor amounts of wetting or
- compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
- the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
- Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin.
- Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
- the formulation should suit the mode of administration.
- composition comprising the
- compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
- the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.
- the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
- the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
- an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
- compositions described herein are formulated as neutral or salt forms.
- Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric,
- compositions described herein which will be effective in the treatment, inhibition and prevention of a disease or disorder associated with aberrant expression and/or activity of a Therapeutic protein can be determined by standard clinical techniques.
- in vitro assays may optionally be employed to help identify optimal dosage ranges.
- the precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses are extrapolated from dose-response curves derived from in vitro or animal model test systems.
- a heteromultimer construct described herein is suitably administered to the patient at one time or over a series of treatments.
- about 1 ⁇ g/kg to 15 mg/kg (e.g. 0.1 mg/kg - 10 mg/kg) of T cell activating bispecific antigen binding molecule can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion.
- One typical daily dosage might range from about 1 ⁇ g/kg to 100 mg/kg or more, depending on the factors mentioned above.
- the treatment would generally be sustained until a desired
- a dosage of the heteromultimer described herein would be in the range from about 0.005 mg/kg to about 10 mg/kg.
- a dose may also comprise from about 1 microgram/kg body weight, about 5 microgram/kg body weight, about 10 microgram/kg body weight, about 50 microgram/kg body weight, about 100 microgram/kg body weight, about 200 microgram/kg body weight, about 350 microgram/kg body weight, about 500 microgram/kg body weight, about 1 milligram/kg body weight, about 5 milligram/kg body weight, about 10
- milligram/kg body weight about 200 milligram/kg body weight, about 350 milligram/kg body weight, about 500 milligram/kg body weight, to about 1000 mg/kg body weight or more per administration, and any range derivable therein.
- a range of about 5 mg/kg body weight to about 100 mg/kg body weight, about 5 microgram kg body weight to about 500 milligram kg body weight, etc. can be administered, based on the numbers described above.
- Such doses may be administered intermittently, e.g. every week or every three weeks (e.g. such that the patient receives from about two to about twenty, or e.g. about six doses of the T cell activating bispecific antigen binding molecule).
- An initial higher loading dose, followed by one or more lower doses may be administered.
- heteromultimers described herein are generally used in an amount effective to achieve the intended purpose.
- a heteromultimer described herein, or pharmaceutical compositions thereof are administered or applied in a therapeutically effective amount. Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.
- a therapeutically effective dose can be estimated initially from in vitro assays, such as cell culture assays.
- a dose can then be formulated in animal models to achieve a circulating concentration range that includes the IC 50 as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.
- Initial dosages can also be estimated from in vivo data, e.g., animal models, using techniques that are well known in the art.
- in vivo data e.g., animal models
- Dosage amount and interval may be adjusted individually to provide plasma levels of the heteromultimer described herein which are sufficient to maintain therapeutic effect.
- Usual patient dosages for administration by injection range from about 0.1 to 50 mg/kg/day, typically from about 0.5 to 1 mg/kg/day.
- Therapeutically effective plasma levels may be achieved by administering multiple doses each day. Levels in plasma may be measured, for example, by HPLC.
- the effective local concentration of the heteromultimer described herein may not be related to plasma concentration.
- One having skill in the art will be able to optimize therapeutically effective local dosages without undue experimentation.
- a therapeutically effective dose of the heteromultimer constructs described herein will generally provide therapeutic benefit without causing substantial toxicity.
- Toxicity and therapeutic efficacy of a heteromultimer described herein can be determined by standard pharmaceutical procedures in cell culture or experimental animals. Cell culture assays and animal studies can be used to determine the LD 50 (the dose lethal to 50% of a population) and the
- ED 50 the dose therapeutically effective in 50% of a population.
- the dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50/ED50.
- T cell activating bispecific antigen binding molecules that exhibit large therapeutic indices are preferred.
- the heteromultimer construct described herein according to the present invention exhibits a high therapeutic index.
- the data obtained from cell culture assays and animal studies can be used in formulating a range of dosages suitable for use in humans.
- the dosage lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
- the dosage may vary within this range depending upon a variety of factors, e.g., the dosage form employed, the route of administration utilized, the condition of the subject, and the like.
- 91 WSGF can be chosen by the individual physician in view of tr
- the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity).
- the magnitude of an administered dose in the management of the disorder of interest will vary with the severity of the condition to be treated, with the route of administration, and the like. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency will also vary according to the age, body weight, and response of the individual patient.
- Also provided is a process for the production of a pharmaceutical composition comprising a eteromutlimer described herein, said process comprising: culturing a host cell under conditions allowing the expression of a heteromultimer; recovering the produced heteromultimer from the culture; and producing the pharmaceutical composition.
- the heteromultimer constructs described herein are administered in combination with one or more other agents in therapy.
- a heteromultimer described herein is co-administered with at least one additional therapeutic agent.
- therapeutic agent encompasses any agent administered to treat a symptom or disease in an individual in need of such treatment.
- additional therapeutic agent may comprise any active ingredients suitable for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
- an additional therapeutic agent is an immunomodulatory agent, a cytostatic agent,
- the additional therapeutic agent is an anti-cancer agent, for example a microtubule disruptor, an antimetabolite, a topoisomerase inhibitor, a DNA intercalator, an alkylating agent, a hormonal therapy, a kinase inhibitor, a receptor antagonist, an activator of tumor cell apoptosis, or an antiangio genie agent.
- an anti-cancer agent for example a microtubule disruptor, an antimetabolite, a topoisomerase inhibitor, a DNA intercalator, an alkylating agent, a hormonal therapy, a kinase inhibitor, a receptor antagonist, an activator of tumor cell apoptosis, or an antiangio genie agent.
- Such other agents are suitably present in combination in amounts that are effective for the purpose intended.
- the effective amount of such other agents depends on the amount of T cell activating bispecific antigen binding molecule used, the type of disorder or treatment, and other factors discussed above.
- the heteromultimers described herein are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate.
- Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate compositions), and separate administration, in which case, administration of the heteromultimer described herein can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent and/or adjuvant.
- Heteromultimer constructs described herein can also be used in combination with radiation therapy.
- an article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the disorders described above is provided.
- the article of manufacture comprises a container and a label or package insert on or associated with the container.
- Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc.
- the containers may be formed from a variety of materials such as glass or plastic.
- the container holds a composition which is by itself or
- the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
- At least one active agent in the composition is a T cell activating bispecific antigen binding molecule of the invention.
- the label or package insert indicates that the composition is used for treating the condition of choice.
- the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises a heteromultimer described herein; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent.
- the article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition.
- the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically- acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate- buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
- Example 1 Bispecific CD3-CD19 scFvs fused to an asymmetric lgG1 Fc.
- V873 belongs to a novel family of CD3- based bispecific azymetric lgG1 antibodies that can be expressed and purified with significantly higher yields in mammalian CHO cells compared to
- V873 demonstrates unexpected effectontarget cell binding, bridging and target cell killing.
- V873 and bispecific CD3-based azymetric antibodies have utility in targeted T cell mediated killing of diseased cells and hence may be useful for treating cancers and autoimmune and inflammatory diseases.
- V873 is a bispecific CD3-CD19 scFvs fused to an azymetric lgG1 Fc.
- v873 represents a novel bispecific azymetric antibody class comprising one anti-CD3 warhead and a second warhead comprising a cell surface antigen of a target cell, and an antibody Fc heterodimer comprising heterodimer lgG1 Fc.
- the fusion of the CD3 warhead to chain A or B of the Fc is important for its druggable properties.
- V874 and V875 are two other Bispecific CD3-CD19 scFvs fused to an asymmetric lgG1 Fc but have different CD3 amino acid compositions.
- V873 shows unexpected good mammalian CHO expression and purification yields compared to the Amgen/Micromet blinatumomab CD3-CD19 BiTE tandem scFvs. V873 bridges T and B cells and results in potent killing of cultured human Burkitt lymphoma cells (Raji B-cell lymphoma line) using resting and IL-2 activated human PBMCs.
- V873 and its related bispecific CD3-Azymetric antibodies differ from
- V873 can bind FcRn and this heterodimeric Fc class shows typical antibody
- V873 and related bispecific CD3-based constructs address known stability issues with tandem scFvs and generally recognized poor
- V873 addresses the short PK of blinatumomab and CNS adverse effects owing to its wildtype FcRn binding affinity and MWT which restricts its distribution to the peripheral compartment.
- the Azymetric heterodimer Fc confers additional tailored FcgR effector ADCC, CDC, and ADCP activities and hence efficacy to drug resistant tumors.
- v873 to mediate PBMC (T cell)- B cell killing is highly unexpected.
- the properties of blinatumomab bscCD19xCD3 and related BiTEs is reported to rely on the use of flexibly linked single-chain variable fragments (scFv) that are arranged in tandem joined by a short linker will allow for a much closer approximation of opposing cells than is possible with larger bispecific formats such as quadroma antibodies.
- the flexible linkage is expected to enable free rotation and kinking of the 2 scFv arms, thereby facilitating the simultaneous recognition of 2 epitopes present on 2 opposing cell membranes and the formation of a cytolytic immunologic synapse.
- bispecific CD3-CD19 construct as described herein with significantly different structural presentations of CD3 and CD19 scFv warheads on the heterodimer Fc can bind, bridge T and B cells, and mediate PBMC (T cell)-B cell killing.
- Other applications include depletion of B cells in B cell driven autoimmune and inflammatory diseases such as RA, lupus, MS, IBD.
- V873 related bispecific CD3-Based azymetric antibodies may be useful for diagnostic purposes.
- Example 2 Design, expression and purification of heteromultimer constructs with a heterodimeric Fc.
- FIG. 1A An exemplary schematic representation of an anti-CD3/anti-CD19 antibody is shown in Figure 1A.
- v873, v874, v875 exemplify bispecific anti-CD3/ anti-CD19 heterodimeric Fc constructs and were prepared and tested as described below.
- the description includes a reference to BiTE, it refers to the antibody construct having an identical amino acid sequence to either the VH or VL of the anti-CD3 anti-CD19 BiTE molecule with or without modifications to variable heavy and light chain orientation (e.g. VH-VL) as indicated below.
- v873 has a anti-CD19 BiTE (VL-VH) scFv on chain A and a CD3 BiTETM (VH-VL) scFv on chain B of the heterodimer Fc with the following mutations L351Y_F405A_Y407V on chain A and T366L_K392M_T394W on chain B.
- VL-VH anti-CD19 BiTE
- VH-VL CD3 BiTETM
- V874 has a anti-CD19 BiTETM (VL-VH) scFv on chain A and the CD3 BiTETM (VLVH) scFv on chain B of the heterodimer Fc with the following mutations L351Y_F405A_Y407V on chain A and T366L_K392M_T394W on chain B.
- VL-VH anti-CD19 BiTETM
- VLVH CD3 BiTETM
- V875 has a anti-CD19 BiTETM (VL-VH) scFv on chain A and the CD3 OKT3 (VL-VH) scFv on chain B of the heterodimer Fc with the following mutations L351Y_F405A_Y407V on chain A and T366L_K392M_T394W on chain B.
- VL-VH anti-CD19 BiTETM
- VL-VH CD3 OKT3
- v1379 has a anti-CD19 BiTETM (VL-VH) scFv on chain A and a anti- CD3 (VH-VL) BiTE on chain B of the heterodimer Fc with the following mutations T350V_L351Y_F405A_Y407V on chain A and
- v865, v866, v867, v868 exemplify monospecific anti-CD3 or CD19 bivalent scFv-Fc constructs and were prepared and tested as described below:
- v865 has a anti-CD19 BiTE (VL-VH) scFv
- v866 has a anti-CD3 BiTETM (VH-VL) on chain A and a anti-CD3 BiTETM (VH-VL) scFv on chain with a WT Fc (105kDa).
- VH-VL anti-CD3 BiTETM
- VH-VL anti-CD3 BiTETM
- WT Fc 105kDa
- v867 has a anti-CD3 BiTETM (VL-VH) scFv on chain A and a anti- CD3 BiTETM (VL-VH) scFv on chain B with a WT Fc (105kDa).
- VL-VH anti-CD3 BiTETM
- VL-VH anti- CD3 BiTETM
- WT Fc 105kDa
- v868 has a anti-CD3 OKT3 (VL-VH) scFv on chain A and a anti- CD3 OKT3 VL-VH scFv on chain with a WT Fc (105 kDa).
- VL-VH anti-CD3 OKT3
- WT Fc 105 kDa
- v869 has a anti-CD19 BiTETM scFv (VL-VH) on chain A and a Fc on chain B of the heterodimer Fc with the following mutations
- v870 has a anti- CD3 BiTETM (VH-VL) scFv on chain B and a Fc on chain A of the heterodimer Fc with the following mutations
- v871 has a anti-CD3 BiTETM (VL-VH) scFv on chain B and a Fc on chain A of the heterodimer Fc with the following mutations
- v872 has a anti-CD3 OKT3 (VL-VH) scFv on chain B and a Fc on chain A of the heterodimer Fc with the following mutations
- v891 has the identical sequence blinatumc
- BiTE scFv and anti-CD19 BiTE scFv 50 kDa [Polypeptide sequence corresponds to SEQ ID No: 90
- v1380 has a anti-CD19 BiTETM (VL-VH) scFv on chain A and a anti-CD3 VHVL BiTETM on chain B of the heterodimer Fc with the following mutations L234A_L235A_T350V_L351Y_F405A_Y407V on chain A and L234A_L235A_T350V_T366L_K392L_T394W on chain B (corresponding to polypeptide sequences SEQ ID NOs:93 and 94).
- v1381 has a anti-CD19 BiTETM (VI-VH) scFv on chain A and a anti-
- N297A_T350V_T366L_K392L_T394W on chain B (corresponding to polypeptide sequences SEQ ID NOs: 93 and 98)
- the following variants contain mutations to the anti-CD3 scFv that include either changes to the linker length, VH-VL orientations, or point mutations to improve stability and yeild.
- v1653 has a anti-CD19 BiTETM (VL-VH) scFv on chain A and a anti- CD3 OKT3 (VL-VH) (with C to S mutation at position 100A of the VH CDR3) on chain B of the heterodimer Fc with the following mutations
- v1654 has a anti-CD19 BiTETM scFv on chain A and a anti-CD3
- OKT3 OKT3 (VH-VL) with 18 amino acid linker chain B of the heterodimer Fc with the following mutations T350V_L351 Y_F405A_Y407V on chain A and
- v1655 has a anti-CD19 BiTE ' scFv on cr
- OKT3 OKT3 (VH-VL) with 10 amino acid linker chain B of the heterodimer Fc with the following mutations T350V_L351 Y_F405A_Y407V on chain A and T350V_T366L_K392L_T394W on chain B. (corresponding to polypeptide sequences SEQ ID NOs: 109 and 1 10)
- v1656 has a anti-CD3 BiTETM VHVL scFv on chain A and a anti- CD19 BiTETM (VL-VH) on chain B of the heterodimer Fc with the following mutations T350V_L351Y_F405A_Y407V on chain A and
- v1657 has a anti-CD3 OKT3 (VL-VH) scFv (with C to S mutation at position 100A of the VH CDR3) on chain A and a anti-CD19 BiTETM (VL-VH) scFv on chain B of the heterodimer Fc with the following mutations
- v1658 has a anti-CD3 OKT3 (VH-VL) scFv with 18 amino acid linker on chain A and a anti-CD19 BiTETM (VL-VH) scFv on chain B of the heterodimer Fc with the following mutations T350V_L351Y_F405A_Y407V on chain A and T350V_T366L_K392L_T394W on chain B. (corresponding to polypeptide sequences SEQ ID NOs: 121 and 122)
- v1659 has a anti-CD3 OKT3 (VH-VL) scFv with 10 amino acid linker on chain A and a anti-CD19 BiTETM (VL_VH) scFv on chain B of the heterodimer Fc with the following mutations T350V_L351Y_F405A_Y407V on chain A and T350V_T366L_K392L_T394W on chain B. (corresponding to polypeptide sequences SEQ ID NOs: 125 and 126)
- v1660 has a anti-CD3 OKT3 (VH-VL) scFv with 19 amino acid linker on chain A and a anti-CD19 BiTETM (VL-VH) scFv on chain B of the heterodimer Fc with the following mutations T350V_L351Y_F405A_Y407V on chain A and T350V_T366L_K392L_T394W on chain B. (corresponding to polypeptide sequences SEQ ID NOs: 129 and 130)
- the following variants are Fc knoock outs that contain mutations to the anti-CD3 scFv that include either changes to the linker length, VH-VL orientations, or point mutations to improve stability and yeild.
- v1661 has a anti-CD19 BiTETM (VL-VH) scFv on chain A and a anti- CD3 OKT3 (VL-VH) scFv on chain B of the heterodimer Fc with the following mutations D265S_L234A_L235A_T350V_L351Y_F405A_Y407V on chain A and D265S_L234A_L235A_T350V_T366L_K392L_T394W on chain B.
- v1662 has a anti-CD19 BiTETM (VL-VH) scFv on chain A and a anti- CD3 OKT3 (VL-VH) scFv (with C to S mutation at position 100A of the VH CDR3) on chain B of the heterodimer Fc with the following mutations
- v1663 has a anti-CD19 BiTETM (VL-VH) scFv on chain A and a anti- CD3 OKT3 (VL-VH) scFv with 18 amino acid linker on chain B of the
- v1664 has a anti-CD19 BiTETM (VL-VH) scFv on chain A and a anti- CD3 OKT3 (VH-VL) scFv with 10 amino acid linker on chain B of the
- v1665 has a anti-CD3 BiTETM (VH-VL) scFv on chain A and a anti-CD3 BiTETM (VH-VL) scFv on chain A and a anti-CD3 BiTETM (VH-VL) scFv on chain A and a anti-CD3 BiTETM (VH-VL) scFv on chain A and a anti-CD3 BiTETM (VH-VL) scFv on chain A and a anti-CD3 BiTETM (VH-VL) scFv on chain A and a anti-CD3 BiTETM (VH-VL) scFv on chain A and a anti-CD3 BiTETM (VH-VL) scFv on chain A and a anti-CD3 BiTETM (VH-VL) scFv on chain A and a anti-CD3 BiTETM (VH-VL) scFv on chain A
- v1666 has a anti-CD3 OKT3 (VH-VL) scFv with a 19 amino acid linker on chain A and a anti-CD19 BiTETM (VL-VH) scFv on chain B of the heterodimer Fc with the following mutations
- v1667 has a anti-CD19 BiTETM (VL-VH) scFv on chain A and a anti- CD3 OKT3 (VL-VH) scFv on chain B of the heterodimer Fc with the following mutations N297A_T350V_L351Y_F405A_Y407V on chain A and
- v1668 has a anti-CD19 BiTETM (VL-VH) scFv on chain A and a anti- CD3 OKT3 (VL-VH) scFv (with C to S mutation at position 100A of the VH CDR3) on chain B of the heterodimer Fc with the following mutations
- v1669 has a anti-CD19 BiTETM (VL-VH) scFv on chain A and a anti- CD3 OKT3 (VH-VL) scFv with 18 amino acid linker on chain B of the
- v1670 has a anti-CD19 BiTETM (VL-VH) scFv on chain A and a anti-
- v1671 has a anti-CD3 BiTE I M (VH-VL) sc
- v1672 has a anti-CD3 OKT3 (VH-VL) scFv with a 19 amino acid linker on chain A and a anti-CD19 BiTETM (VL-VH) scFv on chain B of the heterodimer Fc with the following mutations
- the following variants contain mutations to the anti-CD3 scFv that include either changes to the linker length, VH-VL orientations, or point mutations to improve stability and yeild.
- v1673 has a anti-CD3 OKT3 (VL-VH) scFv (with C to S mutation at position 100A of the VH CDR3) on chain B and a Fc on chain A of the heterodimer Fc with the following mutations T350V_L351Y_F405A_Y407V on chain A and T350V_T366L_K392L_T394W on chain B. (corresponding to polypeptide sequences SEQ ID NOs: 169 and 170)
- v1674 has a anti-CD3 OKT3 (VH-VL) scFv with 18 amino acid linker on chain B and a Fc on chain A of the heterodimer Fc with the following mutations T350V_L351Y_F405A_Y407V on chain A and
- v1798 has a anti-CD3 OKT3 (VH-VL) scFv with 10 amino acid linker on chain B and a Fc on chain A of the heterodimer Fc with the following mutations T350V_L351Y_F405A_Y407V on chain A and
- v1799 has a anti-CD3 OKT3 (VH-VL) scF
- the following variants contain point mutations for disulfide stabilization at position 100 in the variable light and position 44 in the variable heavy chain (denoted 44-100SS).
- v1800 has a anti-CD19 BiTETM (VL-VH) scFv on chain A and anti- CD3 OKT3 (VL-VH) 44-100SS on chain B of the heterodimer Fc with the following mutations T350V_L351Y_F405A_Y407V on chain A and
- v1801 has a anti-CD19 BiTETM (VL-VH) scFv on chain A and anti- CD3 OKT3 (VL-VH) (with C to S mutation at position 100A of the VH CDR3) 44-100SS on chain B of the heterodimer Fc with the following mutations T350V_L351Y_F405A_Y407V on chain A and T350V_T366L_K392L_T394W on chain B. (corresponding to polypeptide sequences SEQ ID NOs: 189 and 190)
- v1802 has a anti-CD3 BiTETM (VH-VL) 44-100SS scFv on chain A and anti-CD19 BiTETM (VL-VH) scFv on chain B of the heterodimer Fc with the following mutations T350V_L351Y_F405A_Y407V on chain A and
- v4541 has a anti-CD3 BiTETM (VH-VL) 44-100SS scFv on chain A and anti-CD19 BiTETM (VL-VH) scFv on chain B of the heterodimer Fc with the following mutations D265S_L234A_L235A_T350V_L351Y_F405A_Y407V on chain A and D265S_L234A_L235A_T350V_T366L_K392L_T394W on chain B. [Polypeptide sequences correspond to SEQ ID No: 50 and 52]
- v4542 has a cyno/human cross-reactive anti-CD3 BiTETM 12C (VH- VL) scFv on chain A and cyno/human cross-reactive anti-CD19 MOR208 (VH- VL) scFv on chain B of the heterodimer Fc with the following mutations
- v4543 has a cyno/human cross-reactive anti-CD3 BiTETM 12C (VH- VL) 44-100SS scFv on chain A and cyno/human cross -reactive anti-CD19 MOR208 (VH-VL) 44-100SS scFv on chain B of the heterodimer Fc with the following mutations D265S_L234A_L235A_T350V_L351Y_F405A_Y407V on chain A and D265S_L234A_L235A_T350V_T366L_K392L_T394W on chain B.
- Polypeptide sequences correspond to SEQ ID No: 58 and 60]
- v4544 has a cyno/human cross-reactive anti-CD3 BiTETM 12C (VH-VL) scFv on chain A and cyno/human cross-reactive anti-CD19 MOR208 (VL-VH) scFv on chain B of the heterodimer Fc with the following mutations D265S_L234A_L235A_T350V_L351Y_F405A_Y407V on chain A and
- v4545 has a cyno/human cross-reactive anti-CD3 BiTETM 12C (VH- VL) 44-100SS scFv on chain A and cyno/human cross -reactive anti-CD19 MOR208 (VL-VH) 44-100SS scFv on chain B of the heterodimer Fc with the following mutations D265S_L234A_L235A_T350V_L351Y_F405A_Y407V on chain A and D265S_L234A_L235A_T350V_T366L_K392L_T394W on chain B.
- Polypeptide sequences correspond to SEQ ID No: 66 and 68]
- v4546 has a cyno/human cross-reactive anti-CD3 BiTETM 12C (VH- VL) scFv on chain A and cyno/human cross-reactive anti-CD19 MDX-1342 (VH-VL) scFv on chain B of the heterodimer Fc with the following mutations D265S L234A L235A T350V L351Y F405A Y407V on chain A and
- v4547 has a cyno/human cross-reactive anti-CD3 BiTETM 12C (VH- VL) 44-100SS scFv on chain A and cyno/human cross-reactive anti-CD19 MDX-1342 (VH-VL) 44-100SS scFv on chain B of the heterodimer Fc with the following mutations D265S_L234A_L235A_T350V_L351Y_F405A_Y407V on chain A and D265S_L234A_L235A_T350V_T366L_K392L_T394W on chain B. [Polypeptide sequences correspond to SEQ ID No: 74 and 76].
- v4548 has a cyno/human cross-reactive anti-CD3 BiTETM 12C (VH- VL) scFv on chain A and cyno/human cross-reactive anti-CD19 MDX-1342 (VL-VH) scFv on chain B of the heterodimer Fc with the following mutations D265S_L234A_L235A_T350V_L351Y_F405A_Y407V on chain A and
- v4549 has a cyno/human cross-reactive anti-CD3 BiTETM 12C (VH- VL) 44-100SS scFv on chain A and cyno/human cross -reactive anti-CD19 MDX-1342 (VL-VH) 44-100SS scFv on chain B of the heterodimer Fc with the following mutations D265S_L234A_L235A_T350V_L351Y_F405A_Y407V on chain A and D265S_L234A_L235A_T350V_T366L_K392L_T394W on chain B.
- Polypeptide sequences correspond to SEQ ID No: 82 and 84]
- the antibodies and antibody controls were cloned and expressed as follows.
- the genes encoding the antibody heavy and light chains were constructed via gene synthesis using codons optimized for human/mammalian expression.
- the Fab sequences were generated from a known Her2/neu binding Ab (Carter P. et al. (1992) Humanization of an anti P185 Her2 antibody for human cancer therapy. Proc Natl Acad Sci 89, 4285.) and the Fc was an lgG1 isotype.
- the scFv-Fc and OAA sequences, were generated from a known anti-CD3 and CD19 scFv Bite antibodies (Kipriyanov et. al., 1998, Int.
- the CHO cells were transfected in exponential growth phase (1 .5 to 2 million cells/mL) with aqueous 1 mg/ml_ 25kDa polyethylenimine (PEI, Polysciences) at a PEhDNA ratio of 2.5:1 .(Raymond C. et al. A simplified polyethylenimine-mediated transfection process for large-scale and high- throughput applications. Methods. 55(1 ):44-51 (201 1 )).
- the DNA was transfected in optimal DNA ratios of the heavy chain A (HC-A), light chain (LC), and heavy chain B that allow for heterodimer formation (e.g.
- HC-A/HC-B/ ratios 50:50% (OAAs; HC/Fc), 50:50%.
- Transfected cells were harvested after 5-6 days with the culture medium collected after centrifugation at 4000rpm and clarified using a 0.45 ⁇ filter.
- the clarified culture medium was loaded onto a MabSelect SuRe (GE Healthcare) protein-A column and washed with 10 column volumes of PBS buffer at pH 7.2.
- the antibody was eluted with 10 column volumes of citrate buffer at pH 3.6 with the pooled fractions containing the antibody neutralized with TRIS at pH 1 1.
- the protein was finally desalted using an Econo-Pac 10DG column (Bio-Rad).
- the protein was futher purified by protein L chromatography by the method as follows. Capto L resin PBS was equilibrated with PBS and protein A purified v875, neutralized with 1 M Tris, was added to resin and incubated at RT for 30 min. Resin washed with PBS and flow through collected, bound protein was eluted with 0.5 ml 0.1 M Glycine, pH 3.
- the protein was further purified by gel filtration,
- the SDS-PAGE in Figure 7 shows that all examplary heteromultimer can be expressed transiently in CH03E7 cells with a cell viability of > 80 %.
- Example 3 Heteromultimer v873 is able to bridge Jurkat CD3 T cells and Raji CD19 B cells.
- Pellets were resuspended in 2 ml of L10 + GS1 + NaN3 to a final concentration 5x x106 cells/ml.
- each cell line was mixed together at the desired ratio, at a final concentration of 1x10 6 cells/ml.
- T:T bridging was assessed with Jurkat-violet + Jurkat-FarRed
- B:B was assessed with RAJI-violet + RAJI-FarRed
- T:B bridging was assessed with Jurkat-violet + RAJI-FarRed.
- Antibodies were diluted to 2x in L10+GS1 +NaN3 at room
- Bridging % was calculated as the percentage of events that are simultaneously labeled violet and Far-red.
- Figure 1 B shows the ability of v873 and blinatumomab CD19-CD3 BiTE (v891 , MT-103) to bridge Jurkat CD3 T cells (Top left quadrant) with Raji CD19 B cells (bottom right quadrant) by FACS. Bridged T-B cells appear in top right quadrant. This result demonstrates that at 300 nM, heteromultimer v873 is able to specifically bridge Jurkat T cells and Raji B cells to a similar extent (23% of total cells) as BiTE (21 % of total cells).
- Example 4 Heteromultimers bind selectively to CD3- and CD19- expressing cells.
- Figure 2 also demonstrates that the one-armed anti-CD3 antibody specifically binds to Jurkat T cells and does not cross-react to CD19 expressing B cells, and that the one- armed anti-CD19 antibody specifically binds to Raji B cells and does not cross- react to Jurkat T cells.
- FIG. 9 shows that the FACS assay shows that v873 binds selectively to Jurkat T-cells and to Raji B-cells (Figure 9 B).
- Figure 9A shows that human IgG (hlgG) does not bind to Jurkat T-cells and has low level binding to Raji B-cells, as expected due to the interactions between the hlgG Fc and CD32B on the Raji B-cells.
- Figure 9A also shows that the anti-CD19 OAA binding selectively to the Raji B-cells and does not cross-react to Jurkat T cells.
- the FACS assay was also carried out to confirm that v873 does not bind to control cell lines that do not express CD3 or CD19.
- Figure 10 shows that v873 does not bind to the K562 cell line, which does not express CD19 or CD3.
- Figure 1 1 shows that v873 also does not bind to mouse lymphoid cells which does not express CD19 or CD3.
- Example 5 Heteromultimers mediate PBMC killing of target Raji B cells
- v873 to mediate T cell cytoxicity against target Raji B-cells was measured using IL-2-stimulated PBMCs as follows.
- CD4+ and CD8+ cells were then labeled and analyzed by cytometry to evaluate the contents of CD69+ cells in the preparations.
- the PBMCs and the enriched fractions from the second day were used in the assay as is without IL- 2 activation or in resting state.
- CFSE was used and tested as a differential label between Raji and autologous B cells.
- Raji target cells were pre-labeled with minimal amounts of CFSE before the incubation with effectors, with or without test items.
- the cell pellets were resuspended in various antibody cocktails for flow cytometry analysis.
- a Guava 8HT flow cytometer was used for analysis of cell
- Each condition tested included appropriate controls; wells with all effector and target cell types separately, for all donors, incubated with all test items at all concentrations used in the potency assay.
- markers used for B and T cell staining were anti- CD20 and anti-CD7, respectively for the study shown in Figure 23. This assay was performed with resting and IL2-activated PBMCs, purified CD4+ and CD8+ T cells.
- the averaged culture medium background signal (OD values) is subtracted from all wells before evaluating cytotoxic response.
- OD values averaged culture medium background signal
- the spontaneous release of effectors and target combined is obtained from wells without any test items, for each effector population tested: the PBMCs, CD4 and CD8 negatively selected populations.
- the background LDH activity of the test system is better evaluated in wells containing the experimental mixture of effectors -target populations without any test item present.
- Figure 3A depicts the ability of v873 to redirect IL-2 activated PBMC to kill target Raji B cells from 3 donors.
- Figure 3B demonstrates that v873 is able to mediate higher redirected T-cell cytotoxicity than v891 in one of the donors.
- IL-2 stimulation of PBMC was tested at 1000-3000 units per mL.
- Example 6 Heteromultimers mediate redirected killing of target Raji B cells with resting and IL-2 activated CD4+ and CD8+ T cells
- Figure 16A depicts the ability of v875, v1379 and v1380 to mediate antibody dependent B cell cytoxicity by redirected CD4+ and CD8+ T cell towards Raji B cells.
- the top panel of Figure 16 illustrates the cytoxicity of resting CD4+ and CD8+ T cells towards Raji B cells. These results illustrate that v875, v1380 and v891 ellicit a concentration dependant cytotoxic response that is more prominent in the CD8+ T cells.
- the lower panel of Figure 16 illustrates that the cytoxicity of IL-2 stimulated CD4+ and CD8+ T cells towards target Raji B cells. These results indicate that v875 and v1379 ellicit similar
- Figure 16 B-E depict representations of the data in Figure 16A normalized to human IgG, for v875 ( Figures 16B and C), and v1379 and v1380
- Figures 16D and E include % cytotoxicity indicated at each test antibody concentration.
- Figure 16B shows the % cytotoxicity to target Raji B cells with v875 with IL-2 activated CD4+ and CD8+ effector T cells.
- Figure 16C shows the % cytotoxicity to target Raji B cells with v875 with resting CD4+ and CD8+ effector T cells.
- Figure 16D and E show direct comparisons of v1379 (WT Fc) and v1380 (L234A_L235A Fc knockout) in IL-2 activated ( Figure 16D), and resting (Figure 16E) CD4+ and CD8+ T cells. The most signicant impact of the
- FIG. 20 shows the target Raji B cell cytotoxicty of v875 with IL-2 activated CD4+ and CD8+ T cells.
- the percent target B cell killing elicited with IL-2 activated CD4+ cells and CD8+ T cells did not increase at v875 concentrations above the 0.06 nM.
- target B cell killing is greater with IL-2 activated CD8+ T cells, compared to IL-2 activated CD4+ T cells.
- Figure 20B shows the target Raji B cell cytotoxicty of v875 with resting CD4+ and CD8+ T cells. The percent target B cell killing elicited with resting CD8+ cells does not increase greatly at v875
- Dose-dependent B cell killing is seen with v875 and v873 when CD4+ and CD8+ effector T cells are used. As expected, target B cell killing is greater with resting CD8+ T cells, compared to resting CD4+ T cells.
- Example 7 The heterodimeric Fc contributes to target Raji B cell cytotoxicity
- Figure 17 depicts v875 and v873 Azymetri
- FIG. 17A illustrates that Fc blocking of IL-2 activated PBMC results in a minor (v875) or no (v873) reduction in the % cytotoxicity of target Raji B cells.
- Figure 17B illustrates that Fc blocking of resting PBMC results in a reduction in the % cytotoxicity of target Raji B cells for v875 and v873.
- Figure 18A illustrates that Fc blocking of IL-2 activated PBMC results in a reduction in the % cytotoxicity of target Raji B cells at all antibody
- Figure 18B illustrates that Fc blocking of resting PBMC results in a reduction in the % cytotoxicity of target Raji B cells at all antibody concentrations tested for v875 and v873.
- Figures 17 and 18 show that the Fc contributes to target Raji B cell cytotoxicity in the
- Example 8 Heteromultimers mediate autologous B cell cytotoxicity
- Figure 21 shows, relative to untreated media and human IgG controls, that v875 and v873 (300 nM) mediate autologous B cell killing in total resting PBMC (left panel) and total IL-2 activated PBMC .
- Example 9 Heteromultimer v875 spares autologous T cell cytotoxicity compared to BiTE
- Figure 22 shows, relative to untreated me ⁇
- v875 has a more selective B cell killing by sparing more autologous T cells compared to v873 and v891.
- Example 10 Design, expression and purification of heteromultimers with an albumin scaffold.
- the sequences for the anti-CD19 and anti-CD3 scFvs were chosen from two molecules that are currently in clinical trials and are well documented and tested for stability and production.
- the anti-CD19 and anti-CD3 scFv were directly adopted from the BiTE molecule blinatumomab.
- the antiCD3 scFv was chosen in the VH-VL orientation, consistent with what used in BiTE.
- the benchmark molecule was an scFv molecule based on BiTE (v891 ).
- AlbuCORE_1 (ABH2) CD3/CD19 fusions were created by attaching the antiCD3 warhead to the natural N terminus of fragment 1 and the antiCD19 to the C terminus of fragment 2 (v1092, polypeptide sequences corresponding to SEQ ID NO:264 and 266).
- the linkers used were identical to the ones used for the multivalent HER2 AlbuCORE experiments: GGGS at the N terminus of fragment 1 and (GGSG) 4 GG at the C terminus of fragment 2.
- a second molecule was created where the warheads were reversed (i.e. anti-CD19 warhead at the natural N terminus of fragment 1 and the anti-CD3 at the C terminus of fragment 2, v1093.
- v1094 was designed to accommodate two different fusions at the natural termini of the albumin polypeptide (polypeptide sequences corresponding to SEQ ID NO:268).
- the scFv fusions were linked to the albumin molecule through a GGS linker at the N terminus and a GGSG linker at the C terminus.
- the length of the linkers reflect the ones used in the MM-1 1 1 molecule, despite having a different sequence type.
- V221 is the albumin-based heteromultimer used to construct v1092, but without the cargo molecules (polypeptide sequences corresponding to SEQ ID NO:269 and 270).
- Example 1 1 Heteromultimers with an albumin scaffold bind specifically to CD3- or CD19-expressing cells
- Example 12 Heteromultimers with heterodimeric Fc or albumin scaffolds show comparable B-cell targeting and T-cell bridging
- Example 13 Exemplary heteromultimers have higher anti-CD3 KD and higher Bmax in binding to T and B cell as determined by FACS.
Abstract
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WO2015143079A1 (en) * | 2014-03-19 | 2015-09-24 | Regeneron Pharmaceuticals, Inc. | Antibody compositions for tumor treatment |
WO2015153765A1 (en) | 2014-04-01 | 2015-10-08 | Adimab, Llc | Multispecific antibody analogs comprising a common light chain, and methods of their preparation and use |
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BR112015000798A2 (en) | 2017-06-27 |
AU2013289883A1 (en) | 2015-02-19 |
CA2878843A1 (en) | 2014-01-16 |
US20190248897A1 (en) | 2019-08-15 |
EP2872170A2 (en) | 2015-05-20 |
EP2872170A4 (en) | 2016-06-22 |
KR20150036606A (en) | 2015-04-07 |
CN104640562A (en) | 2015-05-20 |
WO2014012085A9 (en) | 2015-03-19 |
JP2015528003A (en) | 2015-09-24 |
AU2013289883B2 (en) | 2018-11-01 |
US20210317212A1 (en) | 2021-10-14 |
RU2650868C2 (en) | 2018-04-17 |
WO2014012085A3 (en) | 2014-03-27 |
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HK1207575A1 (en) | 2016-02-05 |
US20140154253A1 (en) | 2014-06-05 |
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