US20140302037A1 - BISPECIFIC-Fc MOLECULES - Google Patents

BISPECIFIC-Fc MOLECULES Download PDF

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US20140302037A1
US20140302037A1 US14/210,178 US201414210178A US2014302037A1 US 20140302037 A1 US20140302037 A1 US 20140302037A1 US 201414210178 A US201414210178 A US 201414210178A US 2014302037 A1 US2014302037 A1 US 2014302037A1
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amino acid
cell
polypeptide chain
seq
chain
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Luis G. Borges
Patrick A. Baeuerle
Wei Yan
Mark L. Michaels
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Amgen Inc
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Amgen Inc
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Assigned to AMGEN INC. reassignment AMGEN INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BAEUERLE, PATRICK A., YAN, WEI, MICHAELS, MARK L., BORGES, LUIS G.
Publication of US20140302037A1 publication Critical patent/US20140302037A1/en
Priority to US15/885,998 priority patent/US11753475B2/en
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    • 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
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
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    • 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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    • C07K16/2803—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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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
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    • C07K2317/565—Complementarity determining region [CDR]
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    • C07K2317/622—Single chain antibody (scFv)
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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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    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
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    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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    • C07K2319/00—Fusion polypeptide

Definitions

  • Bispecific antibodies have promise as therapeutics in a variety of indications.
  • Bispecific antibodies having a standard IgG format can be challenging to produce because they include four different polypeptide chains.
  • the efficacy of a smaller, more easily-produced bispecific molecule has been clinically demonstrated in non-Hodgkin's lymphoma. See, e.g., Bargou et al. (2008), Science 321(5891): 974-977. Daily administration was used to achieve these results, presumably because of the short in vivo half life of this small, single chain molecule. Id.
  • bispecific therapeutics with favorable pharmacokinetic properties, as well as therapeutic efficacy and a format that makes them straightforward to produce.
  • a Bispecific-Fc (Bi-Fc) as described herein molecule can bind to one molecule of each of two different proteins and contains an Fc region of an antibody.
  • a Bi-Fc also can have favorable pharmacokinetic properties relative to a single chain molecule lacking an Fc region.
  • One protein bound by a Bi-Fc can be expressed on an immune effector cell such as a T cell, an NK cell, a neutrophil, or a macrophage, and the other protein can be expressed on a target cell, for example, a cancer cell, a cell infected by a pathogen, or a cell mediating a disease, such as a fibrotic cell.
  • the Bi-Fc molecules described herein can elicit activation an immune effector cell in the presence of a target cell.
  • a Bi-Fc which can comprise: (a) (i) a first polypeptide chain having the formula V1-L1-V2-L2-V3-L3-V4-L4-Fc, wherein Fc is an Fc polypeptide chain, wherein V1, V2, V3, and V4 are each immunoglobulin variable regions that have different amino acid sequences, wherein L1, L2, L3, and L4 are linkers, and wherein L4 can be present or absent, and (ii) a second polypeptide chain that comprises an Fc polypeptide chain; or (b) (i) a first polypeptide chain having the formula Fc-L4-V1-L1-V2-L2-V3-L3-V4, wherein Fc is an Fc polypeptide chain, wherein V1, V2, V3, and V4 are each immunoglobulin variable regions that have different amino acid sequences, wherein L1, L2, L3, and L4 are linkers, and wherein L4 can be present or absent,
  • V1 can be a heavy chain variable (VH) region, and V2 can be a light chain variable (VL) region.
  • V1 can be a VL region and V2 can be a VH region.
  • V3 and V4 can be a VH and a VL region, respectively, or V3 and V4 can be a VL and a VH region, respectively.
  • L1 and L3 can be at least 15 amino acids long, and L2 can be less than 12 amino acids long.
  • V1 and V2 can bind to a target cell or an immune effector cell when they are part of an IgG and/or an scFv antibody
  • V3 and V4 can bind to a target cell or an immune effector cell when they are part of an IgG and/or an scFv antibody.
  • the Fc polypeptide chain in the first polypeptide chain can comprise a heterodimerizing alteration
  • the Fc polypeptide chain in the second polypeptide chain can comprise another heterodimerizing alteration.
  • the heterodimerizing alteration in the first polypeptide chain can be a charge pair substitution
  • the heterodimerizing alteration in the second polypeptide chain can be a charge pair substitution.
  • the first polypeptide chain can comprise the charge pair substitutions K409D or K409E and K392D or K392E
  • the second polypeptide chain can comprise the charge pair substitutions D399K or D399R and D356K or D356R
  • the second polypeptide chain comprises the charge pair substitutions K409D or K409E and K392D or K392E
  • the first polypeptide chain comprises the charge pair substitutions D399K or D399R and D356K or D356R.
  • the Fc polypeptide chains of the first and second polypeptide chains can be human IgG Fc polypeptide chains, such as IgG1, IgG2, IgG3, or IgG4 Fc polypeptide chains.
  • the Fc polypeptide chains of the first and second polypeptide chains can comprise one or more alterations that inhibit(s) Fc gamma receptor (Fc ⁇ R) binding or enhance(s) ADCC.
  • the Fc polypeptide chains of the first and second polypeptide chains comprise, for example, L234A, L235A, and any substitution at N297.
  • a Bi-Fc which can comprise: (i) a first polypeptide chain having following formula: V1-L1-V2-L2-V3-L3-V4-L4-Fc, wherein Fc is an Fc polypeptide chain, wherein V1, V2, V3, and V4 are each immunoglobulin variable regions that have different amino acid sequences, wherein L1, L2, L3, and L4 are linkers, and wherein L4 can be present or absent; and (ii) a second polypeptide chain comprising an Fc polypeptide chain; wherein L1 and L3 are at least 15 amino acids long and L2 is less than 12 amino acids long; wherein either V1 is a VH region and V2 is a VL region or V1 is a VL region and V2 is a VH region; wherein either V3 is a VH region and V4 is a VL region or V3 is a VL region and V4 is a VH region; wherein the F
  • the Fc polypeptide chains can be human IgG Fc polypeptide chains, such as IgG1, IgG2, IgG3, or IgG4 Fc polypeptide chains.
  • the Fc polypeptide chains of the first and second polypeptide chains comprise one or more alteration that inhibits Fc ⁇ R binding, such as one or more of L234A, L235A, and any substitution at N297.
  • a Bi-Fc can comprise: (a) a first polypeptide chain having the formula V1-L1-V2-L2-V3-L3-V4-L4-Fc, wherein Fc is an Fc polypeptide chain, wherein V1, V2, V3, and V4 are each immunoglobulin variable regions that have different amino acid sequences, wherein L1, L2, L3, and L4 are linkers, and wherein L4 can be present or absent; or (b) a first polypeptide chain having the following formula: Fc-L4-V1-L1-V2-L2-V3-L3-V4, wherein Fc is an Fc polypeptide chain, wherein V1, V2, V3, and V4 are each immunoglobulin variable regions that have different amino acid sequences, wherein L1, L2, L3, and L4 are linkers, and wherein L4 can be present or absent; wherein the Bi-Fc is a monomer and wherein the Bi-Fc mediates cytolysis of a target cell
  • the Fc polypeptide chain can be a human IgG Fc polypeptide chain, such as IgG1, IgG2, IgG3, or IgG4 Fc polypeptide chain.
  • the Fc polypeptide chain of (a) or (b) can comprise one or more the following alterations: K392D, K382E, K409D, K409E, Y349T, L351T, L368T, L398T, F405T, Y407T, Y407R.
  • the Fc polypeptide chain of (a) or (b) can comprise one or more alteration that inhibits Fc ⁇ R binding, such as one or more of L234A, L235A, and any substitution at N297.
  • the immune effector cell of any Bi-Fc described herein can be a human T cell and/or a cynomolgus monkey T cell.
  • the effector cell protein of any Bi-Fc described herein can be part of the human and/or cynomolgus monkey TCR-CD3 complex.
  • the effector cell protein of any Bi-Fc described herein can be the human and/or cynomolgus monkey TCR ⁇ , TCR ⁇ , TCR ⁇ , TCR ⁇ , CD3 ⁇ chain, CD3 ⁇ chain, CD3 ⁇ chain, CD3 ⁇ chain, or CD3 ⁇ chain.
  • the Bi-Fc can comprise a VH region and a VL comprising the amino acid sequences of SEQ ID NOs:7 and 8, respectively, or comprising the amino acid sequences of SEQ ID NOs:29 and 31, respectively.
  • the target cell of any Bi-Fc can be a cancer cell, a cell infected by a pathogen, or a cell that mediates disease.
  • the cancer can be a hematologic malignancy or a solid tumor malignancy.
  • the pathogen can be virus, including human immunodeficiency virus, hepatitis virus, human papilloma virus, or cytomegalovirus, or a bacterium of the genus Listeria, Mycobacterium, Staphylococcus , or Streptococcus .
  • the target cell is a cell that mediates a disease
  • the target cell can be a fibrotic cell that mediates a fibrotic disease or an autoimmune or inflammatory disease.
  • composition comprising any of the Bi-Fc molecules described herein and a physiologically acceptable excipient.
  • nucleic acids encoding any of the Bi-Fc described herein and vectors containing such nucleic acids, as well as host cell containing such nucleic acids and/or vectors.
  • a method for making a Bi-Fc comprising culturing the host cell containing the nucleic acids or vector under conditions such that the nucleic acids are expressed, and recovering the Bi-Fc from the cell mass or the culture medium.
  • a method for treating a cancer patient comprising administering to the patient a therapeutically effective dose of any of the Bi-Fc molecules described herein, wherein the target cell of the Bi-Fc is a cancer cell.
  • This method can further comprise administering radiation, a chemotherapeutic agent, or a non-chemotherapeutic, anti-neoplastic agent before, after, or concurrently with the administration of the Bi-Fc.
  • the patient can have a hematologic malignancy or a solid tumor malignancy.
  • a method for treating a patient having a fibrotic disease comprising administering to the patient a therapeutically effective dose of any of the Bi-Fc molecules described herein, wherein the target cell of the Bi-Fc is a fibrotic cell.
  • the fibrotic disease can be atherosclerosis, chronic obstructive pulmonary disease (COPD), cirrhosis, scleroderma, kidney transplant fibrosis, kidney allograft nephropathy, or a pulmonary fibrosis, including idiopathic pulmonary fibrosis.
  • COPD chronic obstructive pulmonary disease
  • described herein is a method for treating a patient having a disease mediated by a pathogen comprising administering to the patient a therapeutically effective dose of any of the Bi-Fc molecules described herein.
  • the pathogen can be a virus, a bacterium, or a protozoan.
  • compositions comprising any of the Bi-Fc molecules described herein.
  • Such compositions can be for the treatment of a cancer, an infectious disease, an autoimmune or inflammatory disease, or a fibrotic disease.
  • FIG. 1 A diagram of a Bi-Fc molecule.
  • Four immunoglobulin variable regions are indicated by ovals and labeled V1, V2, V3, and V4.
  • CH2 and CH3 regions are labeled as such and diagramed as an elongated hexagon. Lines between these regions indicate linkers or a hinge region.
  • Exemplary disulfide bridges are indicated by horizontal lines.
  • FIG. 2 Binding of a Bi-Fc to target cell and immune effector cells. Methods are described in Example 2. Mean fluorescence intensity (MFI) is indicated on the x axis, and the number of cells is indicated on the y axis.
  • the unfilled profiles represent data from cells in the absence of one of the bispecific molecules, and the solidly filled profiles represent data from cells in the presence of one of the bispecific molecules.
  • panels at left represent data from samples containing the anti-HER2/CD3 Bi-Fc
  • panels at right represent data from samples containing the single chain anti-HER2/CD3. Top two panels represent data from samples containing JIMT-1 cells (which express the target cell protein HER2), and bottom two panels represent data from samples containing T cells (which express the effector cell protein CD3 ⁇ ).
  • FIG. 3 Cytolytic activity of an anti-FOLR1/CD3 Bi-Fc and a single chain anti-FOLR1/CD3 molecule. Methods are described in Example 3.
  • the x axis in each panel indicates the concentration of the Bi-Fc or single chain molecule (pM) in each sample.
  • the y axis in each panel indicates the percent specific lysis calculated as described in Example 3.
  • Open circles connected by a dashed line indicate data from samples containing the single chain molecule, and filled circles connected by a solid line indicate data from the Bi-Fc molecule.
  • the top, middle, and bottom panels, as indicated, show data from Cal-51 cells (which express FOLR1), T47D cells (which express FOLR1), and BT474 cells (which do not express FOLR1), respectively.
  • FIG. 4 Cytolytic activity of an anti-HER2/CD3 Bi-Fc and a single chain anti-HER2/CD3 molecule. Methods are described in Example 3.
  • the x axis in each panel indicates the concentration of the Bi-Fc or single chain molecule (pM) in each sample.
  • the y axis in each panel indicates the percent specific lysis calculated as described in Example 3.
  • Open circles connected by a dashed line indicate data from samples containing the single chain molecule, and filled circles connected by a solid line indicate data from the Bi-Fc molecule.
  • the top, middle, and bottom panels, as indicated, show data from JIMT-1 cells (which express HER2), T47D cells (which express HER2), and SHP77 cells (which do not express HER2), respectively.
  • FIG. 5 Cytokine production by T cells in the presence of an anti-FOLR1/CD3 Bi-Fc or single chain molecule. Methods are described in Example 4. Open circles connected by dashed lines indicate data from assays containing the anti-FOLR1/CD3 Bi-Fc, and solidly filled circles connected by solid lines indicate data from the single chain anti-FOLR1/CD3 molecule.
  • the x axis in each panel indicates the concentration of the Bi-Fc or single chain molecule (pM) in each assay.
  • the y axis indicates the concentration and identity of the cytokine detected (pg/mL).
  • Panel A shows data for interferon gamma (IFN ⁇ ).
  • Panel B shows data for tumor necrosis factor alpha (TNF ⁇ ).
  • Panel C shows data for interleukin-10 (IL-10).
  • Panel D shows data for interleukin-2 (IL-2).
  • Panel E shows data for interleukin-13 (IL-13).
  • graphs on the left show data from T47D cells (which express FOLR1), and graphs on the right show data from BT474 cells (which do not express FOLR1).
  • FIG. 6 Cytokine production by T cells in the presence of an anti-HER2/CD3 Bi-Fc or single chain molecule. Methods are described in Example 4. Open circles connected by dashed lines indicate data from assays containing the anti-HER2/CD3 Bi-Fc, and solidly filled circles connected by solid lines indicate data from the single chain anti-HER2/CD3 molecule.
  • the x axis in each panel indicates the concentration of the Bi-Fc or single chain molecule (pM) in each assay.
  • the y axis indicates the concentration and identity of the cytokine detected (pg/mL).
  • Panels A, B, C, D, and E show data for IFN ⁇ , TNF ⁇ , IL-10 IL-2, and IL-13, respectively, as indicated. As indicated, panels on the left show data from JIMT-1 cells (which express HER2), and panels on the right show data from SHP77 cells (which do not express HER2).
  • FIG. 7 Percentage of CD25 + and CD69 + cells in the presence of an anti-HER2/CD3 Bi-Fc or single chain molecule. Methods are described in Example 5.
  • the x axis indicates the concentration (pM) of the anti-HER2/CD3 Bi-Fc or single chain molecule.
  • the y axis indicates the percent of CD3 + T cells that are also CD25 + (left panel) or CD69 + (right panel) cells.
  • Symbols indicate as follows: open squares connected by dashed line, the single chain molecule plus JIMT-1 target cells; solidly filled, downward pointing triangles connected by a solid line, the Bi-Fc molecule plus JIMT-1 target cells; open circles connected by a dashed line, the single chain molecule without JIMT-1 target cells; and solidly filled, upward pointing triangles connected by a solid line, the Bi-Fc without JIMT-1 target cells.
  • FIG. 8 Pharmacokinetic properties of a Bi-Fc and a single chain bispecific molecule in mice. Methods are described in Example 6. In the top panel, a pharmacokinetic profile following an intravenous injection is shown, and below is shown the profile following a subcutaneous injection. Solidly filled circles connected by a solid line indicate data from the anti-HER2/CD3 single chain molecule, and asterisks connected by a solid line indicate data from the anti-HER2/CD3 Bi-Fc molecule.
  • SEQ ID NO Description SEQ ID NO: 1 Amino acid sequence preceding VH CDR1 SEQ ID NO: 2 Amino acid sequence preceding VH CDR2 SEQ ID NO: 3 Amino acid sequence following VH CDR3 SEQ ID NO: 4 Amino acid sequence following light chain CDR3 SEQ ID NO: 5 Amino acid sequence of anti-HER2 VH region SEQ ID NO: 6 Amino acid sequence of anit-HER2 VL region SEQ ID NO: 7 Amino acid sequence of anti CD3 ⁇ VH region SEQ ID NO: 8 Amino acid sequence of anti-CD3 ⁇ VL region SEQ ID NO: 9 Amino acid sequence of a single chain anti-HER2/CD3 (P136629.3) SEQ ID NO: 10 Amino acid sequence of a first polypeptide chain of an anti- HER2/CD3 of a Bi-Fc SEQ ID NO: 11 Nucleic acid sequence of SEQ ID NO: 10 SEQ ID NO: 12 Amino acid sequence of a human IgG1
  • Bi-Fc a new form of bispecific antibody, called herein a Bi-Fc, which contains one polypeptide chain or two different polypeptide chains.
  • One chain comprises two heavy chain variable (VH) regions, two light chain variable (VL) regions, and an Fc polypeptide chain, and an optional second polypeptide chain comprises an Fc polypeptide chain.
  • one of the proteins to which the Bi-Fc binds is expressed on the surface of an immune effector cell, such as a T cell, an NK cell, a macrophage, or a neutrophils
  • the other protein to which the Bi-Fc binds is expressed on the surface of a target cell, for example a cancer cell, a cell infected by a pathogen, or a cell that mediates a disease, such as, for example, a fibrotic disease.
  • a Bi-Fc has only one binding site for each of these proteins (i.e., it binds each protein “monovalently,” as meant herein), its binding, by itself, will not oligomerize the proteins it binds to on a cell surface.
  • CD3 will not be oligomerized on the T cell surface in the absence of a target cell. Oligomerization of CD3 can cause a generalized activation of a T cell, which can be undesirable.
  • the Bi-Fc tethers an immune effector cell to a target cell, thereby eliciting specific cytolytic activity against the target cell, rather than a generalized inflammatory response. Further, the Bi-Fc molecules have favorable pharmacokinetic properties and are not unduly complex to manufacture since they contain only one or only two different polypeptide chains.
  • an “antibody,” as meant herein, is a protein containing at least one VH or VL region, in many cases a heavy and a light chain variable region.
  • the term “antibody” encompasses molecules having a variety of formats, including single chain Fv antibodies (scFv, which contain VH and VL regions joined by a linker), Fab, F(ab) 2 ′, Fab′, scFv:Fc antibodies (as described in Carayannopoulos and Capra, Ch. 9 in F UNDAMENTAL I MMUNOLOGY, 3 rd ed., Paul, ed., Raven Press, New York, 1993, pp.
  • IgG antibodies can be of the IgG1, IgG2, IgG3, or IgG4 isotype and can be human antibodies.
  • the portions of Carayannopoulos and Capra that describe the structure of antibodies are incorporated herein by reference.
  • antibody includes dimeric antibodies containing two heavy chains and no light chains such as the naturally-occurring antibodies found in camels and other dromedary species and sharks. See, e.g., Muldermans et al., 2001, J. Biotechnol.
  • An antibody can be “monospecific” (that is, binding to only one kind of antigen), “bispecific” (that is, binding to two different antigens), or “multispecific” (that is, binding to more than one different antigen). Further, an antibody can be monovalent, bivalent, or multivalent, meaning that it can bind to one, two, or multiple antigen molecules at once, respectively.
  • An antibody binds “monovalent,” to a particular protein when one molecule of the antibody binds to only one molecule of the protein, even though the antibody may also bind to a different protein as well. That is, an antibody binds “monovalently,” as meant herein, to two different proteins when it binds to only one molecule of each protein. Such an antibody is “bispecific” and binds to each of two different proteins “monovalently.”
  • An antibody can be “monomeric,” i.e., comprising a single polypeptide chain.
  • An antibody can comprise multiple polypeptide chains (“multimeric”) or can comprise two (“dimeric”), three (“trimeric”), or four (“tetrameric”) polypeptide chains.
  • an antibody can be a homomulitmer, i.e., containing more than one molecule of only one kind of polypeptide chain, including homodimers, homotrimer, or homotetramers.
  • a multimeric antibody can be a heteromultimer, i.e., containing more than one different kind of polypeptide chain, including heterodimers, heterotrimers, or heterotetramers.
  • An antibody can have a variety of possible formats including, for example, monospecific monovalent antibodies (as described in International Application WO 2009/089004 and US Publication 2007/0105199, the relevant portions of which are incorporated herein by reference) that may inhibit or activate the molecule to which they bind, bivalent monospecific or bispecific dimeric Fv-Fc, scFv-Fc, or diabody Fc, monospecific monovalent scFv-Fc/Fc's, the multispecific binding proteins and dual variable domain immunoglobulins described in US Publication 2009/0311253 (the relevant portions of which are incorporated herein by reference), the heterodimeric bispecific antibodies described herein, and the many formats for bispecific antibodies described in Chapters 1, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 of B ISPECIFIC A NTIBODIES , Kontermann, ed., Springer, 2011 (which chapters are incorporated herein by reference), among many other possible antibody formats.
  • monospecific monovalent antibodies as described in International Application WO
  • a “Bi-Fc,” as meant herein, comprises a first polypeptide chain and, optionally, a second polypeptide chain.
  • a Bi-Fc comprises both a first and a second polypeptide chain.
  • a Bi-Fc is a monomer comprising only the first polypeptide chain.
  • the first polypeptide chain comprises two VH regions and two VL regions separated by linkers and an Fc polypeptide chain.
  • the Fc polypeptide chain can be N-terminal or C-terminal relative to the four immunoglobulin variable regions, and it can be joined to the variable regions via a linker. This linker can be present or absent.
  • the second polypeptide chain if present, comprises an Fc polypeptide chain.
  • a Bi-Fc can be a monomer or a heterodimer.
  • a Bi-Fc can bind to an immune effector cell via an effector cell protein and to a target cell via a target cell protein and can mediate cytolysis of a target cell by an immune effector cell.
  • cancer cell antigen is a protein expressed on the surface of a cancer cell. Some cancer cell antigens are also expressed on some normal cells, and some are specific to cancer cells. Cancer cell antigens can be highly expressed on the surface of a cancer cell. There are a wide variety of cancer cell antigens. Examples of cancer cell antigens include, without limitation, the following human proteins: epidermal growth factor receptor (EGFR), EGFRvIII (a mutant form of EGFR), melanoma-associated chondroitin sulfate proteoglycan (MCSP), mesothelin (MSLN), folate receptor 1 (FOLR1), and human epidermal growth factor 2 (HER2), among many others.
  • EGFR epidermal growth factor receptor
  • EGFRvIII a mutant form of EGFR
  • MCSP melanoma-associated chondroitin sulfate proteoglycan
  • MSLN mesothelin
  • FOLR1 folate receptor 1
  • HER2 human epidermal
  • “Chemotherapy,” as used herein, means the treatment of a cancer patient with a “chemotherapeutic agent” that has cytotoxic or cytostatic effects on cancer cells.
  • a “chemotherapeutic agent” specifically targets cells engaged in cell division and not cells that are not engaged in cell division. Chemotherapeutic agents directly interfere with processes that are intimately tied to cell division such as, for example, DNA replication, RNA synthesis, protein synthesis, the assembly, disassembly, or function of the mitotic spindle, and/or the synthesis or stability of molecules that play a role in these processes, such as nucleotides or amino acids. A chemotherapeutic agent therefore has cytotoxic or cytostatic effects on both cancer cells and other cells that are engaged in cell division.
  • Chemotherapeutic agents are well-known in the art and include, for example: alkylating agents (e.g. busulfan, temozolomide, cyclophosphamide, lomustine (CCNU), methyllomustine, streptozotocin, cis-diamminedi-chloroplatinum, aziridinylbenzo-quinone, and thiotepa); inorganic ions (e.g. cisplatin and carboplatin); nitrogen mustards (e.g. melphalan hydrochloride, ifosfamide, chlorambucil, and mechlorethamine HCl); nitrosoureas (e.g.
  • alkylating agents e.g. busulfan, temozolomide, cyclophosphamide, lomustine (CCNU), methyllomustine, streptozotocin, cis-diamminedi-chloroplatinum, azirid
  • BCNU carmustine
  • anti-neoplastic antibiotics e.g. adriamycin (doxorubicin), daunomycin, mitomycin C, daunorubicin, idarubicin, mithramycin, and bleomycin
  • plant derivatives e.g. vincristine, vinblastine, vinorelbine, paclitaxel, docetaxel, vindesine, VP-16, and VM-26
  • antimetabolites e.g.
  • Alkylating agents and nitrogen mustard act by alkylating DNA, which restricts uncoiling and replication of strands.
  • Methotrexate, cytarabine, 6-mercaptopurine, 5-fluorouracil, and gemcitabine interfere with nucleotide synthesis.
  • Plant derivatives such a paclitaxel and vinblastine are mitotic spindle poisons. The podophyllotoxins inhibit topoisomerases, thus interfering with DNA replication.
  • Other mechanisms of action include carbamoylation of amino acids (lomustine, carmustine), and depletion of asparagine pools (asparaginase).
  • chemotherapeutic agents are those that directly affect the same cellular processes that are directly affected by the chemotherapeutic agents listed above.
  • a drug or treatment is “concurrently” administered with a Bi-Fc if it is administered in the same general time frame as the Bi-Fc, optionally, on an ongoing basis. For example, if a patient is taking Drug A once a week on an ongoing basis and a Bi-Fc once every six months on an ongoing basis, Drug A and the Bi-Fc are concurrently administered, whether or not they are ever administered on the same day. Similarly, if the Bi-Fc is taken once per week on an ongoing basis and Drug A is administered only once or a few times on a daily basis, Drug A and the Bi-Fc are concurrently administered as meant herein. Similarly, if both Drug A and the Bi-Fc are administered for short periods of time either once or multiple times within a one month period, they are administered concurrently as meant herein as long as both drugs are administered within the same month.
  • a “conservative amino acid substitution,” as meant herein, is a substitution of an amino acid with another amino acid with similar properties. Properties considered include chemical properties such as charge and hydrophobicity. Table 1 below lists substitutions for each amino acid that are considered to be conservative substitutions as meant herein.
  • an “Fc region” is a dimer consisting of two polypeptide chains joined by one or more disulfide bonds, each chain comprising part or all of a hinge domain plus a CH2 and a CH3 domain.
  • Each of the polypeptide chains is referred to as an “Fc polypeptide chain.”
  • a chain an “A chain” and the other is referred to as a “B chain.”
  • the Fc regions contemplated for use with the present invention are IgG Fc regions, which can be mammalian, for example human, IgG1, IgG2, IgG3, or IgG4 Fc regions.
  • the amino acid sequences of the two Fc polypeptide chains can vary from those of a mammalian Fc polypeptide by no more than 10 amino acid substitutions, insertions, and/or deletions of a single amino acid per 100 amino acids relative to the sequence of a mammalian Fc polypeptide amino acid sequence.
  • such variations can be “heterodimerizing alterations” that facilitate the formation of heterodimers over homodimers, an Fc alteration that extends half life, an alteration that inhibits Fc gamma receptor (Fc ⁇ R) binding, and/or an alteration that enhances ADCC.
  • Fc alteration that extends half life is an alteration within an Fc polypeptide chain that lengthens the in vivo half life of a protein that contains the altered Fc polypeptide chain as compared to the half life of a similar protein containing the same Fc polypeptide, except that it does not contain the alteration.
  • Such alterations can be included in an Fc polypeptide chain that is part of a Bi-Fc.
  • the alterations M252Y, S254T, and T256E (methionine at position 252 changed to tyrosine; serine at position 254 changed to threonine; and threonine at position 256 changed to glutamic acid; numbering according to EU numbering as shown in Table 2) are Fc alterations that extend half life and can be used together, separately or in any combination. These alterations and a number of others are described in detail in U.S. Pat. No. 7,083,784. The portions of U.S. Pat. No. 7,083,784 that describe such alterations are incorporated herein by reference. Similarly, M428L and N434S are Fc alterations that extend half life and can be used together, separately or in any combination.
  • GGCVFNMFNCGG SEQ ID NO:33
  • GGCHLPFAVCGG SEQ ID NO:34
  • GGCGHEYMWCGG SEQ ID NO:35
  • GGCWPLQDYCGG SEQ ID NO:36
  • GGCMQMNKWCGG SEQ ID NO:37
  • GGCDGRTKYCGG SEQ ID NO:38
  • GGCALYPTNCGG SEQ ID NO:39
  • GGCGKHWHQCGG SEQ ID NO:40
  • GGCHSFKHFCGG SEQ ID NO:41
  • GGCQGMWTWCGG SEQ ID NO:42
  • An “Fc alteration that is unfavorable to homodimer formation,” includes any alteration in an Fc polypeptide chain such that the Fc polypeptide chain has decreased ability to form homodimers compared to a wild type Fc polypeptide chain.
  • Such alterations are described in detail in U.S. Patent Application Publication US2012/0244578. The portions of this publication that described such alteration are incorporated herein by reference.
  • Such alterations can be included in an Fc polypeptide chain that is part of a Bi-Fc, especially in embodiments where the Bi-Fc is a monomer.
  • such alterations occur in the CH3 region of the Fc polypeptide chain and comprise an alteration such that one or more charged amino acids in the wild type amino acid sequence are replaced with amino acids electrostatically unfavorable to CH3 homodimer formation, and/or one or more hydrophobic interface residues are replaced with a small polar amino acid, such as, for example, asparagine, cysteine, glutamine, serine, or threonine. More specifically, for example, a charged amino acid, e.g., lysine at position 392 and/or position 409, can be replaced with a neutral or oppositely charged amino acid, for example aspartate or glutamate. This can also occur at any other charged amino acid within the Fc polypeptide chain.
  • one or more hydrophobic interface residues selected from the group consisting of Y349, L351, L368, V397, L398, F405, and Y407 can be replaced with a small polar amino acid.
  • the Fc polypeptide chain can have one or more mutated cysteine residues to prevent di-sulfide bond formation.
  • Particularly useful cysteine mutations in this regard are those in the hinge region of the Fc polypeptide chain. Such cysteines can be deleted or substituted with other amino acids.
  • Heterodimerizing alterations generally refer to alterations in the A and B chains of an Fc region that facilitate the formation of heterodimeric Fc regions, that is, Fc regions in which the A chain and the B chain of the Fc region do not have identical amino acid sequences. Such alterations can be included in an Fc polypeptide chain that is part of a Bi-Fc. Heterodimerizing alterations can be asymmetric, that is, an A chain having a certain alteration can pair with a B chain having a different alteration. These alterations facilitate heterodimerization and disfavor homodimerization.
  • hetero- or homo-dimers have formed can be assessed by size differences as determined by polyacrylamide gel electrophoresis in some situations or by other appropriate means (such as molecular tags or binding by antibodies that recognize certain portions of the heterodimer) in situations where size is not a distinguishing characteristic.
  • paired heterodimerizing alterations are the so-called “knobs and holes” substitutions. See, e.g., U.S. Pat. No. 7,695,936 and US Patent Application Publication 2003/0078385, the portions of which describe such mutations are incorporated herein by reference.
  • an Fc region that contains one pair of knobs and holes substitutions contains one substitution in the A chain and another in the B chain.
  • knobs and holes substitutions in the A and B chains of an IgG1 Fc region have been found to increase heterodimer formation as compared with that found with unmodified A and B chains: 1) Y407T in one chain and T366Y in the other; 2) Y407A in one chain and T366W in the other; 3) F405A in one chain and T394W in the other; 4) F405W in one chain and T394S in the other; 5) Y407T in one chain and T366Y in the other; 6) T366Y and F405A in one chain and T394W and Y407T in the other; 7) T366W and F405W in one chain and T394S and Y407A in the other; 8) F405W and Y407A in one chain and T366W and T394S in the other; and 9) T366W in one polypeptide of the Fc and T366S, L368A
  • Such alterations in an IgG1 Fc region include, for example, the following substitutions: Y349C in one Fc polypeptide chain and S354C in the other; Y349C in one Fc polypeptide chain and E356C in the other; Y349C in one Fc polypeptide chain and E357C in the other; L351C in one Fc polypeptide chain and S354C in the other; T394C in one Fc polypeptide chain and E397C in the other; or D399C in one Fc polypeptide chain and K392C in the other.
  • substitutions changing the charge of a one or more residue can enhance heterodimer formation as explained in WO 2009/089004, the portions of which describe such substitutions are incorporated herein by reference.
  • Such substitutions are referred to herein as “charge pair substitutions,” and an Fc region containing one pair of charge pair substitutions contains one substitution in the A chain and a different substitution in the B chain.
  • charge pair substitutions include the following: 1) K409D or K409E in one chain plus D399K or D399R in the other; 2) K392D or K392E in one chain plus D399K or D399R in the other; 3) K439D or K439E in one chain plus E356K or E356R in the other; and 4) K370D or K370E in one chain plus E357K or E357R in the other.
  • the substitutions R355D, R355E, K360D, or K360R in both chains can stabilize heterodimers when used with other heterodimerizing alterations. Specific charge pair substitutions can be used either alone or with other charge pair substitutions.
  • single pairs of charge pair substitutions and combinations thereof include the following: 1) K409E in one chain plus D399K in the other; 2) K409E in one chain plus D399R in the other; 3) K409D in one chain plus D399K in the other; 4) K409D in one chain plus D399R in the other; 5) K392E in one chain plus D399R in the other; 6) K392E in one chain plus D399K in the other; 7) K392D in one chain plus D399R in the other; 8) K392D in one chain plus D399K in the other; 9) K409D and K360D in one chain plus D399K and E356K in the other; 10) K409D and K370D in one chain plus D399K and E357K in the other; 11) K409D and K392D in one chain plus D399K, E356K, and E357K in the other; 12) K409D and K370D
  • an “alteration that inhibits Fc ⁇ R binding,” as meant herein, is one or more insertions, deletions, or substitutions within an Fc polypeptide chain that inhibits the binding of Fc ⁇ RIIA, Fc ⁇ RIIB, and/or Fc ⁇ RIIIA as measured, for example, by an ALPHALISA®-based competition binding assay (PerkinElmer, Waltham, Mass.).
  • Such alterations can be included in an Fc polypeptide chain that is part of a Bi-Fc. More specifically, alterations that inhibit Fc gamma receptor (Fc ⁇ R) binding include L234A, L235A, or any alteration that inhibits glycosylation at N297, including any substitution at N297.
  • alterations that inhibit glycosylation at N297 include additional alterations that stabilize a dimeric Fc region by creating additional disulfide bridges.
  • Further examples of alterations that inhibit Fc ⁇ R binding include a D265A alteration in one Fc polypeptide chain and an A327Q alteration in the other Fc polypeptide chain.
  • ADCC antibody dependent cell-mediated cytotoxicity
  • Such alterations can be included in an Fc polypeptide chain that is part of a Bi-Fc. Many such alterations are described in International Patent Application Publication WO 2012/125850. Portions of this application that describe such alterations are incorporated herein by reference. Such alterations can be included in an Fc polypeptide chain that is part of a heterodimeric bispecific antibody as described herein.
  • ADCC assays can be performed as follows. Cell lines that express high and lower amounts of a cancer cell antigen on the cell surface can be used as target cells.
  • target cells can be labeled with carboxyfluorescein succinimidyl ester (CFSE) and then washed once with phosphate buffered saline (PBS) before being deposited into 96-well microtiter plates with V-shaped wells.
  • CFSE carboxyfluorescein succinimidyl ester
  • Purified immune effector cells for example T cells, NK cells, macrophages, neutrophils can be added to each well.
  • a monospecific antibody that binds to the cancer antigen and contains the alteration(s) being tested and an isotype-matched control antibody can be diluted in a 1:3 series and added to the wells. The cells can be incubated at 37° C. with 5% CO 2 for 3.5 hrs.
  • the cells can be spun down and re-suspended in 1 ⁇ FACS buffer (1 ⁇ phosphate buffered saline (PBS) containing 0.5% fetal bovine serum (FBS)) with the dye TO-PRO®-3 iodide (Molecular Probes, Inc. Corporation, Oregon, USA), which stains dead cells, before analysis by fluorescence activated cell sorting (FACS).
  • FACS fluorescence activated cell sorting
  • Total cell lysis is determined by lysing samples containing effector cells and labeled target cells without a bispecific molecule with cold 80% methanol.
  • exemplary alterations that enhance ADCC include the following alterations in the A and B chains of an Fc region: (a) the A chain comprises Q311M and K334V substitutions and the B chain comprises L234Y, E294L, and Y296W substitutions or vice versa; (b) the A chain comprises E233L, Q311M, and K334V substitutions and the B chain comprises L234Y, E294L, and Y296W substitutions or vice versa; (c) the A chain comprises L234I, Q311M, and K334V substitutions and the B chain comprises L234Y, E294L, and Y296W substitutions or vice versa; (d) the A chain comprises S298T and K334V substitutions and the B chain comprises L234Y, K290Y, and Y296W substitutions
  • an “IgG antibody,” as meant herein, is an antibody consisting essentially of two immunoglobulin IgG heavy chains and two immunoglobulin light chains, which can be kappa or lambda light chains. More specifically, the heavy chains contain a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region in that order, while the light chains contain a VL region followed by a CL region. Numerous sequences of such immunoglobulin regions are known in the art. See, e.g., Kabat et al. in S EQUENCES OF I MMUNOLOGICAL I NTEREST , Public Health Service N.I.H., Bethesda, Md., 1991.
  • an “immune effector cell,” as meant herein, is a cell that is involved in the mediation of a cytolytic immune response, including, for example, T cells, NK cells, macrophages, or neutrophils.
  • the heterodimeric bispecific antibodies described herein bind to an antigen that is part of a protein expressed on the surface of an immune effector cell. Such proteins are referred to herein as “effector cell proteins.”
  • immunoglobulin heavy chain consists essentially of a VH region, a CH1 region, a hinge region, a CH2 region, a CH3 region in that order, and, optionally, a region downstream of the CH3 region in some isotypes. Close variants of an immunoglobulin heavy chain containing no more than 10 amino acid substitutions, insertions, and/or deletions of a single amino acid per 100 amino acids relative to a known or naturally occurring immunoglobulin heavy chain amino acid sequence are encompassed within what is meant by an immunoglobulin heavy chain.
  • immunoglobulin light chain consists essentially of a light chain variable region (VL) and a light chain constant domain (CL). Close variants of an immunoglobulin light chain containing no more than 10 amino acid substitutions, insertions, and/or deletions of a single amino acid per 100 amino acids relative to a known or naturally occurring immunoglobulin light chain amino acid sequence are encompassed within what is meant by an immunoglobulin light chain.
  • an “immunoglobulin variable region,” as meant herein, is a VH region, a VL region, or a variant thereof. Close variants of an immunoglobulin variable region containing no more than 10 amino acid substitutions, insertions, and/or deletions of a single amino acid per 100 amino acids relative to a known or naturally occurring immunoglobulin variable region amino acid sequence are encompassed within what is meant by an immunoglobulin variable region.
  • Many examples of VH and VL regions are known in the art, such as, for example, those disclosed by Kabat et al in S EQUENCES OF I MMUNOLOGICAL I NTEREST , Public Health Service N.I.H., Bethesda, Md., 1991.
  • An immunoglobulin variable region contains three hypervariable regions, known as complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2), and complementarity determining region 3 (CDR3). These regions form the antigen binding site of an antibody.
  • the CDR5 are embedded within the less variable framework regions (FR1-FR4).
  • the order of these subregions within an immunoglobulin variable region is as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • Numerous sequences of immunoglobulin variable regions are known in the art. See, e.g., Kabat et al, S EQUENCES OF P ROTEINS OF I MMUNOLOGICAL I NTEREST , Public Health Service N.I.H., Bethesda, Md., 1991.
  • CDR5 can be located in a VH region sequence in the following way.
  • CDR1 starts at approximately residue 31 of the mature VH region and is usually about 5-7 amino acids long, and it is almost always preceded by a Cys-Xxx-Xxx-Xxx-Xxx-Xxx-Xxx-Xxx (SEQ ID NO:1) (where “Xxx” is any amino acid).
  • the residue following the heavy chain CDR1 is almost always a tryptophan, often a Trp-Val, a Trp-Ile, or a Trp-Ala.
  • Fourteen amino acids are almost always between the last residue in CDR1 and the first in CDR2, and CDR2 typically contains 16 to 19 amino acids.
  • CDR2 may be immediately preceded by Leu-Glu-Trp-11e-Gly (SEQ ID NO:2) and may be immediately followed by Lys/Arg-Leu/Ile/Val/Phe/Thr/Ala-Thr/Ser/Ile/Ala. Other amino acids may precede or follow CDR2. Thirty two amino acids are almost always between the last residue in CDR2 and the first in CDR3, and CDR3 can be from about 3 to 25 residues long. A Cys-Xxx-Xxx almost always immediately precedes CDR3, and a Trp-Gly-Xxx-Gly (SEQ ID NO:3) almost always follows CDR3.
  • Light chain CDR5 can be located in a VL region in the following way.
  • CDR1 starts at approximately residue 24 of the mature antibody and is usually about 10 to 17 residues long. It is almost always preceded by a Cys. There are almost always 15 amino acids between the last residue of CDR1 and the first residue of CDR2, and CDR2 is almost always 7 residues long.
  • CDR2 is typically preceded by Ile-Tyr, Val-Tyr, Ile-Lys, or Ile-Phe. There are almost always 32 residues between CDR2 and CDR3, and CDR3 is usually about 7 to 10 amino acids long.
  • CDR3 is almost always preceded by Cys and usually followed by Phe-Gly-Xxx-Gly (SEQ ID NO:4).
  • a “linker,” as meant herein, is a peptide that links two polypeptides, which can be two immunoglobulin variable regions in the context of a heterodimeric bispecific antibody.
  • a linker can be from 2-30 amino acids in length. In some embodiments, a linker can be 2-25, 2-20, or 3-18 amino acids long. In some embodiments, a linker can be a peptide no more than 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 amino acids long. In other embodiments, a linker can be 5-25, 5-15, 4-11, 10-20, or 20-30 amino acids long.
  • a linker can be about, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids long.
  • exemplary linkers include, for example, the amino acid sequences TVAAP (SEQ ID NO:17), ASTKGP (SEQ ID NO:18), GGGGSGGGGS (SEQ ID NO:19), GGGGSAAA (SEQ ID NO:20), GGGGSGGGGSGGGGS (SEQ ID NO:21), and AAA, among many others.
  • Non-chemotherapeutic anti-neoplastic agents are chemical agents, compounds, or molecules having cytotoxic or cytostatic effects on cancer cells other than chemotherapeutic agents.
  • Non-chemotherapeutic antineoplastic agents may, however, be targeted to interact directly with molecules that indirectly affect cell division such as cell surface receptors, including receptors for hormones or growth factors.
  • non-chemotherapeutic antineoplastic agents do not interfere directly with processes that are intimately linked to cell division such as, for example, DNA replication, RNA synthesis, protein synthesis, or mitotic spindle function, assembly, or disassembly.
  • non-chemotherapeutic anti-neoplastic agents include inhibitors of Bcl2, inhibitors of farnesyltransferase, anti-estrogenic agents such as tamoxifen, anti-androgenic compounds, interferon, arsenic, retinoic acid, retinoic acid derivatives, antibodies targeted to tumor-specific antigens, and inhibitors of the Bcr-Abl tyrosine kinase (e.g., the small molecule STI-571 marketed under the trade name GLEEVECTM by Novartis, New York and New Jersey, USA and Basel, Switzerland), among many possible non-chemotherapeutic anti-neoplastic agents.
  • Bcr-Abl tyrosine kinase e.g., the small molecule STI-571 marketed under the trade name GLEEVECTM by Novartis, New York and New Jersey, USA and Basel, Switzerland
  • a “target cell” is a cell that a Bi-Fc binds to and that is involved in mediating a disease.
  • a target cell can be a cell that is ordinarily involved in mediating an immune response, but is also involved in the mediation of a disease.
  • a B cell which is ordinarily involved in mediating immune response, can be a target cell.
  • a target cell is a cancer cell, a cell infected with a pathogen, or a cell involved in mediating an autoimmune or inflammatory disease, for example a fibrotic disease.
  • the Bi-Fc can bind to the target cell via binding to an antigen on a “target cell protein,” which is a protein that is displayed on the surface of the target cell, possibly a highly expressed protein.
  • Tumor burden refers to the number of viable cancer cells, the number of tumor sites, and/or the size of the tumor(s) in a patient suffering from a cancer.
  • a reduction in tumor burden can be observed, for example, as a reduction in the amount of a tumor-associated antigen or protein in a patient's blood or urine, a reduction in the number of tumor cells or tumor sites, and/or a reduction in the size of one or more tumors.
  • a “therapeutically effective amount” of a Bi-Fc or any other drug is an amount that has the effect of, for example, reducing or eliminating the tumor burden of a cancer patient or reducing or eliminating the symptoms of any disease condition that the protein is used to treat.
  • a therapeutically effective amount need not completely eliminate all symptoms of the condition, but may reduce severity of one or more symptoms or delay the onset of more serious symptoms or a more serious disease that can occur with some frequency following the treated condition.
  • Treatment of any disease mentioned herein encompasses an alleviation of at least one symptom of the disease, a reduction in the severity of the disease, or the delay or prevention of disease progression to more serious symptoms that may, in some cases, accompany the disease or lead to at least one other disease. Treatment need not mean that the disease is totally cured. A useful therapeutic agent needs only to reduce the severity of a disease, reduce the severity of one or more symptoms associated with the disease or its treatment, or delay the onset of more serious symptoms or a more serious disease that can occur with some frequency following the treated condition.
  • a named VH/VL pair of immunoglobulin variable regions can bind to a target cell or and/or an immune effector cell “when they are part of an IgG and/or scFv antibody,” it is meant that an IgG antibody that contains the named VH region in both heavy chains and the named VL region in both light chains and/or an scFv antibody containing these VH and VL regions can bind to the target cell and/or the immune effector cell.
  • the binding assay described in Example 2 can be used to assess binding.
  • a Bi-Fc can bind monovalently to two different antigens and comprises one polypeptide chain or two different polypeptide chains having different amino acid sequences.
  • it can bind to the neonatal Fc receptor (FcRn) at slightly acidic pH (about pH 5.5-6.0) via its Fc region. This interaction with FcRn can lengthen the half life of a molecule in vivo.
  • the first polypeptide chain (which, in some cases, is the only polypeptide chain) comprises an Fc polypeptide chain and two VH regions plus two VL regions separated by linkers.
  • the Fc polypeptide chain can be N-terminal or C-terminal relative to the four immunoglobulin variable regions, and it can be joined to the variable regions via a linker.
  • the second polypeptide chain when present, comprises an Fc polypeptide chain.
  • a Bi-Fc can bind to an immune effector cell and a target cell and/or can mediate cytolysis of a target cell by an immune effector cell.
  • FIG. 1 shows an embodiment where the Fc polypeptide chain is C-terminal (at left) and an embodiment where the Fc polypeptide chain in N-terminal (at right).
  • More particular embodiments specify the order of immunoglobulin variable regions and the length of the linkers and specify which immunoglobulin variable regions can associate to form a binding site for an effector cell protein or a target cell protein.
  • the antigen-binding portion of an antibody includes both a VH and a VL region, referred to herein as a “VH/VL pair,” although in some cases a VH or a VL region can bind to an antigen without a partner. See, e.g., US Application Publication 2003/0114659.
  • variable regions can be arranged in the following order: VH1-linker1-VL1-linker2-VH2-linker3-VL2, where VH1/VL1 is an antigen-binding pair and VH2/VL2 is another antigen-binding pair.
  • linker1 and linker3 can be at least 15 amino acids long, and linker2 can be less than 12 amino acids long.
  • the VH1/VL1 pair can bind to a target cell protein, and the VH2/VL2 pair can bind to an effector cell protein.
  • the VH1/VL1 pair can bind to an effector cell protein, and the VH2/VL2 pair can bind to a target cell protein.
  • variable regions can be arranged in the following order: VL1-linker1-VH1-linker2-VL2-linker3-VH2, where VH1/VL1 is an antigen-binding pair and VH2/VL2 is an antigen-binding pair.
  • linker2 can be less than 12 amino acids long, and linker1 and linker3 can be at least 15 amino acids long.
  • the VH1/VL1 pair can bind to a target cell protein, and the VH2/VL2 pair can bind to an effector cell protein.
  • the VH1/VL1 pair can bind to an effector cell protein, and the VH2/VL2 pair can bind to a target cell protein.
  • variable regions can be arranged in the following order: VH1-linker1-VL1-linker2-VL2-linker3-VH2, where VH1/VL1 is an antigen-binding pair and VH2/VL2 is an antigen-binding pair.
  • linker2 can be less than 12 amino acids long, and linker1 and linker3 can be at least 15 amino acids long.
  • the VH1/VL1 pair can bind to a target cell protein, and the VH2/VL2 pair can bind to an effector cell protein.
  • the VH1/VL1 pair can bind to an effector cell protein, and the VH2/VL2 pair can bind to a target cell protein.
  • variable regions can be arranged in the following order: VL1-linker1-VH1-linker2-VH2-linker3-VL2, where VH1/VL1 is an antigen-binding pair and VH2/VL2 is an antigen-binding pair.
  • linker2 can be less than 12 amino acids long, and linker1 and linker3 can be at least 15 amino acids long.
  • the VH1/VL1 pair can bind to a target cell protein, and the VH2/VL2 pair can bind to an effector cell protein.
  • the VH1/VL1 pair can bind to an effector cell protein, and the VH2/VL2 pair can bind to a target cell protein.
  • a Bi-Fc can comprise an Fc polypeptide chain of an antibody.
  • the Fc polypeptide chain can be of mammalian (for example, human, mouse, rat, rabbit, dromedary, or new or old world monkey), avian, or shark origin.
  • the Fc polypeptide chain can be a human IgG1, IgG2, IgG3, or IgG4 Fc polypeptide chain.
  • an Fc polypeptide chain can comprise a limited number of alterations. More particularly, an Fc polypeptide chain can contain no more than 10 insertions, deletions, and/or substitutions of a single amino acid per 100 amino acids relative to a known or naturally-occurring sequence.
  • the two Fc polypeptide chains of a heterodimeric Bi-Fc contain heterodimerizing alterations, which can be, for example, charge pair substitutions.
  • the first polypeptide chain of the Bi-Fc can comprise the substitutions K409D or K409E and K392D or K392E and the second polypeptide chain of the Bi-Fc can comprise D399K or D399R and D356K or D356R.
  • the first polypeptide chain of the Bi-Fc can comprise D399K or D399R and D356K or D356R
  • the second polypeptide chain of the Bi-Fc can comprise K409D or K409E and K392D or K392E.
  • An Fc polypeptide chain can also comprise one or more “Fc alterations unfavorable to homodimer formation” and/or one or more “Fc alterations that extend half life,” as meant herein.
  • the Bi-Fc can comprise one or more “Fc alterations that are unfavorable to homodimer formation,” as defined above.
  • an Fc region included in a Bi-Fc can comprise one or more “alterations that inhibit the binding of an Fc gamma receptor (Fc ⁇ R)” to the Fc region as defined above.
  • an Fc region included in a Bi-Fc can comprise one or more “Fc alterations that extends half life,” as defined above.
  • one or more “alterations that enhance ADCC” can be included in an Fc region that is part of a Bi-Fc.
  • the amino acid sequences of the Fc polypeptides can be mammalian, for example a human, amino acid sequences or variants thereof that comprise not more than 10 deletions, insertions, or substitutions of a single amino acid per 100 amino acids of sequence relative to a human amino acid sequence.
  • the isotype of the Fc polypeptide can be IgA, IgD, IgE, IgM, or IgG, such as IgG1, IgG2, IgG3, or IgG4.
  • Table 2 below shows an alignment of the amino acid sequences of human IgG1, IgG2, IgG3, and IgG4 Fc polypeptide chain sequences.
  • the hinge regions of the IgG1, IgG2, and IgG4 Fc polypeptides extend from about position 216 to about 230. It is clear from the alignment that the IgG2 and IgG4 hinge regions are each three amino acids shorter than the IgG1 hinge. The IgG3 hinge is much longer, extending for an additional 47 amino acids upstream.
  • the CH2 region extends from about position 231 to 340, and the CH3 region extends from about position 341 to 447.
  • Naturally occurring amino acid sequences of Fc polypeptides can be varied slightly. Such variations can include no more than 10 insertions, deletions, and/or substitutions of one amino acid per 100 amino acids of sequence in a known or naturally-occurring amino acid sequence of an Fc polypeptide. If there are substitutions, they can be conservative amino acid substitutions, as defined above.
  • the Fc polypeptides on the first and second polypeptide chains of a Bi-Fc can differ in amino acid sequence.
  • they can include one or more “heterodimerizing alterations,” “alterations that enhance ADCC,” “alterations that inhibit Fc ⁇ R binding,” “Fc alterations that are unfavorable to homodimer formation,” and/or “Fc alterations that extend half life,” as defined above.
  • a Bi-Fc can bind to an immune effector cell through an antigen that is part of an effector cell protein and can bind to a target cell through an antigen that is part of a target cell protein.
  • a number of possible effector cell proteins are described in detail below. Similarly, a number of possible target cell proteins is also described below.
  • a Bi-Fc can bind to any combination of an effector cell protein and a target cell protein.
  • Exemplary amino acid sequences of Bi-Fc's include the following pairs of amino acid sequences: SEQ ID NOs:10 and 12 and SEQ ID NOs:15 and 12.
  • nucleic acids encoding Bi-Fc's are provided.
  • Numerous nucleic acid sequences encoding immunoglobulin regions including VH, VL, hinge, CH1, CH2, CH3, and CH4 regions are known in the art. See, e.g., Kabat et al. in S EQUENCES OF I MMUNOLOGICAL I NTEREST , Public Health Service N.I.H., Bethesda, Md., 1991.
  • nucleic acid sequences and/or other nucleic acid sequences known in the art to create nucleic acid sequences encoding Bi-Fc's.
  • Exemplary pairs of nucleic acids encoding Bi-Fc's include SEQ ID NOs:11 and 13 and SEQ ID NOs:16 and 13.
  • nucleic acid sequences encoding Bi-Fc's can be determined by one of skill in the art based on the amino acid sequences provided herein and elsewhere and knowledge in the art. Besides more traditional methods of producing cloned DNA segments encoding a particular amino acid sequence, companies such as DNA 2.0 (Menlo Park, Calif., USA) and BlueHeron (Bothell, Wash., USA), among others, now routinely produce chemically synthesized, gene-sized DNAs of any desired sequence to order, thus streamlining the process of producing such DNAs.
  • Bi-Fc's can be made using methods well known in the art.
  • nucleic acids encoding the one or two polypeptide chains of a Bi-Fc can be introduced into a cultured host cell by a variety of known methods, such as, for example, transformation, transfection, electroporation, bombardment with nucleic acid-coated microprojectiles, etc.
  • the nucleic acids encoding a Bi-Fc can be inserted into a vector appropriate for expression in the host cells before being introduced into the host cells.
  • vectors can contain sequence elements enabling expression of the inserted nucleic acids at the RNA and protein levels.
  • Such vectors are well known in the art, and many are commercially available.
  • the host cells containing the nucleic acids can be cultured under conditions so as to enable the cells to express the nucleic acids, and the resulting Bi-Fc's can be collected from the cell mass or the culture medium.
  • a Bi-Fc can be produced in vivo, for example in plant leaves (see, e.g., Scheller et al. (2001), Nature Biotechnol. 19: 573-577 and references cited therein), bird eggs (see, e.g., Zhu et al. (2005), Nature Biotechnol. 23: 1159-1169 and references cited therein), or mammalian milk (see, e.g., Laible et al. (2012), Reprod. Fertil. Dev. 25(1): 315).
  • a variety of cultured host cells can be used including, for example, bacterial cells such as Escherichia coli or Bacillus stearothermophilus , fungal cells such as Saccharomyces cerevisiae or Pichia pastoris , insect cells such as lepidopteran insect cells including Spodoptera frugiperda cells, or mammalian cells such as Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, monkey kidney cells, HeLa cells, human hepatocellular carcinoma cells, or 293 cells, among many others.
  • bacterial cells such as Escherichia coli or Bacillus stearothermophilus
  • fungal cells such as Saccharomyces cerevisiae or Pichia pastoris
  • insect cells such as lepidopteran insect cells including Spodoptera frugiperda cells
  • mammalian cells such as Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, monkey kidney cells, HeLa cells, human
  • a Bi-Fc can bind to a molecule expressed on the surface of an immune effector cell (called “effector cell protein” herein) and to another molecule expressed on the surface of a target cell (called a “target cell protein” herein).
  • the immune effector cell can be a T cell, an NK cell, a macrophage, or a neutrophil.
  • the effector cell protein is a protein included in the T cell receptor (TCR)-CD3 complex.
  • the TCR-CD3 complex is a heteromultimer comprising a heterodimer comprising TCR ⁇ and TCR ⁇ or TCR ⁇ and TCR ⁇ plus various CD3 chains from among the CD3 zeta (CD3 ⁇ ) chain, CD3 epsilon (CD3 ⁇ ) chain, CD3 gamma (CD3 ⁇ ) chain, and CD3 delta (CD3 ⁇ ) chain.
  • the effector cell protein can be the human CD3 epsilon (CD3 ⁇ ) chain (the mature amino acid sequence of which is disclosed in SEQ ID NO:22), which can be part of a multimeric protein.
  • the effector cell protein can be human and/or cynomolgus monkey TCR ⁇ , TCR ⁇ , TCR ⁇ , TCR ⁇ , CD3 beta (CD3 ⁇ ) chain, CD3 gamma (CD3 ⁇ ) chain, CD3 delta (CD3 ⁇ ) chain, or CD3 zeta (CD3 ⁇ ) chain.
  • a Bi-Fc can also bind to a CD3 ⁇ chain from a non-human species, such as mouse, rat, rabbit, new world monkey, and/or old world monkey species.
  • a non-human species such as mouse, rat, rabbit, new world monkey, and/or old world monkey species.
  • species include, without limitation, the following mammalian species: Mus musculus, Rattus rattus, Rattus norvegicus , the cynomolgus monkey, Macaca fascicularis , the hamadryas baboon, Papio hamadryas , the Guinea baboon, Papio papio , the olive baboon, Papio anubis , the yellow baboon, Papio cynocephalus , the Chacma baboon, Papio ursinus, Callithrix jacchus, Saguinus Oedipus ; and Saimiri sci
  • the mature amino acid sequence of the CD3 ⁇ chain of cynomolgus monkey is provided in SEQ ID NO:23.
  • SEQ ID NO:23 The mature amino acid sequence of the CD3 ⁇ chain of cynomolgus monkey.
  • the heterodimeric bispecific antibody can bind to an epitope within the first 27 amino acids of the CD3 ⁇ chain, which may be a human CD3 ⁇ chain or a CD3 ⁇ chain from different species, particularly one of the mammalian species listed above.
  • the epitope can contain the amino acid sequence Gln-Asp-Gly-Asn-Glu (SEQ ID NO:24).
  • the advantages of an antibody that binds such an epitope are explained in detail in U.S. Patent Application Publication 2010/183615, the relevant portions of which are incorporated herein by reference.
  • the epitope to which an antibody binds can be determined by alanine scanning, which is described in, e.g., U.S. Patent Application Publication 2010/183615, the relevant portions of which are incorporated herein by reference.
  • effector cell proteins to which a Bi-Fc can bind include, without limitation, the CD3 ⁇ chain, the CD3 ⁇ , the CD3 ⁇ chain, the CD3 ⁇ chain, TCR ⁇ , TCR ⁇ , TCR ⁇ , and TCR ⁇ .
  • an NK cell or a cytotoxic T cell is an immune effector cell
  • NKG2D, CD352, NKp46, or CD16a can, for example, be an effector cell protein.
  • a CD8 + T cell is an immune effector cell
  • 4-1BB or NKG2D for example, can be an effector cell protein.
  • a Bi-Fc could bind to other effector cell proteins expressed on T cells, NK cells, macrophages, or neutrophils.
  • Target Cells and Target Cell Proteins Expressed on Target Cells
  • a Bi-Fc can bind to an effector cell protein and a target cell protein.
  • the target cell protein can, for example, be expressed on the surface of a cancer cell, a cell infected with a pathogen, or a cell that mediates a disease, for example an inflammatory, autoimmune, and/or fibrotic condition.
  • the target cell protein can be highly expressed on the target cell, although high levels of expression are not necessarily required.
  • a heterodimeric bispecific antibody as described herein can bind to a cancer cell antigen as described above.
  • a cancer cell antigen can be a human protein or a protein from another species.
  • a heterodimeric bispecific antibody may bind to a target cell protein from a mouse, rat, rabbit, new world monkey, and/or old world monkey species, among many others.
  • Such species include, without limitation, the following species: Mus musculus, Rattus rattus, Rattus norvegicus , cynomolgus monkey, Macaca fascicularis , the hamadryas baboon, Papio hamadryas , the Guinea baboon, Papio papio , the olive baboon, Papio anubis , the yellow baboon, Papio cynocephalus , the Chacma baboon, Papio ursinus, Callithrix jacchus, Saguinus oedipus , and Saimiri sciureus.
  • the target cell protein can be a protein selectively expressed on an infected cell.
  • the target cell protein in the case of an HBV or HCV infection, can be an envelope protein of HBV or HCV that is expressed on the surface of an infected cell.
  • the target cell protein can be gp120 encoded by human immunodeficiency virus (HIV) on HIV-infected cells.
  • HIV human immunodeficiency virus
  • a target cell can be a cell that mediates an autoimmune or inflammatory disease.
  • human eosinophils in asthma can be target cells, in which case, EGF-like module containing mucin-like hormone receptor (EMR1), for example, can be a target cell protein.
  • EGF-like module containing mucin-like hormone receptor (EMR1) for example, can be a target cell protein.
  • excess human B cells in a systemic lupus erythematosus patient can be target cells, in which case CD19 or CD20, for example, can be a target cell protein.
  • excess human Th2 T cells can be target cells, in which case CCR4 can, for example, be a target cell protein.
  • a target cell can be a fibrotic cell that mediates a disease such as atherosclerosis, chronic obstructive pulmonary disease (COPD), cirrhosis, scleroderma, kidney transplant fibrosis, kidney allograft nephropathy, or a pulmonary fibrosis, including idiopathic pulmonary fibrosis and/or idiotypic pulmonary hypertension.
  • COPD chronic obstructive pulmonary disease
  • FAP alpha fibroblast activation protein alpha
  • FAP alpha can, for example, be a target cell protein.
  • the immune effector cell is a T cell.
  • the following very similar assay can be used where the immune effector cells are NK cells.
  • a target cell line expressing the target cell protein of interest can be labeled with 2 ⁇ M carboxyfluorescein succinimidyl ester (CFSE) for 15 minutes at 37° C. and then washed.
  • An appropriate number of labeled target cells can then be incubated in one or more 96 well flat bottom culture plates for 40 minutes at 4° C., with or without a bispecific protein, a control protein, or no added protein at varying concentrations.
  • NK cells isolated from healthy human donors can be isolated using the Miltenyi NK Cell Isolation Kit II (Miltenyi Biotec, Auburn, Calif.) and then added to the target cells at an Effector:Target ratio of 10:1.
  • the NK cells which are the immune effector cells in this assay, can be used immediately post-isolation or after overnight culture at 37° C. Plates containing tumor target cells, bispecific proteins, and immune effector cells can be cultured for 18-24 hours at 37° C. with 5% CO2. Appropriate control wells can also be set up. After the 18-24 hour assay period, all cells can be removed from the wells. A volume of a 7-AAD solution equal to the volume of the content of the wells can be added to each sample. Samples can then assayed to determine the percentage of live versus dead target cells via flow cytometry as described in the Examples below.
  • Bi-Fc's can be used to treat a wide variety of conditions including, for example, various forms of cancer, infections, autoimmune or inflammatory conditions, and/or fibrotic conditions.
  • compositions comprising Bi-Fc's.
  • Such pharmaceutical compositions comprise a therapeutically effective amount of a Bi-Fc plus one or more additional components such as a physiologically acceptable carrier, excipient, or diluent.
  • additional components can include buffers, carbohydrates, polyols, amino acids, chelating agents, stabilizers, and/or preservatives, among many possibilities.
  • a Bi-Fc can be used to treat cell proliferative diseases, including cancer, which involve the unregulated and/or inappropriate proliferation of cells, sometimes accompanied by destruction of adjacent tissue and growth of new blood vessels, which can allow invasion of cancer cells into new areas, i.e. metastasis.
  • cell proliferative diseases including cancer
  • non-malignant conditions that involve inappropriate cell growth, including colorectal polyps, cerebral ischemia, gross cystic disease, polycystic kidney disease, benign prostatic hyperplasia, and endometriosis.
  • a Bi-Fc can be used to treat a hematologic or solid tumor malignancy.
  • cell proliferative diseases that can be treated using a Bi-Fc are, for example, cancers including mesotheliomas, squamous cell carcinomas, myelomas, osteosarcomas, glioblastomas, gliomas, carcinomas, adenocarcinomas, melanomas, sarcomas, acute and chronic leukemias, lymphomas, and meningiomas, Hodgkin's disease, Sézary syndrome, multiple myeloma, and lung, non-small cell lung, small cell lung, laryngeal, breast, head and neck, bladder, ovarian, skin, prostate, cervical, vaginal, gastric, renal cell, kidney, pancreatic, colorectal, endometrial, and esophageal, hepatobiliary, bone, skin, and hematologic cancers, as well as cancers of the nasal cavity and paranasal sinuses, the nasopharynx, the
  • a Bi-Fc can be added to a therapy regimen using other anti-neoplastic agents in treating a cancer patient.
  • a Bi-Fc can be administered concurrently with, before, or after a variety of drugs and treatments widely employed in cancer treatment such as, for example, chemotherapeutic agents, non-chemotherapeutic, anti-neoplastic agents, and/or radiation.
  • drugs and treatments widely employed in cancer treatment such as, for example, chemotherapeutic agents, non-chemotherapeutic, anti-neoplastic agents, and/or radiation.
  • chemotherapy and/or radiation can occur before, during, and/or after any of the treatments described herein.
  • chemotherapeutic agents include, but are not limited to, cisplatin, taxol, etoposide, mitoxantrone (Novantrone®), actinomycin D, cycloheximide, camptothecin (or water soluble derivatives thereof), methotrexate, mitomycin (e.g., mitomycin C), dacarbazine (DTIC), anti-neoplastic antibiotics such as adriamycin (doxorubicin) and daunomycin, and all the chemotherapeutic agents mentioned above.
  • mitomycin e.g., mitomycin C
  • DTIC dacarbazine
  • anti-neoplastic antibiotics such as adriamycin (doxorubicin) and daunomycin
  • a Bi-Fc can also be used to treat infectious disease, for example a chronic hepatitis B virus (HBV) infection, a hepatitis C virus (HCV) infection, a human immunodeficiency virus (HIV) infection, an Epstein-Barr virus (EBV) infection, or a cytomegalovirus (CMV) infection, among many others.
  • infectious disease for example a chronic hepatitis B virus (HBV) infection, a hepatitis C virus (HCV) infection, a human immunodeficiency virus (HIV) infection, an Epstein-Barr virus (EBV) infection, or a cytomegalovirus (CMV) infection, among many others.
  • HBV chronic hepatitis B virus
  • HCV hepatitis C virus
  • HCV human immunodeficiency virus
  • EBV Epstein-Barr virus
  • CMV cytomegalovirus
  • a Bi-Fc can find further use in other kinds of conditions where it is beneficial to deplete certain cell types. For example, depletion of human eosinophils in asthma, excess human B cells in systemic lupus erythematosus, excess human Th2 T cells in autoimmune conditions, or pathogen-infected cells in infectious diseases can be beneficial. In a fibrotic condition, it can be useful to deplete cells forming fibrotic tissue.
  • Bi-Fc Therapeutically effective doses of a Bi-Fc can be administered.
  • the amount of Bi-Fc that constitutes a therapeutically dose may vary with the indication treated, the weight of the patient, the calculated skin surface area of the patient. Dosing of a Bi-Fc can be adjusted to achieve the desired effects. In many cases, repeated dosing may be required. For example, a Bi-Fc can be dosed twice per week, once per week, once every two, three, four, five, six, seven, eight, nine, or ten weeks, or once every two, three, four, five, or six months.
  • the amount of a Bi-Fc administered on each day can be from about 0.0036 mg to about 450 mg.
  • the dose can calibrated according to the estimated skin surface of a patient, and each dose can be from about 0.002 mg/m 2 to about 250 mg/m 2 .
  • the dose can be calibrated according to a patient's weight, and each dose can be from about 0.000051 mg/kg to about 6.4 mg/kg.
  • a Bi-Fc or a pharmaceutical composition containing such a molecule, can be administered by any feasible method.
  • Protein therapeutics will ordinarily be administered by a parenteral route, for example by injection, since oral administration, in the absence of some special formulation or circumstance, would lead to hydrolysis of the protein in the acid environment of the stomach.
  • Subcutaneous, intramuscular, intravenous, intraarterial, intralesional, or peritoneal bolus injection are possible routes of administration.
  • a Bi-Fc can also be administered via infusion, for example intravenous or subcutaneous infusion. Topical administration is also possible, especially for diseases involving the skin.
  • a Bi-Fc can be administered through contact with a mucus membrane, for example by intra-nasal, sublingual, vaginal, or rectal administration or administration as an inhalant.
  • certain appropriate pharmaceutical compositions comprising a Bi-Fc can be administered orally.
  • Bi-Fc molecules were generated using methods essentially described previously. Löffler et al. (2000), Blood 95(6): 2098-2103.
  • a construct encoding an anti-HER2/CD3 Bi-Fc was made as follows. DNA fragments encoding the VH region (SEQ ID NO:5) and the VL region (SEQ ID NO:6) of an anti-HER2 IgG antibody and the VH region (SEQ ID NO:7) and VL region (SEQ ID NO:8) of anti-human CD3 IgG antibody were amplified by PCR using forward and reverse primers and spliced together with flexible linkers.
  • the resulting DNA fragment which encodes a linear fusion DNA encoding two scFv's joined by a linker is referred to herein as the single chain anti-HER2/CD3 (SEQ ID NO:9).
  • This construct was subcloned into a mammalian expression vector for antibody production.
  • An anti-HER2/CD3 Bi-Fc (SEQ ID NO:10) was constructed by fusing DNA encoding the single chain anti-HER2/CD3 to DNA encoding one of the two chains of an engineered human IgG1 Fc region. Specifically, DNA encoding an Fc polypeptide chain containing two positively charged mutations (D356K/D399K, EU numbering) plus alterations that inhibit Fc ⁇ R binding (L234A and L235A) was fused to the DNA encoding the single chain anti-HER2/CD3 at the 3′ end. The amino acid sequence of this anti-HER/CD3 Bi-Fc and the nucleic acid sequence encoding it are shown in SEQ ID NO:10 and 11, respectively.
  • the second polypeptide chain that was part of the anti-HER2/CD3 Bi-Fc was a human IgG1 Fc polypeptide chain containing two negatively charged mutations (K392D/K409D, EU numbering) plus L234A and L235A, as shown in SEQ ID NO:12.
  • DNA encoding this polypeptide (SEQ ID NO:13) was amplified and inserted into an appropriate vector for expression.
  • a single chain anti-FOLR1/CD3 (SEQ ID NO: 14) and an anti-FOLR1/CD3 Bi-Fc (SEQ ID NO:15) were constructed by replacing DNA encoding the anti-HER2 scFv fragment with DNA encoding an scFv fragment derived from an anti-human FOLR1 IgG antibody.
  • All single chain and Bi-Fc molecules described above were produced by transient transfection in human HEK 293-6E cells.
  • the culture media was harvested after 6 days.
  • the single chain anti-HER2/CD3 and anti-FOLR1/CD3 molecules were purified by nickel HISTRAP® (GE Healthcare Bio-Sciences, L.L.C., Uppsala, Sweden) column chromatography and eluted with a 25 to 300 mM imidizole gradient.
  • the elution pools were further purified by size exchange chromatography (SEC) using a preparative SUPERDEX® 200 (GE Healthcare Bio-Sciences, L.L.C., Uppsala, Sweden) column, concentrated to >1 mg/mL, and stored at ⁇ 70° C.
  • Anti-HER2/CD3 Bi-Fc and anti-FOLR1/CD3 Bi-Fc molecules were purified using MABSELECT SURETM (GE Healthcare Bio-Sciences, L.L.C., Uppsala, Sweden) affinity chromatography, eluting with 50 mM citrate, 1M L-Arginine, pH 3.5.
  • the eluate was buffer-exchanged into formulation buffer by a preparative SEC with 10 mM potassium phosphate, 161 mM L-Arginine, pH 7.6 or with a solution containing acetate and sucrose with 150 mM NaCl, 161 mM L-Arginine, pH 5.2
  • Binding of the anti-HER2/CD3 Bi-Fc and single chain anti-HER2/CD3 to T cells expressing CD3 and JIMT-1 cells expressing HER2 was assessed as follows. Human pan-T cells (purified using Pan T Cell Isolation Kit II, human, Miltenyi Biotec, Auburn, Calif.) or purified JIMT-1 cells were incubated for 16 hrs at 4° C. in the absence or presence of 10 ⁇ g/mL of the anti-HER2/CD3 Bi-Fc or the single chain anti-HER2/CD3. Cell binding of the anti-HER2/CD3 Bi-Fc was detected using an allophycocyanin (APC)-labeled anti-human Fc secondary antibody.
  • APC allophycocyanin
  • the single chain anti-HER2/CD3, which includes a FLAG tag was detected using a mouse anti-FLAG® antibody followed by an APC-labeled mouse Ig-specific antibody.
  • the unfilled profiles represent data from cells in the absence of one of the bispecific molecules
  • the solidly filled profiles represent data from cells in the presence of one of the bispecific molecules, as indicated in the description of FIG. 2 .
  • the anti-HER2/CD3 ⁇ and anti-FOLR1/CD3 ⁇ Bi-Fc's and single chain anti-HER2/CD3 ⁇ and anti-FOLR1/CD3 ⁇ molecules described above were assayed to determine their activity in a T cell-dependent cell cytolysis (TDCC) assay using tumor cells expressing HER2 or FOLR1 as target cells.
  • TDCC T cell-dependent cell cytolysis
  • pan T cells were isolated from healthy human donors using the Pan T Cell Isolation Kit II, human (Miltenyi Biotec, Auburn, Calif.).
  • T cells were incubated with CFSE-labeled tumor target cells at a ratio of 10:1 in the presence or absence of the anti-HER2/CD3 ⁇ or anti-FOLR1/CD3 ⁇ Bi-Fc's or the single chain anti-HER2/CD3 ⁇ or anti-FOR1/CD3 described in Example 1 at the varying concentrations as indicated in FIGS. 2 and 3 .
  • some samples contained T cells and tumor target cells, but no Bi-Fc or single chain molecule.
  • the target cells for the anti-HER2/CD3 ⁇ Bi-Fc and single chain molecule were either Cal-51 cells (expressing about 148,000 molecules of FOLR1 per cell), T47D cells (expressing about 101,000 molecules of FOLR1 per cell), or the control cell line BT474 (which did not express detectable levels of FOLR1).
  • the target cells for the anti-CD3 ⁇ /HER2 Bi-Fc and single chain molecules were JIMT-1 cells (expressing about 181,000 molecules of HER2 per cell), T47D cells (expressing about 61,000 molecules of HER2 per cell), or the control cell line SHP77 (which did not express detectable amounts of HER2).
  • % specific lysis [% tumor lysis with Bi-Fc ⁇ % tumor cell lysis without bispecific/% of total cell lysis ⁇ % tumor cell lysis without bispecific] ⁇ 100.
  • samples containing immune effector and labeled target cells without a Bi-Fc or single chain molecule were lysed with cold 80% methanol.
  • Results for the anti-FOLR1/CD3 ⁇ Bi-Fc and single chain molecule are shown in FIG. 3 . Both the anti-FOLR1/CD3 ⁇ Bi-Fc and single chain molecule exhibited dose dependent lysis of both the Cal-51 and the T47D target cells. The EC 50 for each of these molecules in each of these cell lines is shown in Table 3 below.
  • results for the anti-HER2/CD3 ⁇ Bi-Fc and single chain molecule are shown in FIG. 4 .
  • Both the anti-HER2/CD3 ⁇ Bi-Fc and single chain molecule exhibited dose dependent lysis of both the JIMT-1 and the T47D target cells, but no lysis of the control SHP77 cell line (which does not express HER2).
  • the EC 50 for each of these molecules in each of these cell lines is shown in Table 4 below.
  • the anti-HER2/CD3 ⁇ single chain and Bi-Fc and the anti-FOLR1/CD3 ⁇ single chain and Bi-Fc described above were assayed to determine whether they could stimulate the production of inflammatory cytokines by T cells. Briefly, twenty four hour cell culture supernatants from the TDCC assays like those described in Example 3 were assessed for cytokine concentrations using the Human TH1/TH2 7-Plex and Human Proinflammatory 1 4-Plex ultra Sensitive Kits from Meso Scale Diagnostics, L.L.C. Assays were performed according to the manufacturer's directions.
  • FIGS. 5 and 6 show the T cells secreted cytokines in the presence of the anti-FOLR1/CD3 ⁇ Bi-Fc or single chain in the presence of cells expressing FOLR1 (T47D, graphs on the left), but not in the presence of cells that did not express FOLR1 (BT474, graphs on the right).
  • T47D the anti-FOLR1/CD3 ⁇ Bi-Fc or single chain in the presence of cells expressing FOLR1
  • BT474 graphs on the right
  • IFN- ⁇ interferon gamma
  • TNF- ⁇ tumor necrosis factor alpha
  • IL-10 interleukin-10
  • IL-2 interleukin-2
  • IL-13 interleukin-13
  • the Bi-Fc's had very potent activity in the assay, exhibiting EC 50 's in the pM range as shown in the table below.
  • PBMC peripheral blood mononuclear cells
  • CD25 and CD69 are markers of activation of T cells.
  • the single dose pharmacokinetic profiles of an anti-HER2/CD3 ⁇ Bi-Fc (comprising the amino acid sequences of SEQ ID NOs:10 and 12) and an anti-HER2/CD3 ⁇ single chain (comprising the amino acid sequence of SEQ ID NO:9) was assessed by intravenous and subcutaneous bolus administration in male NOD.SCID mice (Harlan, Livermore, Calif.). These test molecules were injected as a bolus at 1 mg/kg intravenously via the lateral tail vein in some mice or subcutaneously under the skin over the shoulders in others. Serial bleeds of approximately 0.1 mL of whole blood were collected at each time point via retro-orbital sinus puncture.
  • Serum samples were processed to obtain serum ( ⁇ 0.040 mL per sample). Serum samples were analyzed by immunoassay using the technology Gyros AB (Warren, N.J.) to determine the serum concentrations of the anti-HER2/CD3 ⁇ single chain and Bi-Fc. Serum samples were collected at 0, 0.5, 2, 8, 24, 72, 120, 168, 240, 312, 384, and 480 hours. Serum samples were maintained at ⁇ 70° C. ( ⁇ 10° C.) prior to analysis. Pharmacokinetic parameters were estimated from serum concentrations using non-compartmental analysis using Phoenix® 6.3 software (Pharsight, Sunnyvale, Calif.).
  • the single dose pharmacokinetic profiles of the Bi-Fc and the single chain molecule are shown in FIG. 8 .
  • the Bi-Fc showed an extended serum half life (219 hours) compared to the single chain molecule, which was rapidly eliminated and had a half life of only 5 hours. Exposure of the Bi-Fc was characterized by an area under the curve (AUC) of 524 hr* ⁇ g/mL, as compared to 19 hr* ⁇ g/mL for the single chain molecule. The subcutaneous bioavailability of the Bi-Fc was 83%, while that of the single chain molecule was 29%. Thus, the Bi-Fc showed favorable single dose pharmacokinetic properties as compared to the single chain molecule.

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