bispecific protein that can be activated by proteases.

Abstract

To provide protease-activatable bispecific proteins (PABPs), nucleic acids encoding the PABPs, methods of making the PABPs, and methods of using the PABPs.SOLUTION: Described herein are protease-activatable proteins (PABPs), which, when activated, can mediate cytolysis of target cells by effector cells. Also provided are nucleic acids encoding the PABPs and methods of making and using the PABPs. Such PABPs comprise at least a portion that binds to a target cell, a portion that binds to an effector cell, and a protease cleavage site.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] field This invention belongs to the field of protein engineering. [Background technology]

[0002] background Bispecific antibodies show promise as cancer treatments. For example, bispecific antibodies targeting both CD3 and CD19 in the form of Bispecific T cell Engager (BiTE®) have shown remarkable efficacy at low doses. Bargou et al. (2008), Science 321: 974-978 (Non-patent Literature 1). The BiTE® form essentially consists of two scFv linked by a linker, one targeting CD3 and the other targeting a tumor antigen. The resulting antibody has a short in vivo half-life and therefore requires administration by continuous infusion. Bispecific forms with improved pharmacokinetic properties are considered desirable to eliminate the need for continuous infusion. However, since CD3 binding causes T cell activation, forms with longer half-lives can, as can be easily imagined, lead to prolonged T cell activation and insufficient localization, resulting in undesirable side effects. Tsoukas et al. (1985), J. Immunol. 135(3): 1719-1723 (Non-patent Literature 2). Therefore, in this art, there is a need for a bispecific antibody form that has a moderately long half-life but is specifically activated in the disease microenvironment, for example, in the vicinity of tumors. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Bargou et al. (2008), Science 321 : 974-978 [Non-Patent Document 2] Tsoukas et al. (1985), J. Immunol. 135(3): 1719-1723 [Overview of the project]

[0004] overview Generally, bispecific proteins (PABPs) that can be activated by proteases, nucleic acids encoding PABPs, methods for producing PABPs, and methods for using PABPs are described herein. Such PABPs include at least a portion that binds to target cells, a portion that binds to effector cells, and a protease cleavage site.

[0005] More specifically, a protein comprising (a) one or more polypeptide chains that bind to target cells; (b) one or more polypeptide chains that bind to effector cells; (c) a third polypeptide; and (d) a linker containing a protease cleavage site that links the third polypeptide of (c) to the rest of the protein, wherein if the protease cleavage site is essentially completely cleaved, the protein binds to target cells more effectively than the binding observed when the protease cleavage site is not cleaved, and / or the E of the protein in a cell lysis assay when the protease cleavage site is essentially completely cleaved. C 50 is the E of the protein in the same assay when the protease cleavage site is not cleaved. CThe value is less than or equal to 1 / 5 of 50. The polypeptide chain of (a) may include a first pair (VH1 and VL1) of immunoglobulin heavy chain variable regions and light chain variable regions that bind to target cells when they are part of an IgG or scFv antibody, and the polypeptide chain of (b) may include a second pair (VH2 and VL2) of immunoglobulin heavy chain variable regions and light chain variable regions that bind to effector cells when they are part of an IgG or scFv antibody. The effector cells may be T cells or NK cells. VH2 and VL2 may bind to polypeptides that are part of the TCR-CD3 complex, such as human CD3ε, when they are part of an IgG or scFv antibody. VH2 may include heavy chains CDR1, CDR2, and CDR3 containing amino acid sequences of SEQ ID NO: 42, 43, and 44, respectively, and VL2 may include light chains CDR1, CDR2, and CDR3 containing amino acid sequences of SEQ ID NO: 47, 48, and 49, respectively. VH2 and VL2 may contain amino acid sequences of SEQ ID NO: 40 and 45, respectively. In some embodiments, the protease-cleavable site may be cleaved by MMP-2, MMP-9, or MMP-11. In some embodiments, the protease-cleavable site is It may contain an amino acid sequence selected from the group consisting of TIFF0007842528000001.tif17161.

[0006] In one aspect, the protein may include a first polypeptide chain comprising an amino acid sequence having the formula:VH1-L1-VL1-L2-VH2-L3-VL2-X1, where L1, L2, and L3 are linkers, L3 may or may not be present, and X1 is a half-life extension portion, e.g., an Fc polypeptide chain; and a second polypeptide chain comprising an amino acid sequence having the formula:Y-L4-X2, where Y is the polypeptide of (c) above, L4 is a linker comprising the protease cleavage site of (d) above, and X2 is a half-life extension portion, e.g., an Fc polypeptide chain. The first polypeptide chain may include the amino acid sequence of SEQ ID NO:30, and the second polypeptide chain may include the amino acid sequence of SEQ ID NO:36 or SEQ ID NO:38.

[0007] In another aspect, the protein may include a first polypeptide chain comprising an amino acid sequence having the formula VH1-L4-VL2-L5-CL-X1, where L4 and L5 are linkers, which may or may not be present, CL is a light chain constant region, and X1 is a half-life extension region, which may or may not be present; and a second polypeptide chain having the formula Y-L1-VH2-L2-VL1-L3-CH1-X2, where Y is the polypeptide of (c) above, L1 is a linker comprising the protease cleavage site of (d) above, L2 and L3 are linkers, which may or may not be present, CH1 is a first heavy chain constant region, and X2 is a half-life extension region, which may or may not be present. X1 and X2 may be Fc polypeptide chains, and both may or may not be present. The first polypeptide chain may contain the amino acid sequence of SEQ ID NO: 6, and the second polypeptide chain may contain the amino acid sequence of SEQ ID NO: 10, 12, 14, 16, or 18.

[0008] In a further context, the protein may include a first polypeptide chain comprising an amino acid sequence having the formula VH1-L4-VL1-L5-X1 or VL1-L4-VH1-L5-X1, where L4 and L5 are linkers, which may or may not be present, and X1 is an Fc polypeptide chain; and a second polypeptide comprising amino acids having the formula Y-L1-VH2-L2-VL2-L3-X2 or Y-L1-VL2-L2-VH2-L3-X2, where Y is the polypeptide of (c) above, L1 is a linker comprising the protease cleavage site of (d) above, L2 and L3 are linkers, which may or may not be present, and X2 is an Fc polypeptide chain. The first polypeptide chain may contain the amino acid sequence of SEQ ID NO: 20, and the second polypeptide chain may contain the amino acid sequence of SEQ ID NO: 24, 26, or 28.

[0009] Any of the target cells of the PABP described herein may be cancer cells. In this case, VH1 and VL1, when part of an scFv or IgG antibody, may bind to proteins selected from the group consisting of epidermal growth factor receptor (EGFR), EGFRvIII, melanoma-conjugated chondroitin sulfate proteoglycan (MCSP), mesothelin (MSLN), folate receptor 1 (FOLR1), CD33, CDH19, and epidermal growth factor 2 (HER2).

[0010] In some embodiments, the proteins described herein are pairs of polypeptide chains: (a) a first polypeptide chain comprising an amino acid sequence having the formula: VH1-CH1-L1-VH2-CH1, where VH1 and VH2 are immunoglobulin heavy chain variable regions, CH1 is the first heavy chain constant region, and L1 is a linker containing a protease cleavage site; and a second polypeptide chain comprising an amino acid sequence having the formula: VL1-CL-L2-VL2-CL, where VL1 and VL2 are immunoglobulin light chain variable regions, CL is the light chain constant region, and L2 is a linker not containing a protease cleavage site; (b) A first polypeptide chain comprising an amino acid sequence having the following formula: VH1-CH1-L1-VL2-CL, where VH1 is the immunoglobulin heavy chain variable region, VL2 is the immunoglobulin light chain variable region, CH1 is the first heavy chain constant region, CL is the light chain constant region, and L1 is a linker containing a protease cleavage site; and a second polypeptide chain comprising an amino acid sequence having the following formula: VL1-CL-L2-VH2-CH1, where VL1 is the immunoglobulin light chain variable region, VH2 is the immunoglobulin heavy chain variable region, L2 is a linker not containing a protease cleavage site, and CH1 is the first heavy chain constant region; (c) A first polypeptide chain comprising an amino acid sequence having the following formula: VL1-CL-L1-VL2-CL, where VL1 and V2 are immunoglobulin light chain variable regions, CL is a light chain constant region, and L1 is a linker containing a protease cleavage site; and a second polypeptide chain comprising an amino acid sequence having the following formula: VH1-CH1-L2-VH2-CH1, where VH1 and VH2 are heavy chain variable regions, L2 is a linker not containing a protease cleavage site, and CH1 is the first heavy chain constant region;(d) A first polypeptide chain comprising an amino acid sequence having the following formula: VL1-CL-L1-VH2-CH1, wherein VH2 is the immunoglobulin heavy chain variable region, VL1 is the immunoglobulin light chain variable region, CH1 is the first heavy chain constant region, CL is the light chain constant region, and L1 is a protease-cleavable linker; and a second polypeptide chain comprising an amino acid sequence having the following formula: VH1-CH1-L2-VL2-CL, wherein VL VL1 and VH1 may comprise one of the following: 2 is the immunoglobulin light chain variable region, VH1 is the immunoglobulin heavy chain variable region, L2 is a linker that does not contain a protease cleavage site, CH1 is the first heavy chain constant region, and CL is the light chain constant region; where VL1 and VH1 bind to target cells when they are part of an IgG or scFv antibody, and VL2 and VH2 bind to effector cells when they are part of an IgG or scFv antibody. Effector cells may be T cells. VH2 and VL2 may bind to proteins that are part of the TCR-CD3 complex, such as human CD3ε, when they are part of an IgG or scFv antibody. VH2 and VL2 may contain immunoglobulin heavy chains CDR1, CDR2, and CDR3 containing amino acid sequences of SEQ ID NO: 42, 43, and 44, respectively, and immunoglobulin light chains CDR1, CDR2, and CDR3 containing amino acid sequences of SEQ ID NO: 47, 48, and 49, respectively. VH2 and VL2 may contain amino acid sequences of SEQ ID NO: 40 and 45, respectively. The protease cleavage sites are; The amino acid sequence may be selected from the group consisting of TIFF0007842528000002.tif17161. The target cells may be cancer cells. When VH1 and VL1 are part of an IgG or scFv antibody, they may bind to epidermal growth factor receptor (EGFR), EGFRvIII, melanoma-conjugated chondroitin sulfate proteoglycan (MCSP), mesotheline (MSLN), folate receptor 1 (FOLR1), CD33, CDH19, or epidermal growth factor 2 (HER2).

[0011] In another aspect, nucleic acids encoding any of the above or below PABPs are described herein. Vectors and host cells containing such nucleic acids are also provided. Exemplary pairs of nucleic acids encoding PABP include, without limitation, the following sequences: SEQ ID NO:7 and 11; SEQ ID NO:7 and 13; SEQ ID NO:7 and 15; SEQ ID NO:7 and 17; SEQ ID NO:7 and 19; SEQ ID NO:21 and 25; SEQ ID NO:21 and 27; SEQ ID NO:21 and 29; SEQ ID NO:31 and 37; and nucleic acids including SEQ ID NO:31 and 39. Also described herein is a method for making any of the PABPs described herein, including culturing a host cell containing a nucleic acid encoding PABP under conditions such that the PABP is expressed, and recovering the PABP from the culture medium or cell population.

[0012] In a further aspect, a method for treating a cancer patient is described herein, which includes administering a therapeutically effective dose of a PABP described herein. This method includes, in some embodiments, administering radiation, chemotherapeutic agents, and / or non-chemotherapeutic anti-neoplastic agents before, after, and / or simultaneously with the administration of PABP. The cancer cells of the patient may express a protease that can cleave a protease cleavage site that is part of the PABP.

[0013] In another aspect, a method for treating a patient suffering from an infectious disease, a fibrotic disease, a neurodegenerative disease, or an autoimmune or inflammatory disease is described herein, which includes administering a therapeutically effective dose of a PABP described herein. The basic features and various embodiments of the present invention are listed below. [1] (a) One or more polypeptide chains comprising a first pair (VH1 and VL1) of immunoglobulin heavy chain variable regions and light chain variable regions that bind to target cells and, if they are part of an IgG or scFv antibody, bind to target cells, (b) One or more polypeptide chains comprising a second pair (VH2 and VL2) of immunoglobulin heavy chain variable regions and light chain variable regions that bind to effector cells and, if they are part of an IgG or scFv antibody, (c) Third polypeptide; and (d) A linker containing a protease cleavage site that connects the third polypeptide of (c) to the rest of the protein. A protein containing, The first polypeptide chain of the protein comprises an amino acid sequence having the formula: VH1-L1-VL1-L2-VH2-L3-VL2-X1, where L1, L2, and L3 are linkers, L3 may or may not be present, and X1 is a half-life extension portion. The second polypeptide chain of the protein comprises an amino acid sequence having the formula: Y-L4-X2, where Y is the polypeptide of (c), L4 is a linker containing the protease cleavage site of (d), and X2 is the half-life extension portion, and When the protease cleavage site is essentially completely cleaved, the protein either binds more effectively to target cells or more effectively to effector cells compared to the binding observed when the protease cleavage site is not cleaved. The aforementioned protein. [2] (c) The third polypeptide of the protein of [1] inhibits the binding of the protein to effector cells. [3] (a) One or more polypeptide chains comprising a first pair (VH1 and VL1) of immunoglobulin heavy chain variable regions and light chain variable regions that bind to target cells and, if they are part of an IgG or scFv antibody, bind to target cells, (b) One or more polypeptide chains comprising a second pair (VH2 and VL2) of immunoglobulin heavy chain variable regions and light chain variable regions that bind to effector cells and, if they are part of an IgG or scFv antibody, (c) A third polypeptide that inhibits the cytolytic activity of proteins in cell lysis assays; and (d) A linker containing a protease cleavage site that connects the third polypeptide of (c) to the rest of the protein. A protein containing, The first polypeptide chain of the protein comprises an amino acid sequence having the formula: VH1-L1-VL1-L2-VH2-L3-VL2-X1, where L1, L2, and L3 are linkers, L3 may or may not be present, and X1 is a half-life extension portion. The second polypeptide chain of the protein comprises an amino acid sequence having the formula: Y-L4-X2, where Y is the polypeptide of (c), L4 is a linker containing the protease cleavage site of (d), and X2 is the half-life extension portion, and When the protease cleavage site is essentially completely cleaved, the E of the protein in a cell lysis assay. C 50 is the E of the protein in the same assay when the protease cleavage site is not cleaved. C It is less than 1 / 5 of 50. The aforementioned protein. [4] The effector cell is a T cell, and it is one of the proteins [1] to [3]. [5] The effector cell is an NK cell, and it is one of the proteins [1] to [3]. [6] The proteins of [4] that bind to polypeptides that are part of the TCR-CD3 complex when VH2 and VL2 are part of an IgG or scFv antibody. [7] The protein [6] whose polypeptide is human CD3ε, which is part of the TCR-CD3 complex. [8] The protein [7], wherein VH2 comprises heavy chains CDR1, CDR2, and CDR3 having amino acid sequences of SEQ ID NO: 42, 43, and 44, respectively, and VL2 comprises light chains CDR1, CDR2, and CDR3 having amino acid sequences of SEQ ID NO: 47, 48, and 49, respectively. [9] VH2 and VL2 are proteins containing amino acid sequences SEQ ID NO: 40 and 45, respectively.[8]

[10] A protein whose protease-cleavable site can be cleaved by MMP-2, MMP-9, or MMP-11, one of the [1] to [9] proteins.

[11] The sites where protease can be cleaved are, TIFF0007842528000003.tif16140 A protein

[10] containing an amino acid sequence selected from the group consisting of the following.

[12] A protein from any of the above [1] to

[11] , wherein X1 and X2 are Fc polypeptide chains.

[13] A protein having a first polypeptide chain containing the amino acid sequence of SEQ ID NO:30 and a second polypeptide chain containing the amino acid sequence of SEQ ID NO:36 or SEQ ID NO:38,

[12] .

[14] A protein from any of the above [1] to

[13] whose target cells are cancer cells.

[15] If VH1 and VL1 are part of an IgG or scFv antibody, they bind to one of the following proteins: epidermal growth factor receptor (EGFR), EGFRvIII, melanoma-conjugated chondroitin sulfate proteoglycan (MCSP), mesothelin (MSLN), folate receptor 1 (FOLR1), CD33, CDH19, or epidermal growth factor 2 (HER2)

[14] .

[16] The following polypeptide chain pairs: (a) (i) A first polypeptide chain comprising an amino acid sequence having the following formula: VH1-CH1-L1-VH2-CH1, wherein VH1 and VH2 are immunoglobulin heavy chain variable regions, CH1 is the first heavy chain constant region, and L1 is a linker containing a protease-cleavable site, and (ii) A second polypeptide chain comprising an amino acid sequence having the following formula: VL1-CL-L2-VL2-CL, wherein VL1 and VL2 are immunoglobulin light chain variable regions, CL is a light chain constant region, and L2 is a linker that does not contain a protease cleavage site, or (b) (i) A first polypeptide chain comprising an amino acid sequence having the following formula: VH1-CH1-L1-VL2-CL, wherein VH1 is the immunoglobulin heavy chain variable region, VL2 is the immunoglobulin light chain variable region, CH1 is the first heavy chain constant region, CL is the light chain constant region, and L1 is a linker containing a protease cleavage site, and (ii) A second polypeptide chain comprising an amino acid sequence having the following formula: VL1-CL-L2-VH2-CH1, wherein VL1 is the immunoglobulin light chain variable region, VH2 is the immunoglobulin heavy chain variable region, L2 is a linker that does not contain a protease cleavage site, and CH1 is the first heavy chain constant region, or (c) (i) A first polypeptide chain comprising an amino acid sequence having the following formula: VL1-CL-L1-VL2-CL, wherein VL1 and V2 are immunoglobulin light chain variable regions, CL is a light chain constant region, and L1 is a linker containing a protease cleavage site, and (ii) A second polypeptide chain comprising an amino acid sequence having the following formula: VH1-CH1-L2-VH2-CH1, wherein VH1 and VH2 are heavy chain variable regions, L2 is a linker that does not contain a protease cleavage site, and CH1 is the first heavy chain constant region, or (d) (i) A first polypeptide chain comprising an amino acid sequence having the following formula: VL1-CL-L1-VH2-CH1, wherein VH2 is an immunoglobulin heavy chain variable region, VL1 is an immunoglobulin light chain variable region, CH1 is a first heavy chain constant region, CL is a light chain constant region, and L1 is a protease-cleavable linker, and (ii) A second polypeptide chain comprising an amino acid sequence having the following formula: VH1-CH1-L2-VL2-CL, wherein VL2 is the immunoglobulin light chain variable region, VH1 is the immunoglobulin heavy chain variable region, L2 is a linker that does not contain a protease cleavage site, CH1 is the first heavy chain constant region, and CL is the light chain constant region. A protein containing one of the following: VL1 and VH1 bind to target cells when they are part of an IgG or scFv antibody, and VL2 and VH2 bind to effector cells when they are part of an IgG or scFv antibody. The aforementioned protein.

[17] The protein of the effector cell, which is a T cell,

[16] .

[18] The proteins of

[17] that bind to the proteins that are part of the TCR-CD3 complex when VH2 and VL2 are part of an IgG or scFv antibody.

[19] VH2 and VL2 are proteins of

[17] that bind to human CD3ε.

[20] The protein

[19] comprises immunoglobulin heavy chains CDR1, CDR2, and CDR3 having amino acid sequences of SEQ ID NO: 42, 43, and 44, respectively, and immunoglobulin light chains CDR1, CDR2, and CDR3 having amino acid sequences of SEQ ID NO: 47, 48, and 49, respectively.

[21] VH2 and VL2 are proteins containing amino acid sequences with SEQ ID NO: 40 and 45, respectively.

[20]

[22] The protease cleavage site is TIFF0007842528000004.tif16139 A protein containing an amino acid sequence selected from the group consisting of

[16] to

[21] .

[23] A protein whose target cells are cancer cells, one of the

[16] -

[22] proteins.

[24] VH1 and VL1 are proteins that bind to epidermal growth factor receptor (EGFR), EGFRvIII, melanoma-conjugated chondroitin sulfate proteoglycan (MCSP), mesothelin (MSLN), folate receptor 1 (FOLR1), CD33, CDH19, or epidermal growth factor 2 (HER2) when they are part of an IgG or scFv antibody,

[23] .

[25] A nucleic acid that codes for one of the proteins [1] to

[24] .

[26] A vector containing nucleic acid

[25] .

[27]

[26] Host cells containing nucleic acids.

[28] A method for producing any of the proteins [1] to

[24] , The steps include culturing host cells containing nucleic acids encoding the protein under conditions that allow the protein to be expressed, and Steps to recover the protein from the culture medium or cell population. The method, including the method described above.

[29] A method for treating a cancer patient, comprising the step of administering a therapeutically effective dose of any of the proteins [1] to

[24] .

[30] The method of

[29] further comprises the step of administering radiation, a chemotherapeutic agent, and / or a non-chemotherapeutic antineoplastic agent before, after, or simultaneously with the protein.

[31] A method according to

[29] or

[30] wherein the patient's cancer cells express a protease capable of cleaving protease cleavage sites.

[32] A method for treating a patient suffering from an infection, fibrous disease, neurodegenerative disease, or autoimmune or inflammatory disease, comprising the step of administering a therapeutically effective dose of any of the proteins [1]-

[12] and

[16] -

[22] . [Brief explanation of the drawing]

[0014] [Figure 1] An illustrative schematic diagram of a bispecific protein (PABP) that can be activated by a protease is shown. Numbered articles have the following meanings: The oval labeled "1" represents component 1, which binds to the target molecule as defined herein; the oval labeled "2" represents component 2, which binds to the effector cell molecule as defined herein; the oval labeled "3" represents component 3, which is any portion, optionally a polypeptide, that binds to component 1 or 2 and prevents component 1 or 2 from binding to the target cell or effector cell, respectively; the dotted line labeled "4" represents component 4, a protease-cleavable amino acid sequence, which may include a further linker sequence; the rectangle labeled "5" represents component 5, which is any half-life extension portion, which may optionally be a polypeptide; the solid curve extending from the oval labeled "3" is a non-cleavable linker, which may, for example, be a polypeptide. [Figure 2] A schematic diagram of one embodiment of PABP is shown. The ovals labeled VH1 and VL1 represent the immunoglobulin heavy and light chain variable (VH and VL) regions, respectively, which constitute component 1 and bind to target cells when they are part of an IgG or scFv antibody. The ovals labeled VH2 and VL2 represent the VH and VL regions, respectively, which bind to CD3ε when they are part of an IgG or scFv antibody and constitute component 2, respectively. The smaller oval labeled "CD3ε" is all or part of CD3ε, which represents component 3, as shown. The ovals labeled CH2 and CH3 represent the second and third constant domains of the IgG antibody, respectively. Together with part or all of the hinge region, these two domains form an Fc polypeptide chain. The two Fc polypeptide chains represent component 5, as shown. The dotted line labeled "4" represents component 4, as shown, which contains the protease cleavage site. Solid lines represent peptide linkers (curves) or hinge regions (straight lines). [Figure 3] A schematic diagram of one aspect of PABP is shown. All ellipses, solid lines, and dashed lines shown have the same meaning as in Figure 2. The rectangles labeled "CH1" and "CL" represent the immunoglobulin CH1 and CL regions, respectively. [Figure 4] A schematic diagram of one aspect of PABP is shown. All ellipses, solid lines, and dashed lines shown have the same meaning as in Figure 2. [Figure 5] Figure 5A shows a schematic diagram of one aspect of PABP. All ellipses, solid lines, and dashed lines shown have the same meaning as in Figures 2 and 3. Figure 5B shows a schematic diagram of one aspect of PABP. All ellipses, solid lines, and dashed lines shown have the same meaning as in Figures 2 and 3. [Figure 6]This shows the digestion of PABP and a control molecule by MMP-2. The method is described in Example 2, and the digested products were electrophoresed on an SDS-PAGE gel under reducing conditions. The lanes contain the following samples: 1) CD3ε(1-27)-aCD3-aHER2-Xbody, without MMP-2; 2) CD3ε(1-27)-aCD3-aHER2-Xbody, with MMP-2; 3) CD3ε(1-27)-MMP-2csV1-aCD3-aHER2-Xbody, without MMP-2; 4) CD3ε(1-27)-MMP-2csV1-aCD3-aHER2-Xbody, with MMP-2; 5) CD3ε(1-27)-Furin csV1-aCD3-aHER2-Xbody, without MMP-2; 6) CD3ε(1-27)-Furin csV1-aCD3-aHER2-Xbody, with MMP-2; 7) CD3ε(1-27)-MMP-2csV2-aCD3-aHER2-Xbody, without MMP-2; 8) CD3ε(1-27)-MMP-2csV2-aCD3-aHER2-Xbody, with MMP-2; 9) CD3ε(1-27)-Furin csV2-aCD3-aHER2-Xbody, without MMP-2; 10) CD3ε(1-27)-Furin csV2-aCD3-aHER2-Xbody, with MMP-2; 11) CD3ε(1-27)-MMP-2csV3-aCD3-aHER2-Xbody, without MMP-2; and 12) 11) CD3ε(1-27)-MMP-2csV3-aCD3-aHER2-Xbody, with MMP-2. A "+" on the lane indicates a sample treated with MMP2. [Figure 7]This shows the digestion of PABP and a control molecule by MMP-2. The method is described in Example 2, and the digested products were electrophoresed on an SDS-PAGE gel under reducing conditions. The lanes contain the following samples: 1) CD3ε(1-27)-aCD3-aHER2-mxb, without MMP-2; 2) CD3ε(1-27)-aCD3-aHER2-mxb, with MMP-2; 3) CD3ε(1-27)-MMP-2csV1-aCD3-aHER2-mxb, without MMP-2; 4) CD3ε(1-27)-MMP-2csV1-aCD3-aHER2-mxb, with MMP-2; 5) CD3ε(1-27)-MMP-2csV2-aCD3-aHER2-mxb, without MMP-2; 6) CD3ε(1-27)-MMP-2csV2-aCD3-aHER2-Xbody, with MMP-2; 7) CD3ε(1-27)-furin csV2-aCD3-aHER2-Xbody, without MMP-2; and 8) CD3ε(1-27)-furin csV2-aCD3-aHER2-Xbody, with MMP-2. A "+" on the lane indicates a sample treated with MMP2. [Figure 8]This shows the digestion of PABP and a control molecule by MMP-9. The method is described in Example 2, and the digested products were electrophoresed on an SDS-PAGE gel under reducing conditions.The lanes contain the following samples: 1) CD3ε(1-27)-aCD3-aHER2-Xbody, without MMP-2; 2) CD3ε(1-27)-aCD3-aHER2-Xbody, with MMP-2; 3) CD3ε(1-27)-MMP-2csV1-aCD3-aHER2-Xbody, without MMP-2; 4) CD3ε(1-27)-MMP-2csV1-aCD3-aHER2-Xbody, with MMP-2; 5) CD3ε(1-27)-Furin csV1-aCD3-aHER2-Xbody, without MMP-2; 6) CD3ε(1-27)-Furin csV1-aCD3-aHER2-Xbody, with MMP-2; 7) CD3ε(1-27)-MMP-2csV2-aCD3-aHER2-Xbody, without MMP-2; 8) CD3ε(1-27)-MMP-2csV2-aCD3-aHER2-Xbody, with MMP-2; 9) CD3ε(1-27)-FuryncsV2-aCD3-aHER2-Xbody, without MMP-2; 10) CD3ε(1-27)-FuryncsV2-aCD3-aHER2-Xbody, with MMP-2; 11) CD3ε(1-27)-MMP-2csV3-aCD3-aHER2-Xbody, without MMP-2; 12) CD3ε(1-27)-MMP-2csV3-aCD3-aHER2-Xbody, with MMP-2; 13) CD3ε(1-27)-aCD3-aHER2-mxb, without MMP-2;14) CD3ε(1-27)-aCD3-aHER2-mxb, with MMP-2;15) CD3ε(1-27)-MMP-2csV1-aCD3-aHER2-mxb, without MMP-2;16) CD3ε(1-27)-MMP-2csV1-aCD3-aHER2-mxb, with MMP-2;17) CD3ε(1-27)-MMP-2csV2-aCD3-aHER2-mxb, without MMP-2;18) CD3ε(1-27)-MMP-2csV2-aCD3-aHER2-mxb, with MMP-2;19) CD3ε(1-27)-furin csV2-aCD3-aHER2-mxb, without MMP-2; and 20) CD3ε(1-27)-furin csV2-aCD3-aHER2-mxb, with MMP-2. A "+" on the lane indicates a sample treated with MMP2. [Figure 9] Figure 9A shows the lysis of SKOV-3 cells in the presence of pan-T cells and a control molecule. The method is described in Example 3. The x-axis represents the concentration of the control molecule added to the assay, and the y-axis represents the percentage of lysed cells. The symbols represent data from assays performed using the following proteins: solid black circles, aCD3-aHER2-Xbody; and solid black squares, aCD3-aHER2-mxb. Figure 9B shows the percentage of T cells expressing CD25. The method is described in Example 3. The x-axis represents the concentration of the control molecule added to the assay, and the y-axis represents the percentage of cells expressing CD25. The symbols have the same meaning as in Figure 9A. [Figure 10] Figure 10A shows the lysis of pan-T cells and SKOV-3 cells in the presence of PABP or a control molecule. The method is described in Example 3. The x-axis represents the concentration of PABP or the control molecule added to the assay, and the y-axis represents the percentage of lysed cells. The symbols represent data from assays performed using the following proteins: solid black square, CD3ε(1-27)-aCD3-aHER2-Xbody, undigested; solid white square, CD3ε(1-27)-aCD3-aHER2-Xbody, digested by MMP-2; solid black triangle, CD3ε(1-27)-MMP-2csV1-aCD3-aHER2-Xbody, undigested; solid white triangle, CD3ε(1-27)-MMP-2csV1-aCD3-aHER2-Xbody, digested by MMP-2; solid black circle, CD3ε(1-27)-furin csV1-aCD3-aHER2-Xbody, undigested; and solid white circle, CD3ε(1-27)-furin csV1-aCD3-aHER2-Xbody, digested by MMP-2. Figure 10B shows the percentage of T cells expressing CD25. The method is described in Example 3. The x-axis represents the concentration of the control molecule or PABP added to the assay, and the y-axis represents the percentage of cells expressing CD25. The symbols have the same meaning as in Figure 10B. [Figure 11]Figure 11A shows the lysis of SKOV-3 cells in the presence of pan-T cells and PABP. The method is described in Example 3. The x-axis represents the concentration of PABP or control molecule added to the assay, and the y-axis represents the percentage of lysed cells. The symbols represent data from assays performed using the following proteins: solid black square, CD3ε(1-27)-MMP-2csV2-aCD3-aHER2-Xbody, undigested; solid white square, CD3ε(1-27)-MMP-2csV2-aCD3-aHER2-Xbody, digested with MMP-2; solid black triangle, CD3ε(1-27)-furin csV2-aCD3-aHER2 -Xbody, undigested; white triangle with solid line, CD3ε(1-27)-furin csV2-aCD3-aHER2-Xbody, digested with MMP-2; black circle with solid line, CD3ε(1-27)-MMP-2csV3-aCD3-aHER2-Xbody, undigested; and white circle with solid line, CD3ε(1-27)-MMP-2csV3-aCD3-aHER2-Xbody, digested with MMP-2. Figure 11B shows the percentage of T cells expressing CD25. The method is described in Example 3. The x-axis represents the concentration of the control molecule or PABP added to the assay, and the y-axis represents the percentage of cells expressing CD25. The symbols have the same meaning as in Figure 11B. [Figure 12]Figure 12A shows the lysis of pan-T cells and SKOV-3 cells in the presence of PABP or a control molecule. The method is described in Example 3. The x-axis represents the concentration of PABP or the control molecule added to the assay, and the y-axis represents the percentage of lysed cells. The symbols represent data from assays performed using the following proteins: solid black square, CD3ε(1-27)-aCD3-aHER2-mxb, undigested; solid white square, CD3ε(1-27)-aCD3-aHER2-mxb, digested with MMP-2; solid upward black triangle, CD3ε(1-27)-MMP-2csV1-aCD3-aHER2-mxb, undigested; solid upward white triangle, CD3ε(1-27)-MMP-2csV1-aCD3-aHER2 -mxb, digested by MMP-2; black circle with solid line, CD3ε(1-27)-MMP-2csV2-aCD3-aHER2-mxb, undigested; white circle with solid line, CD3ε(1-27)-MMP-2csV2-aCD3-aHER2-mxb, digested by MMP-2; black diamond with solid line, CD3ε(1-27)-furin csV2-aCD3-aHER2-mxb; and white diamond with solid line, CD3ε(1-27)-furin csV2-aCD3-aHER2-mxb. Figure 12B shows the percentage of T cells expressing CD25. The method is described in Example 3. The x-axis represents the concentration of the control molecule or PABP added to the assay, and the y-axis represents the percentage of cells expressing CD25. The symbols have the same meaning as in Figure 12B. [Figure 13]The binding of PABP and control molecules to T cells is shown. The method is described in Example 5. The x-axis represents relative fluorescence intensity (mean fluorescence intensity (MFI)). The Y-axis represents cell number. Each trace is indicated by a number, which indicates the protein incubated with T cells as follows: 1, negative control without added protein; 2, anti-CD3 IgG antibody; 3, aCD3-aHER2-Bi-Fc; 4, CD3ε(1-27)-aCD3-aHER2-BiFc, not cleavable; 5, CD3ε(1-27)-MMP-2cs-aCD3-aHER2-BiFc, undigested; and 6, CD3ε(1-27)-furin cs-aCD3-aHER2-BiFc, possibly digested in the HEK-293 cells from which it was produced. [Figure 14] This shows the lysis of pan-T cells and JIMT-1 cells in the presence of PABP or a control molecule. The method is described in Example 5. The x-axis shows the concentration (pM) of the protein included in the assay, and the y-axis shows the percentage of lysed target cells (JIMT-1 cells). Figure 13 is numbered using the same numbering as described above, indicating the proteins used in the assay.

[0015] A brief explanation of array lists TIFF0007842528000005.tif165166TIFF0007842528000006.tif220165TIFF0007842528000007.tif235166 [Modes for carrying out the invention]

[0016] Detailed explanation Several forms of bispecific proteins, and optionally bispecific antibodies, that can be activated by proteolytic cleavage are described herein. These proteins are referred herein to as protease-activated bispecific proteins (PABPs). PABPs may find applications in disease states where one or more proteases are present in large quantities in the local disease microenvironment, for example, in various cancers, inflammatory diseases, fibrotic diseases, and neurodegenerative diseases such as Alzheimer's disease. See, for example, Broder and Becker-Pauly (2013), Biochem. J. 450: 253-264. In such situations, bispecific proteins can be activated in the presence of disease cells but not in the absence of disease cells. Therefore, the bispecific proteins described herein may be specifically activated in the disease microenvironment and may have lower activity or be inactive in other parts of the body.

[0017] The PABP illustrated in Figure 1 essentially contains three components and may contain two additional optional components. The various components of this molecule do not have to be in the order shown in Figure 1. Component 1 (the oval labeled "1" in Figure 1) can bind to target molecules expressed on the surface of pathogens, infected cells, or disease-mediating cells. Component 2 (the oval labeled "2") can bind to effector cell molecules expressed on the surface of effector cells that play a role in cell death, such as T cells. Component 3 (the smaller oval labeled "3") is an optional component that can bind to component 1 or 2, thereby preventing component 1 or 2 from binding to the target molecule or effector cell molecule, respectively. Therefore, for example, if component 3 is bound to component 2, the bispecific molecule is effectively monospecific or at least less effective in binding to effector cell molecules. In some embodiments, component 3 may be omitted, in which case the binding of component 1 or component 2 to the target molecule or effector cell molecule, respectively, may be blocked or inhibited by the three-dimensional structure of PABP. Component 4 (represented by the dashed line indicated by "4") is a linker containing a protease cleavage site, which is positioned so that cleavage at this site allows both components 1 and 2 to bind to their respective binding partners. In some embodiments, cleavage separates component 3 from the rest of PABP, thereby activating the molecule, i.e., making the molecule completely bispecific. In other embodiments, cleavage may make component 1 or 2 more accessible, and therefore more active. In some embodiments, PABP may further include component 5 (a rectangle indicated by "5") which extends the half-life. Component 5 may be, for example, an Fc polypeptide chain, all or part of a serum albumin protein, or another polypeptide that can extend the in vivo half-life.

[0018] definition As used herein, “antibody” refers to a protein containing at least one immunoglobulin heavy chain variable region (VH) or light chain variable region (VL), often both VH and VL. Therefore, the term “antibody” includes single-chain Fv antibodies (scFv, containing VH and VL regions linked by a linker), Fab, F(ab)2', Fab', scFv:Fc antibodies (Carayannopoulos and Capra, Ch. 9 in FUNDAMENTAL IMMUNOLOGY, 3 rd This term encompasses molecules in various forms, including full-length antibodies containing two full-length heavy chains and two full-length light chains, such as those described in Paul, ed., Raven Press, New York, 1993, pp. 284-286, or natural IgG antibodies found in mammals. Ibid. Such full-length antibodies referred to herein as “IgG antibodies” may be of the IgG1, IgG2, IgG3, or IgG4 isotype and may be human antibodies. The portion by Carayannopoulos and Capra describing the structure of antibodies is incorporated herein by reference. Furthermore, the term “antibody” includes dimer antibodies containing two heavy chains and no light chains, such as natural antibodies found in camels and other dromedary camel species, as well as sharks. See, for example, Muldermans et al., 2001, J. Biotechnol. 74:277-302; Desmyter et al., 2001, J. Biol. Chem. 276:26285-90; Streltsov et al. (2005), Protein Science 14: 2901-2909. Antibodies may be "monospecific" (i.e., bind to only one antigen), "bispecific" (i.e., bind to two different antigens), or "multispecific" (i.e., bind to two or more different antigens). Furthermore, antibodies may be monovalent, bivalent, or polyvalent, meaning that an antibody can bind to one, two, or more antigen molecules at a time.

[0019] As used herein, “immunoglobulin heavy chain” essentially consists of, in this order, VH, the first heavy chain constant region (CH1), the hinge region, the second heavy chain constant region (CH2), the third heavy chain constant region (CH3), and optionally, the region downstream of CH3 in some isotypes. Closely related variants of immunoglobulin heavy chains containing no more than 10 single amino acid substitutions, insertions, and / or deletions per 100 amino acids compared to known or natural immunoglobulin heavy chain amino acid sequences are included in what is meant by immunoglobulin heavy chain.

[0020] As used herein, “immunoglobulin light chain” essentially consists of a VL and a light chain constant domain (CL). Closely related variants of immunoglobulin light chains containing no more than 10 single amino acid substitutions, insertions, and / or deletions per 100 amino acids, compared to known or naturally occurring immunoglobulin light chain amino acid sequences, are included in what is meant by immunoglobulin light chain.

[0021] As used herein, “immunoglobulin variable regions” refer to VH, VL, or their variants. Closely related variants of immunoglobulin variable regions containing no more than 10 single-amino acid substitutions, insertions, and / or deletions per 100 amino acids, compared to known or naturally occurring immunoglobulin variable region amino acid sequences, are included in what is meant by immunoglobulin variable regions. Many examples of VH and VL are known in the art, such as those disclosed in Kabat et al. in SEQUENCES OF IMMUNOLOGICAL INTEREST, Public Health Service NIH, Bethesda, MD, 1991. Based on the broad sequence commonality in smaller portions of the variability of VH and VL, the position of the variability within larger regions of the sequence, and the expected tertiary structure, those skilled in the art can recognize immunoglobulin variable regions by their sequence. See, for example, Honegger and Plueckthun (2001), J. Mol. Biol. 309: 657-670.

[0022] The 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 the antibody. The CDRs are embedded within framework regions (FR1-FR4) with less variability. The order of these sub-regions within the 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, for example, Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, Public Health Service NIH, Bethesda, MD, 1991.

[0023] CDRs can be located within the VH region sequence in the following ways: CDR1 begins around the 31st residue of the mature VH region, is typically about 5-7 amino acids long, and is almost always preceded by Cys-Xxx-Xxx-Xxx-Xxx-Xxx-Xxx-Xxx-Xxx (SEQ ID NO: ) (where "Xxx" is any amino acid). The residue following heavy chain CDR1 is almost always tryptophan, often Trp-Val, Trp-Ile, or Trp-Ala. Almost always, there are 14 amino acids between the last residue in CDR1 and the first residue in CDR2, and CDR2 typically contains 16-19 amino acids. Leu-Glu-Trp-Ile-Gly (SEQ ID NO: ) may precede CDR2, and Lys / Arg-Leu / Ile / Val / Phe / Thr / Ala-Thr / Ser / Ile / Ala may follow immediately after it. Other amino acids may precede or follow CDR2. Almost always, there are 32 amino acids between the last residue in CDR2 and the first residue in CDR3, and CDR3 may be approximately 3 to 25 residues long. Cys-Xxx-Xxx almost always precedes CDR3, and Trp-Gly-Xxx-Gly (SEQ ID NO: ) almost always follows CDR3.

[0024] The light chain CDRs can be located within the VL region in the following ways: CDR1 begins around the 24th residue of the mature antibody and is typically about 10-17 residues long. CDR1 is almost always preceded by Cys. Almost always, there are 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. Almost always, there are 32 residues between CDR2 and CDR3, and CDR3 is typically about 7-10 amino acids long. CDR3 is almost always preceded by Cys and is usually followed by Phe-Gly-Xxx-Gly (SEQ ID NO: ).

[0025] When VH and / or VL are said to "bind" to target cells or immune effector cells "if they are part of an IgG and / or scFv antibody," it means that an IgG or scFv antibody containing the specified VH and VL may bind to target cells and / or immune effector cells. Binding can be evaluated using the binding assay described in Example 5.

[0026] When a polypeptide is said to "inhibit the binding of the polypeptide chain to target cells or effector cells," the inhibition of binding is determined by a binding assay using fluorescence-activated cell sorting (FACS) as described in Example 5, and the results are shown in Figure 13. Similarly, when it is said that "the polypeptide chain binds more effectively to target cells or effector cells when the protease cleavage site is essentially completely cleaved," the improvement in binding is evaluated by the same assay. Essentially complete cleavage of the protease cleavage site is evaluated by Western blotting as described in Example 2, and is shown in Figures 6–8. For example, lanes 4, 8–10, and 12 in Figure 6 show essentially complete cleavage because, in these digested samples, the upper band that would be visible in an undigested sample is barely detectable, if present. While lanes 4 and 8 in Figure 6 may contain trace amounts of this upper band, it should be noted that samples containing such small amounts of uncleaved species are considered essentially completely cleaved as defined herein. In contrast, lanes 4 and 6 in Figure 7 show partial cleavage. The absence of cleavage can be assessed by the same method. For example, lane 2 in Figure 7 appears to be essentially identical to lane 1, which was not digested by MMP2, thus indicating a complete absence of cleavage. Furthermore, "When the protease cleavage site is essentially completely cleaved, the E of the protein in the cell lysis assay" C 50 represents the E in the same assay when the protease cleavage site is not cleaved. C This same definition of a complete cut essentially applies even when it is said to be "less than 1 / 5 of 50".

[0027] As used herein, "cancer cell antigen" refers to molecules, and optionally proteins, expressed on the surface of cancer cells. 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 cancer cells. A wide variety of cancer cell antigens exist. Examples of cancer cell antigens include, but are not limited to, the following human proteins: epidermal growth factor receptor (EGFR), EGFRvIII (mutant EGFR), melanoma-conjugated chondroitin sulfate proteoglycan (MCSP), mesotheline (MSLN), folate receptor 1 (FOLR1), CD33, CDH19, and epidermal growth factor 2 (HER2).

[0028] As used herein, "chemotherapy" means treatment of cancer patients with "chemotherapeutic agents" that have a cytotoxic effect or an inhibitory effect on cell proliferation against cancer cells. "Chemotherapeutic agents" specifically target cells involved in cell division and do not target cells not involved in cell division. Chemotherapeutic agents directly interfere with processes closely related to cell division, such as DNA replication, RNA synthesis, protein synthesis, spindle assembly, dispersion, or function, and / or the synthesis or stability of molecules such as nucleotides or amino acids that play a role in these processes. Therefore, chemotherapeutic agents have a cytotoxic effect or an inhibitory effect on cell proliferation against both cancer cells and other cells involved in cell division.Chemotherapy agents are well known in the art, and among many others known in the art, for example: alkylating agents (e.g., busulfan, temozolomide, cyclophosphamide, lomustine (CCNU), methyllomustine, streptozotocin, cis-diamminedichloroplatin, aziridinylbenzoquinone, and thiotepa); inorganic ions (e.g., cisplatin and carboplatin); nitrogen mustards (e.g., melphalan hydrochloride, ifosfamide, chlorambucil, and mechloretamine HCl); nitrosoureas (e.g., carmustine) (BCNU)); antineoplastic antibiotics (e.g., Adriamycin (doxorubicin), daunomycin, mitomycin C, daunorubicin, idarubicin, mitramycin, and bleomycin); plant derivatives (e.g., vincristine, vinblastine, vinorelbine, paclitaxel, docetaxel, vindesine, VP-16, and VM-26); antimetabolites (e.g., methotrexate with or without leucovorin, 5-fluorouracil with or without leucovorin, 5-fluorodeoxyuridine, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacitidine, hydroxyurea, deoxycoformycin, gemcitabine, and fludarabine); podophyllotoxins (e.g., etoposide, irinotecan, and topotecan); as well as actinomycin D and dacarbazine (DTIC), mMASA, procarbazine, hexamethylmelamine, pentamethylmelamine, L-asparaginase, and mitoxantrone are included. For example, relevant portions of Cancer: Principles and Practice of Oncology, 4 are incorporated herein by reference. thSee Edition, DeVita et al., eds., J.B. Lippincott Co., Philadelphia, PA (1993). Alkylating agents and nitrogen mustards act by alkylating DNA, which restricts strand unwinding and replication. Methotrexate, cytarabine, 6-mercaptopurine, 5-fluorouracil, and gemcitabine interfere with nucleotide synthesis. Plant derivatives such as paclitaxel and vinblastine are spindle poisons. Podophyllotoxin interferes with DNA replication by inhibiting topoisomerase. The antibiotics doxorubicin, bleomycin, and mitomycin interfere with DNA synthesis by intercalating (inhibiting unwinding) between the bases of DNA, causing strand breaks, and alkylating DNA, respectively. Other mechanisms of action include carbamoylation of amino acids (lomustine, carmustine), and depletion of the asparagine pool (asparaginase). Merck Manual of Diagnosis and Therapy, 17 th Edition, Section 11, Hematology and Oncology, 144. Principles of Cancer Therapy, Table 144-2 (1999). Specifically included among chemotherapeutic agents are the chemotherapeutic agents listed above, and chemotherapeutic agents that directly affect the same cellular processes that are directly affected by the chemotherapeutic agents listed above.

[0029] A drug or treatment will be administered "concurrently" with PABP if it is administered optionally on a continuous basis within the same general timeframe as PABP as defined herein. For example, if a patient takes drug A continuously once a week and PABP continuously once every six months, they will be administered concurrently, whether or not they are administered on the same day. Similarly, if PABP is taken continuously once a week and drug A is administered only once or several times a day, drug A and PABP will be administered concurrently as defined herein. Likewise, if both drug A and PABP are administered for a short period, either once or multiple times within a month, they will be administered concurrently as defined herein, as long as both drugs are administered within the same month.

[0030] As used herein, a “conservative amino acid substitution” is a substitution of an amino acid with another amino acid having similar properties. Properties considered include chemical properties such as charge and hydrophobicity. Table 1 below lists substitutions for each amino acid that are considered conservative substitutions as used herein.

[0031] (Table 1) Conservative amino acid substitutions TIFF0007842528000008.tif103128

[0032] As used herein, “effector cells” refer to cells involved in mediating cytolytic immune responses, including, for example, T cells, NK cells, monocytes, macrophages, or neutrophils. The bispecific antibodies activated by the proteases described herein bind to molecules expressed on the surface of effector cells. Such proteins are referred to herein as “effector cell molecules.”

[0033] Where applicable in this specification, “Fc region” is a dimer consisting of two polypeptide chains linked by one or more disulfide bonds, each chain containing CH2 and CH3 in addition to some or all of the hinge domain. Each polypeptide chain is referred to as an “Fc polypeptide chain.” To distinguish between two Fc polypeptide chains, one is optionally referred to herein as “chain A” and the other as “chain B.” More specifically, the Fc region intended for use with the present invention is an IgG Fc region, which may be a mammalian, e.g., human IgG1, IgG2, IgG3, or IgG4 Fc region. Within the human IgG1 Fc region, at least two alleles are known. In other embodiments, the amino acid sequences of two Fc polypeptide chains may differ from the amino acid sequences of mammalian Fc polypeptides by substitutions, insertions, and / or deletions of no more than 10 single amino acids per 100 amino acids in the sequence. In some embodiments, such modifications may be "heterodimerizing modifications" that promote the formation of heterodimers rather than homodimers, Fc modifications that extend the half-life, modifications that inhibit Fcγ receptor (FcγR) binding, and / or modifications that enhance Fcγ receptor binding and enhance ADCC.

[0034] As used herein, “half-life-extending Fc modification” refers to a modification within an Fc polypeptide chain that extends the in vivo half-life of a protein containing a modified Fc polypeptide chain compared to the half-life of a similar protein containing an Fc polypeptide that is otherwise identical. Such modifications may be present in Fc polypeptide chains that are part of PABPs as described herein. Modifications M252Y, S254T, and T256E (methionine at position 252 changed to tyrosine; serine at position 254 changed to threonine; threonine at position 256 changed to glutamic acid; numbered according to the EU numbering shown in Table 2) are half-life-extending Fc modifications and may be used together, separately, or in any combination. These modifications and several other modifications are described in detail in U.S. Patent No. 7,083,784. The portion of U.S. Patent No. 7,083,784 describing such modifications is incorporated herein by reference. Similarly, M428L and N434S are Fc modifications that extend the half-life and can be used together, separately, or in any combination. These modifications and several other modifications are described in detail in U.S. Patent Application Publication 2010 / 0234575 and U.S. Patent No. 7,670,600. The portions of U.S. Patent Application Publication 2010 / 0234575 and U.S. Patent No. 7,670,600 describing such modifications are incorporated herein by reference. In addition, any substitution in any of the following parts can be considered an Fc modification that extends the half-life as meant herein: 250, 251, 252, 259, 307, 308, 332, 378, 380, 428, 430, 434, 436. The half-life of PABP described herein can be extended using each of these modifications or in combination thereof. Other modifications that may be used to extend the half-life are described in detail in International Application PCT / US2012 / 070146, filed on 17 December 2012. The portion of this application describing such modifications is incorporated herein by reference. Some specific embodiments described in this application include, among other things, the following amino acid sequences: The PABP contains inserts that extend the half-life between positions 384 and 385 (EU numbering shown in Table 2), including TIFF0007842528000009.tif30161. PABPs containing such inserts are intended.

[0035] As used herein, “half-life extension portion” refers to a molecule that extends the in vivo half-life of a protein to which it is attached, compared to the in vivo half-life of a protein without the extension portion. Methods for measuring half-life are well known in the art. Methods for confirming half-life are disclosed, for example, in WO 2013 / 096221, the relevant portion of which is incorporated herein by reference. Essentially, the molecule is administered to an animal or human at known doses, and the amount of the molecule in the blood is assayed sequentially after administration. The half-life extension portion may be a polypeptide, such as an Fc polypeptide chain, or a polypeptide capable of binding to albumin. The amino acid sequence of a human fibronectin type III (Fn3) domain engineered to bind to albumin is provided in SEQ ID NO:83, and various human IgG Fc polypeptide sequences are shown in SEQ ID NO:84-87. The Fc polypeptide can be modified, for example, to be more effective in extending the half-life than an unmodified Fc polypeptide chain. Such modifications include, for example, those described above as "Fc modifications that extend the half-life." In an alternative embodiment, the half-life extension portion may be a non-polypeptide molecule. For example, a polyethylene glycol (PEG) molecule may be the half-life extension portion. Other half-life extension portions, including various polypeptides, are intended.

[0036] As used herein, "heterodimer" refers to a dimer containing two polypeptide chains having different amino acid sequences.

[0037] "Heterodimerizing modification" generally refers to modifications in the A and B chains of an Fc region that promote the formation of a heterodimer Fc region, i.e., an Fc region in which the A and B chains do not have the same amino acid sequence. Such modifications may be present in the Fc polypeptide chain that is part of the PABP described herein. Heterodimerizing modifications may be asymmetric, i.e., an A chain with a particular modification may pair with a B chain with a different modification. These modifications promote heterodimerization and detrimental to homodimerization. Whether a heterodimer or homodimer has been formed can be assessed by a size difference, which may be determined by polyacrylamide gel electrophoresis, or by other appropriate means such as different charges or biophysical properties, including binding by an antibody or other molecule that recognizes a particular portion of the heterodimer containing a molecular tag. An example of such a pair of heterodimerizing modifications is the so-called "knob and hole" substitution. For example, see U.S. Patent No. 7,695,936 and U.S. Patent Application Publication No. 2003 / 0078385, the portions describing such mutations which are incorporated herein by reference. Where used herein, an Fc region containing a pair of knob-and-hole substitutions contains one substitution in the A chain and another substitution in the B chain.For example, the following knob and hole substitutions in the A and B chains of the IgG1 Fc region have been found to increase heterodimerization compared to those observed in 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 strand and T366W and T394S in the other strand; and 9) T366W in one polypeptide of Fc and T366S, L368A, and Y407V in the other polypeptide of Fc. This variant notation method can be explained as follows: Using the EU numbering system (presented in Edelman et al. (1969), Proc. Natl. Acad. Sci. 63:78-85; see also Table 2 below), the EU position is followed by the amino acid normally present at a given position in the CH3 region (using a single-letter code), followed by the alternative amino acid present at that position. For example, Y407T means that the tyrosine normally present at EU407 is substituted with threonine. Alternatively, in addition to such modifications, substitutions that create new disulfide bridges may promote heterodimerization. For example, see U.S. Patent Application Publication No. 2003 / 0078385, which is incorporated herein by reference in a portion describing such mutations.Such modifications in the 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. Similarly, for example, C. H 3-C HSubstitutions that alter the charge of one or more residues at the interface can enhance heterodimer formation, as described in WO 2009 / 089004, and the portion of that publication describing such substitutions is incorporated herein by reference. Such substitutions are referred herein to as “charge pair substitutions,” and an Fc region containing a pair of charge pair substitutions contains one substitution in the A chain and a different substitution in the B chain. Common examples of charge pair substitutions include: 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. In addition, substitutions R355D, R355E, K360D, or K360R in both chains can stabilize heterodimers when used in conjunction with other heterodimerization modifications. Certain charge pair substitutions can be used alone or in conjunction with other charge pair substitutions.Specific examples of single charge pair substitutions and their combinations include: 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) 11) K409D and K370D in one strand, plus D399K and E357K in the other strand; 12) K409D and K392D in one strand, plus D399K, E356K, and E357K in the other strand; 13) K409D and K392D on one strand, plus D399K on the other strand; 14) K409D and K392D on one strand, plus D399K and E356K on the other strand; 15) K409D and K370D on one strand, plus D399K and D357K on the other strand; 16) D399K on one strand, plus K409D and K360D on the other strand; and 17) In addition to K409D and K439D on one strand, D399K and E356K are present on the other strand. Any of these heterodimerization modifications can be used in the Fc region of the heterodimer bispecific antibodies described herein.

[0038] As used herein, “modifications that inhibit FcγR binding” refer to one or more insertions, deletions, or substitutions within an Fc polypeptide chain that inhibit the binding of FcγRIIA, FcγRIIB, and / or FcγRIIIA, as measured, for example, by a competitive binding assay based on ALPHALISA® (PerkinElmer, Waltham, MA). Such modifications may be present in the Fc polypeptide chain that is part of the PABP described herein. More specifically, modifications that inhibit Fcγ receptor (FcγR) binding include any modifications that inhibit glycosylation at N297, including any substitutions at L234A, L235A, or N297. In addition, along with modifications that inhibit glycosylation at N297, additional modifications that stabilize the dimeric Fc region by creating additional disulfide crosslinks are also intended. Further examples of modifications that inhibit FcγR binding include the D265A modification in one Fc polypeptide chain and the A327Q modification in the other Fc polypeptide chain.

[0039] As used herein, “ADCC-enhancing modification” refers to one or more insertions, deletions, or substitutions in an Fc polypeptide chain that enhance antibody-dependent cell-mediated cytotoxicity (ADCC). Such modifications may be contained in the Fc polypeptide chain that is part of the PABP described herein. Many such modifications are described in International Patent Application Publication WO 2012 / 125850. The portion of this application describing such modifications is incorporated herein by reference. Such modifications may be contained in the Fc polypeptide chain that is part of the PABP described herein. The ADCC assay can be performed as follows: Cell lines expressing large and small amounts of cancer cell antigens on their cell surface can be used as target cells. These target cells can be labeled with carboxyfluorescein succinimimidyl ester (CFSE), washed once with phosphate-buffered saline (PBS), and then deposited in a 96-well microtiter plate having V-shaped wells. Purified immunoeffector cells, such as T cells, NK cells, macrophages, monocytes, or peripheral blood mononuclear cells (PBMCs), can be added to each well. Monospecific antibodies that bind to cancer antigens and contain the test modification, along with isotype-matched control antibodies, can be added to the wells in a 1:3 series dilution. Cells can be incubated with 5% CO2 at 37°C for 3.5 hours. Cells can be centrifuged and resuspended in 1× FACS buffer (1× phosphate-buffered saline (PBS) containing 0.5% fetal bovine serum (FBS)) containing the dead cell stain TO-PRO®-3 iodide (Molecular Probes, Inc. Corporation, Oregon, USA), and subsequently analyzed by fluorescence-activated cell sorting (FACS). The percentage of cell death can be calculated using the following formula: (Lysis rate of tumor cells in the presence of bispecificity - Lysis rate of tumor cells in the absence of bispecificity) / (Total cytolysis rate - Lysis rate of tumor cells in the absence of bispecificity) Total cell lysis is determined by dissolving a sample containing effector cells and labeled target cells with 80% cold methanol, without the use of bispecific molecules. Exemplary modifications that enhance ADCC include the following modifications in the A and B chains of the Fc region: (a) the A chain contains Q311M and K334V substitutions and the B chain contains L234Y, E294L, and Y296W substitutions, or vice versa; (b) the A chain contains E233L, Q311M, and K334V substitutions and the B chain contains L234Y, E294L, and Y296W substitutions, or vice versa; (c) the A chain contains L234I, Q311M, and K334V substitutions and the B chain contains L234Y, E294L, and Y296W substitutions, or vice versa; (d) the A chain contains S298T and K334V substitutions and the B chain contains L234Y, K290Y, and Y296W substitutions, or vice versa; (f) (f) Chain A contains A330M and K334V substitutions and Chain B contains L234Y, K290Y, and Y296W substitutions, or vice versa; (g) Chain A contains A330F and K334V substitutions and Chain B contains L234Y, K290Y, and Y296W substitutions, or vice versa; (h) Chain A contains Q311M, A330M, and K334V substitutions and Chain B contains L234Y, E294L, and Y296W substitutions, or vice versa; (i) (j) Chain A contains the S298T, A330M, and K334V substitutions and Chain B contains the L234Y, K290Y, and Y296W substitutions, or vice versa; (k) Chain A contains the S298T, A330F, and K334V substitutions and Chain B contains the L234Y, K290Y, and Y296W substitutions, or vice versa; (k) Chain A contains the S239D, A330M, and K334V substitutions and Chain B contains the L234Y, K290Y, and Y296W substitutions, or vice versa;(l) Chain A contains the S239D, S298T, and K334V substitutions and Chain B contains the L234Y, K290Y, and Y296W substitutions, or vice versa; (m) Chain A contains the K334V substitution and Chain B contains the Y296W and S298C substitutions, or vice versa; (n) Chain A contains the K334V substitution and Chain B contains the L234Y, Y296W, and S298C substitutions, or vice versa; (o) Chain A contains the L235S, S239D, and K334V substitutions and Chain B contains the L234Y, K290Y, and Y296W substitutions, or vice versa; (p) (q) Chain A contains L235S, S239D, and K334V substitutions and Chain B contains L234Y, Y296W, and S298C substitutions, or vice versa; (r) Chain A contains Q311M and K334V substitutions and Chain B contains L234Y, F243V, and Y296W substitutions, or vice versa; (s) Chain A contains Q311M and K334V substitutions and Chain B contains L234Y, K296W, and S298C substitutions, or vice versa; (t) (u) Chain A contains S239D and K334V substitutions and Chain B contains L234Y, Y296W, and S298C substitutions, or vice versa; (v) Chain A contains F243V and K334V substitutions and Chain B contains L234Y, K290Y, and Y296W substitutions, or vice versa; (w) Chain A contains E294L and K334V substitutions and Chain B contains L234Y, Y296W, and S298C substitutions, or vice versa; (x) (y) Chain A contains E294L and K334V substitutions and Chain B contains L234Y, Y296W, and S298C substitutions, or vice versa; (y) Chain A contains A330M and K334V substitutions and Chain B contains L234Y and Y296W substitutions, or vice versa;Alternatively, (z) the A chain contains the A330M and K334V substitutions, and the B chain contains the K290Y and Y296W substitutions, or vice versa.

[0040] As used herein, "linker" refers to a peptide that links two polypeptides, which may be two immunoglobulin variable regions in relation to PABP. The linker may be 2 to 30 amino acids long. In some embodiments, the linker may be 2 to 40, 2 to 40, or 3 to 18 amino acids long. In some embodiments, the linker may be a peptide of 40, 30, 20, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 amino acids or less long. In other embodiments, the linker may be 5 to 40, 5 to 15, 4 to 11, 10 to 20, or 20 to 40 amino acids long. In other embodiments, the linker may be approximately 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 acid lengths. Examples of linkers include, among many others, the amino acid sequence (GGGGS) n (In the formula, n is any integer between 1 and 10; SEQ ID NO: 88) This includes TIFF0007842528000010.tif11148 and AAA.

[0041] In the cell lysis assays described herein, PABP is considered to "mediate cell lysis of target cells by immune effector cells" if the addition of PABP in an amount of 0.001 pM to 20000 pM effectively induces cell lysis of target cells. The cell lysis assay is described in Example 3.

[0042] "Non-chemotherapy antineoplastic agents" are chemical agents, compounds, or molecules other than chemotherapeutic agents that have cytotoxic or cell proliferation inhibitory effects on cancer cells. However, non-chemotherapy antineoplastic agents may target molecules that indirectly affect cell division, such as cell surface receptors, including receptors for hormones or growth factors. However, non-chemotherapy antineoplastic agents do not directly interfere with processes closely related to cell division, such as DNA replication, RNA synthesis, protein synthesis, or spindle function, assembly, or dispersal. Examples of non-chemotherapy antineoplastic agents include, among many other possible non-chemotherapy antineoplastic agents, Bcl2 inhibitors, farnesyltransferase inhibitors, anti-estrogens such as tamoxifen, anti-androgen compounds, interferons, arsenic, retinoic acid, retinoic acid derivatives, antibodies targeting tumor-specific antigens, and Bcr-Abl tyrosine kinase inhibitors (e.g., the small molecule STI-571, marketed under the trade name GLEEVEC® by Novartis, New York and New Jersey, USA, and Basel, Switzerland).

[0043] As used herein, "non-cleavable linker" refers to a linker that does not contain a protease cleavage site.

[0044] As used herein, “protease cleavage site” includes all cleavage sites expressly disclosed herein (in Table 2), and any other cleavage sites, encompassing an amino acid sequence cleaved by a protease.

[0045] As used herein, "protein" includes a polypeptide chain of at least 30 amino acids linked by peptide bonds, and may include multiple polypeptide chains. Proteins may further include additional moieties added by post-translational modifications, such as sugars.

[0046] "Target cells" are cells to which PABPs described herein bind and which are involved in the mediation of disease. In some cases, target cells may be cells that are normally involved in mediating an immune response but are also involved in mediating disease. For example, in B-cell lymphoma, B cells that are normally involved in mediating an immune response may be target cells. In some embodiments, target cells are cancer cells, cells infected with pathogens, or cells involved in mediating autoimmune or inflammatory diseases. PABPs may bind to target cells by binding to a "target molecule" presented on the surface of the target cell, which may be, for example, a protein or a sugar, possibly a highly expressed protein, or a protein having a restrictive expression pattern that is abundant in the target cell compared to other types of cells or tissues in the body. The target molecule may also be, for example, a specific type of sugar molecule.

[0047] As used herein, a “therapeutic dose” of PABP is the amount that has the effect of reducing or eliminating, for example, the tumor burden in a cancer patient, or reducing or eliminating the symptoms of any disease condition for which the protein is used for treatment. A therapeutic dose does not need to completely eliminate all symptoms of the condition, but may reduce the severity of one or more symptoms, or delay the onset of more severe symptoms or more serious diseases that may occur with some frequency after the condition has been treated.

[0048] "Treatment" of any disease referred to herein includes alleviation of at least one symptom of the disease, reduction of the severity of the disease, or delay or prevention of disease progression to more severe symptoms that may be associated with the disease or may result in at least one other disease. Treatment does not necessarily mean a complete cure of the disease. A useful treatment only needs to reduce the severity of the disease, reduce the severity of one or more symptoms associated with the disease or its treatment, or delay the onset of more severe symptoms or more severe diseases that may occur with some frequency after the condition has been treated.

[0049] When it is said that a designated VH / VL pair of immunoglobulin variable regions "can bind to target cells or immune effector cells if they are part of an IgG or scFv antibody," it means that an IgG antibody containing a designated VH region in both heavy chains and a designated VL region in both light chains, or an scFv containing a VH / VL pair, can bind to target cells or immune effector cells. A binding assay is described in Example 5. Those skilled in the art can construct an IgG or scFv antibody containing a desired sequence by considering the knowledge in the art.

[0050] Component 1 and target molecule As described above, component 1 of PABP is a portion of PABP that can bind to a target molecule expressed on the surface of a pathogen or endogenous disease-causing cell. The pathogen may be, for example, a virus, a bacterium, or a protozoan. In some embodiments, component 1 includes both heavy-chain and light-chain variable (VH and VL) regions that can bind to the target molecule. The VH and VL regions may be on the same or different polypeptide chains. In other embodiments, component 1 may be a VH region or a VL region, insofar as the VH region or VL region can bind to a disease-causing cell or pathogen on its own. Such a single variable domain antibody is described, for example, in US 2008 / 0008713, the relevant portion of which is incorporated herein by reference. Either of these VH and / or VL regions may be of mammalian origin, for example, a human VH and / or VL region. In other embodiments, component 1 may be a polypeptide that is not part of the antibody. For example, if the target molecule is mesothelin, component 1 may be the whole or a portion of a polypeptide that binds to mesothelin, or a short peptide selected for its ability to bind to mesothelin.

[0051] Disease-mediating cells or pathogens may express target molecules on their surface. Such cells include, for example, endogenous cells that mediate cancer, autoimmune or inflammatory diseases, fibrous diseases, neurodegenerative diseases, or infectious diseases. For example, many proteins are known to be expressed at high levels specifically on cancer cells, cells that mediate autoimmune or inflammatory conditions, or infectious pathogens or infected cells. Such proteins are potential target molecules of PABP described herein.

[0052] As described above, the PABPs described herein bind to effector cell molecules and target molecules. Target molecules may be expressed on the surface of, for example, cancer cells (i.e., cancer cell antigens), cells infected with pathogens, or cells mediating inflammatory, autoimmune, or fibrotic conditions. In some embodiments, the target molecule may be highly expressed on target cells, but this is not required.

[0053] If the target cells are cancer cells, PABP may bind to cancer cell antigens as defined above herein. Cancer cell antigens may be human proteins and / or proteins of another species. For example, PABP may bind to target molecules, which may be proteins, from among many, including, mice, rats, rabbits, New World monkeys, and / or Old World monkey species. Such species include, without limitation, the following: humans (Homo sapiens), house mice (Mus musculus); black rats (Rattus rattus); brown rats (Rattus norvegicus); crab-eating macaques (Macaca fascicularis); hamadryas baboons (Papio hamadryas); guinea baboons (Papio papio); Anubis baboons (Papio anubis); yellow baboons (Papio cynocephalus); chacma baboons (Papio ursinus); common marmosets (Callithrix jacchus); cotton-top tamarins (Saguinus Oedipus); and squirrel monkeys (Saimiri sciureus).

[0054] In some examples, the target molecule may be a protein selectively expressed on infected cells. For example, in the case of hepatitis B virus (HBV) or hepatitis C virus (HCV) infection, the target molecule may be the envelope protein of HBV or HCV expressed on the surface of infected cells. In other embodiments, the target molecule may be gp120, encoded by human immunodeficiency virus (HIV), expressed on HIV-infected cells. Similarly, the target molecule may be a molecule expressed on the surface of pathogens, including, for example, viruses, bacteria (including species of the genera Borrelia, Staphylococcus, and Escherichia, among many other species), fungi (including yeast), Giardia, amoeba, eukaryotic protozoa of the genera Plasmodium, ciliates, trypanosomes, nematodes, and other eukaryotic parasites.

[0055] In conditions where depleting regulatory T cells is desirable, such as cancer or infectious diseases, regulatory T cells can serve as target cells. In such cases, CCR4 may be the target molecule.

[0056] In other contexts, target cells may be cells that mediate autoimmune or inflammatory diseases. For example, human eosinophils in asthma may be target cells, in which case, for example, EGF-like module-containing mucin-like hormone receptor 1 (EMR1) may be the target molecule. Alternatively, excess human B cells in patients with systemic lupus erythematosus may be target cells, in which case, for example, CD19 or CD20 may be the target molecule. In other autoimmune conditions, excess human Th2 T cells may be target cells, in which case, for example, CCR4 may be the target molecule. Similarly, target cells may be fibrocystic cells that mediate diseases such as atherosclerosis, chronic obstructive pulmonary disease (COPD), cirrhosis, scleroderma, renal transplant fibrosis, allogeneic renal transplant nephropathy, or pulmonary fibrosis including idiopathic pulmonary fibrosis and / or idiotypic pulmonary hypertension. In relation to such fibrotic conditions, for example, fibroblast-activating protein α (FAPα) may be the target molecule.

[0057] Specific examples of component 1 include, for example, VH / VL pairs that bind to cancer cell antigens, such as VH / VL pairs containing amino acid sequences of amino acids 20-140 of SEQ ID NO:6 and amino acids 197-303 of SEQ ID NO:8.

[0058] Component 2 and effector cell molecules Component 2 may bind to effector cell molecules. It may include a VH region and a VL region. In some embodiments, component 2 may include a VH region or a VL region that can bind to effector cell molecules individually. Either of these VH and / or VL regions may be of mammalian origin, e.g., a human VH and / or VL region. Alternatively, component 2 may be a non-antibody polypeptide that can bind to effector cell molecules. Component 2 may bind to molecules that are expressed on the surface of effector cells, which may be proteins. Effector cells may be, for example, T cells, NK cells, monocytes, macrophages, or neutrophils.

[0059] In some embodiments, effector cell molecules are proteins contained within the T cell receptor (TCR)-CD3 complex. At least three types of TCRs exist. The αβTCR complex contains a heterodimer consisting of TCRα and TCRβ (αβTCR), a homodimer consisting of two CD3ζ proteins (CD3ζζ), a heterodimer consisting of CD3δ and CD3ε (CD3δε), and a heterodimer consisting of CD3γ and CD3ε (CD3γε). The γδTCR complex contains a heterodimer consisting of TCRγ and TCRδ (γδTCR), as well as CD3δε and CD3γε heterodimers and CD3ζζ homodimers. The pTCR consists of a heterodimer consisting of pTα and TCRβ, as well as CD3δε and CD3γε heterodimers and CD3ζζ homodimers. For example, see Kuhns and Badgandi (2012), Immunological Rev. 250: 120-143, the relevant portion of which is incorporated herein by reference. Component 2 may bind to any of the proteins contained in the TCR-CD3 complex.

[0060] In some embodiments, PABP may bind to a human CD3ε chain (whose mature amino acid sequence is disclosed in SEQ ID NO: 50), which may be part of a multimeric protein. Alternatively, the effector cell molecule may be human and / or cynomolgus monkey TCRα, TCRβ, TCRδ, TCRγ, CD3β, CD3γ, CD3δ, or CD3ζ.

[0061] In some embodiments, PABPs may bind to CD3ε chains derived from other species, such as mice, rats, rabbits, New World monkeys, and / or Old World monkey species. Such species include, but are not limited to, the following mammalian species: house mice; black rats; brown rats; cynomolgus macaques, Macaca fascicularis; hamadryas baboons, Papio hamadryas; guinea baboons, Papio papio; Anubis baboons, Papio anubis; yellow baboons, Papio cynocephalus; chacma baboons, Papio ursinus; common marmosets; cotton-top tamarins; and squirrel monkeys. The mature amino acid sequence of the CD3ε chain of cynomolgus macaques is provided in SEQ ID NO: 51. As is well known in the field of protein therapeutics development, having therapeutics that can have equivalent activity in humans and species such as mice and monkeys, which are commonly used for preclinical trials, can simplify and accelerate drug development. Such advantages can be significant in the long and expensive process of bringing drugs to market.

[0062] In a more specific embodiment, PABP may bind to an epitope within the first 27 amino acids of a CD3ε chain, which may be a human CD3ε chain or a CD3ε chain derived from a different species, in particular one of the mammalian species listed above. The epitope to which the antibody binds may be a portion of an amino acid sequence selected from the group consisting of SEQ ID NO: 52 and SEQ ID NO: 53. The epitope may contain the amino acid sequence Gln-Asp-Gly-Asn-Glu (SEQ ID NO: 54). The advantages of a protein that binds to this amino acid sequence are described in detail in U.S. Patent Application Publication 2010 / 183615, the relevant portion of which is incorporated herein by reference. The portion of a protein to which the antibody or protein binds can be determined by alanine scanning, which is described, for example, in U.S. Patent Application Publication 2010 / 183615, the relevant portion of which is incorporated herein by reference.

[0063] If NK cells or cytotoxic T cells are immune effector cells, NKG2D, CD352, NKp46, or CD16a may be effector cell molecules to which component 2 can bind. CD8 + If T cells are immune effector cells, then 4-1BB, OX40, GITR, CD28, CD27, or ICOS may be effector cell molecules to which component 2 can bind. Alternatively, PABP may bind to other antigens expressed on T cells, NK cells, macrophages, monocytes, or neutrophils.

[0064] The VH and VL regions that can be used as component 2 of PABP include those that can bind to CD3ε or other components of the TCR-CD3 complex, such as those containing the amino acid sequences of SEQ ID NO: 40 and 45. Other VH / VL pairs that can bind to CD3ε or other effector cell molecules expressed on T cells, NK cells, macrophages, monocytes, or neutrophils can also be used as component 2.

[0065] Ingredient 3 Component 3, which is an optional component, is a polypeptide that can bind to component 1 or 2, and, upon binding, can prevent or inhibit component 1 or 2 from binding to effector cells or target cells. In some embodiments, component 3 is part or all of a target molecule to which component 1 can bind or an effector cell molecule to which component 2 can bind. For example, if the effector cell is a T cell, component 3 may be part or all of a polypeptide that is part of the TCR-CD3 complex, such as TCRα, TCRβ, TCRδ, TCRγ, pTα, CD3β, CD3γ, CD3δ, CD3ε, or CD3ζ. Alternatively, if the effector cell is an NK cell or cytotoxic T cell, component 3 may be part or all of NKG2D, CD352, NKp46, or CD16a. Similarly, if the effector cell is a CD8 +If it is a T cell, component 3 may be part or all of 4-1BB, OX40, GITR, CD28, CD27, or ICOS. In some embodiments, component 3 includes a portion of CD3ε. For example, component 3 may be mature human CD3ε (SEQ ID NO: 50), or it may be CD3ε derived from a different species, in particular cynomolgus monkey (SEQ ID NO: 51), or it may include the first 27 amino acids of CD3ε.

[0066] In some embodiments, component 3 may comprise an in vitro selected peptide, which, if it is part of PABP, may block or inhibit the binding of PABP to effector cells or target cells compared to the binding observed with the same PABP when component 3 is separated from the rest of PABP by protease cleavage. Alternatively, component 3 comprising such an in vitro selected peptide may, if it is part of PABP, inhibit the cytolysis of target cells in the presence of effector cells and PABP compared to the cytolysis observed in the presence of the same effector cells and PABP when component 3 is separated from the rest of PABP by protease cleavage.

[0067] Ingredient 4 Component 4 includes a protease cleavage site. The cleavage site may be cleaved by a protease specifically expressed in the physical vicinity of a pathogen, a pathogen-infected cell, or a disease-mediating cell, such as a cancer cell. The protease may be, among many others, a metalloproteinase, a matrix metalloproteinase (MMP), such as MMP2, MMP9, or MMP11, a serine protease, a cysteine ​​protease, furin, plasmin, or a plasminogen activator (such as urokinase-type plasminogen activator (u-PA) or tissue plasminogen activator (tPA)), or fibroblast-activating protein α (FAPα).

[0068] These protease cleavage sites may include, for example, sites cleaved by plasmin. The enzyme precursor plasminogen is activated by proteolytic cleavage by u-PA and converted to the active enzyme, plasmin. Plasmin, a serine protease, may play a role in translocation due to its degradation of the extracellular matrix and its activation of other enzymes, such as type IV collagenase. See, for example, Kaneko et al. (2003), Cancer Sci. 94(1): 43-39, the relevant portion of which is incorporated herein by reference.

[0069] Such protease cleavage sites also include those of the metalloproteinases, meprine α and meprine β, which may be involved in certain diseases such as certain cancers, inflammatory bowel disease, cystic fibrosis, kidney disease, diabetic nephropathy, and cutaneous fibrous tumors. The cleavage sites of meprine α and β are not limited to a single defined sequence for each of these proteases. However, there is strong selectivity for one or a few specific amino acids at certain amino acid positions relative to the cleavage site. For example, see Becker-Pauly et al. (2011), Molecular and Cellular Proteomics 10(9):M111.009233. DOI:10.1074 / mcp.M111.009233, the portion describing specific cleavage sites, including supplemental material, which is incorporated herein by reference. A small selection of known cleavage sites for various proteases, including meprine α and meprine β, is provided in Table 2 below. Component 4 of the present invention as described herein may, in no way, contain cleavage sites of any metalloproteinase including meprin α and meprin β, including any of the cleavage sites listed in Table 2.

[0070] Similarly, matrix metalloproteinases (MMPs), MMP-2 and MMP-9, are overexpressed in various human tumors, including ovarian tumors, breast tumors, and prostate tumors, as well as in melanoma. Furthermore, a correlation between invasive tumor growth and high levels of MMP-2 and / or MMP-9 has been observed in both clinical and experimental studies. See, for example, Roomi et al. (2009), One. Rep. 21: 1323-1333. The cleavage sites of MMP-2 or MMP-9 can be represented as P4-P3-P2-P1|P1'-P2'-P3'-P4', where P1~P4 and P1'~P4' are amino acids and the vertical lines represent the cleavage sites. Some generalizations can be made regarding the MMP-2 cleavage sites. P1 is most likely glycine or proline. P2 is most likely to be proline, and somewhat less likely to be alanine, valine, or isoleucine. P3 is most likely to be alanine, serine, or arginine. P4 is most likely to be alanine, glycine, asparagine, or serine. P1' is most likely to be leucine, and somewhat less likely to be isoleucine, phenylalanine, or tyrosine. P2' is most likely to be lysine, and somewhat less likely to be alanine, valine, isoleucine, or tyrosine. P3' is most likely to be alanine, serine, or glycine. P4' is most likely to be alanine, lysine, or aspartic acid. A somewhat more apparent selectivity exists with respect to the MMP-9 cleavage site. P4 is most likely to be glycine. P3 is most likely to be proline. P2 is most likely to be lysine. P1 is most likely to be glycine or proline. P1' is most likely to be leucine, and somewhat less likely to be isoleucine. P2' is most likely lysine. P3' is most likely glycine or alanine. P4' is most likely alanine, proline, or tyrosine.Any MMP-2 or MMP-9 cleavage site, including those disclosed in Prudova et al. (2010), Mol. Cell. Proteomics 9(5): 894-911, as shown in Table 2 or, for example, relevant portions thereof, incorporated herein by reference, may be contained in component 4 of the present invention as described herein.

[0071] Higher-than-normal levels of u-PA are known to be associated with various cancers, including colorectal cancer, breast cancer, monocytic and myeloid leukemia, bladder cancer, thyroid cancer, liver cancer, gastric cancer, and cancers of the capsule, lung, pancreas, ovaries, and head and neck. See, for example, Skelly et al. (1997), Clin. Can. Res. 3: 1837-1840; Han et al. (2005), Oncol. Rep. 14(1): 105-112; Kaneko et al. (2003), Cancer Sci. 94(1): 43-49; Liu et al. (2001), J. Biol. Chem. 276(21): 17976-17984. Table 2 below reports small specimens of sites that can be resected by u-PA. Component 4 of the present invention as described herein may contain cleavage sites of any serine protease including u-PA and tissue plasminogen activator (tPA), which include any of the cleavage sites listed in Table 2.

[0072] Several cysteine ​​proteases, including cathepsin B, have been found to be overexpressed in tumor tissue and are likely to play a causative role in some cancers. See, for example, Emmert-Buck et al. (1994), Am. J. Pathol. 145(6): 1285-1290; Biniosseek et al. (2011), J. Proteome Res. 10: 5363-5373. The portions of these references describing the protease cleavage sites are incorporated herein by reference. Similar to the cleavage sites of meprin α and meprin β, there is considerable heterogeneity in the cleavage sites of cathepsin B. The cleavage sites of cathepsin B (and other proteases) can be represented as P3-P2-P1|P1'-P2'-P3', where P1~P3 and P1'~P3' are all amino acids and the vertical line represents the cleavage site. Some generalizations apply to cathepsin B cleavage sites. P3 is most often G, F, L, or P (using single-letter amino acid codes). P2 is most often A, V, Y, F, or I. P1 is most often G, A, M, Q, or T. P1' is most often F, G, I, V, or L. P2' is most often V, I, G, T, or A. P3' is most often G. Furthermore, some subsite cooperativity exists. For example, if P2 is F, then P3 is most likely to be G and least likely to be L, and P1' is most likely to be F and least likely to be L. This and other examples of subsite cooperativity are described in detail in Biniossek et al. (2011), J. Proteome Res. 10: 5363-5373. Figures 3 and 5 of Biniossek, as well as the accompanying text and supplementary Table 1, are incorporated herein by reference. All cathepsin B cleavage sites, including, but not limited to, those in Table 2, may be contained in component 4 of the present invention as described herein.

[0073] (Table 2) Examples of protease cleavage sites TIFF0007842528000011.tif186128 * The vertical lines represent the predicted cutting points.

[0074] Component 4 and other parts of PABP may contain non-protease-cleavable "linker" sequences. For example, component 4 may contain a protease-cleavage site and other non-cleavable linker sequences. Alternatively, component 4 may contain only a protease-cleavage site. These non-cleavable linkers include, for example, (G4S) n In the formula, n may be, for example, 1, 2, 3, 4, 5, 6, 7, or 8, and may include amino acid sequences such as G4S. G4S is listed as SEQ ID NO: 88. Other exemplary linkers include, among many others, amino acid sequences, This includes TIFF0007842528000012.tif11154 and AAA.

[0075] Ingredient 5 The half-life extension portion may be, for example, an Fc polypeptide, albumin, an albumin fragment, a portion that binds to albumin or the embryonic Fc receptor (FcRn), a derivative of fibronectin engineered to bind to albumin or a fragment thereof, a peptide, a single-domain protein fragment, or other polypeptides that can increase the serum half-life. In an alternative embodiment, the half-life extension portion may be a non-polypeptide molecule, such as polyethylene glycol (PEG). Sequences of human IgG1, IgG2, IgG3, and IgG4 Fc polypeptides that can be used are provided in SEQ ID NO: 84-87. Variants of these sequences containing one or more heterodimerization modifications, one or more Fc modifications that extend the half-life, one or more modifications that enhance ADCC, and / or one or more modifications that inhibit Fcγ receptor (FcγR) binding are also intended as other closely related varieties containing no more than 10 single amino acid deletions, insertions, or substitutions per 100 amino acids of the sequence.

[0076] The sequence of a derivative of human fibronectin type III (Fn3) engineered to bind to albumin is provided in SEQ ID NO:83. As is known in the art, the loop of the human fibronectin type III (Fn3) domain can be engineered to bind to other targets. Koide (1998), J Mol Biol.: 284(4): 1141-51.

[0077] The half-life extension portion may be the Fc region of the antibody. In this case, the first polypeptide chain may contain the Fc polypeptide chain after the CH1 region, and the second polypeptide chain may contain the Fc polypeptide chain after the CL region. Alternatively, only one polypeptide chain may contain the Fc polypeptide chain. Linkers may, but are not required, be present between the CH1 region and the Fc region, and / or between the CL region and the Fc region. As described above, the Fc polypeptide chain includes the CH2 and CH3 regions following all or part of the hinge region. The Fc polypeptide chain may be of mammalian origin (e.g., human, mouse, rat, rabbit, dromedary, or New World or Old World monkey), avian, or shark origin. In addition, as described above, the Fc polypeptide chain may include a limited number of modifications. For example, the Fc polypeptide chain may include one or more heterodimerization modifications, one or more modifications that inhibit or enhance binding to FcγR, or one or more modifications that increase binding to FcRn.

[0078] In some embodiments, the amino acid sequence of the Fc polypeptide may be that of a mammal, such as a human. The isotype of the Fc polypeptide may be IgG, IgA, IgD, IgE, or IgM, such as IgG1, IgG2, IgG3, or IgG4. Table 2 below shows the amino acid sequence alignment of human IgG1, IgG2, IgG3, and IgG4 Fc polypeptide chains.

[0079] (Table 2) Amino acid sequence of human IgG Fc polypeptide chain TIFF0007842528000013.tif132150

[0080] The numbering shown in Table 2 follows the EU numbering system based on the sequential numbering of the constant region of the IgG1 antibody. Edelman et al. (1969), Proc. Natl. Acad. Sci. 63: 78-85. Therefore, this numbering does not adequately correspond to the additional length of the IgG3 hinge. Nevertheless, this numbering is used herein to specify the position within the Fc region because it is still commonly used in the art to indicate a position within the Fc region. The hinge regions of the IgG1, IgG2, and IgG4 Fc polypeptides extend from approximately position 216 to approximately position 230. Alignment reveals that the hinge regions of IgG2 and IgG4 are 3 amino acids shorter than the IgG1 hinge, respectively. The IgG3 hinge is much longer, with an additional 47 amino acids extending upstream. The CH2 region extends from approximately position 231 to position 340, and the CH3 region extends from approximately position 341 to position 447.

[0081] The native amino acid sequence of Fc polypeptides may vary slightly. Such variations may include insertions, deletions, or substitutions of no more than 10 single amino acids per 100 amino acids in the sequence of the native Fc polypeptide chain. Where substitutions exist, they may be conservative amino acid substitutions as defined above. The amino acid sequences of the Fc polypeptides on the first and second polypeptide chains may differ. In some embodiments, they may include "heterodimerizing modifications" that promote heterodimerization, such as charge-pair substitutions as defined above. Furthermore, the Fc polypeptide portion of PABP may also contain modifications that inhibit or enhance FcγR binding. Such mutations are described above and in Xu et al. (2000), Cell Immunol. 200(1): 16-26, the relevant portions of which are incorporated herein by reference. The Fc polypeptide portion may also include the “Fc modifications that extend half-life” described above, including, for example, those described in U.S. Patents No. 7,037,784, 7,670,600, and 7,371,827, U.S. Patent Application Publication No. 2010 / 0234575, and International Application PCT / US2012 / 070146, all relevant portions thereof are incorporated herein by reference. Furthermore, the Fc polypeptide may include the “ADCC-enhancing modifications” as defined above.

[0082] Various forms of bispecific molecules that can be activated by proteases Figure 2 is a schematic diagram of an example of PABP as described herein. The ovals labeled "VH1" and "VL1" represent heavy and light chain variable (VH and VL) regions that can together bind to target molecules expressed on disease-mediated cells, such as cancer cell antigens, or target molecules expressed on infected cells or pathogens. As shown, VH1 and VL1 together constitute component 1 as discussed above in relation to Figure 1. As shown, the ovals labeled "VH2" and "VL2" represent the VH and VL regions that can together bind to CD3ε and constitute component 2. The smaller oval labeled "CD3ε" represents a portion of CD3ε to which VH2 and VL2 bind, and thus constitutes component 3 as defined above. As discussed above in relation to components 2 and 3, component 3 may be a protein other than CD3ε that is expressed on T cells, NK cells, monocytes, macrophages, or neutrophils. The dashed line and arrow labeled "4" represent the protease cleavage site (corresponding to component 4 discussed above). Other curves represent non-cleavable linkers. The straight line extending upward from the CH2 region, connected by a horizontal line, is the disulfide-bonded hinge region. The ellipses labeled "CH2" and "CH3," together with part or all of the hinge region, represent Fc polypeptide chains that can extend the half-life. As shown, the Fc region is considered component 5.

[0083] Another embodiment is illustrated in Figure 3. As shown, one polypeptide chain includes a fragment of CD3ε (component 3) followed by VH2, a linker, VL1, CH1, and an Fc polypeptide chain. The other polypeptide chain includes VH1 followed by a linker, VL2, CL, and an Fc polypeptide chain. VH2 and VL2 can bind to CD3ε. As shown, the dashed curve represents the protease cleavage site (component 4), and the straight and curved lines represent the hinge region and linker as shown above.

[0084] Further embodiments are illustrated in Figure 4. One polypeptide chain contains an scFv comprising VH1 and VL1 (oval shapes labeled "VH1" and "VL1") derived from antibodies that bind to target cell molecules, an optional linker, and an Fc polypeptide chain (hinge, as well as oval shapes labeled "CH2" and "CH3"). The other polypeptide chain contains a portion of CD3ε, which is component 3 of PABP as shown. This is followed by VH2 and VL2 derived from antibodies that bind to CD3ε, and an scFv comprising an optional linker and an Fc polypeptide chain. The dashed line represents the protease cleavage site, i.e., component 4 as shown. The curve shows the linker sequence. A straight vertical line extending upward from the CH2 region, connected by a horizontal line, represents the hinge region connected by a disulfide bond. As described in relation to Figure 2, component 3 may be a protein other than CD3ε, to which VL2 and VH2 may bind.

[0085] Further other embodiments are shown in Figures 5A and 5B. Figure 5A shows a protein in which one polypeptide contains VH1 followed by a protease cleavage site (component 4), followed by VH2 and CH1. The other polypeptide contains VL1 followed by a linker, VL2, and CL. As shown, VH1 and VL1 represent component 1, and VH2 and VL2 represent component 2.

[0086] Figure 5B shows a protein containing VH2 followed by CH1, a protease cleavage site (component 4), VH1, and a polypeptide containing CH1. The other polypeptide contains VL2, CL, a linker, VL1, and CL. As shown, VH1 and VL1 represent component 1, and VH2 and VL2 represent component 2.

[0087] Nucleic acids encoding PABP Nucleic acids encoding PABP as described herein are provided. Numerous nucleic acid sequences encoding immunoglobulin regions, including the VH, VL, hinge, CH1, CH2, CH3, and CH4 regions, are known in the art. See, for example, Kabat et al. in SEQUENCES OF IMMUNOLOGICAL INTEREST, Public Health Service NIH, Bethesda, MD, 1991. Using the guidelines provided herein, those skilled in the art can combine such nucleic acid sequences and / or other nucleic acid sequences known in the art to create the nucleic acid sequences encoding PABP as described herein.

[0088] In addition, the nucleic acid sequences encoding PABP described herein can be determined by those skilled in the art based on the amino acid sequences provided herein and the knowledge in the art. Beyond more traditional methods of generating cloned DNA segments encoding specific amino acid sequences, companies such as DNA2.0 (Menlo Park, CA, USA) and BlueHeron (Bothell, WA, USA) are now streamlining the production process of such DNA by routinely producing chemically synthesized, gene-sized DNA of any desired sequence on order.

[0089] How to make PABP PABPs described herein can be prepared using methods well known in the art. For example, the nucleic acids encoding the two polypeptide chains of PABP can be introduced into cultured host cells by various known methods, such as transformation, transfection, electroporation, or a microparticle gun using nucleic acid-coated microparticles. In some embodiments, the nucleic acids encoding PABP can be inserted into a vector suitable for expression in the host cell before being introduced into the host cell. Typically, such vectors may contain sequence elements that enable the expression of the inserted nucleic acid at the RNA and protein levels. Such vectors are well known in the art and many are commercially available. Host cells containing nucleic acid can be cultured under conditions that enable the expression of nucleic acid by the cells, and the resulting PABP can be collected from a cell population or culture medium. Alternatively, PABP can be produced in vivo in, for example, plant leaves (see, e.g., Scheller et al. (2001), Nature Biotechnol. 19: 573-577 and the references cited therein), bird eggs (see, e.g., Zhu et al. (2005), Nature Biotechnol. 23: 1159-1169 and the references cited therein), or mammalian milk (see, e.g., Laible et al. (2012), Reprod. Fertil. Dev. 25(1): 315).

[0090] Among the many options, various cultured host cells can be used, including, for example, bacterial cells such as Escherichia coli or Bacilis steorothermophilus; 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.

[0091] Therapeutic methods and compositions PABP as described herein can be used to treat a wide variety of conditions, including, for example, various forms of cancer, infections, fibrous diseases, and / or autoimmune or inflammatory conditions.

[0092] This specification provides pharmaceutical compositions comprising PABP as described herein. Such pharmaceutical compositions include, in addition to a therapeutically effective amount of PABP as described herein, one or more additional components such as physiologically acceptable carriers, excipients, or diluents. Such additional components may include, among many possibilities, buffers, carbohydrates, polyols, amino acids, chelating agents, stabilizers, and / or preservatives.

[0093] In some embodiments, PABP as described herein can be used to treat cell proliferation disorders, including cancer, that involve uncontrolled and / or inappropriate cell growth, often accompanied by destruction of adjacent tissue and neovascularization that can lead to the invasion, i.e., metastasis, of cancer cells into new areas. These conditions include hematological malignancies and solid malignancies. Conditions treatable with PABP as described herein include non-malignant conditions with inappropriate cell growth, such as colorectal polyps, cerebral ischemia, macroscopic cystic diseases, polycystic kidney disease, benign prostatic hyperplasia, and endometriosis. Other cell proliferative diseases that can be treated with PABP of the present invention include, for example, mesothelioma, squamous cell carcinoma, myeloma, osteosarcoma, glioblastoma, glioma, carcinoma, adenocarcinoma, melanoma, sarcoma, acute and chronic leukemia, lymphoma, as well as meningioma, Hodgkin's disease, Sézary syndrome, multiple myeloma, as well as lung cancer, non-small cell lung cancer, small cell lung cancer, pharyngeal cancer, breast cancer, head and neck cancer, bladder cancer, ovarian cancer, skin cancer, Cancers including prostate cancer, cervical cancer, vaginal cancer, gastric cancer, renal cell carcinoma, kidney cancer, pancreatic cancer, colorectal cancer, endometrial cancer, and esophageal cancer, hepatobiliary cancer, bone cancer, skin cancer, and hematological cancers, as well as cancers of the nasal cavity and sinuses, nasopharynx, oral cavity, oropharynx, larynx, inferior larynx, salivary glands, mediastinum, stomach, small intestine, colon, rectum and anal region, ureters, urethra, penis, testes, vulva, endocrine system, central nervous system, and plasma cell carcinomas.

[0094] One textbook providing guidelines for cancer treatment is *Cancer, Principles and Practice of Oncology, 4th Edition*, DeVita et al., Eds. JB Lippincott Co., Philadelphia, PA (1993). Appropriate treatment approaches are selected according to the specific type of cancer and other factors such as the patient's overall condition, as recognized in the relevant field. In the treatment of cancer patients, PABP as described herein may be added to a treatment plan using other antineoplastic agents and / or procedures.

[0095] In some embodiments, PABP can be administered concurrently with, before, or after, a variety of drugs and procedures widely used in cancer treatment, such as chemotherapeutic agents, non-chemotherapeutic antineoplastic agents, and / or radiation. For example, chemotherapy and / or radiation can be administered before, during, and / or after any of the procedures described herein. Examples of chemotherapeutic agents have been discussed above and include, but are not limited to, antineoplastic antibiotics such as cisplatin, taxol, etoposide, mitoxantrone (Novantrone®), actinomycin D, cycloheximide, camptothecin (or its water-soluble derivatives), methotrexate, mitomycin (e.g., mitomycin C), dacarbazine (DTIC), adriamycin (doxorubicin), and daunomycin, as well as all the chemotherapeutic agents mentioned above.

[0096] PABPs described herein can also be used to treat infectious diseases, among others, such as chronic hepatitis B virus (HBV) infection, hepatitis C virus (HPC) infection, human immunodeficiency virus (HIV) infection, Epstein-Barr virus (EBV) infection, or cytomegalovirus (CMV) infection.

[0097] PABPs described herein may find further applications in other types of conditions where depletion of certain cell types is beneficial. 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 may be beneficial. Depletion of myofibroblasts or other pathological cells in pulmonary fibrosis such as idiopathic pulmonary fibrosis (IPF), or in fibrotic conditions such as renal or hepatic fibrosis, is another application of PABPs.

[0098] The therapeutically effective doses of PABP described herein can be administered. The amount of antibody constituting the therapeutic dose may vary depending on the indication being treated, the patient's weight, and the patient's calculated skin surface area. The administration of PABP described herein can be adjusted to achieve the desired effect. Repeated administration may often be necessary. For example, the PABP described herein can be administered three times a week, twice a week, once a week, once every 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks, or once every 2, 3, 4, 5, or 6 months. The daily dose of PABP may range from approximately 0.0036 mg to approximately 450 mg. Alternatively, the dose can be calibrated according to the patient's estimated skin surface area, with each dose being approximately 0.002 mg / m². 2 ~about 250 mg / m 2 This may be the case. In another option, the dose can be calibrated according to the patient's weight, and each dose may range from approximately 0.000051 mg / kg to approximately 6.4 mg / kg.

[0099] PABP or pharmaceutical compositions containing these molecules can be administered by any viable method. Because oral administration, in the absence of certain formulations or circumstances, causes hydrolysis of proteins in the acidic environment of the stomach, protein therapeutics are usually administered via parenteral routes, such as injection. Subcutaneous, intramuscular, intravenous, intra-arterial, intrafocal, or peritoneal injection are possible routes of administration. PABP can also be administered by infusion, such as intravenous or subcutaneous infusion. Topical administration is also possible, especially for diseases affecting the skin. Alternatively, PABP can be administered through contact with mucous membranes, such as intranasal, sublingual, vaginal, or rectal administration, or as an inhalant. Alternatively, specific suitable pharmaceutical compositions containing PABP can be administered orally.

[0100] Although the present invention has been described above using general terms, the following examples are provided as illustrations rather than limitations. [Examples]

[0101] Example 1: Construction and generation of PABP and control proteins PABP was constructed by introducing a linker, i.e., (G4S)3, and / or a protease cleavage site into an existing DNA construct, in addition to the DNA encoding amino acids 1-27 of mature human CD3ε. For example, in the case of CD3ε(1-27)-aCD3-aHER2-Xbody, CD3ε(1-27)-MMP-2csV1-aCD3-aHER2-Xbody, CD3ε(1-27)-Furin csV1-aCD3-aHER2-Xbody, CD3ε(1-27)-MMP-2csV2-aCD3-aHER2-Xbody, CD3ε(1-27)-Furin csV2-aCD3-aHER2-Xbody, and CD3ε(1-27)-MMP-2csV3-aCD3-aHER2-Xbody, the existing DNA construct codes for a bispecific protein (referred to as aCD3-aHER2-Xbody) containing amino acid sequences SEQ ID NO: 6 and 93, which is described in international application PCT / US / 2014 / 026658, the relevant portion of which is incorporated herein by reference. The CD3ε fragment and the insert containing the linker and / or protease cleavage site were introduced by PCR using appropriate primers, and the construct was completed by Gibson assembly as described in Gibson et al. (2009), Nature Methods 6(5): 343-343. The portion of this reference describing how this method is carried out is incorporated herein by reference. Briefly, a double-stranded DNA fragment with a duplicate sequence at the end was incubated at 50°C with T5 exonuclease (which retracts the double-stranded DNA from the 5' end), PHUSION® DNA polymerase (New England Biolabs), and Taq ligase, and then E. coli was transformed using this to obtain colonies containing the DNA construct with the desired sequence.

[0102] DNA constructs encoding the PABPs CD3ε(1-27)-aCD3-aHER2-mxb, CD3ε(1-27)-MMP-2csV1-aCD3-aHER2-mxb, CD3ε(1-27)-MMP-2csV2-aCD3-aHER2-mxb, and CD3ε(1-27)-furin csV2-aCD3-aHER2-mxb were constructed in a similar manner, starting with the DNA construct encoding aCD3-aHER2-mxb, which contains amino acid sequences of SEQ ID NO: 20 and 94.

[0103] Similarly, DNA constructs encoding CD3ε(1-27)-aCD3-aHER2-BiFc, CD3ε(1-27)-MMP-cs-aCD3-aHER2-BiFc, and CD3ε(1-27)-furin cs-aCD3-aHER2-BiFc were constructed starting from the DNA construct encoding aCD3-aHER2-Bi-Fc, which contains the amino acid sequences of SEQ ID NO: 30 and 32.

[0104] Proteins were generated by transient transfection of HEK 293-6e cells, and the proteins were purified from the conditioned medium.

[0105] Example 2: The MMP cleavage site can be digested in vitro. To evaluate the cleavage of various PABPs by MMP-2, the proteins to be assayed were diluted to 100 ng / μl in phosphate-buffered saline (PBS) containing 30 μM ZnCl2. MMP-2 protease (Calbiochem (Cat#PF023)) was added to 20 μl (containing 2000 ng) of PABP-containing solution (0.5 μl at 0.1 mg / ml) and incubated overnight at 37°C. Subsequently, digested proteins from the protease reaction product (0.5 ul (50 ng)), along with undigested proteins, were loaded onto NUPAGE® NOVEX® 4-12% Bis-Tris gels (Life Technologies, Grand Island, New York) and electrophoresed under reducing conditions with MES buffer. The gels were transferred by Western blotting, and bispecific proteins were detected using horseradish peroxidase (HRP)-conjugated anti-human Fc antibody.

[0106] Figure 6 shows some of these results for constructs having the general form shown in Figure 3. The two polypeptide chains of each heterodimer PABP appear as two bands of similar size. Antibodies lacking the MMP2 cleavage site, some of which contain the furin cleavage site, do not change in size when digested with MMP2. See lanes 1 and 2, 5 and 6, and 9 and 10. In PABPs containing the MMP2 cleavage site, digestion with MMP2 reduces the size of one of the two polypeptide chains. See lanes 3 and 4, 7 and 8, and 11 and 12. In addition, PABPs containing the furin cleavage site are recovered from the conditioned medium as either a fully cleaved protein (CD3ε-furin csV2-aCD3-aHER2-Xbody; lanes 9 and 10) or a partially cleaved protein (CD3ε-furin csV1-aCD3-aHER2-Xbody; lanes 5 and 6). As is well known in the art, HEK-293 cells express furin proteases intracellularly, which have been observed to cleave recombinant proteins produced in HEK-293 cells. See, for example, Wu et al. (2003), J. Biol. Chem. 278: 25847-25852. These intracellular furins are likely responsible for the cleavage of PABPs containing furin cleavage sites.

[0107] Similar experiments were conducted to determine whether the MMP2 cleavage site in bispecific scFv-Fc PABP having the general form shown in Figure 4 can be cleaved in vitro. MMP2 digestion and gel electrophoresis were performed as described above. Most of the antibodies, with the exception of one containing a furin site (CD3ε-furin csV2-aCD3-aHER2-mxb; Figure 7, lanes 7 and 8), appeared as two distinct bands of similar size. CD3ε-furin csV2-aCD3-aHER2-mxb appeared as a single band, indicating that the furin cleavage site was cleaved. PABPs without an MMP2 cleavage site did not change in size upon MMP2 digestion. See Figure 7, lanes 1 and 2 and lanes 7 and 8. In antibodies containing an MMP2 site, the upper band became weaker upon MMP2 digestion, and the lower band became stronger compared to the upper band, suggesting that the MMP2 cleavage site was partially cleaved. Please refer to Figure 7, lanes 3 and 4, and lanes 5 and 6.

[0108] Using the PABPs described above, additional experiments were conducted to determine whether the MMP2 cleavage sites in these PABPs could be cleaved in vitro by MMP9. PABPs containing MMP2 cleavage sites were cleaved by digestion with MMP9. See Figure 8, lanes 3 and 4, 7 and 8, 11 and 12, 15 and 16, and 17 and 18. In addition, PABPs containing furin cleavage sites appeared to be at least partially cleaved by MMP9 (Figure 8, lanes 5 and 6), and some MMP9 digestion resulted in smaller bands (Figure 8, lanes 2, 4, 10, 14, 16, 18, and 20). These data suggest that MMP9 may be less selective than MMP2.

[0109] Example 3: Cell lysis activity of heterodimeric bispecific PABP and T cell activation by said PABP In the following experiments, we tested the in vitro cytolytic activity (T cell-dependent cell lysis (TDCC)) and their ability to activate T cells (measured as CD25 expression) of protease-digested and protease-undigested PABP having the general formula illustrated in Figure 3.

[0110] The TDCC assay used HER2-expressing tumor cells, specifically SKOV-3 cells, as target cells (Figures 9A, 10A, 11A, and 12A). SKOV-3 cells express approximately 530,000 molecules of HER2 protein per cell. Briefly, pan-T cells were isolated from healthy human donors using the Pan T Cell Isolation Kit II, human (Miltenyi Biotec, Auburn, CA). As shown in Figures 9A, 10A, 11A, and 12A, T cells were incubated with carboxyfluorescein succinimimidyl ester (CFSE)-labeled tumor target cells in a 10:1 ratio in the presence or absence of various concentrations of PABP. As negative controls, several samples contained T cells and tumor target cells but did not contain the bispecific protein.

[0111] After 40 hours of incubation, cells were harvested, and the lysis rate of tumor cells was monitored by uptake of 7-amino-actinomycin D (7-AAD), which stains double-stranded nucleic acids. While intact cells reject 7-AAD, 7-AAD can penetrate the membranes of dead or dying cells and stain the double-stranded nucleic acids inside these cells. Specific lysis rates were calculated according to the following formula: TIFF0007842528000014.tif10128

[0112] T cell activation was assessed based on CD25 expression by T cells. Pan T cells were isolated from healthy human donors using the Pan T Cell Isolation Kit II, human (Miltenyi Biotec, Auburn, CA). These T cells were incubated with PABP in the presence of HT-29 cells (tumor-derived cells expressing HER2) in a T cell:tumor cell ratio of 10:1. After 40 hours of incubation, non-adherent cells were removed from the wells. All samples were stained with allophycocyanin (APC)-conjugated anti-CD25 antibody, a marker of T cell activation, and analyzed by FACS.

[0113] Figures 9A and 9B show the results of positive control experiments for the TDCC assay and T cell activation assay of aCD3-aHER2-Xbody and aCD3-aHER2-mxb, respectively. These molecules have the general structure illustrated in Figures 3 and 4, respectively, except that they lack the CD3ε(1-27) peptide (component 3) and a linker containing a protease cleavage site that links it to the rest of the molecule. They are predicted to be activated without protease cleavage. Both molecules exhibit potent cytolytic activity against SKOV-3 cells, with an Ec50 of less than 1 ng / mL in this assay. See Figure 9A; Table 3 below. Furthermore, the addition of either molecule increased the percentage of activated T cells in a concentration-dependent manner. Figure 9B.

[0114] In further samples, anti-CD3ε / HER2 PABPs containing CD3ε(1-27) fragments were tested for cytolytic activity and T cell activation with and without digestion by MMP2. In Figures 10A, 10B, 11A, and 11B, all data are from assays using PABPs with the general structure shown in Figure 3 and identical amino acid sequences except for the linker that connects the CD3ε fragment to the rest of the molecule. PABPs are CD3ε(1-27)-MMP-2csV1-aCD3-aHER2-Xbody (linker containing an MMP2 cleavage site), CD3ε(1-27)-furin csV1-aCD3-aHER2-Xbody (linker containing a furin cleavage site), CD3ε(1-27)-aCD3-aHER2-Xbody (non-cleavable linker), CD3ε(1-27)-MMP-2csV2-aCD3-aHER2-Xbody (linker containing an MMP2 cleavage site), CD3ε(1-27)-furin csV2-aCD3-aHER2-Xbody (linker containing a furin cleavage site), and CD3ε(1-27)-MMP-2csV3-aCD3-aHER2-Xbody (linker containing an MMP2 cleavage site). Since these proteins were produced in HEK-293 cells that produce furin intracellularly, it is highly probable that CD3ε(1-27)-furin csV1-aCD3-aHER2-Xbody and CD3ε(1-27)-furin csV2-aCD3-aHER2-Xbody were cleaved during production by HEK-293 cells.

[0115] CD3ε(1-27)-furin csV1-aCD3-aHER2-Xbody and CD3ε(1-27)-furin csV2-aCD3-aHER2-Xbody, regardless of whether they were digested with MMP2, showed an E content of less than 1 ng / mL in the TDCC assay. CIt had 50. Figures 10A and 11A; Table 3. In contrast, CD3ε(1-27)-MMP-2csV1-aCD3-aHER2-Xbody, CD3ε(1-27)-MMP-2csV2-aCD3-aHER2-Xbody, and CD3ε(1-27)-MMP-2csV3-aCD3-aHER2-Xbody had higher E when not digested in MMP2. C It has a value of 50, and when digested with MMP2, the E content is less than 1 ng / mL. C It had 50. Figures 10A and 11A; Table 3. Consistent with the data shown in Figures 7 and 8, these data suggest that PABPs containing furin cleavage sites were cleaved by HEK-293 cells as predicted, and PABPs containing MMP2 cleavage sites were cleaved by MMP2 digestion. Uncleavable CD3ε(1-27)-aCD3-aHER2-Xbody E C E equivalent to 50 C The sample size was 50. In summary, these data strongly suggest that the presence of CD3ε(1-27) fragments reduced PABP activity in TDCC and T cell activation assays, and that the release of CD3ε fragments by protease digestion increased PABP's ability to induce TDCC and T cell activation.

[0116] (Table 3)E C 50 TIFF0007842528000015.tif48128

[0117] Example 4: Cell lysis activity of scFv-Fc PABP and T cell activation by said PABP TDCC assays and T cell activation assays were also performed on anti-HER2 / CD3 PABPs having the general structure shown in Figure 4. These PABPs had identical amino acid sequences except for the linker between the CD3ε fragment and the rest of the molecule, and these PABPs included: CD3ε(1-27)-MMP-2csV1-aCD3-aHER2-mxb (containing a linker with an MMP2 cleavage site), CD3ε(1-27)-MMP-2csV2-aCD3-aHER2-mxb (containing a linker with a different MMP2 cleavage site), CD3ε(1-27)-Furin csV2-aCD3-aHER2-mxb (containing a linker with a Furin cleavage site), and CD3ε(1-27)-aCD3-aHER2-mxb (containing a non-cleavable linker). The results are shown in Figures 12A and 12B.

[0118] PABP samples that remained uncleaved and were therefore expected to retain the CD3ε fragment showed low activity in the cell lysis assay and no detectable activity in the T cell activation assay. These samples included digested and undigested CD3ε(1-27)-aCD3-aHER2-mxb, as well as undigested CD3ε(1-27)-MMP-2csV1-aCD3-aHER2-mxb and CD3ε(1-27)-MMP-2csV2-aCD3-aHER2-mxb. Figures 12A and 12B. In contrast, PABP samples that were cleaved and therefore expected to lack the CD3ε fragment showed significantly higher activity in both assays. These samples included digested CD3ε(1-27)-MMP-2csV1-aCD3-aHER2-mxb and CD3ε(1-27)-MMP-2csV2-aCD3-aHER2-mxb, as well as digested and undigested CD3ε(1-27)-furin csV2-aCD3-aHER2-mxb. Figures 12A and 12B. These data suggest that the presence of CD3ε(1-27) fragments on these PABPs reduces PABP activity in TDCC assays and T cell activation assays, and that this activity can be restored by proteolytic cleavage removing the CD3ε(1-27) fragments.

[0119] Example 5: Binding and cytolytic activity of Bi-Fc PABP to T cells PABP in the form illustrated in Figure 2 was tested for binding to T cells and activity in TDCC assays. Cell lysis activity was determined as described in Example 3, except that the target cells were JIMT-1 cells expressing approximately 181,000 molecules of HER2 protein per cell. Binding to T cells was evaluated by fluorescence-activated cell sorting (FACS) analysis.

[0120] One PABP (CD3ε(1-27)-aCD3-aHER2-BiFc) contained a non-cleavable linker, one (CD3ε(1-27)-MMP-2cs-aCD3-aHER2-BiFc) contained an MMP2 cleavage site, and one (CD3ε(1-27)-furin cs-aCD3-aHER2-BiFc) contained a furin cleavage site, which was predicted to be cleaved intracellularly in the HEK-293 cells used to produce the protein. The control protein (aCD3-aHER2-BiFc) had the form shown in Figure 2, except that it did not contain a CD3ε fragment. This molecule was predicted to bind to T cells and have cytolytic activity. An anti-CD3 IgG antibody was used as a positive control in the binding assay, and a sample without the added protein was used as a negative control (binding data shown by lines 2 and 1 in Figure 13, respectively).

[0121] The data in Figure 13 shows that CD3ε(1-27)-furin cs-aCD3-aHER2-BiFc (line labeled 6) binds to T cells, as do the positive control aCD3-aHER2-BiFc (line labeled 3) and the anti-CD3 antibody (line labeled 2). CD3ε(1-27)-MMP-2cs-aCD3-aHER2-BiFc (line labeled 5) and CD3ε(1-27)-aCD3-aHER2-BiFc (line labeled 4) did not show binding. While CD3ε(1-27)-furin cs-aCD3-aHER2-BiFc was predicted to be cleaved, CD3ε(1-27)-MMP-2cs-aCD3-aHER2-BiFc and CD3ε(1-27)-aCD3-aHER2-BiFc were not predicted to be cleaved. Therefore, these data suggest that the release of CD3ε fragments by protease cleavage enabled binding to T cells.

[0122] Consistent with these results, the data in Figure 14 show that CD3ε(1-27)-furin cs-aCD3-aHER2-BiFc and aCD3-aHER2-BiFc (lines 6 and 3 in Figure 14, respectively) exhibit potent cytolytic activity, while CD3ε(1-27)-MMP-2cs-aCD3-aHER2-BiFc and CD3ε(1-27)-aCD3-aHER2-BiFc (lines 5 and 4 in Figure 14, respectively) exhibit considerably lower activity. These data suggest that the presence of CD3ε fragments can prevent these PABPs from binding to T cells, substantially inhibiting their cytolytic activity.

[0123] Array List SEQ ID NO:1 Amino acid sequence of the MMP-2 cleavage site TIFF0007842528000016.tif4128SEQ ID NO:2 Amino acid sequence of the MMP-2 cleavage site TIFF0007842528000017.tif4128SEQ ID NO:3 Amino acid sequence of the MMP-2 cleavage site TIFF0007842528000018.tif3128SEQ ID NO:4 Amino acid sequence of the furin cleavage site TIFF0007842528000019.tif3128SEQ ID NO:5 Amino acid sequence of the furin cleavage site TIFF0007842528000020.tif4128SEQ ID NO:6 Amino acid sequence (including signal sequence) of the first polypeptide chain of aCD3-aHER2-Xbody, CD3ε(1-27)-aCD3-aHER2-Xbody, CD3ε(1-27)-MMP-2csV1-aCD3-aHER2-Xbody, CD3ε(1-27)-MMP-2csV2-aCD3-aHER2-Xbody, CD3ε(1-27)-MMP-2csV3-aCD3-aHER2-Xbody, CD3ε(1-27)-furin csV1-aCD3-aHER2-Xbody, or CD3ε(1-27)-furin csV2-aCD3-aHER2-Xbody TIFF0007842528000021.tif81128SEQ ID NO:7 SEQ ID NO:6 encoding nucleic acid sequence TIFF0007842528000022.tif234114SEQ ID NO:8 Amino acid sequence of the second polypeptide chain of CD3ε(1-27)-aCD3-aHER2-Xbody (including signal sequence) TIFF0007842528000023.tif81128SEQ ID NO:9 Nucleic acid sequence encoding SEQ ID NO:8 TIFF0007842528000024.tif81128TIFF0007842528000025.tif145128SEQ ID NO:10 Amino acid sequence of the second polypeptide chain of CD3ε(1-27)-MMP-2csV1-aCD3-aHER2-Xbody TIFF0007842528000026.tif80128SEQ ID NO:11 Nucleic acid sequence encoding SEQ ID NO:10 TIFF0007842528000027.tif177128TIFF0007842528000028.tif69128SEQ ID NO:12 Amino acid sequence of the second polypeptide chain of CD3ε(1-27)-MMP-2csV2-aCD3-aHER2-Xbody TIFF0007842528000029.tif87128SEQ ID NO:13 Nucleic acid sequence encoding SEQ ID NO:12 TIFF0007842528000030.tif24128TIFF0007842528000031.tif228114SEQ ID NO:14 Amino acid sequence of the second polypeptide chain of CD3ε(1-27)-MMP-2csV3-aCD3-aHER2-Xbody TIFF0007842528000032.tif81128SEQ ID NO:15 Nucleic acid sequence encoding SEQ ID NO:14 TIFF0007842528000033.tif88128TIFF0007842528000034.tif158128SEQ ID NO:16 Amino acid sequence of the second polypeptide chain of CD3ε(1-27)-furin csV1-aCD3-aHER2-Xbody TIFF0007842528000035.tif80128SEQ ID NO:17 Nucleic acid sequence encoding SEQ ID NO:16 TIFF0007842528000036.tif164128TIFF0007842528000037.tif81128SEQ ID NO:18 Amino acid sequence of the second polypeptide chain of CD3ε(1-27)-furin csV2-aCD3-aHER2-Xbody TIFF0007842528000038.tif87128SEQ ID NO:19 Nucleic acid sequence encoding SEQ ID NO:18 TIFF0007842528000039.tif17128TIFF0007842528000040.tif222114TIFF0007842528000041.tif10128SEQ ID NO:20 Amino acid sequences of the first polypeptide chains of aCD3-aHER2-mxb, CD3ε(1-27)-aCD3-aHER2-mxb, CD3ε(1-27)-MMP-2csV1-aCD3-aHER2-mxb, and CD3ε(1-27)-MMP-2csV2-aCD3-aHER2-mxb TIFF0007842528000042.tif68128SEQ ID NO:21 Nucleic acid sequence encoding SEQ ID NO:20 TIFF0007842528000043.tif94128TIFF0007842528000044.tif100128SEQ ID NO:22 Amino acid sequence of the second polypeptide chain of CD3ε(1-27)-aCD3-aHER2-mxb TIFF0007842528000045.tif68128SEQ ID NO:23 Nucleic acid sequence encoding SEQ ID NO:22 TIFF0007842528000046.tif11128TIFF0007842528000047.tif196114SEQ ID NO:24 Amino acid sequence of the second polypeptide chain of CD3ε(1-27)-MMP-2csV1-aCD3-aHER2-mxb TIFF0007842528000048.tif74128SEQ ID NO:25 SEQ ID NO:24 encoding nucleic acid TIFF0007842528000049.tif132128TIFF0007842528000050.tif81128SEQ ID NO:26 Amino acid sequence of the second polypeptide chain of CD3ε(1-27)-MMP-2csV2-aCD3-aHER2-mxb TIFF0007842528000051.tif74128SEQ ID NO:27 Nucleic acid sequence encoding SEQ ID NO:26 TIFF0007842528000052.tif23128TIFF0007842528000053.tif190128SEQ ID NO:28 Amino acid sequence of the second polypeptide chain of CD3ε(1-27)-furin csV2-aCD3-aHER2-mxb TIFF0007842528000054.tif74128SEQ ID NO:29 Nucleic acid sequence encoding SEQ ID NO:28 TIFF0007842528000055.tif132128TIFF0007842528000056.tif81128SEQ ID NO:30 Amino acid sequences (with signal sequences) of the first polypeptide chains of aCD3-aHER2-Bi-Fc, CD3ε(1-27)-aCD3-aHER2-Bi-Fc, CD3ε(1-27)-MMP-2cs-aCD3-aHER2-Bi-Fc, and CD3ε(1-27)-furin cs-aCD3-aHER2-Bi-Fc TIFF0007842528000057.tif100128SEQ ID NO:31 Nucleic acid sequence encoding SEQ ID NO:30 TIFF0007842528000058.tif215114TIFF0007842528000059.tif75128SEQ ID NO:32 Amino acid sequence of the second polypeptide chain of aCD3-aHER2-Bi-Fc (with signal sequence) TIFF0007842528000060.tif28128SEQ ID NO:33 Nucleic acid sequence encoding SEQ ID NO:32 TIFF0007842528000061.tif78128SEQ ID NO:34 Amino acid sequence of the second polypeptide chain of CD3ε(1-27)-aCD3-aHER2-Bi-Fc (with signal sequence) Nucleic acid sequence encoding TIFF0007842528000062.tif36128SEQ ID NO:35 SEQ ID NO:34 TIFF0007842528000063.tif113128SEQ ID NO:36 Amino acid sequence of the second polypeptide chain of CD3ε(1-27)-MMP-2cs-aCD3-aHER2-Bi-Fc (with signal sequence) TIFF0007842528000064.tif35128SEQ ID NO:37 Nucleic acid sequence encoding SEQ ID NO:36 TIFF0007842528000065.tif113128SEQ ID NO:38 Amino acid sequence of the second polypeptide chain of CD3ε(1-27)-furin cs-aCD3-aHER2-Bi-Fc (with signal sequence) TIFF0007842528000066.tif36128SEQ ID NO:39 Nucleic acid sequence encoding SEQ ID NO:38 TIFF0007842528000067.tif113128SEQ ID NO:40 Amino acid sequence of the anti-CD3ε VH region TIFF0007842528000068.tif10160SEQ ID NO:41 Nucleic acid sequence encoding SEQ ID NO:40 TIFF0007842528000069.tif36160SEQ ID NO:42 Amino acid sequence of heavy chain CDR1 of SEQ ID NO:40 TIFF0007842528000070.tif3128SEQ ID NO:43 Amino acid sequence of heavy chain CDR2 of SEQ ID NO:40 TIFF0007842528000071.tif3128SEQ ID NO:44 Amino acid sequence of heavy chain CDR3 of SEQ ID NO:40 TIFF0007842528000072.tif3128SEQ ID NO:45 Amino acid sequence of the anti-CD3ε VL region TIFF0007842528000073.tif10160SEQ ID NO:46 Nucleic acid sequence encoding SEQ ID NO:45 TIFF0007842528000074.tif29160SEQ ID NO:47 SEQ ID NO:45 Light chain CDR1 amino acid sequence TIFF0007842528000075.tif3128SEQ ID NO:48 SEQ ID NO:45 Light chain CDR2 amino acid sequence Amino acid sequence of light chain CDR3 of TIFF0007842528000076.tif3128SEQ ID NO:49 SEQ ID NO:45 TIFF0007842528000077.tif3128SEQ ID NO:50 Amino acid sequence of mature human CD3ε TIFF0007842528000078.tif17148SEQ ID NO:51 Amino acid sequence of mature CD3ε in cynomolgus monkeys TIFF0007842528000079.tif20150SEQ ID NO:52 Amino acid sequence of the extracellular domain of human CD3ε TIFF0007842528000080.tif12148SEQ ID NO:53 Human CD3ε amino acids 1-27 TIFF0007842528000081.tif4128SEQ ID NO:54 Peptide sequence derived from human CD3ε TIFF0007842528000082.tif4128SEQ ID NO:55 Amino acid sequence of the meprin α or meprin β cleavage site TIFF0007842528000083.tif3128SEQ ID NO:56 Amino acid sequence of the meprin α or meprin β cleavage site TIFF0007842528000084.tif3128SEQ ID NO:57 Amino acid sequence of the meprine α or meprine β cleavage site TIFF0007842528000085.tif3128SEQ ID NO:58 Amino acid sequence of the meprin α or meprin β cleavage site TIFF0007842528000086.tif3128SEQ ID NO:59 Amino acid sequence of the u-PA cleavage site TIFF0007842528000087.tif3128SEQ ID NO:60 Amino acid sequence of the u-PA cleavage site TIFF0007842528000088.tif3128SEQ ID NO:61 Amino acid sequence of the u-PA cleavage site TIFF0007842528000089.tif3128SEQ ID NO:62 Amino acid sequence of the u-PA cleavage site SGRSS SEQ ID NO:63 Amino acid sequence of the u-PA cleavage site SGRRA SEQ ID NO:64 Amino acid sequence of the u-PA cleavage site TIFF0007842528000090.tif3128SEQ ID NO:65 Amino acid sequence of the u-PA cleavage site TIFF0007842528000091.tif3128SEQ ID NO:66 Amino acid sequence of the tPA cleavage site TIFF0007842528000092.tif4128SEQ ID NO:67 Amino acid sequence of the cathepsin B cleavage site TIFF0007842528000093.tif4128SEQ ID NO:68 Amino acid sequence of the cathepsin B cleavage site TIFF0007842528000094.tif3128SEQ ID NO:69 Amino acid sequence of the cathepsin B cleavage site TIFF0007842528000095.tif3128SEQ ID NO:70 Amino acid sequence of the cathepsin B cleavage site TIFF0007842528000096.tif3128SEQ ID NO:71 Amino acid sequence of the cathepsin B cleavage site TIFF0007842528000097.tif3128SEQ ID NO:72 Amino acid sequence of the cathepsin B cleavage site TIFF0007842528000098.tif3128SEQ ID NO:73 Amino acid sequence of the cathepsin B cleavage site TIFF0007842528000099.tif3128SEQ ID NO:74 Amino acid sequence of the cathepsin B cleavage site TIFF0007842528000100.tif3128SEQ ID NO:75 Amino acid sequence of the cathepsin B cleavage site LAAANP SEQ ID NO:76 Amino acid sequence of the cathepsin B cleavage site TIFF0007842528000101.tif4128SEQ ID NO:77 Amino acid sequence of the cathepsin B cleavage site TIFF0007842528000102.tif4128SEQ ID NO:78 Amino acid sequence of the cathepsin B cleavage site TIFF0007842528000103.tif3128SEQ ID NO:79 Amino acid sequence of the cathepsin B cleavage site TIFF0007842528000104.tif3128SEQ ID NO:80 Amino acid sequence of the cathepsin B cleavage site TIFF0007842528000105.tif3128SEQ ID NO:81 Amino acid sequence of the cathepsin B cleavage site TIFF0007842528000106.tif4128SEQ ID NO:82 Amino acid sequence of the furin cleavage site TIFF0007842528000107.tif3128SEQ ID NO:83 Amino acid sequence of a human fibronectin fragment TIFF0007842528000108.tif35144SEQ ID NO:84 Amino acid sequence of human IgG1 Fc polypeptide chain TIFF0007842528000109.tif94144SEQ ID NO:85 Amino acid sequence of human IgG2 Fc polypeptide chain TIFF0007842528000110.tif94144SEQ ID NO:86 Amino acid sequence of human IgG3 Fc polypeptide chain TIFF0007842528000111.tif113144SEQ ID NO:87 Amino acid sequence of human IgG4 Fc polypeptide chain TIFF0007842528000112.tif95144SEQ ID NO:88 Linker amino acid sequence TIFF0007842528000113.tif4128 (where n is any integer between 1 and 10) SEQ ID NO:89 Linker amino acid sequence TIFF0007842528000114.tif3128SEQ ID NO:90 Linker amino acid sequence TIFF0007842528000115.tif3128SEQ ID NO:91 Linker amino acid sequence TIFF0007842528000116.tif3128SEQ ID NO:92 Linker amino acid sequence TIFF0007842528000117.tif3128SEQ ID NO:93 Amino acid sequence of the second polypeptide of aCD3-aHER2-Xbody (excluding signal sequence) TIFF0007842528000118.tif95145TIFF0007842528000119.tif208143TIFF0007842528000120.tif164143SEQ ID NO:94 Amino acid sequence of the second polypeptide chain of aCD3-aHER2-mxb TIFF0007842528000121.tif62128TIFF0007842528000122.tif185128TIFF0007842528000123.tif72128

Claims

1. (a) A polypeptide that binds to a target cell and comprises a first pair (VH1 and VL1) of immunoglobulin heavy chain variable regions and light chain variable regions that bind to the target cell, (b) A polypeptide comprising a second pair (VH2 and VL2) of immunoglobulin heavy chain variable regions and light chain variable regions that bind to effector cells and human CD3ε, wherein the effector cells are T cells, and VH2 comprises heavy chains CDR1, CDR2, and CDR3 having amino acid sequences of SEQ ID NO: 42, 43, and 44, respectively, and VL2 comprises light chains CDR1, CDR2, and CDR3 having amino acid sequences of SEQ ID NO: 47, 48, and 49, respectively. (c) A polypeptide consisting of the first 27 amino acids of mature human CD3ε described in SEQ ID NO:50 that inhibits the binding of the protein to effector cells; and Linker containing a protease cleavage site that links the polypeptide of (d)(c) to the half-life extension portion. A protein containing, The first polypeptide chain of the protein comprises an amino acid sequence having the formula: VH1-L1-VL1-L2-VH2-L3-VL2-X1, where L1, L2, and L3 are linkers, L3 may or may not be present, and X1 is a half-life extension portion. The second polypeptide chain of the protein comprises an amino acid sequence having the formula: Y-L4-X2, where Y is the polypeptide of (c), L4 is a linker containing the protease cleavage site of (d), X2 is a half-life extension region linked by the linker of (d), and the first polypeptide chain and the second polypeptide chain are linked by a hinge region, and When the protease cleavage site is essentially completely cleaved, the protein binds to effector cells more effectively compared to the binding observed when the protease cleavage site is not cleaved. The aforementioned protein.

2. (a) A polypeptide that binds to a target cell and comprises a first pair (VH1 and VL1) of immunoglobulin heavy chain variable regions and light chain variable regions that bind to the target cell, (b) A polypeptide comprising a second pair (VH2 and VL2) of immunoglobulin heavy chain variable regions and light chain variable regions that bind to effector cells and human CD3ε, wherein the effector cells are T cells, and VH2 comprises heavy chains CDR1, CDR2, and CDR3 having amino acid sequences of SEQ ID NO: 42, 43, and 44, respectively, and VL2 comprises light chains CDR1, CDR2, and CDR3 having amino acid sequences of SEQ ID NO: 47, 48, and 49, respectively. (c) A polypeptide consisting of the first 27 amino acids of mature human CD3ε described in SEQ ID NO:50 that inhibits the cytolytic activity of the protein in a cell lysis assay; and Linker containing a protease cleavage site that links the polypeptide of (d)(c) to the half-life extension portion. A protein containing, The first polypeptide chain of the protein comprises an amino acid sequence having the formula: VH1-L1-VL1-L2-VH2-L3-VL2-X1, where L1, L2, and L3 are linkers, L3 may or may not be present, and X1 is a half-life extension portion. The second polypeptide chain of the protein comprises an amino acid sequence having the formula: Y-L4-X2, where Y is the polypeptide of (c), L4 is a linker containing the protease cleavage site of (d), X2 is a half-life extension region linked by the linker of (d), and the first polypeptide chain and the second polypeptide chain are linked by a hinge region, and When the protease cleavage site is essentially completely cleaved, the E of the protein in a cell lysis assay. C 50 is the E of the protein in the same assay when the protease cleavage site is not cleaved. C The value is less than 1 / 5 of 50, and the cell lysis assay is an assay for evaluating the lysis of target cells by immune effector cells to which the protease cleavage site is essentially completely cleaved. The aforementioned protein.

3. The protein according to claim 1 or 2, wherein VH2 and VL2 each contain amino acid sequences of SEQ ID NO: 40 and 45, respectively.

4. The protein according to any one of claims 1 to 3, wherein the protease-cleavable site can be cleaved by MMP-2, MMP-9, or MMP-11.

5. The sites where protease can be cleaved are, 【Chemistry 1】 The protein according to claim 4, comprising an amino acid sequence selected from the group consisting of the following.

6. The protein according to any one of claims 1 to 5, wherein X1 and X2 are Fc polypeptide chains.

7. The protein according to claim 6, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:30, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:36 or SEQ ID NO:

38.

8. The protein according to any one of claims 1 to 7, wherein the target cell is a cancer cell.

9. The protein according to claim 8, which, when VH1 and VL1 are part of an IgG or scFv antibody, binds to one of the following proteins: epidermal growth factor receptor (EGFR), EGFRvIII, melanoma-conjugated chondroitin sulfate proteoglycan (MCSP), mesothelin (MSLN), folate receptor 1 (FOLR1), CD33, CDH19, or epidermal growth factor 2 (HER2).

10. A nucleic acid encoding any of the proteins described in any one of claims 1 to 9.

11. A vector containing the nucleic acid described in claim 10.

12. A host cell containing the nucleic acid described in claim 10.

13. A method for producing the protein according to any one of claims 1 to 9, The steps include culturing host cells containing nucleic acids encoding the protein under conditions that allow the protein to be expressed, and Steps to recover the protein from the culture medium or cell population. The method, including the method described above.

14. A composition for treating cancer patients, comprising a therapeutically effective dose of the protein according to any one of claims 1 to 9.

15. A composition for treating patients suffering from infectious diseases, fibrotic diseases, neurodegenerative diseases, or autoimmune or inflammatory diseases, comprising a therapeutically effective dose of the protein described in any one of claims 1 to 6.

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