WO2019011167A1 - 双特异性重组蛋白 - Google Patents

双特异性重组蛋白 Download PDF

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WO2019011167A1
WO2019011167A1 PCT/CN2018/094447 CN2018094447W WO2019011167A1 WO 2019011167 A1 WO2019011167 A1 WO 2019011167A1 CN 2018094447 W CN2018094447 W CN 2018094447W WO 2019011167 A1 WO2019011167 A1 WO 2019011167A1
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seq
antibody
set forth
recombinant protein
scfv
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PCT/CN2018/094447
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English (en)
French (fr)
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薛彤彤
肖亮
郑勇
刘登念
崔亚敏
刘立平
王利春
王晶翼
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四川科伦博泰生物医药股份有限公司
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Priority to CN201880031493.1A priority Critical patent/CN110959015B/zh
Priority to CN202310772156.0A priority patent/CN117343192A/zh
Publication of WO2019011167A1 publication Critical patent/WO2019011167A1/zh

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Definitions

  • the present invention relates to the field of biomedicine, and in particular to the field of prevention and/or treatment of diseases associated with excessive TNF[alpha] and/or IL-6 activity.
  • the invention relates to recombinant proteins that specifically bind to TNF[alpha] and IL-6R, or TNF[alpha] and IL-6.
  • Autoimmune diseases as the third-biggest killer threatening human health after cardiovascular disease and cancer, have also been included in China's ten major diseases.
  • Autoimmune refers to the phenomenon that the body's immune system responds to the immune response of its own tissue cells. When the body's immune system responds to its own tissue cells and causes cell destruction or tissue damage and clinical symptoms, it is called an autoimmune disease. More than 30 autoimmune diseases have been discovered, including rheumatoid arthritis (RA), insulin-dependent diabetes mellitus, multiple sclerosis, lupus erythematosus, psoriasis, inflammatory bowel disease, ulcerative colitis, and severe disease.
  • RA rheumatoid arthritis
  • Myasthenia gravis polymyositis, dermatomyositis, Crohn's disease, autoimmune cytopenia, vasculitis, systemic lupus erythematosus, etc.
  • rheumatoid arthritis involves a variety of genetic and environmental factors, and the therapeutic effects are therefore different, making it very difficult to treat all patients with complete remission.
  • Many new drugs, especially biologics, used to treat RA help to bring about revolutionary advances in treatment.
  • TNF alpha tumor necrosis factor alpha
  • IL-6 interleukin-6
  • TNF ⁇ activates T cells and induces T cell infiltration and neovascularization, which leads to joint destruction of osteoclast formation by increasing proliferation of fibroblast-like synoviocytes (FLS).
  • FLS fibroblast-like synoviocytes
  • IL-6 causes B cell proliferation and production of antibodies, and induces differentiated T cells to secrete into IL-17 secreting T helper cells (Th17), thereby inhibiting regulatory T cell differentiation.
  • IL-6 stimulates angiogenesis and osteoclastogenesis.
  • IL-6 specifically binds to IL-6R.
  • IL-6 receptor There are two forms of IL-6 receptor: membrane-bound receptor (mIL-6R) and soluble receptor (sIL-6R).
  • IL-6 binds to IL-6R (including mIL-6R and sIL-6R) and forms an IL-6/IL-6R complex that binds to gp130 and initiates downstream IL-including JAK/STAT, ERK and PI3K. 6 signaling pathways, therefore, TNF ⁇ and IL-6 may together cause many pathogenic signals leading to RA.
  • IL-6 receptor IL-6 receptor
  • ACTEMRA monoclonal antibody drug tocilizumab
  • IL-6 pathway inhibitors including the marketed tocilizumab and the researched variety, regardless of whether the target is IL-6 or IL-6R, have comparable clinical effects and adverse reaction rates. There is also no significant difference. Many patients have insufficient response to TNF-inhibiting drugs or have a reduced effect after long-term use. Tocilizumab's multiple phase III clinical trials have shown that patients with insufficient TNF-inhibiting drug response or reduced therapeutic efficacy can achieve good therapeutic effects with tocilizumab.
  • Bispecific antibodies are a class of dual-homophilic combinatorial antibodies, usually bivalent (also tetravalent and hexavalent), ie, two antigen-binding arms with binding to two different specific antigens.
  • Chinese patent application CN102112495A discloses a bispecific antibody DVD279 and DVD280 which specifically bind to TNF ⁇ and IL-6R, which are sequentially linked at the N-terminus of antibodies CH1 and CL to TNF ⁇ and IL-6R, or to IL-6R and TNF ⁇ .
  • variable domain but its ratio of EC50 to the same antigen as the anti-TNF ⁇ parent antibody and the anti-IL-6R parent antibody is as high as 149.2 times (EC50 of the parent antibody against IL-6R is 1.42 nM, and DVD279 for IL-6R The EC50 was 211.9 nM) and 9.13 times (the EC50 of the parent antibody to TNF ⁇ was 0.44 nM, and the EC50 of DVD280 to TNF ⁇ was 4.02 nM), that is, the binding ability of the bispecific antibody was significantly worse than that of the parent antibody, indicating that the bispecificity
  • the binding of an antibody to one antigen (TNF ⁇ or IL-6R) significantly inhibits its binding to another antigen (IL-6R or TNF ⁇ ), and it is not possible to simultaneously inhibit TNF ⁇ and IL-6R efficiently, and it is difficult to ensure therapeutic effects.
  • the inventors have developed a recombinant protein which bispecifically binds TNF ⁇ and IL-6R, or TNF ⁇ and IL-6, a nucleic acid molecule encoding a recombinant protein, a vector containing a nucleic acid molecule, and a recombinant preparation by a large amount of research.
  • a method of protein, a pharmaceutical composition comprising the recombinant protein, a use of the pharmaceutical composition for the preparation of a medicament, a recombinant protein for diagnosing/treating/preventing a disease associated with excessive TNF ⁇ and IL-6 activity (for example, an inflammatory disease or itself) Use or method in an immunological disorder, and a kit comprising the recombinant protein.
  • the invention provides a recombinant protein comprising:
  • a first antibody that specifically binds to a first antigen comprising a heavy chain (HC) and a light chain (LC);
  • an antibody fragment eg, Fv, scFv, di-scFv
  • VH heavy chain variable region
  • VL light chain variable region
  • antibody fragment is linked to the N-terminus or C-terminus of the heavy or light chain of the first antibody
  • the first antigen is TNF ⁇ and the second antigen is IL-6R or IL-6; alternatively, the first antigen is IL-6R or IL-6 and the second antigen is TNF ⁇ .
  • the antibody fragment is a scFv.
  • the recombinant protein comprises 1 of the first antibody and 2 of the scFv; and, the first antibody comprises two HCs and two LCs, wherein the first antibody
  • the heavy chain variable region (VH) of one HC forms an antigen binding site with the light chain variable region (VL) of one LC
  • the heavy chain variable region (VH) of another HC is variable with the light chain of another LC
  • the region (VL) forms an antigen binding site.
  • each of the scFvs is linked to the N-terminus or C-terminus of the two heavy or two light chains of the first antibody, respectively.
  • each of the scFvs is linked to the N-terminus of the two heavy chains of the first antibody, respectively. In certain preferred embodiments, each of the scFvs is linked to the C-terminus of the two heavy chains of the first antibody, respectively.
  • each of the scFvs is linked to the N-terminus of the two light chains of the first antibody, respectively. In certain preferred embodiments, each of the scFvs is linked to the C-terminus of the two light chains of the first antibody, respectively.
  • one of the scFvs is linked to the N-terminus of the heavy or light chain of the first antibody, and the other of the scFvs is linked to the heavy or light chain of the first antibody. end.
  • the heavy chain of the first antibody comprises a heavy chain variable region (VH) and a CH1 domain, and the light chain comprises a light chain variable region (VL) and a light chain constant region (CL).
  • the first antibody can be a Fab fragment, a Fab' fragment or a F(ab') 2 fragment.
  • the heavy chain of the first antibody comprises a heavy chain variable region (VH) and a heavy chain constant region (CH), and the light chain comprises a light chain variable region (VL) and Light chain constant region (CL).
  • the first antibody can be a full length antibody.
  • the heavy chain of the first antibody is an IgG isotype, such as IgGl, IgG2, IgG3 or IgG4; preferably a human IgG isotype. In certain embodiments, the heavy chain of the first antibody is a human IgGl isotype. In certain preferred embodiments, the light chain of the first antibody is a Kappa isoform, preferably a human Kappa isoform.
  • the two HCs of the first antibody comprise the same CDR; and/or the two LCs of the first antibody comprise the same CDR.
  • the two HCs of the first antibody comprise the same VH; and/or the two LCs of the first antibody comprise the same VL.
  • the two HCs of the first antibody have the same amino acid sequence; and/or the two LCs of the first antibody have the same amino acid sequence.
  • the two scFvs have the same or different amino acid sequences. In certain preferred embodiments, the two scFvs have the same amino acid sequence.
  • the recombinant protein comprises two first polypeptide chains and two second polypeptide chains, wherein for each of the polypeptide chains:
  • said first polypeptide chains each independently comprise a heavy chain (HC) of said first antibody and said scFv;
  • said second polypeptide chain each independently comprising a light chain (LC) of said first antibody
  • scFv is linked to the N-terminus or C-terminus of the HC of the first antibody via a linker S1.
  • the recombinant protein comprises two first polypeptide chains and two second polypeptide chains, wherein for each of the polypeptide chains:
  • said first polypeptide chains each independently comprise a light chain (LC) of said first antibody and said scFv;
  • said second polypeptide chain each independently comprising a heavy chain (HC) of said first antibody
  • scFv is linked to the N-terminus or C-terminus of the LC of the first antibody via a linker S1.
  • the recombinant protein comprises two first polypeptide chains and two second polypeptide chains, wherein for each of the polypeptide chains:
  • said first polypeptide chains each independently comprise a heavy chain (HC) of said first antibody and said scFv;
  • said second polypeptide chain each independently comprising a light chain (LC) of said first antibody
  • scFv is linked to the N-terminus of the HC of the first antibody via a linker S1.
  • the N-terminus or C-terminus of the scFv is linked to the C-terminus or N-terminus of the linker S1.
  • the scFv has the structure: NH 2 -VH-S2-VL -COOH or NH 2 -VL-S2-VH- COOH, wherein said S2 is a linker.
  • the linker S1 and/or S2 is a peptide linker, for example having an amino acid sequence as shown by (G m S n ) x , wherein m, n are each independently selected from 1 to 8 An integer (eg, 1, 2, 3, 4, 5, 6, 7, or 8), x is independently selected from an integer from 1 to 20 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20).
  • the linker S1 and/or S2 has an amino acid sequence as shown by (G 4 S) x , and x is independently selected from an integer from 1 to 6.
  • the linker S1 and/or S2 has an amino acid sequence selected from the group consisting of SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35.
  • the linker S2 has an amino acid sequence as shown by (G 4 S) 4 , namely GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 33).
  • the linker S1 when the scFv is linked to the N-terminus of the heavy or light chain of the first antibody, the linker S1 has an amino acid sequence as shown by (G 4 S) 3 , ie GGGGSGGGGSGGGGS (SEQ ID NO: 34); when the scFv is linked to the C-terminus of the heavy or light chain of the first antibody, the linker S1 has an amino acid sequence as shown by (G 4 S) 2 , ie GGGGSGGGGS (SEQ ID NO: 35).
  • a disulfide bond is present between the VH and VL of the scFv.
  • Methods for introducing a disulfide bond between the VH and VL of an antibody are well known in the art, for example, see U.S. Patent Application No. 5,747,654; Rajagopal et al, Prot. Engin. 10 (1997) 1453-1459; Reiter et al.
  • the amino acid at position 44 of VH and the amino acid at position 100 of VL of the scFv are respectively cysteine, wherein the amino acid position referred to is according to the position of the Kabat numbering system;
  • the VH and VL of the scFv are linked by a disulfide bond formed between two cysteine residues at the 44th position of VH and the 100th position of VL, respectively.
  • the first antibody specifically binds to TNF[alpha] and the scFv specifically binds to IL-6R, wherein:
  • the first antibody comprises:
  • HCDR1 as shown in SEQ ID NO: 6; HCDR2 as shown in SEQ ID NO: 7; and HCDR3 as shown in SEQ ID NO: 8; LCDR1 as shown in SEQ ID NO: 2; LCDR2 shown in SEQ ID NO: 3; and LCDR3 as shown in SEQ ID NO: 4;
  • HCDR1 as shown in SEQ ID NO: 14; HCDR2 as shown in SEQ ID NO: 15; and HCDR3 as shown in SEQ ID NO: 16; LCDR1 as shown in SEQ ID NO: 10; LCDR2 shown in SEQ ID NO: 11; and LCDR3 as shown in SEQ ID NO: 12;
  • the scFv contains:
  • HCDR1 as shown in SEQ ID NO: 22; HCDR2 as shown in SEQ ID NO: 23; and HCDR3 as shown in SEQ ID NO: 24; LCDR1 as shown in SEQ ID NO: 18; LCDR2 shown in SEQ ID NO: 19; and LCDR3 as shown in SEQ ID NO: 20;
  • HCDR1 as shown in SEQ ID NO: 30; HCDR2 as shown in SEQ ID NO: 31; and HCDR3 as shown in SEQ ID NO: 32; LCDR1 as shown in SEQ ID NO: 26; LCDR2 shown in SEQ ID NO: 27; and LCDR3 as shown in SEQ ID NO: 28.
  • the first antibody comprises: HCDR1 as set forth in SEQ ID NO: 6; HCDR2 as set forth in SEQ ID NO: 7; and HCDR3 as set forth in SEQ ID NO: ; LCDR1 as shown in SEQ ID NO: 2; LCDR2 as shown in SEQ ID NO: 3; and LCDR3 as shown in SEQ ID NO: 4;
  • the scFv comprises: HCDR1 as set forth in SEQ ID NO: 22; HCDR2 as set forth in SEQ ID NO: 23; and HCDR3 as set forth in SEQ ID NO: 24; LCDR1 as set forth in SEQ ID NO: ; LCDR2 as shown in SEQ ID NO: 19; and LCDR3 as shown in SEQ ID NO: 20.
  • the first antibody comprises: HCDR1 as set forth in SEQ ID NO: 14; HCDR2 as set forth in SEQ ID NO: 15; and HCDR3 as set forth in SEQ ID NO: ; LCDR1 as shown in SEQ ID NO: 10; LCDR2 as shown in SEQ ID NO: 11; and LCDR3 as shown in SEQ ID NO: 12;
  • the scFv comprises: HCDR1 as set forth in SEQ ID NO: 30; HCDR2 as set forth in SEQ ID NO: 31; and HCDR3 as set forth in SEQ ID NO: 32; LCDR1 as set forth in SEQ ID NO: ; LCDR2 as shown in SEQ ID NO: 27; and LCDR3 as shown in SEQ ID NO: 28.
  • the first antibody comprises: HCDR1 as set forth in SEQ ID NO: 6; HCDR2 as set forth in SEQ ID NO: 7; and HCDR3 as set forth in SEQ ID NO: ; LCDR1 as shown in SEQ ID NO: 2; LCDR2 as shown in SEQ ID NO: 3; and LCDR3 as shown in SEQ ID NO: 4;
  • the scFv comprises: HCDR1 as set forth in SEQ ID NO: 30; HCDR2 as set forth in SEQ ID NO: 31; and HCDR3 as set forth in SEQ ID NO: 32; LCDR1 as set forth in SEQ ID NO: ; LCDR2 as shown in SEQ ID NO: 27; and LCDR3 as shown in SEQ ID NO: 28.
  • the first antibody comprises: HCDR1 as set forth in SEQ ID NO: 14; HCDR2 as set forth in SEQ ID NO: 15; and HCDR3 as set forth in SEQ ID NO: ; LCDR1 as shown in SEQ ID NO: 10; LCDR2 as shown in SEQ ID NO: 11; and LCDR3 as shown in SEQ ID NO: 12;
  • the scFv comprises: HCDR1 as set forth in SEQ ID NO: 22; HCDR2 as set forth in SEQ ID NO: 23; and HCDR3 as set forth in SEQ ID NO: 24; LCDR1 as set forth in SEQ ID NO: ; LCDR2 as shown in SEQ ID NO: 19; and LCDR3 as shown in SEQ ID NO: 20.
  • the recombinant protein comprises 1 of the first antibody and 2 of the scFv; and, the first antibody comprises two HCs and two LCs, wherein the first antibody The VH of one HC forms an antigen binding site with the VL of one LC, and the VH of the other HC forms an antigen binding site with the VL of the other LC; each of the scFvs is linked to the two heavy chains of the first antibody, respectively N-terminal; and, the first antibody comprises: HCDR1 as set forth in SEQ ID NO: 6; HCDR2 as set forth in SEQ ID NO: 7; and HCDR3 as set forth in SEQ ID NO: 8; LCDR1 shown as NO: 2; LCDR2 as shown in SEQ ID NO: 3; and LCDR3 as shown in SEQ ID NO: 4; and, the scFv comprises: HCDR1 as shown in SEQ ID NO: HCDR2 as shown in SEQ ID NO: 23; and HCDR3 as
  • each scFv is linked to the N-terminus of each heavy chain of the first antibody via a linker S1. More preferably, the scFv structure is NH 2 -VL-S2-VH- COOH, wherein said S2 is a linker.
  • the amino acid sequence of the heavy chain variable region (VH) of the first antibody has at least 90%, at least 91 of the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO:5. %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; and, the first antibody
  • the amino acid sequence of the light chain variable region (VL) has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO: 1. 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; and,
  • the amino acid sequence of the heavy chain variable region (VH) of the scFv has at least 90%, at least 91%, at least 92% of the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO: 21 or SEQ ID NO: 91 At least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; and, the light chain variable region of the scFv ( The amino acid sequence of VL) has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO: 17 or SEQ ID NO: 90. 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.
  • the heavy chain variable region of the first antibody is selected from the heavy chain variable region set forth in SEQ ID NO: 5; and the light chain variable region of the first antibody is selected a light chain variable region as shown in SEQ ID NO: 1;
  • the heavy chain variable region of the scFv is selected from the heavy chain variable region set forth in SEQ ID NO: 21 or SEQ ID NO: 91; and the light chain variable region of the scFv is selected from the group consisting of SEQ ID NO: 17. Or the light chain variable region set forth in SEQ ID NO:90.
  • the heavy chain variable region of the first antibody is selected from the heavy chain variable region set forth in SEQ ID NO: 13; and the light chain variable region of the first antibody Selected from the light chain variable region set forth in SEQ ID NO: 9;
  • the heavy chain variable region of the scFv is selected from the heavy chain variable region set forth in SEQ ID NO: 29 or SEQ ID NO: 93; and the light chain variable region of the scFv is selected from the group consisting of SEQ ID NO: Or the light chain variable region set forth in SEQ ID NO:92.
  • the heavy chain variable region of the first antibody is selected from the heavy chain variable region set forth in SEQ ID NO: 5; and, the light chain variable region of the first antibody Selected from the light chain variable region set forth in SEQ ID NO: 1;
  • the heavy chain variable region of the scFv is selected from the heavy chain variable region set forth in SEQ ID NO: 29 or SEQ ID NO: 93; and the light chain variable region of the scFv is selected from the group consisting of SEQ ID NO: Or the light chain variable region set forth in SEQ ID NO:92.
  • the heavy chain variable region of the first antibody is selected from the heavy chain variable region set forth in SEQ ID NO: 13; and the light chain variable region of the first antibody Selected from the light chain variable region set forth in SEQ ID NO: 9;
  • the heavy chain variable region of the scFv is selected from the heavy chain variable region set forth in SEQ ID NO: 21 or SEQ ID NO: 91; and the light chain variable region of the scFv is selected from the group consisting of SEQ ID NO: 17. Or the light chain variable region set forth in SEQ ID NO:90.
  • the first antibody comprises: VH as set forth in SEQ ID NO: 5 and VL as set forth in SEQ ID NO: 1;
  • the scFv includes:
  • VH as shown in SEQ ID NO: 21 and VL as shown in SEQ ID NO: 17;
  • the first antibody comprises a VH as set forth in SEQ ID NO: 5 and a VL as set forth in SEQ ID NO: 1; and wherein the scFv comprises as set forth in SEQ ID NO: 91 VH is shown and VL is shown as SEQ ID NO:90.
  • the first antibody comprises VH as set forth in SEQ ID NO: 13 and VL as set forth in SEQ ID NO: 9;
  • the scFv includes:
  • VH as shown in SEQ ID NO: 29 and VL as shown in SEQ ID NO: 25;
  • VH as shown in SEQ ID NO: 93 and VL as shown in SEQ ID NO: 92.
  • the first antibody comprises a VH as set forth in SEQ ID NO: 5 and a VL as set forth in SEQ ID NO: 1; and wherein the scFv comprises:
  • VH as shown in SEQ ID NO: 29 and VL as shown in SEQ ID NO: 25;
  • VH as shown in SEQ ID NO: 93 and VL as shown in SEQ ID NO: 92.
  • the first antibody comprises a VH as set forth in SEQ ID NO: 13 and a VL as set forth in SEQ ID NO: 9; and wherein the scFv comprises:
  • VH as shown in SEQ ID NO: 21 and VL as shown in SEQ ID NO: 17;
  • the first antibody specifically binds to IL-6R and the scFv specifically binds to TNF ⁇ , wherein:
  • the first antibody comprises:
  • HCDR1 as shown in SEQ ID NO: 22; HCDR2 as shown in SEQ ID NO: 23; and HCDR3 as shown in SEQ ID NO: 24; LCDR1 as shown in SEQ ID NO: 18; LCDR2 shown in SEQ ID NO: 19; and LCDR3 as shown in SEQ ID NO: 20;
  • HCDR1 as shown in SEQ ID NO: 30; HCDR2 as shown in SEQ ID NO: 31; and HCDR3 as shown in SEQ ID NO: 32; LCDR1 as shown in SEQ ID NO: 26; LCDR2 shown in SEQ ID NO: 27; and LCDR3 as shown in SEQ ID NO: 28;
  • the scFv contains:
  • HCDR1 as shown in SEQ ID NO: 6; HCDR2 as shown in SEQ ID NO: 7; and HCDR3 as shown in SEQ ID NO: 8; LCDR1 as shown in SEQ ID NO: 2; LCDR2 shown in SEQ ID NO: 3; and LCDR3 as shown in SEQ ID NO: 4;
  • HCDR1 as shown in SEQ ID NO: 14; HCDR2 as shown in SEQ ID NO: 15; and HCDR3 as shown in SEQ ID NO: 16; LCDR1 as shown in SEQ ID NO: 10; LCDR2 shown in SEQ ID NO: 11; and LCDR3 as shown in SEQ ID NO: 12.
  • the first antibody comprises: HCDR1 as set forth in SEQ ID NO: 22; HCDR2 as set forth in SEQ ID NO: 23; and HCDR3 as set forth in SEQ ID NO: ; LCDR1 as shown in SEQ ID NO: 18; LCDR2 as shown in SEQ ID NO: 19; and LCDR3 as shown in SEQ ID NO: 20;
  • the scFv comprises: HCDR1 as shown in SEQ ID NO: 6; HCDR2 as shown in SEQ ID NO: 7; and HCDR3 as shown in SEQ ID NO: 8; LCDR1 as shown in SEQ ID NO: ; LCDR2 as shown in SEQ ID NO: 3; and LCDR3 as shown in SEQ ID NO: 4.
  • the first antibody comprises: HCDR1 as set forth in SEQ ID NO: 30; HCDR2 as set forth in SEQ ID NO: 31; and HCDR3 as set forth in SEQ ID NO: ; LCDR1 as shown in SEQ ID NO: 26; LCDR2 as shown in SEQ ID NO: 27; and LCDR3 as shown in SEQ ID NO: 28;
  • the scFv comprises: HCDR1 as set forth in SEQ ID NO: 14; HCDR2 as set forth in SEQ ID NO: 15; and HCDR3 as set forth in SEQ ID NO: 16; LCDR1 as set forth in SEQ ID NO: ; LCDR2 as shown in SEQ ID NO: 11; and LCDR3 as shown in SEQ ID NO: 12.
  • the first antibody comprises: HCDR1 as set forth in SEQ ID NO: 22; HCDR2 as set forth in SEQ ID NO: 23; and HCDR3 as set forth in SEQ ID NO: ; LCDR1 as shown in SEQ ID NO: 18; LCDR2 as shown in SEQ ID NO: 19; and LCDR3 as shown in SEQ ID NO: 20;
  • the scFv comprises: HCDR1 as set forth in SEQ ID NO: 14; HCDR2 as set forth in SEQ ID NO: 15; and HCDR3 as set forth in SEQ ID NO: 16; LCDR1 as set forth in SEQ ID NO: ; LCDR2 as shown in SEQ ID NO: 11; and LCDR3 as shown in SEQ ID NO: 12.
  • the first antibody comprises: HCDR1 as set forth in SEQ ID NO: 30; HCDR2 as set forth in SEQ ID NO: 31; and HCDR3 as set forth in SEQ ID NO: ; LCDR1 as shown in SEQ ID NO: 26; LCDR2 as shown in SEQ ID NO: 27; and LCDR3 as shown in SEQ ID NO: 28;
  • the scFv comprises: HCDR1 as shown in SEQ ID NO: 6; HCDR2 as shown in SEQ ID NO: 7; and HCDR3 as shown in SEQ ID NO: 8; LCDR1 as shown in SEQ ID NO: ; LCDR2 as shown in SEQ ID NO: 3; and LCDR3 as shown in SEQ ID NO: 4.
  • the amino acid sequence of the heavy chain variable region (VH) of the first antibody has at least 90%, at least 91 of the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO:21. %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; and, the first antibody
  • the amino acid sequence of the light chain variable region (VL) has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, and the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO:17. At least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; and,
  • the amino acid sequence of the heavy chain variable region (VH) of the scFv has at least 90%, at least 91%, at least 92% of the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO: 5 or SEQ ID NO: 87 At least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; and, the light chain variable region of the scFv ( The amino acid sequence of VL) has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO: 1 or SEQ ID NO: 86. 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.
  • the heavy chain variable region of the first antibody is selected from the heavy chain variable region set forth in SEQ ID NO: 21; and the light chain variable region of the first antibody Selected from the light chain variable region set forth in SEQ ID NO: 17;
  • the heavy chain variable region of the scFv is selected from the heavy chain variable region set forth in SEQ ID NO: 5 or SEQ ID NO: 87; and the light chain variable region of the scFv is selected from the group consisting of SEQ ID NO: 1. Or the light chain variable region set forth in SEQ ID NO:86.
  • the heavy chain variable region of the first antibody is selected from the heavy chain variable region set forth in SEQ ID NO: 29; and, the light chain variable region of the first antibody Selected from the light chain variable region set forth in SEQ ID NO: 25;
  • the heavy chain variable region of the scFv is selected from the heavy chain variable region set forth in SEQ ID NO: 13 or SEQ ID NO: 89; and the light chain variable region of the scFv is selected from the group consisting of SEQ ID NO: 9 Or the light chain variable region set forth in SEQ ID NO:88.
  • the heavy chain variable region of the first antibody is selected from the heavy chain variable region set forth in SEQ ID NO: 29; and, the light chain variable region of the first antibody Selected from the light chain variable region set forth in SEQ ID NO: 25;
  • the heavy chain variable region of the scFv is selected from the heavy chain variable region set forth in SEQ ID NO: 5 or SEQ ID NO: 87; and the light chain variable region of the scFv is selected from the group consisting of SEQ ID NO: 1. Or the light chain variable region set forth in SEQ ID NO:86.
  • the heavy chain variable region of the first antibody is selected from the heavy chain variable region set forth in SEQ ID NO: 21; and the light chain variable region of the first antibody Selected from the light chain variable region set forth in SEQ ID NO: 17;
  • the heavy chain variable region of the scFv is selected from the heavy chain variable region set forth in SEQ ID NO: 13 or SEQ ID NO: 89; and the light chain variable region of the scFv is selected from the group consisting of SEQ ID NO: 9 Or the light chain variable region set forth in SEQ ID NO:88.
  • the first antibody comprises: VH as set forth in SEQ ID NO: 21 and VL as set forth in SEQ ID NO: 17;
  • the scFv includes:
  • VH as shown in SEQ ID NO: 5 and VL as shown in SEQ ID NO: 1;
  • the first antibody comprises a VH as set forth in SEQ ID NO: 21 and a VL as set forth in SEQ ID NO: 17; and wherein the scFv comprises as set forth in SEQ ID NO: 87 VH is shown and VL is shown as SEQ ID NO:86.
  • the first antibody comprises: a VH as set forth in SEQ ID NO: 29 and a VL as set forth in SEQ ID NO: 25; and wherein the scFv comprises:
  • VH as shown in SEQ ID NO: 13 and VL as shown in SEQ ID NO: 9;
  • the first antibody comprises a VH as set forth in SEQ ID NO: 21 and a VL as set forth in SEQ ID NO: 17; and wherein the scFv comprises as set forth in SEQ ID NO: 87 VH is shown and VL is shown as SEQ ID NO:86.
  • the first antibody comprises VH as set forth in SEQ ID NO: 29 and VL as set forth in SEQ ID NO: 25;
  • the scFv includes:
  • VH as shown in SEQ ID NO: 5 and VL as shown in SEQ ID NO: 1;
  • the first antibody comprises VH as set forth in SEQ ID NO: 21 and VL as set forth in SEQ ID NO: 17;
  • the scFv includes:
  • VH as shown in SEQ ID NO: 13 and VL as shown in SEQ ID NO: 9;
  • the first antibody comprises:
  • the recombinant protein comprises two identical first polypeptide chains and two identical second polypeptide chains.
  • the first polypeptide chain has an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 38, 44, 46, 50, 52, 58, 60, 64, 66, 70, An amino acid sequence as set forth in any one of 76, 94, 95, 96, 97, 98 and 99; and/or said second polypeptide chain has an amino acid sequence selected from the group consisting of SEQ ID NOs: 42, 48, 56 The amino acid sequences shown in any of 68, 74 and 80.
  • the recombinant protein comprises:
  • a recombinant protein of the invention has antibody-dependent cell-mediated cytotoxicity (ADCC) activity that is comparable to a parent antibody of the first antibody.
  • the recombinant protein of the invention has antibody-dependent cell-mediated cytotoxicity (ADCC) activity comparable to that of the parent antibody of the first antibody, and further has The parent antibody is equivalent to complement-dependent cytotoxicity (CDC) activity.
  • the recombinant protein of the invention has an affinity for TNF ⁇ and IL-6R.
  • the recombinant proteins of the invention have equal or higher affinity for TNF[alpha] and IL-6R than their respective parent antibodies.
  • the recombinant proteins of the invention have a higher affinity for TNF[alpha] and IL-6R on the cell surface than their respective parent antibodies.
  • the recombinant protein of the invention has good thermal stability. In certain preferred embodiments, the recombinant proteins of the invention have substantially the same thermal stability as compared to the parent antibody.
  • the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a recombinant protein of the invention.
  • the isolated nucleic acid molecule encodes a recombinant protein of the invention.
  • the isolated nucleic acid molecule comprises a nucleotide sequence encoding a first polypeptide chain of the invention. In certain preferred embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a second polypeptide chain of the invention. In certain preferred embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a first polypeptide chain of the invention and a nucleotide sequence encoding a second polypeptide chain of the invention.
  • the invention provides a vector (eg, a cloning vector or an expression vector) comprising an isolated nucleic acid molecule of the invention.
  • a vector eg, a cloning vector or an expression vector
  • the vector comprises a nucleotide sequence encoding a first polypeptide chain of the invention. In certain preferred embodiments, the vector comprises a nucleotide sequence encoding a second polypeptide chain of the invention. In certain preferred embodiments, the vector comprises a nucleotide sequence encoding a first polypeptide chain of the invention and a nucleotide sequence encoding a second polypeptide chain of the invention.
  • the vectors of the invention are, for example, plasmids, cosmids, phages, and the like.
  • the vector is capable of expressing a recombinant protein, a first polypeptide chain, or a second polypeptide chain of the invention in a subject (eg, a mammal, eg, a human).
  • the invention provides a host cell comprising an isolated nucleic acid molecule of the invention or a vector of the invention.
  • host cells include, but are not limited to, prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (eg, mammalian cells, such as mouse cells, human cells, etc.).
  • the host cell of the invention is a mammalian cell, such as CHO (eg, CHO-K1, CHO-S, CHO DG44) or HEK293.
  • the invention provides a method of making a recombinant protein of the invention, comprising culturing a host cell of the invention under conditions permitting expression of the recombinant protein, and recovering from the cultured host cell culture Recombinant protein.
  • the method comprises:
  • an expression vector comprising a nucleotide sequence encoding a first polypeptide chain and a nucleotide sequence encoding a second polypeptide chain; or constructing a first nucleotide sequence comprising a first polypeptide chain
  • An expression vector and a second expression vector comprising a nucleotide sequence encoding a second polypeptide chain;
  • the recombinant protein of the present invention can be used to inhibit the activity of TNF ⁇ and IL-6/IL-6R, block the TNF ⁇ and/or IL-6 signaling pathway, and prevent and/or treat and overexpress in vitro or in a subject.
  • a disease associated with TNF ⁇ and/or IL-6 activity eg, an inflammatory disease or an autoimmune disease.
  • the invention provides a pharmaceutical composition comprising a recombinant protein of the invention, and a pharmaceutically acceptable carrier and/or excipient.
  • the pharmaceutical composition may also comprise additional pharmaceutically active agents.
  • the additional pharmaceutically active agent is for use in preventing and/or treating a disease associated with excessive TNF ⁇ and/or IL-6 activity (eg, an inflammatory disease or an autoimmune disease) a drug, such as an anti-inflammatory drug or an immunosuppressive agent, such as a non-steroidal anti-inflammatory drug (eg, ibuprofen, diclofenac, naproxen, indomethacin, piroxicam, meloxicam, nabumetone or nimese) Shuli), steroidal anti-inflammatory drugs (such as prednisone, dexamethasone or hydrocortisone), antibodies or antagonists of inflammatory cytokines (eg, TNF ⁇ , IL-1, IL-6,
  • a drug such as an anti-inflammatory drug or an immuno
  • the invention provides the use of a recombinant protein of the invention or a pharmaceutical composition of the invention for the preparation of a medicament for use in the prevention and/or treatment of a subject (eg, a human)
  • a disease associated with TNF ⁇ and/or IL-6 activity eg, an inflammatory disease or an autoimmune disease
  • TNF ⁇ and IL-6/IL-6R in vitro or in a subject (eg, a human) active.
  • the invention provides a method for preventing and/or treating a disease associated with excessive TNF ⁇ and/or IL-6 activity (eg, inflammatory disease or autoimmune in a subject (eg, a human) A disease, and/or a method for inhibiting the activity of TNF ⁇ and IL-6/IL-6R in vitro or in a subject (eg, a human), wherein the method comprises administering an effective to a subject in need thereof A quantity of the recombinant protein of the invention, or a pharmaceutical composition of the invention.
  • a disease associated with excessive TNF ⁇ and/or IL-6 activity eg, inflammatory disease or autoimmune in a subject (eg, a human) A disease
  • a method for inhibiting the activity of TNF ⁇ and IL-6/IL-6R in vitro or in a subject eg, a human
  • the method comprises administering an effective to a subject in need thereof A quantity of the recombinant protein of the invention, or a pharmaceutical composition of the invention.
  • the diseases associated with excessive TNF ⁇ and/or IL-6 activity include, but are not limited to, inflammatory diseases or autoimmune diseases such as rheumatoid arthritis, insulin-dependent diabetes mellitus, multiple sclerosis Symptoms, psoriasis, inflammatory bowel disease, ulcerative colitis, pancreatitis, myasthenia gravis, polymyositis, dermatomyositis, Crohn's disease, autoimmune cytopenia, vasculitis, systemic lupus erythematosus or adult Still sick and so on.
  • inflammatory diseases or autoimmune diseases such as rheumatoid arthritis, insulin-dependent diabetes mellitus, multiple sclerosis Symptoms, psoriasis, inflammatory bowel disease, ulcerative colitis, pancreatitis, myasthenia gravis, polymyositis, dermatomyositis, Crohn's disease, autoimmune cytopenia, vasculitis, systemic
  • the recombinant protein of the present invention or the pharmaceutical composition of the present invention may be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, Tinctures, lozenges, suppositories, injections (including injections, sterile powder for injection and concentrated solutions for injection), inhalants, sprays, and the like.
  • the preferred dosage form will depend on the intended mode of administration and therapeutic use.
  • the pharmaceutical compositions of the invention should be sterile and stable under the conditions of manufacture and storage.
  • a preferred dosage form is an injectable. Such an injection may be a sterile injectable solution.
  • a sterile injectable solution can be prepared by incorporating the necessary amount of the recombinant protein of the present invention in a suitable solvent, and optionally, incorporating other desired ingredients (including, but not limited to, pH adjustment) A surfactant, a surfactant, an adjuvant, an ionic strength enhancer, an isotonicity agent, a preservative, a diluent, or any combination thereof, followed by filtration sterilization.
  • sterile injectable solutions can be prepared as a sterile lyophilized powder (for example, by vacuum drying or freeze drying) for ease of storage and use. Such sterile lyophilized powders can be dispersed in a suitable vehicle, such as sterile pyrogen-free water, before use.
  • the recombinant protein of the present invention may be present in a pharmaceutical composition in unit dosage form for ease of administration.
  • the unit dosage is at least 1 mg, at least 5 mg, at least 10 mg, at least 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 45 mg, at least 50 mg, at least 75 mg, or at least 100 mg.
  • the pharmaceutical composition is in the form of a liquid (eg, injection) dosage form, it may comprise a concentration of at least 0.1 mg/ml, such as at least 0.25 mg/ml, at least 0.5 mg/ml, at least 1 mg/ml, at least 2.5 mg.
  • /ml at least 5 mg/ml, at least 8 mg/ml, at least 10 mg/ml, at least 15 mg/ml, at least 25 mg/ml, at least 50 mg/ml, at least 75 mg/ml, or at least 100 mg/ml of the recombinant protein of the invention.
  • the recombinant protein or pharmaceutical composition of the invention may be administered by any suitable method known in the art including, but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic. In the trough, in the groin, intravesical, local (eg, powder, ointment or drops), or nasal route.
  • the preferred route/mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular).
  • the route and/or manner of administration will vary depending on the intended purpose.
  • the recombinant protein or pharmaceutical composition of the invention is administered by intravenous infusion or injection.
  • the medicament, pharmaceutical composition or recombinant protein provided by the present invention may be used singly or in combination, or may be used in combination with another pharmaceutically active agent such as an anti-inflammatory drug or an immunosuppressive agent.
  • the recombinant proteins of the invention are used in combination with other anti-inflammatory drugs or immunosuppressive agents to prevent and/or treat diseases associated with excessive TNF[alpha] and/or IL-6 activity (eg, inflammation) sexual or autoimmune disease).
  • additional pharmaceutically active agents can be administered prior to, concurrently with, or subsequent to administration of the recombinant protein of the invention or the pharmaceutical composition of the invention.
  • compositions of the invention may comprise a "therapeutically effective amount” or a “prophylactically effective amount” of a recombinant protein of the invention.
  • prophylactically effective amount is meant an amount sufficient to prevent, arrest, or delay the onset of a disease, such as a disease associated with excessive TNF[alpha] and/or IL-6 activity.
  • therapeutically effective amount is meant an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease.
  • the therapeutically effective amount of the recombinant protein of the present invention may vary depending on factors such as the severity of the disease to be treated, the overall state of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and Other treatments administered at the same time, and the like.
  • the dosage regimen can be adjusted to achieve the best purpose response (e.g., therapeutic or prophylactic response).
  • the best purpose response e.g., therapeutic or prophylactic response
  • it may be administered in a single administration, may be administered multiple times over a period of time, or may be proportionally reduced or increased depending on the urgency of the treatment.
  • a typical non-limiting range of therapeutically or prophylactically effective amounts of the recombinant protein of the invention is from 0.02 to 50 mg/kg, such as from 0.1 to 50 mg/kg, from 0.1 to 25 mg/kg, or from 1 to 10 mg/kg. It should be noted that the dosage may vary depending on the type and severity of the condition to be treated. Moreover, those skilled in the art understand that for any particular patient, the particular dosage regimen should be adjusted over time according to the needs of the patient and the professional evaluation of the physician; the dosage ranges given herein are for illustrative purposes only and are not limiting Use or range of the pharmaceutical compositions of the invention.
  • the subject may be a mammal, such as a human.
  • the recombinant protein of the present invention is capable of specifically binding to TNF ⁇ /IL-6 or TNF ⁇ /IL-6R, thereby being useful for detecting the presence or level of TNF ⁇ /IL-6 or TNF ⁇ /IL-6R in a sample, and diagnosing a subject Whether it has a disease associated with excessive TNF ⁇ and/or IL-6 activity (eg, an inflammatory disease or an autoimmune disease).
  • a disease associated with excessive TNF ⁇ and/or IL-6 activity eg, an inflammatory disease or an autoimmune disease.
  • the invention provides a kit comprising the recombinant protein of the invention.
  • the recombinant proteins of the invention carry a detectable label.
  • the kit further comprises a second antibody that specifically recognizes the first antibody or scFv of the recombinant protein of the invention.
  • the second antibody further comprises a detectable label.
  • the detectable label may be any substance detectable by fluorescence, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. It is particularly preferred that such markers are suitable for use in immunological assays (eg, enzyme-linked immunoassays, radioimmunoassays, fluorescent immunoassays, chemiluminescent immunoassays, etc.).
  • immunological assays eg, enzyme-linked immunoassays, radioimmunoassays, fluorescent immunoassays, chemiluminescent immunoassays, etc.
  • Such labels include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.), radionuclides ( For example, 3 H, 125 I, 35 S, 14 C or 32 P), fluorescent dyes (eg, fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), algae Red protein (PE), Texas Red, rhodamine, quantum dot or cyanine dye derivatives (eg Cy7, Alexa 750), luminescent substances (eg chemiluminescent substances such as acridine esters), magnetic beads (E.g, a heat-sensitive marker such as colloidal gold or colored glass or plastic (for example, polystyrene, polypropylene, latex, etc.) beads, and avidin modified for binding to the above
  • enzymes e
  • markers that teach the use of such markers include, but are not limited to, U.S. Patent Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241 each incorporated herein by reference.
  • the markers encompassed in the present invention can be detected by methods known in the art.
  • the radioactive label can be detected using a photographic film or a scintillation counter
  • the fluorescent label can be detected using a photodetector to detect the emitted light.
  • Enzyme labels are typically detected by providing a substrate for the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, and the calorimetric label is detected by simply visualizing the stained label.
  • a detectable label as described above can be linked to a recombinant protein of the invention by a linker of varying length to reduce potential steric hindrance.
  • the invention provides a method of detecting the presence or level of TNF ⁇ /IL-6 or TNF ⁇ /IL-6R in a sample comprising the step of using a recombinant protein of the invention.
  • the recombinant protein of the invention also carries a detectable label.
  • the method further comprises detecting the recombinant protein segment of the invention using a reagent with a detectable label. The method can be used for diagnostic purposes, or for non-diagnostic purposes (eg, the sample is a cell sample, not a sample from a patient).
  • the invention provides a method of diagnosing whether a subject has a disease associated with excessive TNF ⁇ and/or IL-6 activity, such as an inflammatory disease or an autoimmune disease, comprising: using the invention
  • the recombinant protein detects the presence or level of TNF ⁇ and/or IL-6 in a sample from the subject.
  • the recombinant protein of the invention also carries a detectable label.
  • the method further comprises the step of detecting the recombinant protein of the invention using a reagent with a detectable label.
  • a recombinant protein of the invention in a kit for detecting the presence or level of TNF ⁇ and/or IL-6 in a sample, or for diagnosing a subject Whether it has a disease associated with excessive TNF ⁇ and/or IL-6 activity (eg, an inflammatory disease or an autoimmune disease).
  • a disease associated with excessive TNF ⁇ and/or IL-6 activity eg, an inflammatory disease or an autoimmune disease.
  • antibody refers to an immunoglobulin molecule that is typically composed of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC).
  • Antibody light chains can be classified as kappa (kappa) and lambda (lambda) light chains.
  • Heavy chains can be classified as ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ , and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chain further comprises a "D" region of about 3 or more amino acids.
  • Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region consists of three domains (CH1, CH2 and CH3).
  • Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region consists of one domain CL.
  • the constant region of the antibody mediates binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (C1q) of the classical complement system.
  • the VH and VL regions can also be subdivided into regions with high denaturation (referred to as complementarity determining regions (CDRs)) interspersed with more conserved regions called framework regions (FR).
  • CDRs complementarity determining regions
  • VH and VL each heavy/light chain pair form an antigen binding site, respectively.
  • the assignment of amino acids to regions or domains follows Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901- 917; Chothia et al. (1989) Nature 342: 878-883.
  • CDR complementarity determining region
  • LCDR1 ⁇ , 50-56 ⁇ LCDR2 ⁇ , 89-97 ⁇ LCDR3 ⁇ , and residues 31-35 ⁇ HCDR1 ⁇ , 50-65 ⁇ HCDR2 ⁇ , 95-102 ⁇ HCDR3 ⁇ in the heavy chain variable region see, for example, Kabat et al, Sequences of Proteins of lmmunological lnterest, Fifth Edition, Public Health Service, National Institutes of Health, Bethesda, Maryland (1991), or residues in the light chain variable region 26-32 ⁇ Ll ⁇ , 50-52 ⁇ L2 ⁇ , 91-96 ⁇ L3 ⁇ and residues 26-32 ⁇ H1 ⁇ , 53-55 ⁇ H2 ⁇ , 96-101 ⁇ H3 ⁇ in the heavy chain variable region (see, Chothia) And Lesk J. Mol. Bio
  • framework region or "FR” residue refers to those amino acid residues in the variable regions of an antibody other than the CDR residues as defined above.
  • antibody is not limited by any particular method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies.
  • the antibodies may be antibodies of different isotypes, for example, IgG (eg, IgGl, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • antigen binding site refers to a portion of the heavy and light chain variable regions (VH and VL) that are formed by amino acid residues that participate in antigen binding, including those An amino acid residue that interacts with an antigen and determines its specificity and affinity for the antigen.
  • the antigen binding site may also comprise amino acid residues of the framework regions necessary for maintaining the proper conformation of the amino acid residues directly binding to the antigen described above.
  • full length antibody means an antibody consisting of two “full length heavy chains” and two “full length light chains”.
  • full-length heavy chain refers to a polypeptide chain which is composed of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain in the N-terminal to C-terminal direction.
  • VH heavy chain variable region
  • HR hinge region
  • the constant region CH2 domain, the heavy chain constant region CH3 domain is composed; and, when the full length antibody is of the IgE isotype, optionally further comprises a heavy chain constant region CH4 domain.
  • the "full length heavy chain” is a polypeptide chain consisting of VH, CH1, HR, CH2 and CH3 in the N-terminal to C-terminal direction.
  • a “full length light chain” is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction.
  • VL light chain variable region
  • CL light chain constant region
  • Two pairs of full length antibody chains are joined by a disulfide bond between the disulfide bond between CL and CH1 and the HR of the two full length heavy chains.
  • the full length antibodies of the invention may be from a single species, such as a human; or may be chimeric or humanized antibodies.
  • the full-length antibody of the present invention comprises two antigen-binding sites formed by a pair of VH and VL, respectively, which specifically recognize/bind the same antigen.
  • Fab fragment means an antibody fragment consisting of VL, VH, CL and CH1 domains;
  • Fab'fragment means reducing two residues in a F(ab') 2 fragment.
  • the fragment obtained after the disulfide bond of the strand fragment consists of a complete Fd fragment of the light and heavy chains (consisting of the VH and CH1 domains);
  • F(ab') 2 fragment means the inclusion of the region through the hinge
  • An antibody fragment of two Fab fragments joined by a disulfide bridge Each of the above antibody fragments retains the ability to specifically bind to the same antigen to which the full length antibody binds, and/or compete with the full length antibody for specific binding to the antigen.
  • scFv refers to a single polypeptide chain comprising VL and VH domains, wherein the VL and VH are linked by a linker (see, eg, Bird et al., Science 242: 423). -426 (1988); Huston et al, Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Roseburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994)).
  • Such scFv molecules can have the general structure: NH 2 -VL- linker -VH-COOH or NH 2 -VH- linker -VL-COOH.
  • Disulfide bonds may also be present between the VH and VL of the scFv of the invention. Methods for introducing a disulfide bond between the VH and VL of an antibody are well known in the art, for example, see U.S. Patent Application No. 5,747,654; Rajagopal et al, Prot. Engin. 10 (1997) 1453-1459; Reiter et al.
  • di-scFv refers to an antibody fragment formed by the joining of two scFvs.
  • Fv fragment means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody.
  • parent antibody refers to an anti-TNF ⁇ antibody or an anti-IL-6R/IL-6 antibody used to prepare a recombinant protein of the present invention, which antibody has an amino acid sequence which can be replaced by, for example, amino acid substitution. Or a structural alteration or the like for the preparation of the first antibody or scFv contained in the recombinant protein of the present invention.
  • the "anti-TNF ⁇ parent antibody” may be an antibody having a heavy chain and a light chain variable region as shown in SEQ ID NOs: 1 and 5, respectively, or an antibody as shown in SEQ ID NOs: 9 and 13, respectively;
  • the anti-IL-6R parent antibody may be an antibody of the heavy chain and light chain variable regions as shown in SEQ ID NOs: 17 and 21, respectively, or an antibody as shown in SEQ ID NOs: 25 and 29, respectively.
  • the CDRs, VH, VL, CH, CL, HC, LC contained in the recombinant protein of the present invention may also be derived from other antibodies or antibody fragments thereof known in the art capable of specifically binding TNF ⁇ or IL-6R or IL-6. Or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% with the above-mentioned known antibodies, antibody fragments thereof or CDRs thereof, VH, VL, CH, CL, HC, LC An antibody that is at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.
  • the antibody or protein capable of specifically binding TNF ⁇ comprises: Infliximab, Etanercept, Adalimumab, Certolizumab pegol, Golimumab, Tasonermin, TNF Kinoid, ESBA105, afelimomab, lensercept, nerelimomab, onercept, ozoralizumab, pegsunercept, placulumab, tulinercept;
  • the antibody or protein capable of specifically binding IL-6R or IL-6 includes: clazakizumab, elsilimomab, olamkicept, olokizumab, siltuximab, sirukumab, Tocilizumab, olamkicept, satralizumab, sarilumab, vobarilizumab.
  • linker refers to a linear polypeptide formed by the joining of multiple amino acid residues by peptide bonds.
  • the linker of the invention may be a synthetic amino acid sequence, or a naturally occurring polypeptide sequence, such as a polypeptide having the function of a hinge region.
  • linker polypeptides are well known in the art (see, for example, Holliger, P. et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ et al. (1994) Structure 2: 1121- 1123).
  • the term “specifically binds” refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and the antigen to which it is directed.
  • an antibody that specifically binds to an antigen means that the antibody is less than about 10 -5 M, such as less than about 10 -6 M, 10 - 7 M, Affinity (K D ) of 10 -8 M, 10 -9 M or 10 -10 M or less binds to the antigen.
  • K D refers to a particular antibody - antigen interaction solutions dissociation equilibrium constant, which is used to describe the binding affinity between antibody and antigen.
  • the antibody e.g., an antibody of the invention
  • the antibody is less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10 M or less.
  • the dissociation equilibrium constant (K D ) binds to an antigen (eg, HBsAg), for example, as determined using surface plasmon resonance (SPR) in a BIACORE instrument.
  • vector refers to a nucleic acid vehicle into which a polynucleotide can be inserted.
  • a vector is referred to as an expression vector when the vector enables expression of the protein encoded by the inserted polynucleotide.
  • the vector can be introduced into the host cell by transformation, transduction or transfection, and the genetic material element carried thereby can be expressed in the host cell.
  • Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1 derived artificial chromosomes (PAC).
  • Phage such as lambda phage or M13 phage and animal virus.
  • Animal viruses useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, nipples Multi-tumor vacuolar virus (such as SV40).
  • a vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain an origin of replication.
  • the term "host cell” refers to a cell that can be used to introduce a vector, including, but not limited to, a prokaryotic cell such as Escherichia coli or Bacillus subtilis, such as a fungal cell such as a yeast cell or an Aspergillus.
  • a prokaryotic cell such as Escherichia coli or Bacillus subtilis
  • a fungal cell such as a yeast cell or an Aspergillus.
  • S2 Drosophila cells or insect cells such as Sf9
  • animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • identity is used to mean the matching of sequences between two polypeptides or between two nucleic acids.
  • a position in the two sequences being compared is occupied by the same base or amino acid monomer subunit (for example, a position in each of the two DNA molecules is occupied by adenine, or two
  • Each position in each of the polypeptides is occupied by lysine, and then each molecule is identical at that position.
  • the "percent identity" between the two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions to be compared x 100. For example, if 6 of the 10 positions of the two sequences match, then the two sequences have 60% identity.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match).
  • the comparison is made when the two sequences are aligned to produce maximum identity.
  • Such alignment can be achieved by, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). It is also possible to use the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)) integrated into the ALIGN program (version 2.0), using the PAM 120 weight residue table.
  • the gap length penalty of 12 and the gap penalty of 4 were used to determine the percent identity between the two amino acid sequences.
  • the Needleman and Wunsch (J MoI Biol. 48: 444-453 (1970)) algorithms in the GAP program integrated into the GCG software package can be used, using the Blossum 62 matrix or The PAM250 matrix and the gap weight of 16, 14, 12, 10, 8, 6 or 4 and the length weight of 1, 2, 3, 4, 5 or 6 to determine the percent identity between two amino acid sequences .
  • amino acids are generally represented by single letter and three letter abbreviations as are known in the art.
  • alanine can be represented by A or Ala.
  • pharmaceutically acceptable carrier and/or excipient refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, It is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and includes, but is not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancement. Agents, diluents, agents that maintain osmotic pressure, agents that delay absorption, preservatives.
  • pH adjusting agents include, but are not limited to, phosphate buffers.
  • Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants such as Tween-80.
  • Ionic strength enhancers include, but are not limited to, sodium chloride.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like.
  • Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like.
  • Agents that delay absorption include, but are not limited to, monostearate and gelatin.
  • the term "subject” refers to a mammal, such as a primate mammal, such as a human.
  • the subject eg, a human
  • diseases or disease states are characterized by their benefit from a reduction in TNF[alpha] and/or IL-6 levels or inhibition of TNF[alpha] and/or IL-6 activity resulting in amelioration or cure.
  • an effective amount refers to an amount sufficient to achieve, or at least partially achieve, a desired effect.
  • an effective amount to prevent a disease eg, a disease associated with excessive TNF ⁇ and/or IL-6 activity
  • an amount effective to treat is an amount sufficient to cure or at least partially arrest a disease and a complication thereof in a patient already suffering from the disease. Determination of such an effective amount is well within the capabilities of those skilled in the art.
  • the amount effective for therapeutic use will depend on the severity of the condition to be treated, the overall condition of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments for simultaneous administration. and many more.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • cytotoxic cells eg, natural killer (NK) cells, neutrophils.
  • FcR Fc receptor
  • Methods for detecting ADCC activity of an antibody are known in the art and can be evaluated, for example, by measuring the binding activity between an antibody to be tested and an Fc receptor (e.g., CD16a).
  • complement dependent cytotoxicity refers to the activation of a cytotoxic form of the complement cascade by binding complement component Clq to antibody Fc.
  • Methods for detecting the CDC activity of an antibody are known in the art and can be evaluated, for example, by measuring the binding activity between an antibody to be tested and an Fc receptor (e.g., C1q).
  • the recombinant protein of the present invention not only specifically recognizes/binds TNF ⁇ and IL-6R, but also has affinity for TNF ⁇ and IL-6R at least comparable to that of its respective parent antibody, and can significantly and simultaneously inhibit TNF ⁇ and in vitro and in a subject.
  • the activity of IL-6R blocks the TNF ⁇ and IL-6 signaling pathways.
  • the recombinant protein of the present invention has an excellent thermal stability equivalent to that of the parent antibody of the first antibody.
  • the recombinant protein of the present invention exhibits good therapeutic activity in in vivo experiments.
  • the bispecific recombinant protein of the invention has superior therapeutic activity compared to the parent antibody. Therefore, the recombinant protein of the present invention has potential for treating diseases associated with excessive TNF ⁇ and IL-6 activity, such as inflammatory diseases or autoimmune diseases, and has great clinical value.
  • Figure 1 shows a schematic representation of constructs of recombinant proteins against TNF[alpha] and IL-6R.
  • FIGS. 2A-2B show reduced and non-reduced SDS-PAGE electropherograms of ANT, ACT, TNA, TCA, ALNT, and ALCT.
  • Figure 2A Lane: 1. Protein marker; 2. AB01 non-reducing; 3. AB01 reduction; 4. ANT non-reduction; 5. ANT reduction; 6. ACT non-reduction; 7. ACT reduction; 8. TNA non-reduction; .TNA restore.
  • Figure 2B Lane: 1. Protein marker; 2. AB01 non-reducing; 3. AB01 reduction; 4. TCA non-reduction; 5. TCA reduction; 6. ALNT non-reduction; 7. ALNT reduction; 8. ALCT non-reduction; .ALCT restore. The results showed that the size of each recombinant protein band was in line with expectations, and there was no obvious aggregation degradation, indicating that each bispecific recombinant protein can be efficiently expressed and assembled correctly.
  • Figure 3 shows the results of detection of inhibition of IL-6-induced cell proliferation by the anti-TNF ⁇ and IL-6R recombinant proteins in Example 7. The results showed that the anti-TNF ⁇ and IL-6R recombinant proteins significantly inhibited the proliferation of U266 cells induced by IL-6, and were consistent with the effect of the parent antibody.
  • Fig. 4 is a view showing the joint swelling of the rhesus arthritis model in Example 11.
  • Fig. 5 shows the change in body weight of the rhesus arthritis model in Example 11, and the arrows indicate the time points of administration.
  • Fig. 6 is a graph showing the clinical score change curve of the rhesus arthritis model in Example 11, and the arrow indicates the administration time point.
  • Fig. 7 is a graph showing changes in serum CRP levels of the rhesus arthritis model in Example 11, and arrows indicate the time points of administration.
  • Figure 8 shows the hematology RBC profile of the rhesus arthritis model of Example 11, with arrows indicating the time point of administration. Among them, the normal reference value range is 4.17-6.27.
  • Figure 9 shows the hematology HGB profile of the rhesus arthritis model of Example 11, with arrows indicating the time point of administration. Among them, the normal reference value range is 111.7-152.5.
  • Fig. 10 is a graph showing the hematological HCT curve of the rhesus arthritis model of Example 11, and the arrows indicate the time points of administration. Among them, the normal reference value range is 35.41-49.97.
  • Example 1 Construction of an expression vector encoding an anti-TNF ⁇ /IL-6R bispecific recombinant protein
  • the anti-TNF ⁇ parent antibody (AB01, AB02) and the anti-IL-6R parent antibody (AB03, AB04) shown in Table 2 were first obtained, and then the first polypeptide was encoded by DNA recombination technology.
  • nucleic acid sequences encoding the parental antibodies were all gene-synthesized by Nanjing Kingsray Biotechnology Service Co., Ltd., and the parental antibody variable region and constant region sequences are referred to Table 2.
  • a nucleotide sequence encoding the first polypeptide chain of the recombinant protein and a nucleotide sequence encoding the second polypeptide chain were constructed in accordance with the construction of each recombinant protein shown in Table 3.
  • the VH and VL thereof are linked by a peptide linker (SEQ ID NO: 33, amino acid sequence: GGGGSGGGGSGGGGSGGGGS), and the VH of the parental antibody of the scFv is 44th by a PCR site-directed mutagenesis method.
  • AB01-scFv indicates the parent antibody AB01-derived scFv, the variable region of which differs from AB01 in that the VH at position 44 and the VL at position 100 of the scFv are respectively cysteine; other similar expressions have similar meaning.
  • nucleic acid construct as an example of a partially recombinant protein is constructed as follows:
  • the nucleotide sequence encoding AB01-scFv is ligated to the 5' or 3' end of the nucleotide sequence encoding the heavy chain of AB03 by a nucleotide sequence encoding linker S1 to construct a first coding TNA or TCA, respectively.
  • a nucleotide sequence of the polypeptide chain wherein the sequence of the AB01-scFv from the N-terminus to the C-terminus is VL-linker-VH; the nucleotide sequence of the second polypeptide chain encoding TNA or TCA is the nucleoside of the LC encoding AB03 Acid sequence.
  • the nucleotide sequence encoding AB01-scFv is ligated to the 5' or 3' end of the nucleotide sequence encoding the light chain of AB03 by a nucleotide sequence encoding linker S1 to construct a first coding TLNA or TLCA, respectively.
  • a nucleotide sequence of the polypeptide chain wherein the sequence of the AB01-scFv from the N-terminus to the C-terminus is VL-linker-VH; the nucleotide sequence of the second polypeptide chain encoding TLNA or TLCA is the nucleoside of HC encoding AB03 Acid sequence.
  • the nucleotide sequence encoding AB03-scFv is ligated to the 5' or 3' end of the nucleotide sequence encoding the heavy chain of AB01 by the nucleotide sequence encoding linker S1 to construct the first coding ANT or ACT, respectively.
  • the nucleotide sequence encoding AB03-scFv is ligated to the 5' or 3' end of the nucleotide sequence encoding the heavy chain of AB01 by a nucleotide sequence encoding linker S1 to construct an ANT-2 or ACT- coding, respectively.
  • the nucleotide sequence encoding AB03-scFv is ligated to the 5' or 3' end of the nucleotide sequence encoding the light chain of AB01 by a nucleotide sequence encoding linker S1 to construct a first coding ALNT or ALCT, respectively.
  • a nucleotide sequence of the polypeptide chain wherein the sequence of the AB03-scFv from the N-terminus to the C-terminus is VL-linker-VH; the nucleotide sequence of the second polypeptide chain encoding ALNT or ALCT is a nucleoside encoding HC of AB01 Acid sequence.
  • the nucleotide sequence encoding AB02-scFv is ligated to the 3' end of the nucleotide sequence encoding the heavy chain of AB04 by a nucleotide sequence encoding linker S1 to construct a nucleotide encoding the first polypeptide chain of SCG a sequence; wherein the sequence of the AB02-scFv from the N-terminus to the C-terminus is VL-linker-VH; the nucleotide sequence of the second polypeptide chain encoding the SCG is the nucleotide sequence of the LC encoding AB04.
  • the nucleotide sequence encoding AB04-scFv is ligated to the 3' end of the nucleotide sequence encoding the heavy chain of AB02 by a nucleotide sequence encoding linker S1 to construct a nucleotide encoding the first polypeptide chain of GCS a sequence; wherein the sequence of the AB04-scFv from the N-terminus to the C-terminus is VL-linker-VH; the nucleotide sequence of the second polypeptide chain encoding GCS is the nucleotide sequence of the LC encoding AB02.
  • a nucleotide sequence encoding each of the first polypeptide chain or the second polypeptide chain described above is ligated to a nucleotide sequence encoding a murine IgG-KAPPA signal peptide (amino acid sequence SEQ ID NO: 82, nucleic acid sequence SEQ ID NO: 83
  • the pTT5 plasmid was separately introduced by homologous recombination to construct an expression vector encoding the first polypeptide chain and an expression vector encoding the second polypeptide chain.
  • the amino acid sequences of the finally obtained recombinant proteins are shown in Table 4.
  • Example 2 Expression of anti-TNF ⁇ /IL-6R bispecific recombinant protein
  • CHO-S cells in good growth state and in log phase were centrifuged and inoculated with 200 ml at 2E6 cells/ml, and the density was as long as about 4E6 cells/ml on the next day.
  • the plasmid to be transfected obtained in Example 1 was sterilized by filtration through a 0.22 ⁇ m filter, 100 ⁇ g of the first polypeptide chain recombinant plasmid and 100 ⁇ g of the corresponding second polypeptide chain recombinant plasmid were added, and 20 ml of CHOgro Complex Formation Solution was purchased.
  • Example 3 Purification of anti-TNF ⁇ /IL-6R bispecific recombinant protein
  • the CHO-S cell culture expressed in Example 2 for 5 days was firstly centrifuged to separate the supernatant and the cell pellet; and then centrifuged at high speed to obtain a clear liquid.
  • the recombinant antibody was purified by affinity chromatography (Protein A) and ion exchange two-step method.
  • the medium used in the purification was Mab Select SuRe produced by GE Company and Eshmuno CPX produced by Millipore Company.
  • each bispecific recombinant protein was basically the same, both in the range of 22-45mg/L, and consistent with the expression level of the same condition anti-IL-6R parental antibody AB03, indicating that each recombinant protein can be successfully expressed and has a higher
  • the expression efficiency, the specific expression level is shown in Table 5, wherein ANT and TCA have the highest expression efficiency.
  • the isolated and purified recombinant protein was concentrated by ultrafiltration tube and exchanged into PBS solution. SDS-PAGE electrophoresis showed that the non-reduced band of AB01 was about 150 kDa and 50 kDa (heavy chain) after reduction, as shown in Fig. 2A-2B.
  • scFv linked to the heavy chain bispecific antibody protein non-reducing band size is about 200kDa, reducing band size is 75kDa (heavy chain-scFv) and 25kDa (light chain); scFv is attached to the light chain
  • the bispecific antibody protein non-reducing band size is approximately 200 kDa, the reducing band size is 50 kDa (heavy chain) and 50 kDa (light chain-scFv). The size of the band was as expected, and there was no obvious aggregation degradation, indicating that each bispecific recombinant protein can be efficiently expressed and assembled correctly.
  • Antibody name Obtain protein amount Expression efficiency ACT 4.4mg 22mg/L ANT 9mg 45mg/L TCA 9mg 45mg/L TNA 5.6mg 28mg/L ALCT 4.2mg 21mg/L ALNT 5.1mg 25.5mg/L AB03 5mg 25mg/L
  • Example 4 Detection of antigen binding biological activity of anti-TNF ⁇ /IL-6R bispecific recombinant protein
  • the difference in the affinity of each bispecific recombinant protein to bind to the same antigen by the parent antibody is detected by ELISA, and the relative affinity of the bispecific recombinant protein to simultaneously bind the two antigens is verified to verify the ability to block an antigen alone. Whether it is lower than the parent antibody; whether it can block both antigens at the same time, block the two signaling pathways, and produce a synergistic effect in the treatment of diseases such as rheumatoid arthritis and other immune system diseases.
  • the recombinant TNF ⁇ protein (purchased from Beijing Yishen Shenzhou Co., Ltd.) was added to a 96-well microtiter plate (purchased from Thermo Corporation) at 50 ng/well, and coated at 4 ° C overnight; the next day, the solution in the well was discarded, and the washing buffer (containing 0.05) was used.
  • the recombinant IL6R-mFC protein (obtained from the Cologne Institute) was added to the 96-well microtiter plate at 200 ng/well, and coated at 4 ° C overnight; the next day, the solution in the well was discarded, washed once with washing buffer, patted; 2% BSA in PBS solution, 100 ⁇ l/well, blocked at 37°C for 2h, then patted dry; bispecific recombinant protein, anti-IL-6R parental antibody AB03 and AB04 start at 4000ng/ml, do 4-fold dilution, a total of 11 Gradient, 100 ⁇ l/well; incubate the plate at 37 ° C for 2 h, pat dry, wash buffer 3 times; add HRP-conjugated Goat Anti-Human IgG (H + L) solution, incubate at 37 ° C for 1 h; add TMB The solution was reacted at room temperature for about 5 min; the stop solution was added and placed in a microplate reader to
  • the recombinant TNF ⁇ protein was coated in a 96-well microtiter plate at 50 ng/well at 4° C.; the next day, the solution was discarded and washed once with washing buffer (phosphate buffer containing 0.05% Tween-20).
  • Each of the bispecific recombinant proteins can bind to both TNF ⁇ and IL-6R antigens.
  • ACT and ANT have the highest activity of both TNF ⁇ and IL-6R, and the binding activity of ALCT and ALNT is slightly. It is weaker than other recombinant proteins, but the EC50 is at the pM level.
  • the bispecific recombinant protein of the present invention binds to one antigen and does not affect the binding of the second antigen, and can simultaneously bind the two antigens efficiently, and retains
  • the parental antibody has an equally excellent binding activity, and thus it can be particularly suitable for use by simultaneously neutralizing TNF ⁇ and IL-6R inflammatory factors which play a key role in diseases such as rheumatoid arthritis, thereby simultaneously suppressing two signaling pathways. Treatment of diseases associated with excessive TNF ⁇ and/or IL-6 activity.
  • Example 5 Detection of binding activity of antigen by bispecific recombinant protein at the cellular level
  • CHO cells expressing TNF ⁇ on a cell surface in good condition obtained by introducing a TNF ⁇ expression vector into CHO cells
  • PBS Resuspend in 0.5% BSA in PBS, add 50 ⁇ l/well, 3E6 cells/well to 96-well plates; each bispecific recombinant protein, anti-TNF ⁇ parental antibody AB01 and AB02 were started at 1 ⁇ M and diluted 3 times for 10 Gradient, 50 ⁇ l/well was added to the 96-well plate containing cells, mixed, incubated at 4 ° C for 1 hour; washed once with PBS, 50 ⁇ l of FITC anti-human IgG1 antibody (purchased from Biolegend), incubated at 4 ° C for 30 minutes; PBS washed 3 The test was carried out by flow cytometry (beckmanCyto FLEX).
  • IL-6R-expressing CHO cells (which were obtained by introducing an IL-6R expression vector into CHO cells) were obtained in a well-prested cell surface, centrifuged, and washed once with PBS. Resuspend in PBS containing 0.5% BSA, add 50 ⁇ l/well, 3E6 cells/well to 96-well plate; bispecific recombinant protein, anti-IL-6R parental antibody AB03 and AB04 start at 1 ⁇ M, do 3-fold dilution, total 10 Gradient, 50 ⁇ l/well was added to the 96-well plate containing cells, mixed, incubated at 4 ° C for 1 hour; washed once with PBS, 50 ⁇ l of FITC anti-human IgG1 antibody (purchased from Biolegend), and incubated at 4 ° C for 30 minutes; PBS wash After 3 times, the assay was performed using a flow cytometer (beckmanCyto FLEX).
  • Antibody name EC50(nM) Ratio to AB03 AB03 6.72 1 ANT 4.64 0.69
  • Recombinant protein/parent antibody AB01 AB03 TNA TCA ACT ANT ALCT ALNT Tm (°C) 71.41 69.18 58.83 59.04 71.87 72.09 72.75 73.17
  • Example 7 Cellular biological activity assay of bispecific recombinant protein
  • the inhibitory activity of the bispecific recombinant protein on TNF ⁇ and IL-6R was detected by a cell level assay.
  • Recombinant human TNF ⁇ was induced to be added to L929 cells for 18-24 h to induce cytotoxicity of TNF ⁇ to L929 cells; after addition of bispecific recombinant protein and TNF ⁇ , cytotoxicity was attenuated to elimination.
  • L929 cells in good condition were resuspended in RPMI1640+2% FBS and adjusted to a density of 3 ⁇ 10 5 cells/ml, 100 ⁇ l/well into 96-well plates, overnight at 37 ° C; to RPMI 1640 + 2% FBS Adding Actinomycin D at a final concentration of 0.4 ⁇ g/ml, adding a recombinant TNF ⁇ protein at a final concentration of 6 ng/ml; adjusting the bispecific recombinant protein and the anti-TNF ⁇ parent antibody AB01 to 2 ⁇ g/ml, and performing 3-fold serial dilution.
  • Recombinant protein Recombinant protein EC50 (pM) AB01 EC50(pM) Ratio to AB01 ANT 85.05 64.19 1.33 ACT 79.39 67.76 1.17 ALNT 213.3 74.42 2.87 ALCT 106.0 74.42 1.42
  • U266 cells in good condition were resuspended in RPMI1640+5% FBS, adjusted to 8 ⁇ 10 4 cells/ml, 100 ⁇ l/well into 96-well plates; RPMI1640+5% FBS was added to a final concentration of 10 ng/ml.
  • Recombinant IL6; the bispecific recombinant protein and anti-IL-6R parental antibody AB03 were adjusted to 200 ⁇ g/ml, and serial dilutions were performed in 3 folds, 8 gradients were added, U266 cells were added to 100 ⁇ l per well, and incubated at 37 ° C for 48 h.
  • the cell viability was measured by adding 20 ⁇ l of CCK8 reagent, and the reaction was read for 4 hours.
  • the IC50 value of the antibody neutralizing IL-6R was calculated using Graphpad Prism5.
  • Example 8 In vivo metabolism experiment of bispecific recombinant protein
  • Twenty-five male SD rats were randomly divided into 7 groups. 2.5 mg/kg of ANT and parental antibody AB01 were administered by subcutaneous injection, respectively. Blood was collected from the tail veins before administration, 1h, 4h, 8h, Day1, Day3, Day5, Day8, Day11, Day15, Day22, Day29, Day36, and the blood samples were collected by centrifugation at 1000-3000 ⁇ g 4°C for 10 min. Clear, stored at -80 ° C; blood concentration was measured by ELISA, and the time curve was fitted, and the pharmacokinetic parameters were calculated to evaluate the pharmacokinetic behavior of the bispecific recombinant protein in rats.
  • CHO-S-TNF ⁇ cells Jurkat-NFAT/CD16a cells, CHO-S-TNF ⁇ cells, CHO-S-IL6R cells (all cells were prepared by Sichuan Kelunbotai Biomedical Co., Ltd.), centrifuged, using 1640+1% FBS medium and resuspended cells were diluted adjusted to 2 ⁇ 10 6 /ml,0.5 ⁇ 10 6 /ml,0.5 ⁇ 10 6 /ml,0.5 ⁇ 10 6 / ml after counting.
  • Target cell plating 50 ⁇ l/well of CHO-S-TNF ⁇ cells, 50 ul/well of CHO-S-IL6R cells, and a total of 5 ⁇ 10 4 cells per well.
  • Effector cell plating Jurkat-NFAT/CD16a cells 50 ⁇ l/well, totaling 1 ⁇ 10 5 cells per well.
  • Detection antibody was added: AB01, ACT, ANT, and IgG control antibodies were diluted to 200 ⁇ g/ml, and diluted 2 times in sequence, for a total of 11 concentration points. 50 ⁇ l of the diluted antibody (initial concentration 50 ⁇ g/ml) was added to the well, and the final well was added to the detection Buffer.
  • Detection After the cells were incubated for 5 hours, 40 ⁇ l of One-glo detection reagent (Promega, Cat: E6120) was added to each well of the test area, and the plate was detected by a microplate reader.
  • One-glo detection reagent Promega, Cat: E6120
  • Example 11 Evaluation of in vivo activity of bispecific recombinant protein in rhesus arthritis model
  • This example evaluates the effect of the bispecific recombinant protein of the present invention on bovine type II collagen-induced rhesus arthritis disease progression.
  • the recombinant protein is intravenously administered to the rhesus monkeys with successful arthritis once a week, and the body weight and blood samples are measured once a week to calculate the recombinant protein for the treatment of rhesus arthritis.
  • Pharmacodynamic anti-inflammatory effect.
  • Drug name, source, configuration method ANT, according to the animal's body weight, according to the dose of 5mg / kg, 1ml / kg, dilute the mother liquor to obtain a treatment solution.
  • Bovine type II collagen immuno grade collagen
  • Chondrex Chondrex
  • Freund's complete adjuvant (Sigma).
  • Bovine type II collagen was dissolved in 0.1 M acetic acid solution, and then an equal volume of Freund's complete adjuvant was added, and the mixture was stirred at a low temperature to form a uniform and stable emulsion at a final concentration of 2 mg/ml.
  • the emulsion was injected subcutaneously at the back of 4 rhesus monkeys at 10 points, 0.2 ml/point, ie, the amount of collagen injected was 4 mg.
  • the day of collagen injection was recorded as D1. Two weeks after the first collagen injection (Day 15, D15), the second collagen injection was completed as described above. Weighing and serum collection once a week during the period. According to the monitoring scores in Table 16, the successful model animals showed various symptoms such as weight loss, arthritis swelling, CRP level rise and anemia.
  • the results of the modeling were as shown in Table 17, and 3 monkeys were successfully modeled.
  • Table 16 Clinical scoring criteria for rhesus arthritis disease progression
  • animal No. 16063 developed rapidly, failed to give ANT treatment in time, and died on day D22.
  • animal 16066 was selected for early administration of the development of arthritis, starting from D28; animal 16068 was selected for development in the course of arthritis.
  • dosing is started from D42.
  • the regimen for the administration of ANT recombinant protein to rhesus monkeys was as follows: 5 mg/kg of ANT was administered at a dose of 1 ml/kg, and the administration route was a single intravenous injection, and the frequency of administration was once a week for a total of 4 times.
  • the animal's body weight and blood samples were measured once a week. The swelling of the forefoot and hindfoot joints was observed and clinically scored.
  • the arthritis scores are shown in Table 16, and the serum CRP (C-reactive protein) levels and hematology RBC were analyzed. Red blood cells), HGB (hemoglobin), HCT (hematocrit) indicators to evaluate the therapeutic efficacy of ANT. Animal deaths were recorded daily. See Table 17 and Figure 4 for the model and administration of rhesus monkeys. Changes in body weight, arthritis scores, CRP, and hematologic parameters are shown in Figures 5-10.
  • animal model 16067 was unsuccessful and all monitoring indicators returned to normal values soon after collagen injection.
  • animal No. 16063 was rapidly ill, failed to give ANT treatment in time, and finally died on day D22.
  • Animal 16066 was given ANT treatment in the early stage of the disease (D28 for the first time, each time Once a week, 4 times of administration), arthritis swelling was relieved after administration, body weight gradually recovered, CRP level returned to normal level, anemia symptoms disappeared; animal 16068 was given ANT treatment in the late stage of the disease (D42 days for the first time, each Once a week, 5 times of administration), although the arthritis swelling did not significantly relieve after administration, the body weight did not recover significantly, and the hematological parameters did not return to normal levels, but the CRP level was significantly reduced, and the animals did not die.
  • the above results indicate that the ANT of the present invention has a good anti-inflammatory effect in the rhesus monkey arthritis model.
  • the bispecific recombinant protein of the invention has superior therapeutic activity compared to the parent antibody.

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Abstract

本发明涉及生物医药领域，特别是一种双特异性重组蛋白，编码它们的核酸分子，制备它们的方法，包含它们的药物组合物所述重组蛋白在制备药物组合物中的用途，以及所述重组蛋白在治疗炎性疾病或自身免疫性疾病中的应用。

Description

双特异性重组蛋白 技术领域
本发明涉及生物医药领域，特别是预防和/或治疗与过度的TNFα和/或IL-6活性相关的疾病的领域。具体而言，本发明涉及特异性结合TNFα和IL-6R，或TNFα和IL-6的重组蛋白。
背景技术
自身免疫性疾病作为继心血管疾病、癌症后威胁人类健康的第三大杀手，也被列入我国十类重大疾病。自身免疫是指机体免疫系统对自身组织细胞所发生的免疫应答的现象。当机体免疫系统对自身组织细胞发生免疫应答而造成细胞的破坏或组织的损伤并出现临床症状时，则称为自身免疫性疾病。目前发现的自身免疫性疾病有30多种，包括类风湿性关节炎(RA)、胰岛素依赖型糖尿病、多发性硬化症、红斑狼疮、银屑病、炎症性肠病、溃疡性结肠炎、重症肌无力、多发性肌炎、皮肌炎、Crohn病、自身免疫性血细胞减少、血管炎、系统性红斑狼疮等。
类风湿性关节炎的治疗涉及多种遗传和环境因素，治疗效果因此不同，导致对所有患者完全缓解的治疗有效是非常困难。很多用于治疗RA的新药物特别是生物制剂有助于带来治疗革命性进步。
类风湿性关节的发病涉及许多促炎细胞因子，特别是肿瘤坏死因子α(TNFα)和白细胞介素-6(IL-6)。
TNFα活化T细胞并诱导T细胞浸润和新血管生成，通过增加成纤维细胞样滑膜细胞(FLS)的增殖导致关节破坏破骨细胞的形成。IL-6引起B细胞增殖并产生抗体，并诱导分化的T细胞分泌成IL-17分泌性T辅助细胞(Th17)，从而抑制调节性T细胞分化。
IL-6能刺激血管生成和破骨细胞生成。IL-6可与IL-6R特异结合。IL-6受体有2种形式：膜结合受体(mIL-6R)和可溶性受体(sIL-6R)。IL-6与IL-6R(包括mIL-6R和sIL-6R)结合后，形成IL-6/IL-6R复合体，与gp130结合，启动下游包括JAK/STAT、ERK和PI3K在内的IL-6信号通路，因此，TNFα和IL-6可能共同引起许多致病性信号从而导致RA。
当类风湿关节炎病人接受IL-6受体(IL-6R)的单克隆抗体药物 tocilizumab(ACTEMRA，Roche)治疗后，病情得到明显缓解，更进一步证明了IL-6在类风湿性节炎中的重要作用。
对现有临床结果进行综合比较发现，IL-6通路抑制剂，包括已上市的tocilizumab和在研品种，无论其靶点是IL-6，还是IL-6R，其临床效果相当，且不良反应率也没有明显差异。许多病人对TNF抑制药物响应不足或长期使用后效果下降，tocilizumab的多项III期临床试验表明TNF抑制药物响应不足或治疗效果下降的病人使用tocilizumab可得到良好的治疗效果。
双特异性抗体(BsAb)是一类具有双亲嗜性的组合抗体，通常是双价的(也有四价和六价的)，即有两条抗原结合臂，具有结合两种不同特异性抗原的功能。中国专利申请CN102112495A公开了一种特异性结合TNFα和IL-6R的双特异性抗体DVD279和DVD280，其在抗体CH1和CL的N端依次连接针对TNFα和IL-6R，或针对IL-6R和TNFα的可变结构域，但其与抗TNFα亲本抗体和抗IL-6R亲本抗体对相同抗原的EC50的比值分别高达149.2倍(亲本抗体对IL-6R的EC50为1.42nM，DVD279对IL-6R的EC50为211.9nM)和9.13倍(亲本抗体对TNFα的EC50为0.44nM，DVD280对TNFα的EC50为4.02nM)，也即该双特异性抗体的结合力明显差于亲本抗体，表明该双特异性抗体对一种抗原(TNFα或IL-6R)的结合显著抑制了其对另一种抗原(IL-6R或TNFα)的结合力，无法同时高效抑制TNFα和IL-6R，难以保证治疗效果。
因此，对于自身免疫性疾病或急慢性炎性疾病等疾病仍有未满足的需求，发展创新的、能更为有效、且副作用更小的治疗方法和药物是迫切而必要的。
发明内容
在本申请中，发明人通过大量的研究开发了一种双特异性结合TNFα和IL-6R，或TNFα和IL-6的重组蛋白，编码重组蛋白的核酸分子，包含核酸分子的载体，制备重组蛋白的方法，包含重组蛋白的药物组合物，药物组合物的在制备药物中的用途，重组蛋白在诊断/治疗/预防与过度的TNFα和IL-6活性相关的疾病(例如炎性疾病或自身免疫性疾病)中的用途或方法，以及包含重组蛋白的试剂盒。
重组蛋白
因此，在一个方面，本发明提供了一种重组蛋白，其包含：
1)特异性结合第一抗原的第一抗体，所述第一抗体包括重链(HC)和轻链(LC)；和
2)特异性结合第二抗原的包含重链可变区(VH)和轻链可变区(VL)的抗体片段(例如Fv，scFv，di-scFv)；
其中，所述抗体片段连接于第一抗体的重链或轻链的N端或C端；
所述第一抗原为TNFα，并且所述第二抗原为IL-6R或IL-6；或者，第一抗原为IL-6R或IL-6，并且所述第二抗原为TNFα。
在某些优选的实施方案中，所述抗体片段为scFv。
在某些优选的实施方案中，所述重组蛋白包含1个所述第一抗体和2个所述scFv；并且，所述第一抗体包括两条HC和两条LC，其中所述第一抗体的一条HC的重链可变区(VH)与一条LC的轻链可变区(VL)形成抗原结合部位，另一条HC的重链可变区(VH)与另一条LC的轻链可变区(VL)形成抗原结合部位。
在某些优选的实施方案中，每个所述scFv分别连接于所述第一抗体的两条重链或两条轻链的N端或C端。
在某些优选的实施方案中，每个所述scFv分别连接于所述第一抗体的两条重链的N端。在某些优选的实施方案中，每个所述scFv分别连接于所述第一抗体的两条重链的C端。
在某些优选的实施方案中，每个所述scFv分别连接于所述第一抗体的两条轻链的N端。在某些优选的实施方案中，每个所述scFv分别连接于所述第一抗体的两条轻链的C端。
在某些优选的实施方案中，一个所述scFv连接于所述第一抗体的重链或轻链的N端，另一个所述scFv连接于所述第一抗体的重链或轻链的C端。
在某些优选的实施方案中，所述第一抗体的重链包含重链可变区(VH)和CH1结构域，并且所述轻链包含轻链可变区(VL)和轻链恒定区(CL)。在此类实施方案中，所述第一抗体可以为Fab片段、Fab’片段或F(ab’) 2片段。在某些优选的实施方案中，所述第一抗体的重链包含重链可变区(VH)和重链恒定区(CH)，并且所述轻链包含轻链可变区(VL)和轻链恒定区(CL)。在此类实施方案中，所述第一抗体可以为全长抗体。
在某些优选的实施方案中，所述第一抗体的重链为IgG同种型，例如IgG1、IgG2、IgG3或IgG4；优选为人IgG同种型。在某些实施方案中，所述第一抗体的重链为人IgG1同种型。在某些优选的实施方案中，所述第一抗体的轻链为Kappa同种型，优选为人Kappa同种型。
在某些优选的实施方案中，所述第一抗体的两条HC包含相同的CDR；和/或，所述第一抗体的两条LC包含相同的CDR。
在某些优选的实施方案中，所述第一抗体的两条HC包含相同的VH；和/或，所述第一抗体的两条LC包含相同的VL。
在某些优选的实施方案中，所述第一抗体的两条HC具有相同的氨基酸序列；和/或，所述第一抗体的两条LC具有相同的氨基酸序列。
在某些优选的实施方案中，两个所述scFv具有相同或不相同的氨基酸序列。在某些优选的实施方案中，两个所述scFv具有相同的氨基酸序列。
在某些优选的实施方案中，所述重组蛋白包含两条第一多肽链和两条第二多肽链，其中对于所述每条多肽链：
a)所述第一多肽链各自独立地包含所述第一抗体的重链(HC)和所述scFv；和
b)所述第二多肽链各自独立地包含所述第一抗体的轻链(LC)；
其中，所述scFv通过接头S1与所述第一抗体的HC的N端或C端相连。
在某些优选的实施方案中，所述重组蛋白包含两条第一多肽链和两条第二多肽链，其中对于所述每条多肽链：
i)所述第一多肽链各自独立地包含所述第一抗体的轻链(LC)和所述scFv；和
ii)所述第二多肽链各自独立地包含所述第一抗体的重链(HC)；
其中，所述scFv通过接头S1与所述第一抗体的LC的N端或C端相连。
在某些优选的实施方案中，所述重组蛋白包含两条第一多肽链和两条第二多肽链，其中对于所述每条多肽链：
a)所述第一多肽链各自独立地包含所述第一抗体的重链(HC)和所述scFv；和
b)所述第二多肽链各自独立地包含所述第一抗体的轻链(LC)；
其中，所述scFv通过接头S1与所述第一抗体的HC的N端相连。
在某些优选的实施方案中，所述scFv的N端或C端与接头S1的C端或N端连接。
在某些优选的实施方案中，所述scFv具有结构：NH 2-VH-S2-VL-COOH或NH 2-VL-S2-VH-COOH，其中所述S2为接头。
在某些优选的实施方案中，所述接头S1和/或S2为肽接头，例如具有如(G mS n) x所示的氨基酸序列，其中m、n各自独立地选自1～8的整数(例如，1、2、3、4、5、6、7 或8)，x独立地选自1～20的整数(例如，1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20)。在某些优选的实施方案中，所述接头S1和/或S2具有如(G 4S) x所示的氨基酸序列，x独立地选自1-6的整数。
在某些优选的实施方案中，所述接头S1和/或S2具有选自下列的氨基酸序列：SEQ ID NO:33、SEQ ID NO:34和SEQ ID NO:35。
在某些优选的实施方案中，所述接头S2具有如(G 4S) 4所示的氨基酸序列，即GGGGSGGGGSGGGGSGGGGS(SEQ ID NO:33)。在某些优选的实施方案中，当所述scFv与所述第一抗体的重链或轻链的N端连接时，所述接头S1具有如(G 4S) 3所示的氨基酸序列，即GGGGSGGGGSGGGGS(SEQ ID NO:34)；当所述scFv与所述第一抗体的重链或轻链的C端连接时，所述接头S1具有如(G 4S) 2所示的氨基酸序列，即GGGGSGGGGS(SEQ ID NO:35)。
在某些优选的实施方案中，所述scFv的VH与VL之间存在二硫键。在抗体的VH和VL之间引入二硫键的方法是本领域熟知的，例如可参见美国专利申请US5,747,654；Rajagopal等人，Prot.Engin.10(1997)1453-1459；Reiter等人，Nature Biotechnology 14(1996)1239-1245；Reiter等人，Protein Engineering 8(1995)1323-1331；Webber等人，Molecular Immunology 32(1995)249-258；Reiter等人，Immunity 2(1995)281-287；Reiter等人，JBC 269(1994)18327-18331；Reiter等人，Inter.J.of Cancer 58(1994)142-149；或，Reiter等人，Cancer Res.54(1994)2714-2718；其通过引用并入本文。
在某些优选的实施方案中，所述scFv的位于VH第44位的氨基酸和位于VL第100位的氨基酸分别为半胱氨酸，其中提及的氨基酸位置是根据Kabat编号系统的位置；并且，所述scFv的VH与VL通过分别位于VH第44位和VL第100位的2个半胱氨酸残基之间所形成的二硫键连接。
在某些优选的实施方案中，所述第一抗体特异性结合TNFα，并且所述scFv特异性结合IL-6R，其中：
所述第一抗体包含：
(a)如SEQ ID NO:6所示的HCDR1；如SEQ ID NO:7所示的HCDR2；以及，如SEQ ID NO:8所示的HCDR3；如SEQ ID NO:2所示的LCDR1；如SEQ ID NO:3所示的LCDR2；以及，如SEQ ID NO:4所示的LCDR3；或，
(b)如SEQ ID NO:14所示的HCDR1；如SEQ ID NO:15所示的HCDR2；以及， 如SEQ ID NO:16所示的HCDR3；如SEQ ID NO:10所示的LCDR1；如SEQ ID NO:11所示的LCDR2；以及，如SEQ ID NO:12所示的LCDR3；
和/或，
所述scFv包含：
(i)如SEQ ID NO:22所示的HCDR1；如SEQ ID NO:23所示的HCDR2；以及，如SEQ ID NO:24所示的HCDR3；如SEQ ID NO:18所示的LCDR1；如SEQ ID NO:19所示的LCDR2；以及，如SEQ ID NO:20所示的LCDR3；或，
(ii)如SEQ ID NO:30所示的HCDR1；如SEQ ID NO:31所示的HCDR2；以及，如SEQ ID NO:32所示的HCDR3；如SEQ ID NO:26所示的LCDR1；如SEQ ID NO:27所示的LCDR2；以及，如SEQ ID NO:28所示的LCDR3。
在某些优选的实施方案中，所述第一抗体包含：如SEQ ID NO:6所示的HCDR1；如SEQ ID NO:7所示的HCDR2；以及，如SEQ ID NO:8所示的HCDR3；如SEQ ID NO:2所示的LCDR1；如SEQ ID NO:3所示的LCDR2；以及，如SEQ ID NO:4所示的LCDR3；并且，
所述scFv包含：如SEQ ID NO:22所示的HCDR1；如SEQ ID NO:23所示的HCDR2；以及，如SEQ ID NO:24所示的HCDR3；如SEQ ID NO:18所示的LCDR1；如SEQ ID NO:19所示的LCDR2；以及，如SEQ ID NO:20所示的LCDR3。
在某些优选的实施方案中，所述第一抗体包含：如SEQ ID NO:14所示的HCDR1；如SEQ ID NO:15所示的HCDR2；以及，如SEQ ID NO:16所示的HCDR3；如SEQ ID NO:10所示的LCDR1；如SEQ ID NO:11所示的LCDR2；以及，如SEQ ID NO:12所示的LCDR3；并且，
所述scFv包含：如SEQ ID NO:30所示的HCDR1；如SEQ ID NO:31所示的HCDR2；以及，如SEQ ID NO:32所示的HCDR3；如SEQ ID NO:26所示的LCDR1；如SEQ ID NO:27所示的LCDR2；以及，如SEQ ID NO:28所示的LCDR3。
在某些优选的实施方案中，所述第一抗体包含：如SEQ ID NO:6所示的HCDR1；如SEQ ID NO:7所示的HCDR2；以及，如SEQ ID NO:8所示的HCDR3；如SEQ ID NO:2所示的LCDR1；如SEQ ID NO:3所示的LCDR2；以及，如SEQ ID NO:4所示的LCDR3；并且，
所述scFv包含：如SEQ ID NO:30所示的HCDR1；如SEQ ID NO:31所示的HCDR2；以及，如SEQ ID NO:32所示的HCDR3；如SEQ ID NO:26所示的LCDR1； 如SEQ ID NO:27所示的LCDR2；以及，如SEQ ID NO:28所示的LCDR3。
在某些优选的实施方案中，所述第一抗体包含：如SEQ ID NO:14所示的HCDR1；如SEQ ID NO:15所示的HCDR2；以及，如SEQ ID NO:16所示的HCDR3；如SEQ ID NO:10所示的LCDR1；如SEQ ID NO:11所示的LCDR2；以及，如SEQ ID NO:12所示的LCDR3；并且，
所述scFv包含：如SEQ ID NO:22所示的HCDR1；如SEQ ID NO:23所示的HCDR2；以及，如SEQ ID NO:24所示的HCDR3；如SEQ ID NO:18所示的LCDR1；如SEQ ID NO:19所示的LCDR2；以及，如SEQ ID NO:20所示的LCDR3。
在某些优选的实施方案中，所述重组蛋白包含1个所述第一抗体和2个所述scFv；并且，所述第一抗体包括两条HC和两条LC，其中所述第一抗体的一条HC的VH与一条LC的VL形成抗原结合部位，另一条HC的VH与另一条LC的VL形成抗原结合部位；每个所述scFv分别连接于所述第一抗体的两条重链的N端；且，所述第一抗体包含：如SEQ ID NO:6所示的HCDR1；如SEQ ID NO:7所示的HCDR2；以及，如SEQ ID NO:8所示的HCDR3；如SEQ ID NO:2所示的LCDR1；如SEQ ID NO:3所示的LCDR2；以及，如SEQ ID NO:4所示的LCDR3；并且，所述scFv包含：如SEQ ID NO:22所示的HCDR1；如SEQ ID NO:23所示的HCDR2；以及，如SEQ ID NO:24所示的HCDR3；如SEQ ID NO:18所示的LCDR1；如SEQ ID NO:19所示的LCDR2；以及，如SEQ ID NO:20所示的LCDR3。优选的，所述每个scFv通过接头S1与所述第一抗体的每条重链的N端连接。更优选的，所述scFv的结构为NH 2-VL-S2-VH-COOH，其中所述S2为接头。
在某些优选的实施方案中，所述第一抗体的重链可变区(VH)的氨基酸序列与SEQ ID NO:5所示的重链可变区的氨基酸序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性；并且，所述第一抗体的轻链可变区(VL)的氨基酸序列与SEQ ID NO:1所示的重链可变区的氨基酸序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性；和，
所述scFv的重链可变区(VH)的氨基酸序列与SEQ ID NO:21或SEQ ID NO:91所示的重链可变区的氨基酸序列具有至少90％、至少91％、至少92％、至少93％、至 少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性；并且，所述scFv的的轻链可变区(VL)的氨基酸序列与SEQ ID NO:17或SEQ ID NO:90所示的重链可变区的氨基酸序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性。
在某些优选的实施方案中，所述第一抗体的重链可变区选自SEQ ID NO:5所示的重链可变区；并且，所述第一抗体的轻链可变区选自SEQ ID NO:1所示的轻链可变区；和，
所述scFv的重链可变区选自SEQ ID NO:21或SEQ ID NO:91所示的重链可变区；并且，所述scFv的的轻链可变区选自SEQ ID NO:17或SEQ ID NO:90所示的轻链可变区。
在某些优选的实施方案中，所述第一抗体的重链可变区选自SEQ ID NO:13所示的重链可变区；并且，所述第一抗体的的轻链可变区选自SEQ ID NO:9所示的轻链可变区；和，
所述scFv的重链可变区选自SEQ ID NO:29或SEQ ID NO:93所示的重链可变区；并且，所述scFv的的轻链可变区选自SEQ ID NO:25或SEQ ID NO:92所示的轻链可变区。
在某些优选的实施方案中，所述第一抗体的重链可变区选自SEQ ID NO:5所示的重链可变区；并且，所述第一抗体的的轻链可变区选自SEQ ID NO:1所示的轻链可变区；和，
所述scFv的重链可变区选自SEQ ID NO:29或SEQ ID NO:93所示的重链可变区；并且，所述scFv的的轻链可变区选自SEQ ID NO:25或SEQ ID NO:92所示的轻链可变区。
在某些优选的实施方案中，所述第一抗体的重链可变区选自SEQ ID NO:13所示的重链可变区；并且，所述第一抗体的的轻链可变区选自SEQ ID NO:9所示的轻链可变区；和，
所述scFv的重链可变区选自SEQ ID NO:21或SEQ ID NO:91所示的重链可变区；并且，所述scFv的的轻链可变区选自SEQ ID NO:17或SEQ ID NO:90所示的轻链可变区。
在某些优选的实施方案中，所述第一抗体包含：如SEQ ID NO:5所示的VH和如 SEQ ID NO:1所示的VL；
并且，所述scFv包含：
(1)如SEQ ID NO:21所示的VH和如SEQ ID NO:17所示的VL；或，
(2)如SEQ ID NO:91所示的VH和如SEQ ID NO:90所示的VL。
在某些优选的实施方案中，所述第一抗体包含如SEQ ID NO:5所示的VH和如SEQ ID NO:1所示的VL；并且，所述scFv包含如SEQ ID NO:91所示的VH和如SEQ ID NO:90所示的VL。
在某些优选的实施方案中，所述第一抗体包含如SEQ ID NO:13所示的VH和如SEQ ID NO:9所示的VL；
并且，所述scFv包含：
(1)如SEQ ID NO:29所示的VH和如SEQ ID NO:25所示的VL；或，
(2)如SEQ ID NO:93所示的VH和如SEQ ID NO:92所示的VL。
在某些优选的实施方案中，所述第一抗体包含如SEQ ID NO:5所示的VH和如SEQ ID NO:1所示的VL；并且，所述scFv包含：
(1)如SEQ ID NO:29所示的VH和如SEQ ID NO:25所示的VL；或，
(2)如SEQ ID NO:93所示的VH和如SEQ ID NO:92所示的VL。
在某些优选的实施方案中，所述第一抗体包含如SEQ ID NO:13所示的VH和如SEQ ID NO:9所示的VL；并且，所述scFv包含：
(1)如SEQ ID NO:21所示的VH和如SEQ ID NO:17所示的VL；或，
(2)如SEQ ID NO:91所示的VH和如SEQ ID NO:90所示的VL。
在某些优选的实施方案中，所述第一抗体特异性结合IL-6R，并且所述scFv特异性结合TNFα，其中：
所述第一抗体包含：
(a)如SEQ ID NO:22所示的HCDR1；如SEQ ID NO:23所示的HCDR2；以及，如SEQ ID NO:24所示的HCDR3；如SEQ ID NO:18所示的LCDR1；如SEQ ID NO:19所示的LCDR2；以及，如SEQ ID NO:20所示的LCDR3；或，
(b)如SEQ ID NO:30所示的HCDR1；如SEQ ID NO:31所示的HCDR2；以及，如SEQ ID NO:32所示的HCDR3；如SEQ ID NO:26所示的LCDR1；如SEQ ID NO:27所示的LCDR2；以及，如SEQ ID NO:28所示的LCDR3；
和/或，
所述scFv包含：
(i)如SEQ ID NO:6所示的HCDR1；如SEQ ID NO:7所示的HCDR2；以及，如SEQ ID NO:8所示的HCDR3；如SEQ ID NO:2所示的LCDR1；如SEQ ID NO:3所示的LCDR2；以及，如SEQ ID NO:4所示的LCDR3；或，
(ii)如SEQ ID NO:14所示的HCDR1；如SEQ ID NO:15所示的HCDR2；以及，如SEQ ID NO:16所示的HCDR3；如SEQ ID NO:10所示的LCDR1；如SEQ ID NO:11所示的LCDR2；以及，如SEQ ID NO:12所示的LCDR3。
在某些优选的实施方案中，所述第一抗体包含：如SEQ ID NO:22所示的HCDR1；如SEQ ID NO:23所示的HCDR2；以及，如SEQ ID NO:24所示的HCDR3；如SEQ ID NO:18所示的LCDR1；如SEQ ID NO:19所示的LCDR2；以及，如SEQ ID NO:20所示的LCDR3；并且，
所述scFv包含：如SEQ ID NO:6所示的HCDR1；如SEQ ID NO:7所示的HCDR2；以及，如SEQ ID NO:8所示的HCDR3；如SEQ ID NO:2所示的LCDR1；如SEQ ID NO:3所示的LCDR2；以及，如SEQ ID NO:4所示的LCDR3。
在某些优选的实施方案中，所述第一抗体包含：如SEQ ID NO:30所示的HCDR1；如SEQ ID NO:31所示的HCDR2；以及，如SEQ ID NO:32所示的HCDR3；如SEQ ID NO:26所示的LCDR1；如SEQ ID NO:27所示的LCDR2；以及，如SEQ ID NO:28所示的LCDR3；并且，
所述scFv包含：如SEQ ID NO:14所示的HCDR1；如SEQ ID NO:15所示的HCDR2；以及，如SEQ ID NO:16所示的HCDR3；如SEQ ID NO:10所示的LCDR1；如SEQ ID NO:11所示的LCDR2；以及，如SEQ ID NO:12所示的LCDR3。
在某些优选的实施方案中，所述第一抗体包含：如SEQ ID NO:22所示的HCDR1；如SEQ ID NO:23所示的HCDR2；以及，如SEQ ID NO:24所示的HCDR3；如SEQ ID NO:18所示的LCDR1；如SEQ ID NO:19所示的LCDR2；以及，如SEQ ID NO:20所示的LCDR3；并且，
所述scFv包含：如SEQ ID NO:14所示的HCDR1；如SEQ ID NO:15所示的HCDR2；以及，如SEQ ID NO:16所示的HCDR3；如SEQ ID NO:10所示的LCDR1；如SEQ ID NO:11所示的LCDR2；以及，如SEQ ID NO:12所示的LCDR3。
在某些优选的实施方案中，所述第一抗体包含：如SEQ ID NO:30所示的HCDR1；如SEQ ID NO:31所示的HCDR2；以及，如SEQ ID NO:32所示的HCDR3；如SEQ ID NO:26所示的LCDR1；如SEQ ID NO:27所示的LCDR2；以及，如SEQ ID NO:28所示的LCDR3；并且，
所述scFv包含：如SEQ ID NO:6所示的HCDR1；如SEQ ID NO:7所示的HCDR2；以及，如SEQ ID NO:8所示的HCDR3；如SEQ ID NO:2所示的LCDR1；如SEQ ID NO:3所示的LCDR2；以及，如SEQ ID NO:4所示的LCDR3。
在某些优选的实施方案中，所述第一抗体的重链可变区(VH)的氨基酸序列与SEQ ID NO:21所示的重链可变区的氨基酸序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性；并且，所述第一抗体的的轻链可变区(VL)的氨基酸序列与SEQ ID NO:17所示的重链可变区的氨基酸序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性；和，
所述scFv的重链可变区(VH)的氨基酸序列与SEQ ID NO:5或SEQ ID NO:87所示的重链可变区的氨基酸序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性；并且，所述scFv的的轻链可变区(VL)的氨基酸序列与SEQ ID NO:1或SEQ ID NO:86所示的重链可变区的氨基酸序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性。
在某些优选的实施方案中，所述第一抗体的重链可变区选自SEQ ID NO:21所示的重链可变区；并且，所述第一抗体的的轻链可变区选自SEQ ID NO:17示的轻链可变区；和，
所述scFv的重链可变区选自SEQ ID NO:5或SEQ ID NO:87所示的重链可变区；并且，所述scFv的的轻链可变区选自SEQ ID NO:1或SEQ ID NO:86所示的轻链可变区。
在某些优选的实施方案中，所述第一抗体的重链可变区选自SEQ ID NO:29所示的重链可变区；并且，所述第一抗体的的轻链可变区选自SEQ ID NO:25所示的轻链可变 区；和，
所述scFv的重链可变区选自SEQ ID NO:13或SEQ ID NO:89所示的重链可变区；并且，所述scFv的的轻链可变区选自SEQ ID NO:9或SEQ ID NO:88所示的轻链可变区。
在某些优选的实施方案中，所述第一抗体的重链可变区选自SEQ ID NO:29所示的重链可变区；并且，所述第一抗体的的轻链可变区选自SEQ ID NO:25所示的轻链可变区；和，
所述scFv的重链可变区选自SEQ ID NO:5或SEQ ID NO:87所示的重链可变区；并且，所述scFv的的轻链可变区选自SEQ ID NO:1或SEQ ID NO:86所示的轻链可变区。
在某些优选的实施方案中，所述第一抗体的重链可变区选自SEQ ID NO:21所示的重链可变区；并且，所述第一抗体的的轻链可变区选自SEQ ID NO:17所示的轻链可变区；和，
所述scFv的重链可变区选自SEQ ID NO:13或SEQ ID NO:89所示的重链可变区；并且，所述scFv的的轻链可变区选自SEQ ID NO:9或SEQ ID NO:88所示的轻链可变区。
在某些优选的实施方案中，所述第一抗体包含：如SEQ ID NO:21所示的VH和如SEQ ID NO:17所示的VL；
并且，所述scFv包含：
(1)如SEQ ID NO:5所示的VH和如SEQ ID NO:1所示的VL；或，
(2)如SEQ ID NO:87所示的VH和如SEQ ID NO:86所示的VL。
在某些优选的实施方案中，所述第一抗体包含如SEQ ID NO:21所示的VH和如SEQ ID NO:17所示的VL；并且，所述scFv包含如SEQ ID NO:87所示的VH和如SEQ ID NO:86所示的VL。
在某些优选的实施方案中，所述第一抗体包含：如SEQ ID NO:29所示的VH和如SEQ ID NO:25所示的VL；并且，所述scFv包含：
(1)如SEQ ID NO:13所示的VH和如SEQ ID NO:9所示的VL；或，
(2)如SEQ ID NO:89所示的VH和如SEQ ID NO:88所示的VL。
在某些优选的实施方案中，所述第一抗体包含如SEQ ID NO:21所示的VH和如 SEQ ID NO:17所示的VL；并且，所述scFv包含如SEQ ID NO:87所示的VH和如SEQ ID NO:86所示的VL。
在某些优选的实施方案中，所述第一抗体包含如SEQ ID NO:29所示的VH和如SEQ ID NO:25所示的VL；
并且，所述scFv包含：
(1)如SEQ ID NO:5所示的VH和如SEQ ID NO:1所示的VL；或，
(2)如SEQ ID NO:87所示的VH和如SEQ ID NO:86所示的VL。
在某些优选的实施方案中，所述第一抗体包含如SEQ ID NO:21所示的VH和如SEQ ID NO:17所示的VL；
并且，所述scFv包含：
(1)如SEQ ID NO:13所示的VH和如SEQ ID NO:9所示的VL；或，
(2)如SEQ ID NO:89所示的VH和如SEQ ID NO:88所示的VL。
在某些优选的实施方案中，所述第一抗体包含：
(1)如SEQ ID NO:101所示的CH；和/或，
(2)如SEQ ID NO:100所示的CL。
在某些优选的实施方案中，所述重组蛋白包含两条相同的第一多肽链和两条相同的第二多肽链。
在某些优选的实施方案中，所述第一多肽链具有选自下列的氨基酸序列：SEQ ID NOs:36，38，44，46，50，52，58，60，64，66，70，76，94,95,96,97,98和99任一项所示的氨基酸序列；和/或，所述第二多肽链具有选自下列的氨基酸序列：SEQ ID NOs:42，48，56，68，74和80任一项所示的氨基酸序列。
在某些优选的实施方案中，所述重组蛋白包含：
(1)如SEQ ID NO:36所示的第一多肽链和如SEQ ID NO:42所示的第二多肽链；
(2)如SEQ ID NO:38所示的第一多肽链和如SEQ ID NO:42所示的第二多肽链；
(3)如SEQ ID NO:44所示的第一多肽链和如SEQ ID NO:48所示的第二多肽链；
(4)如SEQ ID NO:46所示的第一多肽链和如SEQ ID NO:48所示的第二多肽链；
(5)如SEQ ID NO:50所示的第一多肽链和如SEQ ID NO:56所示的第二多肽链；
(6)如SEQ ID NO:52所示的第一多肽链和如SEQ ID NO:56所示的第二多肽链；
(7)如SEQ ID NO:58所示的第一多肽链和如SEQ ID NO:56所示的第二多肽链；
(8)如SEQ ID NO:60所示的第一多肽链和如SEQ ID NO:56所示的第二多肽链；
(9)如SEQ ID NO:64所示的第一多肽链和如SEQ ID NO:68所示的第二多肽链；
(10)如SEQ ID NO:66所示的第一多肽链和如SEQ ID NO:68所示的第二多肽链；
(11)如SEQ ID NO:94所示的第一多肽链和如SEQ ID NO:68所示的第二多肽链；
(12)如SEQ ID NO:95所示的第一多肽链和如SEQ ID NO:68所示的第二多肽链；
(13)如SEQ ID NO:96所示的第一多肽链和如SEQ ID NO:42所示的第二多肽链；
(14)如SEQ ID NO:97所示的第一多肽链和如SEQ ID NO:42所示的第二多肽链；
(15)如SEQ ID NO:98所示的第一多肽链和如SEQ ID NO:48所示的第二多肽链；
(16)如SEQ ID NO:99所示的第一多肽链和如SEQ ID NO:48所示的第二多肽链；
(17)如SEQ ID NO:70所示的第一多肽链和如SEQ ID NO:74所示的第二多肽链；或
(18)如SEQ ID NO:76所示的第一多肽链和如SEQ ID NO:80所示的第二多肽链。
在某些实施方案中，本发明的重组蛋白具有与所述第一抗体的亲本抗体相比同等的抗体依赖性细胞介导的细胞毒性(ADCC)活性。在某些实施方案中，本发明的重组蛋白具有与所述第一抗体的亲本抗体相比同等的抗体依赖性细胞介导的细胞毒性(ADCC)活性，并且还具有与所述第一抗体的亲本抗体相比同等的补体依赖的细胞毒性(CDC)活性。
另一方面，本发明的重组蛋白对TNFα和IL-6R具有亲和力。在某些优选的实施方案中，本发明的重组蛋白对TNFα和IL-6R具有与其各自的亲本抗体相比同等或者更高的亲和力。在某些优选的实施方案中，本发明的重组蛋白对细胞表面的TNFα和IL-6R具有与其各自的亲本抗体相比更高的亲和力。
另一方面，本发明的重组蛋白具有良好的热稳定性。在某些优选的实施方案中，本发明的重组蛋白具有与亲本抗体相比基本相同的热稳定性。
重组蛋白的表达
在另一个方面，本发明提供了一种分离的核酸分子，其包含编码本发明的重组蛋白的核苷酸序列。在某些优选的实施方案中，所述分离的核酸分子编码本发明的重组蛋白。
在某些优选的实施方案中，所述分离的核酸分子包含编码本发明的第一多肽链的核苷酸序列。在某些优选的实施方案中，所述分离的核酸分子包含编码本发明的第二多肽链的核苷酸序列。在某些优选的实施方案中，所述分离的核酸分子包含编码本发明的第一多肽链的核苷酸序列和编码本发明的第二多肽链的核苷酸序列。
在另一个方面，本发明提供了一种载体(例如克隆载体或表达载体)，其包含本发明的分离的核酸分子。
在某些优选的实施方案中，所述载体包含编码本发明的第一多肽链的核苷酸序列。在某些优选的实施方案中，所述载体包含编码本发明的第二多肽链的核苷酸序列。在某些优选的实施方案中，所述载体包含编码本发明的第一多肽链的核苷酸序列和编码本发明的第二多肽链的核苷酸序列。
在某些优选的实施方案中，本发明的载体是例如质粒，粘粒，噬菌体等。在某些优选的实施方案中，所述载体能够在受试者(例如哺乳动物，例如人)体内表达本发明的重组蛋白、第一多肽链或第二多肽链。
在另一个方面，本发明提供了一种宿主细胞，其包含本发明的分离的核酸分子或本发明的载体。此类宿主细胞包括但不限于，原核细胞例如大肠杆菌细胞，以及真核细胞例如酵母细胞，昆虫细胞，植物细胞和动物细胞(如哺乳动物细胞，例如小鼠细胞、人细胞等)。在某些优选的实施方案中，本发明的宿主细胞是哺乳动物细胞，例如CHO(例如CHO-K1、CHO-S、CHO DG44)或HEK293。
在另一个方面，本发明提供了制备本发明的重组蛋白的方法，其包括，在允许所述重组蛋白表达的条件下，培养本发明的宿主细胞，和从培养的宿主细胞培养物中回收所述重组蛋白。
在某些优选的实施方案中，所述方法包括：
(1)构建包含编码第一多肽链的核苷酸序列和编码第二多肽链的核苷酸序列的表达载体；或，构建包含编码第一多肽链的核苷酸序列的第一表达载体和包含编码第二多肽链的核苷酸序列的第二表达载体；
(2)将步骤(1)中所述的表达载体转化至宿主细胞；或，将步骤(1)中所述的第一表达载体和第二表达载体转化至宿主细胞；
(3)在允许本发明的重组蛋白表达的条件下，培养步骤(2)中所述的宿主细胞；和
(4)从培养的宿主细胞培养物中回收所述重组蛋白。
治疗方法和药物组合物
本发明的重组蛋白可用于体外或在受试者体内中抑制TNFα和IL-6/IL-6R的活性，阻断TNFα和/或IL-6信号通路，以及用于预防和/或治疗与过度的TNFα和/或IL-6活性相关的疾病(例如炎性疾病或自身免疫性疾病)。
因此，在另一个方面，本发明提供了一种药物组合物，其含有本发明的重组蛋白，以及药学上可接受的载体和/或赋形剂。在某些优选的实施方案中，所述药物组合物还可以包含另外的药学活性剂。在某些优选的实施方案中，所述另外的药学活性剂是用于预防和/或治疗与过度的TNFα和/或IL-6活性相关的疾病(例如炎性疾病或自身免疫性疾病)的药物，例如抗炎药物或免疫抑制剂，例如非甾体抗炎药(如布洛芬、双氯芬酸、萘普生、吲哚美辛、吡罗昔康、美洛昔康、萘丁美酮或尼美舒利)、甾体抗炎药(如强的松、地塞米松或氢化考的松)、致炎性细胞因子的抗体或拮抗剂(例如，TNFα、IL-1、IL-6、IL-8、GM-CSF或PAF的抗体或受体拮抗剂)、抗炎细胞因子(如IL-10、IL-4、IL-11、IL-13或TGFβ)、抗增殖/抗代谢类药物(如环磷酰胺、甲氨蝶呤、硫唑嘌呤、莱氟米特)、钙调磷酸酶抑制剂(如环孢素、他克莫司)、天然提取物(如白芍总苷、雷公藤总苷、青藤碱)等。
在另一个方面，本发明提供了本发明的重组蛋白或本发明的药物组合物在制备药物中的用途，所述药物用于在受试者(例如人)中预防和/或治疗与过度的TNFα和/或IL-6活性相关的疾病(例如炎性疾病或自身免疫性疾病)，和/或用于体外或在受试者(例如人)体内抑制TNFα和IL-6/IL-6R的活性。
在另一个方面，本发明提供了一种用于在受试者(例如人)中预防和/或治疗与过度的TNFα和/或IL-6活性相关的疾病(例如炎性疾病或自身免疫性疾病)，和/或用于体外或在受试者(例如人)体内抑制TNFα和IL-6/IL-6R的活性的方法，其中所述方法包括，给有此需要的受试者施用有效量的本发明的重组蛋白，或者本发明的药物组合物。
在本发明中，所述与过度的TNFα和/或IL-6活性相关的疾病包括但不限于炎性疾病或自身免疫性疾病，例如，类风湿性关节炎、胰岛素依赖型糖尿病、多发性硬化症、银屑病、炎症性肠病、溃疡性结肠炎、胰腺炎、重症肌无力、多发性肌炎、皮肌炎、Crohn病、自身免疫性血细胞减少、血管炎、系统性红斑狼疮或成人Still病等。
本发明的重组蛋白或者本发明的药物组合物可以配制成医学领域已知的任何剂型，例如，片剂、丸剂、混悬剂、乳剂、溶液、凝胶剂、胶囊剂、粉剂、颗粒剂、酏剂、锭剂、栓剂、注射剂(包括注射液、注射用无菌粉末与注射用浓溶液)、吸入剂、喷雾剂等。 优选剂型取决于预期的给药方式和治疗用途。本发明的药物组合物应当是无菌的并在生产和储存条件下稳定。一种优选的剂型是注射剂。此类注射剂可以是无菌注射溶液。例如，可通过下述方法来制备无菌注射溶液：在适当的溶剂中掺入必需剂量的本发明的重组蛋白，以及任选地，同时掺入其他期望的成分(包括但不限于，pH调节剂，表面活性剂，佐剂，离子强度增强剂，等渗剂、防腐剂、稀释剂，或其任何组合)，随后过滤除菌。此外，可以将无菌注射溶液制备为无菌冻干粉剂(例如，通过真空干燥或冷冻干燥)以便于储存和使用。此类无菌冻干粉剂可在使用前分散于合适的载体中，例如无菌无热原水。
此外，本发明的重组蛋白可以以单位剂量形式存在于药物组合物中，以便于施用。在某些实施方案中，所述单位剂量为至少1mg，至少5mg，至少10mg，至少15mg，至少20mg，至少25mg，至少30mg，至少45mg，至少50mg，至少75mg，或至少100mg。在所述药物组合物为液体(例如，注射剂)剂型的情况下，其可以包含浓度至少为0.1mg/ml，如至少0.25mg/ml，至少0.5mg/ml，至少1mg/ml，至少2.5mg/ml，至少5mg/ml，至少8mg/ml，至少10mg/ml，至少15mg/ml，至少25mg/ml，至少50mg/ml，至少75mg/ml，或至少100mg/ml的本发明的重组蛋白。
本发明的重组蛋白或药物组合物可以通过本领域已知的任何合适的方法来施用，包括但不限于，口服、口腔、舌下、眼球、局部、肠胃外、直肠、叶鞘内、内胞浆网槽内、腹股沟、膀胱内、局部(如，粉剂、药膏或滴剂)，或鼻腔途径。但是，对于许多治疗用途而言，优选的给药途径/方式是胃肠外给药(例如静脉注射，皮下注射，腹膜内注射，肌内注射)。技术人员应理解，给药途径和/或方式将根据预期目的而发生变化。在一个优选的实施方案中，本发明的重组蛋白或药物组合物通过静脉输注或注射给予。
本发明所提供的药物、药物组合物或重组蛋白可以单独使用或联合使用，也可以与另外的药学活性剂(例如抗炎药物或免疫抑制剂)联合使用。在某些优选的实施方案中，将本发明的重组蛋白与其它抗炎药物或免疫抑制剂联合使用，以预防和/或治疗与过度的TNFα和/或IL-6活性相关的疾病(例如炎性疾病或自身免疫性疾病)。这种另外的药学活性剂可以在施用本发明的重组蛋白或本发明的药物组合物之前、同时或之后施用。
本发明的药物组合物可以包括“治疗有效量”或“预防有效量”的本发明的重组蛋白。“预防有效量”是指，足以预防，阻止，或延迟疾病(例如与过度的TNFα和/或IL-6活性相关的疾病)的发生的量。“治疗有效量”是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。本发明的重组蛋白的治疗有效量可根据如下因素发生变化：待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄， 体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
在本发明中，可调整给药方案以获得最佳目的反应(例如治疗或预防反应)。例如，可以单次给药，可以在一段时间内多次给药，或者可以随治疗情况的紧急程度按比例减少或增加剂量。
本发明的重组蛋白的治疗或预防有效量的典型非极限范围是0.02～50mg/kg，例如0.1～50mg/kg，0.1～25mg/kg，或1～10mg/kg。应注意的是，剂量可随需要治疗的症状的类型和严重性不同而发生变化。此外，本领域技术人员理解，对于任一特定患者，特定的给药方案应根据患者需要和医生的专业评价而随时间调整；此处给出的剂量范围只用于举例说明目的，而不限定本发明药物组合物的使用或范围。
在本发明中，所述受试者可以为哺乳动物，例如人。
检测/诊断方法和试剂盒
本发明的重组蛋白能够特异性结合TNFα/IL-6或TNFα/IL-6R，从而可用于检测TNFα/IL-6或TNFα/IL-6R在样品中的存在或其水平，以及诊断受试者是否患有与过度的TNFα和/或IL-6活性相关的疾病(例如炎性疾病或自身免疫性疾病)。
因此，在另一个方面，本发明提供了一种试剂盒，其包括本发明的重组蛋白。在某些优选的实施方案中，本发明的重组蛋白带有可检测的标记。在一个优选的实施方案中，所述试剂盒还包括第二抗体，其特异性识别本发明的重组蛋白的第一抗体或scFv。优选地，所述第二抗体还包括可检测的标记。
在本发明中，所述可检测的标记可以是可通过荧光、光谱、光化学、生物化学、免疫学、电学、光学或化学手段检测的任何物质。特别优选的是，此类标记能够适用于免疫学检测(例如，酶联免疫测定法、放射免疫测定法、荧光免疫测定法、化学发光免疫测定法等)。这类标记是本领域熟知的，包括但不限于，酶(例如，辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、脲酶、葡萄糖氧化酶，等)、放射性核素(例如， 3H、 125I、 35S、 14C或 32P)、荧光染料(例如，异硫氰酸荧光素(FITC)、荧光素、异硫氰酸四甲基罗丹明(TRITC)、藻红蛋白(PE)、德克萨斯红、罗丹明、量子点或花菁染料衍生物(例如Cy7、Alexa 750))、发光物质(例如化学发光物质，如吖啶酯类化合物)、磁珠(例如，
Figure PCTCN2018094447-appb-000001
)、测热标记物例如胶体金或有色玻璃或塑料(例如，聚苯乙烯、聚丙烯、乳胶，等)珠、以及用于结合上述标记物修饰的亲和素(例如，链霉亲和素)的生物素。教导该标记物的使用的专利包括，但不限于，美国专利3,817,837；3,850,752； 3,939,350；3,996,345；4,277,437；4,275,149；及4,366,241(全部通过引用并入本文)。本发明中涵盖的标记物可通过本领域已知的方法检测。例如，放射性标记可使用摄影胶片或闪烁计算器检测，荧光标记物可使用光检测器检测，以检测发射的光。酶标记物一般通过给酶提供底物及检测通过酶对底物的作用产生的反应产物来检测，及测热标记物通过简单可视化着色标记物来检测。在某些实施方案中，可通过不同长度的接头将如上所述的可检测的标记连接至本发明的重组蛋白，以降低潜在的位阻。
在另一个方面，本发明提供了检测TNFα/IL-6或TNFα/IL-6R在样品中的存在或其水平的方法，其包括使用本发明的重组蛋白的步骤。在一个优选的实施方案中，本发明的重组蛋白还带有可检测的标记。在另一个优选的实施方案中，所述方法还包括，使用带有可检测的标记的试剂来检测本发明的重组蛋白段。所述方法可以用于诊断目的，或者非诊断目的(例如，所述样品是细胞样品，而非来自患者的样品)。
在另一个方面，本发明提供了诊断受试者是否患有与过度的TNFα和/或IL-6活性相关的疾病(例如炎性疾病或自身免疫性疾病)的方法，其包括：使用本发明的重组蛋白检测TNFα和/或IL-6在来自所述受试者的样品中的存在或其水平。在一个优选的实施方案中，本发明的重组蛋白还带有可检测的标记。在另一个优选的实施方案中，所述方法还包括，使用带有可检测的标记的试剂来检测本发明的重组蛋白的步骤。
在另一个方面，提供了本发明的重组蛋白在制备试剂盒中的用途，所述试剂盒用于检测TNFα和/或IL-6在样品中的存在或其水平，或用于诊断受试者是否患有与过度的TNFα和/或IL-6活性相关的疾病(例如炎性疾病或自身免疫性疾病)。
术语定义
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、生物化学、核酸化学、免疫学实验室等操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，术语“抗体”是指，通常由两对多肽链(每对具有一条轻链(LC)和一条重链(HC))组成的免疫球蛋白分子。抗体轻链可分类为κ(kappa)和λ(lambda)轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链 可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。抗体的恒定区可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各V H和V L由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗原结合部位。氨基酸至各区域或结构域的分配遵循Kabat,Sequences of Proteins of Immunological Interest(National Institutes of Health,Bethesda,Md.(1987 and 1991))，或Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883的定义。
如本文中所使用的，术语“互补决定区”或“CDR”是指抗体可变区中负责抗原结合的氨基酸残基，其通常可包括，轻链可变区中的残基24-34{LCDR1}、50-56{LCDR2}、89-97{LCDR3}以及重链可变区中的残基31-35{HCDR1}、50-65{HCDR2}、95-102{HCDR3}(参见例如，Kabat等，Sequences of Proteins of lmmunological lnterest，第五版，Public Health Service，美国国立卫生研究院，贝塞斯达、马里兰州(1991))，或者轻链可变区中的残基26-32{Ll}、50-52{L2}、91-96{L3}以及重链可变区中的残基26-32{H1}、53-55{H2}、96-101{H3}(参见，Chothia和Lesk J.Mol.Biol.196:901-917(1987))。
如本文中所使用的，术语“构架区”或“FR”残基是指，抗体可变区中除了如上定义的CDR残基以外的那些氨基酸残基。
术语“抗体”不受任何特定的产生抗体的方法限制。例如，其包括，重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
如本文中所使用的，术语“抗原结合部位”是指，由重链以及轻链的可变区(VH和VL)的氨基酸残基所形成的、参与进行抗原结合的部分，其包含那些与抗原相互作用且决定了该抗体对抗原的特异性及亲和力的氨基酸残基。所述抗原结合部位还可以包含那些对维持上述直接结合抗原的氨基酸残基的合适构象所必需的框架区的氨基酸残基。
如本文中所使用的，术语“全长抗体”意指，由两条“全长重链”和两条“全长轻链”组成的抗体。其中，“全长重链”是指这样的多肽链，其在N端到C端的方向上由重链可变区(VH)、重链恒定区CH1结构域、铰链区(HR)、重链恒定区CH2结构域、重链恒定区CH3结构域组成；并且，当所述全长抗体为IgE同种型时，任选地还包括重链恒定区CH4结构域。优选地，“全长重链”是在N端到C端方向上由VH、CH1、HR、CH2和CH3组成的多肽链。“全长轻链”是在N端到C端方向上由轻链可变区(VL)和轻链恒定区(CL)组成的多肽链。两对全长抗体链通过在CL和CH1之间的二硫键和两条全长重链的HR之间的二硫键连接在一起。本发明的全长抗体可以来自单一物种，例如人；也可以是嵌合抗体或人源化抗体。本发明的全长抗体包含分别由VH和VL对形成的两个抗原结合部位，这两个抗原结合部位特异性识别/结合相同的抗原。
如本文中所使用的，术语“Fab片段”意指由VL、VH、CL和CH1结构域组成的抗体片段；术语“Fab'片段”意指还原连接F(ab') 2片段中两个重链片段的二硫键后所获片段，由一条完整的轻链和重链的Fd片段(由VH和CH1结构域组成)组成；术语“F(ab') 2片段”意指包含通过铰链区上的二硫桥连接的两个Fab片段的抗体片段。上述各个抗体片段均保持了特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合。
如本文中所使用的，术语“scFv”是指，包含VL和VH结构域的单个多肽链，其中所述VL和VH通过接头(linker)相连(参见，例如,Bird等人,Science 242:423-426(1988)；Huston等人,Proc.Natl.Acad.Sci.USA 85:5879-5883(1988)；和Pluckthun，The Pharmacology of Monoclonal Antibodies，第113卷，Roseburg和Moore编，Springer-Verlag，纽约，第269-315页(1994))。此类scFv分子可具有一般结构：NH 2-VL-接头-VH-COOH或NH 2-VH-接头-VL-COOH。本发明的scFv的VH与VL之间还可以存在二硫键。在抗体的VH和VL之间引入二硫键的方法是本领域熟知的，例如可参见美国专利申请US5,747,654；Rajagopal等人，Prot.Engin.10(1997)1453-1459；Reiter等人，Nature Biotechnology 14(1996)1239-1245；Reiter等人，Protein Engineering 8(1995)1323-1331；Webber等人，Molecular Immunology 32(1995)249-258；Reiter等人，Immunity 2(1995)281-287；Reiter等人，JBC 269(1994)18327-18331；Reiter等人，Inter.J.of Cancer 58(1994)142-149；或，Reiter等人，Cancer Res.54(1994)2714-2718；其通过引用并入本文。如本文中所使用的，术语“di-scFv”是指，由两个scFv连接形成的抗体片段。
如本文中所使用的，术语“Fv片段”意指由抗体的单臂的VL和VH结构域组成的抗体片段。
如本文中所使用的，术语“亲本抗体”是指，用于制备本发明的重组蛋白的抗TNFα抗体或抗IL-6R/IL-6抗体，该抗体所具有的氨基酸序列可通过例如氨基酸置换或结构改变等方式以用于制备本发明的重组蛋白所包含的第一抗体或scFv。在本发明中，“抗TNFα亲本抗体”可以为重链和轻链可变区分别如SEQ ID NOs:1和5所示的抗体或分别如SEQ ID NOs:9和13所示的抗体；“抗IL-6R亲本抗体”可以为重链和轻链可变区分别如SEQ ID NOs:17和21所示的抗体或分别如SEQ ID NOs:25和29所示的抗体。
本发明的重组蛋白所包含的CDR、VH、VL、CH、CL、HC、LC还可以来自其他被本领域所知晓的能够特异性结合TNFα或IL-6R或IL-6的抗体或其抗体片段，或者与上述已知抗体、其抗体片段或其CDR、VH、VL、CH、CL、HC、LC具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的抗体。作为示例，所述能够特异性结合TNFα的抗体或蛋白包括：Infliximab，Etanercept，Adalimumab，Certolizumab pegol，Golimumab，Tasonermin，TNF Kinoid，ESBA105，afelimomab，lenercept，nerelimomab，onercept，ozoralizumab，pegsunercept，placulumab，tulinercept；所述能够特异性结合IL-6R或IL-6的抗体或蛋白包括：clazakizumab，elsilimomab，olamkicept，olokizumab，siltuximab，sirukumab，Tocilizumab，olamkicept，satralizumab，sarilumab，vobarilizumab。
如本文中所使用的，术语“接头”是指，由多个氨基酸残基通过肽键连接形成的线性多肽。本发明的接头可以为人工合成的氨基酸序列，或天然存在的多肽序列，例如具有铰链区功能的多肽。此类接头多肽是本领域众所周知的(参见例如，Holliger,P.等人(1993)Proc.Natl.Acad.Sci.USA 90:6444-6448；Poljak,R.J.等人(1994)Structure 2:1121-1123)。
如本文中所使用的，术语“特异性结合”是指，两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。在某些实施方式中，特异性结合某抗原的抗体(或对某抗原具有特异性的抗体)是指，抗体以小于大约10 -5M，例如小于大约10 -6M、10 - 7M、10 -8M、10 -9M或10 -10M或更小的亲和力(K D)结合该抗原。在本发明中，术语“K D”是指特定抗体-抗原相互作用的解离平衡常数，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。 通常，抗体(例如，本发明的抗体)以小于大约10 -5M，例如小于大约10 -6M、10 -7M、10 -8M、10 -9M或10 -10M或更小的解离平衡常数(K D)结合抗原(例如，HBsAg)，例如，如使用表面等离子体共振术(SPR)在BIACORE仪中测定的。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞。
如本文中所使用的，术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如，如果两个序列的10个位置中有6个匹配，那么这两个序列具有60％的同一性。例如，DNA序列CTGACT和CAGGTT共有50％的同一性(总共6个位置中有3个位置匹配)。通常，在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用，例如，可通过计算机程序例如Align程序(DNAstar,Inc.)方便地进行的Needleman等人(1970)J.Mol.Biol.48：443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.Appl Biosci.，4:11-17(1988))的算法，使用PAM120权重残基表(weight residue table)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外，可使用已整合入 GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J MoI Biol.48:444-453(1970))算法，使用Blossum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。
本文涉及的二十个常规氨基酸的编写遵循常规用法。参见例如，Immunology-A Synthesis(2nd Edition,E.S.Golub and D.R.Gren,Eds.,Sinauer Associates,Sunderland,Mass.(1991))，其以引用的方式并入本文中。在本发明中，术语“多肽”和“蛋白质”具有相同的含义且可互换使用。并且在本发明中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。
如本文中所使用的，术语“药学上可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂，稀释剂，维持渗透压的试剂，延迟吸收的试剂，防腐剂。例如，pH调节剂包括但不限于磷酸盐缓冲液。表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80。离子强度增强剂包括但不限于氯化钠。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。维持渗透压的试剂包括但不限于糖、NaCl及其类似物。延迟吸收的试剂包括但不限于单硬脂酸盐和明胶。
如本文中使用的，术语“受试者”是指哺乳动物，例如灵长类哺乳动物，例如人。在某些实施方式中，所述受试者(例如人)患有与过度的TNFα和/或IL-6活性相关的疾病，或者，具有患有上述疾病的风险。通常而言，这类疾病或疾病状态的特征是其将受益于TNFα和/或IL-6水平的降低或TNFα和/或IL-6活性的抑制从而得到缓解或治愈。
如本文中所使用的，术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病(例如与过度的TNFα和/或IL-6活性相关的疾病)有效量是指，足以预防，阻止，或延迟疾病(例如与过度的TNFα和/或IL-6活性相关的疾病)的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
如本文中所使用的，术语“抗体依赖性细胞介导的细胞毒性(ADCC)”是指，一种细胞毒性形式，Ig通过与细胞毒性细胞(例如自然杀伤(NK)细胞、中性粒细胞或巨噬细胞)上存在的Fc受体(FcR)结合，使这些细胞毒性效应细胞特异性结合到抗原附着的靶细胞上，然后通过分泌细胞毒素杀死靶细胞。检测抗体的ADCC活性的方法是本领域已知的，例如可通过测定待测抗体与Fc受体(例如CD16a)之间的结合活性来评价。
如本文中所使用的，术语“补体依赖的细胞毒性(CDC)”是指，通过使补体成分C1q与抗体Fc结合来激活补体级联的细胞毒性形式。检测抗体的CDC活性的方法是本领域已知的，例如可通过测定待测抗体与Fc受体(例如C1q)之间的结合活性来评价。
发明的有益效果
与现有技术相比，本发明的技术方案至少具有以下有益效果：
本发明的重组蛋白不仅能够特异性识别/结合TNFα和IL-6R，而且其对TNFα和IL-6R的亲和力至少与其各自的亲本抗体相当，在体外和受试者体内能够显著且同时抑制TNFα和IL-6R的活性，阻断TNFα和IL-6信号通路。并且，本发明的重组蛋白还具有与第一抗体的亲本抗体同等优良的热稳定性。特别地，在体内实验中，本发明的重组蛋白展现出了良好的治疗活性。与亲本抗体相比，本发明双特异性重组蛋白具有更优的治疗活性。因此，本发明的重组蛋白具有用于治疗与过度的TNFα和IL-6活性相关的疾病(例如炎性疾病或自身免疫性疾病)的潜力，具有重大的临床价值。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得可实施。
附图说明
图1显示了抗TNFα和IL-6R重组蛋白的构建体的示意图。
图2A-2B显示了ANT、ACT、TNA、TCA、ALNT、ALCT的还原与非还原SDS-PAGE电泳图。图2A，泳道：1.蛋白marker；2.AB01非还原；3.AB01还原；4.ANT非 还原；5.ANT还原；6.ACT非还原；7.ACT还原；8.TNA非还原；9.TNA还原。图2B，泳道：1.蛋白marker；2.AB01非还原；3.AB01还原；4.TCA非还原；5.TCA还原；6.ALNT非还原；7.ALNT还原；8.ALCT非还原；9.ALCT还原。结果显示，各重组蛋白条带大小符合预期，且无明显聚集降解，表明各双特异性重组蛋白均能高效表达，正确装配。
图3显示了在实施例7中，抗TNFα和IL-6R重组蛋白抑制IL-6诱导的细胞增殖的检测结果。结果显示，抗TNFα和IL-6R重组蛋白可明显抑制由IL-6诱导的U266细胞增殖，且与亲本抗体的效果一致。
图4显示了实施例11中恒河猴关节炎模型的关节肿胀示意图。
图5显示了实施例11中恒河猴关节炎模型的体重变化曲线，箭头指示给药时间点。
图6显示了实施例11中恒河猴关节炎模型的临床评分变化曲线，箭头指示给药时间点。
图7显示了实施例11中恒河猴关节炎模型的血清CRP水平变化曲线，箭头指示给药时间点。
图8显示了实施例11中恒河猴关节炎模型的血液学RBC变化曲线，箭头指示给药时间点。其中，正常参考值范围为4.17-6.27。
图9显示了实施例11中恒河猴关节炎模型的血液学HGB变化曲线，箭头指示给药时间点。其中，正常参考值范围为111.7-152.5。
图10显示了实施例11中恒河猴关节炎模型的血液学HCT变化曲线，箭头指示给药时间点。其中，正常参考值范围为35.41-49.97。
序列信息
本发明涉及的部分序列的信息提供于下面的表1中。
表1：序列的描述
Figure PCTCN2018094447-appb-000002
Figure PCTCN2018094447-appb-000003
Figure PCTCN2018094447-appb-000004
具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
除非特别指明，本发明中所使用的分子生物学实验方法和免疫检测法，基本上参照J.Sambrook等人，分子克隆：实验室手册，第2版，冷泉港实验室出版社，1989，以及F.M.Ausubel等人，精编分子生物学实验指南，第3版，John Wiley&Sons,Inc.，1995中所述的方法进行；限制性内切酶的使用依照产品制造商推荐的条件。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。
实施例1：编码抗TNFα/IL-6R双特异性重组蛋白的表达载体的构建
在本实施例中，首先获得表2中所示的抗TNFα亲本抗体(AB01、AB02)和抗IL-6R亲本抗体(AB03、AB04)，然后通过DNA重组技术，分别构建包含编码第一多肽链的核苷酸序列的表达载体和编码第二多肽链的核苷酸序列的表达载体，以获得本发明的重组蛋白。
各亲本抗体(AB01、AB02、AB03、AB04)的编码核酸序列由南京金斯瑞生物技术服务公司全基因合成，各亲本抗体可变区及恒定区序列参照表2。
表2：亲本抗体的可变区及恒定区序列
Figure PCTCN2018094447-appb-000005
按照表3中所示各重组蛋白的构建方式构建编码该重组蛋白的第一多肽链的核苷酸序列和编码第二多肽链的核苷酸序列。其中，对于各重组蛋白中的scFv，其VH、VL通过肽接头(SEQ ID NO:33，氨基酸序列为：GGGGSGGGGSGGGGSGGGGS)相连接，并且通过PCR定点突变方法将该scFv的亲本抗体的VH第44位和VL第100位氨基酸分别突变为半胱氨酸(Cys，C)，从而使得该scFv的VH与VL之间形成二硫键。表3中所示各重组蛋白的构建方式例举见图1。
表3：各重组蛋白的构建方式
Figure PCTCN2018094447-appb-000006
Figure PCTCN2018094447-appb-000007
注：AB01-scFv表示亲本抗体AB01来源的scFv，其可变区与AB01的区别在于：该scFv的VH第44位和VL第100位氨基酸分别为半胱氨酸；其他类似的表述具有类似的含义。
具体而言，作为示例地部分重组蛋白的核酸构建体的构建方式如下：
将编码AB01-scFv的核苷酸序列通过编码接头S1的核苷酸序列连接于编码AB03的重链的核苷酸序列的5’端或3’端，以分别构建编码TNA或TCA的第一多肽链的核苷酸序列；其中AB01-scFv从N端到C端的顺序为VL-linker-VH；编码TNA或TCA的第二多肽链的核苷酸序列为编码AB03的LC的核苷酸序列。
将编码AB01-scFv的核苷酸序列通过编码接头S1的核苷酸序列连接于编码AB03的轻链的核苷酸序列的5’端或3’端，以分别构建编码TLNA或TLCA的第一多肽链的核苷酸序列；其中AB01-scFv从N端到C端的顺序为VL-linker-VH；编码TLNA或TLCA的第二多肽链的核苷酸序列为编码AB03的HC的核苷酸序列。
将编码AB03-scFv的核苷酸序列通过编码接头S1的核苷酸序列连接于编码AB01的重链的核苷酸序列的5’端或3’端，以分别构建编码ANT或ACT的第一多肽链的核苷酸序列；其中AB03-scFv从N端到C端的顺序为VL-linker-VH；编码ANT或ACT的第二多肽链的核苷酸序列为编码AB01的LC的核苷酸序列。
将编码AB03-scFv的核苷酸序列通过编码接头S1的核苷酸序列连接于编码AB01的重链的核苷酸序列的5’端或3’端，以分别构建编码ANT-2或ACT-2的第一多肽链的核苷酸序列；其中AB03-scFv从N端到C端的顺序为VH-linker-VL；编码ANT-2或ACT-2的第二多肽链的核苷酸序列为编码AB01的LC的核苷酸序列。
将编码AB03-scFv的核苷酸序列通过编码接头S1的核苷酸序列连接于编码AB01的轻链的核苷酸序列的5’端或3’端，以分别构建编码ALNT或ALCT的第一多肽链的核苷酸序列；其中AB03-scFv从N端到C端的顺序为VL-linker-VH；编码ALNT或ALCT的第二多肽链的核苷酸序列为编码AB01的HC的核苷酸序列。
将编码AB02-scFv的核苷酸序列通过编码接头S1的核苷酸序列连接于编码AB04的重链的核苷酸序列的3’端，以构建编码SCG的第一多肽链的核苷酸序列；其中AB02-scFv从N端到C端的顺序为VL-linker-VH；编码SCG的第二多肽链的核苷酸序列为编 码AB04的LC的核苷酸序列。
将编码AB04-scFv的核苷酸序列通过编码接头S1的核苷酸序列连接于编码AB02的重链的核苷酸序列的3’端，以构建编码GCS的第一多肽链的核苷酸序列；其中AB04-scFv从N端到C端的顺序为VL-linker-VH；编码GCS的第二多肽链的核苷酸序列为编码AB02的LC的核苷酸序列。
将编码上述各第一多肽链或第二多肽链的核苷酸序列与编码鼠IgG-KAPPA信号肽的核苷酸序列连接(氨基酸序列SEQ ID NO:82，核酸序列SEQ ID NO:83)，并通过同源重组的方式分别导入pTT5质粒，以构建编码第一多肽链的表达载体及编码第二多肽链的表达载体。最终获得的各重组蛋白的氨基酸序列如表4所示。
表4:各重组蛋白的氨基酸序列
Figure PCTCN2018094447-appb-000008
实施例2：抗TNFα/IL-6R双特异性重组蛋白的表达
将生长状态良好、处于对数期的CHO-S细胞，离心并按2E6cells/ml接种200ml，第二天密度长至约4E6cells/ml。将实施例1中获得的待转染质粒通过0.22μm滤膜除菌过滤，取100μg第一多肽链重组质粒及100μg相应的第二多肽链重组质粒，加入20ml  CHOgro Complex Formation Solution(购自MIRUS公司)，加入1μg/ml PEIMAX(购自polysciencse公司)1.2ml，震荡混匀三次，静置10min，加入到200ml上述细胞培养物中。将培养物置于37℃、5％CO 2摇床培养，24h后加入20ml 10％Sheff-CHO PF ACF(购自KERRY公司)，继续培养4天后收获细胞培养物。
实施例3：抗TNFα/IL-6R双特异性重组蛋白的纯化
取实施例2中表达5天的CHO-S细胞培养物，先低速离心分离上清与细胞沉淀；再高速离心，得到澄清的料液。重组抗体通过亲和层析法(Protein A)和离子交换两步法进行纯化，纯化中使用的介质分别是GE公司生产的Mab Select SuRe和Millipore公司生产的Eshmuno CPX。各双特异性重组蛋白表达效率基本一致，均在22-45mg/L区间，且和同等条件抗IL-6R亲本抗体AB03的表达水平保持一致，说明各重组蛋白均可成功表达，且具有较高的表达效率，具体表达水平见表5，其中ANT和TCA的表达效率最高。分离纯化的重组蛋白通过超滤管浓缩并换液到PBS溶液中，SDS-PAGE电泳显示如图2A-2B所示，AB01非还原条带大小约为150kDa，还原后为50kDa(重链)及25kDa(轻链)；scFv连接在重链的双特异抗体蛋白非还原条带大小约为200kDa,还原条带大小为75kDa(重链-scFv)及25kDa(轻链)；scFv连接在轻链的双特异抗体蛋白非还原条带大小约为200kDa,还原条带大小为50kDa(重链)及50kDa(轻链-scFv)。条带大小符合预期，且无明显聚集降解，说明各双特异性重组蛋白均能高效表达，正确装配。
表5：双特异性重组蛋白的表达水平
抗体名称 获得蛋白量 表达效率
ACT 4.4mg 22mg/L
ANT 9mg 45mg/L
TCA 9mg 45mg/L
TNA 5.6mg 28mg/L
ALCT 4.2mg 21mg/L
ALNT 5.1mg 25.5mg/L
AB03 5mg 25mg/L
实施例4：抗TNFα/IL-6R双特异性重组蛋白的抗原结合生物学活性检测
本实施例以ELISA的方式分别检测各双特异性重组蛋白与其亲本抗体结合相同抗原的亲和力的差异，以及双特异性重组蛋白同时结合两种抗原的相对亲和力高低，以验证单独封闭一种抗原能力比亲本抗体是否有所下降；是否能同时封闭两种抗原，阻断两种 信号通路，在治疗类风湿性关节炎等免疫系统疾病时产生协同作用。
4.1双特异性重组蛋白对TNFα的结合活性检测
将重组TNFα蛋白(购自北京义翘神州公司)以50ng/孔加入96孔酶标板(购自thermo公司)，4℃包被过夜；次日，弃孔内溶液，用洗涤缓冲液(含有0.05％Tween-20的磷酸盐缓冲液)洗1次，拍干；加入含2％BSA的PBS溶液，100μl/孔，37℃封闭2h后拍干；各双特异性重组蛋白以及亲本抗体AB01和AB03以1000ng/ml起始，做3倍稀释，共11个梯度，100μl/孔；将酶标板放入37℃孵育2h，拍干，洗涤缓冲液洗涤3次；加入HRP偶联Goat Anti-Human IgG(H+L)溶液，100μl/孔，37℃孵育1h；100μl/孔加入TMB溶液，室温反应约5min；100μl/孔加入终止液，并放入酶标仪，读OD450吸光值。实验数据通过GraphPad prism5拟合曲线并计算EC50。
结果如表6所示，所检测的各重组蛋白结合TNFα的活性EC50与抗TNFα亲本抗体AB01的EC50相当，表明本发明的重组蛋白整体上保持了与亲本抗体同等优良的对TNFα的结合活性。
表6：重组蛋白对TNFα的结合活性
重组蛋白/亲本抗体 EC50(pM) 与AB01的比值
AB01 95.3 1
ACT 124.9 1.31
ANT 119.6 1.25
TCA 194.2 2.04
TNA 103.8 1.09
4.2双特异性重组蛋白对IL-6R的结合活性检测
将重组IL6R-mFC蛋白(获得自科伦研究院)以200ng/孔加入96孔酶标板，4℃包被过夜；次日，弃孔内溶液，用洗涤缓冲液洗1次，拍干；加入含2％BSA的PBS溶液，100μl/孔，37℃封闭2h后拍干；双特异性重组蛋白、抗IL-6R亲本抗体AB03及AB04以4000ng/ml起始，做4倍稀释，共11个梯度，100μl/孔；将酶标板放入37℃孵育2h，拍干，洗涤缓冲液洗涤3次；加入HRP偶联Goat Anti-Human IgG(H+L)溶液，37℃孵育1h；加入TMB溶液，室温反应约5min；加入终止液，并放入酶标仪，读OD450吸光值。实验数据通过GraphPad prism5拟合曲线并计算EC50。
结果如表7所示，所检测的各重组蛋白结合IL-6R的EC50与抗IL-6R亲本抗体AB03的EC50相当，表明本发明的重组蛋白整体上保持了与亲本抗体同等优良的对IL- 6R的结合活性。
表7：重组蛋白对IL-6R的结合活性
重组蛋白/亲本抗体 EC50(pM) 与AB03的比值
AB03 52.76 1
ACT 124.1 2.35
ANT 59.3 1.12
TCA 80.9 1.53
TNA 60.4 1.14
4.3双特异性重组蛋白同时结合TNFα及IL-6R的活性检测
将重组TNFα蛋白以50ng/孔，4℃过夜包被于96孔酶标板；次日，弃孔内溶液，用洗涤缓冲液(含有0.05％Tween-20的磷酸盐缓冲液)洗1次，拍干；加入含2％BSA的PBS溶液，100μl/孔，37℃封闭2h后拍干；双特异性重组蛋白以2000ng/ml起始，做3倍稀释，共11个梯度，100μl/孔；将酶标板放入37℃孵育2h，拍干，洗涤缓冲液洗涤3次；每孔加IL6R-mFc抗原(科伦)0.8μg/ml，100μl/孔，37℃孵育2h，拍干，洗涤缓冲液洗涤3次；加入HRP偶联Goat Anti-Mouse FC(购自Thermo公司)溶液，37℃孵育1h；加入TMB溶液，室温反应约5min；加入终止液，并放入酶标仪，读OD450吸光值。实验数据通过GraphPad prism5拟合曲线并计算EC50。
结果如表8所示，各双特异性重组蛋白均能同时结合TNFα及IL-6R两种抗原，其中，ACT和ANT同时结合TNFα及IL-6R两种抗原活性最高，ALCT及ALNT结合活性稍弱于其他重组蛋白，但EC50都在pM级，上述结果表明本发明的双特异性重组蛋白结合一种抗原后，不影响第二种抗原结合，能同时高效的结合两种抗原，保留了与亲本抗体同等优良的结合活性，因此其可以通过同时中和在类风湿性关节炎等疾病中的起关键作用的TNFα及IL-6R炎症因子，以同时抑制两条信号通路，从而特别适用于与过度的TNFα和/或IL-6活性相关的疾病的治疗。
表8：双特异性重组蛋白同时结合TNFα及IL-6R的活性
抗体名称 EC50(pM)
ACT 86.8
ANT 104.6
TCA 112.3
TNA 161.9
ALCT 250.0
ALNT 284.3
实施例5：双特异性重组蛋白在细胞水平上对抗原的结合活性检测
本实施例中，通过流式细胞仪检测双特异性重组蛋白是否能正常结合细胞表面抗原。
5.1双特异性重组蛋白对细胞表面TNFα的结合活性检测
取状态良好的细胞表面表达TNFα的CHO细胞(所述细胞通过将TNFα表达载体导入CHO细胞构建获得)，离心，PBS洗涤一次。用含0.5％BSA的PBS重悬，按50μl/孔，3E6cells/孔加入96孔板；各双特异性重组蛋白、抗TNFα亲本抗体AB01及AB02以1μM起始，做3倍稀释，共10个梯度，50μl/孔加入含细胞的96孔板，混匀，4℃孵育1小时；PBS洗涤1次，加入50μl FITC anti-humanIgG1抗体(购自Biolegend公司)，4℃孵育30分钟；PBS洗涤3次后用流式细胞仪(beckmanCyto FLEX)进行检测。
结果显示，经检测的各重组蛋白结合细胞表面TNFα的活性至少与抗TNFα亲本抗体相当，表明本发明的重组蛋白至少保持了与亲本抗体同等优良的对细胞表面TNFα的结合活性。特别地，ANT在细胞水平上结合细胞表面TNFα的EC50值甚至低于亲本抗体(如表9所示)，显示出更优的对TNFα的结合活性。
表9：重组蛋白结合细胞表面TNFα活性
重组蛋白/亲本抗体 EC50(nM) 与AB01的比值
AB01 4.37 1
ANT 3.06 0.70
5.2双特异性重组蛋白对细胞表面IL-6R的结合活性检测
取状态良好的细胞表面表达IL-6R的CHO细胞(所述细胞通过将IL-6R表达载体导入CHO细胞构建获得)，离心，PBS洗涤一次。用含0.5％BSA的PBS重悬，按50μl/孔，3E6cells/孔加入96孔板；双特异性重组蛋白、抗IL-6R亲本抗体AB03及AB04以1μM起始，做3倍稀释，共10个梯度，50μl/孔加入含细胞的96孔板，混匀，4℃孵育1小时；PBS洗涤1次，加入50μl FITC anti-humanIgG1抗体(购自Biolegend公司)，4℃孵育30分钟；PBS洗涤3次后用流式细胞仪(beckmanCyto FLEX)进行检测。
结果显示，经检测的各重组蛋白结合细胞表面IL-6R的活性至少与抗IL-6R亲本抗体相当，表明本发明的重组蛋白至少保持了与亲本抗体同等优良的对细胞表面IL-6R的结合活性。特别地，ANT在细胞水平上结合细胞表面IL-6R的EC50值甚至低于亲本抗体(如表10所示)，显示出更优的对IL-6R的结合活性。
表10：重组蛋白结合细胞表面IL-6R活性
抗体名称 EC50(nM) 与AB03的比值
AB03 6.72 1
ANT 4.64 0.69
实施例6：热变性温度检测
将双特异性重组蛋白及亲本抗体AB01用PBS溶液稀释至0.7mg/ml，将
Figure PCTCN2018094447-appb-000009
Orange Protein Gel Stain(thermo S6651)用蒸馏水稀释至40×。取0.2ml离心管，依次加入稀释样品12.5μl，稀释染料4.2μl，蒸馏水8.3μl。混匀后放入荧光定量PCR仪(thermo 7500),设置反应参数25℃3min,以1％速率上升至95℃，95℃2min。
结果如表11所示，以AB01为第一抗体的ACT、ANT、ALCT及ALNT热稳定性优于AB03为第一抗体的TCA、TNA，与亲本抗体AB01及AB03的TM值更为相近。
表11：双特异性重组蛋白的热变性温度
重组蛋白/亲本抗体 AB01 AB03 TNA TCA ACT ANT ALCT ALNT
Tm(℃) 71.41 69.18 58.83 59.04 71.87 72.09 72.75 73.17
实施例7：双特异性重组蛋白的细胞生物学活性实验
本实施例为验证双特异性重组蛋白功能活性，通过细胞水平实验检测双特异性重组蛋白对TNFα及IL-6R抑制活性。
7.1在L929细胞中中和TNFα诱导的细胞毒性
将重组人TNFα诱导加入L929细胞中孵育18-24h，以诱导TNFα对L929细胞的细胞毒性；加入双特异性重组蛋白中和TNFα后，细胞毒性减弱至消除。
取状态良好的L929细胞，用RPMI1640+2％FBS重悬，并调至密度为3×10 5cells/ml，100μl/孔铺入96孔板,37℃培养箱过夜；向RPMI1640+2％FBS中加入终浓度为0.4μg/ml的Actinomycin D，加入终浓度为6ng/ml的重组TNFα蛋白；将双特异性重组蛋白及抗TNFα亲本抗体AB01调至2μg/ml，并进行3倍连续稀释，共稀释8个梯度，L929细胞每孔加入100μl，37℃培养箱孵育48h；加入20μl CCK8试剂检测细胞活力，反应16-20小时读数。使用GrapadPrism5计算抗体中和TNFα的EC50值。
结果如表12所示，双特异性重组蛋白中和TNFα的效果与抗TNFα亲本抗体基本在同一水平，ANT和ACT活性优于ALNT和ALCT，上述结果表明本发明的重组蛋白保持了亲本抗体的生物学活性。
表12：重组蛋白中和TNFα诱导的L929细胞毒性
重组蛋白 重组蛋白EC50(pM) AB01 EC50(pM) 与AB01的比值
ANT 85.05 64.19 1.33
ACT 79.39 67.76 1.17
ALNT 213.3 74.42 2.87
ALCT 106.0 74.42 1.42
7.2在U266细胞中抑制IL6诱导的细胞增殖
培养U266细胞时，加入适当浓度重组IL-6蛋白，能刺激细胞生长加速；加入双特异性重组蛋白封闭细胞表面IL-6R后，细胞生长将减慢。
取状态良好的U266细胞，用RPMI1640+5％FBS重悬，调整浓度至8×10 4cells/ml，100μl/孔铺入96孔板；向RPMI1640+5％FBS中加入终浓度为10ng/ml的重组IL6；将双特异性重组蛋白及抗IL-6R亲本抗体AB03调至200μg/ml，并进行3倍连续稀释，共稀释8个梯度，U266细胞每孔加入100μl，37℃培养箱孵育48h；加入20μl CCK8试剂检测细胞活力，反应4小时读数。使用GraphpadPrism5计算抗体中和IL-6R的IC50值。
由于不能达到上下平台，无法推算出准确IC50值。由图3中各个重组蛋白与亲本抗体趋势对比可以看出，四种双特异性重组蛋白封闭IL-6R和抗IL-6R的能力与亲本抗体基本一致。上述结果表明本发明的重组蛋白至少保持了亲本抗体的生物学活性。
实施例8：双特异性重组蛋白的体内代谢实验
实验采用25只雄性SD大鼠，随机分为7组。分别经皮下注射给予2.5mg/kg的ANT和亲本抗体AB01。于给药前、给药后1h、4h、8h、Day1、Day3、Day5、Day8、Day11、Day15、Day22、Day29、Day36尾静脉采血，采集血样经1000-3000×g 4℃离心10min，收集上清，置于-80℃条件下保存；通过ELISA法检测血药浓度，并拟合药时曲线，计算药代动力学参数，评价双特异性重组蛋白在大鼠体内的药代动力学行为。
结果如表13所示，由于检测线未达到下平台，无法算出准确半衰期，但由AUC(药物暴露量)可知，ANT在大鼠体内代谢水平和亲本抗体AB01基本一致。
表13：双特异性重组蛋白的大鼠体内代谢结果
Figure PCTCN2018094447-appb-000010
Figure PCTCN2018094447-appb-000011
实施例9：双特异性重组蛋白ADCC活性的检测
细胞处理：取Jurkat-NFAT/CD16a细胞、CHO-S-TNFα细胞，CHO-S-IL6R细胞细胞(所有细胞均由四川科伦博泰生物医药股份有限公司制备)，离心，用1640+1％FBS培养基重悬，计数后分别稀释调整细胞至2×10 6/ml、0.5×10 6/ml、0.5×10 6/ml、0.5×10 6/ml。靶细胞铺板：CHO-S-TNFα细胞50μl/孔，CHO-S-IL6R细胞50ul/孔，每孔总计5×10 4个细胞。效应细胞铺板：Jurkat-NFAT/CD16a细胞50μl/孔，每孔总计1×10 5个细胞。加入检测抗体：稀释AB01、ACT、ANT以及IgG对照抗体至200μg/ml，依次2倍稀释，共11个浓度点。向孔中加入50μl稀释后抗体(初始终浓度50μg/ml)，最后一孔加入检测Buffer。检测：细胞孵育5小时后取出，检测区每孔加入40μl One-glo检测试剂(Promega,Cat:E6120)，酶标仪上机检测。
表14：双特异性重组蛋白ADCC活性的检测
重组蛋白/亲本抗体 EC50(nM) 与AB01的比值
AB01 2.5 1
ACT 18.9 7.56
ANT 3.1 1.24
IgG对照 N/A N/A
结果如表14所示。结果显示，ANT分子的ADCC的活性显著优于ACT，与AB01活性相当，这一结果表明ANT分子保留了亲本抗体完整的ADCC活性。
实施例10：双特异性重组蛋白C1q活性的检测
按下表用包被液CBS(碳酸盐缓冲溶液)包板，100μl每孔，4℃过夜。300μL PBS清洗一次，加入100μL PBS(2％BSA)，37℃封闭2小时。用PBS(2％BSA)稀释C1q(PROSPEC,货号：PRO-554)至5μg/ml，100μL/孔，37℃孵育2小时。300μL PBST清洗3次，用PBS(2％BSA)稀释1:300羊抗C1q-HRP(Abcam，货号Ab46191)，加入100μL对应的孔中，37℃孵育1小时。300μL PBST清洗5次，加入100μL TMB对应的孔中，室温显色3分钟，加入50μL 1M H 2SO 4终止，酶标仪450nm读数。
表15：双特异性重组蛋白C1q活性的检测
重组蛋白/亲本抗体 EC50(nM) 与AB01的比值
AB01 7.2 1
AB03 7.6 --
ACT 6.1 0.85
ANT 5.3 0.74
IgG对照 N/A N/A
结果如表15所示。结果显示，ANT分子的C1q的结合活性略强于ACT，ANT和ACT的结合活性均略优于亲本抗体AB01、AB03，这一结果表明ANT和ACT保留了亲本抗体完整的CDC活性。
实施例11：双特异性重组蛋白在恒河猴关节炎模型中的体内活性评价
本实施例评价本发明的双特异性重组蛋白对牛II型胶原诱导的恒河猴关节炎疾病进程的影响。具体而言，将重组蛋白以每周1次的频率静脉注射给关节炎造模成功的恒河猴，每周1次测定动物体重和采集血样，进而计算重组蛋白对恒河猴关节炎的治疗药效(抗炎疗效)。
受试药物：
药物名称、来源、配置方法：ANT，根据动物体重，按照5mg/kg，1ml/kg的剂量，稀释母液得到处理品溶液。
实验动物及饲养：
恒河猴(四川横竖生物科技股份有限公司，生产许可证号：SCXK(川)2014-029)；
动物房(四川抗菌素研究所，实验动物试用许可证号：SYXK(川)2014-021)。
造模试剂：
牛II型胶原(免疫接种等级胶原)(Chondrex)，弗氏完全佐剂(Sigma)。
关节炎模型的造模方法：
牛II型胶原溶解于0.1M乙酸溶液中，然后加入等体积的弗氏完全佐剂，低温高速搅拌形成均匀稳定的终浓度为2mg/ml的乳剂。在4只恒河猴背部皮下注射乳剂10个点，0.2ml/点，即胶原注射量为4mg。胶原注射当天记为D1。第1次胶原注射后2周(第15天，D15)按上述方法完成第2次胶原注射。期间每周1次称重和血清收集。根据表16监测评分指标，造模成功的动物会出现体重降低、关节炎肿胀、CRP水平上升和贫血等多种症状，造模结果如表17，得到造模成功的恒河猴3只。
表16：恒河猴关节炎疾病进程的临床评分标准
Figure PCTCN2018094447-appb-000012
在关节炎造模成功的动物中，16063号动物发病迅速，未能及时给予ANT治疗，在D22天死亡。对于其他两只造模成功的动物，根据其关节炎发病的进程，16066号动物选择在其关节炎病程发展的早期给药，从D28开始给药；16068号动物选择在其关节炎病程发展的晚期给药，从D42开始给药。对恒河猴给予ANT重组蛋白干预的方案为：给予5mg/kg的ANT，给药体积为1ml/kg，给药途径为单次静脉注射，给药频率为每周1次，共计4次。
表17：恒河猴造模和给药情况
Figure PCTCN2018094447-appb-000013
每周1次测量动物体重和采集血样，通过观察动物前足、后足关节肿胀情况并予以临床评分，关节炎评分标准见表16，以及分析血清中CRP(C反应蛋白)水平和血液学RBC(红细胞)，HGB(血红蛋白)，HCT(红细胞压积)指标来评价ANT的治疗药效。每天观察记录动物死亡情况。恒河猴造模和给药情况见表17、图4。体重、关节炎评分、CRP和血液学指标变化情况见图5-图10。
结果显示，16067号动物造模未成功，所有监测指标在胶原注射后很快恢复正常值。而在关节炎造模成功的动物中，16063号动物发病迅速，未能及时给予发明ANT治疗，最终在D22天死亡；16066号动物在疾病早期及时给予发明ANT治疗(D28天首次给药，每周1次，给药4次)，给药后关节炎肿胀缓解，体重逐渐恢复，CRP水平回归正常水平，贫血症状消失；16068号动物在疾病晚期给予发明ANT治疗(D42天首次给药，每周1次，给药5次)，虽然给药后关节炎肿胀未能明显缓解、体重未明显恢复、以及血液学指 标未回归正常水平，但CRP水平明显降低，而且动物未死亡。
以上结果表明在恒河猴关节炎模型中，本发明的ANT具有良好的抗炎药效。与亲本抗体相比，本发明双特异性重组蛋白具有更优的治疗活性。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。

Claims (25)

  1. 一种重组蛋白，其包含：
    1)特异性结合第一抗原的第一抗体，所述第一抗体包括重链(HC)和轻链(LC)；和
    2)特异性结合第二抗原的scFv；
    其中，所述scFv连接于第一抗体的重链或轻链的N端或C端；
    所述第一抗原为TNFα，并且所述第二抗原为IL-6R或IL-6；或者，第一抗原为IL-6R或IL-6，并且所述第二抗原为TNFα；
    优选地，所述重组蛋白包含1个所述第一抗体和2个所述scFv；并且，所述第一抗体包括两条HC和两条LC，其中所述第一抗体的一条HC的VH与一条LC的VL形成抗原结合部位，另一条HC的VH与另一条LC的VL形成抗原结合部位；
    优选地，一个所述scFv连接于所述第一抗体的重链或轻链的N端，另一个所述scFv连接于所述第一抗体的重链或轻链的C端；
    优选地，每个所述scFv分别连接于所述第一抗体的两条重链或两条轻链的N端；或者，每个所述scFv分别连接于所述第一抗体的两条重链或两条轻链的C端。
  2. 权利要求1所述的重组蛋白，其中所述第一抗体的重链包含重链可变区(VH)和重链恒定区(CH)，并且所述轻链包含轻链可变区(VL)和轻链恒定区(CL)；
    优选地，所述第一抗体为全长抗体。
  3. 权利要求1或2所述的重组蛋白，其中所述第一抗体的重链为IgG同种型，例如IgG1、IgG2、IgG3或IgG4，例如人IgG同种型；和/或，所述第一抗体的轻链为Kappa同种型，例如人Kappa同种型。
  4. 权利要求1-3任一项所述的重组蛋白，其中所述第一抗体的两条HC包含相同的CDR；和/或，所述第一抗体的两条LC包含相同的CDR；
    优选地，所述第一抗体的两条HC包含相同的VH；和/或，所述第一抗体的两条LC包含相同的VL；
    优选地，所述第一抗体的两条HC具有相同的氨基酸序列；和/或，所述第一抗体的 两条LC具有相同的氨基酸序列。
    优选地，两个所述scFv具有相同或不相同的氨基酸序列；更优选地，两个所述scFv具有相同的氨基酸序列。
  5. 权利要求1-4任一项所述的重组蛋白，其中所述重组蛋白包含两条第一多肽链和两条第二多肽链，其中对于所述每条多肽链：
    a)所述第一多肽链各自独立地包含所述第一抗体的重链(HC)和所述scFv；和
    b)所述第二多肽链各自独立地包含所述第一抗体的轻链(LC)；
    其中，所述scFv通过接头S1与所述第一抗体的HC的N端或C端相连；
    或者，
    i)所述第一多肽链各自独立地包含所述第一抗体的轻链(LC)和所述scFv；和
    ii)所述第二多肽链各自独立地包含所述第一抗体的重链(HC)；
    其中，所述scFv通过接头S1与所述第一抗体的LC的N端或C端相连；
    优选地，所述scFv具有结构：NH 2-VH-S2-VL-COOH或NH 2-VL-S2-VH-COOH，其中所述S2为接头；
    优选地，所述重组蛋白包含两条相同的第一多肽链和两条相同的第二多肽链。
  6. 权利要求5所述的重组蛋白，其中所述接头S1和/或S2为肽接头，例如具有如(G mS n) x所示的氨基酸序列，其中m、n各自独立地选自1～8的整数(例如，1、2、3、4、5、6、7或8)，x独立地选自1～20的整数(例如，1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20)；
    优选地，所述所述接头S1和/或S2具有如(G 4S) x所示的氨基酸序列，x独立地选自1-6的整数；
    优选地，所述S1和/或S2具有选自下列的氨基酸序列：SEQ ID NO:33、SEQ ID NO:34和SEQ ID NO:35；
    优选地，所述接头S2具有如SEQ ID NO:33所示的氨基酸序列，且当所述scFv与所述第一抗体的重链或轻链的N端连接时，所述接头S1具有如SEQ ID NO:34所示的氨基酸序列，当所述scFv与所述第一抗体的重链或轻链的C端连接时，所述接头S1如SEQ ID NO:35所示的氨基酸序列。
  7. 权利要求1-6任一项所述的重组蛋白，其中所述scFv的VH与VL之间存在二硫键；
    优选地，所述scFv的位于VH第44位的氨基酸和位于VL第100位的氨基酸分别为半胱氨酸，其中提及的氨基酸位置是根据Kabat编号系统的位置；并且，所述scFv的VH与VL通过分别位于VH第44位和VL第100位的2个半胱氨酸残基之间所形成的二硫键连接。
  8. 权利要求1-7任一项所述的重组蛋白，其中所述第一抗体特异性结合TNFα，并且所述scFv特异性结合IL-6R，其中：
    所述第一抗体包含：
    (1)如SEQ ID NO:6所示的HCDR1；如SEQ ID NO:7所示的HCDR2；以及，如SEQ ID NO:8所示的HCDR3；如SEQ ID NO:2所示的LCDR1；如SEQ ID NO:3所示的LCDR2；以及，如SEQ ID NO:4所示的LCDR3；或，
    (2)如SEQ ID NO:14所示的HCDR1；如SEQ ID NO:15所示的HCDR2；以及，如SEQ ID NO:16所示的HCDR3；如SEQ ID NO:10所示的LCDR1；如SEQ ID NO:11所示的LCDR2；以及，如SEQ ID NO:12所示的LCDR3；
    并且，所述scFv包含：
    (i)如SEQ ID NO:22所示的HCDR1；如SEQ ID NO:23所示的HCDR2；以及，如SEQ ID NO:24所示的HCDR3；如SEQ ID NO:18所示的LCDR1；如SEQ ID NO:19所示的LCDR2；以及，如SEQ ID NO:20所示的LCDR3；或，
    (ii)如SEQ ID NO:30所示的HCDR1；如SEQ ID NO:31所示的HCDR2；以及，如SEQ ID NO:32所示的HCDR3；如SEQ ID NO:26所示的LCDR1；如SEQ ID NO:27所示的LCDR2；以及，如SEQ ID NO:28所示的LCDR3。
  9. 权利要求8所述的重组蛋白，其中，所述重组蛋白包含1个所述第一抗体和2个所述scFv；并且，所述第一抗体包括两条HC和两条LC，其中所述第一抗体的一条HC的VH与一条LC的VL形成抗原结合部位，另一条HC的VH与另一条LC的VL形成抗原结合部位；每个所述scFv分别连接于所述第一抗体的两条重链的N端；且，
    所述第一抗体包含：如SEQ ID NO:6所示的HCDR1；如SEQ ID NO:7所示的HCDR2；以及，如SEQ ID NO:8所示的HCDR3；如SEQ ID NO:2所示的LCDR1； 如SEQ ID NO:3所示的LCDR2；以及，如SEQ ID NO:4所示的LCDR3；并且，所述scFv包含：如SEQ ID NO:22所示的HCDR1；如SEQ ID NO:23所示的HCDR2；以及，如SEQ ID NO:24所示的HCDR3；如SEQ ID NO:18所示的LCDR1；如SEQ ID NO:19所示的LCDR2；以及，如SEQ ID NO:20所示的LCDR3；
    优选的，所述每个scFv通过接头S1与所述第一抗体的每条重链的N端连接；
    优选的，所述scFv的结构为NH 2-VL-S2-VH-COOH，其中所述S2为接头；
    优选地，所述重组蛋白包含两条第一多肽链和两条第二多肽链，其中对于所述每条多肽链：
    a)所述第一多肽链各自独立地包含所述第一抗体的重链(HC)和所述scFv，所述scFv任选地通过接头S1与所述第一抗体的HC的N端连接；和
    b)所述第二多肽链各自独立地包含所述第一抗体的轻链(LC)；
    优选地，所述重组蛋白包含两条相同的第一多肽链和两条相同的第二多肽链。
  10. 权利要求1-9任一项所述的重组蛋白，其中：所述第一抗体包含如SEQ ID NO:5所示的VH和如SEQ ID NO:1所示的VL；并且，所述scFv包含：
    (1)如SEQ ID NO:91所示的VH和如SEQ ID NO:90所示的VL；或，
    (2)如SEQ ID NO:21所示的VH和如SEQ ID NO:17所示的VL。
  11. 权利要求1-8任一项所述的重组蛋白，其中：所述第一抗体包含如SEQ ID NO:13所示的VH和如SEQ ID NO:9所示的VL；并且，所述scFv包含：
    (1)如SEQ ID NO:93所示的VH和如SEQ ID NO:92所示的VL；或，
    (2)如SEQ ID NO:29所示的VH和如SEQ ID NO:25所示的VL。
  12. 权利要求1-7任一项所述的重组蛋白，其中所述第一抗体特异性结合IL-6R，并且所述scFv特异性结合TNFα，其中：
    所述第一抗体包含：
    (1)如SEQ ID NO:22所示的HCDR1；如SEQ ID NO:23所示的HCDR2；以及，如SEQ ID NO:24所示的HCDR3；如SEQ ID NO:18所示的LCDR1；如SEQ ID NO:19所示的LCDR2；以及，如SEQ ID NO:20所示的LCDR3；或，
    (2)如SEQ ID NO:30所示的HCDR1；如SEQ ID NO:31所示的HCDR2；以及， 如SEQ ID NO:32所示的HCDR3；如SEQ ID NO:26所示的LCDR1；如SEQ ID NO:27所示的LCDR2；以及，如SEQ ID NO:28所示的LCDR3；
    并且，
    所述scFv包含：
    (i)如SEQ ID NO:6所示的HCDR1；如SEQ ID NO:7所示的HCDR2；以及，如SEQ ID NO:8所示的HCDR3；如SEQ ID NO:2所示的LCDR1；如SEQ ID NO:3所示的LCDR2；以及，如SEQ ID NO:4所示的LCDR3；或，
    (ii)如SEQ ID NO:14所示的HCDR1；如SEQ ID NO:15所示的HCDR2；以及，如SEQ ID NO:16所示的HCDR3；如SEQ ID NO:10所示的LCDR1；如SEQ ID NO:11所示的LCDR2；以及，如SEQ ID NO:12所示的LCDR3。
  13. 权利要求1-7或12任一项所述的重组蛋白，其中：所述第一抗体包含如SEQ ID NO:21所示的VH和如SEQ ID NO:17所示的VL；并且，所述scFv包含：
    (1)如SEQ ID NO:87所示的VH和如SEQ ID NO:86所示的VL；或，
    (2)如SEQ ID NO:5所示的VH和如SEQ ID NO:1所示的VL。
  14. 权利要求1-7或12任一项所述的重组蛋白，其中：所述第一抗体包含如SEQ ID NO:29所示的VH和如SEQ ID NO:25所示的VL；并且，所述scFv包含：
    (1)如SEQ ID NO:89所示的VH和如SEQ ID NO:88所示的VL；或，
    (2)如SEQ ID NO:13所示的VH和如SEQ ID NO:9所示的VL。
  15. 权利要求1-14任一项所述的重组蛋白，其中所述第一抗体包含：如SEQ ID NO:101所示的CH；和/或，如SEQ ID NO:100所示的CL。
  16. 权利要求1-15任一项所述的重组蛋白，其中所述重组蛋白包含：
    (1)如SEQ ID NO:36所示的第一多肽链和如SEQ ID NO:42所示的第二多肽链；
    (2)如SEQ ID NO:38所示的第一多肽链和如SEQ ID NO:42所示的第二多肽链；
    (3)如SEQ ID NO:44所示的第一多肽链和如SEQ ID NO:48所示的第二多肽链；
    (4)如SEQ ID NO:46所示的第一多肽链和如SEQ ID NO:48所示的第二多肽链；
    (5)如SEQ ID NO:50所示的第一多肽链和如SEQ ID NO:56所示的第二多肽链；
    (6)如SEQ ID NO:52所示的第一多肽链和如SEQ ID NO:56所示的第二多肽链；
    (7)如SEQ ID NO:58所示的第一多肽链和如SEQ ID NO:56所示的第二多肽链；
    (8)如SEQ ID NO:60所示的第一多肽链和如SEQ ID NO:56所示的第二多肽链；
    (9)如SEQ ID NO:64所示的第一多肽链和如SEQ ID NO:68所示的第二多肽链；
    (10)如SEQ ID NO:66所示的第一多肽链和如SEQ ID NO:68所示的第二多肽链；
    (11)如SEQ ID NO:94所示的第一多肽链和如SEQ ID NO:68所示的第二多肽链；
    (12)如SEQ ID NO:95所示的第一多肽链和如SEQ ID NO:68所示的第二多肽链；
    (13)如SEQ ID NO:96所示的第一多肽链和如SEQ ID NO:42所示的第二多肽链；
    (14)如SEQ ID NO:97所示的第一多肽链和如SEQ ID NO:42所示的第二多肽链；
    (15)如SEQ ID NO:98所示的第一多肽链和如SEQ ID NO:48所示的第二多肽链；
    (16)如SEQ ID NO:99所示的第一多肽链和如SEQ ID NO:48所示的第二多肽链；
    (17)如SEQ ID NO:70所示的第一多肽链和如SEQ ID NO:74所示的第二多肽链；或
    (18)如SEQ ID NO:76所示的第一多肽链和如SEQ ID NO:80所示的第二多肽链。
  17. 权利要求1-16任一项所述的重组蛋白，其中所述重组蛋白对TNFα和IL-6R具有与其各自的亲本抗体相比同等或者更高的亲和力；
    优选地，所述重组蛋白对细胞表面的TNFα和IL-6R具有与其各自的亲本抗体相比更高的亲和力。
  18. 权利要求1-17任一项所述的重组蛋白，其中所述重组蛋白具有与亲本抗体相比基本相同的热稳定性。
  19. 一种分离的核酸分子，其包含编码权利要求1-18任一项所述的重组蛋白的核苷酸序列；
    优选地，所述分离的核酸分子包含编码权利要求1-18任一项所述的重组蛋白的第一多肽链的核苷酸序列；
    优选地，所述分离的核酸分子包含编码权利要求1-18任一项所述的重组蛋白的第二多肽链的核苷酸序列。
  20. 一种载体，其包含权利要求19所述的分离的核酸分子。
  21. 宿主细胞，其包含权利要求19所述的分离的核酸分子或权利要求20所述的载体。
  22. 制备权利要求1-18任一项所述的重组蛋白的方法，其包括，在允许所述重组蛋白表达的条件下，培养权利要求21所述的宿主细胞，和从培养的宿主细胞培养物中回收所述重组蛋白。
  23. 药物组合物，其含有权利要求1-18任一项所述的重组蛋白，以及药学上可接受的载体和/或赋形剂；
    优选地，所述药物组合物还包含另外的药学活性剂，例如用于预防和/或治疗与过度的TNFα和/或IL-6活性相关的疾病的药物，例如抗炎药物或免疫抑制剂；
    优选地，所述与过度的TNFα和/或IL-6活性相关的疾病为炎性疾病或自身免疫性疾病，例如，类风湿性关节炎、胰岛素依赖型糖尿病、多发性硬化症、银屑病、炎症性肠病、溃疡性结肠炎、胰腺炎、重症肌无力、多发性肌炎、皮肌炎、Crohn病、自身免疫性血细胞减少、血管炎、系统性红斑狼疮或成人Still病。
  24. 权利要求1-18任一项所述的重组蛋白或权利要求23所述的药物组合物在制备药物中的用途，所述药物用于在受试者(例如人)中预防和/或治疗与过度的TNFα和/或IL-6活性相关的疾病(例如炎性疾病或自身免疫性疾病)，和/或用于体外或在受试者(例如人)体内抑制TNFα和IL-6R的活性；
    优选地，所述与过度的TNFα和/或IL-6活性相关的疾病为炎性疾病或自身免疫性疾病，例如，类风湿性关节炎、胰岛素依赖型糖尿病、多发性硬化症、银屑病、炎症性肠病、溃疡性结肠炎、胰腺炎、重症肌无力、多发性肌炎、皮肌炎、Crohn病、自身免疫性血细胞减少、血管炎、系统性红斑狼疮或成人Still病；
    优选地，所述受试者可以为哺乳动物，例如人。
  25. 一种用于在受试者(例如人)中预防和/或治疗与过度的TNFα和/或IL-6活性相关的疾病(例如炎性疾病或自身免疫性疾病)，和/或用于体外或在受试者(例如人)体内抑制TNFα和IL-6R的活性的方法，其中所述方法包括，给有此需要的受试者施用有效 量的权利要求1-18任一项所述重组蛋白，或者权利要求23所述的药物组合物；
    优选地，所述与过度的TNFα和/或IL-6活性相关的疾病为炎性疾病或自身免疫性疾病，例如，类风湿性关节炎、胰岛素依赖型糖尿病、多发性硬化症、银屑病、炎症性肠病、溃疡性结肠炎、胰腺炎、重症肌无力、多发性肌炎、皮肌炎、Crohn病、自身免疫性血细胞减少、血管炎、系统性红斑狼疮或成人Still病；
    优选地，所述受试者可以为哺乳动物，例如人。
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